Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Dysiarenone, a Dimeric C21 Meroterpenoid with Inhibition of COX‑2 Expression from the Marine Sponge Dysidea arenaria Wei-Hua Jiao,† Bao-Hui Cheng,‡ Guo-Dong Chen,§ Guo-Hua Shi,† Jing Li,† Tian-Yong Hu,‡ and Hou-Wen Lin*,† †
Research Center for Marine Drugs, State Key Laboratory of Oncogene and Related Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P. R. China ‡ Shenzhen Key Laboratory of ENT, Longgang ENT Hospital & Institute of ENT, Shenzhen 518172, P. R. China § Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou 510632, P. R. China S Supporting Information *
ABSTRACT: Dysiarenone (1), a dimeric C21 meroterpenoid featuring an unprecedented 2-oxaspiro[bicyclo[3.3.1]nonane9,1′-cyclopentane] carbon skeleton, was isolated from the marine sponge Dysidea arenaria. The structure of 1 was determined by HRMS and NMR spectroscopic analyses coupled with ECD calculations. Dysiarenone showed inhibitory activities against COX-2 expression and the production of prostaglandin E2 with an IC50 value of 6.4 μM in LPS-stimulated RAW264.7 macrophages.
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Dysiarenone (1) was isolated as a yellowish gum ([α]D20 +9.4). Its molecular formula was determined to be C42H56O4 by HRESIMS data (m/z 623.4105 [M − H]−, calcd for C42H55O4, 623.4100), with 15 degrees of unsaturation. The IR spectrum of 1 showed the presence of hydroxy (3367 cm−1), α,β-unsaturated ketone (1682 and 1665 cm−1), and benzene (1604 and 1459 cm−1) groups. The 1H NMR spectrum (Table 1) displayed two meta-coupling aromatic protons at δH 6.57 (d, J = 3.0 Hz) and 6.54 (d, J = 3.0 Hz), indicative of a 1,2,3,5-substituted benzene, two mutually cis-coupled olefinic protons at δH 6.62 (d, J = 10.2 Hz) and 6.14 (d, J = 10.2 Hz), as well as two olefinic singlets at δH 5.13 and 5.06. A detailed analysis of the 13 C and DEPT NMR spectra revealed the existence of 42 carbon resonances, including eight methyls, nine methylenes, 12 methines (two aromatic at δC 120.4 and 113.6 and four olefinic at δC 146.8, 130.8, 120.2, and 119.7), and 13 nonprotonated carbons (one unsaturated ketone at δC 196.8, four aromatic at δC 149.4, 145.1, 129.0, and 118.4, and two olefinic at δC 144.4 and 141.9). These aforementioned data accounted for seven out of 15 degrees of unsaturation, which is indicative of the presence of eight rings in 1. 1 H−1H COSY correlations of 1 coupled with HSQC spectrum showed the presence of five fragments (Figure 1). In the HMBC spectrum, four groups of HMBC cross-peaks, H3-11/C-3, C-4, and C-5, H3-12/C-4, C-5, C-6, and C-10, H3-13/C-7, C-8, and C-9, and H3-14/C-8, C-9, and C-10, allowed for the linkage of rings A and B and the attachment of the four methyl groups at C-4, C-5, C-8, and C-9, respectively. This assignment was verified by the long-range correlations from H-10 to C-1, C-2,
arine sponge derived C21 meroterpenoids, also known as sesquiterpene quinones/hydroquinones, represent a prominent class of mixed biogenic metabolites that incorporate a sesquiterpene moiety into a benzenoid unit.1 Their structural diversity and pharmaceutical potential have attracted continuing interest from both chemists and biologists.2 More than 200 C21 meroterpenoids have been isolated from various marine sponges,1 whereas only 12 dimeric meroterpenoids have been isolated since the first report of bispuupehenone in 1983 by Scheuer et al.3 Moreover, some dimers showed more intriguing biological activities than monomers.4 Our search for novel C21 meroterpenoids from the South China Sea prompted us to focus on the marine sponges of the Dysidea genus. Although several new C21 meroterpenoids were isolated from our previous studies,5 an assessment of the organic extract of Dysidea arenaria using LC−MS revealed the generation of a uniquely different molecular formula from those previously reported. Formula-guided fractionation yielded a novel dimeric C21 meroterpenoid, dysiarenone (1), in which the central quinone unit was uncommonly rearranged to an unprecedented 2-oxaspiro[bicyclo[3.3.1]nonane-9,1′cyclopentane] carbon skeleton. Herein, we report the structural elucidation, possible biogenetic pathway, and biological activity of this strained heterocyclic metabolite.
Received: April 11, 2018
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.8b01148 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 1. 1H and 13C NMR Data for 1 in CDCl3 no.
δC
1 2α 2β 3 4 5 6α 6β 7 8 9 10 11 12 13 14 15α 15β 16 17 18 19 20 21 17-OH
34.2 28.9 119.7 141.9 38.0 35.7 27.9 45.6 41.2 58.5 16.8 18.9 18.5 18.8 51.9 49.6 97.5 146.8 130.8 196.8 58.8
δH (J, Hz) 2.44, 2.00, 1.90, 5.06,
ddd (14.4, 10.2, 3.0) m m br s
0.88, 1.55, 1.30, 1.15,
m m m m
1.34, 1.57, 0.92, 1.01, 0.98, 0.91, 1.77,
d s s d s d d
(14.4)
(6.0) (14.4) (14.4)
6.62, d (10.2) 6.14, d (10.2) 3.38, s 2.92, s
no.
δC
1′a 1′b 2′ 3′ 4′ 5′ 6′α 6′β 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′a 15′b 16′ 17′ 18′ 19′ 20′ 21′
19.6 26.4 120.2 144.4 38.3 36.9 28.6 36.2 42.0 45.5 18.1 20.0 16.6 17.8 37.2 129.0 145.1 118.4 113.6 149.4 120.4
δH (J, Hz) 1.52, 0.84, 2.08, 5.13,
m m m br s
1.16, 1.67, 1.44, 1.13,
m dt (12.6, 2.4) m m
1.19, 1.50, 0.99, 0.74, 0.84, 2.79, 2.51,
d d s d s d d
(12.6) (1.2) (6.6) (13.8) (13.8)
6.54, d (3.0) 6.57, d (3.0)
confirmed by the HMBC correlation of H-19′/C-21 (Figure S29). Additionally, HMBC correlations from the shielded hydroxy 17-OH (δH 2.92) to C-17 (δC 97.5) and C-18 suggested the hemiketal nature of C-17 (Figure S31) and established a new phenolic ether bond between C-17 and C-17′, thus resulting in the formation of ring E to account for the remaining one degree of unsaturation. Finally, the unusual tricyclic 2-oxaspiro[bicyclo[3.3.1]nonane-9,1′-cyclopentane] scaffold (rings C/D/E) was disclosed in 1, as depicted in Figure 1. The structure of 1 totally possesses 12 stereocenters in two spatially isolated chiral clusters, C-1−C-21 in part A and C-1′− C-15′ in part B. Determination of the relative configuration within part A utilized a combination of J-based configuration analysis and extensive NOESY evidence. The large coupling constant between H-1 and H-10 (J = 14.4 Hz) and the diagnostic chemical shift of C-12 (δC 18.9) suggested the axial orientations of the two protons and the trans fusion of rings A/B/C (Figures 2a,b),7 which was supported by the NOESY correlations of H-1/H3-12 and H-1/H3-14 (Figure S39). Moreover, the NOESY correlations of H-6β/H3-12, H3-12/H3-14, H3-13/H3-14, and H3-14/H-15β indicated the β-orientation of these protons and methyl groups (Figure S41), whereas the α-orientation of H-2α, H-8, H-10, and H-15α was determined by the NOESY correlations from H-10 to H-2α, H-8, and H-15α (Figure 2a and Figure S42). In addition, the correlation of H-10/H-15α implied the envelope-type conformation of ring C. In particular, NOESY correlations from H-18 to H-1 and H3-14 suggested the axial orientation of the carbon bond C-17−C-18 (Figure S43). Furthermore, the correlation between 17-OH and H3-14 supported the β-orientation of 17-OH, while the NOESY correlation of H-21/H-2α, H-10, and H-15α indicated the α-orientation of H-21. In addition, the relative configurations of C-5′, C-8′, C-9′, and C-10′ in part B were determined as 5′S*,8′S*,9′R*,10′S* by the NOESY experiment
Figure 1. Key COSY and HMBC correlations of 1.
C-4, C-5, C-8, C-9, C-12, and C-14 (Figures S33 and S34). Meanwhile, the five-membered ring C was established by the HMBC correlations of H-1/C-17, H3-14/C-9, C-10, and C-15, and H2-15/C-1, C-9, C-10, and C-16 (Figures S31 and S34). Moreover, the HMBC correlations of H2-15/C-17 and C-21, H-18/C-16 and C-20 as well as H-19/C-17 and C-21 indicated the presence of a rare spiro[4.5]dec-8-en-7-one moiety (rings C/D), which was supported by the HMBC correlations of H-21/C-16, C-17, C-19, and C-20. Thus, one part of the C21 meroterpenoid (part A) was revealed. In part B, the bicyclic sesquiterpene moiety (from C-1′ to C-15′, rings G and H) was established by COSY and HMBC correlations (Figure 1). The presence of the p-hydroquinone unit (ring F) was deduced from the HMBC correlations of H-19′/C-17′and C-21′, H-21′/ C-17′, C-19′, and C-20′ (Figure S29) coupled with the chemical shifts of C-17′ (δC 145.1) and C-20′ (δC 149.4). The position of the p-hydroquinone unit at C-15′ was determined by HMBC correlations of H3-14′/C-15′, H2-15′/C-16′, C-17′, and C-21′ as well as H-21′/C-15′. The planar structure of part B is identical to that of avarol.6 Furthermore, H-21 showed key HMBC correlations with C-17′, C-18′, and C-19′, which linked ring D with ring F by an unusual covalent connection of C-21−C-18′, as B
DOI: 10.1021/acs.orglett.8b01148 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 3. Experimental ECD spectrum of 1 and the calculated ECD spectra of two possible absolute configurations of 1 (bandwidth σ = 0.25 eV).
tentatively assigned as 5′S,8′S,9′R,10′S, the same as avarol. Further determination of the full configuration of 1 will likely rely on the stereocontrolled synthesis of both possible diastereomers followed by detailed NMR and optical comparisons with the natural product. Dysiarenone (1) represents the first dimeric C21 meroterpenoid possessing a unique 2-oxaspiro[bicyclo[3.3.1]nonane-9,1′cyclopentane] scaffold from nature. Biogenetically, this dimeric compound might be derived from two monomers isolated from the title marine sponge, avarone and avarol (Scheme 1).
Figure 2. (a) Key NOESY correlations of 1. (b) Configuration and conformation of 1 established on the basis of key coupling constants and NOESY correlations.
(Figure 2). The proposed structure of 1 was confirmed by a comparison between the experimental and GIAO-based calculated 13C NMR chemical shifts (Supporting Information (SI)). Thus, the relative configurations within parts A and B of 1 were unambiguously established as shown in Figure 2. The spatial isolation means that the NMR methods alone are insufficient to unambiguously define the relative configuration between the two chiral clusters. Unfortunately, crystals for 1 were unachievable, likely due to their isolation as a gum, and the limited amount of the sample. As a result, a computational method was used to clarify the absolute configurations of parts A and B in 1 by comparing the experimental ECD spectrum with the time-dependent density-functional theory (TDDFT) calculated ECD spectra. A conformational search for 1 was initially performed using CONFLEX version 7.0 with an MMFF94s force-field, 5 kcal mol−1 above the ground state, and an RMSD cutoff (rmsd) of 0.5 Å. Then each of the acceptable conformers was optimized as part of the HF/6-31G(d) method in the Gaussian09. Further optimization at the APFD/6-31G(d) level provided 15 stable conformers, which were taken for ECD calculations performed with APFD/6-311++G(2d,p). The solvent effects were taken into account by the polarizableconductor calculation model (CPCM, acetonitrile as the solvent). The resulted excitation energies and rotational strengths were Boltzman weighted and fitted to a Gaussian function to generate the computed ECD spectra for the four possible isomers of 1 (SI) and subsequently superimposed on the experimental data. The experimental ECD spectrum of 1 displayed positive Cotton effects at 362 nm and negative at 242 nm, mainly contributed by the strained 2-oxaspiro[bicyclo[3.3.1]nonane-9,1′-cyclopentane] motif in part A. The calculated ECD spectra for two out of the four isomers, 1S,5S,8S,9R,10S,16S,17S,21S,5′S,8′S,9′R,10′S-1a and 1S,5S,8S,9R,10S,16S,17S,21S,5′R,8′R,9′S,10′R-1b, showed that a good match with the experimental spectrum of 1 with 1a was better than 1b (Figure 3); thus, the absolute configurations of eight chiral centers in part A were assigned as 1S,5S,8S,9R,10S,16S,17S,21S; meanwhile the four chiral centers in part B were
Scheme 1. Hypothetical Biosynthetic Pathways for 1
The dehydrogenation between C-1 and C-10 followed by an intramolecular nucleophilic addition from the double bond Δ1,10 to C-16 leads to the formation of a spiro[4.5]dec-8-en-7one unit.5a Subsequent quinone rearrangement and etherization between C-17 and C-17′ afford a key intermediate a. A further intramolecular condensation between C-21 and C-18′ finally forms the new strained tricyclic carbon skeleton in 1. Cyclooxygenase-2 (COX-2) is an inducible isozyme of COX, which plays an important role in the conversion of arachidonic acid to various prostaglandins and promotes cellular proliferation, angiogenesis, cancer invasiveness, and antiapoptosis.8 Selective COX-2 inhibitors have been used as a type of nonsteroidal anti-inflammatory drug (NSAID); however, they are not amenable to prolonged administration since they may cause severe side effects. COX-2 expression is a multistep process, which is highly regulated at transcription and post-transcriptional levels. Targeting COX-2 expression might represent a promising strategy by which the same therapeutic benefits could be gained while avoiding the severe side effects of COX-2 enzymatic inhibition.9 Therefore, LPS-stimulated RAW264.7 C
DOI: 10.1021/acs.orglett.8b01148 Org. Lett. XXXX, XXX, XXX−XXX
Organic Letters
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macrophages were treated by dysiarenone (1) to investigate their inhibitory activity against COX-2 expression. The results show that 1 selectively inhibited the LPS-stimulated COX-2 expression in a dose-dependent manner without having a significant impact on COX-1 expression (Figure 4a). Moreover,
dysiarenone A decreased the production of PGE2 in LPSstimulated macrophages with an IC50 value of 6.4 μM (Figure 4b and SI), about 10 times more potent than that of avarol, which indicated that the incorporation of a 2-oxaspiro[bicyclo[3.3.1]nonane-9,1′-cyclopentane] scaffold could enhance the inhibitory activity of dysiarenone. In summary, dysiarenone (1) is a structurally unique dimeric C21 meroterpenoid isolated from the marine sponge D. arenaria. This metabolite showed potent inhibitory activities against COX-2 expression and PGE2 production in LPS-stimulated RAW264.7 macrophages. The complex structure, natural abundance, and intriguing biological activity of 1 make it a good target for chemical synthesis.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01148. Experimental section, including extraction and isolation; 13 C NMR and ECD spectra calculations; bioassay; 1D and 2D NMR, HRESIMS, IR, UV, and ECD spectra for 1 (PDF)
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REFERENCES
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Figure 4. (a) Compound 1 inhibits COX-2 expression in LPSstimulated RAW 264.7 macrophages without significant impact on COX-1 expression. (b) The inhibitory activity of 1 against the production of PGE2 in LPS-stimulated RAW 264.7 macrophages. Data were derived from three independent experiments and summarized as mean ± SD **P < 0.01, ***P < 0.001 compared to the control group.
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hou-Wen Lin: 0000-0002-7097-0876 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Professor Jin-He Li (Institute of Oceanology, CAS) for identifying the marine sponge. This work was supported by the National Natural Science Foundation of China programs (Nos. 41576130, U1605221, 81773978, 41476121, 81403160, and 81673315). We are also grateful for the high-performance computing platform of Jinan University. D
DOI: 10.1021/acs.orglett.8b01148 Org. Lett. XXXX, XXX, XXX−XXX
