Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
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Alstonlarsines A−D, Four Rearranged Indole Alkaloids from Alstonia scholaris Xu-Xin Zhu,† Yao-Yue Fan,‡ Lei Xu,‡ Qun-Fang Liu,‡ Jiang-Ping Wu,§ Jing-Ya Li,‡ Jia Li,*,‡ Kun Gao,*,† and Jian-Min Yue*,†,‡
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†
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China ‡ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China § Laboratory of Immunology and Cardiovascular Research, Centre Hospitalier de l’Université de Montréal, 900 rue St-Denis, Montréal, Québec H2X 0A9, Canada S Supporting Information *
ABSTRACT: Four indole alkaloids, alstonlarsines A−D (1−4), were isolated from Alstonia scholaris and structurally characterized. Compound 1 possesses a new carbon skeleton with a cage-shaped 9-azatricyclo[4.3.1.03,8]decane motif, and compounds 2−4 feature a rare carbon skeleton that was found in nature for the first time. Plausible biosynthetic routes for 1−4 are proposed. Compound 1 showed DRAK2 inhibitory activity with an IC50 value of 11.65 ± 0.63 μΜ.
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obtained by synthetic methods.9 Herein we present the isolation, structure elucidation, hypothetical biosynthetic routes, and biological evaluation of the four novel alkaloids.
he monoterpenoid indole alkaloids (MIAs) represent an enormous and most diverse class of structures of natural secondary metabolites (approximately 3000 structures), mainly produced by the plant species of Apocynaceae, Rubiaceae, and Loganiaceae.1 The structural diversity of MIAs mainly results from the rearrangements of the highly malleable 10-carbon terpene moiety derived from secologanin.2 Previous research also revealed that MIAs exhibit fascinating bioactive diversity, such as cytotoxic,3 anti-inflammatory,4 antibacterial, and antifungal activities.5 Because of their complex structures and diverse bioactivities, MIAs have drawn great attention from synthetic chemists and pharmacologists.6 Alstonia scholaris (Apocynaceae), widely distributed in the south of China, is well-known to be a rich source of indole alkaloids, and more than 300 compounds with a diverse range of pharmacological activities7 have been identified from this species. In our continuing efforts to discover novel MIAs,8 we undertook an in-depth phytochemical investigation of the roots and bark of Alstonia scholaris, which led to four novel indole alkaloids, alstonlarsines A−D (1−4). Alstonlarsine A (1) is an unprecedented cage-shaped indole monoterpenoid alkaloid bearing a unique 9-azatricyclo[4.3.1.03,8]decane core, and the linkage between C-5 and C-20 is also unprecedented in previously known compounds of strychnine-type alkaloids. Alstonlarsines B−D (2−4) are rare indole alkaloids bearing a 5,5-spirocyclic moiety. The architecture of compounds 2−4 was found in nature for the first time, and there is only one literature report of compounds of this type, which were © XXXX American Chemical Society
Alstonlarsine A (1) was obtained as colorless crystals (mp 226−228 °C). Its molecular formula of C21H24N2O3 was established by 13C NMR (Table S3) and HR-ESI-MS (m/z 353.1864 [M + H]+, calcd 353.1865) data and requires 11 degrees of unsaturation (DOU). The IR spectrum indicated Received: January 18, 2019
A
DOI: 10.1021/acs.orglett.9b00230 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters absorptions for carbonyl groups (1700 and 1673 cm−1), a double bond (1633 cm−1), and an aromatic ring (1608 and 1465 cm−1). The 1H NMR (Table S3), 13C NMR, DEPT, and HSQC spectra displayed signals for a disubstituted indole ring [δH 7.22 (1H, d, J = 7.4 Hz, H-9), 6.96 (1H, dd, J = 7.6, 7.4 Hz, H-10), 7.18 (1H, dd, J = 7.8, 7.6 Hz, H-11), and 6.84 (1H, d, J = 7.8 Hz, H-12); δC 168.3 (C-2), 58.7 (C-7), 134.9 (C-8), 123.5 (C-9), 121.5 (C-10), 128.1 (C-11), 109.3 (C-12), and 144.7 (C-13)].10 Besides the indole ring, the alkaloid possesses a methoxycarbonyl group [δC 168.6 and 51.0; δH 3.74 (3H, s)], an acetyl group [δC 212.5 and 25.2; δH 2.25 (3H, s)], an NCH3 [δC 42.9; δH 2.64 (3H, s)], two nitrogen-bearing methines [δC 67.7 (C-3) and 72.5 (C-5)], and a persubstituted double bond [δC 168.3 (C-16) and 96.7 (C-2) ]. The abovementioned functionalities accounted for eight of the 11 DOU, and the remaining ones thus required the attendance of three additional rings in compound 1. Construction of the planar structure of 1 was accomplished by interpretation of 2D NMR data. Analysis of the 1H−1H COSY data revealed three structural fragments: a (C-9 to C12), b (C-5 to C-6), and c (C-3 to C-15 via C-14), which are drawn with bold bonds in Figure 1A. The coupling fragments,
suggested that CH3-23 and the acetyl group are attached to C20 (1c in Figure 1A). Finally, the gross structure of 1 was thus elucidated as shown in Figure 1. The NOESY interaction pairs of H-9 with H-6a (δH 2.30), H-3, and NCH3 indicated that H-6a, H-3, NCH3 are on the same side, while the cross-peaks of H3-23 with H-6b (δH 2.29) and H-15 indicated that these protons are on the other side, with CH3-23 faced to the indole ring (Figure 1B). The spatial relative configuration of 1 was unambiguously elucidated. To deduct and corroborate the stereochemistry of 1, we took a great effort to obtain X-ray-quality crystals in various solvents. Finally, high-quality crystals that allowed the successful performance of a single-crystal X-ray diffraction experiment were obtained in acetonitrile, and the X-ray data not only confirmed the structure assigned by spectroscopic data but also established the absolute configuration of 1, as shown in Figure 2 [Flack parameter = 0.05 (12)].
Figure 2. ORTEP drawing of compound 1.
Alstonlarsine B (2) {[α]20 D −71.7 (c 0.30, MeOH)} was obtained as colorless crystals (mp 244−245 °C) in MeOH. Its molecular formula of C19H24N2O4 was established by 13C NMR (Table S3) and HR-ESI-MS (m/z 345.1814 [M + H]+, calcd 345.1814) data and requires nine DOU. The 13C NMR spectrum resolved 19 carbon resonances corresponding to two methyls, four methylenes (two nitrogen-bearing), eight methines (four olefinic and one nitrogen-bearing), and five quaternary carbons, which were distinguished with the aid of HSQC and DEPT experiments. The UV spectrum showed absorption maxima at 208, 252, and 287 nm, indicative of an oxindole moiety,11 which was further confirmed by the 1D NMR resonances [δH 7.49 (1H, dd, J = 7.6, 1.2 Hz, H-9), 7.13 (1H, ddd, J = 7.8, 7.6, 1.0 Hz, H-10), 7.31 (1H, td, J = 7.8, 1.2 Hz, H-11), and 6.94 (1H, dd, J = 7.8, 1.0 Hz, H-12); δC 179.7 (C-2), 59.6 (C-7), 128.1 (C-8), 124.3 (C-9), 124.1 (C-10), 130.9 (C-11), 111.4 (C-12), and 143.4 (C-13)].12 Besides the oxindole ring, the alkaloid possesses a carboxylic group (δC 181.0), an NCH3 [δH 3.45 (3H, s) and δC 57.6], two nitrogenbearing methylenes (δC 69.5 and 63.6), and an oxygenated methine (δC 71.2). On the basis of the above-mentioned information, we drew a conclusion that the remaining two DOU accounted for the two additional rings in compound 2. The examination of the 1H−1H COSY and HSQC spectra of compound 2 enabled the construction of three structural fragments drawn with bold bonds in Figure 3A. The HMBC cross-peaks of H2-6 with C-2, C-3, C-7, and C-8 and NCH3 with C-3, C-5, and C-21 established rings C and D. In
Figure 1. Key 1H−1H COSY, HMBC, and NOESY correlations of 1.
quaternary carbons, and heteroatoms were then connected by detailed HMBC analysis (Figure 1A). The HMBC cross-peaks of H-9 with C-7 and C-13, H-10 and H-12 with C-8, and H-11 with C-13 further delineated the typical indole ring system (rings A and B). The HMBC cross-peaks of H-3 with C-8, H214 with C-7, and H-15 with C-2 and C-16 fulfilled the construction of ring C, which is fused with indole moiety via C-2 and C-7. In addition, the attachment of the methoxycarbonyl group at C-16 was achieved by the key HMBC correlations of OCH3 with C-22 and H-15 with C-22 (1a in Figure 1A). The construction of ring D was established by the HMBC cross-peaks of H2-6 with C-2, C-3, and C-7 and NCH3 with C-3 and C-5 (1b in Figure 1A). The HMBC cross-peaks of H3-23 with C-5, C-15, and C-20 and H3-18 with C-19 and C-20 revealed that C-5 and C-15 are connected by a bridge through C-20 to form the unique ring E, and simultaneously B
DOI: 10.1021/acs.orglett.9b00230 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
Alstonlarsine D (4), a colorless gum, was assigned a molecular formula of C19H22N2O3 on the basis of its 13C NMR data (Table S3) and (+)-HR-ESI-MS peak at m/z 327.1709 ([M + H]+, calcd 327.1709). Detailed analysis of the NMR data for 4 revealed that the only difference between 4 and 3 is the location of the double bond, which could be deduced from the 2D NMR data (Figure S2A). Diagnostic HMBC cross-peaks of H2-19 with C-15 (δC 132.3), C-20 (δC 133.0), and C-21 (δC 62.0) and H3-18 with C-20 confirmed a Δ15 double bond in compound 4. The relative configuration of 4 was basically established via the NOESY spectrum (Figure S2B). The NOESY interactions of H-9 with H-6α and H-3 and NCH3 with H-5α, H-3, and H-21α indicated that H-3, H-5α, H-6α, H-21α are α-oriented, while the cross-peak of H-21β with H-14β indicated that these protons are β-oriented. On the basis of biogenetic considerations, the absolute configurations of compounds 3 and 4 were proposed to be identical to that of 2, which was further confirmed by their highly similar electronic circular dichroism (ECD) curves within the wavelength range from 190 to 350 nm (Figure 5). Figure 3. Key 1H−1H COSY, HMBC, and NOESY correlations of 2.
addition, a carboxylic group was located at C-15 on the basis of the HMBC cross-peaks of H2-14, H-15, and H-20 with C-16 (δC 181.0) (Figure 3A). The relative configuration of 2 was assigned on the basis of NOESY data (Figure 3B). The NOESY interactions of H-9 with H-6α and H-3, NCH3 with H-5α, H-3, and H-21α, and H-15 with H-3 indicated that H-3, H-5α, H-6α, H-15, and H21α are α-oriented, and the cross-peaks of H-14β with H-20 and H-21β indicated that those protons are β-oriented. The structure and absolute configuration of 2 was finally confirmed by a single-crystal X-ray diffraction experiment [Flack parameter = 0.06 (4)] (Figure 4).
Figure 5. CD spectra of compounds 2−4.
Plausible biosynthetic routes for 1−4 are proposed in Scheme 1. The biogenetic precursor for 1−4 was considered to be N(4)-demethylalstogustine,10 which is richly produced in this plant. The release of one molecule of water from N(4)demethylalstogustine yields akuammicine, which is then transformed into intermediate i by oxidation at C-21. Intermediate i undergoes a sequence of cleavage of the C21−N bond and formation of an aldehyde moiety at C-21 to produce intermediate ii,13 which is transformed into iii by a cascade of oxidation and N-methylation procedures. Dehydration of iii affords intermediate iv with an imine group, which is subsequently attacked by the Δ19 double bond to form a new carbon bond (C-5−C-20) in v. By an oxidation reaction, intermediate v is transformed into intermediate vi bearing a βketo acid group, which undergoes a decarboxylation reaction14 to afford intermediate vii. Finally, methylation of vii by Sadenosylmethionine (SAM)15 produces compound 1. With regard to biogenetic pathway of compounds 2−4, oxidative cleavage of the Δ2 double bond of N(4)-demethylalstogustine affords diketo intermediate viii, which is then transformed into key intermediate ix via ester hydrolysis and oxidative decarboxylation.16 The N-methylation reaction of ix generates compound 2, which then undergoes dehydration to afford compound 3. Subsequently, the Δ19 double bond in 3 is transferred to Δ15,20 to afford 4. DAP kinase-related apoptosis-inducing protein kinase-2 (DRAK2) is a kind of CaM-dependent serine/threonine
Figure 4. ORTEP drawing of compound 2.
Alstonlarsine C (3), a colorless gum, was assigned a molecular formula of C19H22N2O3 on the basis of its 13C NMR data (Table S3) and the (+)-HR-ESI-MS data (m/z 327.1709 [M + H]+, calcd 327.1709). The 13C NMR spectrum of 3 showed high similarity to that of 2, except for the disappearance of two methines (δC 43.9 and 71.2) and the presence of an additional double bond, which revealed that compound 3 is a dehydrated derivative of 2. This deduction was further confirmed by the HMBC cross-peaks of H3-18 with C-19 (δC 133.6) and C-20 (δC 128.4) and H-19 with C-15 (δC 44.5), C-18 (δC 14.2), and C-21 (δC 67.4) (Figure S1A). The relative configuration of 3 was consistent with that of 2, as deduced by comparison of their coupling constants and analysis of the NOESY spectrum of 3 (Figure S1B). Thus, the structure of 3 was elucidated. C
DOI: 10.1021/acs.orglett.9b00230 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters ORCID
Scheme 1. Hypothetical Biosynthetic Pathway of 1−4
Kun Gao: 0000-0002-3856-3672 Jian-Min Yue: 0000-0002-4053-4870 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (21532007 and 21778027), the Drug Innovation Major Project of China (2018ZX09711001-001005), and the Shanghai Commission of Science and Technology (16430711700) is gratefully acknowledged.
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protein kinase that plays an important role in initiating and inducing programmed cell death.17 Alstonlarsine A−C were tested for DRAK2 inhibitory activity, and alstonlarsine A (1) showed moderate inhibitory activity with an IC50 value of 11.65 ± 0.63 μΜ, whereas alstonlarsine B (2) and alstonlarsine C (3) were inactive.
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REFERENCES
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00230. Experimental procedures; bioassay; X-ray crystallographic data for 1 and 2; tabulated NMR data for 1− 4; selected key 2D NMR cross-peaks for 3 and 4; and 1D and 2D NMR, MS, and IR spectra of 1−4 (PDF) Accession Codes
CCDC 1886045 and 1886049 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 e-mailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (J. Li). *E-mail:
[email protected] (K. Gao). *E-mail:
[email protected] (J. M. Yue). D
DOI: 10.1021/acs.orglett.9b00230 Org. Lett. XXXX, XXX, XXX−XXX
