Cytotoxic Prenylated Stilbenes Isolated from ... - ACS Publications

Article Cite This: J. Nat. Prod. 2017, 80, 2684-2691

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Cytotoxic Prenylated Stilbenes Isolated from Macaranga tanarius Tiphaine Péresse,† Gwenael̈ le Jézéquel,† Pierre-Marie Allard,§ Van-Cuong Pham,‡ Doan T. M. Huong,‡ Florent Blanchard,† Jérôme Bignon,† Hélène Lévaique,† Jean-Luc Wolfender,§ Marc Litaudon,*,† and Fanny Roussi*,† †

Institut de Chimie des Substances Naturelles, CNRS, ICSN UPR2301, University of Paris-Saclay, 91198, Gif-sur-Yvette, France Advanced Center for Bioorganic Chemistry of the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, 8404, Caugiay, Hanoi, Vietnam § School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU−Rue Michel Servet 1, 1211 Geneva 11, Switzerland ‡

S Supporting Information *

ABSTRACT: With the aim of discovering new cytotoxic prenylated stilbenes of the schweinfurthin series, Macaranga tanarius was selected for detailed phytochemical investigation among 21 Macaranga species examined by using a molecular networking approach. From an ethanol extract of the fruits, seven new prenylated stilbenes, schweinfurthins K−Q (7−13), were isolated, along with vedelianin (1), schwenfurthins E−G (2−4), mappain (5), and methyl-mappain (6). The structures of the new compounds were established by spectroscopic data analysis. The relative configurations of compounds 8, 12, and 13 were determined based on ROESY NMR spectroscopic analysis. The cytotoxic activities of compounds 1−13 were evaluated against the human glioblastoma (U87) and lung (A549) cancer cell lines.

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Macaranga, containing 308 species, is a large tropical genus of the Euphorbiaceae family, of which 21 species originating from New Caledonia, Madagascar, Uganda, and Vietnam are part of an in-house plant collection.8 Recently, in order to explore their metabolome, a full molecular network was constructed from UHPLC-HRMS/MS data.9 From a careful analysis of this network, several novel prenylated stilbenes were detected in an extract of M. tanarius fruits. Macaranga tanarius Muell.-Arg. is a medium-sized tree that is widespread in the Southeast Asian and western Pacific regions. In Vietnam, this species has been used in traditional medicine for treatment of furuncles.10 A phytochemical study performed earlier on the leaves of this plant revealed the presence of several flavonoids.11 In the present study, several new and known stilbenes isolated from the fruits of M. tanarius and their cytotoxicity toward U87 human glioblastoma and A549 human lung cancer cells have been reported.

revious chemical investigation of Macaranga species has led to the discovery of several prenylated stilbenes, which exhibited potent cytotoxic activities.1,2 These compounds are promising agents for further development due to their highly selective and potent antiproliferative activities, in particular when their structures possess a hexahydroxanthene moiety resulting from the cyclization of a geranyl chain.3 Vedelianin (1), the first derivative of this chemical series, was isolated in 1992 from the leaves of Macaranga vedeliana in the context of an ethnopharmacological study.4 Then, Beutler et al. and Yoder et al. isolated close structural analogues of 1, namely, schweinfurthins A−J, with or without a hexahydroxanthene moiety, from M. schweinf urthii and M. alnifolia.2−5 These compounds were found to be selectively active in the NCI 60cell-line screening toward human cancer cells. For example, cancer cell lines of the central nervous system (such as SF-295) were found particularly sensitive (in the nanomolar range), whereas various other cell lines, such as different lung cancer cell lines (e.g., A549), were more resistant (micromolar range). Most importantly, the NCI 60-cell mean-graph cytotoxicity profile showed no resemblance to those of any of the profiles in the NCI standard agents database, indicating that this family of compounds acts via a novel mechanism of action.5 Recently, they were found to interact with a protein of the ORP family (oxysterol related proteins), but the precise mechanism of action leading to cell death is still incompletely known.6,7 An extensive study on the genus Macaranga is necessary for the discovery of new and promising stilbenes. © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The crude EtOAc extracts of 21 Macaranga species were used to establish a full molecular network on the GNPS platform.12 In order to easily detect clusters corresponding to schweinfurthin analogues, compounds 1−6 were co-injected in the network (Figure S1, Supporting Information). The molecular network was then annotated using an in silico fragmented Received: May 11, 2017 Published: October 3, 2017 2684

DOI: 10.1021/acs.jnatprod.7b00409 J. Nat. Prod. 2017, 80, 2684−2691

Journal of Natural Products

Article

Table 1. NMR Spectroscopic Data [(CDCl3 (7), CD3CN (8−10), CD3OD (11)] for Compounds 7−11 7 position

δC

1 2 3 4 5 6 7a 7b 8 9 10 11 12 13 14 15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″eq 1″ax 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″ OH-13 OH-2″

142.3 144.7 111.1 137.9 120.9 127.8 30.3 121.9 139.4 16.5 40.0 26.7 124.1 132.6 18.1 26.1 128.9 126.6 130.5 108.1 154.2 106.2 154.9 105.3 26.6 69.7 77.1 24.9 22.6

8

δH (J in Hz)

6.90, s 6.75, s 3.35, dd (7.1) 5.32, t (7.1) 1.77, s 2.09, m 2.11, m 5.06, t (5.9) 1.59, s 1.67, s 6.84, d (16.5) 6.71, d (16.5) 6.57, s

6.46, s 2.70, dd (17.1, 5.2) 2.90, dd (17.1, 5.0) 3.82, dd (5.2, 5.0) 1.30, s 1.36, s

δC 141.6 146.8 111.1 129.2 120.6 121.8 31.8 68.2 80.8 19.4 38.3 22.6 125.4 132.6 17.8 25.9 129.1 127.3 138.3 105.3 156.7 108.3 155.4 107.7 27.3 69.7 77.8 25.7 21.4

9

δH (J in Hz)

6.87, s 6.74, s 2.68, dd (16.2, 6.4) 2.99, dd (16.2, 4.7) 3.85, dd (6.4, 4.7) 1.28, s 1.63, m 2.17, m 5.13, t (5.6) 1.59, s 1.66, s 6.91, d (16.3) 6.82, d (16.3) 6.52, s

6.45, s 2.48, dd (16.3, 6.3) 2.83, dd (16.3, 5.8) 3.74, dd (6.3, 5.8) 1.28, s 1.22, s

δC 143.7 145.3 111.5 130.4 121.1 129.5 29.0 123.7 137.5 16.5 37.7 30.8 78.7 73.3 26.2 24.8 129.3 127.0 137.4 105.3 156.6 108.3 155.4 107.4 27.3 69.8 77.7 25.7 21.4

10

δH (J in Hz)

6.90, s 6.84, s 3.31, d (7.3) 5.37, t (7.3) 1.73, s 2.07, m, 2.27, m 1.33, m, 1.66, m 3.21, d (11.0) 1.09, s 1.07, s 6.91, d (16.5) 6.79, d (16.5) 6.53, d (1.5)

6.45, d (1.5) 2.74, dd (17.0, 7.9) 2.83, dd (17.0, 5.7) 3.73, ddd (7.9, 5.7, 5.3) 1.28, s 1.22, s

δC 143.7 145.4 111.4 130.0 121.1 129.5 29.0 123.5 137.5 16.3 37.7 30.8 78.6 73.4 26.2 24.8 129.0 127.1 137.4 106.1 157.0 115.2 157.0 106.1 23.0 123.8 132.6 18.0 25.8

11

δH (J in Hz)

δC 146.4 146.6 113.8 131.5 120.0 116.6

6.90, s 6.84, s

δH (J in Hz)

6.90, d (2.5) 6.75, d (7.4) 6.68, d (7.4)

3.30, d (7.7) 5.37, t (7.7) 1.72, s 2.07, m, 2.26, m 1.33, m, 1.65, m 3.22, m 1.09, s 1.07, s 6.87, d (15.4) 6.76, d (15.4) 6.51, s

6.51, s 3.24, d (6.7) 5.17, t (6.7) 1.66, s 1.72, s

128.7 127.4 137.7 105.8 157.7 116.0 157.7 105.8 23.4

6.78, d (16.0) 6.64, d (16.0)

124.8 134.0 16.4 41.1 27.7 25.6 135.7 17.8 21.0 28.0 125.7 132.1 16.2 26.0

5.20, t (6.7)

6.39, s

6.39, s 3.24, m

1.72, s 1.91, m 2.02, m 5.03, m 1.51, s 1.85, m 1.95, m 5.01, m 1.52, s 1.58, s

2.79, dd (11.0, 5.3) 3.07, d (5.3)

sequentially purified using silica gel flash chromatography and preparative reversed-phase HPLC. This resulted in the isolation of seven new prenylated stilbenes (7−13) including the dimer 12. The structures of these compounds were determined by spectroscopic analysis. The 1H and 13C NMR data of compounds 7−11, 12, and 13 are reported in Tables 1−3. The molecular formula, C 29 H 36 O 5 , of compound 7 (schweinfurthin K) was established by HRESIMS from the protonated [M + H]+ ion at m/z 465.2643 (calcd for C29H37O5+, 465.2636) together with its 13C NMR data, indicating 12 indices of hydrogen deficiency. The UV spectrum displayed an absorption band at 330 nm, characteristic of a trans-stilbene moiety.13 This was confirmed by NMR data analysis: in the 1H NMR spectrum, the presence of two olefinic protons at δH 6.71 and 6.84 (H-2′ and H-1′, respectively, J =

database limited to Euphorbiaceae secondary metabolites, according to a previously developed dereplication strategy.9 This allowed seven schweinfurthin analogues (A−D and H−J) to be identified within the cluster of interest. In addition, several nodes with MS/MS fragmentation patterns similar to schweinfurthins (according to proximity within the molecular network and variable dereplication results) possibly corresponding to new schweinfurthin analogues were evidenced. The ethanolic extract of the fruits of M. tanarius was subjected to partitioning using water and Et2O followed by CH2Cl2. A silica gel chromatographic separation was performed on the CH2Cl2 extract with a gradient of heptane−EtOAc− methanol. As the stilbene moiety of this chemical series displays a characteristic UV maximum at 330 nm, all fractions were subjected to LC-UV/MS analysis. The selected fractions were 2685



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Table 2. NMR Spectroscopic Data [(CD3)2CO] for Compound 12 position

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″

142.6 144.9 113.2 131.3 121.2 131.0 129.3 128.0 140.9 105.9 159.8 102.9 159.8 105.9 159.2 107.5 159.2 140.2 107.5 103.6 81.6 81.1 121.5 113.3 121.4 128.5 144.1 145.1 28.9 123.3 136.8 16.5 40.6 17.1 123.4 131.7 17.9 25.9 28.8 123.5 136.6 16.5 40.6 27.1 125.4 131.7 25.9 17.9

Table 3. NMR Spectroscopic Data [CD3CN] for Compounds 1 and 13

δH (J in Hz)

1

7.02, d (2.0) 6.99, d (2.0) 6.99, d (16.4) 6.92, d (16.4) 6.55, d (1.9) 6.27, d (1.9) 6.55, d (1.9) 6.21, d (1.9)

6.21, 6.27, 4.82, 4.76,

d d d d

(1.9) (1.9) (8.0) (8.0)

6.66, d (1.8) 6.49, d (1.8)

3.35, d (6.9) 5.37, t (6.9) 1.67, d (2.9) 2.03, m 2.11, m 5.12, t (7.1)

position

δC

1 2 3ax 3eq 4ax 4eq 4a 5 6 7 8 8a 9 9a 10a 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″

13

δH (J in Hz)

δC

39.3 78.2 71.8

3.30, dd (7.3, 3.6)

39.6 75.8 36.7

44.6

4.10, m 1.93, m 2.32, dd (14.2, 2.9)

75.7

78.7 147.5 110.9 130.8 120.8 124.6 23.8 48.7 141.8 16.9 29.7 22.6 129.3 127.3 138.2 106.4 157.5 115.5 157.5 106.4 23.4 124.2 132.6 18.3 26.2

6.86, s 6.78, s 2.75, m 1.79, dd (10.7, 7.6) 1.03, s 1.07, s 1.38, s 6.87, d (16.1) 6.80, d (16.1) 6.51, s

6.51, s 3.24, d (7.3) 5.17, t (7.3) 1.74, s 1.66, s

82.9 146.9 110.8 131.0 120.7 124.2 23.2 45.8 141.3 15.2 27.9 14.5 128.9 127.4 137.7 106.2 156.9 115.4 156.9 106.2 23.2 124.0 132.5 18.1 26.1

δH (J in Hz) 3.41, m 1.60, m 1.93, m 3.79, dd (12.0, 3.7)

6.89, s 6.78, s 2.75, m 1.60, m 0.81, s 1.02, s 1.15, s 6.87, d (16.2) 6.81, d (16.2) 6.50, s

6.50, s 3.23, d (6.9) 5.15, t (6.9) 1.73, s 1.65, s

1.58, d (4.2) 1.64, d (4.2) 3.27, d (8.2) 5.27, t (8.2) 1.67, d (2.5) 2.03, m 2.11, m 5.12, t (7.1)

Figure 1. (a) Key 1H−1H COSY (bold) and HMBC 1H → 13C (blue arrows) correlations of compounds 7 and 8. (b and c) Key ROE correlations (red arrows) to determine the relative configurations of 2,2-dimethylpyran-3-ol moieties of compounds 7 and 8 and the dihydropyranol moiety of compound 8, respectively.

1.64, d (4.2) 1.58, d (4.2)

16.5 Hz) was indicative of an E-configured double bond at C1′/C-2′. In the HMBC spectrum, cross-peaks from these protons to the aromatic carbons at δC 130.5 and 137.9, C-3′ and C-4, respectively, suggested that the trans double bond is linked to two aromatic rings (A and B), thus corresponding to a trans-stilbene moiety. Other HMBC correlations were indicative of two 1,2,3,5-tetrasubstituted benzene ring systems, as depicted in Figure 1. In ring A, resonances at δC 142.3 and 144.7 (C-1 and C-2, respectively) supported the presence of two phenolic substituents. A geranyl side chain could be located

at C-6 from the following evidence: the resonances of three methyl groups attached to two olefinic quaternary carbons were observed at δC 16.5, 26.1, and 18.1, as well as three methylenes at δC 30.3, 40.0, and 26.7 (Table 1), and HMBC correlations from the proton at δH 3.35 (H-7) to aromatic carbons C-1 and C-5. The 1H NMR data of 7 also revealed the presence of a 2,2dimethyldihydropyranol moiety: two singlet methyl protons at δH 1.30 and 1.36, one methylene group as an ABX spin system resonating at δH 2.70 (H-1″eq, dd, J = 17.1 and 5.2 Hz) and 2686



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Chart 1. Structures of Compounds 1−13

2.90 (H-1″ax, dd, J = 17.1 and 5.0 Hz), and one doublet of doublets at δH 3.82 (H-2″, J = 5.2 and 5.0 Hz). The location of this pyran ring was confirmed by the HMBC correlations from H2-1″ to the oxygenated tertiary carbons C-5′ and C-7′ at δC 154.2 and 154.9, respectively, the oxymethine carbon C-2″ at δC 69.7, and the oxygenated tertiary carbon C-3″, and crosspeaks from the aromatic protons H-4″ and H-8″ to C-6″ at δC 106.2. The dihydropyranol ring resulted from the intramolecular addition of one phenolic hydroxy group on the isoprenic chain for which the double bond was previously epoxidized. This was confirmed from the comparison of NMR data with those reported for schweinfurthin H, which possesses the same moiety.2 The relative configuration of C-2″ was established by proton coupling constant analysis. Since H-2″ has two gauche coupling constants (J = 5.0 and 5.2 Hz), it has thus a pseudoequatorial orientation. In addition, the strong ROE correlations between H-1″ax/H-2″/H3-4″ suggested that these protons are oriented in the same plane (Figure 1). Compound 8 was assigned the molecular formula C29H36O6 on the basis of HRESIMS from the protonated [M + H]+ ion at m/z 481.2593 (calcd for C29H37O6+, 481.2585), indicating 12 indices of hydrogen deficiency and suggesting the presence of an additional oxygen atom when compared with compound 7. Its UV spectrum showed the same maximum at 330 nm as compound 7, characteristic of a trans-stilbene pattern. The 1H and 13C NMR spectroscopic data of compound 8 were closely comparable to those of compound 7, but revealed also the presence of an additional substituted dihydropyranol ring (Table 1). From the substitution pattern of the trans-stilbene moiety of 8, similar to compound 7, it could be deduced that the additional dihydropyranol ring is fused to ring A via C-1 and C-6. The HMBC correlations from H-8 to C-10 and C-11

and from H2-7 to C-1 and C-5 confirmed the location of this ring as depicted in Figure 1. In the 1H NMR spectrum, the splitting pattern of H-8 appeared as a doublet of doublets with two gauche coupling constants (J = 6.4 and 4.7 Hz), indicating its pseudoequatorial disposition. In the ROESY spectrum, strong ROEs were observed between the C-10 methyl at δH 1.28 and protons H-8 (δH 3.85, dd) and H-7a (δH 2.68, dd). The H-8 proton signal, in turn, showed an ROE with both H7a,b methylene protons. These NMR data were found to be very similar to those obtained for 8-benzoyl-2-(4-methylpenten-3-yl)-chromane-3,5,7-triol, a benzophenone isolated from the leaves of Tovomita longifolia possessing a structurally close chromane moiety and for which molecular modeling calculations were performed.14 From these results, it could be deduced that the only compatible 3D structure for the chromane moiety of compound 8 is the trans-diaxial arrangement between OH-8 and CH3-10 (Figure 1). Finally, the relative configuration and conformation of the 2,2-dimethylpyran-3-ol moiety of compound 8, identical to compound 7, could be deduced from ROE correlations between H-1″b, H-2″, and H3-5″ and the coupling constant values measured for H2-1″ and H-2″. Compound 8, which was named schweinfurthin L, possesses the structure shown in Chart 1. It should be noted that optical rotation values of both 7 and 8 were found to be zero, which led to the postulation that they were obtained as racemic mixtures. The HRESIMS of compound 9 (schweinfurthin M), which displayed a [M + H]+ ion at m/z 499.2685 corresponding to the molecular formula C29H37O7, with 11 indices of hydrogen deficiency, indicated the presence of two additional oxygen atoms when compared with compound 7. Comparison of its NMR spectroscopic data with those of compound 7 suggested 2687



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Figure 2. Key 1H−1H COSY (bold) and HMBC 1H → 13C (blue arrows) correlations of compound 12.

Hz) for H-7 and H-8. The presence of two 1,2,3,5tetrasubstituted and 1,3,5-trisubstituted benzene ring systems was deduced from the observation of respectively two (H-3 and H-5) and three (H-10, H-12, and H-14) meta-coupled aromatic protons in the 1H NMR spectrum of 12. The 13C NMR resonances for four oxygenated tertiary carbons at δC 142.6, 144.9, 159.8, and 159.8 (C-1, C-2, C-11, and C-13, respectively) and the HMBC correlations from the methylene protons H2-1′ to carbons C-1 and C-5 allowed the subunit I to be determined as depicted in Figure 2. Subunit II was constituted of two 1,2,4-trisubstituted and 1,2,3,4-tetrasubstituted benzene ring systems, as indicated by the presence of three ortho-meta-coupled protons (H-16, H-19, and H-20) on one hand and two ortho-coupled protons (H-24 and H-25) on the other hand. In addition, subunit II was found to possess four oxygenated tertiary carbons at δC 159.2, 159.2, 144.1, and 145.1 (C-15, C-17, C-27, and C-28, respectively) and an O− CH−CH−O moiety, as indicated by two coupled methine protons resonating at δH 4.82 and 4.76 (each d, J = 8.0 Hz, H21 and H-22, respectively). The four parts of subunit II (two substituted benzene ring systems, the geranyl, and the O−CHCH−O fragments) could be connected from the COSY and HMBC correlations as depicted in Figure 2. The formation of the dihydrobenzofuran ring probably results from a double oxidation of the trans double bond followed by an intramolecular cyclization. Six hydroxy groups were located at C-1, C-2, C-13, C-21, C-27, and C-28, according to the chemical shifts observed of their respective carbons. From the molecular formula and the chemical shifts of the oxygenated tertiary carbons C-11 and C-15 at δC 159.8 and 159.2, respectively, it could be inferred that subunits I and II are connected through an ether bridge linking C-11 and C-15. The C-(21)H/C-(22)H cis configuration was suggested by the vicinal coupling constant between H-21 and H-22 (J = 8.0 Hz).16 Other prenylated stilbene dimers have already been isolated from various plants in which they act as phytoalexins.17−19 The HREISIMS of compound 13 exhibited a [M + H]+ ion at m/z 481.2587, consistent with the molecular formula C 29 H 36O 6 , identical to that of vedelianin. The NMR spectroscopic data of compound 13 is similar to that of vedelianin 1 but with a different substitution pattern on ring A of the hexahydroxanthene moiety. Indeed, a −CH(O)−CH2− CH(O)− spin system can be deduced from the COSY spectrum of 13, suggesting that a hydroxy group is located at position 4 in the vicinity of the 13-methyl group, whereas the two hydroxy groups are located at positions 2 and 3 in the case of vedelianin (1). This substitution pattern was confirmed by the correlations from H-2 to C-11, H2-3 to C-1, C-4a and C-9a, and H-4 to C-4a, C-9a and C-13 observed in the HMBC spectrum (Figure 3). The relative configuration of compound 13 was determined by analysis of coupling constants and from the ROESY correlations between H-2 and H-4 and between H9a and H-12 on one hand and between H3a and H-11 and H-

that both compounds have a similar structure but with a dihydroxylated C10 geranyl side chain in the case of 9. Indeed, the absence of an olefin proton corresponding to H-13 and the upfield shifts of carbons C-13 and C-14, from 134.1 and 132.6 to 78.7 and 73.3, respectively, suggested the presence of a 1,2 diol function, resulting from an oxidation of the double bond. From the ROE correlations observed between H-1″ax, H-2″, and H3-4″, and the pseudoequatorial orientation of H-2″ deduced from the two gauche coupling constants between H-2″ and H2-1″ (J = 7.9 and 5.7 Hz), it could be assumed that the C2″ configuration and the conformation of the 2,2-dimethylpyran-3-ol moiety of compound 9 are identical to those of compounds 7 and 8. The configuration of carbon C-13 was not determined (Chart 1). The HRESIMS of compound 10 (schweinfurthin N) showed a [M + H]+ ion at m/z 483.2711, consistent with the molecular formula, C29H38O6. The comparison of its NMR spectroscopic data with those of compound 9 indicated that both compounds have a similar structure, but with a prenyl side chain attached at C-6′ on ring B in compound 10, instead of the dihydropyranol ring system in the case of 9. This was confirmed by COSY correlations between the olefinic proton H-2″ at δH 5.21 and H1′ methylene protons and the HMBC correlations from methyl protons H3-4″ and H3-5″ (δH 1.02 and 1.09, respectively) to C2″ and C-3″ (δC 102.3 and 102.9, respectively) and from H-2′ to C-6′. The configuration of carbon C-13 in compound 10 was not determined, (Chart 1). Compound 11 (schweinfurthin O) exhibited a [M + H]+ ion at m/z 449.2681 corresponding to the molecular formula C29H38O6, with 12 indices of hydrogen deficiency. The 1H and 13 C NMR spectroscopic data of this compound were closely comparable to those of pawhuskin C,15 but with a farnesyl side chain in compound 11 instead of a geranyl side chain. In the 1H NMR spectrum, the resonances of four methyl groups attached to three olefinic quaternary carbons were observed at δC 16.4, 17.8, 16.2, and 26.0 (C-4″, C-9″, C-14″, and C-15″, respectively) as well as five methylenes at δC 23.4, 41.1, 27.7, 21.0, and 28.0 (C-1″, C-5″, C-6″, C-10″, and C-11″, respectively). HMBC correlations from the methylene protons H2-1″ to C-3 and C-5 confirmed the location of the side chain at C-4 in compound 11 (Chart 1). Compound 12 (schweinfurthin P) was assigned the molecular formula C48H54O8 on the basis of its 13C NMR data and the [M + H]+ ion at m/z 759.3920, indicating 22 indices of hydrogen deficiency. Analysis of the 1H NMR data revealed the presence of two geranyl chains (C-1′ to C-10′ and C-1″ to C-10″) with six methyl groups attached to six olefinic quaternary carbons (Table 2). To facilitate the structural elucidation, the molecule has been divided into two subunits, I and II (Figure 2). In subunit I, the trans-stilbene moiety was identified from the characteristic proton resonances at δH 6.99 and 6.92 (d, J = 16.4 2688



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Table 4. Cytotoxic (EC50, μM) Activities of Compounds 1− 13 on the Glioblastoma (U87) and Lung Cancer (A549) Cell Lines

Figure 3. Key 1H−1H COSY (bold), HMBC 1H → 13C (blue arrows), and ROESY (red arrows) correlations of compounds 1 and 13.

13 on the other hand (Figure 3). From the co-occurrence of compound 13 and vedelianin (1) in M. tanarius and the similarity of their structure the absolute configuration of 13 is assumed to be 2R, 4S, 4aS, and 9aR (Figure 3). Single crystals of schweinfurthin E (2) suitable for X-ray diffraction analysis were obtained from the slow evaporation of a 95:5 CH 2 Cl 2 −MeOH solution of 2. The absolute configuration of compound 2 was assigned as 2S, 3R, 4aR, and 9aR. An ORTEP view of 2 is shown in Figure 4, and a detailed study is presented in the Supporting Information.

compound

U87a

A549b

1 2 3 4 5 6 7 8 9 10 11 12 13

0.030 ± 0.009 0.20 ± 0.01 0.045 ± 0.005 0.24 ± 0.01 5.4 ± 0.8 7.0 ± 0.2 0.25 ± 0.08 0.45 ± 0.03 0.50 ± 0.04 0.10 ± 0.01 0.025 ± 0.006 >10 0.050 ± 0.003

0.190 ± 0.002 0.26 ± 0.01 0.80 ± 0.04 1.50 ± 0.02 7.0 ± 0.8 >10 0.73 ± 0.01 0.24 ± 0.12 >10 >10 >10 >10 >10

a EC50 (μM) of monomethyl auristatin E used as a positive control: 0.00020 ± 0.00003. bEC50 (μM) of monomethyl auristatin E used as a positive control: 0.00050 ± 0.00005.

were on the same order of magnitude, which is not the case for vedelianin 1 and schweinfurthins E, F, and G (2−4), which are devoid of a pyranol moiety. This suggests a different mechanism of action for both structural types.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured at 24 °C on an Anton Paar MCP 300 polarimeter. UV spectra were recorded on a Varian Cary 100 UV−vis spectrophotometer. IR spectra were recorded on a PerkinElmer BX FT-IR spectrometer. 1D and 2D NMR spectra were recorded in CD3OD, (CD3)2CO, CD3CN, and CDCl3 on a Bruker 600 MHz NMR instrument (Avance 600) using a 1.7 mm microprobe for compound 6, on a Bruker 500 MHz NMR instrument (Avance 500) for plant extracts, fractions, and compounds 2−5, 6−10, and 12 and 13, and on a Bruker 300 MHz NMR instrument (Avance 300) for compound 11. Chemical shifts (relative to the residual solvent) are in ppm, and coupling constants are in hertz. High-resolution mass spectra were obtained on a Waters LCT Premier XE spectrometer in electrospray ionization mode by direct infusion of the purified compounds. Kromasil analytical and preparative C18 columns (150 × 4.6 mm and 250 × 21.1 mm i.d.; 5 μm Thermo) were used for HPLC separation using a Waters autopurification system equipped with a sample manager (Waters 2767), a UV−vis diode array detector (190−600 nm, Waters 2996), and a PL-ELS 1000 ELSD Polymer Laboratory detector. All solvents and silica gel 60 (6−35 μm) were purchased from SDS (Messia sur Sorne, France), and preparative and analytical TLC plates (Si gel 60 F254) were from Merck (Saint Quentin Fallavier, France). Prepacked GraceResolv silica cartridges were used for flash chromatography using a Teledyne Isco Combiflash Rf 200i. Plant Material. The green fruits of M. tanarius were collected in June 2014 at A Luoi, Thua Thien Hue Province, Vietnam, and authenticated by N. T. Cuong and D. D. Cuong. A voucher specimen (VN-2371) has been deposited at the Herbarium of the Institute of Ecology and Biological Resources of the Vietnam Academy of Science and Technology, Hanoi, Vietnam. Extraction and Isolation. The isolation of compounds 1 to 6 has already been described in a previous article.9,20 Isolation of compounds 7−13 was performed as described below. The dried fruits (3 kg) of M. tanarius were extracted with ethanol (2 × 10 L). This extract was subjected to sequential liquid/liquid partitioning using water (1 L), Et2O (1 L), and CH2Cl2 (700 mL) to yield a CH2Cl2 extract (35 g) after concentration in vacuo at 40 °C. This extract (30 g) was subjected to silica gel column chromatography using a gradient of nheptane−EtOAc−MeOH (4:6:0 to 0:8:2) of increasing polarity to

Figure 4. ORTEP view of the X-ray analysis of schweinfurthin E (2).

Compounds 6−13 were evaluated for their antiproliferative activities against U87 glioblastoma and A549 human lung cancer cell lines, using the known compounds vedelianin (1), schweinfurthin G (3), and mappain (5) as references. The results are presented in Table 4. All new compounds, except dimer 12, showed cytotoxic activities in the micromolar range (5 and 6) or in the nanomolar to sub-micromolar range (1−4, 7−11, and 13) on the U87 gliobastoma cell line, but only compounds 7 and 8 were active on the A549 cell line in the sub-micromolar range. As expected, schweinfurthin Q (13), which possess a hexahydroxanthene moiety, was found to be highly cytotoxic, with a similar activity to that of schweinfurthin G (3). Compounds 7 and 8, possessing respectively one or two pyranol moieties in their structure (which may be regarded as half of a hexahydroxanthene moiety), were much more potent than mappain (5) or methyl-mappain (6). Interestingly, the sub-micromolar cytotoxic activities of 7 and 8 on both cell lines 2689



Article

afford eight fractions, FA1−FA8, according to their TLC profiles. Purification of fraction FA2 (13.6 g) was carried out by normal-phase silica gel flash chromatography, using gradient mixtures from 1:0 to 2:8 of toluene−AcOH, yielding 11 fractions (FB1−FB11). Fraction FB8 was purified by reversed-phase silica gel flash chromatography with the eluent system CH3OH−H2O from 1:1 to 0:1, to obtain nine fractions, FC1−FC9. Fractions FC5 (274 mg) and FC7 (1.35 g) were selected based on their respective LC-MS profile and UV spectrum, and both fractions were subjected to preparative reversed-phase HPLC (C18 column, Kromasil, 250 × 21.2 mm, 5 μm, CH3CN−H2O 70:30 + 0.1% formic acid at 21 mL/min) to afford compounds 7 (453 mg), 8 (30 mg), 9 (4.5 mg), 10 (75 mg), and 11 (114 mg). Fraction FA6 (2.1 g) was subjected to silica gel column flash chromatography using a gradient of n-heptane−EtOAc (3:7 to 0:1) of increasing polarity to afford five fractions, FD1−FD5. Compound 13 (4.5 mg) was obtained by preparative reversed-phase HPLC (C18 column, Kromasil, 250 × 21.2 mm, 5 μm, CH3CN−H2O, 45:55 or 55:65, + 0.1% formic acid at 21 mL/min) of fraction FD4 (103 mg). Compound 12 (2.8 mg) was finally purified by preparative reversed-phase HPLC (C18 column, Kromasil, 250 × 21.2 mm, 5 μm, CH3CN−H2O, 70:30, + 0.1% formic acid at 21 mL/min) of fraction FA7 (218 mg). A detailed isolation scheme is given in the Supporting Information. Schweinfurthin K (7): orange oil; [α]24 D 0 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 325 (4.18) nm; IR νmax 959, 1146, 1318, 1606, 2929, 3374 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z 465.2643 [M + H]+ (calcd for C29H37O5+, 465.2636). Schweinfurthin L (8): orange oil; [α]24 D 0 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 325 (4.20) nm; IR νmax 1067, 1145, 1436, 1622, 2980, 3387 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z 481.2593 [M + H]+ (calcd for C29H37O6+, 481.2585). Schweinfurthin M (9): orange oil; [α]24 D −1 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 329 (4.62) nm; IR νmax 1064, 1132, 1593, 2919, 3380 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z 499.2685 [M + H]+ (calcd for C29H39O7+, 499.2690). Schweinfurthin N (10): orange oil; [α]24 D −10.5 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 329 (4.40) nm; IR νmax 850, 960, 1047, 1161, 1221, 1305, 1435, 1497, 1592, 2923, 2970, 3352 cm−1; 1H and 13 C NMR see Table 1; HRESIMS m/z 483.2711 [M + H]+ (calcd for C29H39O6+, 483.2741). Schweinfurthin O (11): orange oil; UV (CH3OH) λmax (log ε) 329 (4.49) nm; IR νmax 824, 1054, 1267, 1439, 1517, 1664, 2921, 2970, 3390 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z 449.2681 [M + H]+ (calcd for C29H37O4+, 449.2686). Schweinfurthin P (12): orange oil; [α]24 D −15.3 (c 0.2, CH3OH); UV (CH3OH) λmax (log ε) 308 (4.06) nm; IR νmax 3414 (br), 1729, 1608, 1528, 1438, 1366, 1295, 1243 cm−1; 1H and 13C NMR see Table 2; HRESIMS m/z 759.3920 [M + H]+ (calcd for C48H55O8+, 759.3891). Schweinfurthin Q (13): orange oil; [α]24 D +72.1 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 330 (4.35) nm; IR νmax 879, 892, 1057, 1074, 1230, 1250, 1394, 1406, 1930, 2901, 2972, 2988, 3675 cm−1; 1H and 13C NMR see Table 2; HRESIMS m/z 481.2587 [M + H]+ (calcd for C29H37O6+, 481.2585). X-ray Crystallographic Analysis of Compound 2. Orthorhombic, space group P212121 (no. 19), a = 10.1039(3) Å, b = 11.7521(3) Å, c = 21.9408(15) Å, V = 2605.3(2) Å3, Z = 4, T = 293 K, μ(Cu Kα) = 0.698 mm−1, Dcalc = 1.261 g/cm3, 11 972 reflections measured (8.06° ≤ 2θ ≤ 136.454°), 4643 unique (Rint = 0.0328, Rsigma = 0.0334), which were used in all calculations. The final R1 was 0.0396 (I > 2σ(I)) and wR2 was 0.1340 (all data). Absolute structure parameter, Flack x = −0.02(7) using 1493 quotients.21 Crystallographic data for compound 2 have been deposited in the Cambridge Crystallographic Data Centre (deposit no. CCDC 1499383). Copies of the data can be obtained, free of charge, from the Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/data_request/cif. Cell Culture and Proliferation Assay. Cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and were cultured according to the supplier’s instructions. Human A549 lung cancer cells were grown in RPMI 1640

supplemented with 10% fetal calf serum (FCS) and 1% glutamine. Human U87 glioblastoma cells were grown in DMEM supplemented with 10% FCS and 1% glutamine. Cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cell growth inhibition was determined by an MTS assay according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, the cells were seeded in 96-well plates (2.5 × 103 cells/well) containing 200 μL of growth medium. After 24 h of culture, the cells were treated with the test compounds at 1 and 10 μM final concentrations. After 72 h of incubation, 40 μL of resazurin was added for 2 h before recording the absorbance at 490 nm with a spectrophotometric plate reader. The percent cytotoxicity index [(OD490treated/OD490control) × 100] was calculated from three experiments. Schweinfurthin G (4) (EC50: 8 × 10−9 and 2 × 10−8 M for U87 and A549 cell lines) and monomethyl auristatin E were used as positive control substances.

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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.7b00409. Molecular network of Macaranga species, crystal data of compound 2, extraction procedure scheme, and 1D and 2D NMR spectra and HRESIMS for compounds 7−13 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: + 33 1 69 82 30 85. Fax: + 33 1 69 07 72 47. E-mail: [email protected]. *Tel: + 33 1 69 82 31 21. Fax: + 33 1 69 07 72 47. E-mail: [email protected]. ORCID

Marc Litaudon: 0000-0002-0877-8234 Fanny Roussi: 0000-0002-5941-9901 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the International Associated Laboratory (LIA: NATPROCHEMLAB) between the Centre National de la Recherche Scientifique (CNRS, ICSN, France) and the Vietnam Academy of Science and Technology (Institute of Marine Biochemistry, VAST, Vietnam), which supports the project, as well as an Investissement d’Avenir grant of the Agence Nationale de la Recherche (CEBA: ANR-10LABX-25-01). We are also grateful to the doctoral school “Innovation Thérapeutique” from Paris Saclay University for a Ph.D. fellowship for T.P. and G.J.

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REFERENCES

(1) van der Kaaden, J. E.; Hemscheidt, T. K.; Mooberry, S. L. J. Nat. Prod. 2001, 64, 103−105. (2) Yoder, B. J.; Cao, S.; Norris, A.; Miller, J. S.; Ratovoson, F.; Razafitsalama, J.; Andriantsiferana, R.; Rasamison, V. E.; Kingston, D. G. I. J. Nat. Prod. 2007, 70, 342−346. (3) Kuder, C. H. Schweinfurthins as Novel Anticancer Agents. Ph.D. Thesis, University of Iowa, Iowa City, IA, 2009; pp 19−25. (4) Thoison, O.; Hnawia, E.; Guéritte-Voegelein, F.; Sévenet, T. Phytochemistry 1992, 31, 1439−1442. (5) (a) Beutler, J. A.; Shoemaker, R. H.; Johnson, T.; Boyd, M. R. J. Nat. Prod. 1998, 61, 1509−1512. (b) Klausmeyer, P.; Van, Q. N.; Jato, J.; McCloud, T. G.; Beutler, J. A. J. Nat. Prod. 2010, 73, 479−481. 2690



Article

(6) Burgett, A. W. G.; Poulsen, T. B.; Wangkanont, K.; Anderson, D. R.; Kikuchi, C.; Shimada, K.; Okubo, S.; Fortner, K. C.; Mimaki, Y.; Kuroda, M.; Murphy, J. P.; Schwalb, D. J.; Petrella, E. C.; CornellaTaracido, I.; Schirle, M.; Tallarico, J. A.; Shair, M. D. Nat. Chem. Biol. 2011, 7, 639−647. (7) Bao, X.; Zheng, W.; Sugi, N. H.; Agarwala, K. L.; Xu, Q.; Wang, Z.; Tendyke, K.; Lee, W.; Parent, L.; Li, W.; Cheng, H.; Shen, Y.; Taylor, N.; Dezso, Z.; Du, H.; Kotake, Y.; Zhao, N.; Wang, J.; Postema, M.; Woodall-Jappe, M.; Takase, Y.; Uenaka, T.; Kingston, D. G. I.; Nomoto, K. Cancer Biol. Ther. 2015, 16, 589−601. (8) Macaranga  The Plant List. http://www.theplantlist.org/1.1/ browse/A/Euphorbiaceae/Macaranga/ (accessed Mar 5, 2017). (9) Allard, P.-M.; Péresse, T.; Bisson, J.; Gindro, K.; Marcourt, L.; Pham, V. C.; Roussi, F.; Litaudon, M.; Wolfender, J.-L. Anal. Chem. 2016, 88, 3317−3323. (10) Chi, V. V. Vietnamese Medical Plant Dictionary; Publishing House Medicine: Ho Chi Minh City, 1997; Vol. 1, p 59. (11) Kawakami, S.; Harinantenaina, L.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Takeda, Y. J. Nat. Prod. 2008, 71, 1872−1876. (12) Wang, M.; Carver, J. J.; Phelan, V. V.; Sanchez, L. M.; Garg, N.; Peng, Y.; Nguyen, D. D.; Watrous, J.; Kapono, C. A.; Luzzatto-Knaan, T.; Porto, C.; Bouslimani, A.; Melnik, A. V.; Meehan, M. J.; Liu, W.-T.; Crüsemann, M.; Boudreau, P. D.; Esquenazi, E.; Sandoval-Calderón, M.; Kersten, R. D.; Pace, L. A.; Quinn, R. A.; Duncan, K. R.; Hsu, C.C.; Floros, D. J.; Gavilan, R. G.; Kleigrewe, K.; Northen, T.; Dutton, R. J.; Parrot, D.; Carlson, E. E.; Aigle, B.; Michelsen, C. F.; Jelsbak, L.; Sohlenkamp, C.; Pevzner, P.; Edlund, A.; McLean, J.; Piel, J.; Murphy, B. T.; Gerwick, L.; Liaw, C.-C.; Yang, Y.-L.; Humpf, H.-U.; Maansson, M.; Keyzers, R. A.; Sims, A. C.; Johnson, A. R.; Sidebottom, A. M.; Sedio, B. E.; Klitgaard, A.; Larson, C. B.; Boya, P. C. A.; TorresMendoza, D.; Gonzalez, D. J.; Silva, D. B.; Marques, L. M.; Demarque, D. P.; Pociute, E.; O’Neill, E. C.; Briand, E.; Helfrich, E. J. N.; Granatosky, E. A.; Glukhov, E.; Ryffel, F.; Houson, H.; Mohimani, H.; Kharbush, J. J.; Zeng, Y.; Vorholt, J. A.; Kurita, K. L.; Charusanti, P.; McPhail, K. L.; Nielsen, K. F.; Vuong, L.; Elfeki, M.; Traxler, M. F.; Engene, N.; Koyama, N.; Vining, O. B.; Baric, R.; Silva, R. R.; Mascuch, S. J.; Tomasi, S.; Jenkins, S.; Macherla, V.; Hoffman, T.; Agarwal, V.; Williams, P. G.; Dai, J.; Neupane, R.; Gurr, J.; Rodríguez, A. M. C.; Lamsa, A.; Zhang, C.; Dorrestein, K.; Duggan, B. M.; Almaliti, J.; Allard, P.-M.; Phapale, P.; Nothias, L.-F.; Alexandrov, T.; Litaudon, M.; Wolfender, J.-L.; Kyle, J. E.; Metz, T. O.; Peryea, T.; Nguyen, D.-T.; VanLeer, D.; Shinn, P.; Jadhav, A.; Müller, R.; Waters, K. M.; Shi, W.; Liu, X.; Zhang, L.; Knight, R.; Jensen, P. R.; Palsson, B. Ø.; Pogliano, K.; Linington, R. G.; Gutiérrez, M.; Lopes, N. P.; Gerwick, W. H.; Moore, B. S.; Dorrestein, P. C.; Bandeira, N. Nat. Biotechnol. 2016, 34, 828−837. (13) Pailee, P.; Sangpetsiripan, S.; Mahidol, C.; Ruchirawat, S.; Prachyawarakorn, V. Tetrahedron 2015, 71, 5562−5571. (14) Pecchio, M.; Solís, P. N.; López-Pérez, J. L.; Vásquez, Y.; Rodríguez, N.; Olmedo, D.; Correa, M.; San Feliciano, A.; Gupta, M. P. J. Nat. Prod. 2006, 69, 410−413. (15) Belofsky, G.; French, A. N.; Wallace, D. R.; Dodson, S. L. J. Nat. Prod. 2004, 67, 26−30. (16) Pappas, S. P.; Zehr, R. D.; Alexander, J. E. J. Heterocycl. Chem. 1970, 7, 1215−1217. (17) Sobolev, V. S.; Neff, S. A.; Gloer, J. B. J. Agric. Food Chem. 2010, 58, 875−881. (18) Syah, Y. M.; Achmad, S. A.; Ghisalberti, E. L.; Hakim, E. H.; Iman, M. Z.; Makmur, L.; Mujahiddin, D. Fitoterapia 2000, 71, 630− 635. (19) Yao, C.-S.; Huang, K.-S.; Lin, M.; Yang, Q.-Y. J. Asian Nat. Prod. Res. 2013, 15, 693−695. (20) Topczewski, J. J.; Wiemer, D. F. Tetrahedron Lett. 2011, 52, 1628−1630. (21) Parsons, S.; Flack, H. Acta Crystallogr., Sect. A: Found. Crystallogr. 2004, 60, 61−61.

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