Dioncophyllines C2, D2, and F and Related ... - ACS Publications

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Dioncophyllines C2, D2, and F and Related Naphthylisoquinoline Alkaloids from the Congolese Liana Ancistrocladus ileboensis with Potent Activities against Plasmodium falciparum and against Multiple Myeloma and Leukemia Cell Lines Jun Li,†,‡,§ Raina Seupel,†,§ Doris Feineis,† Virima Mudogo,⊥ Marcel Kaiser,∥,¶ Reto Brun,∥,¶ Daniela Brünnert,○ Manik Chatterjee,# Ean-Jeong Seo,□ Thomas Efferth,□ 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 ⊥ Faculté des Sciences, Université de Kinshasa, B.P. 202, Kinshasa XI, Democratic Republic of the Congo ∥ Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland ¶ University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland ○ Comprehensive Cancer Center and #Department of Internal Medicine II, Translational Oncology, University Hospital of Würzburg, Versbacher Straße 5, D-97078 Würzburg, Germany □ Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Biology, University of Mainz, Staudinger Weg 5, D-55128 Mainz, Germany ‡

S Supporting Information *

ABSTRACT: Dioncophylline F (1), the first 5,8′-coupled dioncophyllaceous alkaloid (i.e., lacking an oxygen function at C-6 and possessing an R-configuration at C-3), was isolated from the recently described Congolese liana Ancistrocladus ileboensis. Two further, likewise Dioncophyllaceae-type, alkaloids, the dioncophyllines C2 (2) and D2 (3), were identified, along with the Ancistrocladaceae-type compound ancistrocladisine B (4), which is oxygenated at C-6 and Sconfigured at C-3. The structures of the new compounds were determined by spectroscopic, chemical, and chiroptical methods. The stereostructure of 1 was further confirmed by total synthesis. As a consequence of the lack of a methyl group ortho to their biaryl axes, both dioncophylline F (1) and the 7,8′-coupled dioncophylline D2 (3) occur as pairs of configurationally semistable and, thus, slowly interconverting atropo-diastereomers, whereas dioncophylline C2 (2), with its 5,1′linkage, is configurationally stable at the axis. Eight further known naphthylisoquinolines were isolated from A. ileboensis, among them dioncophylline A (P-10), its 4′-O-demethyl analogue P-11, and 5′-O-methyldioncophylline D (7), which were found to display strong cytotoxic activities against multiple myeloma INA-6 cells (P-10 even stronger than the standard drug melphalan) and against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ ADR5000. Moreover, the dioncophyllines 1, 3, and 7 showed highand specificactivities against the malaria parasite Plasmodium falciparum.

T

he Ancistrocladaceae1 from palaeotropical Africa and Asia and the closely related Dioncophyllaceae,2 endemic to coastal West Africa, are phytochemically characterized by the presence of structurally and biosynthetically unique naphthylisoquinoline alkaloids.3−6 Due to their occurrence exclusively in these two small plant families, they are excellent phylogenetic markers.3−5 More than 180 representatives of thesein most casesaxially chiral biaryls have so far been identified.3−6 Despite the broad structural variety of naphthylisoquinoline © 2017 American Chemical Society and American Society of Pharmacognosy

alkaloids, the plants obviously follow strict synthetic principles, which are a useful criterion for the taxonomic classification of different plant species.3−5 Thus, the West African Dioncophyllaceae solely produce alkaloids with R-configuration at C-3, always lacking an oxygen function at C-6 (so-called “Dioncophyllaceae-type”).5 From Southeast Asian and East Received: October 20, 2016 Published: January 25, 2017 443

DOI: 10.1021/acs.jnatprod.6b00967 J. Nat. Prod. 2017, 80, 443−458

Journal of Natural Products

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the first 5,8′-linked dioncophyllaceous naphthylisoquinoline found in nature. This novel combination made this minor alkaloid an attractive synthetic target. Herein, we describe the total synthesis of 1 by Pd-catalyzed Suzuki−Miyaura crosscoupling of its two molecular portions. Moreover, five well-known dioncophyllaceaeous naphthylisoquinolines were isolated from the root bark extracts, viz., dioncophylline A (P-10)3,5,11,12 and its 4′-O-demethyl analogue P-11,3,13 together with the respective atropo-diastereomers M103 and M-11,3 and 5′-O-methyldioncophylline D (7).14 Furthermore, three Ancistrocladaceae-type compounds, ancistrocladine (P-6),3,15 ancistrocladisine A (8),7 and ancistrobertsonine D (9)16 (Figure 2), and a further hybrid-type alkaloid,

African Ancistrocladaceae, by contrast, nearly exclusively 3Sconfigured and 6-oxygenated (so-called “Ancistrocladaceaetype”) naphthylisoquinolines have so far been isolated.3,4 Some of the West and Central African Ancistrocladus species additionally produce mixed Dioncophyllaceae/Ancistrocladaceae hybrid-type compounds (with R-configuration at C-3 and an oxygen function at C-6).3,4,6 In 2000, we discovered the new liana Ancistrocladus ileboensis.7,8 First phytochemical investigations on its root bark led to the discovery of five Ancistrocladaceae-type naphthylisoquinoline alkaloids, along with one hybrid-type compound.7 In this paper, we report the isolation and structural elucidation of 12 further naphthylisoquinolines from the root bark and leaves of A. ileboensis, including four new alkaloids (Figure 1) exhibiting four different coupling types (viz., 5,8′,

Figure 2. Known naphthylisoquinoline alkaloids discovered in related African Dioncophyllaceae and Ancistrocladaceae plants,3,11−15,17,25,27−29 now identified for the first time in A. ileboensis.

ancistrobrevine C (5)3,13,17 (Figure 1), were identified. Thus, A. ileboensis is one of the rare plants that simultaneously produce Ancistrocladaceae-, Dioncophyllaceae-, and hybridtype alkaloids. So far, only A. abbreviatus3,9,17 and A. barteri3,18 and a botanically yet unknown Ancistrocladus liana13 from the Central Congo Basin are known to contain representatives of all three of these subclasses of naphthylisoquinoline alkaloids. The close chemotaxonomic relationship of A. ileboensis to both the Ancistrocladaceae and the Dioncophyllaceae plants from West Africa, in particular to A. abbreviatus,3,9,17 A. barteri,3,18 and Triphyophyllum peltatum,3,5,11,12,14 will be discussed in this paper, as well as the special phylogenetic and chemotaxonomic position of A. ileboensis8,19 with respect to the other Ancistrocladus species from the Congo Basin.13,20−24 The new dioncophyllines 1−3 and the known,7,14,17 but so far not yet tested, alkaloids 5, 7, and 8 were evaluated for their antiprotozoal activities against the pathogens causing malaria tropica, leishmaniasis, Chagas’ disease, and African sleeping sickness. Furthermore, since little is known about the cytotoxic properties of naphthylisoquinoline alkaloids,25,26 the dioncophyllines 1, 3, 7, 8, P-10, P-11, and M-11 were also investigated for their activities against human multiple myeloma INA-6 cells and against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ ADR5000.

Figure 1. Naphthylisoquinoline alkaloids from A. ileboensis: the new dioncophyllaceous alkaloids dioncophylline F (1), dioncophylline C2 (2), and dioncophylline D2 (3) and the new ancistrocladaceous compound ancistrocladisine B (4) and ancistrobrevine C (5); the latter is a hybrid-type compound already known from A. abbreviatus.17 Note: For an easier comparison of naphthylisoquinoline alkaloids of different coupling types, a 2-methyl-4,5-dioxy substitution pattern is applied in the numbering of the naphthalene moiety, regardless of the coupling site.3−6,15

5,1′, 7,1′, and 7,8′). Dioncophylline F (1) and the dioncophyllines C2 (2) and D2 (3) belong to the subclass of Dioncophyllaceae-type compounds. The fourth metabolite, ancistrocladisine B (4), is an Ancistrocladaceae-type alkaloid. While 5,8′-coupled naphthylisoquinolines3,4,6 have so far exclusively been found in both the subclasses of Ancistrocladaceae- and hybrid-type alkaloids such as ancistrobrevine B3,9 and korupensamine A,3,10 respectively, dioncophylline F (1) is 444



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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of the Two Atropo-Diastereomers of Dioncophylline F (1) in Methanol-d4 (δ in ppm) P-1 position

■

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

δH (J in Hz) 4.80, 3.32, 2.56, 2.29,

q (6.6) m dd (17.6, 12.3) dd (17.6, 4.8)

7.00, d (8.4) 6.86, d (8.4)

6.69, br s 6.80, br s

6.91, d (7.8) 7.15, d (7.8)

1.82, 1.25, 2.30, 3.93, 3.95,

d (6.6) d (6.6) s s s

M-1 δC, type

δH (J in Hz)

δC, type

52.4, CH 51.0, CH 33.9, CH2

4.75, 3.28, 2.42, 2.40,

52.4, CH 50.9, CH 33.3, CH2

132.9, 132.3, 114.6, 155.3, 121.3, 133.7, 118.5, 138.0, 109.9, 158.7, 158.2, 106.3, 129.6, 130.8, 137.4, 117.3, 19.3, 18.7, 22.0, 56.9, 56.8,

RESULTS AND DISCUSSION Isolation and Structural Elucidation of Naphthylisoquinoline Alkaloids. Root bark and leaves of A. ileboensis were collected in South-Central DR Congo, along the Kasai ̈ River near its junction with the Sankuru River. Air-dried 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 further purified by liquid/liquid partition with CH2Cl2 to separate the polar and nonpolar metabolites. After evaporation to dryness, the water extracts were dissolved in MeOH and then directly subjected to preparative HPLC, which permitted isolation of ancistrocladisine A (8). This alkaloid had been reported in the first phytochemical study on A. ileboensis.7 Moreover, seven further naphthylisoquinoline alkaloids (Figure 2) were identified, which had previously been isolated from other African and Asian Ancistrocladus species3,13,15,17,25,27 or were known from Triphyophyllum peltatum11,12,14 and related Dioncophyllaceae28,29 plants. Naphthylisoquinoline Alkaloids 5−11. The two major metabolites of A. ileboensis were readily identified as the wellknown 7,1′-coupled alkaloids dioncophylline A (P-10)3,5,11,12 and its 4′-O-demethyl analogue P-11.3,13 Both were isolated together with their known3 atropo-diastereomers, M-10 and M11. This is the first report on the identification of these two pairs of atropo-diastereomers in the same plant. Dioncophylline A (P-10) is a main constituent of the West African lianas T. peltatum11,12 and A. abbreviatus17 and was likewise found to dominate the alkaloid pattern of a botanically as yet undescribed Central Congolese Ancistrocladus species.13 Prior to this phytochemical study on A. ileboensis, 7-epi-dioncophylline A (M-10) had been known to occur only in A. barteri.3 In

C CH CH C C C CH C CH C C CH CH C C C CH3 CH3 CH3 CH3 CH3

q (6.6) m br s d (6.6)

7.06, d (8.4) 6.87, d (8.4)

6.74, br s 6.80, br s

6.91, d (7.8) 7.13, d (7.8)

1.85, 1.23, 2.34, 3.93, 3.95,

d (6.6) d (6.6) s s s

133.4, 131.8, 114.9, 155.4, 121.0, 134.0, 118.8, 138.0, 109.8, 158.8, 158.2, 106.4, 129.1, 131.5, 137.4, 117.1, 19.5, 18.6, 22.1, 56.9, 56.8,

C CH CH C C C CH C CH C C CH CH C C C CH3 CH3 CH3 CH3 CH3

that West African species it was apparently produced in an atropisomerically pure form, i.e., without any co-occurring dioncophylline A (P-10).3 Previous phytochemical investigations had revealed 4′-O-demethyldioncophylline A (P-11) to be a major constituent of A. abbreviatus, accompanied by its atropisomer M-11.3 These findings were thus similar to those obtained here for A. ileboensis. Ancistrocladus barteri, by contrast, had been found to produce 4′-O-demethyl-7-epi-dioncophylline A (M-11) in a stereochemically homogeneous form, albeit as a minor alkaloid.3 5′-O-Methyldioncophylline D (7), which had recently been discovered in callus cultures of T. peltatum (but not in the plant itself) as the first example of a 7,8′-coupled dioncophyllaceous naphthylisoquinoline alkaloid,14 was now identified in the roots of A. ileboensis, too, and, thus, for the first time in an intact plant taken from its natural habitat. Furthermore, two known Ancistrocladaceae-type alkaloids were isolated from A. ileboensis, viz., the 5,1′-coupled ancistrocladine (P-6), which was initially discovered in A. heyneanus from India15 and also found to widely occur in Asian and African Ancistrocladus species,3,6,13,25,27 and the 7,1′linked ancistrobertsonine D (9), earlier identified in A. robertsoniorum from Kenya.16 Ancistrobrevine C (5) (Figure 1), previously detected in A. abbreviatus17 and in a botanically yet undescribed Congolese Ancistrocladus species,13 is the second mixed Ancistrocladaceae/Dioncophyllaceae-type alkaloid isolated from the root bark of A. ileboensis. In addition, four new naphthylisoquinoline alkaloids were discovered in CH2Cl2/MeOH extracts obtained from the root bark and leaves of A. ileboensis. Dioncophylline F (1). The first new compound isolated from the root bark extracts exhibited the typical NMR appearance of a naphthyl-1,3-dimethyltetrahydroisoquinoline 445



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(Table 1), yet showing two sets of signals in a ratio of ca. 1:1.2, with identical coupling patterns and constants, except for those of H-4, thus indicating the presence of two very similar compounds. The two metabolites both corresponded to the molecular formula C24H27NO3, as deduced from HRESIMS. The lack of one oxygen function, as compared to an Ancistrocladaceae-type compound, suggested the new compound to be a Dioncophyllaceae-type naphthylisoquinoline alkaloid. This assumption was corroborated by the appearance of a spin system of six aromatic protons with two singlets and four doublets, indicating the presence of two protons (δ 6.69 and 6.80) meta to each other, and the occurrence of two pairs of adjacent aromatic protons, one resonating at δ 7.00 and 6.86 and one at δ 6.91 and 7.15. The two methoxy groups at δ 3.93 and 3.95 showed NOESY interactions with H-6′ (δ 6.91) and H-3′ (δ 6.80), respectively, being located at C-5′ and C-4′ in the naphthalene half. The third oxygen function thus had to be a hydroxy group at C-8. The “normal”, not high-field-shifted signal of Me-2′ (δ 2.30) excluded the biaryl axis from being located at C-1′ or C-3′, thus leaving only C-8′ or C-6′ as the coupling position. Of these, the latter was ruled out due to NOESY interactions between H-6′ (doublet at δ 6.91) and the normal-shifted methoxy group at C5′ (δ 3.95), thus clearly revealing that the naphthalene portion was coupled via C-8′. This assignment was supported by an HMBC interaction between H-1′ and the quaternary carbon atom C-8′. The attribution of H-1′ (δ 6.69) and H-3′ (δ 6.80), in turn, was deduced from the NOESY correlation sequence {H-1′ ↔ Me-2′ ↔ H-3′ ↔ OMe-4′} and from HMBC interactions, each, from H-1′ and H-3′ to Me-2′ (Figure 3A). With the spin system of the two remaining contiguous protons, H-6 and H-7, giving rise to two doublets (δ 6.86 and 7.00) and from HMBC interactions from H-6 to C-8′ and from H-7 to C5, the position of the biaryl axis in the tetrahydroisoquinoline moiety was deduced to be C-5. In conclusion, the new naphthylisoquinoline alkaloid 1 had to be 5,8′-coupled, which was also in agreement with an HMBC interaction between H-7′ and C-5. The lack of an ortho-methyl group next to the biaryl axis gave rise to a quite low rotational barrier, resulting in a slow interconversion of the two respective atropo-diastereomers at room temperature. The NMR data (Table 1) of the M- and the P-atropisomers of the alkaloid were assigned on the basis of their 2D NMR spectra (see Supporting Information). The absolute configuration at C-3 in the tetrahydroisoquinoline moiety was determined by ruthenium-mediated oxidative degradation.30 The formation of R-3-aminobutyric acid proved the alkaloid to be R-configured at C-3. The relative configuration at C-1 versus C-3 was evidenced to be cis from a NOESY interaction between H-1 (δ 4.80) and H-3 (δ 3.32) (Figure 3B), which, given the above-determined absolute Rconfiguration at C-3, indicated C-1 to be S-configured. The absolute configuration at the biaryl axis was deduced from NOESY interactions both between H-4eq and H-1′ and between H-4ax and H-7′, clearly assigning the minor atropodiastereomer of the alkaloid to be P-configured (Figure 3B). For the M-configured atropisomer, long-range interactions were detected, too (Figure 3B), yet less unambiguously for the assignment of the axial configuration due to the insufficient resolution of the signals for the two diastereotopic protons at C-4 (Table 1). The alkaloid thus had the structure 1, occurring in its two slowly interconverting atropisomeric forms, M-1 and P-1. Being the very first 5,8′-coupled Dioncophyllaceae-type naphthylisoquinoline alkaloid, it was henceforth named

Figure 3. Selected NMR data of dioncophylline F (1): (A) NOESY (double red arrows) and HMBC (single blue arrows) interactions relevant for the constitution of 1 and (B) NOESY correlations indicative of the relative configurations at the biaryl axes and the stereogenic centers in P-1 and M-1; the correlations of the Mconfigured atropo-diastereomer are given in dotted lines, due to the insufficiently resolved signals for the two protons at C-4.

dioncophylline F, in continuation of the series of the other dioncophyllaceous alkaloids,3−6 possessing 7,1′- (type A), 7,6′(type B), 5,1′- (type C), 7,8′- (type D), or 7,3′-linked (type E) biaryl axes. In contrast to known relatedyet Ancistrocladaceae-typenaphthylisoquinoline alkaloids, it is, due to the lack of an oxygen function at C-6, the first such alkaloid with a configurationally semistable biaryl axis. Total Synthesis of Dioncophylline F (1). Since this novel compound 1 occurred in A. ileboensis only as a minor alkaloid, a total synthesis route was developed, not only for a further structural confirmation but also, in particular, for the evaluation of its biological properties. Moreover, the presence of a novel, unprecedented coupling type for Dioncophyllaceae-type alkaloids made the total synthesis of dioncophylline F (1) an important objective. Numerous naphthylisoquinoline alkaloids, even with a high degree of steric hindrance at the coupling sites, have already been efficiently synthesized, e.g., by applying the “lactone concept”,31 a strategy developed in our group for the regio- and stereoselective construction of biaryl axes. A prominent example is the atropo-divergent total synthesis of the 5,8′coupled korupensamines A and B.32 For a first total synthesis access to dioncophylline F (1), with its low steric load next to the axis and its configurational instability, however, an atroposelective construction of the biaryl axis would not have made sense. Thus, a direct, intermolecular coupling technique seemed favorable, similar to the route previously applied by Hoye et al.33 for the non-atroposelective total synthesis of 5,8′446



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Scheme 1. Total Synthesis of Dioncophylline F (1) by Pd-Catalyzed Suzuki−Miyaura Cross-Coupling of 13 and 17a

a Reagents and conditions: (a) Pd(PPh3)4, KOAc, (Bpin)2, DMF, 150 °C, 12 h, 77%; (b) NaBH4, 0 °C, MeOH, 2 h, 92%; (c) BBr3, 0 °C → rt, CH2Cl2, 12 h, > 99%; (d) BnBr, Cs2CO3, DMF, rt, 12 h, 84%; (e) Pd2(dba)3, S-Phos, K3PO4, toluene, 100 °C, 12 h, 76%; (f) H2, Pd/C (10%), CH2Cl2/MeOH, 2:1, rt, 2 h, 84%.

linked naphthylisoquinoline alkaloids such as ancistrobrevine B and the korupensamines. The key step in the synthesis of 1 (Scheme 1) was the Suzuki−Miyaura cross-coupling of the two building blocks 13 and 17, with the coupling positions activated by bromine in the tetrahydroisoquinoline moiety and boronic acid ester in the naphthalene part. The naphthalene precursor 13 was obtained from the known 8-bromonaphthalene 12,33 which was converted to the respective boronic acid ester by Miyaura borylation.34 The synthesis of the isoquinoline building block 17 started from the known35 5-bromo-8-methoxy-dihydroisoquinoline 14, which was prepared via chiral aziridines as described by Hoye et al.33 and further improved by our group.36 Reduction of 14 with NaBH4 selectively gave the respective cis-configured tetrahydroisoquinoline 15 (d.s. > 95%).37 This intermediate, after 8-O-demethylation to give 16 and O,N-dibenzylation, afforded the specifically protected building block 17. Pd-catalyzed cross-coupling of the two molecular portions, 13 and 17, provided the biaryl 18 in a good yield of 76%. Cleavage of the benzyl protecting groups afforded the target molecule 1 as a mixture of its two interconverting atropo-diastereomers, P-1 and M-1, in a ratio of ca. 1:1.1, thusas for the natural alkaloid 1 isolated from A. ileboensis (see above)revealing a slight preference for the Mdiastereomer. The synthesized material 1 proved to be identical in all its chromatographic, physical, and spectroscopic data with the sample of dioncophylline F from A. ileboensis. Online-Chiroptical Investigations on Dioncophylline F (1). HPLC resolution of dioncophylline F (1) on an XSelect high-strength silica PFP column in hyphenation with electronic circular dichroism (ECD) spectroscopy38 gave rise to two baseline-separated peaks, which exhibited two nearly mirrorimaged ECD spectra, thus corroborating 1 to indeed represent a mixture of two atropo-diastereomers (Figure 4). Their absolute axial configurations were deduced by comparison of the ECD curves (recorded in the stopped-flow mode) with the ECD spectrum of the related 5,1′-linked dioncophylline C (19), a well-known,39 P-configured constituent of the roots of T. peltatum. The structure of this naphthylisoquinoline differs from that of the new 5,8′-coupled dioncophylline F (1) only by the position of the methyl group in the naphthalene moiety and by a hydroxy group instead of a methoxy function at C-5′, but the influence of these two substituents on the ECD behavior

Figure 4. Assignment of the absolute axial configuration of the two configurationally semistable atropo-diastereomers of dioncophylline F, P-1, and M-1, by LC-ECD coupling and by comparison of the LCECD spectra of peak A (left) and peak B (right) with the ECD curve of dioncophylline C (19; for its structure, see Figure 5). Onlinechiroptical analysis of the synthesized material of 1 afforded ECD spectra of the two atropo-diastereomers P-1 and M-1 (data not shown) that were essentially identical with those obtained from LCECD investigations on 1 isolated from A. ileboensis.

can be assumed as small compared with that of the large naphthalene chromophore. The ECD spectrum of the slower eluting isomer (= peak B) showed a curve nearly perfectly opposite that of the known P-configured naphthylisoquinoline dioncophylline C (19; for the structure, see Figure 5), thus giving rise to assignment of the slower eluting isomer as M-1, while the faster one (= peak A), which was in quite good agreement with that of 19, was assigned to be P-1. The ECD spectra of P-1 and M-1, however, showed different, not fully opposite curve shapes. In most cases, the ECD curves of atropo-diastereomers are virtually opposite to each other,3,4,24,25,38,40 because the ECD behavior is largely dominated by the stronger biaryl chromophore and, thus, by the axial configuration. On the other hand, P-1 and M-1 are of 447



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Figure 5. Elucidation of the full absolute stereostructure of dioncophylline C2 (2): (A) NOESY (double red arrows) and HMBC (single blue arrows) interactions indicative of the constitution; (B) configuration at the biaryl axis relative to the stereogenic centers through NOESY interactions and (C) absolute axial configuration of 2 assigned by comparison of its ECD spectrum with that of dioncophylline C (19).

Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Data of Dioncophylline C2 (2) and Ancistrocladisine B (4) in Methanol-d4 (δ in ppm) 2 position 1 3 4ax 4eq 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 1-CH3 3-CH3 6-OCH3 2′-CH3 4′-OCH3 5′-OCH3

4

δH (J in Hz) 4.79, 3.30, 2.38, 2.20,

q (6.6) m dd (17.4, 12.0) dd (17.4, 3.0)

6.90, br s 6.90, br s

6.90, br s

6.87, dd (7.8, 1.2) 7.20, dd (8.4, 7.8) 6.69, dd (8.4, 1.2)

1.83, d (6.6) 1.23, d (6.0) 2.14, s 3.96, s 3.93, s

δC, type 52.4, CH 50.9, CH 33.1, CH2 131.6, 132.1, 115.3, 155.3, 121.6, 133.8, 129.5, 136.6, 110.1, 157.8, 158.8, 107.1, 127.8, 119.1, 137.9, 117.6, 19.4, 18.7,

C CH CH C C C C C CH C C CH CH CH C C CH3 CH3

20.6, CH3 56.8, CH3 56.9, CH3

δH (J in Hz) 4.68, 3.52, 3.03, 3.03, 6.53,

q (6.6) m overlapped overlapped s

6.91, s

6.87, d (7.8) 7.19, dd (7.8, 7.8) 6.87, d (7.8)

1.75, 1.53, 3.60, 2.08, 3.97, 3.93,

d (6.6) d (6.0 s s s s

δC, type 52.4, CH 51.2, CH 35.6, CH2 103.8, 159.1, 115.5, 153.8, 114.4, 134.8, 121.7, 138.6, 110.4, 158.3, 158.7, 107.1, 127.6, 119.0, 138.3, 118.0, 19.7, 18.8, 56.1, 20.5, 56.8, 57.0,

CH C C C C C C C CH C C CH CH CH C C CH3 CH3 CH3 CH3 CH3 CH3

Dioncophylline C2 (2). The second new alkaloid exhibited the same molecular weight as dioncophylline F (1) (m/z 377), and its HRESIMS gave a molecular formula of C24H27NO3, identical to that of 1, again revealing the lack of an oxygen, as compared to an Ancistrocladaceae-type naphthylisoquinoline alkaloid. As for 1, two of the three oxygen functions were identified as methoxy groups from their three-proton singlets at δ 3.96 and 3.93. They were deduced to be located at C-4′ and C-5′, respectively, due to their characteristic 1H NMR shifts (Table 2) and the NOESY correlations of OMe-4′ to H-3′ and OMe-5′ to H-6′. Thus, the third oxygen function had to be a hydroxy group at C-8 (Figure 5A). The 1H NMR data of the new alkaloid (Table 2) showed six aromatic protons, in contrast to 1, with a spin system of three contiguous protons in the naphthalene portion, viz., H-6′ (δ 6.87), H-7′ (pseudotriplet at δ 7.20), and H-8′ (δ 6.69), which

course not enantiomers, but diastereomers, with stereogenic centers that may influence the dihedral angles at the axis, thus leading to ECD spectra that may not be opposite to each other,41 so that the non-mirror-image-like ECD behavior of P-1 and M-1 was not totally surprising. All measured ECD curves were fully reproducible over several experiments and entirely identical for the natural and synthesized material. Thus, the structure of this new 5,8′-coupled dioncophyllaceous naphthylisoquinoline 1, including the absolute configurations of its two slowly interconverting atropo-diastereomers, P-1 and M-1, was comprehensively confirmed (1) due to the chromatographic and spectroscopic identity of 1 from natural and synthetic origin, (2) by the online-chiroptical investigations on 1 (Figure 4), through HPLC-ECD coupling, and (3) from the total synthesis as illustrated in Scheme 1. 448



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Figure 6. Elucidation of the full absolute stereostructure of ancistrocladisine B (4): (A) NOESY (double red arrows) and HMBC (single blue arrows) interactions indicative of the constitution; (B) configuration at the biaryl axis relative to the stereogenic centers through NOESY interactions; (C) absolute axial configuration assigned by comparison of the ECD spectrum of 4 with that of ancistrocladisine A (8, for its structure, see Figure 2).

was further evidenced by the NOESY sequence in the series {OMe-5′ ↔ H-6′ ↔ H-7′ ↔ H-8′}. From an HMBC interaction between H-8′ and C-1′, showing C-1′ (129.5 ppm) to be quaternary, C-1′ was deduced to be the axis-bearing carbon atom. This was further corroborated by 3J HMBC interactions of H-8′ and H-3′ with the quaternary C-10′ (117.6 ppm). The coupling position in the isoquinoline moiety was determined to be C-5, due to the presence of two contiguous protons, giving rise to two doublets (δ 6.87 and 7.00), monitored in acetone-d6 (see Supporting Information), and, due to HMBC cross-peaks to C-5 (131.6 ppm) observed for both, the signals of Heq-4 and H-7. In conclusion, the new Dioncophyllaceae-type naphthylisoquinoline alkaloid was established to be 5,1′-coupled, and it had to possess the constitution 2 as shown in Figure 5A. The relative configuration of the 1,3-dimethyl functionality was assigned as cis due to a NOESY correlation between H-1 (δ 4.79) and H-3 (δ 3.30) (Figure 5B). The ruthenium-catalyzed oxidative degradation30 determined the absolute configuration at C-3 as R by formation of R-3-aminobutyric acid, which, in combination with the relative cis-configuration of the tetrahydroisoquinoline portion, established the stereocenter at C-1 to be S-configured. A NOESY interaction between H-4eq and H-8′ revealed that these two spin systems were cofacial, thus permitting assignment of the axis to be P-configured as outlined in Figure 5B. This result was corroborated by the similarity of the ECD spectrum of 2 (Figure 5C) with that of the structurally closely related P-configured alkaloid dioncophylline C (19).39 The new compound thus possessed the full stereostructure 2 and was, hence, the 1-epi-5′-O-methyl analogue of 19; it was named dioncophylline C2. Besides dioncophylline C (19) itself, the new naphthylisoquinoline 2 is the only other example of a “C-type”, i.e., 5,1′-coupled Dioncophyllaceae-type, alkaloid known to date. Ancistrocladisine B (4). The third new compound isolated from the root bark of A. ileboensis had a molecular formula of C25H29NO4, as evidenced by HRESIMS. The 1H NMR data (Table 2) exhibited the typical appearance of an Ancistrocladaceae-type naphthyl-1,3-dimethyltetrahydroisoquinoline, showing the presence of five aromatic protons (two singlets, two doublets, and one triplet) and three O-methyl groups at δ 3.60, 3.93, and 3.97 (three protons each) (Figure 6A). The methoxy group resonating at δ 3.93 was located at C-5′ in the naphthalene portion, because of a NOESY correlation to H-6′ (δ 6.87) and an HMBC interaction with C-5′. The position of the second methoxy function was assigned to be at C-4′, due to a NOESY interaction between H-3′ (δ 6.91) and OMe-4′ (δ

3.97). The third methoxy function (δ 3.60) was found to be at C-6 in the tetrahydroisoquinoline portion, as corroborated by a NOESY interaction between H-5 (δ 6.53) and OMe-6 (δ 3.60). In the isoquinoline moiety, the NOESY correlation sequence {OMe-6 ↔ H-5 ↔ H-4eq} revealed C-7 as the coupling position. With a spin system of three contiguous aromatic protons, giving rise to two doublets (δ 6.87 and 6.87) and one pseudotriplet (δ 7.19), and from the NOESY correlation sequence {OMe-5′ ↔ H-6′ ↔ H-7′ ↔ H-8′}, which excluded the naphthalene moiety from being connected to the isoquinoline part via its “methyl-free” ring, the position of the biaryl axis had to be at C-1′. This assumption was in agreement with the HMBC interactions from H-8′ (δ 6.87) to C-1′ (δ 121.7) and from Me-2′ (δ 2.08) to C-1′, thus confirming C-1′ to be quaternary and hence the axis-bearing carbon. In conclusion, the new compound was a 7,1′-coupled naphthylisoquinoline with the constitution shown in Figure 6A, with a free hydroxy group at C-8 and three methoxy functions at C-6, C-4′, and C-5′. From a NOESY interaction between H-1 (δ 4.68) and H-3 (δ 3.52) (Figure 6B), the relative configuration of the two stereogenic centers at C-1 and C-3 was deduced to be cis. The ruthenium-catalyzed oxidative degradation30 procedure afforded S-3-aminobutyric acid, thus establishing the absolute configuration at C-3 to be S, which, given the relative cisconfiguration, showed that the absolute configuration at C-1 had to be R. On the basis of this absolute 1R,3S-stereoarray in the tetrahydroisoquinoline part, the configuration at the axis was determined to be P due to a long-range NOESY cross-peak detected between H-8′ in the naphthalene part and Me-1 in the isoquinoline portion (Figure 6B), revealing that these two spin systems were cofacial. This stereochemical assignment was further confirmed by the nearly mirror-image-like ECD spectrum of the new alkaloid compared to that of ancistrocladisine A (8) (Figure 6C). As mentioned above, this structurally closely related alkaloid 8 had previously been identified in the root bark of A. ileboensis.7 The isolated compound had nearly the same constitution as 8, but possessed a hydroxy group at C-8 (instead of the methoxy function in 8); at both of the stereocenters in the isoquinoline portion, the isolated alkaloid and 8 showed the same configurations (1R, 3S), but had opposite axial configurations (for formal reasons, however, 8 and the isolated alkaloid have the same P-descriptor according to the Cahn−Ingold−Prelog denotion). The new alkaloid thus possessed the structure 4. Because of its close structural similarity to ancistrocladisine A (8), it was named ancistrocladisine B. 449



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Table 3. 1H (600 MHz) and 13C (150 MHz) NMR Data of the Two Atropo-Diastereomers of Dioncophylline D2 (3) in Methanol-d4 (δ in ppm) P-3

M-3

no.

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

1 3 4ax 4eq 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 1-CH3 3-CH3 N-CH3 2′-CH3 4′-OCH3 5′-OCH3

4.86, m 4.18, m 3.01, m 3.19, m 6.87, d (7.8) 7.08/7.09, d (7.8)

60.3, CH 50.7, CH 30.4, CH2

4.80, m 4.20, m 3.01, m 3.19, m 6.87, d (7.8) 7.08/7.09, d (7.8)

60.3, CH 50.7, CH 30.4, CH2

6.83, s 6.80, s

6.93, d (8.0) 7.24, d (8.0)

1.75, 1.54, 2.89, 2.31, 3.92, 3.95,

d (6.8) d (6.8) s s s s

121.0, 132.8, 128.0, 152.8, 120.1, 131.5, 118.8, 137.8, 110.0, 158.7, 158.8, 106.6, 130.2, 127.6, 137.3, 117.5, 18.7, 16.8, 34.3, 22.0, 57.0, 56.8,

CH CH C C C C CH C CH C C CH CH C C C CH3 CH3 CH3 CH3 CH3 CH3

6.79, s 6.80, s

6.93, d (8.0) 7.23, d (8.0)

1.79, 1.54, 2.84, 2.31, 3.92, 3.95,

d (6.8) d (6.8) s s s s

121.0, 132.8, 128.0, 152.8, 121.0, 131.5, 118.8, 137.8, 110.0, 158.7, 158.8, 106.6, 130.2, 127.6, 137.3, 117.5, 18.6, 16.8, 34.3, 22.0, 57.0, 56.8,

CH CH C C C C CH C CH C C CH CH C C C CH3 CH3 CH3 CH3 CH3 CH3

7,6′-coupling site is significantly higher (ca. 8.4−8.6 Hz),42 thus again confirming the isolated alkaloid to be 7,8′-coupled. A relative trans-configuration between the two stereocenters at C-1 and C-3 was evident from a NOESY interaction of Me-1 (δ 1.75) with the pseudoaxial proton at C-3 (δ 4.18) (Figure 7B). The oxidative degradation30 of the new metabolite, affording R-3-aminobutyric acid, established the absolute configuration at C-3 in the tetrahydroisoquinoline portion as 3R. Thus, given the relative trans-configuration assigned above, the isolated compound was R-configured at C-1, too. Owing to the known configuration at C-1, the assignment of the absolute configuration at the biaryl axis as M for one of the two atropodiastereomers was possible by the NOESY correlation between H-1′ (δ 6.79) and Me-1 (δ 1.79) (Figure 7B). In conclusion, the new compound had the structure 3 and, thus, was the Nmethyl analogue of the co-occurring 5′-O-methyldioncophylline D (7), which had previously been discovered in callus cultures of T. peltatum, likewise consisting of its two slowly interconverting atropo-diastereomers, P-7 and M-7.14 The new alkaloid 3 is one of the rare dioncophyllaceous alkaloids that exhibits a 7,8′-linkage. It was henceforth named dioncophylline D2. Another D-type coupled alkaloid, dioncophylleine D43 from the Malaysian liana Ancistrocladus benomensis (Ancistrocladaceae), has a fully unsaturated isoquinoline portion, so that it is not clear whether it was a true Dioncophyllaceae-type alkaloid (i.e., with R-configuration at C-3) prior to dehydrogenation. The structurally related, but synthetic dioncophylline D (an O-demethyl analogue of 7) had previously been found to be a mixture of two interconverting atropo-diastereomers, too.42 As in the case of that unnatural compound, and in contrast to the successful resolution of 5′-O-methyldioncophylline D (7), on

Dioncophylline D2 (3). The fourth new alkaloid of A. ileboensis was isolated from the leaves. According to HRESIMS, it had a molecular formula of C25H29NO3 and displayed NMR signals typical of an N-methylated Dioncophyllaceae-type naphthyltetrahydroisoquinoline alkaloid. Similar to dioncophylline F (1), the compound exhibited two sets of proton signals with identical coupling patterns and constants in the 1H NMR data (Table 3), thus, again suggesting the presence of atropo-diastereomers. The spectrum showed four methyl groups resonating at δ 1.54, 1.75, 2.31, and 2.89, the latter one being characteristic of an N-methyl group. This was confirmed by its NOESY correlations to H-1 (δ 4.86) and Me-3 (δ 1.54) and by its HMBC interactions with C-1 and C-3. From a NOESY correlation series {H-6 ↔ H-5 ↔ Heq-4 ↔ Hax-4 ↔ Me-3} and from HMBC interactions of H-5 (δ 6.87), both to C-7 (128.0 ppm) and C-4 (30.4 ppm), the coupling position of the biaryl axis was deduced to be C-7 in the tetrahydroisoquinoline moiety. The 1H NMR showed the presence of two methoxy groups (δ 3.92 and 3.95), which gave NOESY interactions with H-3′ (δ 6.80) and H-6′ (δ 6.93), thus being located at C-4′ and C-5′ in the naphthalene portion, respectively. The NOESY correlation sequence in the series {OMe-5′ ↔ H-6′ ↔ H-7′} excluded C-6′ for the position of the biaryl axis, leaving only C-8′ as the axis-bearing carbon atom. This assignment was corroborated by HMBC interactions between H-7′ (δ 7.24) and C-7 (128.0 ppm) and between H-6 (δ 7.08) and C-8′ (127.6 ppm) and by the coupling constants of H-7′ with H-6′, showing a J value of 8.0 Hz for these protons, which is typical of a 7,8′-coupling.14,42 Of note, the coupling constants of H-7′ with H-8′ in the case of a 450



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1−3 and three of the known, but so far not yet tested, constitutents of A. ileboensis, viz., 5′-O-methyldioncophylline D (7),14 ancistrocladisine A (8)7 (Figure 2), and ancistrobrevine C (5)3,13,17 (Figure 1), were investigated for their in vitro activities against protozoan parasites (Table 4). All of these compounds exhibited good to moderate activities against Plasmodium falciparum, the pathogen causing malaria tropica. The strongest effects against the NF54 strain of P. falciparum (sensitive to all known drugs) were displayed by dioncophylline F (1) and by the D-type (i.e., 7,8′-coupled) alkaloids 3 and 7, with half-maximum inhibitory concentrations (IC50) of 0.090 μM (for 1), 0.107 μM (for 3), and 0.228 μM (for 7). Dioncophylline D2 (3) showed only low cytotoxic activities toward L6 cells, which led to a good selectivity index of >580. Thus, 3 and 7 exhibited antiplasmodial activities comparable to that of the synthesized 7,8′-coupled dioncophylline D (IC50 = 0.16 μM),42 not yet found in nature. Their inhibitory potentials were significantly better (by a factor of 6 for 7 and 12 for 3) than that of the related dioncophylleine D (IC50 = 1.34 μM),43 which possesses an isoquinoline portion and is, given the configurational semistability and the absence of stereogenic centers, racemic. Interestingly, dioncophylline C2 (2) exhibited only moderate antiplasmodial activities (IC50 = 0.702 μM), whereas the parent compound, dioncophylline C (19)39 (Figure 5) from T. peltatum, is regarded as a promising antimalarial lead with interesting in vitro activities against P. falciparum (IC50 = 0.01 μM), even displaying a high curative potential in vivo.5,6,44 For all of the evaluated alkaloids, only moderate to weak (for the compounds 1 and 8) or no antitrypanosomal activities were obtained against the pathogens causing African sleeping sickness, Trypanosoma brucei rhodesiense, or against T. cruzi (Chagas’ disease). Furthermore, the naphthylisoquinolines outlined in Table 4 showed virtually no activities against Leishmania donovani, the pathogen of visceral leishmaniasis, showing the measured activities to be clearly structure-dependent and, thus, specific. Naphthylisoquinoline Alkaloids as Agents against Multiple Myeloma. In view of the cytotoxic properties of naphthylisoquinolines against tumor cell lines described earlier,25,26 some of the alkaloids of A. ileboensis (1, 3, 7, 8, P-10, P-11, and M-11) were tested against the human tumor cell line INA-6 derived from a patient suffering from multiple

Figure 7. Selected NMR data of dioncophylline D2 (3): (A) HMBC (single blue arrows) and NOESY (double red arrows) interactions relevant for the constitution of 3 and (B) for the two atropodiastereomers, M-3 and P-3, and long-range NOESY correlations indicative of their relative configurations at centers and (in the case of M-3) axis.

which we reported earlier,14 all attempts to resolve the atropodiastereomers of 3 failed under most different chromatographic conditions, even when using chiral HPLC phases. Antiprotozoal Activities. Some of the naphthylisoquinoline alkaloids previously discovered in other African or Asian Ancistrocladaceae or in West African Dioncophyllaceae have attracted attention as potential therapeutic agents for the treatment of severe, widespread tropical diseases such as Chagas’ disease, African sleeping sickness, leishmaniasis, and, in particular, malaria.3−6,44,45 For this reason, the new compounds

Table 4. Antiparasitic Activities of the Naphthylisoquinoline Alkaloids from A. ileboensis 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 1i 2 3 7 8 5

P. falciparumc 0.009 0.291 0.090 0.045 0.702 0.107 0.228 0.726 6.51

(NF54) (K1)d (NF54) (K1)

d

T. cruzi 2.317

e

T. brucei rhodesiense 0.0075

f

L. donovani 0.432

g

L6 cells (cytotoxicity) 0.017

14.62

0.585

106.7

14.52

44.51 55.04 26.31 12.40 104.4

4.835 13.69 8.08 0.313 22.70

228.4 136.3 >265 25.44 146.4

43.31 62.84 4.016 30.01 34.85

h

selectivity indexb 21.6 (NF54) 667 (K1) 161 (NF54) 323 (K1) 61.7 586 17.6 41.0 5.4

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 calculated as the ratio of the IC50 values for the L6 cells to the IC50 values of P. falciparum. cFor dioncophylline F (1), the activities against P. falciparum were measured on both of the strains NF54 and K1. dChloroquine. eBenznidazole. fMelarsoprol. gMiltefosine. hPodophyllotoxin. iThe material of 1 submitted to the antiprotozoal assays was obtained by total synthesis. 451



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A, which can be isolated from T. peltatum,47 and the synthesized N-acetyldioncophylline A,48 were also found to display strong anti-MM activities (Figure 8C and D), while lacking cytotoxic effects in nonmalignant PMBCs. Among the other alkaloids with different coupling patterns, the 7,8′-linked (i.e., D-type) naphthylisoquinoline 7 showed an anti-MM activity similar to that of 4′-O-demethyldioncophylline A (P11), whereas the 5,1′-coupled dioncophylline C (19)39 exhibited lower activities against MM cells, but exerted distinct cytotoxic effects toward normal blood cells. A further metabolite of A. ileboensis, which was found to exhibit high and specific anti-MM activities, was the 7,1′-coupled Ancistrocladaceae-type alkaloid ancistrocladisine A (8) (Table 4). In previous anti-MM tests on Ancistrocladaceae-type 5,1′-linked naphthylisoquinolines, ancistrocladine (P-6) (Figure 2), also identified in A. ileboensis, had been found to be inactive, while its atropo-diastereomer hamatine (M-6, not shown) had displayed only moderate effects against the MM cell lines (EC50 = 32 μM).25 In summary, the high anti-MM activities of dioncophyllaceous naphthylisoquinolines, in particular the excellent and specific activity of the parent compound, dioncophylline A (P10) (even 10-times better than melphalan), demonstrate the great potential of these biaryl natural products for their further development as anti-MM agents. Naphthylisoquinoline Alkaloids as Agents against Acute Lymphoblastic Leukemia Cells. In a preliminary screening assay, the compounds 1, 3, 7, 8, P-10, P-11, and M11 from A. ileboensis and dioncophylline C (19) from T. peltatum were likewise tested for their ability to inhibit the proliferation of the drug-sensitive leukemia CCRF-CEM cells. As presented in Figure 9, six of the compounds showed only weak to moderate effects on cell viability at a concentration of 10 μM. They caused a reduced growth of the CCRF-CEM tumor cells of, in most cases, less than 50%. Two of the alkaloids, however, 5′-O-methyldioncophylline D (7) and

myeloma (MM) (Table 5). Dioncophylline C2 (2) and ancistrocladisine B (4) were not evaluated due to lack of Table 5. EC50 Values (μM) of INA-6 Multiple Myeloma Cells and Peripheral Mononuclear Blood Cells (PMBCs) Treated with Naphthylisoquinolines or Melphalan compound b

melphalan 1 3 7 8 P-10 P-11 M-11 19 5′-O-demethyl-dioncophylline A N-acetyl-dioncophylline A

INA-6a 2.0 21.0 32.0 2.6 4.8 0.22 2.7 16.0 7.5 1.5 0.8

± ± ± ± ± ± ± ± ± ± ±

0.7 3.0 3.0 0.3 0.3 0.02 0.2 1.0 0.6 0.1 0.1

PMBCs 3.0 16 NRc 19 NRc NRc NRc 50 9.0 NRc NRc

± 0.5 ± 1.0 ± 1.0

± 15 ± 1.0

a

Multiple myeloma cells were treated with different concentrations of the respective naphthylisoquinoline or with melphalan. The viable fractions of the treated cells were determined by annexin V-FITC/PI staining using flow cytometry. bUsed as reference compound. cNR: not reached.

material. Although all of the compounds tested showed significant antitumoral activities, the 7,1′-coupled dioncophylline A (P-10) was the most potent one (Figure 8A). Its 4′-Odemethyl analogue P-11 (Figure 8B), one of the major metabolites of A. ileboensis, was likewise very active. Of note, the effective concentration ranges of P-10 and P-11 were similar to those of melphalan, a well-known DNA-alkylating agent used in a standard therapy against multiple myeloma.46 Normal peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, by contrast, were not affected by the dioncophyllines P-10a and P-11. Remarkably, two structurally closely related compounds, viz., 5′-O-demethyldioncophylline

Figure 8. Anti-MM effects of the highly active naphthylisoquinoline alkaloid (A) dioncophylline A (P-10) and the structurally related compounds (B) 4′-O-demethyldioncophylline (P-11) (from A. ileboensis), (C) 5′-O-demethyldioncophylline A (from T. peltatum), and (D) the synthesized analogue N-acetyldioncophylline A. INA-6 multiple myeloma cells were treated with different concentrations of P-10, P-11, and 5′-O-demethyl- or N-acetyldioncophylline A for 3 d. The viable fractions of the treated cells were determined by annexin V-FITC/PI staining using flow cytometry. Error bars indicate the range of values derived from three independent experiments. 452



Article

resistance to 7 (2.9-fold) and P-10 (2.1-fold) (Table 5), thus indicating that drug-sensitive and -resistant cells were inhibited with similar efficacies. These two candidate compounds, 7 and P-10, deserve further investigations in preclinical and clinical settings. Table 6. IC50 Values (μM) of Human Lymphoblastic CCRFCEM and Multidrug-Resistant CEM/ADR5000 Leukemia Cells Treated with 5′-O-Methyldioncophylline D (7) and Dioncophylline A (P-10) or Doxorubicin Figure 9. Growth percentage (%) of drug-sensitive lymphoblastic leukemia CCRF-CEM cells treated with the naphthylisoquinoline alkaloids 1, 3, 7, 8, P-10, P-11, M-11, and 19 and with doxorubicin as the reference drug at a concentration of 10 μM. Results of three independent experiments with six parallel measurements each are shown.

a

dioncophylline A (P-10), were found to strongly inhibit cell proliferation of CCRF-CEM cells in a concentration range similar to that of the standard antileukemic drug doxorubicin. Subsequent investigations to determine dose−response curves (Figure 10) revealed good to excellent IC50 values of 1.86 μM

compound

CCRF-CEM

CEM/ADR5000

degree of resistance

doxorubicina 7 P-10

0.017 ± 0.002 1.857 ± 0.140 0.243 ± 0.042

30.07 ± 11.81 5.31 ± 0.37 0.52 ± 0.008

1769 2.9 2.1

Used as reference compound.

The lymphoblastic leukemia cells were treated with different concentrations of the respective naphthylisoquinoline or with doxorubicin. Cell viability was assessed by the resazurin assay. Mean values and standard deviation of three independent experiments with six parallel measurements each are shown. The degrees of resistance were calculated by division of the IC50 values of the compounds for CEM/ADR5000 by the corresponding IC50 values for CCRF/CEM cells. In conclusion, the newly discovered Congolese liana A. ileboensis has become a rich source of structurally diverse naphthylisoquinoline alkaloids, among them the first 5,8′coupled dioncophyllaceous naphthylisoquinoline alkaloid dioncophylline F (1) and the new ancistrocladisine B (4). Unprecedented is also the discovery of the dioncophyllines C2 (2) and D2 (3), since Dioncophyllaceae-type alkaloids like 2 and 3 with 5,1′- (type C) or 7,8′-biaryl (type D) linkages are rare in nature. Prior to our isolation work on A. ileboensis, only two other naturally occurring naphthyltetrahydroisoquinolines of these two subtypes had been known, viz., the 5,1′-linked dioncophylline C (19)5,39 from the roots of T. peltatum and 5′O-methyldioncophylline D (7)14 from callus cultures of T. peltatum. The naphthylisoquinoline profile of A. ileboensis differs completely from those of the three other accepted Congolese Ancistrocladus taxa, A. congolensis,20 A. ealaensis,21 and A. likoko,22 which are widely distributed in the Western and Central Congo Basin.1,19 These plants predominantly produce 5,8′-coupled hybrid-type alkaloids, along with some Ancistrocladaceae-type compounds. The 5,8′-coupling site is also frequently found in other Central17,23,24 and East African16 species and is in particular well-known for the Cameroonian liana A. korupensis,3,6,10,49 which produces such 5,8′-linked naphthylisoquinolines (most of them hybrid-type representatives) nearly exclusively, among them also dimers.3,6,10,49 The alkaloid pattern of A. ileboensis, by contrast, mainly consists of 7,1′and 5,1′-linked Dioncophyllaceae- and Ancistrocladaceae-type naphthylisoquinolines. Thus, similar to A. abbreviatus3,9,17 and A. barteri3,18 from the Ivory Coast, and to an as yet botanically undescribed Central Congolese Ancistrocladus species,13 A. ileboensis seems to be more closely related to the Dioncophyllaceae plants from West Africa than to most of the Central African Ancistrocladus lianas with respect to its constitutents, since it predominantly produces typical Dioncophyllaceae-type alkaloids such as 4′-O-demethyldioncophylline A (P-11), along with some Ancistrocladaceae-type compounds

Figure 10. Cytotoxic activities of (A) 5′-O-methyldioncophylline D (7) and (B) dioncophylline A (P-10) toward parental drug-sensitive CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000. The compounds were dissolved in DMSO (99%). Data for 16: yellowish-brown oil; [α]22 D −115.6 (c 0.10, MeOH); νmax 3710, 3237, 2949, 2820, 2704, 2523, 2474, 1595, 1578, 1375, 1450, 1418, 1372, 1326, 1288, 1243, 1207, 1094, 1052, 1033, 1019, 890, 863, 813, 743, 685, 619 cm−1; 1H NMR (methanol-d4, 400 MHz) δ 7.36 (1H, dd, J = 8.7, 0.7 Hz, Ar−H), 6.70 (1H, dd, J = 8.6, 0.8 Hz, Ar−H), 4.67 (1H, q, J = 6.6 Hz, CH), 3.45− 3.33 (1H, m, CH), 3.35 (2H, s, NH, OH), 3.10 (1H, dd, J = 17.4, 3.5 Hz, CH2), 2.71 (1H, dd, J = 17.4, 11.8 Hz, CH2), 1.74 (3H, d, J = 6.6 Hz, CH3), 1.52 (3H, d, J = 6.5 Hz, CH3); 13C NMR (methanol-d4, 101 MHz) δ 155.5 (Ar−Cq), 134.3 (Ar−Cq), 133.1 (Ar−CH), 124.1 (Ar− Cq), 116.6 (Ar−CH), 114.3 (Ar−Cq), 52.2 (CH), 50.6 (CH), 36.3 (CH2), 19.1 (CH3), 18.8 (CH3); EIMS (70 eV) m/z 243 (11), 242 (98), 241 (13), 240 [M − CH3]+ (100); HRESIMS m/z 256.03330 (calcd for C11H15BrNO, 256.03315). 5-Bromo-8-benzyloxy-N-benzyl-1R,3R-dimethyl-1,2,3,4-tetrahydroisoquinoline (17). A suspension of 16 (430 mg, 1.68 mmol, 7.5 equiv), benzyl bromide (2.15 g, 12.6 mmol, 7.5 equiv), and Cs2CO3 (2.73 g, 8.39 mmol, 5.0 equiv) in dry DMF (50 mL) was stirred at room temperature overnight. After filtration, the solvent was removed 455



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under reduced pressure, and the crude residue was purified by column chromatography on silica gel, using n-hexane/Et2O (9:1, with 1% NEt3) as the eluent, to afford compound 17 (614 mg, 1.41 mmol, 84% yield). Data for 17: yellow oil; [α]22 D −53.9 (c 0.09, MeOH); IR (ATR) νmax 3028, 2964, 2924, 1723, 1680, 1574, 1495, 1449, 1372, 1278, 1255, 1187, 1158, 1109, 1085, 1065, 1027, 949, 906, 841, 796, 731, 694 cm−1; 1H NMR (methanol-d4, 400 MHz) δ 7.38−7.20 (11H, m, Ar−H), 6.81 (1H, dd, J = 8.8, 0.6 Hz, Ar−H),5.13−4.97 (2H, m, OCH2), 4.31 (1H, q, J = 6.8 Hz, CH), 3.88 (1H, d, J = 13.9 Hz, NCH2), 3.62 (1H, d, J = 13.9 Hz, NCH2), 3.02 (1H, dd, J = 16.0, 4.7 Hz, CH2), 2.88−2.76 (1H, m, CH), 2.54 (1H, dd, J = 16.0, 8.7 Hz, CH2), 1.29 (3H, d, J = 6.4 Hz, CH3), 1.19 (3H, d, J = 6.8 Hz, CH3); 13 C NMR (methanol-d4, 101 MHz) δ 155.5 (Ar−Cq), 141.3 (Ar−Cq), 138.5 (Ar−Cq), 136.8 (Ar−Cq), 132.8 (Ar−Cq), 131.2 (Ar−CH), 130.1 (Ar−CH), 129.5 (Ar−CH), 129.2 (Ar−CH), 128.9 (Ar−CH), 128.2 (Ar−CH), 127.9 (Ar−CH), 115.8 (Ar−Cq), 112.6 (Ar−CH), 71.1 (OCH2), 60.0 (NCH2), 54.7 (CH), 52.7 (CH), 37.0 (CH2), 23.3 (CH3), 22.8 (CH3); EIMS (70 eV) m/z 422 (77), 420 [M − CH3]+ (75), 331 (12), 329 (13), 91 (100); HRESIMS m/z 436.12631 (calcd for C25H27BrNO, 436.12705). O,N-Dibenzyldioncophylline F (18). A solution of 13 (83.3 mg, 0.254 mmol, 1.7 equiv), 17 (65 mg, 0.149 mmol, 1.0 equiv), and K3PO4 (126 mg, 0.596 mmol, 4.0 equiv) in toluene (15 mL) was degassed. After addition of Pd2(dba)3 (13.6 mg, 14.9 μmol, 0.1 equiv) and S-Phos (24.5 mg, 59.6 μmol, 0.4 equiv), the reaction mixture was again degassed, and the reaction was allowed to proceed overnight with vigorous stirring at 100 °C. After cooling to room temperature, the mixture was diluted in EtOAc and filtered through a short pad of Celite. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on deactivated silica gel, using n-hexane/Et2O (9:1 → 7:1) as the eluent, affording 18 (63.0 mg, 0.113 mmol, 76% yield). Data for 18: colorless foam; [α]25 D −55.3 (c 0.11, CH2Cl2); νmax 2920, 1620, 1582, 1485, 1450, 1372, 1317, 1268, 1237, 1169, 1140, 1110, 1086, 1041, 1028, 965, 904, 806, 732, 696, 618 cm−1; 1H NMR (methanol-d4, 400 MHz) δ 7.44−7.20 (10H, m, Ar−H), 7.13 and 7.13 (1H, d, J = 7.9 Hz, Ar−H), 7.02 and 6.98 (1H, d, J = 8.3 Hz, Ar−H), 6.94 and 6.91 (1H, d, J = 8.5 Hz, Ar−H), 6.88 and 6.86 (1H, d, J = 5.0 Hz, Ar−H), 6.79−6.73 (2H, m, Ar−H), 5.11 (2H, s, NCH2), 4.41−4.59 (1H, m, CH), 3.93 and 3.92 (3H, s, OCH3), 3.92 (3H, s, OCH3), 3.91−3.82 (1H, m, OCH2), 3.71−3.58 (1H, m, OCH2), 2.72−2.53 (1H, m, CH), 2.29 and 2.28 (3H, m, CH3), 2.21−2.27 (2H, m, CH2), 1.28 and 1.22 (3H, d, J = 6.8 Hz, CH3), 1.08 and 1.06 (3H, d, J = 6.3 Hz, CH3); 13C NMR (methanold4, 101 MHz) δ 158.5 (Ar−Cq), 157.8 (Ar−Cq), 155.4 (Ar−Cq), 138.9 (Ar−Cq), 137.6 (Ar−Cq), 137.5 and 134.4 (Ar−Cq), 134.1 (Ar−Cq), 133.9 and 133.4 (Ar−Cq), 132.2 and 132.0 (Ar−Cq), 130.4 (Ar−CH), 130.2 (Ar−Cq), 129.9 (Ar−CH), 129.5 (Ar−CH), 129.3(Ar−CH), 129.2 (Ar−CH), 129.0 (Ar−CH), 128.8 (Ar−CH), 128.5 (Ar−CH), 128.3 and 128.2 (Ar−Cq), 128.1 and 128.0 (Ar−Cq), 119.5 and 119.2 (Ar−CH), 117.0 (Ar−Cq), 110.7 and 110.5 (Ar−CH), 109.7 (Ar− CH), 106.4 (Ar−CH), 71.0 (OCH2), 60.6 and 60.1 (OCH2), 56.9 (OCH3), 56.8 (OCH3), 55.9 and 55.4 (CH), 53.1 and 52.7 (CH), 35.0 and 34.7 (CH2), 24.0 (CH3), 23.0 (CH3), 22.1 (CH3); EIMS (70 eV) m/z 543 (42), 542 [M − CH3]+ (100), 451 (16), 450 (24), 91 (23); HRESIMS m/z 558.30003 (calcd for C38H40NO3, 558.30027). Dioncophylline F (1). To a solution of 18 (51 mg, 91.4 μmol, 1.0 equiv) in 9 mL of dry MeOH/CH2Cl2 (2:1) was added 20.0 mg of Pd/C (10%). After stirring for 2 h at room temperature under a hydrogen atmosphere (1 atm), the catalyst was removed by filtration through Celite. Evaporation of the solvent led to a crude residue, which was purified by column chromatography on deactivated silica gel using CH2Cl2/MeOH (9:1) as the eluent, to give 1 (29.0 mg, 76.8 μmol, 84% yield). Data for 1: colorless solid, [α]25 D −113.2 (c 0.10, MeOH); IR (ATR) νmax 3149, 2949, 2833, 2360, 1588, 1453, 1427, 1379, 1318, 1287, 1272, 1238, 1169, 1142, 1127, 1090, 1028, 993, 975, 831, 813, 492, 765, 670, 613 cm−1; 1H NMR (methanol-d4, 400 MHz) δ 7.15 and 7.13 (1H, d, J = 8.0 Hz, 7′-CH), 7.06 and 7.00 (1H, d, J = 8.2 Hz, 6-CH), 6.92 and 6.90 (1H, d, J = 8.0 Hz, 6′-CH), 6.88 and 6.86 (1H, d, J = 8.2 Hz, 7-CH), 6.80 (1H, d, J = 1.5 Hz, 3′-CH), 6.75 and 6.69 (1H, t, J = 1.5 Hz, 1′-CH), 4.83−4.68 (1H, m, 1-CH), 3.95

(3H, s, 5′-OCH3), 3.93 (3H, s, 4′-OCH3), 3.29−3.18 (1H, m, 3-CH), 2.58 (1H, dd, J = 17.1 Hz, 11.9 Hz, 4ax-H), 2.43−2.39 (2H, m, 4-Hax, 4-Heq), 2.34 and 2.30 (3H, s, 2′-CH3), 2.27 (1H, dd, J = 17.1 Hz, 3.3 Hz, 4-Heq), 1.86 and 1.82 (3H, d, J = 6.6 Hz, 1-CH3), 1.26 and 1.24 (3H, d, J = 6.5 Hz, 3-CH3); 13C NMR (methanol-d4, 101 MHz) δ 158.8 and 158.7 (4′-C), 158.2 and 158.2 (5′-C), 155.4 and 155.3 (8C), 138 (2′), 137.4 (9′-C), 134.0 and 133.7 (10-C), 133.4 and 132.9 (5-C), 132.3 and 131.7 (6-CH), 131.5 and 130.8 (8′-C), 129.7 and 129.1 (7′-CH), 121.4 and 121.0 (9-C), 118.8 and 118.5 (1′-CH), 117.3 and 117.1 (10′-C), 114.9 and 114.6 (7-CH), 109.9 (3′-CH), 106.3 (6′-CH), 56.9 (4′-OCH3), 56.8 (5′-OCH3), 52.4 (1-CH), 51.0 and 50.9 (3-CH), 33.9 and 33.3 (4-CH2), 22.1 and 22.0 (2′-CH3), 19.5 and 19.4 (1-CH3), 18.8 and 18.7 (3-CH3); EIMS (70 eV) m/z 378 [M + H]+ (13), 377 [M]+ (23), 364 (19), 363 (67), 362 [M − CH3]+ (100); HRESIMS m/z 378.20687 (calcd for C24H28NO3, 378.20637). The compound was found to be fully identical with the authentic natural product isolated from A. ileboensis. Stereochemical Analysis of 1. The two atropo-diastereomers of dioncophylline F (1), P-1 and M-1, were resolved at an analytical scale by HPLC, using a Waters XSelect HSS PFP column (4.6 × 250 mm, 4 μm), with 90% H2O (0.05% TFA)/10% MeOH (0.05% TFA) (solvent A) and 10% H2O (0.05% TFA)/90% MeOH (0.05% TFA) (solvent B) as the eluents, using a linear gradient (0 min 50% A, 30 min 25% A), at a flow rate of 1 mL/min, UV detection at 230 nm, and analyzed online, by hyphenation with ECD spectroscopy. Atropodiastereomer P-1 (retention time 22.1 min): ECD (MeOH, c 0.1) λmax (Δε), 200 (−5.79), 210 (−3.48), 228 (1.45), 239 (3.35), 261 (0.63), 275 (0.49), 282 (−0.21), 312 (1.23), 342 (0.08) cm2 mol−1. Atropodiastereomer M-1 (retention time 23.4 min): ECD (MeOH, c 0,1) λmax (Δε), 200 (11.7), 225 (−14.9), 260 (−0.45), 283 (−1.38), 309 (0.63), 322 (−0.18) cm2 mol−1. Antiprotozoal Evaluation. The antiparasitic activities of the compounds 1−3, 5, 7, and 8 against the pathogens P. falciparum (NF54 strain), T. cruzi, T. b. rhodesiense, and L. donovani and the cytotoxicity against mammalian host cells (rat skeletal myoblast L6 cells) were determined in vitro as described previously.53 Viability Analyses in MM Cells and PBMCs. The effects of the compounds 1, 3, 7, 8, P-10, P-11, and M-11 (from A. ileboensis), dioncophylline C (19)39 and 5′-O-demethyldioncophylline A47 (from T. peltatum), and N-acetyldioncophylline A48 (semisynthetic compound) on cell survival were analyzed either in nonmalignant cells (mononuclear cells derived from the peripheral blood of healthy donors) or in malignant cells of the human multiple myeloma cell line INA-6.54 Cells were incubated for 3 d prior to harvesting and viability assessment. Both apoptotic and viable cell fractions were determined by staining with annexin V-FITC and propidium iodide (PI) according to the instructions of the manufacturer (Bender MedSystems, Vienna, Austria). In brief, cells in phosphate-buffered saline, incubated for 10 min in 100 mL of binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) containing 2.5 mL of annexin V-FITC and 1 mg/mL PI, were subsequently diluted with 300 mL of binding buffer and analyzed by flow cytometry (FACSCalibur/CELLQuest; Becton Dickinson, Heidelberg, Germany). In early stages of the apoptotic process, phosphatidylserine translocates from the internal to the external membrane and can be detected by annexin V-FITC. Cells in a late apoptotic stage lose their membrane integrity, and the DNAbinding agent PI can be incorporated. Thus, the cell fraction that is negative for both annexinV-FITC and PI is considered viable. On the basis of the respective viable cell fractions, the means, standard deviations, dose−response curves, and EC50 values were calculated using the Prism software (GraphPad Software Inc., La Jolla, USA). Antileukemic Evaluation. The cytotoxic effects of the naphthylisoquinoline alkaloids 1, 3, 7, 8, P-10, P-11, and M-11 (from A. ileboensis) and dioncophylline C (19)39 (from T. peltatum) on drugsensitive leukemia CCRF-CEM and multidrug-resistant P-glycoprotein-overexpressing subline CEM/ADR5000 cells55 were monitored by the resazurin assay as previously described.56 All compounds were tested at a single concentration of 10 μM (Figure 9) against CCRFCEM cells. Two of these compounds (7 and P-10), inducing less than 30% growth, were further investigated for IC50 determinations in both 456



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CCRF-CEM and CEM/ADR5000 cell lines (Figure 10). Doxorubicin (Sigma-Aldrich, Munich, Germany) was used as the positive control, while DMSO, used to dissolve the compounds, was applied as the negative control. The highest concentration of DMSO was less than 1.0%. Fluorescence was measured on an Infinite M2000 Pro plate reader (Tecan, Crailsheim, Germany), using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. All experiments were performed in triplicate. The viability was evaluated based on a comparison with untreated cells. IC 50 values represent the concentrations of the compounds required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel.57

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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.6b00967. NMR (1H, 13C, 1H,1H−COSY, HSQC, HMBC, NOESY, and ROESY), HRESIMS, IR and ECD spectra, and GCMSD chromatograms obtained from the oxidative degradation of 1−4, and synthetic procedures giving rise to 1 together with the physical and spectroscopic data of the building blocks 13 and 15−18 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49-931-318-5323. Fax: +49-931-318-4755. E-mail: [email protected]. ORCID

Gerhard Bringmann: 0000-0002-3583-5935 Author Contributions §

J. Li and R. Seupel contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Clinical Research Unit 216 “Characterization of the Oncogenic Signaling Network in Multiple Myeloma: Development of Targeted Therapies”, subproject 7 (grant Br 699/16-2) “Design, Characterization, and Optimization of HSP70 Inhibitors, HSF-1 Inhibitors, and Anticancer Naphthoquinones and Naphthylisoquinoline Alkaloids for the Treatment of Multiple Myeloma”; Individual Research Grant Br699/14-2 “Molecular Phylogeny and Chemotaxonomy of the Ancistrocladaceae Plant Family”; SFB 630 “Recognition, Preparation, and Functional Analysis of Agents against Infectious Diseases”, project A2). We thank Dr. M. Büchner, Mr. F. Dadrich, and Mr. J. Wendrich for recording the mass spectra, Dr. M. Grüne, Mrs. E. Ruckdeschel, and Mrs. P. Altenberger for performing the NMR experiments, and Dr. F. Witterauf for analytical support (HPLC-ECD). Further thanks are due to Mrs. M. Michel for the degradation experiments and to Mr. S. Specker and Dr. M. Dreyer for previous phytochemical work on A. ileboensis.

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REFERENCES

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