Fluvirosaones A and B, Two Indolizidine Alkaloids with a Pentacyclic

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

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Fluvirosaones A and B, Two Indolizidine Alkaloids with a Pentacyclic Skeleton from Flueggea virosa Xiang-Kun Luo,† Jie Cai,† Zhi-Yong Yin,† Pan Luo,† Chan-Juan Li,† Hang Ma,‡ Navindra P. Seeram,‡ Qiong Gu,*,† and Jun Xu† †

Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China ‡ Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States S Supporting Information *

ABSTRACT: Fluvirosaones A (1) and B (2), together with virosecurinine (3), were isolated from Flueggea virosa. Their structures were determined by physical, spectroscopic, and X-ray analysis and confirmed through comparison of the calculated and experimental 13C NMR and electronic circular dichroism (ECD) data. Compounds 1 and 2 represent the first examples of a pentacyclic Securinega alkaloid containing a pentacyclic system and an α,β-unsaturated ketone. Plausible biogenetic pathways of compounds 1 and 2 are proposed.

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he Flueggea genus (Euphorbiaceae family) is characterized by diverse Securinega alkaloids whose structures and range of biological activities have attracted wide interest from several research groups. Recent studies have focused on synthetic efforts 1−3 and the search for diverse monomeric, 4−11 dimeric,5,12−17 trimeric,17 and tetrameric18 Securinega alkaloids. The Flueggea genus consists of 12 species, four of which are found in China and distributed throughout the country with the exception of Northwest China. These plants have been used as Chinese herbal agents against rheumatic arthritis and pustular eruption.19 Flueggea virosa Roxb. ex Willd has been widely studied due to the many Securinega alkaloids it contains.20 In continuation of our group’s interest in structurally unique bioactive compounds from the Euphorbiaceae family,21−23 we studied the leaves and twigs of F. virosa, which led to the discovery of two novel pentacyclic indolizidine alkaloids, fluvirosaones A (1) and B (2), together with the known compound virosecurinine (3) (Figure 1). Herein, we report the isolation and structure identification of compounds 1 and 2. In addition, feasible biosynthetic pathways for these compounds are proposed. This is first report of compounds 1 and 2 from nature. Compounds 1 and 2 contain a similar pentacyclic α,βunsaturated ketone incorporated with skeletons of virosecurinine24 and secu’amamine A,7 respectively. Moreover, compound 1 is the first example of a 5,6,5,6,5 cyclic Securinega alkaloid, while compound 2 is the first reported alkaloid containing a 5,5,6,6,5 ring system. Fluvirosaone A (1) was obtained as colorless crystals from acetone. Its molecular formula was determined as C16H15NO3 by the HRESIMS ion at m/z 270.1127 [M + H]+ (calcd for C16H16NO3, 270.1130), indicating 10 degrees of unsaturation. © XXXX American Chemical Society

Figure 1. Structure of compounds 1−3.

The IR absorption bands at 1752 and 1626 cm−1 suggested the presence of an unsaturated γ-lactone.11 The NMR data for 1 (Table S1) demonstrated the presence of a carbonyl (δc 203.1), a lactone (δc 171.2), two trisubstituted double bonds (δc 165.7, 108.9, 174.0 and 134.4; δH 5.70 and 6.01), and a disubstituted double bond (δc 148.5 and 122.6; δH 7.10 and 6.68), and 8 sp3 carbons (CH2 × 5, CH × 1, and C × 2 at δc 91.7 and 73.3). The above spectral data were similar to those of virosecurinine which has a molecular formula of C13H15NO2 and seven degrees of unsaturation,24 with the exception of three additional carbon signals. These included one carbonyl carbon (δc 203.1), one olefinic tertiary carbon (δc 134.4), and one methylene (δc 52.4). The additional signals from fluvirosaone A accounted for two degrees of unsaturation, which indicated the presence of an extra ring system in compound 1. The 1H−1H COSY correlations (Figure 2) revealed the existence of C4−C5−C6 and C8−C7−C15−C14 fragments. The aforementioned data revealed that compound 1 is a Securinega alkaloid with a fiveReceived: December 18, 2017

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DOI: 10.1021/acs.orglett.7b03935 Org. Lett. XXXX, XXX, XXX−XXX

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data of 2 (Table S1) were similar to those of 1 and included one carbonyl carbon (δc 203.1), one lactone (δc 172.7), two trisubstituted double bonds (δc 166.6, 108.5, 173.7, and 129.9; δH 5.77, s, and 5.97, s), one disubstituted double bonds (δc 141.5 and 120.1; δH 6.37 and 6.37 overlap), and eight sp3 carbons (CH2 × 5, CH × 1, and C × 2 at δc 80.1 and 71.2). The 1H−1H COSY correlations of H-7/H-15 and H-7/H-8, H-4/H-3 and H-4/H-5 of fluvirosaone B revealed the presence of the two spin systems C8−C7−C15−C14 (CH2−CH−CH CH) and C3−C4−C5 (CH2−CH2−CH2) which were present in compound 1. Further analysis of the HMBC spectrum (Figure 2) revealed that compound 2 is possibly a derivative of secu’amamine A.7 The HMBC cross peaks from H-8 to C-9a/ C-9/C-13/C-15, H-12 to C-9/C-13/C-14, from H-14 to C-9/ C-13, and from H-1′ to C-9/C-9a allowed the assignment of an unsaturated carbon (δc 129.9) attached to C-9a. In addition, the HMBC cross peaks between H-3 and C-2/C-3′ and between H-3′ and C-2′/C-1′/C-9a allowed for the assignment of a methylene (δc 57.6) attached to C-2. Thus, the planar structure of 2 was determined as shown in Figure 2. The relative configuration of 2 was determined by NOESY correlations (Figure 2) between H-3 (δH 2.19) and H-8 (δH 2.79), which indicated that C-8 and the disubstituted propyl group were on the same side of a plane. NMR chemical shift calculations have been used to predict the planar structure and relative configuration of new natural products.25−27 Therefore, in the present study, the 13C NMR chemical shifts of 2 were calculated at the B3LYP/6-311 + G (2d, p) level. The predicted 13 C NMR spectral data for the suggested configuration of 2 were consistent with the experimental data, the correlation coefficient (R2) between experimental and calculated data was 0.9986 (data obtained by linear analysis, Figure 4), and the

Figure 2. Key 1H−1H COSY (blue) and HMBC (red arrows) cross peaks of 1 and 2, and the NOESY correlations of 2 (double arrows).

membered ring sharing one quaternary carbon (δc 73.3) and one unsaturated tertiary carbon (δc 174.0). This deduction was further confirmed by the HMBC analysis (Figure 2). The cross peaks from H-12 to C-9, from H-14 to C9/C-13, from H-8 to C-2/C-9/C-13, and from H-1′ to C-2/C9/C-2′ allowed the assignment of a methylene group (δc 52.4) attached to C-2. In addition, the HMBC cross peaks between H-4 and C-2/C-3/C-3′ and between H-3′ and C-1′/C-2/C-4 allowed for the identification of an unsaturated carbon (δc 134.4) attached to C-3. Accordingly, the planar structure of 1 for fluvirosaone A was deduced as shown in Figure 2. Due to the signal overlapping the NOESY spectrum (Figure S8) failed to indicate the relative configuration of 1. Therefore, the structure of 1 was determined by X-ray crystallography and its absolute configuration was unambiguously determined as a 2R,7R,9R-configuration by an X-ray diffraction experiment with Cu Kα radiation [Figure 3; Flack parameter: 0.00(5)]. Fluvirosaone B (2) was obtained as a colorless oil. Its molecular formula was determined to be C16H15NO3 by HRESIMS at m/z 270.1122 [M + H]+ (calcd for C16H16NO3, 270.1130), which requires 10 degrees of unsaturation. The IR spectrum of 2 showed absorption bands at 1738 and 1627 cm−1 consistent with an α,β-unsaturated γ-lactone.6,11 The NMR

Figure 4. Linear regression analysis of experimental vs calculated 13C NMR chemical shifts of 2. Linear fitting is shown as a line.

relative chemical shift error was within 5.0 ppm (Table S1). Taken together, these data confirmed the relative configuration of 2. Since compound 2 was obtained as a colorless oil, which could not be crystallized for X-ray crystallographic analysis, its absolute configuration was elucidated by comparing the overall predicted ECD spectra of (2R,7R,9R)-2, (2S,7S,9S)-2, (2S,7R,9R)-2, and (2R,7S,9S)-2 with the experimental ECD spectrum. The calculated ECD data of (2R,7R,9R)-2 were in agreement with the experimental data (Figure 5), and thus, the absolute structure of 2 was determined as 2R,7R,9R. Biosynthetic pathways for 1 and 2 are proposed in Scheme 1. Virosecurinine (3), first identified from F. virosa,28 and also isolated in this study, is postulated as the biosynthetic precursor of compounds 1 and 2. The intermediates I and II could be

Figure 3. X-ray crystallographic structure of 1. B

DOI: 10.1021/acs.orglett.7b03935 Org. Lett. XXXX, XXX, XXX−XXX

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compounds 1 and 2 showed weak effects in reducing triglyceride accumulation in 3T3-L1 cells (Figure 6).

Figure 5. Calculated and experimental ECD spectra of 2. The calculated ECD spectra were computed at the B3LYP/6-311+G(2d,p) level in MeOH.

Figure 6. Effects of compounds 1−3 on lipid accumulation in 3T3-L1 cells. 3T3-L1 preadipocytes were treated with compounds 1−3 for 6 days during the differentiation of cells. (a) Lipid content was evaluated by triglyceride (TG) assay. (b) Lipid accumulation was visualized by Oil Red O staining, and representative microscopy images were captured with 10× magnification. UND: undifferentiated cells, control: differentiated cells without compounds. Data are expressed as the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 vs control.

obtained from 3 by different oxidation reactions. Intermediate I could then be transformed into secu’amamine A by formation of an aziridinium ion.29 A Claisen condensation reaction between acetyl-CoA and malonyl-CoA would yield acetoacetylCoA, which could then react with the oxidized secu’amamine A by nucleophilic addition followed by decarboxylation and elimination of water to give intermediate III. Compound 2 could then be generated from intermediate III through a series of oxidations, dehydrations, and nucleophilic additions. Similarly, compound 1 could be obtained from intermediate IV through the same reactions proposed for compound 2. Compounds 1−3 were assayed for their potential lipidlowering effects in 3T3-L1 cells at nontoxic concentrations of 10 and 20 μM (Figure S17). Compound 3 exhibited the most potent inhibition of triglyceride (TG) accumulation, while

In conclusion, we have isolated two novel indolizidine alkaloids, 1 and 2, with uncommon skeletons from F. virosa. Compounds 1 and 2 represent the first examples of pentacyclic Securinega alkaloids containing an α,β-unsaturated ketone pentacyclic system. Compound 2, with an extended B ring, is the fourth secu’amamine A skeleton alkaloid containing a 5,6,6,5 cyclic system reported in nature.

Scheme 1. Proposed Biogenetic Pathways for 1 and 2

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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.7b03935. Experimental details, IR, HRESIMS, NMR, and computational data of 1 and 2 (PDF) Accession Codes

CCDC 1811960 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 data_ [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 Author

*E-mail: [email protected]. ORCID

Navindra P. Seeram: 0000-0001-7064-2904 Qiong Gu: 0000-0001-6011-3697 Jun Xu: 0000-0002-1075-0337 Author Contributions §

X.-K.L. and J.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by the National Natural Science Foundation of China (81473138, 81573310, 81603030), the National Science and Technology Major Project of the Ministry of Science and Technology of China (2017ZX09305010), and the Medical Scientific Research Foundation of Guangdong Province (No. A2014212).

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DOI: 10.1021/acs.orglett.7b03935 Org. Lett. XXXX, XXX, XXX−XXX