Chlojapolactone A, an unprecedented 1,3-dioxolane linked-lindenane sesquiterpenoid dimer from Chloranthus japonicus

Abstract

Chlojapolactone A (1), a novel lindenane sesquiterpenoid dimer with an unprecedented 1,3-dioxolane linkage, was isolated from Chloranthus japonicus. Its structure and absolute configuration was elucidated by combined spectral, computational, and chemical approaches. Compound 1 exhibited potential inhibitory effects on nitric oxide production in RAW 264.7 cells.

---

Lindenane sesquiterpenoid dimers are a class of highly complex natural products mainly occurring in plants of the genus Chloranthus (Chloranthaceae).^1,2 Biosynthetically they are proposed to be adducted from two lindenanes via Diels–Alder cycloaddition, with the fusing sites of C-6/C-8′ and C-15/C-9′ to form an additional six-membered carbon ring linkage.^1,2 So far, more than 50 lindenane dimers have been isolated.^1,2 Some of them exhibited significant biological activities, such as anticancer,³ anti-HIV,⁴ and inhibition of tyrosinase⁵ and the delayed rectifier (I_K) K⁺ current.^6,7 In recent years, their fascinating structures and important biological activities have attracted great interests of both natural product and synthetic chemists.^8–11

C. japonicus Sieb., a perennial herbaceous plant growing in southern China, has been applied in the Traditional Chinese Medicine (TCM) for the treatment of rheumatic arthralgia, bone fracture, pulmonary tuberculosis, and neurasthenia.¹² Previous investigations on this plant have proved that it was a rich source of structurally diverse lindenane sesquiterpenoid dimers.^3,4 In our efforts toward novel nitric oxide (NO) inhibitors from medicinal plants,¹³ a fraction of the ethanolic extract of C. japonicus showed an inhibitory activity of 61.21% at a concentration of 10 μg ml⁻¹ against the lipopolysaccharide (LPS) induced NO production in RAW 264.7 macrophages. Subsequent chemical investigation led to the isolation of chlojapolactone A (1), a lindenane dimer with a unique 1,3-dioxolane linkage and comprising a rare dimaleate featured 8,9-seco-lindenane monomer (Fig. 1). Compound 1 represents a novel dimerization pattern of the lindenane dimer class, which is proposed to be biosynthetically formed by an aldol condensation of a rare 8,9-seco-lindenane and a lindenane sesquiterpenoids. Bioassay verified that compound 1 had inhibition against NO production in LPS-stimulated RAW 264.7 macrophages with IC₅₀ value of 14.87 μM, being comparable to the positive control quercetin (IC₅₀ = 15.90 μM). Herein, the isolation, structure elucidation, biogenetic origin, and NO inhibitory activity of 1 are described.

Fig. 1 The structure of compound 1.

The air-dried powder of the whole plant of C. japonicus (1.0 kg) was extracted with 95% EtOH at room temperature to give a crude extract, which was suspended in H₂O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. Various column chromatographic separations of the EtOAc extract afforded compound 1 (15 mg).

Chlojapolactone A (1) was obtained as colorless oil. The HRESIMS displayed a pseudo-molecular ion at m/z 535.2331 [M − H]⁻ (calcd for 535.2337), which was consistent with a molecular formula of C₃₁H₃₆O₈, corresponding to 14 degrees of unsaturation. The IR spectrum exhibited absorption bands for carbonyl (1751 and 1711 cm⁻¹) functionalities. The ¹H NMR spectrum showed signals for a terminal double bond [δ_H 5.03 (1H, d, J = 2.0 Hz) and 4.73 (1H, brs)], five methyl singlets [δ_H 1.96, 1.85, 1.62, 1.14, and 0.51 (each, 3H)], an O-methyl [δ_H 3.75 (3H, s)], two oxygenated methines [δ_H 5.15 (1H, s) and 4.30 (1H, s)], and four highly upfield-shifted protons [δ_H 0.85, 0.72, 0.69, and 0.58 (each, 1H, m)] characterized for two cyclopropane methylenes.^6,7 The ¹³C NMR spectrum, in combination with DEPT experiments, resolved 31 carbon resonances attributable to three carbonyls (δ_C 171.0, 170.1, 167.1), three double bonds (one exocyclic and two tetrasubstituted), six methyls (one O-methyl), four sp³ methylenes, eight sp³ methines (two oxygenated), and four sp³ quaternary carbons (two oxygenated). The aforementioned information (Table 1) implied that compound 1 consisted of two lindenane sesquiterpenoid units.^1,6

Table 1 ¹H and ¹³C NMR data of compound 1 (in CDCl₃)

No.	δH^a	δC^b	No.	δH^a	δC^b
a Recorded at 400 MHz.b Recorded at 100 MHz, chemical shifts are in ppm, coupling constant J is in Hz.
1	1.70, m	27.4	1′	1.81, m	23.6
2α	0.72, m	7.7	2′α	0.85, m	15.6
2β	0.58, m		2′β	0.69, m
3	1.61, m	29.3	3′	1.95, m	23.5
4		92.5	4′		151.1
5	2.33, dd (12.5, 6.9)	48.1	5′	3.43, m	50.3
6α	2.45, dd (12.5, 6.9)	27.2	6′α	2.51, dd (18.0, 6.7)	22.1
6β	2.03, dd (12.5, 12.5)		6′β	2.26, dd (18.0, 12.5)
7		128.2	7′		154.1
8		167.1	8′		109.5
9	5.15, s	109.7	9′	4.30, s	85.2
10		48.9	10′		42.6
11		137.9	11′		127.8
12		170.1	12′		171.0
13	1.96, s	16.2	13′	1.85, s	8.7
14	1.14, s	16.4	14′	0.51, s	17.1
15	1.62, s	31.1	15′a	4.73, brs	106.6
OMe	3.75, s	52.6	15′b	5.03, d (2.0)

---

Detailed 2D NMR studies (HSQC, ¹H–¹H COSY, and HMBC experiments) afforded the gross structures of these two units (a and b) as depicted in Fig. 2. Unit b (in blue) was readily determined to be the typical lindenane-type sesquiterpenoid, chloranthalactone E,^14,15 by direct comparison of their 1D NMR data. This was further supported by ¹H–¹H COSY and HMBC correlations (Fig. 2). As for unit a (in red), two structural fragments [a cyclopropane ring (C-1–C-2–C-3) and C-5–C-6] were first established by the ¹H–¹H COSY correlations (Fig. 2). The connectivities of these fragments, quaternary carbons, and other functional groups were mainly achieved by analysis of the HMBC spectrum (Fig. 2). The HMBC correlations from H₃-14 to C-1, C-5, C-9, and C-10 allowed the connection of C-1, C-5, C-9, and C-14 to the quaternary carbon C-10, while the HMBC correlations from H₃-15 to C-3, C-4, and C-5 linked C-3, C-5, and C-15 to the quaternary carbon C-4. Thus, ring A of unit a (Fig. 2) was established. The linkages of C-6–C-7–C-8 were revealed by HMBC correlations of H₂-6/C-7 and C-8, while the correlations from H₃-13 to C-7, C-11, and C-12 and from H₂-6 to C-7 and C-11 further attached C-7, C-12, and C-13 to C-11. The upfield-shifted carbonyl of C-8 (δ_C 167.1) and still “loose end” oxygenated quaternary carbon of C-4 (δ_C 92.5) required the existence of a lactone bridge between C-4 and C-8, which generated ring B of unit a (Fig. 2). Finally, the methoxy group was located at C-12 by the correlation from the O-methyl to C-12. Unit a represented a rare 8,9-seco-lindenane skeleton.¹

Fig. 2 ¹H–¹H COSY (bold lines) and selected HMBC (arrows) correlations of 1.

As above-mentioned structure elucidation already accounted for 13 out of the 14 degrees of unsaturation, the remaining one thus required the presence of an additional ring to link units a and b. In HMBC spectrum, a strong correlation from H-9 (δ_H 5.15) to the ketal carbon at C-8′ was observed, suggesting the presence of an oxygen bridge between C-9 and C-8′. Based on the acetal nature of C-9 (δ_C 109.7) and still “loose end” oxygenated methine C-9′ (δ_C 85.2), the existence of the other oxygen bridge between C-9 and C-9′ was proposed to form the additional 1,3-dioxolane ring. However, in regular HMBC experiment [delay time = 62.5 ms, J_(C,H) = 8 Hz], even recorded in different deuterated solvents, the expected correlations between CH-9 and CH-9′ were not observed (ESI S15 and S20†). Thus, a modified HMBC experiment (delay time = 250 ms, J_(C,H) = 2 Hz) was employed,¹⁶ which generated the correlations of H-9/C-9′ and H-9′/C-9 (ESI S9†). This was also collaborated by a long rang ¹H–¹H COSY correlation¹⁷ between H-9 and H-9′ (ESI S6†). Thus, the gross structure of 1 was elucidated as depicted, with a 1,3-dioxolane ring linking the two lindenane units.

The relative configuration of 1 was established by ROESY experiment and by comparison of its 1D NMR data with those of known lindenane monomers. In unit a, the correlations of H-2β/H-6β and H₃-14/H-2β and H-6β indicated that these protons were co-facial and were arbitrarily assigned as β-orientation (Fig. 3). In consequence, the ROESY cross-peaks of H-5/H-6α and H₃-15 revealed that these protons were α-oriented, which was further supported by the large coupling constant between H-5 and H-6β (J = 12.5 Hz). As for unit b, the ROESY correlations of H₃-14′/H-2′β, H-6′β, and H-9′ indicated that they were co-facial and were arbitrarily assigned as β-oriented (Fig. 3). Consequently, the ROESY cross-peaks of H-5′/H-6′α indicated that these protons were α-oriented, which was further supported by the large coupling constant between H-5′ and H-6′β (J = 12.5 Hz). In addition, the correlation of H-9/H-9′ indicated that these protons were co-facial on the 1,3-dioxolane ring (Fig. 4). The 1D NMR data of most of the chiral centers in units a and b were consistent with those reported in their corresponding monomers.^1,2

Fig. 3 Key ROESY correlations () of unit a and b.

Fig. 4 Internuclear distances of H-5′/H-5 and H-5′/H₃-14 in isomers 1a (C-8′–O–C-12′ in β-orientation) and 1b (C-8′–O–C-12′ in α-orientation).

The configuration of C-8′ could not be directly assigned owing to its ketal nature, resulting in two possible isomers of 1 (1a with C-8′–O–C-12′ in β-orientation and 1b with C-8′–O–C-12′ in α-orientation, in Fig. 4). Thus, a Chem3D molecular modeling study considering the rotatable C-9–C-10 bond was employed to rationalize the ROESY correlations crossing the two lindenane units. In the energy minimized conformation of 1a, after optimization of the conformation around C-9–C-10 bond, the internuclear distances between H-5′ and H-5 or H₃-14 could reach the range within 3.0 Å, while these distances were greater than 4.0 Å in 1b no matter how to rotate the two units around this bond (ESI S32†). As the strong cross-peaks of H-5′/H-5 and H-5′/H₃-14 were observed in the ROESY spectrum, 1a was selected with favorable stereochemistry.

The absolute configuration of 1 was determined by the exciton chirality method.¹⁸ The UV spectrum of 1 showed a strong absorption at λ_max 223 nm (log ε 4.20) corresponding to two moieties of α,β-unsaturated ketones. Consistent with this UV maximum, the CD spectrum of 1 showed a positive Cotton effect at 251 nm (Δε + 0.52, n → π* transition) and a negative Cotton effect at 222 nm (Δε −1.60, π → π* transition) due to the transition interaction between two α,β-unsaturated ketone chromophores (Fig. 5), indicating a positive chirality for 1. The positive chirality of 1 revealed that the transition dipole moments of two chromophores were oriented in clockwise direction, and the absolute stereochemistry of 1 was assigned as depicted.

Fig. 5 CD and UV spectra of 1 (in MeOH, red lines), the stereo view of 1 in exciton chirality method model (arrows denote the electric transition dipole of the coupling chromophores), the calculated ECD spectra for 1a (1R, 3S, 4S, 5R, 9S, 10S, 1′R, 3′S, 5′S, 8′S, 9′S, 10′S, black line), and the calculated ECD spectra for the enantiomer of 1a (dashed line).

To verify the absolute configuration assigned by the CD exciton chirality method, ECD calculations using time dependent density functional theory (TDDFT) were carried out on compound 1 (ESI S33†). The experimental ECD spectrum of 1 showed first positive and second negative Cotton effects at 251 and 222 nm respectively, which matched the calculated ECD curve for 1a (Fig. 5), an isomer with 1R, 3S, 4S, 5R, 9S, 10S, 1′R, 3′S, 5′S, 8′S, 9′S, 10′S configuration, indicating that 1 possessed the same absolute configuration.

This assignment was consistent with the biogenetic origin of lindenane-type sesquiterpenoids from the genus Chloranthus. Thus, compound 1 was assigned as depicted.

Based on the co-isolated known lindenanes 2–4,^14,15,19 a plausible biosynthesis of chlojapolactone A was proposed in Scheme 1. Chloranthalactone A (2), a major component isolated in the study, was considered as the precursor. 2 was modified by several steps of transformations, involving epoxidation of Δ,⁸ epoxy ring-opening, and oxidative cleavage of C-8–C-9 diol, to generate intermediate i and ii. Intermediate ii underwent hydrolysis and hydration then followed by intramolecular esterification to produce chloranerectuslactone V (5). Finally, the aldehyde 5 was acetalized with diol i to afford 1. To support the proposed biosynthetic pathway and to further secure the structure of unit a, compound 5 was biomimetically semisynthesized from 3 (Scheme 2). Interestingly, just after we finished the synthetic work, chloranerectuslactone V (5) was reported as a novel natural product from C. erectus by other group.²⁰ The efforts to semisynthesize i for further condensation with 5 was failed, as i was very labile, which could only be detected in the acid-catalyzed epoxy ring-opening solution of 3 and quickly isomerized to 4 during the purification process. The existence of i was demonstrated by isolation of its 8-O-methyl derivative in a TsOH/MeOH/H₂O reaction system (ESI S2.5†). Above chemical evidences together with the absence of 5 in the crude extract of this plant suggested that 1 was a genuine natural product.

Scheme 1 Proposed biosynthetic pathway for 1.

Scheme 2 Semisynthesis of chloranerectuslactone V (5).

Chlojapolactone A (1) was evaluated for its inhibitory effect on nitric oxide (NO) production of LPS-activated RAW 264.7 macrophages. Under the subtoxic concentration (100 μM), 1 exhibited potential inhibition against NO production with IC₅₀ value of 14.87 μM, being comparable to the positive control quercetin (IC₅₀ = 15.90 μM).

Conclusions

In summary, our bioassay guided investigation into the chemistry of the traditional Chinese medicinal plant Chloranthus japonicus led to the discovery of chlojapolactone A (1), a novel lindenane dimer featuring a rare 1,3-dioxolane linkage between a 8,9-seco-lindenane and a lindenane sesquiterpenoid. To the best of our knowledge, dimers incorporating a 1,3-dioxolane linkage are rare in nature, known examples of which are limited to the sesquiterpene dimers volvalerelactone B from Valeriana officinalis var. latifolia²¹ and cinnafragrin A and B and capsicodendrin from Cinnamosma fragrans.²² Lindenane-type dimers reported previously were exclusively formed through direct carbon–carbon coupling of two monomers. Chlojapolactone A comprising a rare 8,9-seco-lindenane unit represented the first example of lindenane dimer coupled via an acetal bridge. A plausible biosynthetic route for 1 was proposed and partially mimicked by biomimetic semisynthesis. Compound 1 exhibited potential inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 cells, which may account for the inhibitory effect of the crude extract and explain the anti-inflammatory function of C. japonicus in folk medicine. Further studies of compound 1 ²³ regarding its effects on other anti-inflammatory signal pathways are under investigation.

Acknowledgements

The authors thank the National High Technology Research and Development Program of China (863 Project, No. 2015AA020928), the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306047), and the National Natural Science Foundation of China (No. 81402813) for providing financial support to this work.

Notes and references

1. A. R. Wang, H. C. Song, H. M. An, Q. Huang, X. Luo and J. Y. Dong, Chem. Biodiversity, 2015, 12, 451.
2. Y. J. Xu, Chem. Biodiversity, 2013, 10, 1754.
3. O. E. Kwon, H. S. Lee, S. W. Lee, K. H. Bae, K. Kim, M. Hayashi, M. C. Rho and Y. K. Kim, J. Ethnopharmacol., 2006, 104, 270.
4. P. L. Fang, Y. L. Cao, H. Yan, L. L. Pan, S. C. Liu, N. B. Gong, Y. Lu, C. X. Chen, H. M. Zhong, Y. Guo and H. Y. Liu, J. Nat. Prod., 2011, 74, 1408.
5. B. Wu, J. Chen, H. B. Qu and Y. Y. Cheng, J. Nat. Prod., 2008, 71, 877.
6. S. P. Yang, Z. B. Gao, F. D. Wang, S. G. Liao, H. D. Chen, C. R. Zhang, G. Y. Hu and J. M. Yue, Org. Lett., 2007, 9, 903.
7. S. P. Yang, Z. B. Gao, Y. Wu, G. Y. Hu and J. M. Yue, Tetrahedron, 2008, 64, 2027.
8. X. F. He, S. Zhang, R. X. Zhu, S. P. Yang, T. Yuan and J. M. Yue, Tetrahedron, 2011, 67, 3170.
9. X. F. He, S. Yin, Y. C. Ji, Z. S. Su, M. Y. Geng and J. M. Yue, J. Nat. Prod., 2010, 73, 45.
10. C. C. Yuan, B. Du, L. Yang and B. Liu, J. Am. Chem. Soc., 2013, 135, 9291.
11. S. Qian and G. Zhao, Chem. Commun., 2012, 48, 3530.
12. Nanjing University of Chinese Medicine, in Dictionary of Chinese Traditional Medicine (Zhong Yao Da Ci Dian), Shanghai Science and Technology Press, Shanghai, 2006, p. 3028.
13. (a) J. Y. Zhu, B. Cheng, Y. J. Zheng, Z. Dong, S. L. Lin, G. H. Tang, Q. Gu and S. Yin, RSC Adv., 2015, 5, 12202; (b) Z. Dong, Q. Gu, B. Cheng, Z. B. Cheng, G. H. Tang, Z. H. Sun, J. S. Zhang, J. M. Bao and S. Yin, RSC Adv., 2014, 4, 55036.
14. Y. Takeda, H. Yamashita, T. Matsumoto and H. Terao, Phytochemistry, 1993, 33, 713.
15. X. Li, Y. F. Zhang, L. Yang, Y. Feng, Y. M. Liu and X. Zeng, Acta Pharm. Sin. B, 2011, 46, 1349.
16. X. C. Li, H. N. Elsohly, C. D. Hufford and A. M. Clark, Magn. Reson. Chem., 1999, 37, 856.
17. W. H. Zhang, H. Q. Zhao, I. Carmichael and A. S. Serianni, Carbohydr. Res., 2009, 344, 1582.
18. N. Harada, K. Nakanishi and S. Tatsuoka, J. Am. Chem. Soc., 1969, 91, 5896.
19. M. Uchida, G. Kusano, Y. Kondo, S. Nozoe and T. Takemoto, Heterocycles, 1978, 9, 139.
20. T. T. Huong, T. N. Van, T. T. Minh, L. H. Tram, N. T. Anh, H. D. Cuong, C. P. Van and D. V. Ca, Lett. Org. Chem., 2014, 11, 639.
21. P. C. Wang, X. H. Ran, H. R. Luo, J. M. Hu, R. Chen, Q. Y. Ma, H. F. Dai, Y. Q. Liu, M. J. Xie, J. Zhou and Y. X. Zhao, Org. Lett., 2011, 13, 3036.
22. L. Harinantenaina and S. Takaoka, J. Nat. Prod., 2006, 69, 1193.
23. Chlojapolactone A (1). colorless oil; [α]²⁰_D −67.20 (c 0.1, CHCl₃); UV (MeOH) λ_max (log ε) 223 (4.20) nm; IR (KBr) ν_max 1751, 1711, 1648, 1080, 960 cm⁻¹; ¹H and ¹³C NMR data, see Table 1 and ESI Table S1;† positive ESIMS m/z 559.3 [M + Na]⁺, negative ESIMS m/z 535.5 [M − H]⁻; HRESIMS m/z 535.2331 [M − H]⁻ (calcd for C₃₁H₃₅O₈, 535.2337).

---

Footnote

† Electronic supplementary information (ESI) available: NMR spectra of 1–5, detail information for ECD calculation, isolation, purification, semisynthesis, and bioassay protocols. See DOI: 10.1039/c5ra22145e

---