Article pubs.acs.org/jnp Constituents of the Rhizomes of Boesenbergia pandurata and Their Antiausterity Activities against the PANC‑1 Human Pancreatic Cancer Line Nhan Trung Nguyen,*,† Mai Thanh Thi Nguyen,† Hai Xuan Nguyen,† Phu Hoang Dang,† Dya Fita Dibwe,§ Hiroyasu Esumi,‡ and Suresh Awale*,§ † Faculty of Chemistry, University of Science, Vietnam National University, Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam ‡ Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba 278-8510, Japan § Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan S Supporting Information * ABSTRACT: Human pancreatic cancer cell lines have a remarkable tolerance to nutrition starvation, which enables them to survive under a tumor microenvironment The search for agents that preferentially inhibit the survival of cancer cells under low nutrient conditions represents a novel antiausterity strategy in anticancer drug discovery In this investigation, a methanol extract of the rhizomes of Boesenbergia pandurata showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions, with a PC50 value of 6.6 μg/mL Phytochemical investigation of this extract led to the isolation of 15 compounds, including eight new cyclohexene chalcones (1−8) The structures of the new compounds were elucidated by NMR spectroscopic data analysis Among the isolated compounds obtained, isopanduratin A1 (14) and nicolaioidesin C (15) exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutritiondeprived conditions, with PC50 values of 1.0 and 0.84 μM, respectively P reported preferential cytotoxicity against PANC-1 cells in nutrient-deprived medium (NDM).4,7 In the present investigation, it was found that a methanol extract of the rhizomes of B pandurata displayed potent preferential cytotoxicity against PANC-1 cells under nutrientdeprived conditions, with a PC50 value of 6.6 μg/mL Purification of this extract led to the isolation of eight new secondary metabolites (1−8), together with seven known compounds (9− 15) Reported herein are the isolation, stucture determination, and antiausterity activities of these compounds ancreatic cancer is one of the deadliest forms of malignancy and is associated with the lowest five-year survival rates known for cancer.1 It shows resistance to conventional anticancer agents in clinical use.2 Pancreatic cancers are hypovascular in nature, resulting in an inadequate supply of nutrition and oxygen to aggressively proliferating cells However, pancreatic cancer cells show an extraordinary tolerance to starvation, enabling them to survive in hypovascular (austerity) conditions.3 Thus, the development of test compounds aimed at countering this tolerance to nutrient deprivation is a novel antiausterity strategy in anticancer drug discovery Working under this hypothesis, medicinal plants of different origin have been screened for the discovery of antiausterity agents, using the PANC-1 human pancreatic cancer cell line.4−12 Boesenbergia pandurata (Roxb.) Schltr is a perennial medicinal herb belonging to the Zingiberaceae family It is cultivated in some tropical countries in Southeast Asia including Vietnam, Thailand, Myanmar, Indonesia, and Malaysia In Vietnam, it is known as “Ngai bun”, and the fresh rhizomes are mainly used as a spice.13 The rhizomes are also used as traditional medicine to cure flatulence, fatigue, and dysmenorrhea and to promote the discharge of bile in Vietnam, Cambodia, Laos, and the People’s Republic of China.14 This plant contains prenylated chalcones and other flavonoids as the major bioactive constituents, with © XXXX American Chemical Society and American Society of Pharmacognosy ■ RESULTS AND DISCUSSION A methanol-soluble extract of the rhizomes of B pandurata was partitioned between CHCl3 and water to give a CHCl3-soluble fraction The CHCl3 fraction was subjected to a series of column chromatographic separation steps and preparative TLC to afford eight new secondary metabolites (1−8), together with seven known compounds The known compounds nicolaioidesin A (9),15 panduratin A (10),16 isopanduratin A (11),17 4hydroxypanduratin A (12),18 nicolaioidesin B (13),15 isopandurReceived: August 26, 2016 A DOI: 10.1021/acs.jnatprod.6b00784 J Nat Prod XXXX, XXX, XXX−XXX Journal of Natural Products Article Chart Figure Connectivities (bold lines) deduced by the COSY and HSQC spectra and significant HMBC correlations (solid arrows) of compounds 1−8 atin A1 (14),17 and nicolaioidesin C (15)15 were identified by comparing their spectroscopic data with literature values Compound was isolated as a yellowish, amorphous solid, and its molecular formula was found to be C25H28O4 by HRESIMS The IR spectrum of showed absorptions due to hydroxy (3500 cm−1), carbonyl (1640 cm−1), and phenyl (1450 cm−1) groups The 1H NMR spectrum displayed signals corresponding to a phenyl group (δH 7.25, 7.07, 6.97), two magnetically equivalent aromatic protons (δH 5.74), two olefinic methine protons (δH 5.58, 5.13), three aliphatic methines (δH 4.85, 3.06, 2.84), two allylic methylenes (δH 2.31, 2.23, 2.15, 2.14), and three vinyl methyls (δH 1.71, 1.51, 1.41) Its 13C NMR spectrum revealed 25 B DOI: 10.1021/acs.jnatprod.6b00784 J Nat Prod XXXX, XXX, XXX−XXX Journal of Natural Products Article Figure Key NOESY correlations observed for compounds 1−8 shift for a vinyl methyl carbon C-5″ (δC 14.0) along with NOESY correlations of H-2″ with H-4″ and of H-1″ with H-5″ (Figure 2) Moreover, the relative configuration of the cyclohexenyl unit of was established by coupling constant data and NOESY spectroscopic analysis The large coupling constant between H1′ and H-6′ (J = 11.8 Hz) indicated that they are in a trans-diaxial orientation, and the small coupling constant between H-1′ and H-2′ (J = 4.6 Hz) showed their cis relationship This was confirmed by the NOESY correlations between H-1′ and H-2′, H-1′ and H-2‴/H-6‴, H-1′ and H-5′α, H-6′ and H-1″, and H-6′ and H-5′β (Figure 2) Therefore, the structure of compound was concluded to be 3″-hydroxymethylpanduratin A Panduratin J (3) was obtained as a yellowish, amorphous solid having the molecular formula C26H30O5, as determined by HRESIMS The IR spectrum of showed absorptions due to hydroxy, carbonyl, and phenyl groups The 1H and 13C NMR data resembled those of panduratin A (10),16 isolated from the same plant extract, and indicated the presence of a substituted cyclohexene ring, a phenyl ring, two magnetically equivalent aromatic protons, and a methoxy group However, showed signals corresponding to exomethylene (δH 4.81, 4.61; δC 150.1, 109.6) and oxymethine (δH 3.50; δC 75.4) groups instead of signals corresponding to olefinic methine and vinyl methyl groups as in the prenyl unit in compound 10 Thus, the presence of a 3-methyl-2-hydroxybut-3-enyl moiety rather than a prenyl moiety was proposed The HMBC correlations of the H-4″ exomethylene protons (δH 4.81, 4.61) with the C-2″ oxymethine carbon (δC 75.4) and the C-5″ methyl carbon (δC 18.2) indicated that the exomethylene group occurs at C-4″ Similarly, the location of the hydroxy group was determined to be C-2″ based on HMBC correlations from the H-2″ oxymethine proton (δH 3.50) to the C-1″ methylene carbon (δC 37.4) and the C-4″ exomethylene carbon (δC 109.6) and from the H-4″ exomethylene protons (δH 4.81, 4.61) and the H-5″ methyl proton (δH 1.62) to the C-2″ oxymethine carbon (δC 75.4) (Figure 1) Moreover, the partial structure C-1″−C-2″ was deduced from the COSY and HSQC spectra and the downfield shift of C-2″ (δH 3.50; δC 75.4) Finally, the coupling constants and NOESY correlations suggested as having the same relative configuration as in the cyclohexenyl chalcone unit Therefore, the structure of panduratin J (3) was elucidated as shown carbon signals including those for a ketone carbonyl carbon (δC 210.4), 12 aromatic carbons, four olefinic carbons (δC 137.2, 132.7, 123.5, 123.1), three methine carbons (δC 54.8, 47.3, 46.0), two methylenes (δC 35.5, 29.8), and three vinyl methyls (δC 26.0, 21.7, 18.0) These data were similar to those of nicolaioidesin A (9),15 an isolate obtained from the same extract, except for the disappearance of signals due to a methoxy group at C-4 in (δH 3.70; δC 55.7) Thus, compound was assigned tentatively as 4hydroxynicolaioidesin A, which was confirmed by the HMBC spectrum (Figure 1) The relative configuration of was determined from the coupling constant data and NOESY analysis The large coupling constant between H-1′ and H-6′ (J = 11.4 Hz) and between H-1′ and H-2′ (J = 10.2 Hz) indicated that they are in a trans-diaxial orientation This was supported by the NOESY correlations between H-1′ and H-1″, H-1′ and H-2‴/H6‴, H-1′ and H-5′α, H-2′ and H-6′, and H-6′ and H-5′β (Figure 2) Therefore, the structure of compound was assigned as 4hydroxynicolaioidesin A Compound was obtained as a yellowish, amorphous solid, and its molecular formula was determined as C26H30O5 by HRESIMS The IR spectrum of exhibited absorption bands for hydroxy (3600 cm−1), carbonyl (1640 cm−1), and phenyl (1460 cm−1) groups The 1H NMR spectrum showed signals due to a phenyl ring (δH 7.22, 7.18, 7.05), two magnetically equivalent aromatic protons (δH 5.95), two olefinic methine protons (δH 5.43, 5.19), an oxymethylene (δH 3.74), three aliphatic methines (δH 4.84, 3.45, 2.34), two allylic methylenes (δH 2.38, 2.34, 2.13, 2.00), two vinyl methyls (δH 1.78, 1.56), and a methoxy group (δH 3.77) The 13C NMR spectrum displayed 26 carbon signals including a ketone carbonyl carbon (δC 207.5), 12 aromatic carbons, four olefinic carbons (δC 137.8, 136.4, 125.3, 121.9), one oxymethylene (δC 68.7), three methine carbons (δC 54.7, 43.3, 37.8), two methylenes (δC 36.8, 29.2), two vinyl methyls (δC 23.1, 14.0), and one methoxy (δC 55.8) These data closely resembled those of panduratin A (10),16 a major compound of B pandurata, except for the appearance of signals for a hydroxymethyl group in instead of one of the vinyl methyls in 10 The hydroxymethyl group was determined to be at C-4″ based on the HMBC correlations of H-2″ and H-5″ with C-4″ (Figure 1) and the downfield shift of C-4″ (δH 3.74; δC 68.7) The double-bond geometry at C-2″ was assigned in the Econfiguration based on the upfield-shifted 13C NMR chemical C DOI: 10.1021/acs.jnatprod.6b00784 J Nat Prod XXXX, XXX, XXX−XXX Journal of Natural Products Article Figure Morphology of PANC-1 cells under the control and following treatment with nicolaioidesin C (15, 1.5 μM) in NDM at 24 h and stained by ethidium bromide (EB)/acridine orange (AO) Live cells were stained with AO and emitted a bright green fluorescence, while dead cells were stained with EB and emitted a red fluorescence Treatment with nicolaioidesin C (15) at 1.5 μM led to dramatic alteration of PANC-1 cell morphology and total death of PANC-1 cells within 24 h ring, respectively, and were very different from those of 1−5 The relative configuration of was established from the coupling constant data and the NOESY spectrum The large coupling constant between H-1′ and H-6′ (J = 11.4 Hz) indicated that they have a trans-diaxial orientation, while the small coupling constant between H-1′ and H-2′ (J = 5.2 Hz) is cis-oriented Furthermore, in the NOESY spectrum, correlations between H1′ and H-2′, H-1′ and H-5′α, H-6′ and H-2‴/H-6‴, H-6′ and H1″, and H-6′ and H-5′β (Figure 2) were observed, suggesting their proximity, as in nicolaioidesin B (13) Therefore, the structure of panduratin M (6) was concluded as shown Panduratins N (7) and O (8) were both obtained as yellowish, amorphous solids, and they were found to possess the same molecular formula, C26H30O4, as determined by HRESIMS and HRFABMS, respectively The 1H and 13C NMR data of and were similar to those of nicolaioidesin B (13) 15 and isopanduratin A1 (14),17 respectively Also apparent were the methoxy groups at C-4 in and at C-6 in 8, as confirmed by the HSQC and HMBC spectra (Figure 1) However, and were observed to differ from 13 and 14 from a variation in the stereoconfiguration at C-2′ of the cyclohexenyl moiety Both the large coupling constants between H-1′ and H-6′ (J = 11.0−11.2 Hz) and between H-1′ and H-2′ (J = 10.9 Hz) indicated that they are oriented in a trans-diaxial manner This was also supported by the NOESY correlations between H-1′ and H-1″, H-1′ and H5′α, H-2′ and H-2‴/H-6‴, H-2′ and H-6′, H-6′ and H-2‴/H-6‴, andH-6′ and H-5′β (Figure 2) Therefore, the structures of panduratins N (7) and O (8) were assigned as shown All isolated compounds were tested for their preferential cytotoxic activity against the PANC-1 human pancreatic cancer cell line, according to an antiausterity strategy.2 Their PC50 values (the 50% preferential cell death in NDM without cytotoxicity in DMEM) are listed in Table Among the compounds tested, isopanduratin A1 (14) and nicolaioidesin C (15) exhibited the most potent preferential cytotoxicity, with PC50 values of 1.0 and 0.84 μM, respectively, which is comparable to that of arctigenin, a positive control (PC50 value, 0.8 μM) The activity of the isolates was found to greatly depend on the nature of the substituents in the cyclohexene chalcone unit In general, compounds having a phenyl group at C-1′ and a benzoyl substituent at C-6′ of the cyclohexenyl moiety were found to have potent activity (6 > 4, 13 > 10 and 9, 14 > 11) Interestingly, at C-1′ and C-2′ of the cyclohexenyl substituent, prenyl and benzoyl groups or prenyl and phenyl groups on the same side of the ring are more favorable than when on different sides (12 > 1, 13 > 7, 14 > 8) Moreover, at C-2′ and C-3′ of the cyclohexenyl unit, it was observed that the position of the prenyl moiety or its modified form leads to a change of activity (2 > > 10 > 3, 15 > Panduratin K (4) was isolated as a yellowish, amorphous solid, and its molecular formula was found to be C26H30O5 by HRESIMS The IR spectrum of displayed absorbances for hydroxy, carbonyl, and phenyl groups The 1H and 13C NMR data of also resembled analogous data for panduratin A (10).16 However, they differed in the signals due to the prenyl side chain The 1H NMR and HSQC spectra showed the signals of a pair of trans-coupled double bond [δH 5.54 dd (J = 15.4 and 9.4 Hz); δC 126.1 (C-1″) and δH 5.37 d (J = 15.4 Hz); δC 142.0 (C-2″)], a quaternary oxygenated carbon [δC 70.2 (C-3″)], and two tertiary methyl groups [δH 1.18; δC 30.6 (C-4″) and δH 1.17; δC 30.6 (C5″)] In the HMBC spectrum, the two H3-4″ and H3-5″ tertiary methyl groups showed correlations with the C-3″ quaternary oxygenated carbon and the C-2″ olefinic methine carbon, suggesting the linkage of C-4″ and C-5″ with C-2″ of the double bond via the C-3″ quaternary oxygenated carbon (Figure 1) The relative configuration of the cyclohexenyl unit of was found to be the same as those of and based on the coupling constant data and the NOESY spectroscopic analysis Therefore, the structure of panduratin K (4) was determined as shown Panduratin L (5) was obtained as a yellowish, amorphous solid It showed a sodiated molecular ion at m/z 459.2163 [M + Na]+, corresponding to the molecular formula, C27H32O5Na, in the HRESIMS The 1H and 13C NMR data of were similar to those of compound 4, except for the appearance of one more methoxy group (δH 3.00; δC 50.4), and two meta-coupled aromatic proton signals at δH 6.05 and 5.85 (J = 1.9 Hz) instead of the singlet signal of two magnetically equivalent aromatic protons The aromatic methoxy group was located at C-6 based on the HMBC correlation between the methoxy proton at δH 3.99 and the C-6 oxygenated quaternary aromatic carbon at δC 164.0 The location of the aliphatic methoxy group was determined to be at C-3″ based on the HMBC correlations observed between the methoxy proton (δH 3.00) and the C-3″ quaternary oxygenated carbon (δC 75.1) (Figure 1) Analysis of the NOESY correlations together with the coupling constants indicated the relative configuration of the cyclohexenyl moiety to be the same as in 2−4 Therefore, the structure of panduratin L (5) was established as shown Panduratin M (6) was isolated as a yellowish, amorphous solid Its molecular formula was assigned as C26H30O5 by HRESIMS The 1H and 13C NMR spectra were similar to those of nicolaioidesin B (13),15 a compound isolated from the same plant extract This compound showed the presence of a trans-3methyl-3-hydroxybutenyl group at C-2′ of the cyclohexenyl ring instead of the prenyl side chain (Figure 1) On the basis of the HMBC spectrum, the phenyl and 2,6-dihydroxy-4-methoxybenzoyl units were assigned at C-1′ and C-6′ of the cyclohexenyl D DOI: 10.1021/acs.jnatprod.6b00784 J Nat Prod XXXX, XXX, XXX−XXX Journal of Natural Products Article Table 1H NMR Spectroscopic Data (500 MHz) of Compounds 1−5 in Acetone-d6 (δ in ppm, Multiplicities, J in Hz) position 1′ 2′ 4′ 5′α 5′β 6′ Me-3′ 1″ 2″ 4″ 5″ 2‴, 6‴ 3‴, 5‴ 4‴ OH-2 4-OH-4 OH-2″ OH-4″ OMe-4 OMe-6 OMe-3″ 5.74 s 5.74 s 4.85 dd (11.4, 10.2) 2.84 brd (10.2) 5.58 d (4.4) 2.31 dd (18.2, 11.3) 2.14 ddd (18.2, 4.6, 4.4) 3.06 ddd (11.4, 11.3, 4.6) 1.71 s 2.23 brd (16.7) 2.15 brd (16.7) 5.13 t (6.5) 1.51 s 5.95 s 5.95 s 4.84 dd (11.8, 4.6) 2.73 ddd (10.3, 5.0, 4.6) 5.43 br 2.13 dd (18.2, 11.2) 2.38 ddd (18.2, 6.4, 4.2) 3.45 ddd (11.8, 11.2, 6.4) 1.78 s 2.00 ddd (15.5, 6.9, 5.0) 2.34 ddd (15.5, 10.3, 6.9) 5.19 t (6.9) 3.74 d (5.8) 5.94 s 5.94 s 4.89 dd (11.7, 4.5) 2.98 ddd (10.5, 4.5, 4.3) 5.36 br 2.07 dd (18.0, 11.3) 2.32 ddd (18.0, 6.0, 4.6) 3.30 ddd (11.7, 11.3, 6.0) 1.80 s 1.34 ddd (17.6, 10.6, 4.3) 2.00 ddd (17.6, 10.5, 4.0) 3.50 ddd (10.6, 3.7, 3.0) 4.61 s 4.81 s 1.62 s 7.22 d (7.8) 7.17 dd (7.8, 7.5) 7.05 t (7.5) 11.90 s 5.94 s 5.94 s 4.93 dd (11.6, 5.0) 3.16 dd (9.4, 5.0) 5.51 br 2.07 dd (17.8, 11.5) 2.37 ddd (17.8, 4.6, 4.4) 3.40 ddd (11.6, 11.5, 4.6) 1.66 s 5.54 dd (15.4, 9.4) 5.85 d (1.9) 6.05 d (1.9) 4.71 dd (11.8, 4.9) 3.13 dd (9.0, 4.9) 5.55 br 2.08 dd (18.0, 11.6) 2.37 ddd (18.0, 5.1, 5.0) 3.40 ddd (11.8, 11.6, 5.1) 1.71 s 5.47 dd (15.7, 9.0) 5.37 d (15.4) 1.18 s 5.18 d (15.7) 1.15 s 1.41 s 7.25 d (7.8) 7.07 dd (7.8, 7.4) 6.97 t (7.4) 1.56 s 7.22 d (7.4) 7.18 dd (7.4, 7.2) 7.05 t (7.2) 11.86 s 9.08 s 1.12 s 7.22 d (7.4) 7.18 dd (7.4, 7.2) 7.06 t (7.2) 13.8 s 9.45 s 3.6 d (3.7) 3.25 t (5.8) 3.77 s 3.77 s 3.76 s 3.99 s 3.00 s 680 g of a dry extract The MeOH extract was suspended in H2O (1.5 L) and then partitioned successively with CHCl3 (3 × 1.5 L) and EtOAc (3 × 1.5 L) to give CHCl3 (470 g), EtOAc (10 g), and H2O (150 g) extracts, respectively A part of the CHCl3-soluble extract (450 g) was subjected to silica gel column chromatography (9 × 120 cm), eluted with EtOAc−n-hexane gradient mixtures (0−50%), to yield 15 fractions (fr-1, 22.0 g; fr-2, 197.5 g; fr-3, 22.0 g; fr-4, 26.0 g; fr-5, 6.0 g; fr-6, 24.0 g; fr-7, 15.0 g; fr-8, 20.0 g; fr-9, 22.0 g; fr-10, 21.0 g; fr-11, 11.0 g; fr-12, 18.0 g; fr-13, 14.0 g; fr-14, 10.0 g; fr-15, 22.0 g) Fraction (22.0 g) was subjected to further silica gel column chromatography (7.5 × 120 cm), eluted with EtOAc−n-hexane gradient mixtures (0−80%), to give seven subfractions (fr-3-1, 160 mg; fr-3-2, 2.2 g; fr-3-3, 8.1 g; fr-3-4, 6.3 g; fr-35, 3.3 g; fr-3-6, 1.5 g; fr-3-7, 1.3 g) Subfraction 3-2 was rechromatographed on silica gel with a CHCl3−n-hexane gradient system to yield four subfractions, fr-3-2-1−4 Subfraction 3-2-1 (379 mg) was chromatographed on ODS silica gel with MeOH−H2O gradient mixtures (0−50%) to give 10 (300 mg), followed by normal-phase preparative TLC with EtOAc−n-hexane (20:80) to afford (6.1 mg) Subfraction 3-2-3 (377 mg) was chromatographed on ODS silica gel with MeOH−H2O gradient mixtures (0−50%) and then purified by normal-phase preparative TLC with EtOAc−CHCl3 −n-hexane (5:25:70) to give (5.0 mg), 13 (5.0 mg), and 14 (6.6 mg) Subfraction 3-3 was dissolved in CHCl3−n-hexane and left overnight to give crystals of 11 (6.0 g) Subfraction 3-6 was subjected to silica gel column chromatography with an acetone−n-hexane gradient system, to yield five subfractions, fr-3-6-1−5 Subfraction 3-6-4 (190 mg) was again separated by silica gel column chromatography with a further acetone− n-hexane gradient system, followed by reversed-phase preparative TLC with MeOH−CH3CN−H2O (10:70:20), to afford 14 (10.0 mg) Fraction (26.0 g) was subjected to silica gel column (7.5 × 120 cm) chromatography, eluted with an acetone−n-hexane gradient system, to yield 14 subfractions (fr-4-1, 18 mg; fr-4-2, 113 mg; fr-4-3, 127 mg; fr-44, 199 mg; fr-4-5, 65 mg; fr-4-6, 34 mg; fr-4-7, 616 mg; fr-4-8, 20−23 g; fr-4-9, 269 mg; fr-4-10, 32 mg; fr-4-11, 31 mg; fr-4-12, 850 mg; fr-4-13, 303 mg; fr-4-14, 2−8 g) Subfraction 4-13 was chromatographed by silica gel column chromatography, with CHCl3−n-hexane gradient mixtures (0−100%), to obtain (6.5 mg) Fraction (24.0 g) was further separated by silica gel column (7.5 × 120 cm) chromatography, 13 > 6) Furthermore, the presence of a methoxy group at C-6 of the benzoyl moiety was found to result in more potent activity than when a methoxy group at C-4 or a hydroxy group at C-6 is present (11 > 10, 14 > 13, > 7, 11 > 12) At C-4, a hydroxy group was favored over a methoxy group (1 > 9, 12 > 10) Nicolaioidesin C (15) was studied further for its effects on the morphological changes of PANC-1 using an ethidium bromide and acridine orange (EB/AO) staining assay.12 Cells treated with nicolaioidesin C (15, 1.5 μM) showed round morphology of PANC-1 cells and emitted a red fluorescence of EB, indicative of dead cells In contrast, the control cells showed intact morphology and gave a bright green fluorescence of AO, suggestive of live cells (Figure 3) ■ 1.17 s 7.23 d (7.4) 7.18 dd (7.4, 7.2) 7.06 t (7.2) 11.82 s EXPERIMENTAL SECTION General Experimental Procedures Optical rotations were recorded on a JASCO DIP-140 digital polarimeter IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3 solution NMR spectra were taken on a Bruker Advance III 500 spectrometer (Bruker Biospin) with tetramethylsilane as an internal standard, and chemical shifts are expressed in δ values HRESIMS and HRFABMS measurements were carried out on a Bruker micrOTOFQII mass spectrometer and JEOLJMS-AX505HAD mass spectrometer, respectively Silica gel 60, 40−63 μm (230−400 mesh ASTM), for column chromatography was purchased from Scharlau Analytical and preparative TLC was carried out on precoated Merck Kieselgel 60F254 or RP-18F254 plates (0.25 or 0.5 mm thickness) Plant Material The rhizomes of Boesenbergia pandurata were collected in Tinh Bien District of An Giang Province, Vietnam, in April 2013, and this species was identified by Ms Hoang Viet, Faculty of Biology, University of Science, Vietnam National University, Ho Chi Minh City (VNU-HCM) A voucher specimen (MCE0043) has been deposited at the Division of Medicinal Chemistry, Faculty of Chemistry, University of Science, VNU-HCM Extraction and Isolation Dried powdered rhizomes of B pandurata (5.5 kg) were extracted with MeOH (15 L, reflux, h × 3) to yield E DOI: 10.1021/acs.jnatprod.6b00784 J Nat Prod XXXX, XXX, XXX−XXX Journal of Natural Products Article Table 1H NMR Spectroscopic Data (500 MHz) of Compounds 6−8 in Acetone-d6 (δ in ppm, multiplicities, J in Hz) position 1′ 2′ 4′ 5′α 5′β 6′ Me-3′ 1″ 5.95 s 5.95 s 3.53 dd (11.4, 5.2) 2.75 dd (5.2, 5.0) 5.56 br 2.12 dd (17.1, 11.2) 2.65 ddd (17.1, 5.4, 5.2) 4.92 ddd (11.4, 11.2, 5.4) 1.67 s 5.27 dd (15.0, 5.0) 5.84 s 5.84 s 3.14 dd (11.0, 10.9) 2.45 ddd (10.9, 8.2, 6.4) 5.66 d (4.5) 2.15 dd (16.5, 11.3) 2.45 ddd (16.5, 4.7, 4.5) 4.65 ddd (11.3, 11.0, 4.5) 1.72 s 1.94 ddd (16.2, 8.2, 5.0) 2.15 ddd (16.2, 6.4, 5.0) 5.05 t (5.0) 1.71 s 1.45 s 7.18 d (7.9) 7.14 dd (7.9, 7.2) 7.04 t (7.2) 11.71 s 3.72 s 5.74 d (2.0) 5.94 d (2.0) 3.11 dd (11.2, 10.9) 2.48 ddd (10.9, 7.9, 6.4) 5.67 d (4.8) 2.15 dd (16.7, 11.3) 2.36 ddd (16.7, 4.8, 4.5) 4.65 ddd (11.3, 11.2, 4.5) 1.72 s 1.93 ddd (16.1, 7.9, 4.9) 2.14 ddd (16.1, 6.4, 4.9) 5.05 t (4.9) 1.71 s 1.48 s 7.15 d (7.9) 7.18 dd (7.9, 7.2) 7.04 t (7.2) 13.58 s 2″ 4″ 5″ 2‴, 6‴ 3‴, 5‴ 4‴ OH-2 OMe-4 OMe-6 5.27 d (15.0) 1.08 s 1.07 s 7.12 m 7.12 m 7.03 m 11.86 s 3.78 s 3.88 s Table 13C NMR Spectroscopic Data (125 MHz) of Compounds 1−8 in Acetone-d6 position >CO 1′ 2′ 3′ 4′ 5′ 6′ Me-3′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴ OMe-4 OMe-6 OMe-3″ 107.6 165.0 95.7 165.0 95.7 165.0 210.4 54.8 46.0 137.2 123.1 35.5 47.3 21.7 29.8 123.5 132.7 26.0 18.0 144.9 128.5 128.8 126.8 107.0 166.6 94.6 166.6 94.6 166.6 207.5 54.7 43.3 137.8 121.9 36.8 37.8 23.1 29.2 125.3 136.4 68.7 14.0 148.2 128.1 129.0 126.3 55.8 106.9 166.8 94.6 166.8 94.6 166.8 208.1 54.3 39.1 139.2 120.7 37.0 38.6 22.3 37.4 75.4 150.1 109.6 18.2 147.9 128.2 129.0 126.3 55.8 106.6 166.5 94.4 166.5 94.4 166.5 206.6 55.1 46.7 135.5 122.2 36.9 37.9 22.4 126.1 142.0 70.2 30.6 30.6 147.8 128.1 129.0 126.3 55.7 106.7 168.8 97.0 165.4 92.0 164.0 206.4 55.3 47.1 135.1 122.6 36.9 38.1 22.4 129.9 138.7 75.1 26.4 26.2 147.7 128.1 129.0 126.4 106.4 166.9 94.5 166.9 94.5 166.9 210.1 47.6 50.1 136.6 121.3 32.1 44.2 22.5 125.7 142.4 70.2 30.4 30.4 144.2 128.4 129.3 126.3 55.8 106.3 166.7 94.3 166.7 94.3 166.7 209.6 47.7 46.9 136.8 123.6 31.2 51.4 21.7 26.1 121.4 133.2 27.1 18.3 145.3 128.6 129.5 126.7 55.7 105.5 167.3 95.9 165.3 91.3 163.2 207.9 47.2 45.8 135.8 122.7 30.3 50.8 20.9 26.2 120.5 132.2 25.2 17.4 144.3 127.7 128.6 125.8 56.4 50.4 with a MeOH−CHCl3 gradient system, to yield 13 subfractions (fr-6-1, 464 mg; fr-6-2, 388 mg; fr-6-3, 1.6 g; fr-6-4, 3.8 g; fr-6-5, 5.0 g; fr-6-6, 576 mg; fr-6-7, 1.7 g; fr-6-8, 3.3 g; fr-6-9, 1.1 g; fr-6-10, 1.7 g; fr-6-11, 794 mg; fr-6-12, 815 mg; fr-6-13, 442 mg) Subfraction 6-6 was also chromatographed on silica gel with an acetone−n-hexane gradient system, followed by normal-phase preparative TLC with acetone−nhexane (20:80), to give (0.8 mg) Subfraction 6-7 was subjected to silica gel chromatography, with an EtOAc−n-hexane gradient system, to give four subfractions, fr-6-7-1−4 Of these, fr-6-7-2 (115 mg) was chromatographed on ODS silica gel, with acetone−H2O gradient mixtures (0−80%), and followed by normal-phase preparative TLC with 55.3 EtOAc−n-hexane (20:80), to afford (18.3 mg) Subfraction 6-9 was also chromatographed on silica gel with an EtOAc−n-hexane gradient system to give three subfractions, fr-6-9-1−3, and then fr-6-9-2 was dissolved in EtOAc−n-hexane and left overnight to give 12 (300.0 mg) Fraction (20.0 g) was chromatographed on silica gel (7.5 × 120 cm) with MeOH−CHCl3 gradient mixtures (0−50%) to give 20 subfractions (fr-8-1, 12 mg; fr-8-2, 30 mg; fr-8-3, 40 mg; fr-8-4, 22 mg; fr-8-5, 6.9 mg; fr-8-6, 140 mg; fr-8-7, 4.2 g; fr-8-8, 4.2 g; fr-8-9, 402 mg; fr-8-10, 752 mg; fr-8-11, 216 mg; fr-8-12, 2.43 g; fr-8-13, 4.43 g; fr-8-14, 538 mg; fr-8-15, 89 mg; fr-8-16, 119 mg; fr-8-17, 414 mg; fr-8-18, 639 mg; fr-8-19, 116 mg; fr-8-20, 158 mg) Subfraction 8-9 was subjected to silica gel column F DOI: 10.1021/acs.jnatprod.6b00784 J Nat Prod XXXX, XXX, XXX−XXX Journal of Natural Products Article chromatography, eluted with EtOAc−n-hexane gradient mixtures (0− 30%) and then acetone−n-hexane gradient mixtures (0−50%), to afford (5.0 mg) Subfraction 8-10 was subjected to silica gel column chromatography with acetone−n-hexane gradient mixtures (0−50%) to yield three subfractions, fr-8-10-1−3 Both fr-8-10-1 (143 mg) and fr-810-3 (315 mg) were subjected to silica gel column chromatography, eluted with acetone−n-hexane gradient mixtures (0−50%), to afford six subfractions, fr-8-10-1-1−3 and fr-8-10-3-1−3, respectively Subfraction 8-10-1-1 (36.3 mg) was chromatographed over ODS silica gel with MeOH−H2O gradient mixtures (0−80%), followed by reversed-phase preparative TLC with MeOH−H2O (20:80), to give (6.7 mg) Subfraction 8-10-3-2 (45.1 mg) was subjected to normal-phase preparative TLC with EtOAc−n-hexane (4:96) to give two subfractions Of these, fr-8-10-3-2-1 (25.6 mg) was recrystallized with MeOH− CHCl3 to afford (13.1 mg), while fr-8-10-3-2-2 (9.3 mg) was purified by reversed-phase preparative TLC with acetone−H2O (30:70) to afford (6.0 mg) Compound 1: yellow, amorphous solid; [α]D25 +35.6 (c 1, CH3COCH3); IR νmax (CHCl3) 3500, 1640, 1450, 1100 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRESIMS m/z 415.1885 [M + Na]+ (calcd for C25H28O4Na, 415.1885) purchased from the Riken BRC cell bank and maintained in standard Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum supplemented and stored at 37 °C under a humidified atmosphere of 5% CO2 and 95% air Briefly, human pancreatic cancer cells were seeded in 96-well plates (1.5 × 104/well) and incubated in fresh DMEM at 37 °C under 5% CO2 and 95% air for 24 h After the cells were washed twice with phosphate-buffered saline (PBS), the medium was changed to serially diluted test samples in both nutrient-rich medium (DMEM) and nutrient-deprived medium (NDM)2 with a control and blank in each test plate The composition of the NDM was as follows: 265 mg/L CaCl2 (2 H2O), 0.1 mg/L Fe(NO3)3 (9 H2O), 400 mg/L KCl, 200 mg/L MgSO4 (7 H2O), 6400 mg/L NaCl, 700 mg/L NaHCO3, 125 mg/L NaH2PO4, 15 mg/L phenol red, 25 mM/L HEPES buffer (pH 7.4), and MEM vitamin solution (Life Technologies, Inc., Rockville, MD, USA); the final pH was adjusted to 7.4 with 10% NaHCO3 Arctigenin, the positive control in this study, was isolated from the seeds of Arctium lappa.2 After 24 h of incubation with each test compound in DMEM and NDM, the cells were washed twice with PBS and replaced with 100 μL of DMEM containing a 10% WST-8 cell counting kit solution After h of incubation, the absorbance at 450 nm was measured (PerkinElmer EnSpire multilabel reader) Cell viability was calculated from the mean values of data from three wells by using the following equation: Table Preferential Cytotoxicity of Compounds 1−15 against the PANC-1 Human Pancreatic Cancer Cell Line in Nutrient-Deprived Medium (NDM) compound PC50, μMa compound PC50, μMa 5.8 6.3 18.1 6.6 10.5 3.3 7.8 7.4 10 11 12 13 14 15 arctigeninb 7.2 7.8 2.8 3.1 2.1 1.0 0.84 0.83 Cell viability (%) = [Abs(test sample) − Abs(blank)/Abs(control) − Abs(blank)]× 100% Morphological Assessment of Cancer Cells PANC-1 cells were seeded in 24-well plates (6 × 104/well) and incubated in fresh DMEM at 37 °C under 5% CO2 and 95% air for 24 h After the cells were washed twice with PBS, the medium was changed to NDM (control) or nicolaioidesin C (15, 1.5 μM) in NDM (treated) After a 24 h incubation, μL of EB/AO reagent was added to the each test well and incubated for min, and the morphology was captured using an EVOS FL cell imaging system (20× objective) under fluorescent and phase contrast mode ■ a Concentration at which 50% of cells were killed preferentially in NDM bPositive control ASSOCIATED CONTENT S Supporting Information * The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00784 Copies of spectroscopic data for 1−8 (PDF) Compound 2: yellow, amorphous solid; [α]D25 +31.4 (c 1, CH3COCH3); IR νmax (CHCl3) 3600, 1640, 1460, 1090 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRESIMS m/z 445.1991 [M + Na]+ (calcd for C26H30O5Na, 445.1976) Compound 3: yellow, amorphous solid; [α]D25 +77.5 (c 1, CH3COCH3); IR νmax (CHCl3) 3600, 1640, 1445, 1090 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRESIMS m/z 445.1991 [M + Na]+ (calcd for C26H30O5Na, 445.1991) Compound 4: yellow, amorphous solid; [α]D25 +27.9 (c 1, CH3COCH3); IR νmax (CHCl3) 3600, 1640, 1450, 1100 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRESIMS m/z 445.1997 [M + Na]+ (calcd for C26H30O5Na, 445.1991) Compound 5: yellow, amorphous solid; [α]D25 +33.8 (c 1, CH3COCH3); IR νmax (CHCl3) 3500, 1640, 1440, 1090 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRESIMS m/z 459.2163 [M + Na]+ (calcd for C27H32O5Na, 459.2147) Compound 6: yellow, amorphous solid; [α]D25 +27.5 (c 1, CH3COCH3); IR νmax (CHCl3) 3600, 1650, 1450, 1100 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRESIMS m/z 445.1980 [M + Na]+ (calcd for C26H30O5Na, 445.1991) Compound 7: yellow, amorphous solid; [α]D25 +37.5 (c 1, CH3COCH3); IR νmax (CHCl3) 3500, 1650, 1460, 1100 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRESIMS m/z 405.2075 [M − H]− [calcd for C26H29O4, 405.2066] Compound 8: yellow, amorphous solid; [α]D25 +23.6 (c 1, CH3COCH3); IR νmax (CHCl3) 3500, 1650, 1450, 1090 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Tables and 3); HRFABMS m/z 407.22253 [M + H]+ (calcd for C26H31O4, 401.22224) Preferential Cytotoxicity Assay against PANC-1 Cells The PANC-1 (RBRC-RCB2095) human pancreatic cancer cell line was ■ AUTHOR INFORMATION Corresponding Authors *E-mail (N T Nguyen): ntnhan@hcmus.edu.vn Tel: +84-907426-331 Fax: +84-838-353-659 *E-mail (S Awale): suresh@inm.u-toyama.ac.jp Tel: +81-76434-7640 Fax: +81-76-434-7640 ORCID Nhan Trung Nguyen: 0000-0001-5142-4573 Notes The authors declare no competing financial interest ■ ACKNOWLEDGMENTS This research was supported by a grant from Vietnam’s National Foundation for Science and Technology Development (No 104.01-2013.72) to N.T.N and by a Grant in Aid for Scientific Research (16K08319) from the Japan Society for the Promotion of Science (JSPS), Japan, to S.A ■ REFERENCES (1) Izuishi, K.; 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Grimm, C.; Wray, V.; Witte, L.; Proksch, P Phytochemistry 1993, 34, 415−419 (18) Trakoontivakorn, G.; Nakahara, K.; Shinmoto, H.; Takenaka, M.; Onishi-Kameyama, M.; Ono, H.; Yoshida, M.; Nagata, T.; Tsushida, T J Agric Food Chem 2001, 49, 3046−3050 H DOI: 10.1021/acs.jnatprod.6b00784 J Nat Prod XXXX, XXX, XXX−XXX ... between the methoxy proton at δH 3.99 and the C-6 oxygenated quaternary aromatic carbon at δC 164.0 The location of the aliphatic methoxy group was determined to be at C-3″ based on the HMBC correlations... (Figure 1) The relative configuration of the cyclohexenyl unit of was found to be the same as those of and based on the coupling constant data and the NOESY spectroscopic analysis Therefore, the structure... correlations with the C-3″ quaternary oxygenated carbon and the C-2″ olefinic methine carbon, suggesting the linkage of C-4″ and C-5″ with C-2″ of the double bond via the C-3″ quaternary oxygenated