Jozilebomines A and B, Naphthylisoquinoline Dimers from the

Article Cite This: J. Nat. Prod. 2017, 80, 2807-2817

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Jozilebomines A and B, Naphthylisoquinoline Dimers from the Congolese Liana Ancistrocladus ileboensis, with Antiausterity Activities against the PANC‑1 Human Pancreatic Cancer Cell Line Jun Li,†,‡ Raina Seupel,† Torsten Bruhn,†,§ Doris Feineis,† Marcel Kaiser,⊥,∥ Reto Brun,⊥,∥ Virima Mudogo,▽ Suresh Awale,*,○ and Gerhard Bringmann*,† †

Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, and Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, People’s Republic of China § 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 ▽ Faculté des Sciences, Université de Kinshasa, B.P. 202, Kinshasa XI, Democratic Republic of the Congo ○ Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan ‡

S Supporting Information *

ABSTRACT: Two new naphthylisoquinoline dimers, jozilebomines A (1a) and B (1b), were isolated from the roots of the Congolese plant Ancistrocladus ileboensis, along with the known dimer jozimine A2 (2). These compounds are Dioncophyllaceae-type metabolites, i.e., lacking oxygen functions at C-6 and with an R-configuration at C-3 in their tetrahydroisoquinoline moieties. The dimers 1a and 1b consist of two 7,1′-coupled naphthylisoquinoline monomers linked through an unprecedented 3′,6″-coupling in the binaphthalene core and not, as in 2, via the C-3-positions of the two naphthalene units. Thus, different from the C2-symmetric jozimine A2 (2), the new jozilebomines are constitutionally unsymmetric. The central biaryl axis of each of the three dimers is rotationally hindered, so that 1a, 1b, and 2 possess three consecutive chiral axes. The two jozilebomines have identical constitutions and the same absolute configurations at all four stereogenic centers, but differ from each other in their axial chirality. Their structural elucidation was achieved by HRESIMS, 1D and 2D NMR, oxidative degradation, and experimental and calculated ECD data. They exhibited distinct and specific antiplasmodial activities. All dimers showed potent cytotoxicity against HeLa human cervical cancer cells and preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions. Furthermore, these dimers significantly inhibited the colony formation of PANC-1 cells, even when exposed to noncytotoxic concentration for a short time. Jozilebomines A (1a) and B (1b) and jozimine A2 (2) represent novel potential candidates for future drug development against pancreatic cancer.

N

aphthylisoquinoline alkaloids1 are structurally and biosynthetically extraordinary bioactive natural products possessing both stereogenic centers and chiral axes. Depending on their individual structures, they display strong inhibitory effects against Plasmodium2−5 and Leishmania2,5−7 parasites, and they show promising antileukemic8,9 activities. As indicated by their name, they consist of a naphthalene and an isoquinoline portion linked by a biaryl axis. The structural variety of naphthylisoquinolines was further enlarged by the discovery of 23 dimers with unique molecular architectures.4,10−14 In total, about 180 such alkaloids1,2 have been identified, all of them exclusively from woody lianas and © 2017 American Chemical Society and American Society of Pharmacognosy

scandent shrubs belonging to the palaeotropical Ancistrocladaceae15 and Dioncophyllaceae16 families. The evergreen rain forests and swamp regions in Central Africa, in particular in the Democratic Republic of the Congo (DR Congo), are considered to be the main centers of distribution of Ancistrocladus plants. Presently, 13 accepted Ancistrocladus species are known from Africa, among them five from the Congo Basin.15,17 One of these plants is the newly discovered liana Ancistrocladus ileboensis Heubl, Mudogo & G. BringReceived: July 27, 2017 Published: October 18, 2017 2807

DOI: 10.1021/acs.jnatprod.7b00650 J. Nat. Prod. 2017, 80, 2807−2817

Journal of Natural Products

Article

Figure 1. Dioncophyllaceae-type dimeric naphthylisoquinoline alkaloids isolated from the root bark of A. ileboensis: the new jozilebomines A (1a) and B (1b) and the structurally closely related known4 jozimine A2 (2). For comparison, see the structure of the anti-HIV-active 6′,6″-coupled dimer michellamine B (3) from A. korupensis.10 Likewise presented are the corresponding monomers 4′-O-demethyldioncophylline A (5a) and its atropodiastereomer 4′-O-demethyl-7-epi-dioncophylline A (5b) (both occurring in A. ileboensis8) as well as the regioisomeric 5′-O-demethyldioncophylline A (4).

mann17 from South-Central DR Congo. We have recently reported on the isolation and structural elucidation of a series of monomeric Dioncophyllaceae-type naphthylisoquinoline alkaloids (i.e., lacking an oxygen function at C-6 and possessing an R-configuration at C-3)1 from the roots and leaves of A. ileboensis, exhibiting unusual structural features and promising antiplasmodial, antileukemic, and anti-multiple myeloma activities.8 Further isolation work on the roots of A. ileboensis has now led to the identification of the first constitutionally unsymmetric Dioncophyllaceae-type naphthylisoquinoline dimers. These two alkaloids, named jozilebomines A (1a) and B (1b) (Figure 1), possess a novel 3′,6″-coupling in the central binaphthalene core. They were isolated along with the known,4 structurally related jozimine A2 (2) (Figure 1). Prior to the phytochemical work presented here, jozimine A2 had been the only dioncophyllaceous dimer discovered in nature, isolated from another botanically yet undescribed Congolese Ancistrocladus species.4 The jozilebomines are cross-coupling products of two different 7,1′-coupled monomeric naphthylisoquinoline moieties, both with 5′-O-demethyldioncophylline A (4) as one of the molecular units and with 4′-O-demethyldioncophylline A (5a) as the second part in the case of 1a and 4′-O-demethyl-7epi-dioncophylline A (5b) in the case of 1b (Figure 1). These molecular halves are linked via C-3′ and C-6″ of their naphthalene units, so that the central axes are flanked by three ortho-substituents, with sufficient steric hindrance for the central biaryl linkage to be rotationally hindered and, thus, chiral. The known jozimine A2 (2), by contrast, is C2symmetric. 4 It is built up from two fully identical naphthylisoquinoline halves, viz., two molecules of 4′-Odemethyldioncophylline A (5a) (Figure 1). In 2, these moieties are coupled via the 3′,3″-positions in the naphthalene portions, so that the central axis is even flanked by four orthosubstituents.4 As a consequence, 1a, 1b, and 2 possess three

consecutive chiral biaryl axes each and, together with the four stereogenic centers, seven elements of chirality. Most of the other known dimeric naphthylisoquinoline alkaloids such as the anti-HIV active michellamine B (3)10 are coupled via C-6′ in both of the naphthalene units, i.e., in the least hindered positions, so that the central biaryl axis of those quateraryls is configurationally unstable and can freely rotate. The jozilebomines A (1a) and B (1b) were found to display remarkable activities against the malaria parasite Plasmodium falciparum, but only low or no inhibitory effects against Leishmania or Trypanosoma pathogens. Furthermore, jozimine A2 (2) and jozilebomines A (1a) and B (1b) showed strong antiproliferative activities toward the HeLa human cervical cancer cell line and against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions.

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RESULTS AND DISCUSSION Isolation and Structural Elucidation of Naphthylisoquinoline Dimers. Roots of A. ileboensis were collected in South-Central DR Congo, along the Kasai ̈ River near its junction with the Sankuru River. Air-dried root bark material was ground and sequentially extracted with MeOH/CH2Cl2 (1:1) and MeOH/CH2Cl2/HCl (1:1:0.5). The crude extracts were macerated with water, and after evaporation to dryness, they were redissolved in MeOH and then directly subjected to preparative HPLC, which permitted isolation of three alkaloids. One of them, a major constituent of the roots, was readily identified as the known4 dimeric naphthylisoquinoline jozimine A2 (2) (Figure 1). The identity of this metabolite was proven by HPLC coelution with an authentic sample and by its HRESIMS, NMR, and electronic circular dichroism (ECD) data, which were fully in accordance with those reported earlier.4 Jozimine A2 had previously been discovered as a trace metabolite occurring in the roots of a botanically yet unexplored Ancistrocladus species from Central DR Congo,4 2808



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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of Jozilebomines A (1a) and B (1b) in Methanol-d4 (δ in ppm)

displaying an extraordinary antiplasmodial activitiy (IC50 = 1.4 nM).4 The two further alkaloids isolated from the root bark of A. ileboensis were new. Their UV and 1H NMR spectra indicated that they were dimeric naphthylisoquinolines, too. Jozilebomine A (1a). The molecular formula of the first new compound, a minor metabolite, was C46H48N2O8, as deduced from HRESIMS, i.e., the same as that of jozimine A2 (2). In contrast to 2, however, the new dimer revealed a full set of 1H and 13C NMR signals (Table 1), thus evidencing the isolated metabolite to be unsymmetric. The 1H NMR spectrum of its “northwestern” naphthylisoquinoline portion revealed chemical shifts typical of a Dioncophyllaceae-type naphthyltetrahydroisoquinoline bearing methyl groups at C-1 (δ 1.69), C-3 (δ 1.53), and C-2′ (δ 1.81), one methoxy function (δ 4.08), and five aromatic protons, with a two-proton spin system in the tetrahydroisoquinoline moiety (δ 6.87 and 6.98) and a spin system of three contiguous protons in the naphthalene portion (δ 6.94, 7.20, and 6.87). The methoxy group was assigned at C-5′ in the naphthalene moiety, because of a ROESY cross-peak to H-6′ (δ 6.94) and an HMBC interaction with C-5′ (δ 157.9). The high-field shift of the signal of Me-2′ (δ 1.81) and HMBC long-range couplings (Figure 2A) from H-8′ (δ 6.87) to C-1′ (δ 124.7) and C-10′ (δ 115.2), from H-7′ (δ 7.20) to C-9′ (δ 137.1), from H-6′ (δ 6.94) to C-10′, and from Me-2′ to C-1′ established the biaryl axis to be located at C-1′. The two diastereotopic protons at C4 had “normal” chemical shifts (δ 2.95 and 3.22), i.e., no shielding effect from the naphthalene portion, indicating that the biaryl axis was not linked to C-5, but to C-7 in the tetrahydroisoquinoline moiety. In conclusion, the “northwestern” part of the new dimer had to be 7,1′-coupled, which was also in agreement with the two NOESY correlation sequences {OMe-5′ ↔ H-6′ ↔ H-7′ ↔ H-8′} and {Me-2′ ↔ H-6 ↔ H-5 ↔ H-4eq} (Figure 2B). The “southeastern” naphthylisoquinoline part of the new dimer displayed five aromatic protons in the 1H NMR spectrum (Table 1), with one aromatic singlet, H-3″ (δ 7.01), and four doublets suggesting the presence of two pairs of adjacent aromatic protons, one resonating at δ 7.11 and 6.88 and one at δ 6.92 and 7.02. This portion furthermore showed the signals of one aromatic methyl group, Me-2″ (δ 2.20), of one methoxy function, OMe-4″ (δ 4.16), and of two diastereotopic protons, Hax-4‴ (δ 2.95) and Heq-4‴ (δ 3.25), two methyl groups, Me-1‴ (δ 1.72) and Me-3‴ (δ 1.54), and two multiplets, H-1‴ (δ 4.87) and H-3‴ (δ 3.93). Moreover, this moiety exhibited NOESY correlations between H-3″ and CH3O-4″ and between H-7″ (δ 7.11) and H-8″ (δ 6.88) (Figure 2B), which excluded the naphthalene moiety from being connected to the tetrahydroisoquinoline part via its methyl-free ring. Consequently, the biaryl axis had to be located at C-1″ (δ 127.1). This finding was in agreement with the highfield shift of the Me-2″ signal (δ 2.20, see above) and with HMBC couplings from the aromatic protons H-8″ and H-6‴ (δ 7.02) to C-1″ and from Me-2″ to C-1″ (Figure 2A). In the isoquinoline moiety, the NOESY correlation sequence {H-6‴ ↔ H-5‴ ↔ Heq-4‴} revealed C-7‴ (δ 125.6) as the coupling position, which was confirmed by an HMBC interaction from H-5‴ (δ 6.92) with C-7‴. The HMBC correlation between H-7″ and a quaternary carbon atom (C-3′) belonging to a different part of the molecule, viz., the “northwestern” naphthylisoquinoline moiety, established C-6″ (120.1 ppm) to be the coupling position of the central biaryl axis for this “southeastern” naphthylisoquino-

1a position 1 3 4ax 4eq 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 1‴ 3‴ 4‴ax 4‴eq 5‴ 6‴ 7‴ 8‴ 9‴ 10‴ 1-CH3 3-CH3 2′-CH3 5′-OCH3 2″-CH3 4″-OCH3 1‴-CH3 3‴-CH3

δH (J in Hz) 4.87, m 3.89, m 2.95, dd (18.0, 12.0) 3.22, dd (18.0, 4.8) 6.87, d (7.8) 6.98, d (7.8)

6.94, d (7.8) 7.20, dd (9.0, 7.8) 6.87, d (9.0)

7.01, s

7.11, d (8.4) 6.88, d (8.4)

4.87, m 3.93, m 2.95, dd (18.0, 12.0) 3.25, dd (18.0, 4.8) 6.92, d (7.8) 7.02, d (7.8)

1.69, 1.53, 1.81, 4.08, 2.20, 4.16, 1.72, 1.54,

d d s s s s d d

(6.6) (6.0)

(6.6) (6.0)

1b δC, type 49.9, CH 45.2, CH 34.5, CH2

121.1, 132.6, 126.0, 152.7, 121.9, 132.4, 124.7, 138.4, 122.0, 153.2, 157.9, 105.2, 127.1,

CH CH C C C C C C C C C CH CH

120.4, CH 137.1, C 115.2, C 127.1, C 137.2, C 108.7, CH 157.4, C 152.0, C 120.1, C 131.7, CH 118.1, CH 137.5, C 115.2, C 49.8, CH 45.2, CH 34.5, CH2

121.4, CH 132.6, CH 125.6, C 152.4, C 122.2, C 132.5, C 17.9, CH3 19.3, CH3 18.2, CH3 56.9, CH3 20.7, CH3 57.0, CH3 18.0, CH3 19.3, CH3

δH (J in Hz) 4.85, m 3.94, m 2.91, dd (18.0, 11.4) 3.25, dd (18.0, 4.2) 6.87, d (7.8) 6.98, d (7.8)

6.95, d (7.8) 7.24, dd (9.0, 7.8) 6.88, dd (9.0, 1.2)

7.02, s

7.11, d (9.0) 6.87, d (9.0)

4.90, q (6.6) 3.92, m 2.95, dd (18.0, 11.4) 3.25, dd (18.0, 4.2) 6.89, d (7.8) 7.00, d (7.8)

1.72, 1.52, 1.82, 4.08, 2.20, 4.16, 1.73, 1.54,

d d s s s s d d

(6.6) (6.6)

(6.6) (6.6)

δC, type 49.8, CH 45.2, CH 34.5, CH2

121.1, 132.5, 126.0, 152.7, 122.0, 132.4, 124.9, 138.6, 121.9, 153.1, 157.8, 105.3, 127.3,

CH CH C C C C C C C C C CH CH

120.4, C 137.0, C 115.1, C 127.2, C 137.2, C 108.7, CH 157.4, C 152.0, C 120.0, C 131.7, CH 118.1, CH 137.5, C 115.1, C 49.8, CH 45.2, CH 34.5, CH2

121.3, CH 132.5, CH 125.6, C 152.4, C 122.3, C 132.5, C 18.0, CH3 19.3, CH3 18.4, CH3 57.0, CH3 20.7, CH3 57.0, CH3 18.0, CH3 19.3, CH3

line half (Figure 2A). The extremely upfield-shifted signal of the Me-2′ group (δ 1.81, see above), hinting at the proximity of even two aryl substituents next to this methyl group, and a ROESY correlation between H-7″ (δ 7.11) and Me-2′ (Figure 2809



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Figure 2. (A) HMBC and (B) NOESY interactions indicative of the constitution (including the position of the central biaryl axis and the two outer axes) of jozilebomine A (1a) and ROESY interactions defining the relative configuration at stereogenic centers versus the axis within the two molecular halves of 1a: (C) for the “northwestern” molecular portion (dotted line: interaction seen only by selective 1D NOESY experiments) and (D) for the “southeastern” molecular half.

The central biaryl axis of the new dimer was flanked by three ortho-substituents and was, thus, rotationally hindered. Its central binaphthalene core closely resembles that of the C2symmetric jozimine A2 (2), which possesses a 3′,3″-coupled, Mconfigured central biaryl axis (see Supporting Information). Previous investigations4,13 had shown that the chiroptical properties of dimeric naphthylisoquinolines with a 3′,3″-linkage are dominated by the orientation of the two naphthalene chromophores to each other and, thus, by the configuration at the central biaryl linkage. Therefore, the absolute configuration at the central axis in the new dimer was assigned by comparison of its ECD spectrum with that of 2, which was found to be nearly opposite, thus suggesting that the central axis of the new alkaloid was P-configured. The absolute configuration at the central axis in this noveltype naphthylisoquinoline dimer with its unprecedented 3′,6″coupling type was unequivocally confirmed by experimental and computational ECD investigations. The ECD spectrum quantum-chemically calculated for the P,P,P-atropo-diastereomer at the TD-CAM-B3LYP/def2-TZVP level showed a good agreement with the measured spectrum of the natural product (Figure 3, left), whereas the spectrum for the P,M,Patropisomer was, expectedly, virtually opposite (Figure 3, right), because the chiroptical properties of such jozimine-type naphthylisoquinoline dimers are dominated by the orientation of the two strong naphthalene chromophores to each other and, thus, by the configuration at the central biaryl linkage.4,13 The new dimer was, thus, 1R,3R,7P,3′P,7‴P,3‴R,1‴Rconfigured and, consequently, possessed the full absolute stereostructure 1a as outlined in Figure 1. Owing to its close structural resemblance to the co-occurring jozimine A2 (2) and its isolation from A. ileboensis, it was given the name jozilebomine A (1a). Jozilebomine B (1b). HRESIMS analysis of the second dimer again gave a molecular formula of C46H48N2O8, i.e., the same as that of jozilebomine A (1a). Its 1H and 13C NMR spectroscopic data (Table 1) and specific HMBC and ROESY interactions (see Supporting Information) again corresponded to a Dioncophyllaceae-type naphthyltetrahydroisoquinoline dimer and again with a 3′,6″-coupled central biaryl axis.

2B) confirmed the two monomeric halves of the dimer to be connected by a 3′,6″-coupling in the two naphthalene portions. From ROESY correlations between the protons of Me-1 and H-3 and between the protons of Me-1‴ and H-3‴, the relative configurations of the stereogenic centers in both of the isoquinoline portions were established to be trans (Figure 2C and D). The absolute configurations at C-3 and C-3‴ were determined to be R by ruthenium-mediated oxidative degradation,18 exclusively providing (R)-3-aminobutyric acid. Considering the relative trans-orientation of the two methyl groups in the tetrahydroisoquinoline units as deduced from NMR measurements, the absolute configurations at C-1 and C1‴ too had to be R. Based on the long-range ROESY interaction from Me-2″ to Me-1‴, the axial configuration of the naphthalene-tetrahydroisoquinoline linkage in the “southeastern” molecular half was assigned to be P (Figure 2D). Hence, this monomeric portion of the new dimer was identical with 5′-O-demethyldioncophylline A (4),19 a well-known main alkaloid from the West African liana Triphyophyllum peltatum, but so far not found in A. ileboensis. In the “northwestern” part of the dimer, however, no significant long-range ROESY interactions indicative of the axial configuration of the second “outer” biaryl axis, i.e., in the “northwestern” part of the dimer, were detected: no cross-peak between H-8′ (δ 6.87) and the protons of Me-1 (δ 1.69), which would have been decisive for the configuration at the biaryl axis, was observed. The chemical shifts of the protons of Me-2′ (δ 1.81) and Me-1 (δ 1.69, see above) were close to each other, so that it was not possible to unambiguously attribute a ROESY interaction suggesting the axial configuration of this second outer axis. Therefore, selective 1D NOESY experiments were performed. Irradiation of Me-2′ led to a significant enhancement of the signals of Me-1, thus confirming the biaryl axis of the “northwestern” molecular half of the new dimer to be Pconfigured (Figure 2C). Hence, this molecular part was identical to 4′-O-demethyldioncophylline A (5a), a monomeric naphthylisoquinoline alkaloid likewise occurring in the roots of A. ileboensis and also representing the molecular half of jozimine A2 (2).4,8 2810



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Figure 3. Assignment of the absolute configuration of jozilebomine A (1a), by comparison of its experimental ECD curve with the spectra calculated for the P,P,P- and the P,M,P-atropo-diastereomers by using TD-CAM-B3LYP/def2-TZVP.

Figure 4. ROESY interactions evidencing the relative configuration at stereogenic centers versus axes within the two molecular halves of jozilebomine B (1b): (A) for the “northwestern” molecular portion and (B) for the “southeastern” molecular half. (C) Assignment of the absolute configuration of the central biaryl axis of 1b, by comparison of its ECD spectrum with that of jozilebomine A (1a).

ROESY investigations (Figure 4A,B) and the chemical degradation18 hinted at a structure most similar to that of 1a, including the relative trans-configurations of the two tetrahydroisoquinoline units, and only the configuration at the biaryl axis in the “northwestern” part of the new dimer was different from that of 1a. Based on the long-range ROESY correlation between H-8′ (δ 6.88) and the protons of Me-1 (δ 1.72), it was established to be M-configured. Assignment of the absolute configuration at the central biaryl axis was based on the fact that the ECD spectrum of the new dimer was virtually identical to that of jozilebomine A (1a), thus evidencing that the central axis in the new alkaloid was P-configured, too (Figure 4C). Therefore, the full absolute stereostructure of this new Dioncophyllaceae-type dimer 1b was 1R,3R,7M,3′P,7‴P,3‴R,1‴R, as shown in Figure 1, and thus, this alkaloid was the 3′,6″ cross-coupling product of 4′-Odemethyl-7-epi-dioncophylline A (5b) and 5′-O-demethyldioncophylline A (4). Hence, it was the 7-epi analogue of the cooccurring jozilebomine A (1a). The new alkaloid 1b was named jozilebomine B. Antiprotozoal Activities. Jozimine A2 (2) has recently attracted attention due to its excellent antiplasmodial activity against the NF54 strain (sensitive to all known drugs) in vitro (IC50 = 1.4 nM),4 with an inhibitory effect stronger than that of any other of the numerous naturally occurring mono- and dimeric naphthylisoquinoline2,3,5 alkaloids tested so far. Moreover, 2 showed an excellent selectivity index of ca. 11 400, due to its extremely weak cytotoxicity against L6 cells, so that this dimer can be considered as a promising lead compound according to the TDR/WHO guidelines.4,20 Because of this finding and due to the promising antiparasitic activities of some

of the naphthylisoquinoline alkaloids,2−7,21 the two new dimers 1a and 1b were evaluated for their in vitro effects against protozoan parasites causing tropical diseases such as malaria, leishmaniasis, Chagas’ disease, and African sleeping sickness. Jozilebomines A (1a) and B (1b) displayed good to moderate antiplasmodial activities, with half-maximum inhibitory concentration (IC50) values of 0.043 μM (for 1a) and 0.102 μM (for 1b), showing only weak cytotoxicities against rat skeletal myoblast (L6) cells (Table 2). The inhibitory effects against P. falciparum were, however, drastically weaker compared to that of jozimine A2 (2), by a factor as high as 31 in the case of 1a and by a factor of 73 in the case of 1b. These results give an important contribution to our ongoing structure−activity relationship investigations within this class of compounds, clearly corroborating the strong impact of axial chirality on the bioactivities of dimeric naphthylisoquinoline alkaloids.10−12,22 The new alkaloids 1a and 1b displayed only moderate activities against Trypanosoma brucei rhodesiense, the pathogen of African sleeping sickness, and were virtually inactive against T. cruzi (Chagas’ disease) and Leishmania donovani (visceral leishmaniasis) (Table 2). The results reveal the antiparasitic activities of the jozilebomines A (1a) and B (1b) to be significantly structure-dependent and, thus, specific. Cytotoxic Activities against HeLa Cells. The first results on the promising cytotoxic effects of naphthylisoquinoline alkaloids against cancer cell lines have only recently been 2811



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Table 2. Antiparasitic Activities of Jozilebomines A (1a) and B (1b) against Plasmodium falciparum (Strain: NF54), Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani and Cytotoxicities against Rat Skeletal Myoblast (L6) Cells IC50 [μM]a compound standard 1a 1b

P. falciparumc 0.009 0.043 0.102

c

T. cruzi 2.317 23.81 21.88

d

T. brucei rhodesiense 0.0075 0.732 1.035

L. donovani

e

0.432 89.74 55.77

f

L6 cells (cytotoxicity) 0.017 6.85 5.33

f

selectivity indexb 21.6 160 52.3

a

The IC50 values are the means of two independent assays; the individual values vary by a factor of less than 2. bThe selectivity index is the ratio of the IC50 values for the L6 cells to the IC50 data relative to P. falciparum. cChloroquine. dBenznidazole. eMelarsoprol. fMiltefosine. gPodophyllotoxin.

reported.8,9 The jozilebomines A (1a) and B (1b) and jozimine A2 (2) were therefore investigated for their cytotoxic activities against the HeLa human cervical cancer cell line. As shown in Figure 5, all three dimers showed concentration-dependent

pancreatic tumors unresponsive to drugs routinely applied in chemotherapeutic regimens.26 Moreover, anticancer agents in clinical use are often accompanied by severe side effects on normal cells, due to a nonselective toxicity.26,27 The antiausterity approach aims at the identification and development of improved chemotherapeutic agents23,28 that are selectively toxic to pancreatic cancer cells under nutrientdeficient conditions as typical of the hypovascular (austerity) tumor microenvironment,23,26 but without affecting normal cells. Owing to an altered metabolism, PANC-1 cells show a high tolerance to nutrition starvation, enabling them to survive under a low-nutrient state. Compounds displaying preferential cytotoxicity in NDM without exhibiting cytotoxicity in DMEM are therefore considered to be antiausterity agents.23,24 The jozimine-type dimers 1a, 1b, and 2 exhibited remarkable preferential cytotoxicities against PANC-1 cells in a concentration-dependent manner (Figure 6). Their PC50 values (i.e., the concentrations at which 50% of the cells were preferentially killed in NDM without cytotoxicity in DMEM) range from 2.24 to 0.1 μM (Table 3). Among these, the activity of 1b (PC50,

Figure 5. Cytotoxic activities of jozilebomine A (1a), jozilebomine B (1b), and jozimine A2 (2) toward HeLa human cervical cancer cells.

cytotoxicities against HeLa cells. Within this series of dioncophyllaceous dimers, the activity of 2 (IC50, 0.22 μM) was the most potent, followed by those of 1b (IC50, 0.68 μM) and 1a (IC50, 1.08 μM). Preferential Cytotoxicity against PANC-1 Cells. The naphthylisoquinoline dimers 1a, 1b, and 2 were tested for their cytotoxic activity against the PANC-1 human pancreatic cancer cell line in normal nutrient-rich medium (Dulbecco’s modified Eagle’s medium, DMEM) and nutrient-deprived medium (NDM), following the antiausterity strategy.23,24 Since some cancer cells show the ability to rapidly develop multidrug resistance,25 current research aims at the discovery of novel cytotoxic agents, in particular for the treatment of aggressive

Table 3. Cytotoxicity Assay Results of Jozilebomines A (1a) and B (1b) and Jozimine A2 (2) against Human Tumor Cells compound b

paclitaxel arctigeninc 1a 1b 2

HeLa (IC50 in μM)

PANC-1 (PC50 in μM)a

0.001

>100 0.83 2.24 0.87 0.10

1.08 0.68 0.22

a

Concentration at which 50% of cells were killed preferentially in NDM. b,cUsed as reference compounds.

Figure 6. Preferential cytotoxic activity of jozilebomine A (1a), jozilebomine B (1b), and jozimine A2 (2) against the PANC-1 human pancreatic cancer cell line in nutrient-deprived medium (NDM) and Dulbecco’s modified Eagle’s medium (DMEM). 2812



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Figure 7. Morphological changes of PANC-1 cells induced by (B) jozilebomine A (1a), (C) jozilebomine B (1b), and (D) jozimine A2 (2) in comparison to untreated cells (A): PANC-1 cells were treated with 5 μM of the dimer 1a, 1b, or 2 in 12-well plates and incubated for 24 h. Cells were stained with ethidium bromide (EB) and acridine orange (AO). The cells were photographed under fluorescence (red and green) and phase contrast (gray) modes using an EVOS FL digital inverted microscope.

0.87 μM) was comparable to that of the positive control arctigenin (PC50, 0.83 μM), while the activity of 2 (PC50, 0.10 μM) was the most potent, even stronger than that of arctigenin. The new compounds were further evaluated for their effects on cell morphology under nutrient-deprived conditions (NDM) using the ethidium bromide (EB) and acridine orange (AO) double staining assay. AO is a cell-permeable dye and emits bright green fluorescence in live cells. EB is permeable only in the dead cells and gives predominant red fluorescence. As shown in Figure 7, the control cells emitted bright green fluorescence, and the morphology remained intact. However, treatment with 5 μM 1a, 1b, or 2 caused cellular catastrophe in PANC-1 cancer cells, leading to membrane rupture and disintegration of cell organelles, as illustrated by the exclusive red fluorescence (Figure 7B−D). These data prompted us to investigate the further potential of the new dimers against colony formation of PANC-1 cells. In this assay, PANC-1 cells were exposed to the new compounds 1a, 1b, and 2 for 24 h in nutrient-rich DMEM medium at noncytotoxic concentrations. The cells were washed with phosphate-buffered saline (PBS), the medium was replaced by fresh DMEM, and the PANC-1 cells were allowed to grow for 10 d. The cells were finally stained with crystal violet to observe

the PANC-1 cell growth and colony formation. This assay provides potential information on whether the test compound can inhibit cellular proliferation even after exposure to noncytotoxic concentrations for a short time under nutrientrich conditions. As shown in Figure 8, in the absence of compound exposure (control), PANC-1 cells grew exponentially to form a large number of colonies stained with blue color. When treated with the test compounds at the lowest noncytotoxic dose for 24 h in DMEM, the dimers 1a, 1b, and 2 were found to inhibit colony formation significantly in a concentration-dependent manner (see Supporting Information). Most evidently, 2 inhibited colony formation completely even at the exposure of 1.25 μM. Therefore, the jozimine-type dimers 1a, 1b, and 2, in particular, and related naphthylisoquinoline alkaloids, in general, seem to be highly promising leads for the development of drugs against pancreatic cancer. In conclusion, three Dioncophyllaceae-type dimeric naphthylisoquinoline alkaloids, each containing three consecutive chiral biaryl axes, were isolated from A. ileboensis17 from the lowlands in the South of the Congo Basin. Jozimine A2 (2), known from previous isolation work on a related Congolese Ancistrocladus liana,4 consists of two 4′-O-demethyldioncophylline A halves coupled through the sterically quite constrained 2813



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Figure 8. Effect of jozilebomine A (1a), jozilebomine B (1b), and jozimine A2 (2) on colony formation of PANC-1 cells. The figure shows mean values of three independent experiments; ** p < 0.01, * p < 0.05 when compared with the untreated control group. temperature. Organic solvents were dried and distilled prior to use. (S)-MTPA was purchased from Sigma-Aldrich (Steinheim, Germany). Plant Material. Root bark of Ancistrocladus ileboensis was collected by one of us (V.M.) in the Congo Basin, in April 2003, near ̈ Bambange, north of the town Ilebo (Province Kasai-Occidental) in the South-Central part of the Democratic Republic of the Congo. A voucher specimen (no. 57) has been deposited at the Herbarium Bringmann, University of Würzburg. Extraction and Isolation. Air-dried root bark of A. ileboensis (300 g) was ground to a fine powder and sequentially extracted at room temperature with CH2Cl2/MeOH (1:1) and CH2Cl2/MeOH/HCl (1:1:0.05). The combined CH2Cl2/MeOH/HCl layers were concentrated at room temperature in vacuo to give ca. 36.4 g of a crude residue, which was ground and macerated with H2O, then filtered and lyophilized, affording 24.0 g of a brown solid. A portion (2.0 g) of this extract was resuspended in MeOH, and then directly subjected to preparative HPLC on a SymmetryPrep C18 column (19 × 300 mm, 7 μm, Waters) using an eluent system consisting of solvent A [H2O (0.05% TFA)] and solvent B [MeCN (0.05% TFA)], with a linear gradient (0 min 10% B, 10 min 25% B, 45 min 45% B, 50 min 100% B), at a flow rate of 8 mL min−1, providing 24.5 mg of jozimine A2 (2) (retention time 33.8 min), along with two additional dimer-containing subfractions (retention times 32.2 and 34.7 min). The first one was further purified by preparative HPLC, again on a SymmetryPrep C18 column, using a linear gradient: A/B (0 min 45% B, 30 min 60% B, 45 min 60% B, 47 min 100% B, 50 min 100% B), in which A was H2O (0.05% TFA) and B MeOH (0.05% TFA), at a flow rate of 8 mL min−1, yielding 5.8 mg of jozilebomine B (1b) (retention time 38.0 min). In a similar manner, the second subfraction was resolved by preparative HPLC on a Chromolith C18 column, using the following gradient: A/B (0 min 20% B, 10 min 35% B, 12 min 65% B, 13 min 100% B, 14 min 100% B), in which A was H2O (0.05% TFA) and B MeCN (0.05% TFA), at a flow rate of 8 mL min−1, yielding 4.5 mg of jozilebomine A (1a) (retention time 4.9 min). Jozilebomine A (1a): brown, amorphous powder; [α]20 D −70.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 338 (2.85), 324 (2.05), 309 (0.15), 286 (0.15), 245 (0.22), 228 (0.11) nm; ECD (c 0.50; MeOH) λmax (Δε) 191 (−4.95), 199 (+6.31), 212 (−9.30), 222 (−1.23), 230 (−13.2), 243 (+15.9), 263 (+3.30), 279 (+5.69), 298 (−7.65), 319 (+1.23), 339 (+4.67), 364 (−0.01), 445 (+0.01) cm2/mol; IR (ATR) νmax 3364, 1675, 1437, 1390, 1301, 1198, 1138, 1089, 843, 797, 724

C-3′ position in each of the naphthalene units. The new jozilebomines A (1a) and B (1b), by contrast, are the first Dioncophyllaceae-type dimers with an unsymmetrically coupled central biaryl axis. The two naphthalene portions of these dimers are connected via an unprecedented 3′,6″-linkage. Besides their unusual structural features, the dimers 1a, 1b, and, in particular, jozimine A2 (2) are of special interest because of their promising activities against the malaria parasite Plasmodium falciparum and against cervical HeLa cells and PANC-1 human pancreatic cancer cells. Owing to the fact that cancer cells rapidly develop resistance toward drugs routinely used in chemotherapeutic regimens,25 the search for novel cytotoxic agents is an urgent task. The promising activities of jozimine A2 and the two ilebojozimines warrant further investigations on the anticancer potential of naphthylisoquinoline alkaloids in general. More in-depth studies toward this goal are presently in progress.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-1020 polarimeter (JASCO, Gross-Umstadt, Germany), and UV/vis spectra were taken on a Cary 50 Conc spectrophotometer (Varian). IR spectra were measured on a JASCO FT/IR-410 spectrometer. ECD spectra were determined on a JASCO J-715 spectropolarimeter at room temperature, using a 0.1 cm standard cell and spectrophotometric-grade MeOH. 1H NMR (600 MHz) and 13 C NMR (150 MHz) spectra were recorded on a DMX 600 instrument at ambient temperature, using methanol-d4 (δ 3.31 and 49.15 ppm) as the solvent and as the internal 1H and 13C standard. Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J) are given in hertz (Hz). Multiplicities are denoted as singlet (s), doublet (d), doublet of doublets (dd), quartet (q), or multiplet (m). For ROESY experiments, the mixing time was set to 1 s. Proton-detected, heteronuclear correlations were analyzed using HMQC (optimized for 1JHC = 145 Hz) and HMBC (optimized for nJHC = 7 Hz). High-resolution electrospray ionization mass spectra (HRESIMS) were taken on a Bruker Daltonics micrOTOF-focus mass instrument. Preparative HPLC was carried out on a JASCO System (PU-1580 Plus) in combination with UV/vis detection at 190−600 nm (JASCO MD-2010 Plus diode array detector) at room 2814



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cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 725.3574 [M + H]+ (calcd for C46H49N2O6, 725.3585). Jozilebomine B (1b): brown, amorphous powder; [α]20 D +19.5 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 338 (2.60), 322 (2.00), 308 (0.11), 284 (0.11), 244 (0.11), 228 (0.11) nm; ECD (c 0.50; MeOH) λmax (Δε) 192 (−4.66), 201 (+5.74), 212 (−9.30), 222 (−1.23), 230 (−13.2), 244 (+16.1), 268 (+3.88), 279 (+5.72), 298 (−7.65), 319 (+1.23), 339 (+4.67), 364 (−0.01), 412 (+0.02) cm2/mol; IR (ATR) νmax 3437, 3345, 2927, 2852, 1671, 1577, 1436, 1390, 1182, 1132, 1088, 1022, 839, 798, 722 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 725.3583 [M + H]+ (calcd for C46H49N2O6, 725.3585). Oxidative Degradation. Following a miniaturized procedure described earlier,18 ca. 1.0 mg of jozilebomine A (1a) and jozilebomine B (1b) were subjected to ruthenium(III)-catalyzed periodate degradation to give the expected amino acids. After their conversion to the respective methyl esters with MeOH/HCl, followed by Moshertype derivatization with (R)-α-methoxy-α-trifluoromethylphenyl-acetyl chloride [(R)-MTPA-Cl, prepared from (S)-MTPA], the absolute configurations in the tetrahydroisoquinoline portions of 1a and 1b were assigned by gas chromatography coupled to mass-selective detection (GC-MSD) and comparison with the corresponding derivatives of the authentic amino acids of known configuration. Computational Details. To elucidate the absolute configuration of 1a using ECD calculations,29,30 conformational analyses of 1a with a P,P,P and a P,M,P configuration were performed using B3LYP-D3/ def2-SVP (using Becke−Johnson damping)31,32 and the chain-ofspheres approximation.33 As it is known that stereogenic centers in the monomers of naphthylisoquinoline dimers have no significant influence on their ECD spectra due to the chiroptical dominance of the axes,13 the conformations of the isoquinoline moieties were frozen to a geometry that corresponds to a minimum for the monomeric structure. Thus, only the dihedral angles at the axes were investigated, giving just one minimum structure for each configuration, both being fully consistent with the NOEs observed for 1a. To evaluate the suitability of functionals for the subsequent excited states, computations BHLYP, CAM-B3LYP, and ωB97X-D334,35 and def2TZVP32 as the basis set were tested with the simplified TDDFT (sTD)36 approach. According to the ΔESI values,37 the best results were achieved by sTD CAM-B3LYP, and, thus, full TDDFT CAMB3LYP/def2-TZVP (RIJCOSX) calculations34,38 were performed including an increased DFT grid (GRID6, GRIDX5) to finally determine the absolute configuration of the central axis of 1a. All calculations were performed with ORCA,39 and the further processing of the results was done with SpecDis.40 For the TDCAM-B3LYP spectra, a σ value of 0.16 eV and a UV correction of 30 nm were applied. Antiprotozoal Evaluation. The antiparasitic activities of 1a and 1b against the pathogens Plasmodium falciparum (NF54 strain), Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani and the cytotoxicity against mammalian host cells (rat skeletal myoblast L6 cells) were determined in vitro as described previously.41 Cytotoxicity Assay against HeLa Cells. The dimers 1a, 1b, and 2 were tested for their cytotoxic activities in vitro using the human cervical HeLa cell line.42 Cell viability in the presence or absence of tested compounds was determined using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). The HeLa cell line was purchased from the Riken BRC cell bank (Tsukuba, Japan) and maintained in standard DMEM with 10% fetal bovine serum (FBS) supplement, 0.1% NaHCO3, and 1% antibiotic antimycotic solution. For cytotoxicity evaluation, exponentially growing cells were harvested and plated in 96-well plates (2 × 103/ well) in DMEM at 37 °C under an atmosphere of humidified 5% CO2 and 95% air for 24 h. After the cells had been washed with PBS, the medium was changed to serially diluted test samples in DMEM, with the control and blank in each plate. Paclitaxel was used as the positive control in this study. After 72 h of incubation, the cells were washed twice with PBS, and 100 μL of DMEM containing 10% WST-8 cell counting kit solution was added to each well. After incubation for 3 h,

the absorbance at 450 nm was measured on an EnSpire multimode plate reader (PerkinElmer, Inc., Waltham, MA, USA). Cell viability was calculated from the mean values from three wells using the following equation:

Cell viability (%) = [Abs(test sample) − Abs(blank)/Abs(control) − Abs(blank)] × 100% Preferential Cytotoxicity Assay against PANC-1 Cells. The dimers 1a, 1b, and 2 were evaluated for their preferential cytotoxicity against PANC-1 human pancreatic cancer cells.43 The PANC-1 (RBRC-RCB2095) human pancreatic cancer cell line was purchased from the Riken BRC cell bank, maintained in standard DMEM with 10% FBS supplement, and stored at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. Briefly, human pancreatic cancer cells were seeded in 96-well plates (1.5 × 104/well) and incubated in fresh DMEM at 37 °C under 5% CO2 and 95% air for 24 h. After the cells had been washed twice with PBS, the medium was changed to serially diluted test samples in both nutrient-rich medium (DMEM) and nutrient-deprived medium (NDM)23 with a control and a blank in each test plate. The composition of the NDM was as follows: 0.1 mg L−1 Fe(NO3)3 (9 H2O), 265 mg L−1 CaCl2 (2 H2O), 400 mg L−1 KCl, 200 mg L−1 MgSO4 (7 H2O), 6400 mg L−1 NaCl, 700 mg L−1 NaHCO3, 125 mg L−1 NaH2PO4, 15 mg L−1 phenol red, 25 mM L−1 HEPES buffer (pH 7.4), and MEM vitamin solution (Life Technologies, Inc., Rockville, MD, USA); the final pH was adjusted to 7.4 with 10% aqueous NaHCO3. Arctigenin, the positive control, had been isolated from the seeds of Arctium lappa.23 After 24 h of incubation with each test compound in DMEM and NDM, the cells were washed twice with PBS and replaced with 100 μL of DMEM containing 10% WST-8 cell counting kit solution. After 3 h of incubation, cell viability was measured and calculated as described above. Morphological Assessment of Cancer Cells. For studies on morphological changes, PANC-1 cells were seeded in 12-well plates (1 × 106) and incubated in a humidified CO2 incubator for 24 h for the cell attachment. The cells were then washed twice with PBS and treated with vehicle control or test compounds at a concentration of 5 μM in NDM. After 24 h of incubation, 5 μL of EB/AO reagent (dye mixture: 100 μg mL−1 AO and 100 μg mL−1 EB in PBS) was added to each test well and incubated for 5 min. The morphology was visualized in the fluorescent and phase contrast modes, using an EVOS FL digital microscope (20× objective). Images were taken in phase contrast (gray) and fluorescent (red and green) channels. Colony Formation Assay. PANC-1 cells were plated in 12-well plates at a density of 500 cells/well in DMEM (1 mL/well) and incubated at 37 °C under humidified 5% CO2 for 24 h for the cell attachment. The medium was then changed to DMEM containing test compounds (1.25 and 2.5 μM) or DMEM only (control) and exposed for 24 h. The cells were washed twice with PBS, which was replaced with fresh DMEM media, and were allowed to grow for 10 d. The cells were again washed with PBS, fixed with 4% formaldehyde, and stained with crystal violet for 10 min. Each experiment was repeated three times. Finally, the colony area was measured by the ImageJ plugin “Colony Area”,44 and the data were analyzed by GraphPad Prism 6 software.

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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.jnatprod.7b00650. NMR (1H, 13C, 1H,1H−COSY, HSQC, HMBC, NOESY, and ROESY), HRESIMS, IR, and ECD spectra of compounds 1a and 1b (PDF) 2815



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Ndjoko Ioset, K.; Schmitz, W.; Ngoc, L. H. Phytochemistry 2011, 72, 89−93. (8) Li, J.; Seupel, R.; Feineis, D.; Mudogo, V.; Kaiser, M.; Brun, R.; Brünnert, D.; Chatterjee, M.; Seo, E.-J.; Efferth, T.; Bringmann, G. J. Nat. Prod. 2017, 80, 443−458. (9) (a) Jiang, C.; Li, Z.-L.; Gong, P.; Kang, S.-L.; Liu, M.-S.; Pei, Y.H.; Jing, Y.-K.; Hua, H.-M. Fitoterapia 2013, 91, 305−312. (b) Bringmann, G.; Seupel, R.; Feineis, D.; Zhang, G.; Xu, M.; Wu, J.; Kaiser, M.; Brun, R.; Seo, E.-J.; Efferth, T. Fitoterapia 2016, 115, 1− 8. (10) (a) Boyd, M. R.; Hallock, Y. F.; Cardellina, J. H., II; Manfredi, K. P.; Blunt, J. W.; McMahon, J. B.; Buckheit, R. W., Jr.; Bringmann, G.; Schäffer, M.; Cragg, G. M.; Thomas, D. W.; Jato, J. G. J. Med. Chem. 1994, 37, 1740−1745. (b) McMahon, J. B.; Currens, M. J.; Gulakowski, R. J.; Buckheit, R. W., Jr; Lackman-Smith, C.; Hallock, Y. F.; Boyd, M. R. Antimicrob. Agents Chemother. 1995, 39, 484−488. (11) (a) Hallock, Y. F.; Manfredi, K. P.; Dai, J. R.; Cardellina, J. H., II; Gulakowski, R. J.; McMahon, J. B.; Schäffer, M.; Stahl, M.; Gulden, K. P.; Bringmann, G.; François, G.; Boyd, M. R. J. Nat. Prod. 1997, 60, 677−683. (b) Bringmann, G.; Steinert, C.; Feineis, D.; Mudogo, V.; Betzin, J.; Scheller, C. Phytochemistry 2016, 128, 71−81. (12) (a) Hallock, Y. F.; Cardellina, J. H., II; Schäffer, M.; Bringmann, G.; François, G.; Boyd, M. R. Bioorg. Med. Chem. Lett. 1998, 8, 1729− 1734. (b) Bringmann, G.; Wohlfarth, M.; Rischer, H.; Heubes, M.; Saeb, W.; Diem, S.; Herderich, M.; Schlauer, J. Anal. Chem. 2001, 73, 2571−2577. (13) (a) Xu, M.; Bruhn, T.; Hertlein, B.; Brun, R.; Stich, A.; Wu, J.; Bringmann, G. Chem. - Eur. J. 2010, 16, 4206−4216. (b) Bringmann, G.; Lombe, B. K.; Steinert, C.; Ndjoko Ioset, K.; Brun, R.; Turini, F.; Heubl, G.; Mudogo, V. Org. Lett. 2013, 15, 2590−2593. (14) Lombe, B. K.; Bruhn, T.; Feineis, D.; Mudogo, V.; Brun, R.; Bringmann, G. Org. Lett. 2017, 19, 1342−1345. (15) (a) Cheek, M. Kew Bull. 2000, 55, 871−882. (b) Taylor, C. M.; Gereau, R. E.; Walters, G. M. Ann. Missouri Bot. Gard. 2005, 92, 360− 399. (16) Airy Shaw, H. K. Kew Bull. 1951, 6, 327−347. (17) Heubl, G.; Turini, F.; Mudogo, V.; Kajahn, I.; Bringmann, G. Plant Ecol. Evol. 2010, 143, 63−69. (18) Bringmann, G.; God, R.; Schäffer, M. Phytochemistry 1996, 43, 1393−1403. (19) Bringmann, G.; Saeb, W.; God, R.; Schäffer, M.; François, G.; Peters, K.; Peters, E. M.; Proksch, P.; Hostettmann, K.; Aké Assi, L. Phytochemistry 1998, 49, 1667−1673. (20) Nwaka, S.; Ramirez, B.; Brun, R.; Maes, L.; Douglas, F.; Ridley, R. PLoS Neglected Trop. Dis. 2009, 3, e440. (21) (a) Bringmann, G.; Dreyer, M.; Faber, J. H.; Dalsgaard, P. W.; Stærk, D.; Jaroszweski, J. W.; Ndangalasi, H.; Mbago, F.; Brun, R.; Reichert, M.; Maksimenka, K.; Christensen, S. B. J. Nat. Prod. 2003, 66, 1159−1165. (b) Bringmann, G.; Hamm, A.; Günther, C.; Michel, M.; Brun, R.; Mudogo, V. J. Nat. Prod. 2000, 63, 1465−1470. (c) Izumi, E.; Ueda-Nakamura, T.; Dias-Filho, B. P.; Veiga Júnior, V. F.; Nakamura, C. V. Nat. Prod. Rep. 2011, 28, 809−823. (d) Salem, M. M.; Werbovetz, K. A. Curr. Med. Chem. 2006, 13, 2571−2598. (22) (a) Bringmann, G. Bull. Soc. Chim. Belg. 1996, 105, 601−613. (b) Bringmann, G.; Tasler, S. Tetrahedron 2001, 57, 331−34310.1016/ S0040-4020(00)00940-6. (c) Hemberger, Y.; Zhang, G.; Brun, R.; Kaiser, M.; Bringmann, G. Chem. - Eur. J. 2015, 21, 14507−14518. (23) Awale, S.; Lu, J.; Kalauni, S. K.; Kurashima, Y.; Tezuka, Y.; Kadota, S.; Esumi, H. Cancer Res. 2006, 66, 1751−1757. (24) Izuishi, K.; Kato, K.; Ogura, T.; Kinoshita, T.; Esumi, H. Cancer Res. 2000, 60, 6201−6207. (25) Housman, G.; Byler, S.; Heerboth, S.; Lapinska, K.; Longacre, M.; Synder, N.; Sarkar, S. Cancers 2014, 6, 1769−1792. (26) Feig, C.; Gopinathan, A.; Neesse, A.; Chan, D. S.; Cook, N.; Tuveson, D. A. Clin. Cancer Res. 2012, 18, 4266−4276. (27) Conroy, T.; Desseigne, F.; Ychou, M.; Bouché, O.; Guimbaud, R.; Bécouarn, Y.; Adenis, A.; Raoul, J.-L.; Gourgou-Bourgade, S.; de la Fouchardière, C.; Bennouna, J.; Bachet, J.-B.; Khemissa-Akouz, F.; Péré-Vergé, D.; Delbaldo, C.; Assenat, E.; Chauffert, B.; Michel, P.;

AUTHOR INFORMATION

Corresponding Authors

*E-mail (S. Awale): [email protected]. Tel: +81-76434-7640. Fax: +81-76-434-7640. *E-mail (G. Bringmann): [email protected]. Tel: +49-931-318-5323. Fax: +49-931-318-4755. ORCID

Torsten Bruhn: 0000-0002-9604-1004 Suresh Awale: 0000-0002-5299-193X Gerhard Bringmann: 0000-0002-3583-5935 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (SFB 630 “Agents against Infectious Diseases”, project A2). The biological evaluation against cancer cell lines was supported by the Japanese Society for the Promotion of Science (JSPS), Japan, Kakenhi (16K08319) to S.A. We thank Dr. M. Büchner, Mr. F. Dadrich, and Mr. J. Wendrich for recording the mass spectr, and Dr. M. Grüne, Mrs. E. Ruckdeschel, and Mrs. P. Altenberger for performing the NMR measurements. Further thanks are due to Mrs. M. Michel for the degradation experiments.

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DEDICATION We dedicate this paper to Professor Günther Heubl, Systematic Botany and Mycology, Faculty of Biology, Department 1, Ludwig-Maximilians-Universität München (LMU Munich), on the occasion of his 65th birthday.

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REFERENCES

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