Naphthylisoindolinone alkaloids: the first ring-contracted naphthylisoquinolines, from the tropical liana Ancistrocladus abbreviatus, with cytotoxic activity

Abstract

The West African liana Ancistrocladus abbreviatus is a rich source of structurally most diverse naphthylisoquinoline alkaloids. From its roots, a series of four novel representatives, named ancistrobrevolines A-D (14-17) have now been isolated, displaying an unprecedented heterocyclic ring system, where the usual isoquinoline entity is replaced by a ring-contracted isoindolinone part. Their constitutions were elucidated by 1D and 2D NMR and HR-ESI-MS. The absolute configurations at the chiral axis and at the stereogenic center were assigned by using experimental and computational electronic circular dichroism (ECD) investigations and a ruthenium-mediated oxidative degradation, respectively. For the biosynthetic origin of the isoindolinones from 'normal' naphthyltetrahydroisoquinolines, a hypothetic pathway is presented. It involves oxidative decarboxylation steps leading to a ring contraction by a benzilic acid rearrangement. Ancistrobrevolines A (14) and B (15) were found to display moderate cytotoxic effects (up to 72%) against MCF-7 breast and A549 lung cancer cells and to reduce the formation of spheroids (mammospheres) in the breast cancer cell line.

Fig. 1. Secondary metabolites produced by A. abbreviatus: the naphthyltetrahydroisoquinolines ancistrobrevine A (1) and its 6-O-methyl analogue 2, ancistrobrevine D (3), dioncophylline A (4) and its N-methyl congener 5, dioncoline A (6), and ancistrobrevine M (7), the dihydroisoquinoline ancistrobrevidine C (8), the non-hydrogenated ancistrobreveine D (9), the naphthoquinone derivative ancistrobreviquinone A (10), the dimer jozimine A₂ (11), the 1-unsubstituted 1-nor-8-O-demethylancistrobrevine H (12), the ring-cleaved ancistrosecoline D (13), and the novel ancistrobrevolines A–D (14–17). – Ancistrocladaceae-type alkaloids (with 6-OR and 3S) are labeled in “blue–blue”, Dioncophyllaceae-type representatives (6-H and 3R) are marked in “red–red”, the hybrid-type (6-OR and 3R) ancistrobrevine M (7) in “blue-red”, and the only known inverse-hybrid-type (6 H, 3S) dioncoline (6) in “red-blue”.

Fig. 2. 1D and 2D NMR data of the naphthalene moiety and the phenyl part of the heterocyclic molecular half of ancistrobrevoline A (14) closely resembling those of conventional naphthylisoquinoline alkaloids, while the novel-type, yet unknown structural elements of 14 are overlaid in pale blue. (A) ¹H and ¹³C NMR data (in methanol-d₄, δ in ppm). (B) HMBC (single blue arrows) and NOESY (double red arrows) interactions evidencing the constitution of the molecular units of 14 and the position of the biaryl axis between them. – For better comparability, the atom numbering follows the one usually applied for naphthylisoquinoline alkaloids.

Fig. 3. Decisive ¹H and ¹³C NMR shifts (in methanol-d₄, δ in ppm) and key NOESY (double red arrows) and HMBC (single blue arrows) interactions in structures I, III, and V, evidencing the structural elements of the heterocyclic ring in ancistrobrevoline A (14). The two geminal protons at C-4, the proton and the methyl group at C-3 (underlaid in pink in structure II), typical of usual naphthyltetrahydroisoquinoline alkaloids, were not observed in the ¹H NMR spectrum of 14. Deletion of the carbon atom C-3 (marked in pink in structure IV) leads to the formation of the isoindolinone subunit of 14 as shown in structure V.

Fig. 4. Assignment of the absolute configuration of ancistrobrevoline A (14) by comparison of its experimental ECD spectrum (in black, taken in methanol) with the ones calculated (A) for the (1R,7P)-isomer (ECD curve in red) and (B) for 1R,7M (in pale blue) using TDωB97XD3/def2-TZVP//B3LYP-D3/def2-TZVP.

Fig. 5. (A) ¹H and ¹³C NMR data (in methanol-d₄, δ in ppm) of ancistrobrevoline C (16). (B) Key HMBC (single blue arrows) and NOESY (double red arrows) interactions indicative of the constitution of 16. (C) Decisive 1D and 2D NMR data of ancistrobrevoline D (17) evidencing the presence of an additional aromatic proton (highlighted in yellow). (D) Assignment of the absolute axial configuration in 16 by comparison of its experimental ECD spectrum (in pale blue, full line) with the ECD curve (in black, dotted line) of ancistrobrevoline A (14, for its structure, see Fig. 4). (E) Comparison of the ECD spectrum of 17 (in red, full line) with that of the likewise 7,1′-coupled ancistrobrevoline C (16) (in pale blue, full line).

Fig. 6. (A) Structures of the conventional, naphthalene-devoid isoindolinones, aristoyagonine (18) and aristolactam (19). (B) From ‘normal’ naphthyltetrahydroisoquinolines (I) to ancistrobrevolines (IV): Proposed biosynthetic pathway, with oxidation, decarboxylation, and ring contraction reactions.

Fig. 7. Treatment of lung cancer (A549) cells with ancistrobrevolines A (14) and B (15). Determination of IC₅₀ and log IC₅₀ values, and regression analysis of the effects of (A) 14 and (B) 15 (10, 30, 50, 70, and 100 μM each) on the A549 cells after 48 h of incubation; the results are expressed as mean ± SD.

Fig. 8. Cytotoxic effects of ancistrobrevolines A (14) and B (15) on the formation of MCF-7-derived breast cancer stem-like cells (mammosphere). (A) Mammospheres derived from untreated MCF-7 cells as a control. (B–F) MCF-7 cells derived from mammospheres treated with 14 at concentrations of 10 (B), 30 (C), 50 (D), 70 (E), and 100 μM (F). (G-K) MCF-7 cells derived from mammospheres treated with 15 at concentrations of 10 (G), 30 (H), 50 (I), 70 (J), and 100 μM (K). The mammospheres were cultured for 5 d to form spheres in ultra-low attachment surface plates. The images of the mammospheres were taken at 10 × resolution using phase-contrast microscopy.