(+)- and (−)-Ecarlottones, Uncommon Chalconoids ... - ACS Publications

Nov 21, 2017 - Department of Chemistry, Faculty of Science, University Malaya, Kuala Lumpur 50603, Malaysia. •S Supporting Information. ABSTRACT: Fo...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/jnp

(+)- and (−)-Ecarlottones, Uncommon Chalconoids from Fissistigma latifolium with Pro-Apoptotic Activity Charlotte Gény,† Alma Abou Samra,† Pascal Retailleau,† Bogdan I. Iorga,† Hristo Nedev,† Khalijah Awang,‡ Fanny Roussi,† Marc Litaudon,*,† and Vincent Dumontet*,† †

Institut de Chimie des Substances Naturelles, CNRS-ICSN UPR2301, Université Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France ‡ Department of Chemistry, Faculty of Science, University Malaya, Kuala Lumpur 50603, Malaysia S Supporting Information *

ABSTRACT: Four new compounds, (+)- and (−)-ecarlottone (1), (±)-fislatifolione (5), (±)-isofislatifolione (6), and (±)-fislatifolic acid (7), and the known desmethoxyyangonin (2), didymocarpin-A (3), and dehydrodidymocarpin-A (4) were isolated from the stem bark of Fissistigma latifolium, by means of bioassay-guided purification using an in vitro affinity displacement assay based on the modulation of Bcl-xL/Bak and Mcl-1/Bid interactions. The structures of the new compounds were elucidated by NMR spectroscopic data analysis, and the absolute configurations of compounds (+)-1 and (−)-1 were assigned by comparison of experimental and computed ECD spectra. (−)-Ecarlottone 1 exhibited a potent antagonistic activity on both protein−protein associations with Ki values of 4.8 μM for Bcl-xL/Bak and 2.4 μM for Mcl-1/Bid.

N

been associated with resistance to anticancer drugs.16 Targeting both proteins simultaneously appeared as an attractive apoptosis-based strategy to discover novel potential anticancer agents. The genus Fissistigma comprises about 65 species mainly distributed from East India to Northeast Australia. The species F. latifolium var. ovoideum occurs only in the Malay Peninsula.17 Many plants of this genus are widely used in traditional Chinese medicine to treat inflammation, tuberculosis, infections, wounds, bone fractures, hemorrhages, rheumatoid arthritis, and inflammatory disease and to enhance blood circulation.18−20 Previous phytochemical investigations of Fissistigma species (including the study of the MeOH extract of F. latifolium) revealed the presence of aporphines, protoberberins, and aristolactams alkaloids, 21−23 together with various flavonoids such as furanones, cyclopentenones, chalconoids, and Diels−Alder adduct chalconoids.19,24−26

aturally occurring chalcone derivatives represent an interesting class of compounds from the standpoint of their ability to act as dienophiles in enzyme-catalyzed Diels− Alder reactions.1 Panduratins,2−4 crinatusins,5 fissistins,6 schefflerin,7 krachaizins,8 and nicolaiodesins,4,8,9 resulting from Diels−Alder cycloaddition between chalcones and monoterpenes as a diene such as myrcene or β-ocimene, were mainly isolated from species belonging to the families Zingiberaceae and Annonaceae (Fissistigma, Cyathocalyx, and Uvaria genera). In the field of oncology, some of these prenylated chalcones exhibited cytotoxic activity against the KB,6 NCI-H1299, MDA-MB-231, and HCT-116 cell lines and PANC-1 human pancreatic cancer cells under nutritiondeprived conditions,9 and some of them inhibited cell growth and induced apoptosis of HT-29 and A549 cancer cells.10,11 For example, panduratin A was shown to inhibit aminopeptidase N, which plays an important role in tumor-cell invasion, extracellular matrix degradation by tumor cells, and tumor metastasis.8 The biological potency of these molecules has raised interest in the synthesis of analogues using Diels−Alder reactions.12 As part of a continuing search for novel plant-derived anticancer agents,13−15 the EtOAc stem bark extract of Fissistigma latifolium var. ovoideum (King) J. Sinclair was found to significantly disrupt interactions between the antiapoptotic proteins Bcl-xL and Mcl-1 with pro-apoptotic proteins Bak and Bid, respectively. Overexpression of Bcl-xL and Mcl-1 plays a decisive role in cancer development and has © 2017 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION In the present investigation, the bioassay- and 1H NMR-guided fractionation of the stem bark EtOAc extract of F. latifolium var. ovoideum led to the isolation, structural characterization, and biological evaluation of four new Diels−Alder chalconemyrcene adducts, (+)- and (−)-ecarlottone (1), (±)-fislatifolione (5), (±)-isofislatifolione (6), and (±)-fislatifolic acid (7), Received: June 9, 2017 Published: November 21, 2017 3179

DOI: 10.1021/acs.jnatprod.7b00494 J. Nat. Prod. 2017, 80, 3179−3185

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR Spectroscopic Data for 1 (600 MHz, CDCl3) and 5, 6, and 7 (500 MHz, CDCl3)

and the known desmethoxyyangonin (2), didymocarpin-A (3), and dehydrodidymocarpin-A (4). Their structures were elucidated based on NMR and HRMS data, and the absolute configurations of compounds (+)-1 and (−)-1 were assigned by comparison of experimental and computed ECD spectra. Approximately 500 EtOAc extracts, previously filtered on polyamide cartridges, of plants obtained from different tropical rain forests, were screened for their ability to modulate Bcl-xL/ Bak and Mcl-1/Bid interactions at 10 μg/mL. Among the active extracts, the stem bark of F. latifolium var. ovoideum showed significant binding affinities to both proteins Bcl-xL and Mcl-1 with Ki values of 44% and 65%, respectively. Two different isolation procedures were then executed. The first one, based on a bioassay-guided isolation procedure using fluorescentbased binding assays according to reported methodologies,13,14 afforded racemic mixtures of (±)-ecarlottone (1), (±)-fislatifolione (5), and compounds 2−4. Based on structural features of the bioactive (±)-ecarlottone (1), a 1H NMR-guided isolation was carried out on the second part of the extract, leading to compounds 5−7, also as racemic mixtures. Compound 1 was assigned the molecular formula C26H28O6 on the basis of the HRESIMS protonated [M + H]+ ion at m/z 437.1962 (calcd [M + H]+, m/z 437.1959) along with its 13C NMR data, indicating 13 indices of hydrogen deficiency. The IR spectrum showed a broad absorption band at 3033 cm−1 referring to hydroxy groups and sharp bands at 1622, 1596, and 1538 cm−1 suggesting the presence of carbonyl functions. The UV spectra of 1 showed an absorption maximum at 304 nm consistent with a conjugated system. Several NMR experiments were carried out at various temperatures in order to determine the best resolved NMR spectrum for hydroxy groups involved in keto−enol tautomerism. A temperature of 243 K, which favored one form of the keto−enol equilibrium in CDCl3, was selected (Figure S1, Supporting Information). The analysis of 1 H, 13C, and HSQC NMR data of 1 (Tables 1 and 2, Figures S2−S4, Supporting Information) revealed the presence of an aromatic moiety (δH 7.16 3H, 7.24 2H; δC 126.9, 127.1, 128.8, 143.9), two methyl groups (δH 1.62 3H, 1.69 3H; δC 17.9, 26.1), a methoxy group (δH 3.90 3H; δC 60.8), four methylene groups (δH 1.98 2H, 2.07 2H, 2.22 2H, 2.29, 2.42; δC 29.9, 30.8, 37.2, 37.8), four methines including two olefinic (δH 5.14, 5.54; δC 117.9, 123.8), one hydroxy group (δH 17.6), four quaternary carbons, and six oxygenated carbons, four of which were deshielded (δC 175.4, 178.6, 179.7, 204.8). HMBC and COSY correlations (Figure S5, Supporting Information) allowed the

1 position 1 2 3 4 5 6 7 8 9 11 12 2′/4′/6′ 3′/5′ 2″ OMe-4″ OH-1″

5

6

7

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

3.22, ddd (11.2, 9.6, 6.2) 4.55, ddd (11.2, 9.6, 6.2) 2.29, m 2.42, m 5.54, br s

3.06, ddd (11.2, 9.6, 6.2) 3.00, ddd (11.2, 9.2, 6.2) 2.31, m

2.93, ddd (11.0, 10.7, 5.6) 3.01, ddd (11.0, 10.6, 5.2) 2.09, m

3.05, td (10.7, 5.4) 2.86, td (10.7, 8.0) 2.42, br s

5.53, s

5.48, s

2.22, m

2.25, m

5.48, s 2.23, m

1.98, 2.07, 5.14, 1.62, 1.69, 7.16, 7.24,

2.04, m 2.12 m 5.13, m 1.64, s 1.73, m 7.23, m 7.30, m 1.88, s

2.01, 2.09, 5.09, 1.60, 1.68, 7.17, 7.25, 1.81,

m m t (7.0) s s m m

m m m s m m m s

2.24, td (17.5, 5.5) 2.02, m 2.10, m 5.11, m 1.61, s 1.71, s 7.23, m 7.30, m

3.90, s 16.61, s

2D structure of 1 to be determined as depicted in Figure 1. However, the substitution pattern of the quinonoid moiety still needs to be established. A close examination of the COSY spectrum revealed two spin systems (C-7 to C-9 and C-4 to C-6) as depicted in Figure 1. Based on HMBC correlations between H-7 (δH 1.98) and C4 (δC 117.9) and C-6 (37.8), the prenyl side chain is most probably connected to the cyclohexene moiety at C-5 (δC 137.9), while correlations between H-1 (δH 3.22) and C-2′/6′ (δC 127.1) suggested that a benzene ring is attached to the cyclohexene moiety at C-1 (δC 42.2). The structure of the quinonoid moiety was deduced from the molecular formula, the remaining six indices of hydrogen deficiency, and analysis of the 13 C NMR and X-ray crystallographic data (Figure 2). HMBC correlations from HO-1″ (δH 17.61) to C-2 (δC 45.0), C-1″ (204.8), C2″ (108.4), C-3″ (179.7), and C-7″ (175.4) supported the quinonoid moiety to be located at C-2 of the cyclohexene moiety. The relative configuration of the stereogenic carbons was determined from the analysis of the vicinal coupling constant 3180

DOI: 10.1021/acs.jnatprod.7b00494 J. Nat. Prod. 2017, 80, 3179−3185

Journal of Natural Products

Article

Table 2. 13C NMR Spectroscopic Data for 1 (175 MHz, CDCl3) and 5, 6, 7, and 8 (125 MHz, CDCl3) 1

5

6

7

position

δH, type

δH, type

δH, type

δH, type

1 2 3 4 5 6 7 8 9 10 11 12 1′ 2′/6′ 3′/5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ OMe-4″

42.2, CH 45.0, CH 30.8, CH2 117.9, CH 137.9, C 37.8, CH2 37.2, CH2 29.9, CH2 123.8, CH 132.3, C 17.9, CH3 26.1, CH3 143.9, C 127.1, CH 128.8, CH 126.9, CH 204.8, C 108.4, C 179.7, C 139.3, C 143.9, C 178.6, C 175.4, C 60.8, CH3

43.3 53.4 29.1 118.8, CH 137.7, C 37.3 37.5 26.6 124.2 131.9 17.9 25.9 144.5 127.7 128.8 126.8 212.5 29.9, CH3

43.1 53.8 32.0 136.1, C 120.5, CH 34.2 37.6 26.6 124.2 132.0 17.9 25.9 144.3 127.7 128.8 127.2 212.4 30.0, CH3

42.9 45.7 29.5 118.5, CH 137.7, C 36.9 37.5 26.5 124.2 131.9 17.9 25.9 144.2 127.6 128.7 126.8 179,0

The racemic mixture (1) was subjected to chiral-phase supercritical fluid chromatography to afford enantiomers (+)-1 and (−)-1, whose absolute configurations were assigned by comparing the experimental and computed electronic circular dichroism (ECD) spectra.27 The experimental ECD spectrum of (+)-ecarlottone showed a negative Cotton effect (CE) in the n → π* region (300−320 nm) and a positive CE in the π → π* region (260−280 nm). As expected, Cotton effects are opposite in the case of (−)-ecarlottone (Figure S7, Supporting Information). The computed ECD spectrum starting from the X-ray structure of the (S,S)-enantiomer agreed well with the experimental spectrum of (+)-ecarlottone, whereas the computed spectrum of the (R,R)-enantiomer was similar to the experimental spectrum of (−)-ecarlottone. Thus, the absolute configuration was assigned as (1S,2S) and (1R,2R) for compounds (+)-1 and (−)-1, respectively. Compounds (+)-1 and (−)-1 were given the trivial names (+)-ecarlottone and (−)-ecarlottone, respectively, according to their scarlet red crystal color. Compounds (+)-1 and (−)-1 are structurally close to fissistine isolated from F. lanuginosum6 and to panduratins,2−4 crinatusins,5 schefflerins,7 krachaizins,8 and nicolaiodesins,4,9 but none of these compounds contain such a substituted o,pquinone moiety. Yet, several compounds carrying an o,pquinone moiety have been described from natural sources, e.g., dehydro-α-lapachone and picrasidine V from the plants Dolichandrone crispa (Bignoniaceae)28 and Picrassma quassioides (Simaroubaceae), respectively,29 elisapterosin from the sponge Pseudopterogorgia elisabethae (Gorgoniidae),30 and islandoquinone (Cetraria islandica, Parmeliaceae),31 fredericamycin E32 (Streptomyces griseus, Streptomycetaceae), chaetoglobinol A (Chaetomium globosum, Chaetomiaceae),33 and 2-acetoxyrhizopogone (Rhizopogon pumilionus, Rhizppogonaceae)34 from lichens or microorganisms. Fredericamycin E displayed cytotoxic activity against a selected set of cancer cells,32 while chaetoglobinol A exhibited antibacterial activities and showed free radical scavenging capacity.33 The structure of compound 2, desmethoxyyangonin, was elucidated through comparison with literature data.35 It should be noted that the 1H and 13C NMR data of 2 are similar to those of methyl-5-styrylfuran-2-carboxylate isolated from Renealmia nicolaioide (Piperaceae).9 The structure proposed by Gu and collaborators9 is probably wrong due to the furan moiety derived from the molecular formula and the number of indices of hydrogen deficiency. The 1H and 13C NMR chemical shifts are not consistent with experimental and calculated data. The structure of compound 3, didymocarpin-A, was elucidated through comparison of its physical data with

Figure 1. COSY (bold) and HMBC (blue arrows) correlations of compound 1.

pattern of JH‑1/H‑2 displayed in the 1H NMR spectrum recorded in CDCl3 at room temperature (298 K). H-1 and H-2 (δH 2.22 and 1.98, respectively) resonances appeared as ddd (11.2, 9.6, and 6.2 Hz) and supported a trans-diaxial orientation for these two protons. This configuration was further confirmed by X-ray crystallographic analysis (Figure 2, structure refinement details, Figure S6, Supporting Information).

Figure 2. (a) X-ray ORTEP plot drawing of compound (−)-1 (1R,2R) and ellipsoids drawn at 30% probability shown for clarity. (b) 3D representation of (+)-1 and (−)-1 in the crystal mesh corresponding to a monoclinic crystal system (α = 90°, β = 108°, γ = 90°). 3181

DOI: 10.1021/acs.jnatprod.7b00494 J. Nat. Prod. 2017, 80, 3179−3185

Journal of Natural Products

Article

literature data.36 Dydimocarpin-A spontaneously converted into dehydrodidymocarpin-A (4) via autoxidation after 2 days of air exposure at room temperature. Dehydrodidymocarpin-A (4) was synthesized previously,37 but we report here its 1H NMR data for the first time (Figure S8, Supporting Information). Compounds 5−7 were isolated as optically inactive mixtures. Compounds 5 and 6 were assigned the molecular formula C20H26O on the basis of the same HRESIMS protonated [M + H]+ at m/z 283.2062 (calcd [M + H]+, m/z 283.2056), indicating eight indices of hydrogen deficiency. A sharp absorption band at 1709 cm−1 was observed in the IR spectra suggesting the presence of only one carbonyl function. The spectroscopic data of compounds 5 and 6 were similar to those of compound 1, but carbon resonances corresponding to the o,p-quinone moiety were absent in 5 and 6. Analysis of the 1D (Tables 1 and 2, Figures S8−S16, Supporting Information) and 2D NMR data (COSY, edited HSQC, and HMBC) revealed a close structural similarity between compounds 5 and 6. Indeed, both compounds possess an aromatic moiety (δH 7.23 3H, 7.30; δC 126.8, 127.7, 128.8, 144.5) for 5 and (δH 7.17 3H, 7.25; δC 127.2, 127.7, 128.8, 144.3) for 6, three methyl groups (δH 1.64 3H, 1.73 3H, 1.88 3H; δC 17.9, 25.9, 29.9) for 5 and (δH 1.60 3H, 1.68 3H, 1.81 3H; δC 17.9, 25.9, 30.0) for 6, four methylene groups (δH 2.31 2H, 2.04 2H, 2.12 2H, 2.25 2H; δC 26.6, 29.1, 37.3, 37.5) for 5 and (δH 2.01 2H, 2.09 4H, 2.23 2H; δC 26.6, 32.0, 34.2, 37.6) for 6, four methines including two olefinic protons (δH 5.13, 5.53; δC 118.8, 124.2) for 5 and (δH 5.09, 5.48; δC 120.5, 124.2) for 6, and three quaternary carbons and a ketocarbonyl (δC 212.5) for 5 and (δC 212.4) for 6. The main structural difference between compounds 5 and 6 involved the location of the prenyl side chain on the cyclohexene moiety, at C-5 (5) or C-4 (6). Examination of both COSY spectra indicated the presence of two spin systems, the first one (C-7 to C-9) being identical for both compound, whereas the second one displayed two different carbon fragments, C-4 to C-6 in 5 and C-3 to C-5 in 6, as depicted in Figure 3.

and H-2 was deduced from the multiplicity of proton signals (ddd) and the magnitude of the 3J1,2 values. Compounds 5 and 6 were given the trivial names (±)-fislatifolione and (±)-isofislatifolione, respectively. Compound 7 was assigned the molecular formula C20H26O on the basis of its HRESIMS protonated [M + H]+ ion at m/z 285.1850 (calcd [M + H]+, m/z 285.1849), from which eight indices of hydrogen deficiency can be deduced. The NMR data of compound 7 (Figures S17−S20, Supporting Information) are reminiscent of those of compound 5, but with a hydroxycarbonyl group (δC 179.0) at C-2 in 7, instead of an acetyl group in compound 5. The HMBC spectrum of 7 showed correlations from H-1 and H-2 to C-1″, thus confirming the attachment of the hydroxycarbonyl group at C-2 of the cyclohexene moiety. As previously described for compounds 1, 5, and 6, a trans-diaxial relationship of the vicinal protons H-1 and H-2 was established. Compound 7 was given the trivial name (±)-fislatifolic acid. The biosynthesis of compounds 1 and 5−7 could involve an enzymatic catalyzed Diels−Alder reaction between myrcene (8) and the quinochalcone subunit (9) for (±)-ecarlottones (1) or between myrcene (8) and cinnamic acid (10) for (±)-fislatifolic acid (7) (Figure 4).

Figure 4. Putative biosynthetic pathway proposed for (±)-ecarlottone (1) and (±)-fislatifolic acid (7).

The ability of compounds 1−7 to antagonize Bcl-xL/Bak and Mcl-1/Bid association was determined using a fluorescence polarization assay (Table 3, Figure S21, Supporting InformaTable 3. Biological Evaluation of Compounds 1, (+)-1 and (−)-1, and 5−7 in Bcl-xL/Bak and Mcl-1/Bid Displacement Assays

Figure 3. COSY (bold) and HMBC (blue arrows) correlations of compounds 5 and 6. a

In the HMBC spectrum of compound 5, correlations from H-7 to C-4 and C-6 suggested the attachment of the prenyl chain at C-5, the same as in 1, whereas in compound 6, correlations from H-7 to C-3 and C-5 connected the prenyl chain at C-4. In addition, HMBC correlations from H-2 to the ketocarbonyl C-1″ and from methyl protons H3-2″ to C-2 indicated that an acetyl group is attached at C-2 of the cyclohexene moiety. Thus, the structures of regioisomers 5 and 6 were established as shown. These compounds have never been reported, but they share a common carbon skeleton with the prenylated chalcones, fissistine, and isofissistine. The trans-diaxial orientation of H-1

compound

Bcl-xL/Bak Ki (μM)

1 (+)-1 (−)-1 2 3/4 5 6 7 meiogynine Aa ABT−737a

17.5 ± 0.2 >23 4.8 ± 0.1 >23 >23 >23 >23 >23 2.6 ± 0.3 33 >33 >33 >33 >33 8.6 20.8

± 0.1 ± 0.3 ± 0.1

± 0.5 ± 2.9

Reference compounds.

tion).13 All compounds were found inactive except for (+)- and (−)-ecarlottones (1). The levorotatory enantiomer (−)-1 exhibited the most potent binding affinity for proteins Bcl-xL and Mcl-1 with Ki values of 4.8 and 2.4 μM, respectively, while the dextrorotatory enantiomer (+)-1 only showed binding affinity for Mcl-1 with a Ki of 7.4 μM. Since compounds 5−7 were inactive, the prominent role of the o,p-quinone moiety can be inferred. In addition, it can be postulated that the spatial position of this unit is essential for a dual inhibiting activity on both proteins. The cytotoxicity of (±)-ecarlottone (1) was further investigated on the HUVEC and KB cell lines, but no 3182

DOI: 10.1021/acs.jnatprod.7b00494 J. Nat. Prod. 2017, 80, 3179−3185

Journal of Natural Products

Article

μm) using CO2−2-propanol (92:8) plus 0.2% tetrafluoroacetic acid (TFA) (100 bar, 298 K, 40 injections) followed by purification on reversed-phase flash chromatography using MeCN−MeOH−2-propanol to afford (+)-1 (2 mg) and (−)-1 (3 mg). A second part of the air-dried bark (917 g) was extracted with EtOAc (3 × 4.5 L) to yield a crude extract (22.3 g) after concentration in vacuo at 40 °C. The extract was filtered on polyamide to afford a filtered extract (7.9 g), which was subjected to silica gel flash chromatography (Interchim puriFlash, 120 g) using a gradient of n-heptane−CH2Cl2 (100:0 to 0:100), CH2Cl2−MeOH (100:0 to 50:50), then CH2Cl2−MeOH (50:50) + 0.1% TFA to afford fraction F′11. Fraction F′11 (52 mg) was purified by preparative HPLC using MeCN−H2O (70:30) + 0.1% formic acid to afford 5 (5 mg) and 6 (2 mg). (±)-Ecarlottone (1): scarlet red crystal; [α]25D 0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 304 (4.73) nm; IR (neat) νmax 3303, 2914, 2853, 1622, 1596, 1538, 1448, 1323, 1241, 1188, 1082, 1011 cm−1; 1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 175 MHz) see Table 1; HRESIMS m/z [M + H]+ 437.1962 (calcd for C19H31O3 437.1959). (+)-Ecarlottone (1): dark orange oil; [α]25D −32 (c 0.1, CHCl3); ECD (c 0.1 × 10−3, CHCl3) λmax (Δε) 369 (0.10), 310 (−5.30), 270 (5.38), 235 (0.04). (−)-Ecarlottone (1): dark orange oil; [α]25D +35 (c 0.1, CHCl3); ECD (c 0.1 × 10−3, CHCl3) λmax (Δε) 347 (1.31), 306 (3.60), 270 (−7.02), 236 (0.26). (±)-Fislatifolione (5): pale oil; [α]25D 0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 260 (2.51) nm; IR (neat) νmax 2916, 1709, 1356, 1159, 758, 700 cm−1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 1; HRESIMS m/z [M + H]+ 283.2062 (calcd for C19H31O3 283.2056). (±)-Isofislatifolione (6): pale oil; [α]25D 0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 260 (2.51) nm; IR (neat) νmax 2916, 1709, 1356, 1159, 758, 700 cm−1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 1; HRESIMS m/z [M + H]+ 283.2062 (calcd for C19H31O3 283.2056). (±)-Fislatifolic acid (7): yellowish oil; [α]25D 0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 259 (2.72) nm; IR (neat) νmax 3030, 2914, 2846, 1703, 1494, 1435, 1289, 1231, 1201, 943, 759, 698 cm−1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 1; HRESIMS m/z [M + H]+ 285.1850 (calcd for C19H31O3 285.1849). Biological Assays. The modulations of the interaction between Bcl-xL and Bak and between Mcl-1 and Bid were evaluated by competition against a fluorescent-labeled reference compound (fluorescent-tagged BH3 domain of the protein Bak or Bid), as described earlier.14 Meiogynine A and ABT-737 were used as positive controls. Results are expressed as Ki, the concentration corresponding to 50% of such inhibition, and corrected for experimental conditions according to the Kenakin rearranged equation.38 The assays were run in triplicate. The cytotoxic activities of the pure compounds were evaluated against the cancer cell lines HUVEC and KB. Cytotoxicity assays were performed according to a published procedure.39 X-ray Crystallography. Complete crystal and structure refinement data are given in Table S7, Supporting Information. A suitable red single crystal of 1 obtained in a mixture of MeCN−MeOH solvent was irradiated at low temperature (193 K). The racemic compound is found in the asymmetric unit in a head-to-tail dimeric assembly. Crystal data for 1: C26H28O6, M = 436.48, size 0.28 × 0.23 × 0.08 mm3, monoclinic, space group P21, a = 24.2616(18) Å, b = 7.3548(5) Å, c = 26.7947(19) Å, β = 108.147(8)°, V = 4543.4(6) Å3, T = 193(2) K, Z = 8, d = 1.276 Mg/cm3, λ(Cu Kα) = 1.541 78 Å, F(000) = 1856, reflections collected/unique 27 198/8181 [R(int) = 0.0458], h (−21/ 29), k (−8/32), l (−32.30), final R indices R1 = 0.0667 and wR2 = 0.1387, GOF = 1.031. Computation of ECD Spectra. The two enantiomers (+)-1 and (−)-1 extracted from the racemic crystal structure were optimized at the B3LYP/6-311+G(d,p) level; then the ECD spectrum was computed at the B3LYP/TZVP level (60 excited states), using Gaussian09.40 The computed and experimental ECD spectra were superimposed using SpecDis.41 The conformations from the crystal

cytotoxicity was observed at a concentration of up to 10 μM. The highly hydrophilic nature of these compounds can lead to poor penetration through the cell membrane and thus could explain the absence of cytotoxicity. In conclusion, the study of F. latifolium var. ovoideum led to the identification of four new Diels−Alder adducts (1 and 5−7) along with desmethoxyyangonin (2), didymocarpin-A (3), and its oxidation product, dehydrodidymocarpin-A (4). (−)-Ecarlottone (1), possessing an uncommon o,p-quinone moiety, exhibited potent binding affinity for both anti-apoptotic proteins Bcl-xL and Mcl-1, making this compound a good candidate for further studies.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with an Anton Paar MCP200 polarimeter equipped with a sodium lamp and a 1 cm cell. The UV spectra were recorded on a Varian Cary 100 Scan spectrophotometer using CaryWing software. The ECD spectra were measured in CHCl3 on a Jasco J-810 ECD spectrometer, in a 1 cm quartz cell, and were analyzed with the Spectro Manager software. The IR spectra were obtained on a Nicolet FTIR 205 spectrophotometer. The NMR spectra were recorded on a Bruker 500 Avance III operating at 500 (1H) and 125 MHz (13C) or on a Bruker Avance III 600US2 spectrometer equipped with a 1 mm TXI microprobe (1H and 2D NMR) or a 5 mm BBO probe (13C) operating at 600 (1H) and 150 MHz (13C). 2D NMR experiments were performed using standard Bruker programs. All measurements were carried out at 293 K unless specified otherwise. Chemical shifts are reported as δ values (ppm) with the residual solvent signal as internal reference, J in Hz. Standard pulse sequences from the Topspin 2.1 software package were used, and data were analyzed using NMRnotebook V2.7. For HRESIMS, a Waters LCT Premier XE equipped with ESI was used. The mass spectra were recorded in the range m/z 50−1500 in positive ion mode with the aid of Empower control software (Waters). Kromasil analytical, semipreparative, and preparative C18 columns (250 × 4.5 mm; 250 × 10 mm and 250 × 21,2 mm i.d. 5 μm Thermo) were used for preparative HPLC separations using a Waters autopurification system equipped with a binary pump (Waters 2525), a UV−vis diode array detector (190−600 nm, Waters 2996), and a PL-ELS 1000 ELSD Polymer Laboratory detector. All solvents and analytical TLC plates (Si gel 60 F 254) were purchased from SDS (France). Polyamide DC 6 was purchased from Macherey-Nagel (Chromabond PA, 1 g). The X-ray diffraction experiments on compound X were performed on a Rigaku MM007 HF rotating-anode diffractometer, equipped with a Rapid II curved image plate detector and using Cu Kα radiation (λ = 1.541 87 Å). Plant Material. The bark of F. latifolium was collected in Dungun (Terengganu Province), Malaysia, in October 1995. The plant was identified by T. Leong Eng from the University of Malaya. A voucher specimen (KL-4426) was deposited in the herbarium of the Department of Chemistry of the Science Faculty, University of Malaya, Kuala Lumpur (Malaysia). Extraction and Isolation. The air-dried bark (1.5 kg) of F. latifolium was extracted with EtOAc (3 × 3 L) to yield a crude extract (24.6 g) after concentration in vacuo at 40 °C. The extract was dissolved in a mixture of EtOAc−MeOH (80:20) and filtered on polyamide to afford a dried filtered extract (16.4 g), which was subjected to silica gel flash chromatography (RediSep Teledyn Isco, 120 g) using a gradient of n-heptane−CH2Cl2 (100:0 to 0:100), then CH2Cl2−MeOH (100:0 to 80:20), to afford three fractions, F9, F10, and F12. Fractions F9 (500 mg) and F10 (200 mg) were purified separately by preparative HPLC using MeCN−H2O (65:35) + 0.1% formic acid to afford 1 (170 mg) and 7 (13 mg). Fraction F12 (84 mg) was purified by preparative HPLC using MeCN−H2O (50:50) to afford 2 (1.5 mg) and 3 (1 mg). The compounds (−)-1 and (+)-1 (30 mg) were separated by chiral SFC (AD-H Chiralpak 4.6 × 150 mm, 5 3183

DOI: 10.1021/acs.jnatprod.7b00494 J. Nat. Prod. 2017, 80, 3179−3185

Journal of Natural Products

Article

structure were confirmed to be the lowest energy conformations by a protocol involving a conformational search using MacroModel (Schrödinger LLC, https://www.schrodinger.com/) and the geometry optimization of the resulting conformers using Gaussian09.39

■

(5) Shibata, K.; Tatsukawa, A.; Umeoka, K.-I.; Lee, H. S.; Ochi, M. Tetrahedron 2000, 56, 8821−8824. (6) Alias, Y.; Awang, K.; Hadi, A. H. A.; Thoison, O.; Sévenet, T.; Païs, M. J. Nat. Prod. 1995, 58, 1160−1166. (7) Nkunya, M. H. H.; Achenbach, H.; Renner, C.; Waibel, R.; Weenen, H. Phytochemistry 1990, 29, 1261−1264. (8) Morikawa, T.; Funakoshi, K.; Ninomiya, K.; Yasuda, D.; Miyagawa, K.; Matsuda, H.; Yoshikawa, M. Chem. Pharm. Bull. 2008, 56, 956−962. (9) Gu, J.-Q.; Park, E. J.; Vigo, J. S.; Graham, J. G.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 2002, 65, 1616−1620. (10) Yun, J. M.; Kwon, H. J.; Mukhtar, H.; Hwang, J. K. Planta Med. 2005, 71, 501−507. (11) Cheah, S. C.; Appleton, D. R.; Lee, S. T.; Lam, M. L.; Hadi, A. H. A.; Mustafa, M. R. Molecules 2011, 16, 2583−2598. (12) Han, J. G.; Jones, A. X.; Lei, X. G. Synthesis 2015, 47, 1519− 1533. (13) Gény, C.; Rivière, G.; Bignon, J.; Birlirakis, N.; Guittet, E.; Awang, K.; Litaudon, M.; Roussi, F.; Dumontet, V. J. Nat. Prod. 2016, 79, 838−844. (14) Apel, C.; Geny, C.; Dumontet, V.; Birlirakis, N.; Roussi, F.; Pham, V. C.; Doan Thi Mai, H.; Nguyen, V. H.; Chau, V. M.; Litaudon, M. J. Nat. Prod. 2014, 77, 1430−1437. (15) Azmi, M. N.; Geny, C.; Leverrier, A.; Litaudon, M.; Dumontet, V.; Birlirakis, N.; Gueritte, F.; Leong, K. H.; Halim, S. N.; Mohamad, K.; Awang, K. Molecules 2014, 19, 1732−1747. (16) Warr, M. R.; Shore, G. C. Curr. Mol. Med. 2008, 8, 138−147. (17) Irawan, B. URL: http://pustaka.unpad.ac.id/wp-content/ uploads/2010/05/malesian_species_of_fissistigma.pdf, 2005. (18) Yang, Z.; Lu, W.; Ma, X.; Song, D. Phytomedicine 2012, 19, 301−305. (19) Lien, T. P.; Porzel, A.; Schmidt, J.; Van Sung, T.; Adam, G. Phytochemistry 2000, 53, 991−995. (20) Hwang, T. L.; Li, G. L.; Lan, Y. H.; Chia, Y. C.; Hsieh, P. W.; Wu, Y. H.; Wu, Y. C. Free Radical Biol. Med. 2009, 46, 520−528. (21) Alias, A.; Hazni, H.; Mohd Jaafar, F.; Awang, K.; Hadiani Ismail, N. Molecules 2010, 15, 4583−4588. (22) Vichien, J.; Rapepol, B.; Porntip, T.; Rungruedee, R.; Kittisak, L. ScienceAsia 1999, 25, 031−033. (23) Chia, Y.-C.; Chang, F.-R.; Teng, C.-M.; Wu, Y.-C. J. Nat. Prod. 2000, 63, 1160−1163. (24) Chia, Y.-C.; Chang, F.-R.; Wu, Y.-C. Tetrahedron Lett. 1999, 40, 7513−7514. (25) Chia, Y.-C.; Wu, J.-B.; Wu, Y.-C. Tetrahedron Lett. 2000, 41, 2199−2201. (26) Porzel, A.; Phuong Lien, T.; Schmidt, J.; Drosihn, S.; Wagner, C.; Merzweiler, K.; Van Sung, T.; Adam, G. Tetrahedron 2000, 56, 865−872. (27) Harada, N.; Nakanishi, K.; Berova, N. Electronic CD Exciton Chirality Method: Principles and Applications. In Comprehensive Chiroptical Spectroscopy; John Wiley & Sons: New York, 2012; pp 115−166. (28) Prakash, L.; Singh, R. Pharmazie 1980, 35, 122−123. (29) Koike, K.; Ohmoto, T.; Ikeda, K. Phytochemistry 1990, 29, 3060−3061. (30) Shi, Y.-P.; Rodríguez, I. I.; Rodríguez, A. D. Tetrahedron Lett. 2003, 44, 3249−3253. (31) Stepanenko, L. S.; Krivoshchekova, O. E.; Dmitrenok, P. S.; Maximov, O. B. Phytochemistry 1997, 46, 565−568. (32) Chen, Y.; Luo, Y.; Ju, J.; Wendt-Pienkowski, E.; Rajski, S. R.; Shen, B. J. Nat. Prod. 2008, 71, 431−437. (33) Xu, G. B.; He, G.; Bai, H. H.; Yang, T.; Zhang, G. L.; Wu, L. W.; Li, G. Y. J. Nat. Prod. 2015, 78, 1479−1485. (34) Lang, M.; Jagers, E.; Polborn, K.; Steglich, W. J. Nat. Prod. 2009, 72, 214−217. (35) Dharmaratne, H. R.; Nanayakkara, N. P.; Khan, I. A. Phytochemistry 2002, 59, 429−433. (36) Patra, A.; Mitra, A. K.; Bhattacharyya, A.; Adityachaudhury, N. Org. Magn. Reson. 1982, 18, 241−242.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00494. Experimental and computed ECD spectra and structure refinement details of compounds (+)-1 and (−)-1; 1H, 13 C, HSQC, and HMBC spectra of compounds 1 and 5− 7; 1H NMR spectra of compound 4 and raw data used for Table 3 and their graphical representation (PDF) X-ray crystallographic data for (+)-1 and (−)-1 (CIF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel: +33(0)169823038. Fax: +33(0)169077247. E-mail: marc. [email protected]. *Tel: +33(0)169823038. Fax: +33(0)169077247. E-mail: [email protected]. ORCID

Bogdan I. Iorga: 0000-0003-0392-1350 Fanny Roussi: 0000-0002-5941-9901 Marc Litaudon: 0000-0002-0877-8234 Vincent Dumontet: 0000-0002-1770-6566 Notes

The authors declare no competing financial interest. The crystallographic data of 1 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 1539889. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44- (0)1223-336033 or e-mail: deposit@ ccdc.cam.ac.uk).

■

ACKNOWLEDGMENTS We are grateful to the Agence Nationale de la Recherche (ANR), contract no. ANR-10-JCJC-7021, to the ARC Foundation for a grant for one of us (A.A.S.), Labex Program ANR-10-LABX-25-01, ICSN, CNRS, and Malaysian Ministry of Higher Education (grant no. UM.C/HIR/MOHE/SC/37) for financial support. This work was carried out in the framework of the International French Malaysian Natural Product Laboratory (IFM-NatPro-Lab) established between CNRSICSN and the University Malaya. The authors thank D. M. Nor, R. Syamsir, and T. Leong Eng (UM) for the collection and identification of plant material. We are thankful to L. Eloy and M. C. Garcia (ICSN) for in vitro cellular assays and N. Birlirakis (ICSN) for protein production.

■

REFERENCES

(1) Andersen, Ø. M.; Markham, K. R. Flavonoids: Chemistry, Biochemistry and Applications; CRC Taylor & Francis Group: Boca Raton, 2006; p 1212. (2) Tuntiwachwuttikul, P.; Pancharoen, O.; Reutrakul, V.; Byrne, L. T. Aust. J. Chem. 1984, 37, 449−453. (3) Win, N. N.; Awale, S.; Esumi, H.; Tezuka, Y.; Kadota, S.; Panduratins, D.-I. Chem. Pharm. Bull. 2008, 56, 491−6. (4) Nguyen, N. T.; Nguyen, M. T. T.; Nguyen, H. X.; Dang, P. H.; Dibwe, D. F.; Esumi, H.; Awale, S. J. Nat. Prod. 2017, 80, 141−148. 3184

DOI: 10.1021/acs.jnatprod.7b00494 J. Nat. Prod. 2017, 80, 3179−3185

Journal of Natural Products

Article

(37) Bhattacharyya, A.; Chaudhuri, A.; Adityachaudhury, N. Indian J. Chem. B 1980, 19, 428−429. (38) Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.; Wang, S. Anal. Biochem. 2004, 332, 261−273. (39) Treǵuier, B.; Lawson, M.; Bernadat, G.; Bignon, J.; Dubois, J.; Brion, J.-D.; Alami, M.; Hamze, A. ACS Comb. Sci. 2014, 16, 702−710. (40) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09; Gaussian, Inc.: Wallingford, CT, USA, 2009. (41) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. Chirality 2013, 25, 243−249.

3185

DOI: 10.1021/acs.jnatprod.7b00494 J. Nat. Prod. 2017, 80, 3179−3185