Letter pubs.acs.org/OrgLett
Xylopiana A, a Dimeric Guaiane with a Case-Shaped Core from Xylopia vielana: Structural Elucidation and Biomimetic Conversion Ya-Long Zhang, Xu-Wei Zhou, Xiao-Bing Wang, Lin Wu, Ming-Hua Yang, Jun Luo, Yong Yin, Jian-Guang Luo,* and Ling-Yi Kong* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Xylopiana A (1), a dimeric guaiane with an unprecedented pentacyclo[5.2.1.01,2.04,5′.05,4′]decane-3,2′-dione core, and three biosynthetically related intermediates, compounds 2−4, were isolated from the leaves of Xylopia vielana. Their structures and absolute configurations were determined by a combination of spectroscopic data, X-ray crystallography, electronic circular dichroism calculations, and chemical conversion. The structure of known vielanin A was revised to be compound 3. Compound 4 exerted a 3.7-fold potentiation effect on doxorubicin susceptibility at the tested concentration of 10 μM.
D
imeric sesquiterpenoids (DSs) are widely distributed in the plant kingdom, and they display diverse structures and a variety of biological activities.1 In recent decades, DSs have attracted the attention of not only organic chemists but also pharmacologists.2 Guaiane-type sesquiterpenoid dimers are one of the main classes of DSs, predominantly composed of dimeric guaianolides. However, only six dimeric guaianes have been isolated from plant origin, all of which occur in the genus Xylopia from the family Annonaceae.3 As a part of our search for structurally diverse DSs from natural sources,2a investigations on the chemical constituents of the leaves of Xylopia vielana Pierre, the only Xylopia species in the People’s Republic of China,4 were carried out. A unique dimeric guaiane, xylopiana A (1), possessing an unusual case-shaped pentacyclo[5.2.1.01,2.04,5′.05,4′]decane-3,2′-dione core, and three biosynthetically related intermediates, compounds 2−4 (Figure 1), were isolated. Biomimetic conversion of 2 to 1 was successfully conducted by a photochemical [2 + 2] cycloaddition reaction. Additionally, the structure of known vielanin A was revised to be compound 3. Herein we describe the structure elucidation, chemical conversion, hypothetical biogenetic pathway, and multidrug resistance (MDR) reversal activity evaluation of 1−4. Xylopiana A (1) was obtained as a white amorphous powder. Its molecular formula was established to be C30H36O4 by HRESI-MS (m/z 483.2509 [M + Na]+; calcd 483.2506), corresponding to an index of hydrogen deficiency of 13. The UV spectrum of 1 showed an absorption maximum at 254 nm. The IR spectrum displayed the characteristic absorptions for carbonyls (1750 and 1676 cm−1). The 1H NMR spectrum showed the signals of six tertiary methyls and two secondary methyls. The 13C NMR and DEPT spectra revealed the presence of 30 carbons, including four ketonic, four olefinic, six quaternary, four methinic, four methylenic, and eight methylic © 2017 American Chemical Society
Figure 1. Structures of compounds 1−4.
carbons. The 1D NMR data (Table S1) featured two guaiane units (A and B) in the structure of 1, which was further confirmed by 2D NMR experiments (Figure 2). In unit A, the HMBC cross-peaks from H3-15 to C-3/C-4/C-5 and from H-2 to C-1/C-3/C-4/C-5 established the presence of a fivemembered ring (I). A seven-membered ring (II) fused with ring I in unit A at C-1 and C-5 was revealed by the HMBC cross-peaks from H2-6 to C-1/C-4/C-5/C-7/C-8, from H2-9 to C-1/C-7/C-8/C-10/C-14, and from H-10 to C-1/C-8/C-9. The HMBC cross-peaks from H3-12/H3-13 to C-7/C-11 suggested that a methylethylidene was linked to ring II at CReceived: April 27, 2017 Published: May 24, 2017 3013
DOI: 10.1021/acs.orglett.7b01276 Org. Lett. 2017, 19, 3013−3016
Letter
Organic Letters
Figure 3. Key HMBC and ROESY correlations of 2. Figure 2. Key (A) HMBC and (B) ROESY correlations of 1.
that H3-14 is β-oriented. Because of the ambiguous ROESY correlations, however, the relative configuration at C-10′ could not be determined with confidence. In order to completely determine its relative configuration, compound 2 was reduced under NaBH4. Fortunately, the major product 2a was obtained under an excess of NaBH4, and its structure was elucidated by HR-ESI-MS and NMR experiments (see the Supporting Information). In the ROESY spectrum of 2a recorded in DMSO-d6, the key ROESY correlations from OH-2′ to H-2/H-10 and H3-14′ to H-2′/OH-2′ (Figure 4) revealed that H3-14′ is α-oriented. Thus, the structure of 2 was elucidated as shown.
7. On the basis of the above spectral analysis, unit A was deduced to be a tetrasubstituted guaia-7(11)-en-3,8-dione moiety. Similar to unit A, unit B was assigned as a tetrasubstituted guaia-7(11)-en-2,8-dione moiety with the aid of key HMBC cross-peaks. Therefore, compound 1 was elucidated as a rare dimeric guaiane. The connection between units A and B through four direct C−C bonds (C-1 to C-3′, C-2 to C-1′, C-4 to C-5′, and C-5 to C-4′) constructing a case-shaped core was established by the key HMBC cross-peaks from H-3′ to C-2/C-5/C-10, from H-2 to C-5′/C-10′, from H3-15 to C-5′, and from H3-15′ to C-5 (Figure 2); this was also supported by the index of hydrogen deficiency. Thus, the planar structure of 1 possessing an intriguing pentacyclo[5.2.1.01,2.04,5′.05,4′]decane-3,2′-dione core was established as shown. On the basis of the rigidity of the pentacyclic core, the relative configurations at C-1, C-2, C-4, C-5, C-1′, C-3′, C-4′, and C-5′ were set up as shown. The relative configurations of the remaining chiral centers at C-10 and C-10′ were determined on the basis of a ROESY experiment. As shown in Figure 2, the ROESY correlations of H3-14/H-2 and H-10/ H-3′ indicated that H3-14 is β-oriented, while the ROESY correlations of H-10′/H-2, H3-14′/H-2, H3-14′/H-9′a, H-9′a/ H-6′b, and H-6′a/H3-15, H3-12′, H3-15′ combined with the unobserved correlation of H-10′/H-6′b revealed that H3-14′ is α-oriented. Thus, the relative configuration of 1 was established as depicted. The molecular formula of vielanin F (2) was assigned as C30H36O4 on the basis of its HR-ESI-MS peak at m/z 483.2504 [M + Na]+ (calcd for C30H36NaO4, 483.2506). In the 1H NMR spectrum of 2, the presence of eight methyl signals, which was identical to that of 1, suggested that compound 2 might also be a dimeric guaiane. In a comparison of the 13C NMR data for 2 (Table S1) with those for 1, four extra olefinic carbon resonances occurred in the downfield region while four quaternary carbon resonances in the upfield region disappeared, indicating that the connectivity of the two guaiane moieties in 2 is different from that in 1. According to the HSQC and HMBC experiments, the two guaiane moieties in 2 were deduced to be a disubstituted guaia-4,7(11)-dien-3,8-dione and a disubstituted guaia-4,7(11)-dien-2,8-dione. The connection of these two guaiane moieties through two direct C−C bonds (C-1 to C-3′ and C-2 to C-1′) constructing a bicyclo[2.2.1]heptane moiety was determined by the key HMBC cross-peaks from H-3′ to C2/C-10 and from H-2 to C-5′/C-10′ (Figure 3). The relative configuration of the bicyclic system with the endo orientation of the five-membered ring at C-1 and C-2 was assigned as shown by the key ROESY correlation of H3-15′/H-6b (Figure 3). The ROESY correlations of H3-14/H-2 and H-10/H-3′ suggested
Figure 4. Reduction of 2 under NaBH4.
Compound 3 was obtained as colorless crystals. Its HR-ESIMS and NMR data were identical to those of vielanin A (Table S3), which had been isolated from the title plant by Adam and co-workers.3b In the previous literature,3b the relative configuration at C-10 was deduced according to the NOE effect between H3-14 and H-2′. However, not only the correlation between H3-14 and H-2′ but also a correlation between H-10 and H-2′ were observed in the ROESY spectrum of 3 (Figure 5). Therefore, the relative configuration at C-10 could not be determined confidently according to these ROESY correlations. Finally, the stereochemistry of 3 was unambiguously determined by X-ray crystallography (CCDC 1545840) using Cu Kα radiation (Figure 5) and comparison of its experimental electronic CD (ECD) spectrum with the calculated curve (Figure 6). In the literature, the relative configuration at C-10 in vielanin A was misassigned. The structure of vielanin A was thus revised to compound 3. The 3014
DOI: 10.1021/acs.orglett.7b01276 Org. Lett. 2017, 19, 3013−3016
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Organic Letters
Figure 5. Selected ROESY correlations and single-crystal X-ray structure of 3.
Figure 7. Chemical correlation between 3 and 4.
Scheme 1. Plausible Biogenetic Pathway for 1−4
Figure 6. Calculated and experimental ECD spectra of 2 and 3.
ECD curve of compound 2 showed Cotton effects similar to those of compound 3 (Figure 6). Furthermore, the experimental ECD spectrum of 2 is in good accordance with the calculated one for the (1R,2R,10R,1′S,3′S,10′R) enantiomer (Figure 6). Therefore, the stereochemistry of 2 was established as depicted. The NMR spectra of compound 4 displayed pairs of close signals due to an isomeric mixture at OH-8, which were highly identical to those of vielanin E (Table S5).3c Vielanin E was also obtained by Adam and co-workers from the title plant. From biosynthetic considerations, the absolute configuration of 4 should be the same as that of 3, except for C-8 in the peroxide ring of 4. In order to determine the absolute configuration of 4, the chemical correlation between 3 and 4 was investigated (Figure 7). Interestingly, the deacetylated peroxide product 3a was obtained when 3 was treated with K2CO3. Compound 3a was also obtained when 4 was treated with K2CO3. Thus, the absolute configuration of 4 except for C-8 was established as depicted. A plausible biosynthetic pathway for compounds 1−4 is proposed as shown in Scheme 1. Starting from farnesyl pyrophosphate (FPP), two monomeric guaiane intermediates (i and ii) are generated by cyclization and oxidation reactions. The reactive cyclopentadienone moieties in i and ii enable an endo Diels−Alder cycloaddition of these two monomers to afford 2, which is hypothesized to be the biosynthetic intermediate of 1. Subsequently, an intramolecular [2 + 2] cycloaddition in 2 furnishes 1, while reduction and acetylation of 2 affords 3 and further peroxidation affords 4. In order to define the absolute configuration of 1 and validate its postulated biosynthetic pathway, the biomimetic conversion from 2 to 1 was carried out. Guided by the biosynthetic
hypothesis, the photochemical [2 + 2] cycloaddition reaction was conducted on 2. Fortunately, irradiating 2 under a nitrogen atmosphere generated 1 in 45% yield (Figure 8) with recovery of 2 in 13% yield.5 Therefore, the absolute configuration of 1 was established as depicted.
Figure 8. Biomimetic conversion from 2 to 1.
Compounds 1−4 were evaluated for cytotoxicity in doxorubicin-resistant human breast carcinoma cells (MCF-7/ DOX). None of the compounds showed any cytotoxicity at the tested concentration against the drug-sensitive and multidrugresistant cells (IC50 > 30 μM). Subsequently, screening of their MDR reversal activities in MCF-7/DOX cells showed that the noncytotoxic 4 exerted a 3.7-fold potentiation effect on doxorubicin susceptibility at the tested concentration of 10 3015
DOI: 10.1021/acs.orglett.7b01276 Org. Lett. 2017, 19, 3013−3016
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Organic Letters μM (Table S6), while no obvious activity was observed for 1−3 at the tested concentration of 30 μM.6 This result indicated that the peroxide ring in 4 is essential for the MDR reversal activity. In summary, xylopiana A (1), a novel case-shaped guaiane dimer, and three biosynthetically related intermediates, compounds 2−4, were isolated from the leaves of X. vielana. Chemical conversion of these compounds not only confirmed their structures but also validated their biosynthetic relationship. The MDR reversal activities of guaiane dimers were evaluated for the first time. Our study provides a new structural template for discovering potential MDR reversal agents from natural products.
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R. R.; Wang, J.; Ma, Y.; Li, W. X.; Jiang, R. W.; Cai, S. H. Oncotarget 2016, 7, 31466−31483.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01276. Details of isolation, chemical conversion, and biological experimental procedures; MDR reversal activities of 1− 4; 1D NMR data and NMR spectra for 1−4, 2a, and 3a (PDF) Crystallographic data for 3 (CIF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (L.-Y. Kong). *E-mail:
[email protected] (J.-G. Luo). ORCID
Ling-Yi Kong: 0000-0001-9712-2618 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was financially supported by the National Natural Sciences Foundation of China (Program 81430092), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63).
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DOI: 10.1021/acs.orglett.7b01276 Org. Lett. 2017, 19, 3013−3016
