Chloraserrtone A, a Sesquiterpenoid Dimer from Chloranthus serratus

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Chloraserrtone A, a Sesquiterpenoid Dimer from Chloranthus serratus Bai Bai,† Shao-Xia Ye,† De-Po Yang,†,§ Long-Ping Zhu,†,§ Gui-Hua Tang,†,§ Yun-Yun Chen,† George Qian Li,‡ and Zhi-Min Zhao*,†,§ †

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China Centre for Complementary Medicine Research, University of Western Sydney, Sydney, NSW 1797 Australia § Guangdong Technology Research Center for Advanced Chinese Medicine, Guangzhou, Guangdong 510006, People’s Republic of China Downloaded via LMU MUENCHEN on February 6, 2019 at 16:57:07 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Chloraserrtone A (1), a new sesquiterpenoid dimer with two lindenane-type sesquiterpenoid monomers bridged by two six-membered rings, was obtained from Chloranthus serratus. A combination of UV, IR, NMR, HRESIMS, and X-ray diffraction data were used to elucidate the structure of 1. Compound 1 represents the first lindenane-type sesquiterpenoid dimer with extremely unique C-15−C-15′, C-4−C-6′, and C-6−C-11′ linkages to form two six-membered rings between the monomeric units. A plausible biosynthesis toward chloraserrtone A is proposed. This new compound (1), together with the known lindenane dimers (2−11), were assessed for their inhibitory effects on lipopolysaccharide-induced NO production in RAW264.7 cells. Compound 6 showed activity with an IC50 value of 3.7 μM. Chloranthus serratus (Chloranthaceae) is widely distributed throughout southern China in areas such as Zhejiang, Fujian, and Guangdong provinces.1 It has been used as a traditional Chinese medicine, called “si-kuai-wa”, to treat blood stasis and inflammatory swelling and for drainage and detoxification.2 Sesquiterpenoids, especially the sesquiterpenoid dimers, are the characteristic secondary metabolites of the genus Chloranthus and exhibit a number of bioactivities, such as anti-inflammatory and antifungal activities, blockade of the delayed rectifier (IK) K+ current, and inhibition of HIV-1 integrase.3−7 Over the last 30 years, about 70 lindenane-type sesquiterpenoid dimers have been isolated from the genus Chloranthus, and most of the sesquiterpenoid dimers are biosynthesized from two lindenane-type sesquiterpenoid monomers connected via a six-membered ring (C-4−C-15− C-9′−C-8′−C-6−C-5) by an endo Diels−Alder cycloaddition reaction.8 Compounds with novel skeletons are being discovered continually from this genus, and the structural diversity of sesquiterpenoid dimers has attracted the attention of an increasing number of researchers.9,10 As part of an investigation into sesquiterpenoid dimers, chloraserrtone A, a new rearranged sesquiterpenoid dimer with unique C-15−C15′, C-4−C-6′, and C-6−C-11′ linkages forming two sixmembered rings between the monomeric units, together with 10 known lindenane dimers, henriol C (2),11 shizukaol F (3),12 shizukaol B (4),13 chololactone F (5),14 shizukaol D (6),15 chlorahololide D (7),16 chloramultilide B (8),6 8-Omethyltianmushanol (9),17 spicachlorantin A (10),18 and sarcandrolide D (11),19 were isolated from an ethanol extract of C. serratus roots. The isolated compounds were assessed for © XXXX American Chemical Society and American Society of Pharmacognosy

their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. Herein, the isolation, structure determination, biosynthetic origin, and anti-inflammatory effects of these compounds are discussed.

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RESULTS AND DISCUSSION Chloraserrtone A (1) was obtained as colorless crystals with a specific rotation of [α]20 D −39 (c 0.2, CH2Cl2) [UV (MeOH) λmax (log ε) 266 (3.55), 204 (3.73) nm]. The molecular formula of 1 was established as C32H36O9 by the HRESIMS ion at m/z 587.2243 [M + Na]+ (calcd 587.2252), implying 15 indices of hydrogen deficiency. IR absorption bands at 3352, 1730, and 1636 cm−1 suggested the presence of hydroxy, carbonyl, and olefinic functionalities. The 1H NMR data of 1 (Table 1) showed signals for the protons of four methyl [δH 0.84 (s), 1.02 (s), 1.43 (s), and 1.86 (s)] and two methoxy groups [δH 3.60 (s) and 3.85 (s)]. The 13C NMR spectrum, complemented by DEPT experiments, showed 32 signals including four carbonyls, three double bonds, four methyls, two methoxy groups, four sp3 methylenes, seven sp3 methines (two of which contained oxygen), one oxygenated tertiary carbon, and four sp3 quaternary carbons. It suggested that 1 contained two lindenane-type sesquiterpenoid units.20 A range of 2D NMR spectroscopic techniques were used for analyzing and identifying the structure of 1. The two typical methylene signals at δH 0.47, 0.64 (H2-2) and 0.44, 1.03 (H2Received: May 25, 2018

A

DOI: 10.1021/acs.jnatprod.8b00418 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

The two units in 1 are rarely connected by two sixmembered rings (Figure 1, in red). Specifically, the HMBC correlations of H2-15/C-4′ and C-6′, H2-15′/C-4, and H-3/C6′, combined with the 1H−1H COSY correlations of H2-15/ H2-15′, verified that carbons C-4, C-15, C-4′, C-5′, C-6′, and C-15′ formed the six-membered D-ring. The HMBC correlations of H-6/C-11′, C-12′, and C-13′ indicated that C-4, C-5, C-6, C-6′, C-7′, and C-11′ formed the six-membered E-ring, which was connected to the D-ring via a C-4−C-6′ bridge. The 2D structure of 1 was thus defined as an unprecedented lindenane-type sesquiterpenoid dimer. Compound 1 is the first lindenane-type sesquiterpenoid dimer in which two monomeric units are linked with two rings, and the presence of the E-ring is the main novelty of 1. The connecting positions (C-4−C-15−C-15′−C-4′−C-5′−C-6′) of the two monomeric units constituting the D-ring are completely different from those (C-4−C-15−C-9′−C-8′−C-6−C-5) of most lindenane-type sesquiterpenoid dimers of the genus Chloranthus. In most lindenane-type sesquiterpenoid dimers, the relative configuration of the two monomeric units is the same, such as the relative configurations of C-1 and C-1′, C-3 and C-3′, C-9 and C-9′, C-10 and C-10′, and C-14 and C-14′. However, compared with these compounds, the skeletons of the two monomeric units in compound 1 are enantiomeric, which means the relative configurations of these carbons in unit 1 and 2 were opposite. The NOESY correlations (Figure 2a) confirmed this, and H-1, H-3, H-9, and H3-14′ were randomly assigned α-orientations, while H-1′, H-3′, H-9′, and H3-14 were assigned β-orientations. The C-4−C-15 bond was also βoriented because of the correlation of H-3/H-15′α. The Me-

2′), indicating the presence of two lindenane-type sesquiterpenoids, units 1 and 2, with 1,2-disubstituted cyclopropane rings (rings A and A′), was supported by the 1H−1H COSY correlations of H-1/H2-2/H-3 and H-1′/H2-2′/H-3′ (Figure 1). In unit 1, the HMBC correlations of H3-14/C-1, C-5, and C-10 and H-3/C-5 indicated the linkages of C-1−C-10−C-5 and C-3−C-4−C-5, respectively. Thus, a five-membered ring (B) was present and connected to ring A via the C-1−C-3 bridge. Similarly, the HMBC correlation networks of H-9/C-5, C-8, C-10, and C-14 and H-6/C-7, C-8, and C-10 revealed the linkages of C-8−C-9−C-10−C-5 and C-8−C-7−C-6−C-5, respectively. Thus, a six-membered ring (C) was present and connected to ring B via the C-5−C-10 bridge. The locations of the oxygenated tertiary carbon with a chemical shift of δC 84.4 and the oxygenated methine with chemical shift of δC 77.4 confirmed that hydroxy groups were attached to C-5 and C-9, and the presence of the C-8 carbonyl group was confirmed (δC 198.9). In addition, a senecioyl moiety connected to ring C at C-7 was suggested by the HMBC correlation networks of H-6/ C-11 and CH3-13/C-7, C-11, and C-12. A methylene group was connected to ring B at C-4 according to the HMBC correlations of H2-15/C-3 and C-5. Therefore, the structure of unit 1 was confirmed as a lindenane-type sesquiterpenoid (Figure 1). Unit 2 was similar to unit 1 except that rings B′ and C′ each contained a double bond, according to the chemical shifts of C-4′ (δC 150.1), C-5′ (136.9), C-6′ (153.6), and C-7′ (125.4); an isovaleryl moiety rather than a senecioyl moiety was connected to ring C′ based on a C-7′−C-11′ single bond; and C-5′ (δC 136.9) was a quaternary carbon instead of an oxygenated tertiary carbon in unit 1. B

DOI: 10.1021/acs.jnatprod.8b00418 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data and HMBC Correlations for 1 in CDCl3 (δ in ppm) position

δC, type a

1 2

26.6, CH 8.2, CH2

3 4 5 6

25.3,b CH 52.0, C 84.4, C 50.8, CH

7 8 9 10 11 12 13 14 15

127.7, C 198.9, C 77.4, CH 54.3, C 145.8, C 171.2, C 17.5, CH3 15.4, CH3 33.6, CH2

12-OMe 1′

52.8, CH3 26.5,a CH

2′

14.4, CH2

3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′

26.2, CH 150.0, C 136.9, C 153.5, C 125.4, C 199.4, C 81.1, CH 54.7, C 48.1, C 176.5, C 23.4, CH3 15.8, CH3 25.3,b CH2

12′-OMe

52.9, CH3

δH, multi. (J in Hz)

HMBC

1.80, m α 0.47, dd (9.0, 4.7) β 0.64, m 1.46, m

3, 1, 1, 5,

3.27, s

5, 7, 8, 10, 11, 11′, 12′, 13′

4.52, s

1, 5, 8, 10, 14

1.86, s 0.84, s α 2.27, dd (13.2, 4.7) β 1.85, m 3.85, s 2.10, ddd (10.2, 7.9, 5.4) α 1.03, m β 0.44, dd (7.8, 3.5) 1.90, m

7, 8, 11, 12 1, 5, 9, 10 3, 4, 5, 4′, 6′, 15′ 3, 4, 5, 4′, 6′, 15′ 12 4′, 5′, 9′

4.17, s

1′, 8′, 10′, 14′

1.43, s 1.02, s α 2.42, dd (18.6, 5.2) β 2.67, m 3.60, s

4, 5, 9 3, 4, 10 3, 4, 10 6′

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

1′, 3′, 4′, 10′ 1′, 3′, 4′, 10′ 5′, 10′, 15′

H2O-induced Michael addition. Finally, compound 1 is obtained by the oxidation of iii (Scheme 1). To evaluate their anti-inflammatory effects, compounds 1− 11 were assessed for inhibition of the release of NO using LPSinduced RAW264.7 cells as a model system. The IC50 value of compound 6 was 3.7 μM. Compounds 2 and 5 were less active, with IC50 values of 25.5 and 23.1 μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Materials and instruments for the experiments (isolation, identification, and bioactivity assays) are shown in the Supporting Information. Plant Material. Chloranthus serratus was collected in September 2016, in Wenzhou, Zhejiang Province, P.R. China, and was authenticated by one of the authors, D.Y. A voucher specimen (accession number: GDCS201609) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. Extraction and Isolation. The C. serratus roots (9.6 kg) were airdried, powdered, and extracted with 95% EtOH (3 × 40 L, 7 d each). The extract (578 g) was obtained by filtering the supernatant, removing the solvents, and partitioning into petroleum ether and EtOAc to afford fractions P (63 g) and E (268 g). Fraction E was purified by silica gel chromatography using gradient elution with CH2Cl2/MeOH (100:0 to 0:100) to afford nine fractions (E.1−E.9). Fraction E.8 was divided into 12 parts (fractions E.8.1−E.8.12) using an MCI column using gradient elution with H2O/MeOH (70:30 to 0:100). Fraction E.8.7 was chromatographed on silica gel using gradient elution with petroleum ether/EtOAc (90:10 to 25:75) to obtain 11 (20 mg). Fraction E.8.8 was separated using a reversedphase column using gradient elution with H2O/MeOH (50:50 to 20:80) and further purified by HPLC with H2O/CH3CN (30:70) to afford 1 (6 mg). Fraction E.8.8 was purified using an MCI column with H2O/MeOH (40:60 to 0:100) to give three fractions (E.8.8.1− E.8.8.3). Fraction E.8.8.1 was purified by HPLC using isocratic elution with H2O/MeOH (20:80) to give 2 (65 mg), 3 (10 mg), and 4 (10 mg). Fraction E.8.8.3 was purified on a Sephadex LH-20 column using isocratic elution with CH2Cl2/MeOH (0:100) and then purified by HPLC with H2O/MeOH (15:85) to afford 5 (25 mg) and 6 (14 mg). Repeated silica gel chromatography of fraction E.8.12 using a CH2Cl2/MeOH gradient (95:5 to 50:50), followed by purification by semipreparative HPLC, eluted with H2O/CH3CN (35:65), afforded 7 (12 mg), 8 (9 mg), 9 (100 mg), and 10 (175 mg).

6, 7′, 11′, 12′ 1′, 5′, 9′, 10′ 4, 15, 3′, 4′, 5′ 15, 4′, 5′ 12′

a,b

Assignments are interchangeable.

13′ was assigned an α-orientation because of the correlation of H-9/Me-13′. The orientations of HO-5 and H-6 were both assigned as β, which was confirmed by X-ray diffraction. In addition, the correlation of H-6/Me-13 confirmed a Z configuration of the Δ7(11) double bond. Finally, the absolute configuration of 1 was unambiguously determined as (1R, 3S, 4S, 5S, 6R, 9R, 10R, 1′R, 3′S, 9′R, 10′S, 11′R) by X-ray diffraction data analysis, with a Flack absolute structure parameter of −0.07(15) (Figure 2b). The biosynthetic origin of compound 1 is likely to be intermediate i, which is a monomeric lindenane-type sesquiterpenoid involved in the formation of numerous sesquiterpenoid dimers isolated from the genus Chloranthus.21 The terminal double bond of i (in red) is subject to a Diels− Alder cycloaddition as a dienophile with the conjugated diene (in red) of a second molecule of i, yielding sesquiterpenoid dimer intermediate ii with a new six-membered D-ring (in red). Intermediate ii is then converted to intermediate iii with construction of the six-membered E-ring (in purple), by a C

DOI: 10.1021/acs.jnatprod.8b00418 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. (a) NOESY correlations of 1. (b) ORTEP drawing of 1. fetal bovine serum (FBS) and 1% penicillin−streptomycin solution [penicillin (500U/mL) and streptomycin (500 μg/mL)]. The cytotoxicity of compounds 1−11 was measured using an MTT assay.22 The RAW264.7 cells were treated with solutions of compounds 1−11 at 50 μM in DMSO for 48 h in 96-well culture plates (5 × 104 cells/mL). MTT was added to each well (20 μL/well, 5 mg/mL in sterile phosphate-buffered saline), and the samples were incubated in a cell culture incubator. After 4 h, the samples were dissolved by DMSO (100 μL/well). Finally, the absorbance was detected using a microplate reader at 490 nm. Each treatment was repeated three times. Nitric Oxide Production Assay. After treatment with compounds 2−6, the production of NO induced by LPS was measured by a colorimetric method based on the Griess reaction.23 Cells were cultured in a 5% CO2, 37 °C atmosphere in a humidified cell culture incubator for 24 h. The cells were treated with compounds 2−6 in different concentrations for 30 min before stimulation with or without 1 μg/mL LPS. After 24 h, the supernatant was collected (50 μL/well). The supernatant (50 μL/well) was mixed with Griess reagent I (50 μL/well) and Griess reagent II (50 μL/well). Finally, the absorbance was detected by a microplate reader at 560 nm. L-NIL was used as a positive control. Each treatment was repeated three times.

Scheme 1. Putative Biosynthetic Pathway to the Formation of 1

Chloraserrtone A (1): colorless crystals (CH2Cl2); mp 168−171 °C; [α]20 D −39 (c 0.2, CH2Cl2); UV (MeOH) λmax (log ε) 266 (3.55), 204 (3.73) nm; IR (neat) Vmax 3352, 2924, 1730, 1636, 1061, 1021 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 587.2243 [M + Na]+ (calcd for C32H36NaO9, 587.2252). Crystal data for 1: C32H36O9 (M = 564.61 g/mol); colorless block, 0.3 × 0.2 × 0.1 mm3, space group P212121 (No. 19), V = 2790.00(8) Å3, Z = 4, Dc = 1.344 g/cm3, F(000) = 1200, Cu Kα radiation, λ = 1.541 84 Å, T = 100 K, μ = 0.808 mm−1; 2θmax = 144.3°, 26 858 reflections collected, 5516 unique (Rint = 0.1029); final GooF = 1.073, R1 = 0.0481 [I > 2σ(I)], wR2 = 0.1184; absolute structure parameter = −0.07(15). Crystallographic data of chloraserrtone A (CCDC 1835682) can be obtained from the Cambridge Crystallographic Data Center. Cytotoxicity Assay. The murine macrophage cell line, RAW264.7, was cultured in a 5% CO2, 37 °C atmosphere in a humidified cell culture incubator for 24 h. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), which contained 10%

Table 2. Inhibitory Effects of Compounds 1−11 on the LPSInduced NO Production in RAW264.7 Cellsa compound

cell activity (%) at an initial concentration of 50 μM

IC50 (μM)

2 3 4 5 6 L-NILb

66.5 53.1 55.4 71.5 97.0 93.3

25.5 >50 >50 23.1 3.7 7.0

a The IC50 values of compounds with cell activity < 50% at 50 μM were not tested. bPositive control.

D

DOI: 10.1021/acs.jnatprod.8b00418 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Note

(16) Yang, S. P.; Gao, Z. B.; Wu, Y.; Hu, G. Y.; Yue, J. M. Tetrahedron 2008, 64, 2027−2034. (17) Wu, B.; Chen, J.; Cheng, Y. Y. J. Nat. Prod. 2008, 71, 877−880. (18) Kim, S. Y.; Kashiwada, Y.; Kawazoe, K.; Murakami, K.; Sun, H. D.; Li, S. L.; Takaishi, Y. Phytochem. Lett. 2009, 2, 110−113. (19) He, X. F.; Yin, S.; Ji, Y. C.; Su, Z. S.; Geng, M. Y.; Yue, J. M. J. Nat. Prod. 2010, 73, 45−50. (20) Yang, S. P.; Gao, Z. B.; Wang, F. D.; Liao, S. G.; Chen, H. D.; Zhang, C. R.; Hu, G. Y.; Yue, J. M. Org. Lett. 2007, 9, 903−906. (21) Kawabata, J.; Fukushi, E.; Mizutani, J. Phytochemistry 1998, 47, 231−235. (22) Salminen, A.; Lehtonen, M.; Suuronen, T.; Kaarniranta, K.; Huuskonen, J. Cell. Mol. Life Sci. 2008, 65, 2979−2999. (23) Tsikas, D. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 851, 51−70.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00418.

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Experimental details including IR, HRESIMS, and NMR (1H, 13C, COSY, HMBC, HSQC, and NOESY) spectra of compound 1 and 1D NMR spectra of 2−11 (PDF) X-ray data for compound 1 (CIF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

De-Po Yang: 0000-0001-6823-4135 Gui-Hua Tang: 0000-0002-8831-7154 Zhi-Min Zhao: 0000-0002-3384-2365 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Key R&D Program of China (No. 2017YFC1701101), Natural Science Foundation of Guangdong Province of China (2018A030313732), and the Fundamental Research Funds for the Central Universities (No. 17ykpy22) for providing financial support for this work, and Prof. Q.-J. Li of the School of Pharmaceutical Sciences, Sun Yat-sen University, for assisting with the hypothetical biogenetic pathway of 1.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00418 J. Nat. Prod. XXXX, XXX, XXX−XXX