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

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.


Results and discussion
Isolation and structural elucidation of ancistrobrevolines A-D Air-dried root bark material of A. abbreviatus collected in the Parc National de Taï in the Southwestern Côte d'Ivoire (Ivory Coast) was repeatedly extracted with MeOH. Aer ltration of the crude extract and evaporation of the solvent, the residual material was macerated with MeOH-H 2 O (9 : 1, v/v), followed by liquid-liquid partitioning with n-hexane and fractionation by column chromatography on silica gel. HPLC-UV guided analysis of an alkaloid-rich fraction showed the presence of metabolites exhibiting UV spectra closely similar to those of naphthylisoquinoline alkaloids. They showed a rst UV maximum at 224-229 nm, yet with an additional second UV maximum at 259-261 nm, resembling that of alkaloids with a fully dehydrogenated ring like ancistrobreveine D (9).
Ancistrobrevoline A (14). The rst isolated compound had a molecular formula of C 25 (Table 1) showed predominantly signals typical of conventional naphthylisoquinoline alkaloids ( Fig. 2A), in particular for the naphthalene part, with its usual 4 ′ ,5 ′ -dimethoxy-2 ′ -methyl substitution pattern, coupled via C-8 ′ . The NMR data revealed  2B). Even the heterocyclic unit displayed, in part, the usual features as in other naphthylisoquinoline alkaloids, like the isocyclic benzene ring with the axis at C-7 (for the atom numbering applied, see ref. 26) and two methoxy functions at C-6 and C-8, as deduced from two singlets at d H 3.67 and 3.24, each corresponding to three protons, and from an aromatic singlet appearing at d H 7.21 (H-5) ( Fig. 2A). The coupling pattern suggested the presence of a phenyl unit with no substituent at C-5, thus leaving C-7 (d C 128.1) as the axis-bearing carbon atom. This assumption was corroborated by HMBC interactions of H-5 The rest of the molecule, however, as overlaid in light blue (see Fig. 2 and structure I in Fig. 3), seemed substantially different from that of all other known naphthylisoquinoline alkaloids.
It was in particular the aliphatic region of the 1 H NMR spectrum that diverged massively from that of a usual naphthyltetrahydroisoquinoline alkaloid, characterized by the lack of resonances for the methyl group at C-3 (which normally appears around d H 0.9 and 1.3), the missing usual multiplet of H-3 in the region between d H 3.2 and 4.0, and the likewise absent signals of the diastereotopic geminal protons at C-4, which usually appear as doublets of doublets resonating around d H 1.7-3.2 (see structure II in Fig. 3). The observation of a downeld-shied three-proton singlet (d H 3.15) suggested the presence of an N-methyl group, which was in accordance with the observation of a NOESY correlation to the protons of the methyl group at C-1 and with an HMBC interaction from the protons of that methyl group (resonating at d H 3.15) to the aliphatic carbon atom C-1 (d C 58.4) (see structure III in Fig. 3).
A further most notable difference compared to usual naphthylisoquinolines was the presence of an additional oxygen function in that eastern part of the molecule. The 13 C NMR data displayed a downeld-shied signal at d C 170.0, typical of a carbonyl function. The observation of an HMBC cross-peak from the N-methyl group at d H 3.15 to that carbonyl C-atom, however, did not match with the structure of a six-membered tetrahydropyridinone as in structure IV (Fig. 3), since the distances between the two structural elements, N-Me and C-4, would have been two far for such an interaction.
The observed HMBC and NOESY interactions, while not tting with the presence of an isoquinoline unit, gave conclusive evidence of a contracted ring in the new alkaloid, as a consequence of the loss of the carbon atom C-3, thus giving rise to a ve-membered isoindolinone skeleton. This structural assignment presented in Fig. 3 (see structure V) was in agreement with the lack of the signal for C-3 in 13 C NMR (normally resonating at d C 45-53) and with the appearance of a signal at 1673 cm −1 in the IR spectrum, which is characteristic of a carbonyl group as part of an amide function. In conclusion, the new alkaloid had to be a 7,8 ′ -coupled biaryl alkaloid, structurally differing from a conventional naphthylisoquinoline by its unprecedented isoindolinone part.
The absolute conguration at the stereocenter of the new alkaloid at C-1 in the isoindolinone ring system was determined by a ruthenium-mediated oxidative degradation procedure (see the ESI †) developed by us earlier. 6,27 Although this periodate oxidation method had initially been designed for the degradation of 1,3-disubstituted tetra-or dihydroisoquinolines, not for isoindolinones, with their stable C,N-bond, the reaction luckily succeeded in this case, too, affording the simple and easy-toanalyze chiral amino acid N-methyl-D-alanine, hence establishing the absolute conguration at the stereocenter as R.
Unfortunately, it was not possible to assign the conguration at the axis relative to the stereocenter at C-1 by NMR, because no signicant long-range NOESY interactions across the biaryl axis were observed, like between Me-1 and/or H-1 in the isoindolinone ring and H-1 ′ or H-7 ′ in the naphthalene half. Apparently the small ring size of the heterocycle leads to enlarged distances between the substituent at C-1 and the respective H atoms attached to the naphthalene part. Therefore, quantum-chemical electronic circular dichroism (ECD) calculations were performed for both possible atropo-diastereomers.
The experimental ECD spectrum of the isolated naphthylisoindolinone ( Fig. 4) was very similar to the one calculated for the P-isomer (in red) at the TDuB97XD3/def2-TZVP//B3LYP-D3/def2-TZVP level, while being virtually opposite to the one predicted for M (in blue), which unambiguously showed that the new compound was P-congured and, thus, had the full absolute stereostructure 14, as presented in Fig. 1. This novel natural product was given the name ancistrobrevoline A.
Ancistrobrevoline B (15). The second new minor root bark metabolite was spectroscopically almost identical to ancistrobrevoline A (14), with only slightly different chemical shis (Table 1), thus evidencing the presence of another naphthylisoindolinone, again 7,8 ′ -coupled, as attributed by the observed key HMBC and NOESY interactions (see the ESI †). The 1 H NMR spectrum displayed a three-proton singlet at d H 3.13, hinting at an N-Me group, and the 13 C NMR data revealed the presence of a downeld-shied signal at d C 169.9, typical of the amidic carbonyl entity of the isoindolinone unit. This was in accordance with a carbonyl signal at 1678 cm −1 in the IR spectrum of the new metabolite. HR-ESI-MS showed a protonated molecular-ion peak [   respectively, similar as for ancistrobrevoline A (14) (Fig. 2). Thus, different from 14, one of the oxygen functions in the new metabolite was a free hydroxy group at C-6. The ECD spectrum of the new compound (see the ESI †) matched well that of ancistrobrevoline A (14), proving that the absolute conguration at the biaryl axis was again P, as for 14. Likewise as in the case of 14, the degradation 6,27 established the absolute conguration at the stereocenter at C-1 as R by the formation of N-methyl-D-alanine. The new alkaloid thus had the absolute stereostructure 15 as presented in Fig. 1, hence being the 6-Odemethyl analogue of 14. It was named ancistrobrevoline B. Ancistrobrevoline C (16). The third new compound was obtained as a yellow amorphous powder. According to HRESIMS and 13 C NMR, it had a molecular formula of C 24 H 26 NO 5 , as deduced from its monoprotonated molecular ion, [M + H] + , at m/z 408.17974, identical to that of ancistrobrevoline B (15). Again, the 1 H NMR spectrum showed signals for three C-methyl groups (d H 1.55, 2.09, and 3.14), for three methoxy functions (d H 3.64, 3.92, and 3.97), and for one aliphatic proton (d H 4.58) and ve aromatic ones (d H 6.81, 6.85, 6.91, 7.01, and 7.18) (Fig. 5A). This, together with an IR band at 1675 cm −1 and a downeld-shied signal at d C 170.4 in 13 C NMR, characteristic of an amidic carbonyl entity, indicated the new minor metabolite from the root bark of A. abbreviatus to be yet another naphthylisoindolinone alkaloid. Different from 15, the new compound exhibited a spin pattern of the aromatic protons consisting of two singlets, one doublet, and two doublets of doublets. This was in agreement either with a 5,1 ′ -or a 7,1 ′coupling, of which the 5,1 ′ -linkage was excluded due to HMBC interactions of H-5 (d H 7.01) to C-4 (d C 170.4) and C-7 (d C 120.5) and a NOESY correlation between H-5 and MeO-6 (d H 3.64) in the isoindolinone part (Fig. 5B). This assignment was conrmed by NOESY correlation sequences in the series {H-8 ′ 4 H-7 ′ 4 MeO-5 ′ } and {MeO-4 ′ 4 H-3 ′ 4 Me-2 ′ } and by HMBC cross-peaks of Me-2 ′ (d H 2.09) and H-8 ′ (d H 6.81) to C-1 ′ (d C 58.3) in the naphthalene moiety. The three methoxy groups were determined to be located at C-6, C-4 ′ , and C-5 ′ , based on NOESY interactions with their neighboring protons, H-5, H-3 ′ , and H-6 ′ , respectively (Fig. 5B). Another oxygenated carbon atom was found to be located in the phenyl ring, at C-8 (d C 151.9), bearing a free hydroxy function. The ruthenium-mediated oxidative degradation 6,27 of the new compound afforded the D-enantiomer of N-methylalanine, establishing the absolute conguration at the stereocenter as R.
The absolute axial conguration of the new alkaloid was deduced by comparison of its experimental ECD spectrum (full line in blue) with that of ancistrobrevoline A (14, dotted line in black). The new alkaloid differed from 14 only by the position of the methyl group in the naphthalene part or, in other words, by the coupling type (7,1 ′ versus 7,8 ′ ), and by the substitution pattern at C-6 and C-8 (OMe/OH versus OH/OMe in 14). The ECD spectrum of the new alkaloid was virtually opposite to the one of 14 (Fig. 5D), suggesting that the larger part of the naphthalene unit should be directed down, i.e., with an opposite stereoarray as compared to 14 and 15. According to the Cahn-Ingold-Prelog priority rules, however, the descriptor was again assigned as P, i.e., formally the same as for 14 and 15. The new compound thus had the full stereostructure 16 as presented in Fig. 5D. It was named ancistrobrevoline C.
Ancistrobrevoline D (17). The fourth compound was isolated as a yellow amorphous solid with a molecular formula of   (Table 1) revealed the new metabolite to be yet another 7,1 ′linked naphthylisoindolinone alkaloid, with a constitution strongly resembling that of the above-described ancistrobrevoline C (16). Similar to 16, it displayed a 4 ′ ,5 ′ -dimethoxysubstituted naphthalene portion, as deduced by NOESY crosspeaks between MeO-4 ′ and H-3 ′ and between MeO-5 ′ and H-6 ′ . Likewise observed in 1D and 2D NMR were the typical structural features of the heterocyclic ring in the isoindolinone moiety, as outlined in Fig. 5C. The molecular weight of the new metabolite, however, was lower than those of 14-16, hinting at the presence of only four oxygen atoms. This gave rise to the assumption that the new alkaloid might be derived from the subclass of dioncophyllaceae-type compounds, which are characterized by the lack of an oxygen function at C-6 (and the presence of an R-conguration at C-3, which, however, does not apply here). Indeed, the most signicant difference in the NMR spectrum of the new metabolite compared to that of 16 was the appearance of an additional aromatic proton, which was supposed to be the hydrogen atom at C-6. The coupling pattern of the aromatic protons showed one singlet (H-3 ′ ), two doublets (H-6 ′ and H-8 ′ ), and one doublet of doublets (H-7 ′ ) in the naphthalene part (Table 1), and two doublets resonating at d H 7.10 and d H 7.37, located in the isoindolinone portion. This assignment was in accordance with COSY interactions between H-5 (d H 7.37) and H-6 (d H 7.10), which showed ortho-coupling to each other (J ¼ 7.5 Hz), and it was also conrmed by HMBC cross-peaks from H-6 to C-1' (d C 126.1), C-10 (d C 133.6), and C-8 (d C 151.5) (Fig. 5C). Oxidative degradation 6,27 established the absolute conguration at the stereocenter at C-1 as R. The ECD spectrum of the new alkaloid (Fig. 5E) was virtually opposite to that of ancistrobrevoline C (16), while resembling that of ancistrobrevolines A (14) and B (15) (see ESI †), hence establishing the absolute axial conguration as P. The new alkaloid consequently had the full absolute stereostructure 17 as presented in Fig. 5E. It was henceforth named ancistrobrevoline D.

Proposed biosynthetic origin of naphthylisoindolinone alkaloids
The structures of the discovered four novel alkaloids 14-17, with their isoindolinone parts connected to the naphthalene moieties via a stereogenic axis, are unprecedented, and differ substantially from previously described articial naphthylisoindolinones. [28][29][30] In the eld of 'normal', amino-acid derived benzylisoquinoline alkaloids, some few other, yet naphthalene-devoid isoindolinones are known like aristoyagonine (18) 30-32 and aristolactam I (19) 30,33,34 (Fig. 6A). Biosynthetically, 18 and 19 are assumed to be formed from the corresponding benzyltetrahydroisoquinoline alkaloids, by 3,4dioxygenation, followed by a benzilic acid rearrangement, with loss of a carbon atom and ring contraction. [30][31][32][33][34] In an analogous way, the four novel naphthylisoindolinones 14-17 (general partial structure IV, see Fig. 6B) might arise from the respective naphthyltetrahydroisoquinoline alkaloids I, via the corresponding 3,4-diketones IIIyet with the additional necessity of eliminating the methyl group at C-3, maybe via II, or possibly via the 3-oxo analogues. The loss of the C-3 methyl group might occur by a stepwise oxidation to the respective carboxylic acid followed by decarboxylation. A similar elimination of a C-methyl group in naphthylisoquinoline alkaloids had already been observed at C-1 (see the structure of 1-nor-8-Odemethylancistrobrevine H, 12, in Fig. 1), as found to be produced by the same plant, A. abbreviatus, 18 and also oxygenations at Me-3 (see ref. 5, 35 and 36) and at C-4 (see ref. 5, 37 and 38) are known to occur in the biosynthesis of naphthylisoquinoline alkaloids. As in the case of 18 and 19, the resulting diketones III could then undergo a benzilic acid rearrangement, with loss of CO 2 , to provide the isoindolinone unit IV.

Cytotoxic activity of ancistrobrevolines A and B against breast and lung cancer cells
Some of the mono-and dimeric naphthylisoquinoline alkaloids isolated from the West African liana A. abbreviatus display strong cytotoxic activities against various cancer cell lines, among them multiple myeloma, 9,39 leukemia, 16,39 cervical, 15,18,40 brosarcoma, 17 colon, 17 and pancreatic 14,15,40 cancer cells. Investigations regarding the specicity and effectiveness of the compounds on the viability of those cancer cells indicated that the alkaloids may have a substantial therapeutic potential and can thus be regarded as promising candidates for drug development. And only recently, dioncophylline A (5), a main constituent of A. abbreviatus, and related compounds have been reported to exhibit pronounced antiproliferative activities in the submicromolar range against two breast cancer cell lines, MCF-7 and MDA-MB-231. 41 Breast 42,43 and lung 44,45 cancer are among the most commonly diagnosed lethal cancer types worldwide. Although the death rate caused by cancer has been decreasing over the past years, 46 as a result of improved diagnostic and therapeutic approaches to many cancer types, treatment of lung and breast cancer still suffers from a poor prospect to completely eradicate the tumor cells, which in turn leads to a recurrent and metastatic manifestation of the disease, and thus to high mortality rates. [42][43][44][45][46][47][48] Therefore, the search for novel therapeutic agents to efficiently combat lung and breast cancer cells still remains an important task.
Given the pronounced antiproliferative activities of some of the naphthylisoquinoline alkaloids such as dioncophylline A (5), 17,39,41 jozimine A 2 (11), 17,40 or ancistrobrevidine C (8), 15 with their usual six-membered heterocyclic ring system, it seemed rewarding to likewise study the cytotoxic potential of the new ring-contracted naphthylisoindolinones. This was also promising in view of the fact that aristolactams (Fig. 6A) and some of their non-natural analogues, 28,33 all equipped with an isoindolinone moiety similar to the one of ancistrobrevolines A-D (14)(15)(16)(17), are known to exhibit remarkable anticancer properties, too. 34,49,50 We here, therefore, report on the evaluation of ancistrobrevolines A (14) and B (15) for their growth-inhibitory effects against MCF-7 breast adenocarcinoma cells and against the non-small cell lung cancer (NSCLC) cell line A549. Due to the lack of isolated material of ancistrobrevines C (16) and D (17), only 14 and 15 were assessed.
These two alkaloids exhibited only low cytotoxic effects against MCF-7 breast cancer cells, as determined by the MTT assay (see Experimental Section). The results showed that exposure of MCF-7 cells to varying concentrations (10,30,50,70, and 100 mM) of 14 and 15 resulted in growth-inhibitory activities of 19-38% (for 14) and 3.7-43% (for 15) (see Table  2). Although 14 and 15 did not reach the pronounced growthinhibitory activities of the previously investigated naphthylisoquinoline alkaloids on breast cancer cells, 41 the results are important contributions to our ongoing investigations on structure-activity (SAR) relationships.
Against A549 lung cancer cells, the cytotoxic activities of 14 and 15 were signicantly higher, reaching ca. 8-72% and 36-68%, respectively (see Table 3), with IC 50 values of 34.6 mM (for 14) and 9.05 mM (for 15) (Fig. 7). Thus, the IC 50 value of ancistrobrevoline B (15) was markedly lower than that of ancistrobrevoline A (14), showing a higher sensitivity of A549 lung cancer cells to the growth inhibition induced by 15, which further evidenced the pronounced structure-function diversity in naphthylisoquinoline alkaloids. Studies on the immortalized human mammary epithelial cell line MCF-10A 51 revealed ancistrobrevoline B (15) to display moderate cytotoxicity on these non-tumorigenic cells, which led to a selectivity index of 1.4 regarding the A549 cancer cell line, whereas ancistrobrevoline A (14) showed no selectivity towards the cancer cells compared to the normal epithelial MCF-10A cells (SI z 1).

Cytotoxic activity of ancistrobrevolines A and B against breast and lung cancer cells
The manifestation of breast cancer is strongly associated with the occurrence of a small atypical population of tumorinitiating cells that possess the capacity of unlimited propagation and multipotent differentiation. [52][53][54] These cancer-stem-like cells have been identied in a variety of tumors and are under suspicion to be a causative factor of tumor growth and metastasis, eventually facilitating resistance to chemotherapies, and thus, leading to treatment failure and tumor recurrence. 55 Therefore, targeting these cancer stem cells in breast cancer has become a promising approach. 56 Cultivation of human breast epithelial adenocarcinoma cells MCF-7 under non-adherent non-differentiating conditions as described in the Experimental section results in the generation  of discrete clusters of spheroids, termed mammospheres (Fig. 8A), which undergo asymmetric division and self-renewal, making them aggressive and highly metastatic. 57,58 Before this background, we studied the effect of ancistrobrevines A (14) and B (15) on the sphere formation potential of MCF-7 cells aer exposure to 10,30,50,70, and 100 mM of the new naphthylisoindolinone alkaloids. Aer 5 d of treatment, the size of the spheroids was captured using a phase-contrast microscope. As shown in Fig. 8, compounds 14 and 15 signicantly reduced the size of the spheres in MCF-7 culture compared to the nontreated control group, where large spheroids were observed. A dose-dependent spheroid inhibitory activity of ancistrobrevolines A (14) and B (15) was found in MCF-7-derived mammospheres. The results indicate the reduction of stemness and self-renewal of MCF-7 cells in the presence of 14 and 15.

General experimental procedures
Optical rotations were recorded using a JASCO P-1020polarimeter (JASCO, Gross-Umstadt, Germany) operating with a sodium light source (l ¼ 589 nm). UV spectra were measured on a Shimadzu UV-1800 spectrophotometer, and ECD spectra were recorded under nitrogen atmosphere on a JASCO J-715 spectrometer at room temperature, using a standard cell (0.02 cm), then processed using the SpecDis soware. 59,60 IR spectra were measured on a JASCO FT/IR-410 spectrometer. 1D and 2D NMR spectra were taken on a Bruker DMX 600 instrument at ambient temperature, using methanol-d 4 as the solvent, with the 1 H and 13

Extraction and purication of naphthylisoindolinones
The air-dried powdered root bark material of A. abbreviatus (ca. 420 g) was macerated in methanol (2 Â 3 L) at 40 C, followed by ultrasonication for 1 h. The extract was ltered, and the solvent was evaporated under reduced pressure to give a brownish solid residue, which was re-dissolved in 90% aqueous methanol and partitioned with n-hexane. The aqueous methanolic layer was dried under reduced pressure to yield 17.5 g of an alkaloid-rich fraction, which was directly subjected to fractionation over a silica gel column using a linear solvent system consisting of CH 3 OH and CH 2 Cl 2 with a gradient increase of CH 3 OH (0 / 90%), giving rise to ve naphthylisoindolinone-enriched fractions (F 1 -F 5 ). The alkaloid-containing subfractions were puri-  Ancistrobrevoline D (17). Yellow, amorphous powder;  Table 1; HR-ESI-MS m/z 400.1503 [M + Na] + (calcd for C 23 H 23 NNaO 4 , 400.1519).

Oxidative degradation
The naphthylisoindolinone alkaloids 14-17 (ca. 0.5 mg each) were subjected to a ruthenium(VIII)-mediated periodate degradation, 6,27 followed by derivatization of the resulting amino acids with CH 3 OH/HCl and (R)-a-triuoromethylphenylacetylchloride [(R)-MTPA-Cl, prepared from (S)-MTPA]. The absolute congurations were assigned by GC on a dimethylpolysiloxane-coated capillary column coupled to a mass-selective detector and comparison with the corresponding derivatives of authentic amino acids of known absolute conguration.

Computational details
For the calculation of the ECD spectra, a simplied approach was selected. Only the main conformers of the possible congurations of compound 14 were optimized with B3LYP-D3//def2-TZVP. [61][62][63] The conformational freedom of the methoxy groups was not taken into account because their inuence on the ECD should be negligible. Excited states results were achieved utilizing TDuB97XD3/def2-TZVP(-f). 64,65 All calculations were done using ORCA 4.1.0.2. 66 For processing of the data, SpecDis 1.71 was used 59,60 with a UV shi of 18 nm, nally providing the computed ECD spectra, which were compared with the experimental one of 14.
Cytotoxicity study using the MTT assay Approximately 10 4 cells/well were seeded in 96-well plates and kept in the incubator at 37 C and 5% CO 2 . The cells were kept overnight to allow them to adhere and were then fed with fresh medium containing different concentrations (10,30,50,70, and 100 mM) of the test compounds, ancistrobrevolines A (15) and B (16). Aer incubation for 48 h, cell viability was assessed using the MTT assay. 67 At the stipulated time following the treatment of the test sample, medium was aspirated; MTT (5 mg mL −1 ) was added in each well and incubation was continued at 37 C for 2 h. The plates were spun, supernatants were discarded, and purple-colored precipitates of formazan were dissolved in 100 mL of dimethylsulfoxide (DMSO). The color absorbance was recorded at 590 nm using a microplate reader (BioTek Instruments, Inc. USA). IC 50 calculations and regression analysis were performed using the GraphPad Prism 5.0 soware.
The selectivity index (SI) of the test compounds was calculated using the following equation, where MCF-10A represents human normal cells, while A549 denotes lung cancer cells: SI ¼ IC 50 normal cells /IC 50 cancer cells . The IC 50 value indicates the concentration of the test drug required for 50% inhibition of the cancer cells. SI values > 1 indicate that the compound is selective against the respective cancer cells. 68 Mammosphere formation and drug treatment Mammospheres derived from MCF-7 cells were formed by seeding the cells in 6-well ultra-low-attachment surface plates (Corning, Tewksbury, MA, USA) at a density of 2 Â 10 4 cells/well and cultured in serum-free DMEM-Ham's F12 nutrient mixture (1 : 1, v/v), supplemented with 5 mg mL −1 insulin, 0.5 mg mL −1 hydrocortisone, 2% B-27 supplements, 10 ng mL −1 broblast growth factor, and 20 ng mL −1 epidermal growth factor. The cells were incubated at 37 C and 5% CO 2 for 5 d to form mammospheres. 69 To study the effect of ancistrobrevolines A (14) and B (15) on mammosphere formation, the cells were treated with different concentrations (10,30,50,70, and 100 mM) of the agents and then allowed to form spheres for the next 5 d. The compound solutions were prepared using DMSO with its nal non-toxic concentration (0.1%). An equal amount of DMSO was added to the non-treated control group. Aer 5 d, the mammospheres were photographed using phase-contrast microscopy, and the images were analyzed using the ImageJ analysis soware. 69
Moreover, A. abbreviatus excels by the highest number of metabolic follow-up variations, resulting frommostly oxidativesecondary modications. Besides the phenol-oxidative coupling via the naphthalene portion, e.g. to give jozimine A 2 (11), 17 with its three stereogenic axes, and a nearly complete series of its numerous atropo-diastereomers, it is the only plant that produces naphthylisoquinoline alkaloids whose isocyclic moiety is further oxygenated to form an ortho-naphthoquinone as in ancistrobreviquinone A (10). 15 Even more thrilling are the downstream variations of the tetrahydroisoquinoline portion, which can be dehydrogenated leading to the otherwise less frequently occurring 3,4-dihydroisoquinolines and to the (normally very rare) fully dehydrogenated naphthylisoquinoline alkaloids, with no less than seven representatives. Totally unprecedented even is the occurrence of 1-demethylated naphthylisoquinolines like 1-nor-8-O-demethylancistrobrevine H (12) 18 and ring-cleaved representatives like ancistrosecoline D (13) 18 (Fig. 1) and, as described here, the discovery of oxidatively ring-contracted alkaloids, delivering new molecular scaffolds within the class of naphthylisoquinoline alkaloids.
These rst four naphthylisoindolinones, ancistrobrevolines A-D (14)(15)(16)(17), are all N-methylated, and they are all fully Omethylated in the naphthalene part and all are R-congured at C-1. In that respect they t with the structural properties of the presumable precursors to 14, 15, 16, and 17, viz., ancistrobrevine A (1), its 6-O-demethyl analogue 2, ancistrobrevine D (3), and N-methyldioncophylline A (5) (Fig. 1), respectively, which, in agreement with our biosynthetic concept, all co-occur in A. abbreviatus, where they are even among the main alkaloids. Despite their structural similarities, the new metabolites do vary by their oxygenation degree at C-6, by the O-methylation degree at C-8, and by the stereo-orientation at the biaryl axis. This suggests that the structural requirements for the ring contraction are not particularly specic, evidencing that isoindolinone formation in naphthylisoquinoline alkaloids is possibly a more general principle. The work shows that naphthylisoquinoline alkaloids are not just metabolically stable bioactive end products, but can also be substrates for furthermainly oxidative -modications.
Among all investigated naphthylisoquinoline-producing plants, A. abbreviatus shows the broadest diversity of secondary metabolites. It thus occupies a phytochemicallyand phylogeneticallyoutstanding position within the Ancistro-cladaceae family, with a close relationship to Dioncophyllaceae lianas such as Triphyophyllum peltatum on the one hand and to geographically neighboring West and Central African Ancistrocladaceae plants on the other. Ancistrobrevolines A (14) and B (15) exhibited moderate antiproliferative activities against breast (MCF-7) and lung (A549) cancer cell lines. Furthermore, 14 induced a signicant reduction of the formation of highly metastatic spheroid clusters in breast cancer-derived cancer-stem-like cells, worth further, more in-depth investigations on the antitumoral potential of naphthylisoquinoline alkaloids. The results presented here render phytochemical studies on the numerous further, as yet unidentied, trace compounds of Ancistrocladus plants and their biological evaluation rewarding goals.

Conflicts of interest
There are no conicts to declare.