Anacardic Acids from Knema hookeriana as Modulators of Bcl-xL

Article pubs.acs.org/jnp

Anacardic Acids from Knema hookeriana as Modulators of Bcl-xL/Bak and Mcl-1/Bid Interactions Charlotte Gény,† Gwladys Rivière,† Jérome Bignon,† Nicolas Birlirakis,† Eric Guittet,† Khaljah Awang,‡ Marc Litaudon,† Fanny Roussi,*,† 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: Proteins of the Bcl-2 family are key targets in anticancer drug discovery. Disrupting the interaction between antiand pro-apoptotic members of this protein family was the approach chosen in this study to restore apoptosis. Thus, a biological screening on the modulation of the Bcl-xL/Bak and Mcl-1/Bid interactions permitted the selection of Knema hookeriana for further phytochemical investigations. The ethyl acetate extract from the stem bark led to the isolation of six new compounds, three acetophenone derivatives (1−3) and three anacardic acid derivatives (4−6), along with four known anacardic acids (7−10) and two cardanols (11, 12). Their structures were elucidated by 1D and 2D NMR analysis in combination with HRMS experiments. The ability of these compounds to antagonize Bcl-xL/Bak and Mcl-1/Bid association was determined, using a protein−protein interaction assay, but only anacardic acid derivatives (4−10) exhibited significant binding properties, with Ki values ranging from 0.2 to 18 μM. Protein−ligand NMR experiments further revealed that anacardic acid 9, the most active compound, does not interact with the anti-apoptotic proteins Bcl-xL and Mcl-1 but instead interacts with pro-apoptotic protein Bid.

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active extracts on Bcl-xL/Bak interactions, along with 480 additional plant extracts led to the isolation of the dual inhibitors ferrugineic acids B, C, and J and a biologically active ethyl acetate extract from the Knema hookeriana Warb. (Myristicaceae).5 The stem bark extract of this species displayed high-affinity binding to Bcl-xL and Mcl-1 (97% and 92% at 10 μg/mL, respectively) and prompted the search for secondary metabolites that regulate those anti-apoptotic proteins. Knema hookeriana Warb. (Myristicaceae) is a tree that can be found in Indonesia, Malaysia, Singapore, and Thailand. Traditionally, the leaves of this plant are used as a stomach remedy, and the sap is also useful for dying casting nets and cloths.6 Additionally, this plant is used as a preservative surface coating material for valuable wooden, porcelain, and metallic wares.6 Only one chemical investigation to find antinematodal components has been published.6 However, other Knema

he Bcl-2 family of proteins, comprising both anti- and proapoptotic members, are key players in apoptosis. The antiapoptotic proteins disable the pro-apoptotic ones by binding in a hydrophobic cleft through protein−protein interactions. The overexpression of anti-apoptotic proteins such as Bcl-xL and Mcl-1 plays a decisive role in cancer development and can be correlated with resistance to cancer therapeutics.1 These proteins are considered to be challenging targets for the development of novel anticancer treatments, but it was shown that selective inhibition of Bcl-xL results in apoptosis “escape” through the Mcl-1 pathway.2 Thus, identification of small molecules capable of binding to the hydrophobic cleft of both anti-apoptotic proteins Bcl-xL and Mcl-1, releasing the proapoptotic proteins such as Bax, Bad, or Bid, and ultimately restoring apoptosis, is a promising but challenging strategy in the fight against cancer. Recently, an extensive biological screening with 9000 plant extracts was conducted to investigate small molecules modulating Bcl-xL/Bak interactions, leading to the isolation of new active compounds such as meiogynin A3 and kingianin G.4 A second screening on Mcl-1/Bid interactions of the most © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 14, 2015

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DOI: 10.1021/acs.jnatprod.5b00915 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of compounds 1−12 from the ethyl acetate extract of K. hookeriana.

the HMBC and COSY spectra led to definition of the structure of compound 1 as shown in Figure 2.

species have been employed as traditional medicines to treat sores, pimples, rheumatism, and cancer, and some of these activities have been verified in several studies.7−9 For example, all the compounds found in Knema glomerata exhibited moderate cytotoxic activities (ranging from 2 to 35 μM) against the three tumor cell lines A-549, MCF-7, and HT-29.8 According to previous phytochemical investigations, cardanols, flavonoids, acetophenones, lignans, acylphloroglucinols, acylresorcinols, and anacardic acids are the main compounds isolated from the Knema genus.6−13 The anacardic acids were found to be the most biologically active compounds in cancer assays, via inhibition of histone acetyltransferase and NFκB kinase, as well as reducing the expression of various gene products that mediate survival, such as Bcl-2 and Bcl-xL.14,15 In ongoing efforts aimed at the search for plant-derived proapoptotic agents by their binding to Bcl-xL and Mcl-1, the isolation and structural characterization of six new compounds, three acetophenone derivatives (1−3) and three anacardic acid derivatives (4−6), and four known anacardic acids (7−10) and two cardanols (11, 12) from K. hookeriana were delineated in this study. These metabolites were evaluated for modulating the interaction potential of the anti-apoptotic proteins with proapoptotic proteins using a protein−protein interaction bioassay. Binding mechanisms of compound 9 with Bcl-xL and Mcl-1 and then with Bid were also scrutinized through protein−ligand NMR experiments to define the type of interactions observed in bioassays.

Figure 2. COSY (bold) and HMBC (blue arrows) correlations of compound 1.

An alkyl chain was connected to the aromatic ring at C-6 (δC 148.1) based on the HMBC correlations between H-1′ (δH 2.81) and C-1 (δC 115.5) and C-5 (δC 110.8). The correlation from the acetyl methyl protons (δH 2.62) to C-1 supported the location of the acetyl group at C-1. The 1D and 2D NMR data of compound 1 displayed similarities with those of kneglomeratanone B,8 indicating the same resacetophenone moiety, substituted with an aliphatic side chain comprising 10 methylene groups, instead of 12 for kneglomeratanone B. Thus, compound 1 (khookerianone A) was identified as 2,4dihydroxy-6-undecylacetophenone. Compounds 2 and 3 were respectively assigned the molecular formulas C19H28O3 and C21H32O3, on the basis of the HRESIMS protonated [M + H]+ ions at m/z 305.2115 and 333.2421 (calcd [M + H]+, 305.2111 and 333.2424) along with their 13C NMR data, both indicating six indices of hydrogen deficiency. These formulas and their spectroscopic data were similar to those of 1, suggesting unsaturated side chains for compounds 2 and 3. The superimposable resonances at δH 5.33 (2H, m) and the presence of resonances at δC 129.9 and 130.2 implied that their side chains possess one double bond. The chemical shift values of δC 27.1 and 27.4 for the allylic carbons of each double bond indicated a Z-configuration for both olefinic moieties.16,17 To determine the positions of the olefinic functionalities, ozonolysis of compounds 2 and 3 followed by a reduction using dimethyl sulfide was carried out to generate the corresponding aldehydes, which could be analyzed by UPLCHRESIMS. For compound 2, the HRESIMS exhibited a protonated [M + H]+ ion at m/z 251.1865 (calcd [M + H]+ 251.1878), implying the molecular formula of the generated formyl compound is C14H18O4. This confirmed that the location of the olefinic moiety is at C-6′ in the side chain of 2. A protonated [M + H]+ ion at m/z 279.1603 (calcd [M +

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RESULTS AND DISCUSSION Preparative C18 HPLC applications of the ethyl acetate extract of the K. hookeriana stem bark yielded 12 phenolic compounds (Figure 1). The HRESIMS data of compound 1 exhibiting a protonated [M + H]+ ion at m/z 307.2270 (calcd [M + H]+, m/z 307.2268) together with its 13C NMR data led to its molecular formula being established as C19H30O3, suggesting five indices of hydrogen deficiency. The 1H and 13C NMR data (Figures S1 and S2, Supporting Information) associated with edited HSQC NMR data indicate the presence of a tetrasubstituted aromatic moiety (δH 6.22, 6.24; δC 101.9, 110.8, 115.5, 148.1, 161.0, 166.2) with the two protons in meta positions based on the coupling constant of 2.4 Hz, two hydroxy groups (δH 5.55, 13.0; δC 161.1, 166.2), two methyl groups (δH 0.86, 2.62; δC 14.3, 32.5), 10 methylene groups (δH 1.24 8H, 1.57 2H, 2.81 2H; δC 22.9, 29.5, 29.6, 29.7, 29.8, 29.9, 32.1, 32.4, 36.6), and a carbonyl group (δC 204.5). The correlations emanating from B



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H]+ 279.1591) in the HRESIMS data of 3 was consistent with the molecular formula C16H22O4, verifying the presence of its double bond at C-8′. Consequently, compounds 2 and 3 were identified as (Z)-2,4-dihydroxy-6-(undec-6-en-1-yl)acetophenone and (Z)-2,4-dihydroxy-6-(tridec-8-en-1-yl)acetophenone, respectively, and assigned the trivial names khookerianones B (2) and C (3). The HRESIMS data of 5 showed a protonated [M + H]+ ion at m/z 291.1958 (calcd [M + H]+, 291.1955). In conjunction with the 13C NMR data, the molecular formula of 5 was established as C18H26O3, indicating six indices of hydrogen deficiency. The spectroscopic data of compound 5 were similar to those of anacardic acids and compound 4, which was indicative of 5 being structurally based on the anacardic acid scaffold possessing one double bond in the side chain. The Zconfiguration and position of the olefinic functionality at C-6′ were established as for compounds 2 and 3. Thus, compound 5 was identified as (Z)-2-hydroxy-6-(undec-6-en-1-yl)benzoic acid and given the trivial name khookerianic acid B (5). The HRESIMS data of compound 6 producing a protonated molecular ion at m/z 327.1974 (calcd [M + H]+, 291.1955) along with 13C NMR analysis permitted its molecular formula to be assigned as C21H26O3. The NMR data shared significant similarities with those of 2-hydroxy-6-(10′-phenyldecyl)benzoic acid acquired from Knema laurina,10 substantiating the presence of the bisphenyl skeleton in 6. The reduction of 28 amu in the MS data of 6 compared to 2-hydroxy-6-(10′-phenyldecyl)benzoic acid implied that 6 possesses an aliphatic chain composed of eight methylene groups instead of 10 for the known benzoic acid. Thus, compound 6 was identified as 2hydroxy-6-(8′-phenyloctyl)benzoic acid and given the trivial name khookerianic acid C (6). The structures of the seven other compounds were elucidated by comparing their observed and reported spectroscopic data and ozonolysis followed by reduction using dimethyl sulfide for compounds possessing acyclic double bonds. Their structures were assigned as the 3-tridecylphenol (11),18 (Z)-3-(tridec-8-en-1-yl)phenol (12),6 anagigantic acid (7),19 (Z)-2-hydroxy-6-(tridec-8-en-1-yl)benzoic acid (8),20 2hydroxy-6-tridecylbenzoic acid (9),18,21 and (Z)-2-hydroxy-6(pentadec-10-en-1-yl)benzoic acid (10).22 In addition, compound 4, khookerianic acid A, was previously synthesized but never reported as a natural product.23 The ability of compounds 1−12 to antagonize Bcl-xL/Bak and Mcl-1/Bid association was determined using a fluorescence polarization assay adapted from Qian and co-workers (Table 1, Figure S27, Supporting Information).24 The principle of this biological test is based on the competition of interaction between a small-molecule inhibitor and a fluorescent proapoptotic peptide (BH3 domain of BAK protein or BID protein) with the anti-apoptotic proteins Bcl-xL and Mcl-1. Results are given by the K i value, the concentration corresponding to 50% of the binding of the labeled reference compound, and corrected for experimental conditions according to Cheng and Prusoff.25 None of the compounds with a cardanol (11, 12) or resacetophenone (1−3) skeleton were able to displace Bcl-xL/ Bak and Mcl-1/Bid interactions. On the contrary, anacardic acid derivatives (4−10) are highly potent antagonists of both Bcl-xL/Bak and Mcl-1/Bid association. Therefore, the acid functional group seems essential for inducing activity that is modulated by the length of the side chain. Compounds with a C13 chain (8, 9) are active at a submicromolar range, while

Table 1. Biological Evaluation of Compounds 1−12 in BclxL/Bak and Mcl-1/Bid Displacement Assays

a

compound

Bcl-xL/Bak Ki (μM)

Mcl-1/Bid Ki (μM)

1 2 3 4 5 6 7 8 9 10 11 12 meiogynine Aa ABT-737a,b

>23 >23 >23 >23 3.2 ± 0.1 16.3 ± 0.5 1.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 5.7 ± 0.6 >23 >23 2.6 ± 0.3 57 ± 10 nM

>33 >33 >33 17.7 ± 3.1 5.8 ± 0.3 3.7 ± 2.0 0.7 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 1.2 ± 0.9 >33 >33 8.6 ± 0.5 >33

Reference compounds. bResults are expressed in IC50 values.

compounds with a C8 (6), C9 (4), C11 (5, 7), or C15 (10) chain have binding affinities in the micromolar range. However, the binding activity does not seem to be influenced by the degree of unsaturation or the presence of a terminal phenyl group. In addition, despite their significant binding affinities (these are some of the most potent natural compounds reported so far), they were not found to be cytotoxic on the HCT-116 cell line. Protein−ligand NMR experiments were carried out in order to confirm that the promising activities of anacardic acid derivatives were due to a direct binding to Bcl-xL and Mcl-1. Thus, HSQC 1H−15N experiments of both Bcl-xL and Mcl-1 were performed alone or in combination with 1.5 equiv of compound 9, the most potent compound. The protein HN resonances were not disturbed upon addition of the ligand, indicating that compound 9 does not bind to these antiapoptotic proteins (Figures S25 and S26, Supporting Information). In addition, 1D 1H NMR and 2D 1H−1H TOCSY experiments of Bid (pro-apoptotic partner of Mcl-1) alone (at 5 mM: see Figure S26, Supporting Information) or in combination with increasing amounts (0.25, 0.5, 1.0, 2.0, and 5.0 equiv) of compound 9 were investigasted. The 1H NMR spectra (Figure 3) clearly show that the broadness of the Bid signals increases significantly and proportionally to the concentration of 9 (at higher concentrations, compound 9 precipitates in the NMR tube). These results indicate either a nonspecific interaction between Bid and 9, via the formation of micelles, or a direct interaction of high affinity between both partners. Amphiphilic structure and detergent properties reported for anacardic acids suggest that their interaction with compound 9 is not selective and results from the formation of micelles.26 In fact various studies use detergents to mimic membrane environment and characterized conformational modifications of anti- and pro-apoptotic protein complexes.27,28 For example, it has been shown that detergents readily enable pro-apoptotic Bax hetero- and homodimerization and most importantly that Bid exhibits binding to dodecylphosphocholine micelles.29,30 Nevertheless, analysis of the titration data shows that there are significant chemical shift perturbations of the amine and aliphatic protons of Bid peptide in the presence of 9 (Figure 4), suggesting a direct interaction. This direct interaction was confirmed using 2D 1H−1H TOCSY experiments carried out with increasing amounts of 9. C



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Figure 3. 1H NMR spectra of Bid peptide (5 mM) with increasing amounts of 9 showing the effect of the addition of 9 on the decrease of the signalto-noise ratio of the of Bid peptide. equipped with a 5 mm z-gradient TCI (H/C/N) cryoprobe. TOCSY and NOESY data were processed using TOPSIN and analyzed using Mestrenova. The titration experiments of the Bcl-xL with 9 were performed on a Bruker Avance III 800US2 spectrometer, equipped with a 5 mm zgradient TCI (H/C/N) cryoprobe. The 1H−15N HSQC data were processed using TOPSIN and analyzed using SPARKY. HRESIMS spectra and LC chromatograms were obtained on a UPLC Acquity (Waters) using a BEH C18, 2.1 × 50 mm, 1.7 μm column coupled with an LCT Premier XE (Waters) TOF spectrometer. Kromasil analytical and preparative C18 columns (250 × 4.6 mm and 250 × 21.2 mm i.d.; 5 μm Thermo) were used for HPLC separations using a Dionex autopurification system equipped with a Gilson 215 liquid handler, a Dionex UltiMate HPG-3200BX binary pump (Thermo Scientific), a UV−vis diode array detector (200−600 nm, Dionex UVD340U), and a PL-ELS 1000 ELSD Polymer Laboratory detector. All solvents were purchased from SDS (France); analytical and preparative plates (Si gel 60 F−254), from Merck (France). Plant Material. The bark of K. hookeriana Warb. was collected at Gunung Bujang Melaka, Perak, Malaysia, in April 1996. The plant was identified by T. Leong Eng, botanist at the University of Malaya. A voucher specimen (KL-4584) has been deposited at the Herbarium of the Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. Extraction and Isolation. The air-dried bark and stem (100 g) of K. hookeriana were extracted with EtOAc (3 × 300 mL) to yield a crude extract (2.5 g) after concentration in vacuo at 40 °C. A portion (350 mg) of the extract was subjected to preparative HPLC (Kromasil C18 column, 250 × 21.2 mm, 5 μm, at 21 mL/min) using a gradient of MeCN−H2O (8:2 to 9:1) plus 0.1% formic acid to afford three fractions (F1, F7, and F14) and compounds 1 (6 mg), 2 (4 mg), 4 (2 mg), 5 (25 mg), 6 (3 mg), 7 (96 mg), 8 (74 mg), 9 (31 mg), 10 (13 mg), 11 (4 mg), and 12 (3 mg). Fraction F7 (5 mg) was purified by preparative TLC (Merck, Si gel 60 F-254, 2 mm, n-heptane−EtOAc, 8:2) to obtain compound 3 (3 mg).

From the Bid assignment and these TOCSY experiments (Figure S26, Supporting Information), it is evident that 9 interacts specifically with the last five residues of the Bid Cterminal, namely, Asp16, Ser17, Met18, Asp19, and Arg20 (Figure 5). In conclusion, the chemical study of K. hookeriana led to the identification of three new resacetophenones (1−3), two new anacardic acid derivatives (5, 6), an anacardic acid (4) obtained naturally for the first time, four known anacardic acids, and two known cardanols. Of this alkylphenol series, only the anacardic acids exhibited strong disruption of the anti- (Bcl-xL, Mcl-1) and pro-apoptotic protein (Bak, Bid) interactions, based on a fluorescent polarization assay. From the NMR studies, anacardic acid 9 showed binding affinity for pro-apoptotic protein Bid and not for anti-apoptotic proteins Bcl-xL and Mcl1, and we could establish specifically to which amino acids it binds. The objective for finding small-molecule inhibitors of anti-apoptotic proteins Bcl-xL and Mcl-1 was not achieved, but anacardic acids were identified for the first time as modulators of Bcl-xL/Bak and Mcl-1/Bid interactions and could be used as chemical tools for studying these protein−protein interactions.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded on a Perkin-Elmer Lambda 5 spectrophotometer. IR spectra were performed on a PerkinElmer Spectrum 100 FT-IR spectrometer. NMR spectra were recorded in CDCl3-d on a Bruker 600 MHz instrument (Avance 600) using a 1.7 mm microprobe for compound 2, on a Bruker 500 MHz instrument (Avance 500) for plant extracts, fractions, and compounds 4 and 11, and on a Bruker 300 MHz instrument (Avance 300) for compounds 1, 3, 5, 6−10, and 12. The assignment and Chemical Shift Perturbation (CSP) experiments of the Bid peptide were recorded on a Bruker Avance III 600US2 spectrometer, D



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Figure 4. Effect of the addition of 9 on the chemical shifts of Bid protons. Khookerianone A (1): colorless gum; UV (CHCl3) λmax (log ε) 292 (3.65) nm; IR (neat) νmax 3315, 2922, 2853, 1615, 1588, 1454, 1360, 1259, 1160 cm−1; 1H NMR (CDCl3, 300 MHz) δ 13.00 (1H, s, OH2), 6.24 (1H, d, J = 2.4 Hz, H-5), 6.22 (1H, d, J = 2.4 Hz, H-3), 5.55 (1H, br s, OH-4), 2.81 (2H, t, J = 8.1 Hz, H-1′), 2.62 (3H, s, COCH3), 1.57 (2H, m, H-2′), 1.24 (16H, m, H-3′−H-10′), 0.86 (3H, t, J = 6.6 Hz, H-11′); 13C NMR (CDCl3, 75 MHz) δ 204.5 (CO), 166.2 (C-2), 161.0 (C-4), 148.1 (C-6), 115.5 (C-1), 110.8 (C-5), 101.9 (C-3), 36.6 (C-1′), 32.5 (COCH3), 32.4 (C-2′), 32.1 (C-9′), 29.5−29.9 (C-3−C8′), 22.9 (C-10′), 14.3 (C-11′); HRESIMS m/z [M + H]+ 307.2270 (calcd for C19H31O3 307.2268). Khookerianone B (2): yellowish oil; UV (CHCl3) λmax (log ε) 294 (3.60) nm; IR (neat) νmax 3335, 2915, 2850, 1617, 1590 cm−1; 1H NMR (CDCl3, 600 MHz) δ 12.98 (1H, s, OH-2), 6.23 (1H, d, J = 2.4 Hz, H-5), 6.22 (1H, d, J = 2.4 Hz, H-3), 5.66 (1H, br s, OH-4), 5.33 (2H, m, H-6′, H-7′), 2.82 (2H, t, J = 8.0 Hz, H-1′), 2.63 (3H, s, COCH3), 2.00 (2H, m, H-5′, H-8′), 1.58 (2H, m, H-2′), 1.30 (8H, m, H-3′, H-4′, H-9′, H-10′), 0.87 (3H, t, J = 7.1 Hz, H-11′); 13C NMR (CDCl3, 150 MHz) δ 204.5 (CO), 166.3 (C-2), 161.0 (C-4), 148.0 (C-6), 129.5−130.5 (C-6′, C-7′), 115.6 (C-1), 110.7 (C-5), 101.9 (C3), 36.5 (C-1′), 32.5 (COCH3), 32.4 (C-2′), 32.1 (C-9′), 29.6−29.9 (C-3′, C-4′), 27.2−27.3 (C-5′, C-8′), 22.6 (C-10′), 14.2 (C-11′); HRESIMS m/z [M + H]+ 305.2115 (calcd for C19H29O3 305.2111). Khookerianone C (3): yellowish oil; UV (CHCl3) λmax (log ε) 294 (3.75) nm; IR (neat) νmax 3330, 2920, 2853, 1619, 1588 cm−1; 1H

NMR (CDCl3, 500 MHz) δ 12.97 (1H, s, OH-2), 6.24 (1H, d, J = 2.5 Hz, H-5), 6.22 (1H, d, J = 2.5 Hz, H-3), 5.70 (1H, br s, OH-4), 5.33 (2H, m, H-8′, H-9′), 2.81 (2H, t, J = 7.7 Hz, H-1′), 2.62 (3H, s, COCH3), 1.99−2.00 (2H, m, H-7′, H-10′), 1.57 (2H, m, H-2′), 1.29− 1.30 (12H, m, H-3′−H-6′, H-11′, H-12′), 0.87 (3H, t, J = 7.1 Hz, H13′); 13C NMR (CDCl3, 125 MHz) δ 204.5 (CO), 166.1 (C-2), 161.1 (C-4), 148.0 (C-6), 129.9−130.2 (C-8′, C-9′), 115.6 (C-1), 110.8 (C5), 101.9 (C-3), 36.6 (C-1′), 32.5 (COCH3), 32.4 (C-2′), 32.2 (C11′), 29.4−29.9 (C-3′−C-6′), 27.1−27.4 (C-7′, C-10′), 22.6 (C-12′), 14.2 (C-13′); HRESIMS m/z [M + H]+ 333.2421 (calcd for C21H33O3 333.2424). Khookerianic acid B (5): colorless gum; UV (CHCl3) λmax (log ε) 311 (3.55), 243 (3.70) nm; IR (neat) νmax 2923, 2851, 1647, 1603, 1576, 1444, 1297, 1216 cm−1; 1H NMR (CDCl3, 300 MHz) δ 10.62 (1H, s, OH-2), 7.34 (1H, t, J = 7.9 Hz, H-4), 6.85 (1H, dd, J = 7.9 Hz, 1.1 Hz, H-3), 6.75 (1H, dd, J = 7.9 Hz, 1.1 Hz, H-5), 5.34 (2H, m, H6′, H-7′), 2.96 (2H, t, J = 8.0 Hz, H-1′), 2.00 (2H, m, H-5′, H-8′), 1.59 (2H, m, H-2′), 1.30−1.36 (8H, m, H-3′, H-4′, H-9′, H-10′), 0.87 (3H, t, J = 6.9 Hz, H-11′); 13C NMR (CDCl3, 75 MHz) δ 176.0 (COOH), 163.8 (C-2), 147.9 (C-6), 135.6 (C-4), 129.9−130.2 (C-6′, C-7′), 123.0 (C-5), 116.1 (C-3), 110.7 (C-1), 36.7 (C-1′), 32.2 (C-2′), 32.1 (C-9′), 29.6−29.8 (C-3′, C-4′), 27.1−27.4 (C-5′, C-8′), 22.6 (C-10′), 14.2 (C-11′); HRESIMS m/z [M + H]+ 291.1958 (calcd for C18H27O3 291.1955). E



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Figure 5. Superimposition of the 2D 1H−1H TOCSY spectrum of Bid alone (reference with DMSO: blue) and Bid in the presence of 5.0 equiv of 9 (red). Zoom-in on the HN/αH fingerprint region. The spin systems of the last five residues of the Bid C-terminal are shown.

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Khookerianic acid C (6): brown powder; UV (CHCl3) λmax (log ε) 308 (3.46), 242 (3.60) nm; IR (neat) νmax 3450, 2920, 2850, 1651, 1600, 1590 cm−1; 1H NMR (CDCl3, 500 MHz) 11.07 (1H, s, OH-2), 7.37 (1H, t, J = 7.8 Hz, H-4), 7.29 (2H, m, H-2″, H-6″), 7.20 (3H, m, H-3″−H-5″), 6.89 (1H, dd, J = 7.8, 1.0 Hz, H-3), 6.79 (1H, dd, J = 7.8, 1.0 Hz, H-5), 2.98 (2H, t, J = 7.6 Hz, H-1′), 2.62 (2H, t, J = 7.6 Hz, H8′), 1.62 (4H, m, H-2′, H-7′), 1.35 (8H, m, H-3′−H-6′); 13C NMR (CDCl3, 125 MHz) δ 174.9 (COOH), 163.9 (C-2), 147.8 (C-6), 143.1 (C-1″), 135.6 (C-4), 128.6 (C-3″, C-5″), 128.4 (C-2″, C-6″), 125.8 (C-4″), 122.9 (C-5), 116.1 (C-3), 110.5 (C-1), 36.7 (C-1′), 36.2 (C-8′), 32.3 (C-2′), 31.7 (C-7′), 29.5−30.0 (C-3′−C-6′); HRESIMS m/z [M + H]+ 327.1974 (calcd for C21H27O3 327.1955). Ozonolysis of Compounds 2, 3, 5, 8, 10, and 12. A portion of each compound (2 to 6 mg) was dissolved in CH2Cl2, and the solution was cooled to −78 °C. Ozone gas, generated by a BMT 802x ozone generator, flow rate 0.2 mL/min, was passed into the solution until the color turned blue. An excess of dimethylsulfide was added, and the reaction mixture was stirred for 4 h at room temperature. The solvent was evaporated under reduced pressure. The residue was dissolved in MeOH and injected in UPLC/HRESIMS. Biological Assays. The modulations of the interaction between Bcl-xL/Bak and between Mcl-1/Bid were evaluated by competition against a fluorescent-labeled reference compound (fluorescent-tagged BH3 domain of the protein Bak or Bid), as described earlier.5,31 Meiogynine A3 and ABT-73732 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.33 Assays were run in triplicate. The cytotoxic activities of the compounds were evaluated against the cancer cell line HCT-116 (human colon carcinoma). Cytotoxicity assays were performed according to a published procedure.34

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00915. 1 H and 13C NMR spectra for compounds 1−12, HSQC 1 H−13C NMR spectra of Bcl-xL and Mcl-1 with compound 9, proton assignments for Bid peptide, as well as additional CSP experiments (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (F. Roussi). *Tel: +33169823038. Fax: +33169077247. E-mail: vincent. [email protected] (V. Dumontet). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was carried out in the framework of the International French Malaysian Natural Product Laboratory (IFM-NatProLab) established between CNRS-ICSN 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 thank Dr. O. Nosjean (Institut de Recherches Servier) for his scientific advice, for the assay, and for the Bcl-xL F



Article

(28) Losonczi, J. A.; Olejniczak, E. T.; Betz, S. F.; Harlan, J. E.; Mack, J.; Fesik, S. W. Biochemistry 2000, 39, 11024−33. (29) Hsu, Y.-T.; Youle, R. J. J. Biol. Chem. 1998, 273 (17), 10777−83. (30) Denisov, A. Y.; Chen, G.; Sprules, T.; Moldoveanu, T.; Beauparlant, P.; Gehring, K. Biochemistry 2006, 45, 2250−56. (31) Azmi, M.; Gény, C.; Leverrier, A.; Litaudon, M.; Dumontet, V.; Birlirakis, N.; Guéritte, F.; Leong, K.; Halim, S.; Mohamad, K.; Awang, K. Molecules 2014, 19 (2), 1732−47. (32) Zhai, D.; Jin, C.; Satterthwait, A. C.; Reed, J. C. Cell Death Differ. 2006, 13 (8), 1419−21. (33) 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 (2), 261−73. (34) Tréguier, B.; Lawson, M.; Bernadat, G.; Bignon, J.; Dubois, J.; Brion, J.-D.; Alami, M.; Hamze, A. ACS Comb. Sci. 2014, 16 (12), 702−10.

plasmid and Dr. J. Oh (University of Mississippi) for his scientific skills and helpful discussions. We are grateful to the Agence National de la Recherche (ANR), contract number ANR-10-JCJC-7021, 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.

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REFERENCES

(1) Shore, G. C.; Warr, M. R. Curr. Mol. Med. 2008, 8 (2), 138−47. (2) Tse, C.; Shoemaker, A. R.; Adickes, J.; Anderson, M. G.; Chen, J.; Jin, S.; Johnson, E. F.; Marsh, K. C.; Mitten, M. J.; Nimmer, P.; Roberts, L.; Tahir, S. K.; Xiao, Y.; Yang, X.; Zhang, H.; Fesik, S.; Rosenberg, S. H.; Elmore, S. W. Cancer Res. 2008, 68 (9), 3421−8. (3) Litaudon, M.; Bousserouel, H.; Awang, K.; Nosjean, O.; Martin, M.-T.; Dau, M. E. T. H.; Hadi, H. A.; Boutin, J. A.; Sévenet, T.; Guéritte, F. J. Nat. Prod. 2009, 72 (3), 480−3. (4) Leverrier, A.; Awang, K.; Guér itte, F.; Litaudon, M. Phytochemistry 2011, 72 (11−12), 1443−52. (5) Apel, C.; Gény, 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 (6), 1430−7. (6) Alen, Y.; Nakajima, S.; Nitoda, T.; Baba, N.; Kanzaki, H.; Kawazu, K. Z. Naturforsch., C: J. Biosci. 2000, 55 (3−4), 300−303. (7) Kijjoa, A.; Gonzalez, M. J.; Pinto, M. M.; Monanondra, I. O.; Herz, W. Planta Med. 1991, 57 (6), 575−7. (8) Zeng, L.; Gu, Z.-M.; Fang, X.-P.; McLaughlin, J. L. J. Nat. Prod. 1994, 57 (3), 376−81. (9) Phadungkit, M.; Rattarom, R.; Rattana, S. J. Med. Plants Res. 2010, 4 (13), 1269−72. (10) Gonzalez, M. J. T. G.; DeOliveira, C. J. C.; Fernandes, J. O.; Kijjoa, A.; Herz, W. Phytochemistry 1996, 43 (6), 1333−7. (11) Gonzaléz, M. J. T. G.; Pinto, M. M. M.; Kijjoa, A.; Anantachoke, C.; Herz, W. Phytochemistry 1993, 32 (2), 433−8. (12) Pinto, M. M. M.; Kijjoa, A.; Mondranondra, I.-o.; Gutiérrez, A. B.; Herz, W. Phytochemistry 1990, 29 (6), 1985−8. (13) Akhtar, M. N.; Lam, K. W.; Abas, F.; Maulidiani; Ahmad, S.; Shah, S. A. A.; Atta-ur-Rahman; Choudhary, M. I.; Lajis, N. H. Bioorg. Med. Chem. Lett. 2011, 21 (13), 4097−103. (14) Hemshekhar, M.; Sebastin Santhosh, M.; Kemparaju, K.; Girish, K. S. Basic Clin. Pharmacol. Toxicol. 2012, 110, 122. (15) Sung, B.; Pandey, M. K.; Ahn, K. S.; Yi, T.; Chaturvedi, M. M.; Liu, M.; Aggarwal, B. B. Blood 2008, 111 (10), 4880−91. (16) Vysotskii, M. V.; Imbs, A. B.; Popkov, A. A.; Latyshev, N. A.; Svetashev, V. I. Tetrahedron Lett. 1990, 31 (30), 4367−70. (17) Anderson, J. E.; Ma, W.; Smith, D. L.; Chang, C.-J.; McLaughlin, J. L. J. Nat. Prod. 1992, 55 (1), 71−83. (18) Kazlauskas, R.; Mulder, J.; Murphy, P.; Wells, R. Aust. J. Chem. 1980, 33 (9), 2097−101. (19) Asakawa, Y.; Masuya, T.; Tori, M.; Campbell, E. O. Phytochemistry 1987, 26 (3), 735−8. (20) Yalpani, M.; Tyman, J. H. P. Phytochemistry 1983, 22 (10), 2263−6. (21) Zehnter, R.; Gerlach, H. Liebigs Ann. 1995, 1995 (12), 2209− 20. (22) Schultz, D. J.; Olsen, C.; Cobbs, G. A.; Stolowich, N. J.; Parrott, M. M. J. Agric. Food Chem. 2006, 54 (20), 7522−9. (23) Pereira, J. M.; Severino, R. P.; Vieira, P. C.; Fernandes, J. o. B.; da Silva, M. F. G. F.; Zottis, A.; Andricopulo, A. D.; Oliva, G.; Corrêa, A. G. Bioorg. Med. Chem. 2008, 16 (19), 8889−95. (24) Qian, J.; Voorbach, M. J.; Huth, J. R.; Coen, M. L.; Zhang, H.; Ng, S. C.; Comess, K. M.; Petros, A. M.; Rosenberg, S. H.; Warrior, U.; Burns, D. J. Anal. Biochem. 2004, 328 (2), 131−8. (25) Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22 (23), 3099−108. (26) Stasiuk, M.; Kozubek, A. Cell. Mol. Life Sci. 2010, 67 (6), 841− 60. (27) Liu, Q.; Gehring, K. J. Biol. Chem. 2010, 285 (52), 41202−10. G


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