Letter pubs.acs.org/OrgLett
Xishacorenes A−C, Diterpenes with Bicyclo[3.3.1]nonane Nucleus from the Xisha Soft Coral Sinularia polydactyla Fei Ye,†,‡ Zheng-Dan Zhu,†,‡ Jun-Sheng Chen,§ Jing Li,§ Yu-Cheng Gu,∥ Wei-Liang Zhu,†,‡ Xu-Wen Li,*,† and Yue-Wei Guo*,†,‡ †
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China § Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yu-Shan Road, Qingdao 266003, China ∥ Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom S Supporting Information *
ABSTRACT: Three new diterpenes, xishacorenes A−C, featuring an undescribed bicyclo[3.3.1]nonane nucleus bearing 1-vinyl and 13-[(E)-4-methylpenta-1,3-dien-1-yl] alkyl chains, and a related monocyclic known compound, were isolated from the Xisha soft coral Sinularia polydactyla. The structures of xishacorenes A−C, including their absolute configurations, were elucidated by extensive spectroscopic analysis and TDDFT ECD calculations. The new compounds exhibit an interesting dose-dependent promotion effect on the ConA-induced T lymphocyte proliferation. A plausible biosynthetic pathway of xishacorenes A−C is proposed.
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oft corals of the genus Sinularia (order Alcyonacea, family Alcyoniidae) consist of approximately 140 species, which are widely distributed from the East Africa Sea to the Western Pacific, including the South China Sea. More than 50 species of Sinularia have been chemically investigated, with over 500 diverse and complex metabolites being discovered, such as sesquiterpenoids, diterpenoids, polyhydroxylated steroids, alkaloids, polyamines, etc. A large number of these metabolites exhibit broad biological properties, including cytotoxic, antimicrobial, and anti-inflammatory activities.1 Among all the species of Sinularia, the title animals Sinularia polydactyla of the Red Sea have been extensively studied, and numerous diverse diterpenoids such as casbanes, cembranes, and cembrane− africanane hybrids were isolated and characterized.2 However, there is only one report regarding the chemical constituents of S. polydactyla from the South China Sea, with only two sesquiterpenoids being isolated.3 In the course of our continued effort to explore bioactive secondary metabolites from Chinese Sinularia,1a,4 the title animals were collected off the coast of Xisha islands, and chemically investigated, resulting in the discovery of three novel diterpenes, namely xishacorenes A−C (1−3), and a known related compound, fuscol (4) (Figure 1). Herein, we report the isolation, structure determination, biological evaluation, and plausible biosynthetic pathway of the new compounds. The acetone extract of the title animals was partitioned between Et2O and H2O. The Et2O-soluble portion was chromatographed repeatedly over silica gel, Sephadex LH-20, and RP-HPLC to yield pure diterpenes 1 (5.6 mg), 2 (3.4 mg), 3 (1.1 mg), and 4 (4.7 mg), respectively. The known compound 4 was readily identified as the lobane-type © XXXX American Chemical Society
Figure 1. Structures of compounds 1−4.
diterpenoid, fuscol, which was first isolated from a Caribbean gorgonian Eunicea f usca, by comparing its NMR data and specific rotation with those reported in the literature.5 Interestingly, HR-EIMS analysis of the new compounds 1−3 showed the same molecular formula C20H30, indicating the diterpene nature of these isomers and also suggested the presence of six degrees of unsaturation. Furthermore, the IR spectra of 1−3 showed the typical absorptions (around 1640, 985, and 909 cm−1) indicative of terminal double bonds, whereas their UV spectra displayed absorptions at λmax around 250 nm (log ε = 3−5), suggesting the presence of π−π conjugated diene systems. These structural features mentioned above were reminiscent of those of co-occurring fuscol (4). Xishacorene A (1) was obtained as a colorless oil. Its 13C NMR spectrum and HSQC experiments disclosed 20 carbon Received: June 8, 2017
A
DOI: 10.1021/acs.orglett.7b01716 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data (δ in ppm, J in Hz) for Compounds 1−3 in CD3OD 1 δH, mult (J, Hz)
no. 1 2 3ax 3eq 4 5ax 5eq 6ax 6eq 7 8 9a 9b 10 11 12ax 12eq 13 14 15 16 17 18 19 20
1.87 1.67 1.60 1.44 1.67 1.69 1.51 1.41 0.91 5.92 5.05 5.04
t (2.9) dt (12.5, 2.9) ddd (12.5, 3.2, 2.9) t (2.9) m m ddd (13.6, 13.5, 5.6) dt (13.5, 2.8) s dd (17.7, 11.0) dd (17.7,11.0) dd (11.0, 1.3)
1.85 d (1.3) 5.27 t (1.3)
1.07 5.39 6.01 5.72
s d (15.2) dd (15.2, 10.7) brd (10.7)
1.73 s 1.70 s
2 δC 41.8 45.7 27.9 36.5 25.6 30.7 31.1 148.8 112.1 136.8 26.4 133.8 42.0 24.3 143.2 124.5 126.6 133.4 26.0 18.2
δH, mult (J, Hz) 1.85 t (2.9) 1.81 dt (12.5, 2.9) 1.67 dt (12.5, 3.2) 1.52 dd (3.2, 2.9) 1.53−1.57 m 1.53−1.57 m 1.44 ddd (13.6, 13.5, 6.1) 1.24 dd (13.5, 3.4) 0.90 s 5.91 dd (17.7, 11.0) 5.03 (17.7, 1.4) 5.02 dd (11.0, 1.4)
s d (15.4) dd (15.4, 10.7) brd (10.7)
1.76 s 1.74 s
resonances, including five sp3 methyls, three sp3 methylenes, two sp3 methines, two sp3 quaternary carbons, one sp2 methylene, five sp2 methines, and two sp2 quaternary carbons. The diagnostic 1H and 13C NMR resonances, as well as coupling constants of the connected protons (Table 1), indicated the presence of a monosubstituted terminal double bond (δH 5.05, dd, J = 17.7, 11.0 Hz, 1H, H-9a; 5.04, dd, J = 11.0, 1.3 Hz, 1H, H-9b; 5.92, dd, J = 17.7, 11.0 Hz, H-8; δC 112.1, CH2, C-9; 148.8, CH, C-8), a trisubstituted olefinic bond (δH 5.27, t, J = 1.3 Hz, H-12; δC 133.8, CH, C-12; 136.8, C, C10), and a conjugated diene moiety (δH 5.39, d, J = 15.2 Hz, 1H, H-15; 6.01, dd, J = 15.2, 10.7 Hz, 1H, H-16; 5.72, brd, J = 10.7 Hz, 1H, H-17; δC 143.2, CH, C-15; 124.5, CH, C-16; 126.6, CH, C-17; 133.4, C, C-18). The above olefinic bonds accounted for four degrees of unsaturation; thus, the remaining two degrees should be ascribed to a bicyclic system in the molecule. Further analysis of the 1H−1H COSY spectrum of compound 1 revealed three structural fragments a−c as shown in Figure 2A by the clear correlations of H-8/H2-9, H-2/H2-3/H-4/H25/H2-6, and H-15/H-16/H-17, respectively. These subunits, together with two methyls at δc 31.1 and 24.3, three vinylic methyls at δc 18.2, 26.0 and 26.4, and two quaternary olefinic carbons (δc 133.8, 136.8), were connected by careful interpretation of the well resolved HMBC correlations from H3-7 to C-1/C-2/C-6/C-8; from H3-14 to C-4/C-12/C-13/C15; from H3-11 to C-2/C-10/C-12; and from H3-19/H3-20 to C-17/C-18. Finally, the presence of the bicyclo[3.3.1]nonane skeleton and its connection with the vinyl and the 4methylpenta-1,3-dien-1-yl substitutions at C-1 and C-13, were deduced by the HMBC correlations from H-2 (δH 1.87) to C-3 (δC 27.9), H-4 (δH 1.44) to C-3, and H2-3 (δH 1.67, 1.60) to C10/C-13 (δC 42.0), respectively.
41.4 45.6 28.5 38.1 27.0 29.6 30.9 148.9 112.1 136.6 26.8 133.4
1.84 d (1.4) 5.43 brs
1.01 5.52 6.26 5.81
3 δC
43.4 30.3 140.3 124.3 126.7 133.2 26.0 18.3
δH, mult (J, Hz) 2.08 t (2.9) 1.81 m 1.78 m 1.41 brs 1.74 m 1.63 m 1.60−1.65 m 1.60−1.65 m 0.85 s 5.82 dd (17.6, 11.0) 5.04 dd (17.6, 1.2) 5.02 dd (11.0, 1.2) 4.77 d (2.4) 2.75 d (14.8) 2.03 d (14.8) 1.04 5.61 6.20 5.81
s d (15.5) dd (15.5, 10.6) brd (10.6)
1.76 s 1.75 s
δC 39.8 50.1 29.3 39.7 26.5 31.7 29.4 149.4 111.8 149.7 112.3 43.8 41.5 28.1 143.2 123.7 126.8 133.4 26.0 18.3
Figure 2. (A) 1H−1H COSY and key HMBC correlations of compounds 1−3; (B) key NOESY cross peaks of 1−3.
The relative configuration of compound 1 was determined by the analysis of its 1H−1H coupling constants and NOE spectrum. On the basis of the small coupling constants of H-2 (δH 1.87, t, J = 2.9 Hz) and H-4 (δH 1.44, t, J = 2.9 Hz) on the chair conformation of the cyclohexane ring, both of the two protons were determined to be of equatorial orientation (β configuration). The large coupling constants of H-15 and H-16 (J = 15.2 Hz) suggested the E configuration of the double bond at Δ15/16. Furthermore, the clear NOE cross peaks (Figure 2B) between H-8 and H-6eq, H-6ax (δH 1.51) and H3-14, H3-7 and H3-11, and H-3eq (δH 1.60) and H-15, indicated the cis configuration of CH3-7 and CH3-14, and the trans configuration of CH3-7 and H-2. In view of the above evidence, the relative configuration of compound 1 was determined as 1R*,2R*,4S*,13S*. B
DOI: 10.1021/acs.orglett.7b01716 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
for 2, and 8 ones for 3 above 1% population for further reoptimization [Supporting Information (SI), Figures S8−S10]. The resulting geometries were reoptimized at the B3LYP/6311G(d,p) level with IEFPCM solvent model for CH3CN, and frequency analysis was performed as well to confirm that the reoptimized geometries were at the energy minima.7 Finally, the Boltzmann-averaged ECD spectra of (1R, 2R,4S,13S)-1, (1R,2R,4S,13R)-2 and (1R,2R,4S,13R)-3 appeared similar curves which highly matched to the experimental ones, whereas those of their enantiomers showed completely opposite curves. In fact, due to the similar positive CEs of 3 and 1, while different ACs at C-13 position, the ECD spectrum of (1R,2R,4S,13S)-3 was also calculated, which showed nearly opposite curve to the experimental one (SI, Figure S11), confirming the 13R configuration of 3 and also revealed the C13 connected diene moiety as a main chromophore to impact the CD spectrum of such compounds. The CD exciton chirality method8 was also applied to verify the CEs of the three new compounds (Figure 3), especially that of 3, of which the 3D models showed identical CEs with the experimental ones. Consequently, the ACs of xishacorenes A (1), B (2) and C (3) were determined to be (1R,2R,4S,13S), (1R,2R,4S,13R), and (1R,2R,4S,13R), respectively. Although xishacorenes A−C (1−3) formally displayed a quite different skeleton from the co-occurred fuscol (4), they are actually structurally related by sharing some common moieties, such as the cyclohexane ring bearing a vinyl group and a conjugated diene side chain. To explain their possible biogenetic relationship, a hypothetical pathway is proposed as shown in Scheme 1. Compound 4 could first undergo an acid
The 1D (Table 1) and 2D NMR spectra of xishacorene B (2) showed great similarity to those of 1, with the only differences of the chemical shifts on C-13 (δC 42.0 for 1 and 43.4 for 2) and its nearest protons and carbons, which suggested that they should be a pair of stereoisomers with the opposite configuration at C-13 position. Further 2D NMR experiments confirmed the structure and the relative configuration of 2, with characteristic correlations shown in Figure 2. In particular, the clear NOE cross peaks of H-6α/H-15, and H-3β/H3-14, supported the β configuration of CH3-14 (Figure 2B). 2 is the C-13 epimer of 1. Compound 3 possessed 1D NMR data extremely similar to those of 2 (Table 1). The only differences between them is that the trisubstituted olefin and related sp3 methyl (CH3-11) in 2 was replaced by an exocyclic double bond in 3, with δH at 4.77 (d, J = 2.4 Hz, 2H, H2-11) and δC at 112.3 (sp2 CH2, C-11), and 149.7 (sp2 C, C-10). Further aided by 1H−1H COSY and HMBC experiments (Figure 2A), the planar structure of 3 was suggested to be the double bond migration of 2 from Δ10/12 to Δ10/11 (Figure 1). Its related stereochemistry was confirmed to be the same as that of 2 by the analysis of proton coupling constants and NOESY spectrum, with the characteristic correlations shown in Figure 2B. Thus, compound 3 was determined as depicted in Figure 1, namely xishacorene C. The absolute configurations of 1−3 was determined by the time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculation, since this approach has been proved to be a reliable and powerful tool for the determination of the ACs of chiral natural products.6 As shown in Figure 3, the ECD spectra (MeCN) of compounds 1 and 3
Scheme 1. Proposed Biosynthetic Pathway for Compounds 1−3
Figure 3. Experimental ECD spectrum of xishacorenes A−C (1−3) (black), the calculated ECD spectra of 1−3 (red), and their enantiomers (blue), respectively. The 3D model of each compound revealed the identical CE by CD exciton chirality analysis and ECD calculation.
activation of its terminal hydroxyl, followed by an electron delivery from the Δ10/12 olefinic head to the activated hydroxyl tail, accomplishing the cyclization of a six-member ring and the elimination of the terminal hydroxyl. Since the nucleophilic attack from C-12 to C-13 was not selective, both 1a and 2a carbon cations could be generated, and abstraction of H-12 from both of them produced 1 and 2, respectively, whereas abstraction of H-11 from 2a produced 3 (Scheme 1). In order to determine whether the new compounds 1−3 are isolation artifacts, fuscol (4) was resubjected to the acidic silica gel column chromatography (the only possible connection of fuscol and acid during our isolation process) without any trace of the new compounds being produced, and 1−3 were also detected by HPLC in the original Et2O soluble extract (see SI for the detailed results, Figures S1−S6). Both the above
displayed a positive π−π* Cotton effect (CE) at 239 nm (Δε 7.24) and 232 nm (Δε 4.21), respectively, while that of 2 exhibited a negative π−π* CE at 228 nm (Δε −2.41). The initial torsional sampling (MCMM) and OPLS_2005 force field conformational searches of (1R,2R,4S,13S)-1, (1R,2R,4S,13R)-2, and (1R,2R,4S,13R)-3 afforded 22, 16, and 23 conformers, respectively, within the 21 kJ/mol energy window. The Boltzmann populations of the conformers were obtained based on the potential energy provided by the OPLS_2005 force field, leading to 8 conformers for 1, 6 ones C
DOI: 10.1021/acs.orglett.7b01716 Org. Lett. XXXX, XXX, XXX−XXX
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evidence confirmed that 1−3 are naturally generated, probably by the biosynthetic pathway that we proposed. It is worth mentioning that, although bicyclo[3.3.1]nonane-containing natural products are uncommon, such skeleton was found to be rich in plants derived polycyclic polyprenylated acylphloroglucinols (PPAPs),9 which have attracted great attention for their synthetical and biological studies. Therefore, the discovery of xishacorenes A−C as novel bicyclo[3.3.1]nonane-containing diterpenes with hydrocarbon property would captivate many chemists and biologists for their further investigation. All of the new compounds 1−3 were subjected to cytotoxic and immunological assays, and none of them showed cytotoxic effect against several cancer and normal cell lines, whereas some of them displayed weak proliferation effect on certain cancer cells, such as MCF-7/ADR cells (SI, Table S1). Interestingly, in the immunological activity test, xishacorenes A−C (1−3) were found to promote the ConA-induced T lymphocytes proliferation with dose-dependency (Figure 4), while they
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was financially supported by the Natural Science Foundation of China (Nos. 81520108028, 41676073, 81603022), SCTSM Project (No. 15431901000), the SKLDR/SIMM Projects (SIMM 1705ZZ-01). X.-W.L. is thankful for financial support from the “Youth Innovation Promotion Association” (Grant No. 2016258) from Chinese Academy of Sciences, “Young Elite Scientists Sponsorship” from China Association for Science and Technology (2016QNRC001), and Shanghai “Pujiang Program” (No. 16PJ1410600). F.Y. is thankful for the financial support of Syngenta-SIMM-PhD Studentship Project. We thank Prof. Ernesto Mollo from Institute of Biomolecular Chemistry (ICB) of the National Research Council (CNR), Italy, for the biological sample collection and Prof. Hui Huang from South China Sea Institute of Oceanology, CAS, for the identification of the soft coral material.
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Figure 4. Effects of xishacorenes A−C (1−3) on ConA-induced T lymphocyte proliferation; graphs show the mean ± SD, #P < 0.05 vs Crtl, *P < 0.05, and **P < 0.01 vs ConA. Control means without Con A and compound; 0 μM means with Con A while without compound.
had no activity on LPS induced B lymphocytes proliferation (SI, Figure S7). These results indicated that compounds of such a scaffold could be developed as novel and specific immunopotentiator for T lymphocytes. This is the first chemical investigation of S. polydactyla from the Xisha islands in the South China Sea. Three novel diterpenes xishacorenes A−C (1−3), with uncommon bicyclo[3.3.1]nonane structure features, were isolated and fully characterized. The discovery of 1−3 has expanded the diversity and complexity of marine diterpenes, especially those of hydrocarbon characters such as the famous healthcare product squalene. Due to the scarcity of the new products, biomimetic total synthesis and chemical correlation should be conducted to understand their biosynthetic origins and their biological role in the life cycle of the soft coral, which would be helpful for further specific pharmacological studies.
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Letter
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01716. General information on the experiment, experimental procedures, characterization data, biological activity assays, and NMR spectra for all the new compounds (PDF) D
DOI: 10.1021/acs.orglett.7b01716 Org. Lett. XXXX, XXX, XXX−XXX
