Garsubelone A, the First Dimeric Polycyclic Polyprenylated

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

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Garsubelone A, the First Dimeric Polycyclic Polyprenylated Acylphloroglucinols with Complicated Heptacyclic Architecture from Garcinia subelliptica Yong-Ling Wang,† Yan-Song Ye,‡,§ Wen-Wei Fu,† Rong Wu,† Qian Xiang,† Yuan-Zhi Lao,† Jin-Ling Yang,† Hong-Sheng Tan,† Xing-Wei Yang,‡ Bai-Can Yang,† Hong-Xi Xu,*,† and Gang Xu*,‡ Org. Lett. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 02/18/19. For personal use only.

†

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, and Engineering Research Centre of Shanghai Colleges for TCM New Drug Discovery, Shanghai, 201203, People’s Republic of China ‡ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, and Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming 650201, People’s Republic of China § University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Garsubelone A (1), the first dimeric polycyclic polyprenylated acylphloroglucinols type metabolite featuring a complicated 6/6/6/6/6/6/6 heptacyclic architecture containing 10 stereogenic centers, was isolated from Garcinia subelliptica. Biogenetically, this compound was constructed by the plausible monomeric precursor, garsubelone B (2) and secohyperforin, via a key Diels−Alder cycloaddition to form an unique 2oxabicyclo[3.3.1]nonane core. Their structures and absolute configurations were determined by comprehensive spectroscopic and X-ray diffraction techniques. The cytotoxic activities of these isolates were also evaluated.

P

samples were collected for further phytochemical studies. As a result, garsubelone A (1), which is the first dimeric PPAP-type metabolite featuring an unprecedented 6/6/6/6/6/6/6 heptacyclic system containing 10 chiral centers (Figure 1), was obtained together with garsubelone B (2), which is a new monomeric PPAP with a bicyclo[3,3,1]nonane-2,4,9-trione core. Biogenetically, the architecture of this unique dimer should be assembled by two bicyclic PPAPs (garsubelone B and secohyperforin 8 ) through a newly formed 2oxabicyclo[3.3.1]nonane core fused to a α-pyran ring via a key Diels−Alder cycloaddition. It was also noteworthy that 1 possessed extraordinary molecular weight which have never been reported in the field of PPAPs. The structures and absolute configurations of 1 and 2 were determined by comprehensive spectroscopic and X-ray diffraction analysis with Cu Kα radiation. In addition, compound 2 exhibited moderate cytotoxic activities against Hela, A549, and HepG2. Herein, the isolation, structural elucidation, proposed biosynthetic pathway, and cytotoxic activities of the new isolates are described. Garsubelone A (1) was obtained as colorless crystals. Its molecular formula, C60H84O8, was established by positive HRESIMS (m/z 933.6239, [M + H]+, calcd for 933.6239).

olycyclic polyprenylated acylphloroglucinols (PPAPs) are a special class of acylphloroglucinol derivatives containing bicyclic, adamantane, homo-adamantane, spirocyclic, and other related complicated architectures, which were exclusively isolated from the plants of Guttiferae family, especially genera Garcinia and Hypericum.1 Since the first report of PPAP-type natural products, that of hyperforin, in 1971,2 more than 540 PPAPs have been reported until now, many of which exhibit diverse and significant bioactivities, such as cytotoxicity, antiinflammatory, antidepressant, antioxidant, anti-HIV, and antibacterial activity.3 In the past 10 years, the study of PPAP has made rapid progress in phytochemistry, synthesis, and pharmacology.4 Particularly, structurally and biologically fascinating PPAPs exemplified by garcinielliptone FC,5a garcinol,5b guttiferone A,5c and clusianone5d have recently attracted wide attentions. Our group has focused on the systematic research of PPAP-type metabolites for more than 10 years; a total of 14 Garcinia plants have been studied6a and several PPAPs with unique skeletons and diverse bioactivities such as oblongifolin C,6b guttiferone K,6c hyphenrone A,6d hyperuralone A,6e and hyperjapone B6f have been reported. Garcinia subelliptica Merr. is an evergreen tree that is mainly distributed in tropical and subtropical Asia.7a Previous studies indicated that this plant is also a rich source of PPAPs.7b−e In our preliminary HPLC-MS experiments, a few trace constituents with a molecular ion peak of m/z >900 were found, which may represent novel compounds. Therefore, more © XXXX American Chemical Society

Received: January 30, 2019

A

DOI: 10.1021/acs.orglett.9b00388 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Figure 1. Structures of compounds 1 and 2.

Table 1. 13C (150 MHz) and 1H (600 MHz) NMR Spectroscopic Data of 1 in Chloroform-d6 (J in Hz) no.

δC

1 2 3 4 5 6

83.3 190.7 118.1 170.7 56.3 38.9

7 8 9 10 11 12 13 14 15

44.0 46.3 206.9 208.3 42.6 21.6 20.4 24.7 53.8

16 17

82.5 19.4

18 19 20 21 22 23 24

27.6 29.6 119.0 133.4 25.7 18.2 26.3

25 26 27 28 29 30

122.6 133.4 25.8 17.9 22.9 15.9

δH

no.

δC

1′ 2′ 3′ 4′ 5′ 6′

83.1 187.7 125.1 170.9 56.7 39.3

2.04, overlap 1.06, d (6.6) 1.13, overlap 2.32, overlap 0.91, dd (12.8, 4.4)

7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′

43.9 46.7 206.9 208.6 42.7 21.6 20.7 22.4 31.2

1.13, s

16′ 17′

81.1 40.3

18′ 19′ 20′ 21′ 22′ 23′ 24′

29.4 29.9 119.7 133.5 25.4 18.1 26.3

25′ 26′ 27′ 28′ 29′ 30′

122.5 133.3 25.7 17.8 23.0 15.7

a 1.81, overlap b 1.35, overlap 1.32, m

1.47, s 2.41, m 4.86, overlap 1.56, s 1.57, s a 2.09, m b 1.62, m 4.89, overlap 1.67, 1.54, 1.24, 0.98,

s s s s

Figure 2. Key HMBC and 1H−1H COSY correlations of 1.

δH

a 1.83, overlap b 1.33, overlap 1.35, m

1.98, sept (6.5) 1.02, d (6.5) 1.18, d (6.5) 3.05, m a 2.04, overlap b 1.13, overlap

Figure 3. X-ray crystallographic structure of 1.

a 3.27, dd (14.8, 4.9) b 0.99, overlap 1.51, s 2.35, overlap 4.91, overlap

Figure 4. Key HMBC and 1H−1H COSY correlations, X-ray crystallographic structure of 2.

1.49, s 1.60, s a 2.09, overlap b 1.62, overlap 4.88, overlap 1.66, 1.55, 1.21, 1.01,

(δC 56.3, C-5; 46.3, C-8 and 56.7, C-5′; 46.7, C-8′) and downfield quaternary carbons (δC 83.3, C-1 and 83.1, C-1′), as well as two distinguished methines (δC 44.0, C-7 and 43.9, C7′) and two methylenes (δC 38.9, C-6 and 39.3, C-6′) indicated the presence of two PPAP type units with the reported bicyclo[3.3.1]nonane-2,4,9-trione core.9 In addition, two isobutyryl groups were attached to C-1 and C-1′ by the HMBC correlations from H-11 to C-1 and from H-11′ to C-1′. Two pairs of gem-methyls were located at C-8 and C-8′ by the HMBC correlations from H3-29/H3-29′ and H3-30/H3-30′ to C-1/C-1′, C-7/C-7′, and C-8/C-8′. Four isoprenyl groups were attached to C-5, C-5′, C-7 and C-7′, respectively, on the basis of the HMBC correlations from H-19/H-19′ to C-4/C4′, C-5/C-5′, C-6/C-6′ and C-9/C-9′; from H-24/H-24′ to C6/C-6′, C-7/C-7′ and C-8/C-8′ along with 1H−1H COSY cross peaks of H-25 (25′)/H-24 (24′)/H-7 (7′)/H-6 (6′). Hence, the planar structures of units A and C were elucidated as shown in Figure 2.

s s s s

This extraordinary molecular formula and molecular weight comparing with reported PPAPs indicated a dimeric natural of 1, which was further confirmed by the observation of a number of paired signals in the 1H and 13C spectra (Figures S1 and S2). Detailed analysis of carbon signals revealed that 1 possessed 60 carbon atoms including 10 individual and 25 pairs of signals (Table 1). For the 25 paired signals, two nonconjugated ketones (δC 206.9, C-9 and 206.9, C-9′), two enolized 1,3-diketo groups (δC 190.7, C-2; 118.1, C-3; 170.7, C-4 and 187.7, C-2′; 125.1, C-3′; 170.9, C-4′), paired upfield B

DOI: 10.1021/acs.orglett.9b00388 Org. Lett. XXXX, XXX, XXX−XXX

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data undoubtedly confirmed the proposed structure and determined its absolute configuration as 1R,5S,7R (Figure 4). Obviously, garsubelone A (1) should biogenetically originate from two bicyclic PPAPs: garsubelone B (2) and secohyperforin (Scheme 1). It is well-known that the two precursors should be generated through the “mixed” prenylation/polyketide biosynthetic pathway.1b For compound 1, the isoprenyl side chain located at C-3 of secohyperforin may form intermediate i through dehydrogenation.11 The double bond in i serves as a dienophile in a key Diels−Alder cycloaddition with α-pyran ring in compound 2, resulting in the formation of intermediate ii.12 This dimeric PPAP was finally generated by the further cyclization of intramolecular double bond and hydroxyl.13 Detailed literature research shows that the polymers of PPAP have never been described previously,1 possibly because of the difficulties in the polymerization caused by the highly oxygenated phloroglucinol core and high steric hindrance of PPAPs. Therefore, the isoprenyl side chains may provide the maximum possibility of polymerization, which really evidenced by the characterization of 1 in this study. Therefore, it is expectable that more polymers of PPAPs formed by isoprenyl side chains can be found in the future. In addition, the inhibitory activities of isolates against the three human cancer cell lines (Hela, A549 and HepG2) were examined.14 And the result indicated that 2 exhibited moderate cytotoxicities against Hela, A549 and HepG2 with IC50 values of 6.0, 11.6, and 7.3 μM, respectively. The cytotoxic activity of compound 1 was also tested but showed no cytotoxicities with IC50 > 40 μM. In conclusion, garsubelone A (1), the first dimeric PPAP, featuring an unprecedented 6/6/6/6/6/6/6 heptacyclic architecture, and its biosynthetic precursor garsubelone B (2) were obtained from G. subelliptica. The discovery of 1 will enrich the chemical diversity of PPAPs. Being the first dimer of PPAP, the report of garsubelone A represents a milestone work during decades of the research on PPAP and might provide a clue on further research from biosynthetic, synthetic, and pharmacological communities.

Besides the 25 paired carbon signals for units A and C, the remained 10 carbons might be the connection of these two units (unit B). Moreover, its structure was evidenced by the HMBC correlations from H3-18′ (δH, 1.51) to C-15′ (δC, 31.2), C-16′ (δC, 81.1) and C-17′ (δC, 40.3); from H3-18 (δH, 1.47) and H3-17 (δH, 1.13) to C-15 (δC, 53.8) and C-16 (δC, 82.5) coupled with the spin−spin coupling system of Ha-17′ (δH, 3.27)\H-14 (δH, 2.32)\H-15 (δH, 0.91)\H-14′ (δH, 3.05)\Ha-15′ (δH, 2.04) observed in the 1H−1H COSY spectrum. The connection of units A and C through B was deduced by the HMBC correlations from H-14 to C-2, C-3, and C-4, from H-14′ to C-2′, C-3′, and C-4′, from H-15 and H-17′ to C-3, and H-15 and H-15′ to C-3′, respectively. In addition, two six membered ether rings were proposed by the HMBC correlations from H3-18′ to C-4′ and H3-18 to C-4, the downfield chemical shifts C-4 (170.7), C-16 (82.5), and C-4′ (170.9), C-16′ (81.1), as well as the requirement of the degrees of its unsaturation. Hence, the gross structure of 1 was defined as an unprecedented dimeric PPAP assembled by two bicyclic PPAPs through unit B, the newly formed 2oxabicyclo[3.3.1]nonane core constructed by the isoprenyl groups located at C-3 and C-3′, respectively. Since the complication and congestion of its structure and NMR spectral signals, the determination of the configurations was really a challenging task. After attempts with various solvent systems, a single crystal suitable for X-ray analysis was finally obtained. The planar structure of 1 was further confirmed and the absolute configuration was unambiguously determined as 1R,5S,7R,14S,15R,1′R,5′S,7′R,14′R,16′S (Figure 3). Garsubelone B (2) was obtained as colorless crystals. Its molecular formula of C30H42O4 was established by 13C NMR data and HRESIMS (m/z 467.3163 [M + H]+, calcd for 467.3156). This molecular formula indicated 68 mass units less than that of pyrano[7,28-b]hyperforin,10 in accordance with the absence of an isoprenyl group. Detailed comparison of their NMR data (Table S1) revealed that 2 should be the deisoprenylated derivative at C-29 of pyrano[7,28-b]hyperforin,10 which was further confirmed by the HMBC correlations from H3-29 (δH, 1.22) and H3-30 (δH, 1.00) to C1 (δC, 83.5), C-7 (δC, 43.4), and C-8 (δc, 46.8). The ROESY correlations of H-6a/H-19b and H-6a/H-24 displayed that 2 had the same relative configurations of C-1, C-5, and C-7 as pyrano[7,28-b]hyperforin. Finally, the X-ray crystallographic

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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.9b00388. C

DOI: 10.1021/acs.orglett.9b00388 Org. Lett. XXXX, XXX, XXX−XXX

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1D NMR data, general experimental procedures, physical data of the new compounds, original MS, 1D and 2D spectra for compounds 1 and 2 (PDF) Accession Codes

CCDC 1885446 and 1885656 contain 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] (H.-X. Xu). *E-mail: [email protected] (G. Xu). ORCID

Xing-Wei Yang: 0000-0002-9578-2986 Hong-Xi Xu: 0000-0001-6238-4511 Gang Xu: 0000-0001-7561-104X Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This is work was supported by grants from the three-year development plan project for Traditional Chinese Medicine (No. ZY (2018-2020)-CCCX-2001-02), foundations from Kunming Institute of Botany (No. KIB2017001) and Southeast Asia Biodiversity Research Institute (No. 2017CASSEABRIQG003), CAS, Yunnan Key Laboratory of Natural Medicinal Chemistry (No. S2017-ZZ11).

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DOI: 10.1021/acs.orglett.9b00388 Org. Lett. XXXX, XXX, XXX−XXX