One new unusual sesterterpenoid and four new sesquiterpene dimers from Inula britannica

Abstract

One new unusual sesterterpenoid (1), four new sesquiterpene dimers (2–5) together with nine known sesquiterpenes (6–14) were isolated from the aerial parts of Inula britannica. The structures of the new compounds were elucidated by detailed spectroscopic analysis, including HR-ESIMS and 2D-NMR spectroscopic methods. In addition, compounds 1–8 and 10–11 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages.

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Inula genus (Asteraceae) is an important genus of which there are more than 100 species distributed in Asia, Europe and Africa.¹ As one of the most popular traditional Chinese medicines (TCMs) of this genus, Inula britannica has been reported to treat bronchitis, digestive disorders and inflammation.^2,3 Various bioactive secondary metabolites, such as sesquiterpene lactones, have been isolated from this species.^4–6 Our pursuit of biologically active sesquiterpenoids from I. britannica resulted in the isolation of one new unusual sesterterpenoid (1), four new sesquiterpene dimers (2–5), together with nine known sesquiterpenes (6–14). In this paper, we described the isolation and structure elucidation of these new sesquiterpene dimers. In addition, anti-inflammatory activities of these isolates against LPS-induced NO production in RAW264.7 macrophages were also evaluated (Fig. 1).

Fig. 1 Structures of compounds 1–5.

Dibritannilactone A (1) was obtained as orthorhombic crystals. Its molecular formula C₂₇H₃₂O₆ was established by HRESIMS peak at m/z 453.2292 [M + H]⁺ (calcd for C₂₇H₃₃O₆, 453.2272), indicating twelve degrees of unsaturation. The IR spectrum showed bands characteristic of hydroxyl groups (3422 cm⁻¹), carbonyl groups (1765 and 1736 cm⁻¹) and olefinic bonds (1618 cm⁻¹). All the 27 carbon signals in ¹³C NMR spectrum (Table 1) were classified by DEPT and HMQC experiments as five methyls, three methylenes, ten methines and nine quarternary carbons, from which typical signals of two ester carbonyls, eight olefinic carbons and three oxygen-bearing carbons were identified. The ¹³C NMR spectrum also suggested the presence of an acetoxyl group (δ_C 170.3 and 21.2, δ_H 2.10), whose position was determined by HMBC experiment to be at C-2′ (Fig. 2). Besides, detailed analysis of 1D and 2D NMR data of the remaining 25 carbon signals indicated that they were assigned to two units, one sesquiterpene unit (A) and one monoterpene unit (B). The ¹H–¹H COSY spectrum of unit A showed the following correlations: H-2′/H-3′, H₂-6′/H-7′/H-8′/H₂-9′/H-10′/H₃-14′ and H-7′/H-11′/H₃-13′. In addition, HMBC correlations from H₃-13′ to C-7′, C-11′ and C-12′, H₃-14′ to C-1′, C-9′ and C-10′, H₃-15′ to C-1′, C-3′, C-4′ and C-5′ suggested the presence of a partial structure of sesquiterpene unit. The remaining signals of 1 were assigned to a methyl (C-10), a 1,3,4-trisubstituted aromatic ring, a methylene (C-9), an oxygenated methine (C-8) and a quarternary carbon (C-7). In the HMBC spectrum, correlations between H₃-10/C-1, C-2 and C-6, H-6/C-2, C-4, C-5 and C-10, H-3/C-1, C-5 and C-7, H-2/C-4 and C-10 were observed. These correlations suggested the existence of another partial structure of monoterpene unit. The connecting positions of the two units were established according to the following key correlations: in ¹H–¹H COSY spectrum, H-9 correlated to H-3′; in HMBC spectrum, correlations between H-8/C-7 and C-1′, H₂-9/C-1′, C-3′, C-4′, C-4 and C-7, H-3′/C-7, C-1′ and C-5′, which disclosed a new hexatomic ring (–C-7–C-9–C-3′–C-4′–C-5′–C-1′–). Therefore, the planar structure of 1 was constructed as shown in Fig. 2.

Table 1 ¹H and ¹³C NMR data for compounds 1 and 2

No.	1^a		2^b
	δH	δC	δH	δC
a Measured at 400 and 100 MHz respectively in CDCl3.b Measured at 400 and 100 MHz respectively in CD3OD.; δ in ppm; j in Hz within parentheses.
1		139.9 s	4.00 m; 3.95 m	65.6 t
2	6.65 s	111.7 d	1.55 m; 1.35 m	28.2 t
3		158.1 s	1.33 m; 1.09 m	33.4 t
4		131.0 s	2.73 m	35.4 d
5	6.48 d (7.6)	125.2 d		138.1 s
6	6.61 d (7.6)	121.7 d	4.23 s	64.7 d
7	2.25 s	21.5 q	2.74 m	52.9 d
8		59.7 s	5.07 m	78.5 d
9	6.25 s	104.0 d	2.46 m; 2.42 m	35.1 t
10	2.86 dd (12.8, 4.1); 1.55 m	35.5 t		131.3 s
11				56.5 s
12				181.3 s
13			2.08 m; 1.88 m	37.9 t
14			1.74 s	20.7 q
15			1.13 d (7.0)	20.0 q
1′		68.5 s		63.9 s
2′	4.58 s	84.7 d	4.52 brs	83.6 d
3′	2.75 m	47.3 d	2.93 d (1.2)	59.3 d
4′		139.2 s		134.4 s
5′		135.1 s		138.9 s
6′	2.48 brd (16.0)	24.5 t	2.72 m; 2.15 m	25.5 t
7′	0.58 m	41.8 d	2.41 m	44.3 d
8′	4.10 dt (11.5, 3.3)	80.8 d	4.57 dt (11.4, 3.6)	82.9 t
9′	2.00 m; 1.50 m	35.6 t	2.33 m; 1.88 m	37.3 t
10′	2.55 m	26.6 d	2.17 m	31.1 d
11′	2.20 m	39.9 d	2.20 m	41.8 d
12′		179.6 s		182.4 s
13′	1.05 d (7.8)	9.7 q	1.19 d (7.8)	10.1 q
14′	0.97 d (7.1)	19.5 q	1.06 d (7.3)	17.3 q
15′	1.88 s	13.4 q	1.58 d (1.0)	14.3 q
1′′		170.3 s		172.2 s
2′′	2.11 s	21.2 q	2.12 s	21.3 q
1′′′				172.7 s
2′′′			1.96 s	20.9 q

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Fig. 2 Key ¹H–¹H COSY and HMBC correlations of 1.

The stereochemistry of 1 was further confirmed by detailed analysis of NOESY spectra and an X-ray diffraction study (Fig. 3 and 4). In the NOESY spectrum, the key correlations of H-13′/H-8′/H-14′ and H-2′ and H-3′/H-15′ were in good agreement with the X-ray diffraction study. The absolute configuration was determined by X-ray crystallographic analysis (Fig. 4). All relevant chiral centers in 1 were assigned as 8R,2′S,7′R,8′S,10′S,11′S. Hence, compound 1 was given the name (8R,2′S,7′R,8′S,10′S,11′S)-dibritannilactone A.

Fig. 3 Key NOESY correlations of compound 1.

Fig. 4 Single-crystal X-ray structure (copper radiation) of 1.

Dibritannilactone B (2) was obtained as white amorphous powder. Its molecular formula C₃₄H₄₆O₉ was established from its HRESIMS peak at m/z 599.3187 [M + H]⁺ (calcd for C₃₄H₄₇O₉, 599.3215), accounting for twelve degrees of unsaturation. The IR spectrum showed the presence of hydroxyl groups (3440 cm⁻¹), carbonyl groups (1750 cm⁻¹) and olefinic bonds (1635 cm⁻¹). The ¹³C NMR and DEPT spectroscopic data of 2 showed great similarity to those of a known sesquiterpene dimer, inulanolide A (ref. 7) except for the α-methylene lactone functionality (Table 1). The absence of the Δ^11,13 exocyclic methylene group was confirmed by the upfield shifts of C-11′ and C-13′ and the downfield shift of C-12 in 2 compared with those of inulanolide A. NOESY correlations (Fig. S1†) of H-13′/H-8′ and H-14′ were observed. Other observed NOEs correlations suggested that 2 shared the same relative configuration with inulanolide A.

Dibritannilactone C (3) was obtained as white amorphous powder. Its molecular formula C₃₂H₄₄O₈ was established from its HRESIMS peak at m/z 579.2719 [M + Na]⁺ (calcd for C₃₂H₄₅O₈Na, 579.2723), accounting for eleven degrees of unsaturation. The IR spectrum showed the presence of hydroxyl groups (3439 cm⁻¹), carbonyl groups (1762 cm⁻¹) and olefinic bonds (1630 cm⁻¹). The ¹H and ¹³C NMR spectra of 3 were all comparable to those of 2 except for the presence of an hydroxyl group instead of the acetoxyl group which was attached to C-1 in 2 (Tables S1 and S2†).

The ¹H and ¹³C NMR spectra of dibritannilactone D (4) were also comparable to those of 2 except for the absence of the acetoxyl group which was attached to C-2′ in 2 (Tables S1 and S2†).

The ¹³C NMR and DEPT spectroscopic data of 5 were similar to those of a known sesquiterpene dimer, japonicone I,⁸ except for the α-methylene lactone functionality (Tables 1 and 2). The absence of the Δ^11,13 exocyclic methylene group was confirmed by the upfield shifts of C-11′ and C-13′ and the downfield shift of C-12 in 5, compared with those of japonicone I. NOESY (Fig. S2†) correlations of H-13′/H-8′ and H-14′ were also observed. Other observed NOEs correlations suggested that 5 shared the same relative configuration with japonicone I.

Table 2 Inhibitory effects of compounds 1–8 and 10–11 against LPS-induced NO production in RAW264.7 macrophages

Compounds	IC50^a (μM)
a Inhibitory effects of compounds 1–8 and 10–11 against LPS-induced NO production in RAW264.7 macrophages.b Positive control.
1	14.60
2	43.77
3	49.44
4	25.08
5	29.18
6	1.63
7	2.07
8	>50
10	3.80
11	10.86
Aminoguanidine^b	7.90

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The relative configuration of C-4 in compounds 2–5 could not be determined due to the rotatory nature of side chains. However, as one of the monomeric sesquiterpenoids composing compounds 2–5, britannilactone and 1-acetoxy-6α-hydroxyeriolanolide⁷ are abundant constituents in Inula britannica. And their absolute configurations were previously assigned since their single crystals were obtained. We have reasons to deduce that C-4 of compounds 2–5 possesses identical configurations to those of britannilactone and 1-acetoxy-6α-hydroxyeriolanolide because they probably were produced through same biosynthesis pathway. Nevertheless, the possibility of the existence of enantiomers cannot be excluded. Therefore, this is tentative assignment because of the absence of direct evidence.

Compounds 1–8 and 10–11 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages with aminoguanidine as positive control. As shown in Table 2, compounds 6, 7 and 10 exhibited significant inhibitory activities with IC₅₀ values of 1.63, 2.07 and 3.80 μM, respectively. Whereas, compound 1–5 and 8 showed moderate inhibitory effects with IC₅₀ values ranged from 10.86 to 49.44 μM.

In summary, dibritannilactones A–E (1–5), including one new unusual sesterterpenoid (1), four new sesquiterpene dimers (2–5) together with nine known ones (6–14) were obtained from aerial parts of I. britannica. By comparing physical and spectroscopic data with those reported in literatures, structures of known compounds were identified as 6α-(2-methybutyryloxy)-deacetylinulicin (6),⁹ 14-hydroxyinulicin (7),⁹ eupatolide (8),¹⁰ 3β-hydroxyivangustin (9),⁹ 3α-hydroxyivangustin (10),⁹ desacetyl-β-cyclopyrethrosin (11),¹¹ bigelovin (12),¹² 8-epi-helenali (13),¹² and aromaticin (14).¹³ Compounds 1–8 and 10–11 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages and the result displayed that 6, 7 and 10 exhibited significant inhibitory activities with IC₅₀ values of 1.63, 2.07 and 3.80 μM, respectively. 1–5 and 8 showed moderate inhibitory effects with IC₅₀ values ranged from 10.86 to 49.44 μM.

Acknowledgements

This work was supported by program NCET Foundation, NSFC (81230090 and 81102778), partially supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University, Shanghai Leading Academic Discipline Project (B906), FP7-PEOPLE-IRSES-2008 (TCMCANCER Project 230232), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300) and the Scientific Foundation of Shanghai China (10DZ1971700, 12401900501). National Major Project of China (2011ZX09307-002-03 and 2011ZX09102-006-02). National Key Technology R&D Program of China (2012BAI29B06).

Notes and references

1. R. Lin, D. J. Yu and Z. Y. Wu, Flora of China, Science Press, Beijing, 1989, pp. 263–265.
2. Jiangsu New Medical College, Dictionary of Traditional Chinese Materia Medica, Shanghai People’s Press, Shanghai, 1977, vol. 2, pp. 2216–2219.
3. D. Bensky, A. Gamble, T. J. Kaptchuk and I. L. Bensky, Chinese herbal medicine: Materia Medica, Eastland Press, Seattle, 1993, pp. 193–194.
4. B. N. Zhou, N. S. Bai, L. Z. Lin and G. A. Cordell, Phytochemistry, 1993, 34, 249–252.
5. A. L. Khan, J. Hussain, M. Hamayun, S. A. Gilani, S. Ahmad, G. Rehman, Y. H. Kim, S. M. Kang and I. J. Lee, Molecules, 2010, 15, 1562–1577.
6. J. L. Yang, R. Wang, L. L. Liu and Y. P. Shi, Planta Med., 2011, 77, 362–367.
7. H. Z. Jin, D. Lee, J. H. Lee, Y. S. Hong, Y. H. Kim and J. J. Lee, Planta Med., 2006, 72, 40–45.
8. J. J. Qin, H. Z. Jin, X. X. Zhu, J. J. Fu, X. J. Hu, X. H. Liu, Y. Zhu, S. K. Yan and W. D. Zhang, Planta Med., 2010, 76, 278–283.
9. J. J. Qin, H. Z. Jin, J. X. Zhu, J. J. Fu, Z. Qi, X. R. Cheng, Y. Zhu, L. Shan, S. D. Zhang, Y. X. Pan and W. D. Zhang, Tetrahedron, 2010, 66, 9379–9388.
10. T. Uchiyama, T. Miyase, A. Ueno and K. Usmanghani, Phytochemistry, 1989, 28, 3369–3372.
11. M. Konstantinopoulou, A. Karioti, S. Skaltsas and H. Skaltsa, J. Nat. Prod., 2003, 66, 699–702.
12. J. P. Eun and K. Jinwoong, Planta Med., 1998, 64, 752–757.
13. J. Romo, P. Joseph-Nathan and A. F. Diaz, Tetrahedron, 1964, 20, 79–85.

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Footnotes

† Electronic supplementary information (ESI) available: 1D and 2D NMR, MS, IR spectra and data for 1–5, crystallographic data for 1. CCDC 1025166. For ESI and crystallographic data in CIF or other electronic format See DOI: 10.1039/c4ra11171k

‡ These authors contributed equally to this work.

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