Antiprotozoal Spirombandakamines A1 and A2, Fused


Nov 30, 2017 - From the leaves of a yet undescribed Congolese Ancistrocladus species, two novel naphthylisoquinoline dimers, spirombandakamines A1 (1)...
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Letter Cite This: Org. Lett. 2017, 19, 6740−6743

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Antiprotozoal Spirombandakamines A1 and A2, Fused Naphthylisoquinoline Dimers from a Congolese Ancistrocladus Plant Blaise Kimbadi Lombe,†,‡ Torsten Bruhn,§ Doris Feineis,† Virima Mudogo,‡ Reto Brun,⊥,∥ and Gerhard Bringmann*,† †

Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany Faculté des Sciences, Université de Kinshasa, B.P. 202, Kinshasa XI, Democratic Republic of the Congo § Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, D-10589 Berlin, Germany ⊥ Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland ∥ University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland ‡

S Supporting Information *

ABSTRACT: From the leaves of a yet undescribed Congolese Ancistrocladus species, two novel naphthylisoquinoline dimers, spirombandakamines A1 (1) and A2 (2), were isolated, together with a new, but “classical” dimer, mbandakamine B2 (3). The cage-like stereostructures of 1 and 2 were established by combining spectroscopic, chemical, and chiroptical methods with quantum-chemical ECD calculations. Their unique molecular frameworks may originate from “open-chain” dimers, such as 3, by an oxidation-induced cascade of reactions. They possess strong antiprotozoal properties.

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aphthylisoquinoline alkaloids1 are polyketide-derived natural biaryls. Depending on their individual structures, they display potent antiprotozoal or antiviral activities.2 They have so far been found mainly in tropical lianas belonging to the small plant genus Ancistrocladus.1 The vast tropical rain forest of the Democratic Republic of the Congo (DRC) is known to host four botanically accepted Ancistrocladus species,3 mainly distributed in the northwestern area of the country. Genetic analyses on plant material collected at different sites in DRC, however, hinted at the occurrence of a potentially new species in the region near the town of Mbandaka.4 Investigations on the leaves of this Ancistrocladus taxon resulted in the isolation of two antiplasmodial dimeric naphthylisoquinoline alkaloids, mbandakamines A and B,5 which possess a highly unsymmetric, severely hindered central axis, positioned peri to one of the outer axes. Most recently, two structurally even more thrilling dimers, among them cyclombandakamine A2 (4), have been isolated from the same plant material (Figure 1). 6 They are the very first dimeric naphthylisoquinolines with oxygen bridges, leading to a rigid architecture of eight consecutive condensed rings and a total of eight stereogenic elements. Herein we report on the discovery of two additional unique dimers in the leaves of this Congolese liana. These novel metabolites, named spirombandakamines A1 (1) and A2 (2), feature a complex molecular architecture displaying an unprecedented cage-like molecular framework, with a fivemembered carbon ring and five- and seven-membered oxygen heterocycles. Likewise isolated was another new, yet “classical” © 2017 American Chemical Society

Figure 1. Previously reported cyclombandakamine A2 (4).6

dimer, mbandakamine B2 (3), possibly a biogenetic precursor to both 1 and 4. We discuss a presumable biosynthetic pathway from 3 to 1 and 4. Furthermore, the bioactivities of 1−3 are presented. Resolution of the alkaloid-enriched dichloromethane subfraction of the leaf extract by preparative HPLC provided the optically active compound 1 as a colorless powder. Its molecular formula, C50H55N2O8, as deduced from HRESIMS (m/z at 811.3973, [M + H]+), was the same as that of 4.6 Likewise very similar were its 1H and 13C NMR data (signals for seven aromatic protons, three methylene groups with diastereotopic protons, two N-methyl groups, four methoxy groups, six methyl groups, and a carbonyl carbon)as if it was a diastereomer of 4. The UV Received: November 8, 2017 Published: November 30, 2017 6740

DOI: 10.1021/acs.orglett.7b03473 Org. Lett. 2017, 19, 6740−6743

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Organic Letters

3.49 (1H, d, J = 19.5 Hz)]} and by joint HMBC correlations from H-6″ and both Ha-3″ and Hb-3″ to C-10″ (δC 125.7). A weak, four-bond HMBC interaction from CH3-2″ [δH 1.94 (1H, s)] to C-3″, together with an unusually high chemical shift of C-9″ (δC 157.8) and an HMBC interaction from Ha-3″ to C-9″, indicated the presence of a cyclopentenone ring (Figure 2). HMBC interactions from H-7′, CH3-2″, and both Ha-3″ and Hb-3″ to a carbon atom at δC 58.7 showed this atom to be C-1″ and, at the same time, evidenced a 6′,1″-coupling of the two molecular halves of 1 (Figure 2). This connection was further supported by an HMBC correlation from Ha-3 to C-6′, as also shown and highlighted in green in Figure 2. The chemical shift of C-2″ (δC 125.6) suggested it to be doubly oxygenated. This shift, together with 4J HMBC from CH3-2″ to both C-5′ (δC 154.4) and C-6‴ (δC 154.3) and a ROESY interaction between CH3-2″ and H-7‴ [δH 6.90 (1H, s)], indicated C-2″ to be linked to the oxygen at C5′ and the one at C-6‴ (see interactions colored in cyan, Figure 2), thus revealing the presence of an unprecedented spiro-fused molecular skeleton. H-3 [δH 3.02 (1H, m)] exhibited a ROESY correlation with H1, while Heq-4 revealed a cross-peak with H-1′ and Hax-4 with H7′ (Figure 3a). These relationships, showing a cis configuration of

and electronic CD spectra of 1, however, were not comparable to those of 4 at all, showing that the two metabolites were structurally most different. 1D and 2D NMR measurements (Table S1) revealed the first, “southeastern” part of 1, denoted as 1-A (Figure 2),7 to be an N-

Figure 2. Key HMBC (single arrows) and ROESY (double arrows) interactions of 1, indicating the constitutions of the molecular moieties, 1-A and 1-B,7 including the key HMBC correlations between them (in green) that prove their C,C-connection, and 4J HMBC and ROESY correlations (in cyan) evidencing the acetal linkages.

methylated 5,8′-coupled naphthylisoquinoline, with C-6′ (δC 126.3) being the coupling position to the other half, 1-B, as in 4. Key interactions indicative of the coupling site within 1-A included HMBC correlations from H-7′ [δH 6.41 (1H, s)], H-7 [δH 6.55 (1H, s)], and H-4ax [δH 2.32 (1H, dd, J = 17.3, 11.4 Hz)] to C-5 (δC 119.4) and ROESY correlations of H-7′ with H-4ax and of H-4eq [δH 1.88 (1H, dd, J = 17.3, 2.9 Hz)] with H-1′ [δH 6.53 (1H, s)]. The location of a methoxy group at C-8 (δC 157.5) was evidenced by HMBC interactions from H-7 and OCH3-8 [δH 3.87 (3H, s)] to C-8, together with a ROESY effect between OCH3-8 and both CH3-1 [δH 1.61 (3H, d, J = 6.6 Hz)] and H-1 [δH 4.58 (1H, q, J = 6.6 Hz)]. The other methoxy group of this moiety was at C-4′ (δC 157.4), as deduced from the HMBC interaction of its protons [δH 4.01 (3H, s)] and of H-3′ [δH 6.70 (1H, s)] to this carbon atom, in conjunction with the ROESY interactions between its protons and H-3′. The N-methyl group [δH 2.96 (3H, s)] exhibited HMBC correlations with both C-1 and C-3 (δC 60.5) (Figure 2). The isoquinoline portion of the second, “northwestern” part of 1, 1-B (Figure 2), was constitutionally similar to the one of 4, as it possessed a methoxy group [δH 3.78 (3H, s)] at C-8‴ (δC 157.6), an N-methyl group [δH 2.10 (3H, s)], and C-5‴ (δC 123.4) as the connecting point to the northern naphthalene-derived portion. In ROESY experiments, the N-methyl group of this isoquinoline moiety was found to interact with both the isoquinoline and the naphthalene portions of the first half, via H-7 and H-7′, as in 4. In contrast to 4, this N-methyl showed an additional, unusual interaction with H-1′ (Figure 2), hinting at the presence of an unprecedented structure. An AB spin system {H-7″ [δH 7.86 (1H, d, J = 8.7 Hz)], H-6″ [δH 7.23 (1H, d, J = 8.7 Hz)]}, together with an HMBC correlation of H-7″ to C-5‴, indicated that the second half (1-B) was 5‴,8″-coupled, like in the first half, 1-A (Figure 2). HMBC interactions of H-7″ and of a methoxy group [δH 4.03 (3H, s)] jointly to C-5″ (δC 158.4), in conjunction with a ROESY interaction of this O-methyl group with H-6″, established it to be linked to C-5″. The aforementioned carbonyl group thus had to be C-4″ (δC 203.2). Adjacent to it, at C-3″ (δC 52.4), there was a methylene group with diastereotopic protons, as indicated by their typical6,8 chemical shifts {Ha [δH 2.66 (1H, d, J = 19.5 Hz)] and Hb [δH

Figure 3. (a) Key ROESY interactions indicating the relative configuration of 1. The blue arrows indicate correlations within the molecular halves, and the green ones show interactions between them. (b) Comparison of the experimental ECD spectrum with the calculated one, evidencing the absolute configuration of 1.

C-1 versus C-3 (δC 60.5), indicated that the southeastern half (1A) was either 1R,3S,5P-configured or 1S,3R,5M. In the other northwestern part of 1 (1-B), the relative configuration at the stereocenters C-1‴ (δC 58.6) and C-3‴ (δC 50.6) was determined to be trans by a ROESY interaction of CH3-1‴ [δH 1.54 (3H, d, J = 6.8 Hz)] with the axial H-3‴ [δH 4.00 (1H, m)] (Figure 3a). This correlation, in conjunction with the one between Heq-4‴ [δH 3.51 (1H, dd, J = 18.3, 4.9 Hz)] and H-7″, revealed 1-B to possess either a 1‴R,3‴R,5‴P or a 1‴S,3‴S,5‴M configuration. The interactions of the N-methyl group of 1-B with both H-7 and H-7′, together with the one of Hax-4‴ [δH 2.03 (1H, dd, J = 18.3, 11.4 Hz)] with H-7′ (Figure 3a, green arrows), indicated that a P configuration of the eastern biaryl axis would necessitate a P configuration at the northwestern biaryl axis, thus leading to a 1R,3S,5P,1‴R,3‴R,5‴P configuration. An Mconfigured eastern axis would, in turn, lead to a 1S,3R,5M,1‴S,3‴S,5‴M configuration, that is, to the enantiomer. With the relative configurations at the “classical” stereogenic elements for naphthylisoquinoline alkaloids thus determined, it remained to assign the stereoarrays at C-1″ and C-2″ in the northwestern part of the molecule in order to complete the relative configuration. The five- and seven-membered oxygencontaining rings were determined to be cis-fused, from ROESY interactions between CH3-2″ and H-7‴ (Figure 3a), which 6741

DOI: 10.1021/acs.orglett.7b03473 Org. Lett. 2017, 19, 6740−6743

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Organic Letters Scheme 1. Hypothetical Biosynthetic Pathways of 1 and 4 from Joint Precursor 3

graphical abstract, that is, with 1R,3S,5P,1″R,2″S,1‴R,3‴R,5‴P configuration. Further screening of the same plant extract by LC-UV-MS led to the discovery of a very similar minor metabolite, compound 2, isolated by reversed-phase preparative HPLC. It had the molecular formula C49H52N2O8 as deduced from HRESIMS (m/z = 797.3753 [M + H]+), hinting at an O- or N-demethylated derivative of 1. Its 1H and 13C NMR spectra (Table S2), closely related to those of 1, suggested the missing methyl group to be the one linked to the nitrogen atom in the northwestern portion of 1, which was confirmed by the 1D and 2D NMR data (Table S2 and Figure S2). Key evidence for this was provided by the upfield shifts of C-1‴ (δC 48.3) and C-3‴ (δC 45.6) compared to those in 1 (δC 58.6 and 50.6, respectively). The relative configurations at the stereocenters in the isoquinoline portions in 2 were the same as those in 1: cis in the southeastern portion and trans in the northwestern part. Its oxidative degradation provided both (R)- and (S)-3-aminobutyric acid, thus, as for 1, evidencing opposite configurations at C-3 and C-3‴. The concomitant formation of N-methyl-3aminobutyric acid only as its S enantiomer, however, proved that the S-configured center was the one near the N-methyl group (i.e., C-3), and C-3‴ was, thus, R-configured. This, together with the above-stated cis configuration at C-3 versus C-1 and the trans array at C-3‴ relative to C-1‴, indicated that both C-1 and C-1‴ were R-configured. Thus, for 2 (and in contrast to 1), an unambiguous assignment of the full absolute configurations including all stereocenters in the isoquinoline portions was even possible without quantum-chemical ECD calculations. At the axes and at the stereocenters C-1″ and C-2″, the ROESY interactions (Table S2) were all similar to those in 1, which, as described in the case of 1 (see above), demonstrated that both axes of 2 were P-configured, and C-1″ and C-2″ were R- and S-

indicated the northwestern portion to be either 1″R,2″S- or 1″S,2″R-configured. The configuration at the acetal carbon C-2″ correlated with that at the northwestern biaryl axis, as a consequence of the rigidity created by the acetal linkages C5′−O−C-2″−O−C-6‴. An R configuration at C-2″ would be associated with an M configuration at the northwestern biaryl axis, which, given the configurations at other stereogenic elements as described above, would lead to a 1S,3R,5M,1″S,2″R,1‴S,3‴S,5‴M configuration for the whole molecule, and, vice versa, with S configuration at C-2″, the overall configuration would be fully opposite, that is, 1R,3S,5P,1″R,2″S,1‴R,3‴R,5‴P. Ruthenium-mediated oxidative degradation9 provided both (R)- and (S)-N-methyl-3-aminobutyric acid, which proved opposite configurations at C-3 and C-3‴, yet leaving open whether the molecule was 3R,3‴S- or 3S,3‴R-configured. This, combined with the failure of all crystallization attempts, made quantum-chemical ECD calculations the method of choice for the determination of the absolute stereostructure of 1. Of the two remaining possible stereoisomerswhich were, thus, enantiomersarbitrarily the 1S,3R,5M,1″S,2″R,1‴S,3‴S,5‴M enantiomer (here briefly denoted as MM-1) was modeled and optimized with B3LYP-D3/def2-TZVP. For this configuration, the ECD spectrum was calculated using ωB97XD3/def2-TZVP(-f) with the full time-dependent DFT approach.10 The predicted ECD curve of its enantiomer (1R,3S,5P,1″R,2″S,1‴R,3‴R,5‴P, here named PP-1) was obtained by reflecting the calculated spectrum of MM-1 with respect to the zero line. As a result, the experimental ECD spectrum matched well with the one computed for PP-1 (Figure 3b), while being virtually opposite to the one calculated for the MM-1 (Figure S1). Hence, the full absolute stereostructure of spirombandakamine A1 (1) was established as presented in the 6742

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configured, respectively, given the absolute S configuration at C3 and R at C-3‴ as established by the oxidative degradation. Therefore, 2 was unambiguously assigned to be 1R,3S,5P,1″R,2″S,1‴R,3‴R,5‴P-configured, like 1, and had the absolute stereostructure shown in the graphical abstract. In accordance with the close structural similarities of 1 and 2, their ECD spectra closely matched (Figure S3), thus providing further independent confirmation of the absolute stereostructure of 1 as determined by computational investigations. The new alkaloid was henceforth named spirombandakamine A2. The search for possible “open-chain” congeners of 1 or 2 in the same plant led to the isolation of a new naphthylisoquinoline dimer, which was (see the Supporting Information) assigned the absolute stereostructure 3. Given its resemblance to mbandakamine B,5 it was named mbandakamine B2. Although being, at first sight, so different from each other, spirombandakamine A1 (1), mbandakamine B2 (3), and cyclombandakamine A2 (4) share a lot: they have the same southeastern half and similar northwestern isoquinoline portion, and the intact northern aryl ring is identical, too. These similarities and the occurrence in the same extract suggested a possible biosynthetic relationship between them. As shown in Scheme 1, 1 and 4 might both result from 3 by an epoxidation in the highly strained C-1″−C-2″ region, leading to a decrease of steric hindrance. For stereochemical reasons, the configuration of the epoxide 5 (“oxygen down”, i.e., 5a, or “up”, 5b) should be of importance for the further course of the reaction. The epoxide with the oxygen being “down”, 5a, should be cleaved through an attack by O-6‴, leading to 6 and subsequently to 4,11 while 5b (with the oxygen “up”) would be opened by the C-2″−C-3″ electron pair to provide 7 and then 1. In view of the high specificity of enzymes, it is also imaginable that the epoxidation of 3 by a monoxygenase takes place stereoselectively, possibly only from the bottom side, to give 5a. In that case, the resulting intermediate 6 might, alternatively, also lead to 7, with the correct configuration at C-1″, but then the rearrangement would not profit from the epoxide opening as a driving force, as in the direct reaction of 5b to 7. Spirombandakamines A1 (1) and (to a smaller degree) A2 (2) exhibited very good activities against both the chloroquineresistant Plasmodium falciparum K1 strain (7 and 94 nM, respectively) and the nonresistant NF54 strain (IC50 = 40 and 226 nM, respectively). Even more impressive were the activities of mbandakamine B2 (3), with IC50 values of 4 nM (K1) and 17 nM (NF54), with the K1 value being better than that of any naphthylisoquinoline alkaloid tested so far.2,12 Furthermore, 3 strongly inhibited the growth of Trypanosoma brucei rhodesiense, with an excellent IC50 value of 5 nM, while the activities of 1 and 2 (IC50 = 691 and 296 nM, respectively) were again much weaker than that of 3. Thus, the biosynthetic cyclization process from 3 to 1 in the plant diminishes those antiprotozoal properties (Table S4). In conclusion, two novel naphthylisoquinoline dimers, possessing an unprecedented spiro-fused molecular skeleton, were discovered, spirombandakamines A1 (1) and A2 (2). The structural similarities of 1 with cyclombandakamine A2 (4) and also with a coisolated open-chain new dimer, mbandakamine B2 (3), suggest that 1 and 4 may be oxidation-induced cyclization products of 3.

Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03473. Experimental details and additional data (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] ORCID

Torsten Bruhn: 0000-0002-9604-1004 Gerhard Bringmann: 0000-0002-3583-5935 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Br 699/14-2; SFB 630 “Agents against Infectious Diseases”). B.K.L. thanks the Deutscher Akademischer Austauchdienst and the Excellence Scholarship Program BEBUC (www.foerderverein-uni-kinshasa.de) for generous support. We acknowledge assistance from Dr. M. Grüne and Mrs. P. Altenberger (NMR), Dr. M. Büchner and Mrs. J. Adelmann (MS), and Mrs. M. Michel (oxidative degradation), at the University of Würzburg.

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DEDICATION Dedicated to the memory of Professor Friedemann Schneider. REFERENCES

(1) Bringmann, G.; Pokorny, F. In The Alkaloids; Cordell, G. A., Ed.; Academic Press: New York, 1995; Vol. 46, pp 127−271. (2) Bioactivities of naphthylisoquinolines: Ibrahim, S. R. M.; Mohamed, G. A. Fitoterapia 2015, 106, 194−225. (3) (a) Taylor, C. M.; Gereau, R. E.; Walters, G. M. Ann. Mo. Bot. Gard. 2005, 92, 360−399. (b) Heubl, G.; Turini, F.; Mudogo, V.; Kajahn, I.; Bringmann, G. Plecevo 2010, 143, 63−69. (4) Turini, F.; Steinert, C.; Heubl, G.; Bringmann, G.; Lombe, B. K.; Mudogo, V.; Meimberg, H. Taxon 2014, 63, 329−341. (5) Bringmann, G.; Lombe, B. K.; Steinert, C.; Ndjoko-Ioset, K.; Brun, R.; Turini, F.; Heubl, G.; Mudogo, V. Org. Lett. 2013, 15, 2590−2593. (6) Lombe, B. K.; Bruhn, T.; Feineis, D.; Mudogo, V.; Brun, R.; Bringmann, G. Org. Lett. 2017, 19, 1342−1345. (7) The atomic numbering follows that of “normal” mono- and dimeric naphthylisoquinolines (including biosynthetic considerations). (8) Krohn, K.; Michel, A.; Flörke, U.; Aust, H.-J.; Draeger, S.; Schulz, B. Liebigs Ann. Chem. 1994, 1994, 1093−1097. (9) Bringmann, G.; God, R.; Schäffer, M. Phytochemistry 1996, 43, 1393−1403. (10) Pescitelli, G.; Bruhn, T. Chirality 2016, 28, 466−474. (11) For a possible biosynthetic pathway via phenoxy radicals, yet only to 4, see ref 6. (12) Tshitenge, D. T.; Feineis, D.; Mudogo, V.; Kaiser, M.; Brun, R.; Bringmann, G. Sci. Rep. 2017, 7, e5767.

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DOI: 10.1021/acs.orglett.7b03473 Org. Lett. 2017, 19, 6740−6743