Chlotrichenes A and B, Two Lindenane Sesquiterpene Dimers with

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Chlotrichenes A and B, Two Lindenane Sesquiterpene Dimers with Highly Fused Carbon Skeletons from Chloranthus holostegius Jun Chi, Wenjun Xu, Shanshan Wei, Xiaobing Wang, Jixin Li, Hongliang Gao, Lingyi Kong,* and Jun Luo* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, P. R. China Org. Lett. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/17/19. For personal use only.

S Supporting Information *

ABSTRACT: Two unprecedented lindenane sesquiterpene dimers, chlotrichenes A (1) and B (2), were obtained from Chloranthus holostegius var. trichoneurus. Their structures were characterized by NMR, ECD, and X-ray diffraction analysis. They possess a new type of spirocarboncyclic dimeric framework formed by endo-Diels−Alder reaction, and the structure of 1 features a unique 3/5/6/6/6/6/5/3-fused octacyclic skeleton by subsequently plausible epoxidation− cyclization reactions of 2. Compound 2 showed synergetic cytotoxicity with DOX on U2 OS cells (CI: 0.94 ± 0.03).

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indenane sesquiterpene dimers (LSDs), a group of natural products regarded as the chemotaxonomic characters of the family Chloranthaceae, are becoming one of the hot research topics in recent decades due to their great structural specificities and wide ranges of bioactivities, such as cytotoxic, anti-inflammatory, antimalarial actions.1 An overview of the molecular structures of LSDs reveals that they biosynthetically originate from the convergence of two molecular lindenane sesquiterpenes, which are formed mostly by intermolecular Diels−Alder reaction between Δ15(4),5(6) and Δ9′(8′), and several by [2 + 2] cycloaddition, Michael addition, or acetalization.2 Several typical LSDs members have been recently synthesized successfully by Liu’s and Peng’s groups using biomimetic strategy.3 Highly conjugated olefinic bonds and α,β-unsaturated ketones of the lindenane precursors provide key reactive sites for reported LSDs, which also reveals great potential for intriguing frameworks. Encouraged by our previous research on the sesquiterpenes from the Chloranthaceae plants,4 the chemical investigation of the roots of Chloranthus holostegius var. trichoneurus, an endemic species occurring in the Southwest of China,5 was conducted in our current project. Unexpectedly, chlotrichenes A (1) and B (2), two novel LSDs with highly fused-ring skeletons, were discovered (Figure 1). Biogenetically, an unreported endoDiels−Alder addition between Δ15(4),5(6) and Δ15′(4′) of two lindenane units may be the key step to build their basic spirofused dimeric frameworks, and subsequent epoxidation and intermolecular cyclization by tandem nucleophilic attack and epoxy opening on 2 are supposed to form the surprising 3/ 5/6/6/6/6/5/3-fused octacyclic ring core of 1. Herein, we report the isolation, structural elucidation, possible biogenetic pathway, and bioactivities of these two novel LSDs. © XXXX American Chemical Society

Figure 1. Structures of chlotrichenes A (1) and B (2).

Chlotrichene A (1) was obtained as colorless cluster crystals, and its molecular formula C32H36O9 was established by positive HR-ESI-MS at m/z 587.2248 [M + Na] + (calcd for C32H36O9Na, 587.2252). From the 1H NMR and HSQC spectra (Figure S6) of 1 (Table 1), two pairs of specific upfield protons at δH 0.45 (H-2α, td, J = 4.4, 3.2 Hz)/1.06 (H-2β, dt, J = 8.5, 4.0 Hz) and 0.67 (H-2′α, td, J = 8.5, 5.3 Hz)/0.48 (H2′β, q, J = 4.5 Hz) were the characteristic cyclopropane methylenes of lindenane sesquiterpenes.6 The structure of 1 thus may be a lindenane sesquiterpene dimer that was also supported by the carbon numbers from both its molecular formula and 13C NMR data (Table 1). Two obvious methoxyl signals at δH 3.61 (s) and 3.86 (s), key carbonyl signals at δC 176.4 and 171.1 and keto-carbonyl signals at δC 199.4 and 198.9, along with two oxymethylenes at δH 4.17 (s) and 4.52 (d, J = 3.5 Hz), and substituted hydroxyl protons at δH 3.72 (s) and 3.64 (d, J = 3.5 Hz) were also observed, indicating that the lactone rings of lindenanes were opened and two characteristic Received: December 19, 2018

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DOI: 10.1021/acs.orglett.8b04046 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of 1 and 2 in CDCl3 unit A

unit B

1 δH (J in Hz)

no. 1 2α 2β 3 4 5 6 7 8 9 10 11 12 13 14 15α 15β 16 9-OH

2.11 0.45 1.06 1.91

(ddd, 8.5, 5.5, 2.9) (td, 4.4, 3.2) (dt, 8.5, 4.4) (ddd, 8.5, 5.8, 3.2)

4.17 (s)

1.44 1.02 2.67 2.42 3.61 3.72

(s) (s) (ddd, 18.5, 11.1, 5.1) (dd, 18.5, 5.7) (s) (s)

2 δC 26.2 14.5 27.4 150.0 136.9 153.5 125.4 199.4 81.1 54.8 48.1 176.4 23.4 15.8 25.3 52.9

1.96 0.93 0.25 1.69

δC

no.

(dt, 8.4, 5.0, 3.5) (td, 7.5, 4.1) (dd, 7.5, 3.5) (m)

26.3 15.5

1′ 2′α 2′β 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′α 15′β 16′ 9′-OH

3.66 (s)

3.55 (s)

2.14 1.10 2.47 2.23 3.74

1

δH (J in Hz)

(s) (s) (dd, 17.7, 6.0) (m) (s)

23.4 145.0 132.5 46.1 136.1 203.2 79.6 50.5 142.0 171.4 20.1 14.8 23.2 52.7

δH (J in Hz) 1.80 0.67 0.48 1.46

(ddd, 8.6, 6.7, 4.5) (td, 8.6, 5.3) (q, 4.5) (ddd, 8.0, 6.6, 3.9)

3.27 (s)

4.52 (d, 3.5)

1.87 0.85 2.26 1.86 3.86 3.64

(s) (s) (ddd, 13.4, 5.1, 1.5) (m) (s) (d, 3.5)

2 δC

δH (J in Hz)

δC

26.5 8.2

1.88 (m) 0.67(td, 8.6, 5.8) 0.60 (dd, 9.4, 3.6) 1.55 (m)

25.0 10.1

29.2 52.0 84.4 50.8 127.6 198.9 77.1 54.3 145.9 171.1 17.5 15.4 33.6 52.8

6.17 (s)

4.28 (s)

1.91 0.85 2.03 1.84 3.81

(s) (s) (m) (dd, 13.9, 6.0) (s)

31.4 52.9 161.3 117.9 126.7 198.9 77.0 51.7 136.7 171.5 17.2 21.1 32.4 52.7

was connected between C-11 and C-6′ and formed another hexene ring between units A and B. Thus, the planar structure of 1 was established as an unprecedented LSD embracing a unique 3/5/6/6/6/6/5/3-fused octacyclic framework. The relative configuration of 1 was deduced from interpretation of the observed ROESY correlations (Figure 2). The correlations of H-1/H-3 and H-9 and H3-14/OH-9 of unit A and H-1′/H-3′ and H-9′ and H3-14′/OH-9′ of unit B suggested that the stereochemistry of common chiral carbons in units A and B were identical to those of the lactone-opened lindenane moiety of general LSDs,7 and thus, H-1 was assigned as α-oriented. The strong correlations between H2-15/H2-15′ and H-3/H-15α distinguished the orientations of two protons of C15 and C15′, respectively. The relative configuration of newly generated spiral atom (C-4′) was secured through the observation of key cross-peak of H-15′β/H-2′α, resulting that the C-15−C-15′ branch was α-oriented. The strong correlation between H3-13 of unit A and H-9′ rather than H-6′ of unit B indicated that CH3-13 and H-6′ were in opposite directions and identified as β- and α-configurations, respectively. Finally, the α-orientation of OH-5′ was tentatively assigned on the basis of the obvious correlation between H3-13 and H-9′ and absent correlations of adjacent protons H-6′α/H-14′α/H2-15′. In order to solve the above uncertainty and establish the stereochemistry, the single crystals with good quality of 1 were obtained in a methanol/water (10:1) system and subjected to an X-ray diffraction experiment with Cu Kα radiation. The crystal data [CCDC no. 1880242, Flack parameter 0.07(13)] not only confirmed our deduction about the planar and relative structure of 1 but also unambiguously gave its absolute configuration as 1R,3S,9R,10S,11R,1′R,3′S,4′S,5′S,6′R,9′R,10′R (Figure 3). Chlotrichene B (2), a white amorphous powder, was assigned to a molecular formula of C32H36O8 by positive HRESIMS (m/z 571.2309 [M + Na] +, calcd for C32H36O8Na, 571.2302). The 1H NMR and 13C NMR data (Table 1) of 2 displayed high similarity to those of 1, suggesting that 2 was also a LSDs with opened-lactone moieties.6 The presence of

methyl ester side chains with CO-8−H-9/CO-8′−OH-9′ were present like in many conventional LSDs.7 Further analysis of the NMR spectra resolved the detailed assignments of two lindenane sesquiterpene units (A and B, Figure 2) in Table 1. For unit A, the HMBC correlations

Figure 2. Key HMBC and ROESY correlations of 1.

(Figure 2) from H-2 to C-4 (δC 150.0), H3-14 to C-5 (δC 136.9), and H-9 to C-7 (δC 125.4) and the presence of ketocarbonyl C-8 (δC 199.4), located an α,β,γ,δ-unsaturated lactone at C-4/5/6/7/8 and assigned the chemical shift of C-6 as δC 153.5. For unit B, the key HMBC cross-peaks from H3-14′ to C-5′ (δC 84.4), H-2′ to C-4′ (δC 52.0), and from H6′ [δH 3.27 (s)] to C-5′/C-7′/C-8′/C-10′ allowed the assignments of C-4′, C-5′, and C-6′ respectively. Compared with the reported LSDs, the most noticeable difference was the presence of a spin coupling fragment of CH2-15−CH2-15′ at δH 2.67 (ddd, J = 18.5, 11.1, 5.1 Hz)/2.42 (dd, J = 18.5, 5.7 Hz) and δH 2.26 (ddd, J = 13.4, 5.1, 1.5 Hz)/1.86 (m) in 1. Meanwhile, the H2-15 from unit A showed obvious HMBC correlations to both the C-4/C-5 in unit A and C-4′ in unit B; H2-15′ from unit B exhibited key HMBC correlations to both C-4′ in unit B and C-4/C-6 in unit A. These aforementioned data revealed that a new cyclohexene ring was formed between the Δ15(4), 5(6) of unit A and Δ15′(4′) of unit B via Diels−Alder addition.7 Additionally, the strong HMBC correlations from H-6′ of unit B to C-11/C-7/C-12 of unit A, and H3-13 of unit A to C-6′ of unit B revealed that an unparalleled C−C bond B

DOI: 10.1021/acs.orglett.8b04046 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

Figure 3. X-ray structure of 1.

common spin coupling fragment of CH2-15-CH2-15′ at δH 2.47 (ddd, J = 17.7, 6.0 Hz)/2.23 (m) and 2.03 (m)/1.84 (dd, J = 13.9, 6.0 Hz) indicated that the dimerization mode of 2 was also a Diels−Alder addition between Δ15(4), 5(6) and Δ15′(4′) as 1, which was confirmed by the key HMBC correlations (Figure 4) between H2-15 and H2-15′, from H-6 [δH 3.66 (m)] to C-

Figure 5. ECD and UV spectra of 2. The bold lines denote the electric transition dipole of the chromophores.

configuration of 2 was established as 1R,3S,6R,9R,10S,1′R,3′S,4′S,9′R,10′S. In this effort, the structures of 1 and 2 were elucidated to be two LSDs with unusual highly fused polycyclic carbon skeletons, which encouraged us to explore their plausible biosynthetic pathway (Scheme 1). According to their Scheme 1. Plausible Biosynthesis for 1 and 2

Figure 4. Key HMBC and ROESY correlations of 2.

4′/C-3′/C-5′, H2-15′ to C-4/C-6, and H2-15 to C-4′. An additional olefinic proton at δH 6.17 (s) and a down-shifted methyl proton at δH 2.14 (s), combined with their detailed HMBC correlations of H3-13 to C-7/C-11/C-12 in unit A and H-6′ to C-4′/C-5′/C-7′/C-8′/C-10′ in unit B, indicated that these two carbons were present as olefinic carbons and the C6′−C-11 connection in 1 was nonexistent. Accordingly, the planar structure of 2 was determined as an undescribed spirofused LSD, which was a plausible precursor of 1. The relative structure of 2 was resolved on account of its ROESY data (Figure 4) and referencing to those of 1. The observation of key cross-peaks of H-15′β /H-2′α and spatially close H-9/H-6′ and H3-13′ indicated the α-orientation of the CH2-15−CH2-15′ branch and same configuration of spiro carbon C-4′, which thus demonstrated that 2 had a same endoDiels−Alder addition mode as that of 1. The orientation of H6 was assigned as β with the aid of the strong ROESY correlations of H-6/H3-14 and H-3′. The subsequent experimental ECD spectrum (Figure 5) of 2 showed a split Cotton effects with a positive Cotton effect at 234 nm (Δε + 3.24) and a negative Cotton effect at 208 nm (Δε −5.32), indicating a clockwise mode of two coupling chromophores of the twisted π-electron systems C-5′−C-8′/C-11′−C-12′ of unit B and C-7−C-8/C-11−C-12 of unit A.8 Accordingly, the absolute configuration of 2 was established as depicted in Figure 1. Furthermore, the good agreement of experimental and calculated ECD spectra for 1R,3S,6R,9R,10S,1′R,3′S,4′S,9′R,10′S-2 (Figure S18 in Supporting Information) confirmed the above results of exciton chirality. Therefore, the absolute

structures, chloranthalactone A (i), largely existing in Chloranthaceae plants, is considered as the precursor of lindenane units.9 The sequential olefination of allylic position at C5/6, stereoselective epoxidation of the active Δ8,9 olefinic bond, epoxide cleavage, and lactone opening of i generate the universal intermediate v with the required conjugated C

DOI: 10.1021/acs.orglett.8b04046 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Δ15(4),5(6) system.3a,b,10 An endo-selective [4 + 2] cycloaddition of two molecules of v between Δ15(4),5(6) and Δ15′(4′) produces the spiro-fused lindenane homodimer framework of 2. Further, the terminal Δ5′,6′ of unsaturated ester of 2 is subjected to another epoxidation to give epoxide vi.11 The possibly allylic dehydrogenation of H-6 (vii) generates the evolving carbanion (viii). A following intramolecular cyclization involving tandem nucleophilic attack between C-11 and C-6′, and the protonated epoxide opening is accounted for forming the new C-11−C-6′ bond to afford the 3/5/6/6/6/6/5/3-fused octacyclic skeleton of 1.12 Besides, the good agreement of the stereochemistry of related chiral centers of 1 and 2, especially the key spiro carbon C-4′, also brings to light the rationality that 2 can be recognized as the precursor of 1. On the basis of the structural novelty and complexity, the bioactivities of compounds 1 and 2 were screened. Both of them showed no significant inhibitions on NO production by LPS-induced RAW264.7 cells,4b cytotoxicity on MCF-7 and U2 OS cells,13 and multidrug resistance reversal effects on MCF-7/DOX cells.14 The synergetic effects for 1 and 2 with DOX on U2 OS cells were also evaluated, and the combined indexes (CIs) at inhibition of 50% were 1.12 ± 0.07 and 0.94 ± 0.03, respectively, which indicated that 2 had an additive cytotoxic activity.15 Taken altogether, their fascinating skeletons, formed by a new kind of endo-Diels−Alder addition between Δ15(4),5(6) and Δ15′(4′) and subsequently plausible epoxidation−cyclization reactions, supply inspiration for the exploration of more interesting LSDs molecules by discovery from the plant kingdom and biomimetic synthesis.

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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 “Double First-Class” University project (CPU2018GY08). Part of the ECD calculations was supported by the High Performance Computing Center of China Pharmaceutical University.

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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.8b04046. Detailed isolation procedures, biological activity experiments, and full spectroscopic spectra (MS, UV, IR, 1D and 2D NMR, and ECD) for compounds 1 and 2 (PDF) Accession Codes

CCDC 1880242 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Lingyi Kong: 0000-0001-9712-2618 Jun Luo: 0000-0002-7892-011X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research was supported in part by the National Natural Science Foundation of China (81430092), the Outstanding D

DOI: 10.1021/acs.orglett.8b04046 Org. Lett. XXXX, XXX, XXX−XXX