Cerasoidine, a Bis-aporphine Alkaloid Isolated from - ACS Publications

Aug 4, 2016 - ABSTRACT: A new bis-aporphine alkaloid, cerasoidine (1), was isolated from the root extract of Polyalthia cerasoides together with the k...
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Cerasoidine, a Bis-aporphine Alkaloid Isolated from Polyalthia cerasoides during Screening for Wnt Signal Inhibitors Takumi Shono,† Naoki Ishikawa,† Kazufumi Toume,† Midori A. Arai,† Hyuma Masu,‡ Takashi Koyano,§ Thaworn Kowithayakorn,⊥ and Masami Ishibashi*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Center for Analytical Instrumentation, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan § Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164-0012, Japan ⊥ Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ‡

S Supporting Information *

ABSTRACT: A new bis-aporphine alkaloid, cerasoidine (1), was isolated from the root extract of Polyalthia cerasoides together with the known bis-aporphine bidebiline E (2) during screening for compounds with Wnt signal inhibitory activities. The structure of cerasoidine (1) was established by X-ray analysis and shown by chiral HPLC analyses and electronic circular dichroism to be a 57:43 mixture of R(−)- and S(+)atropisomers. Bidebiline E (2) exhibited inhibition of transcriptional activity of TCF/β-catenin with an IC50 value of 20.2 μM and was also found to inhibit Wnt signaling by decreasing nuclear β-catenin.

W

(CCTTTGATC) and is activated by the addition of LiCl, an inhibitory agent of GSK3β (glycogen synthase kinase 3β). The Wnt signal inhibitory activity of the sample is assessed based on the decrease in luciferase activity. SuperFOPFlash is a plasmid with a distorted TCF binding site (CCTTTGGCC), and its luciferase activity (FOP activity) is evaluated to exclude nonselective Wnt signal inhibition. We have examined hundreds of plant extracts using this assay system and identified a promising extract from the roots of P. cerasoides. The dried roots of P. cerasoides were successively extracted with MeOH and CHCl3. The MeOH- and CHCl3-soluble fractions both inhibited TOP activity (65% and 80% inhibition at 10 μg/mL, respectively). The CHCl 3 fraction was fractionated by silica gel and ODS (octadesylsilane) chromatography, and the constituent compounds were purified by reversed-phase HPLC (Develosil ODS-SR-5 column, Nomura Chemical, Seto, Aichi, Japan). A new bis-aporphine alkaloid (1), cerasoidine, was isolated, together with the known bisaporphine bidebiline E (2), as well as octadeca-9,11,13-triynoic acid (3) and its methyl ester (4) (Chart 1). The known compounds (2, 3, and 4) were identified by comparison of their observed and reported physical data.5 Similar purification of the MeOH extract afforded four new 8-oxoberberine alkaloids (5− 8), three known 8-oxoberberine alkaloids [8-oxodehydrodiscretamine (9),6 8-oxodiscretamine (10),6 oxypalmatine (11)],7 and two known monomeric aporphine alkaloids, liriodenine

nt signaling, which is a highly conserved signal transduction pathway, is known to be involved in the development of tissue homeostasis1,2 and is implicated in tumorigenesis, diabetes, and schizophrenia. New treatment strategies that target Wnt pathways are, therefore, being developed to control the replication, survival, and differentiation of stem cells. Consequently, there is interest in identifying Wnt signaling inhibitors from small-molecule natural products. Our ongoing natural-product-based screening studies on Wnt pathways have identified and isolated several unique natural products that affect different steps in Wnt pathways.3 Herein is described a screening study of the root extract of Polyalthia cerasoides (Annonaceae) on Wnt pathways in which a new bis-aporphine alkaloid, cerasoidine (1), was isolated along with bidebiline E (2), a known bis-aporphine. Several bioactive natural products such as alkaloids, sesquiterpenoids, and acetylenic fatty acids have been isolated from P. cerasoides and were found to exhibit antimalarial or antimycobacterial activities.

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RESULTS AND DISCUSSION For the evaluation of the inhibition of TCF/β-catenin-mediated transcriptional activity (TOP activity), a cell-based reporter luciferase assay system was used. The complex of TCF/βcatenin is a transcriptional factor downstream of the Wnt pathway.4 The transcriptional activity (TOP activity) of TCF/ β-catenin is assessed via the STF/293 cell line, a 293 human embryonic kidney (HEK) cell line transfected steadily with SuperTopflash. SuperTopflash is a luciferase reporter plasmid that contains eight copies of the TCF-binding site © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 12, 2016

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Table 1. 1H and 13C NMR Spectroscopic Data of 1 in CDCl3

Chart 1. Structures of the Compounds Isolated from Polyalthia cerasoides

δH (J in Hz)

position 1, 1′ 1a, 1′a 1b, 1′b 2, 2′ 3, 3′ 3a, 3′a 4, 4′ 5, 5′ 6a, 6′a 7, 7′ 7a, 7′a 8, 8′ 9, 9′ 10, 10′ 11, 11′ 11a, 11′a OCH2O− 9, 9′-OMe CO

δC

7.02 s 3.67 m and 2.94 br d (14.6) 4.39 ddd (12.9, 5.3, 1.4) and 3.33 td (13.2, 3.1)

6.66 d (2.8) 7.10 dd (9.1, 2.8) 9.01 d (9.1) 6.28 d (1.7) and 6.23 d (1.7) 3.36 s

141.4 117.2 118.8 145.7 108.4 128.3 29.3 43.2 138.7 121.8 132.3 109.4 157.7 115.4 128.6 118.8 101.1 55.0 166.8

C C C C CH C CH2 CH2 C C C CH C CH CH C CH CH3 C

X-ray analysis revealed that the crystal of cerasoidine (1) was a racemic mixture of two enantiomers (Figure S1, Supporting Information). However, the optical rotation and electronic circular dichroism (ECD) data of isolated cerasoidine (1) suggested that it was optically active: [α]24D −43 (c 0.2, CHCl3); ECD λmax (Δε) (CH3CN) 340 (+0.29), 281 (−7.09), 257 (+0.15), 240 (−3.08), and 217 (+3.58). Chiral HPLC analysis of cerasoidine (1) revealed that it was a 57:43 mixture of atropo-enantiomers (Figure S2, Supporting Information). Comparison of the ECD curve of 1 with that of (S)-(+)-9,10dihydrodibenzo[c,g]phenanthrene suggested that the major enantiomer of 1 was the (R)-(−)-form (Figure S3, Supporting Information).10 Compound 5 was a pale yellow solid, and its molecular formula was determined by HRESIMS to be C20H19NO6 (m/z 392.1124, calcd for [C20H19NO6Na]+, 392.1110). The 1H and 13 C NMR data of compound 5 (Tables S1 and S3, Supporting Information) were similar to those of 8-oxodehydrodiscretamine (9), whereas the 1H NMR spectrum of 5 showed signals indicative of one less aromatic hydrogen and one more methoxy group than in 9, suggesting that one aromatic hydrogen of 9 was replaced by a methoxy group in 5. The position of the methoxy group was deduced to be at C-4 from HMBC correlations from MeO (δH 3.72, 3H, s) to C-4 (δC 144.2) and from H2-5 (δH 2.84, 2H, t, J = 6.2 Hz) to C-4 (Figure S5B). Thus, the structure of compound 5 was revealed to be 4-methoxy-8-oxodehydrodiscretamine. Compound 6, a pale yellow solid, was shown by HRESIMS to have the same molecular formula as compound 5 (C20H19NO6, m/z 392.1130, calcd for [C20H19NO6Na]+, 392.1110), and the 1H and 13C NMR spectra of compound 6 were closely related to those of compound 5. In the HMBC spectrum of 6, correlations from H2-5 (δH 2.80, 2H, t, J = 5.9 Hz) to C-4 (δC 146.7), from 4-OH (δH 9.20, 1H, s) to C-4a (δC 116.0) and C-3 (δC 136.9), and from MeO (δH 3.71, 3H, s) to C-3 (Figure S5C, Supporting Information) suggested that the positions of the OH and MeO groups at C-3 and C-4, respectively, in 5 were reversed in 6. Thus, the structure of compound 6 was defined as 4-hydroxy-3-O-methyl-8-oxodehydrodiscretamine.

(12)8 and lanuginosine (13).9 The spectroscopic data of 9 and 10 were not reported6 and are listed here for the first time. Cerasoidine (1) was obtained as a brown solid, and the molecular formula C37H26N2O7 was determined by HRESITOFMS from the sodium adduct ion observed at m/z 633.1595 [M + Na]+ (calcd for C37H26N2O7Na, 633.1638). The IR spectrum of 1 showed no characteristic OH or NH absorption at 3000−3500 cm−1, but showed bands due to aromatic rings (1609 cm−1). The 1H and 13C NMR spectroscopic data of 1 (Table 1) were closely related to those of bidebiline E (2),5 suggesting that compound 1 has a dimeric structure. The 13C NMR spectrum of 1 afforded 19 peaks, suggesting that the monomeric part of 1 consists of 18 carbons, similar to bidebiline E (2), and one additional amide-like carbonyl carbon at δC 166.8. The molecular formula of 1 indicated that compound 1 has one more carbon, one more oxygen, and two fewer hydrogen atoms (+CO − 2H) than 2. Consequently, compound 1 was deduced to have a structure in which the two nitrogen atoms (N-6 and N-6′) of bidebiline E (2) are connected through a ureido bond. The HMBC spectrum of 1 consistently showed long-range connectivity from H-5 (or H5′) at δH 3.33 to the carbonyl carbon at δC 166.8 (Figure S5A, Supporting Information). This proposed structure of cerasoidine (1) was unequivocally established using X-ray crystallographic analysis (Figure 1). The dihedral angle of the biphenyl bond was 54 degrees (Figure 1). B

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Figure 1. ORTEP drawing of cerasoidine (1) (left: front view, right: side view). The dihedral angle of the biphenyl bond is 54 deg.

394.1287, calcd for [C20H21NO6Na]+, 394.1267). For 8, the HMBC spectrum showed correlations from H2-6 (δH 4.78/ 2.68) to C-4a (δC 115.6), from 4-OH (δH 8.96, 1H, s) to C-4a, C-4 (δC 147.0), and C-3 (δC 134.5), and from MeO (δH 3.65, 3H, s) to C-3 (Figure S5E, Supporting Information), indicating that the positions of the 3-OH and 4-OMe groups in 7 were reversed in 8. Thus, the structure of compound 8 was defined as 4-hydroxy-3-O-methyl-8-oxodiscretamine. The absolute configurations of compounds 7, 8, and 10 were deduced to be 14S based on comparison of their ECD data with those of (S)- and (R)-protoberberine alkaloids11 (Figure S4, Supporting Information). The inhibition of Wnt signaling by compounds 1−13 was evaluated using the STF/293 cell line (vide supra). Among these compounds, compounds 2, 9, 11, 12, and 13 significantly

Compound 7 was a pale yellow solid, and HRESIMS showed its molecular formula to be C20H21NO6 (m/z 394.1285, calcd for [C20H21NO6Na]+, 394.1267). Compound 7 was optically active ([α]24D −794 (c 0.1, MeOH)), and its 1H and 13C NMR data (Tables S2 and S3, Supporting Information) resembled those of 8-oxodiscretamine (10). The 1H NMR spectrum of 7 suggested that H-4 in 10 was replaced by a methoxy group (δH 3.69, 3H, s) in 7, consistent with the HMBC connectivities between MeO/C-4 (δC 144.6), 3-OH (δH 8.62, 1H, s)/C-4, and 3-OH/C-2 (δ C 147.6) (Figure S5D, Supporting Information). Therefore, the structure of compound 7 was defined as 4-methoxy-8-oxodiscretamine. Compound 8, a pale yellow solid, was optically active ([α]24D −609 (c 0.1, MeOH)) and had the same molecular formula as compound 7 based on HRESIMS data (C20H21NO6, m/z C

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that liriodenine (12) augments β-catenin degradation by the proteasome system. In conclusion, fractionated extracts of the root of P. cerasoides were examined using a cell-based luciferase assay specific for Wnt signaling activity, resulting in the isolation of one new bisaporphine alkaloid, cerasoidine (1), four new protoberberine alkaloids (5−8), the known bis-aporphine bidebiline E (2), triynoic fatty acid (3) and its methyl ester (4), three known protoberberine alkaloids (9−11), and two known monomeric aporphine alkaloids (12 and 13). The structure of cerasoidine (1) was established by X-ray analysis and shown by chiral HPLC analyses to be a 57:43 enantiomeric mixture. Although bis-aporphine alkaloids with a ureido bond are known,12,13 cerasoidine (1) has a new dimeric structure with two linkages (a C-7−C-7′ bond and a ureido bond between N-6 and N-6′). The Wnt signaling inhibitory activities of the isolated compounds were evaluated and showed that five compounds (2, 9, 11, 12, and 13) exhibited significantly activity. Of these, bidebiline E (2) apparently inhibits Wnt signaling by suppressing the accumulation of β-catenin in the nucleus, whereas liriodenine (12) appears to up-regulate the proteasome system’s degradation of β-catenin. Bidebiline E (2) and octadeca-9,11,13-triynoic acid (3) have been reported to exhibit antimalarial and antimycobacterial activity,5 while protoberberine alkaloids are weakly cytotoxic toward cancer cells.14 The relationships between these activities and the Wnt signal inhibitory activities of these compounds remain to be investigated.

inhibited TOP activity but had a negligible effect on FOP activity and cell viability (Figure S6, Supporting Information). The IC50 values of compounds 2, 9, 11, 12, and 13 on TOP activity were 20.2, 8.7, 0.6, 0.6, and 7.1 μM, respectively. Of these, the effects of bidebiline E (2) and liriodenine (12) on βcatenin were examined using Western blot analysis. In SW480 colon cancer cells, bidebiline E (2) showed no effects on βcatenin in either whole cell lysate or cytosol lysate, whereas it significantly decreased β-catenin in nuclear lysate. The effects of 2 on the level of c-myc, a TCF/β-catenin target protein, were also examined and revealed dose-dependent decreases (Figure 2). These results suggest that bidebiline E (2) inhibits Wnt

Figure 2. Western blot analysis of β-catenin and c-myc in SW480 colon cancer cells treated with 2. After obtaining full, cytoplasmic, and nuclear lysates from cells, they were subjected to Western blot analysis with anti-β-catenin and anti-c-myc antibodies. β-Actin served as a protein control for the cytoplasmic fraction, and histone H1 served as a control for the nuclear fraction.

signaling by suppressing the β-catenin accumulation in the nucleus. The effects of liriodenine (12) on β-catenin and the Wnt target proteins c-myc and PPARδ were examined in STF/ 293 cells by Western blot analysis, and liriodenine (12) was found to dose-dependently decrease the levels of all three proteins (Figure 3A). The possible involvement of the

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EXPERIMENTAL SECTION

General Experimental Procedures. Spectroscopic instruments used in this study were the same as those in the previous paper15 except ECD (JASCO J-1100 spectrophotometer) and NMR (JEOL ECZ600 and ECZ400 spectrometers). Preparative HPLC was performed using a Develosil ODS-SR-5 column (Nomura Chemical Co., Ltd., Seto, Japan). Plant Materials. Roots of Polyalthia cerasoides (Annonaceae) were obtained from Khon Kaen in Thailand and identified by T. Kowithayakorn. A specimen (KKP335) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Activity-Guided Extraction and Isolation. The air-dried roots of P. cerasoides (148 g) were extracted with MeOH overnight at ambient temperature, homogenized, and filtered, and the solvent was removed under reduced pressure to obtain a crude MeOH extract (15.3 g). The residue was extracted with CHCl3 overnight at ambient temperature and processed as above to obtain a crude CHCl3 extract (1.2 g), which was fractionated by ODS column chromatography (φ 4.5 × 13 cm) using a MeOH/H2O solvent system (8:2−1:0 and washed with 0.1% TFA in MeOH) to afford fractions 12A to 12G. The components of fraction 12C (10.5 mg) were separated by preparative HPLC [Develosil ODS-SR-5 column, φ 10 × 250 mm, MeOH/H2O (95:5), flow rate 2.0 mL/min, detection RI and UV at 254 nm] to provide 2 (2.5 mg, tR 46 min) and 1 (0.4 mg, tR 77 min). Fraction 12A (16.6 mg) was separated using an ODS column (φ 4.5 × 12 cm) and a MeOH/H2O solvent system (6:4−1:0 and washed with 0.1% TFA in MeOH) to afford fractions 22A to 22F. Purification of fraction 22D was performed by preparative HPLC [Develosil ODS-SR-5, φ 10 × 250 mm, MeOH/H2O (95:5), flow rate 2.0 mL/min, detection RI and UV at 254 nm] to give 3 (3.1 mg, tR 17 min) and 4 (2.2 mg, tR 25 min). The MeOH extract (15.2 g) was partitioned between 10% aqueous MeOH (100 mL) and n-hexane (700 mL × 3), followed by extraction with EtOAc and BuOH (700 mL × 3) to give n-hexane (2.4 g), EtOAc (2.5 g), BuOH (8.2 g), and aqueous (6.7 g) soluble fractions. The EtOAc-soluble fraction (2.5 g) was subjected to silica gel column

Figure 3. (A) Western blot analysis of β-catenin, c-myc, and PPARδ in STF/293 cells treated with 12. (B) Western blot analysis of β-catenin in STF/293 cells treated with 12 and MG132 for 24 h. Whole lysates from cells were subjected to Western blot analysis with anti-β-catenin. β-Actin served as a protein control.

proteasome system in the observed decrease in β-catenin level was assessed by examining the effects of liriodenine (12) on β-catenin in the presence of the well-known proteasome inhibitor MG132. MG132 inhibits proteasome-mediated βcatenin degradation. The results shown in Figure 3B indicate that the addition of MG132 inhibits the decrease in β-catenin levels in STF/293 cells caused by liriodenine (12), suggesting D

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chromatography (PSQ100B, φ 4.5 × 15 cm) by elution with a CHCl3/ MeOH solvent system (1:0−0:1 and washed with 0.1% TFA in MeOH) to afford fractions 10A to 10H. Fraction 10A (315 mg), which was eluted with CHCl3/MeOH (1:0), was subjected to silica gel column chromatography (PSQ100B, φ 3 × 33 cm) by elution with an n-hexane/EtOAc solvent system (1:0−0:1, then washed with 0.1% TFA in MeOH) to afford fractions 13A to 13L. Fraction 13K (82 mg), which was eluted with n-hexane/EtOAc (0:1), was subjected to ODS column chromatography (φ 4.5 × 12 cm) by elution with a MeOH/ H2O solvent system (4:6−1:0 and washed with 0.1% TFA in MeOH) to afford fractions 15A to 15N. Fraction 15C (9.8 mg), which was eluted with MeOH/H2O (1:1), was separated by preparative HPLC [Develosil ODS-SR-5, φ 10 × 250 mm, MeOH/H2O (55:45), flow rate 2.0 mL/min, detection RI and UV at 254 nm] to give 10 (0.3 mg, tR 33 min), 7 (0.2 mg, tR 35 min), and 8 (0.4 mg, tR 47 min). Fraction 15D (16.8 mg) was separated by preparative HPLC [Develosil ODSSR-5, φ 10 × 250 mm, MeOH/H2O (60:40), flow rate 2.0 mL/min, detection RI and UV at 254 nm] to give 9 (4.0 mg, tR 34 min), 5 (6.4 mg, tR 37 min), and 6 (2.4 mg, tR 52 min). Fraction 15F (3.4 mg) was separated by preparative HPLC [Develosil ODS-SR-5, φ 10 × 250 mm, MeOH/H2O (65:35), flow rate 2.0 mL/min, detection RI and UV at 254 nm] to give 11 (0.2 mg, tR 52 min). Fraction 15G (1.8 mg) was further separated by preparative HPLC [Develosil ODS-SR-5, φ 10 × 250 mm, MeOH/H2O (75:25), flow rate 2.0 mL/min, detection RI and UV at 254 nm] to give 12 (0.7 mg, tR 27 min). Further separation of fraction 15I (2.9 mg) was performed by preparative HPLC [Develosil ODS-SR-5, φ 10 × 250 mm, MeOH/H2O (85:15), flow rate 2.0 mL/min, detection RI and UV at 254 nm] to give 13 (0.7 mg, tR 19 min). HPLC Separation Using a Chiral Column. A sample of 1 was subjected to HPLC separation [CHIRALPACK IB, φ 4.6 × 250 mm, n-hexane/CHCl3 [100/0−75/25 (linear gradient, 0−30 min), 75/25 (30−90 min), flow rate 0.2 mL/min, detector wavelength 358 nm]. The compound that eluted first was identified as the (R)(−)-enantiomer (tR 73 min). X-ray Diffraction Analysis. Crystals were obtained from a MeOH/CHCl3 (1:1) solution of compound 1 at room temperature in the dark. Single-crystal X-ray diffraction data were collected using a CCD diffractometer with monochromatic Cu Kα (α = 1.541 78 Å) radiation. Data collection was conducted at 173 K under liquid nitrogen cooling. Direct methods (SHELXS-97) revealed the crystal structure, which was refined by full-matrix least-squares (SHELXL2014).16 All non-hydrogen atoms were refined anisotropically, and hydrogen atoms were included at their calculated positions. Crystal data are shown in Figure S1 and Tables S4−S8 in the Supporting Information. The crystallographic data have been deposited in the Cambridge Crystallographic Data Centre under deposition number CCDC 1473176. Cell Cultures. SW480 cells were derived from the Institute of Development, Aging, and Cancer, Tohoku University. The STF/293 cell line was generously provided by Prof. Jeremy Nathans (John Hopkins Medical School). Cell cultures were performed according to the procedures described in the previous paper.15 Viability Assay. Viability assays followed the procedures described previously.15 Luciferase Assay. Luciferase assays were carried out according to the procedures described in the previous paper.15 Preparation of Whole Cellular, Cytosolic, and Nuclear Proteins. Preparation of whole cellular, cytosolic, and nuclear proteins followed the procedures described previously.4,15 Western Blot Analysis. Western blot analyses were performed according to the procedures described in the previous paper.15 Cerasoidine (1): brownish solid; [α]24 D −43 (c 0.2, CHCl3); UV (CH3CN) λmax (log ε) 537 (3.0), 358 (4.0), 266 (4.6), 210 (4.3); IR (ATR) νmax 3844, 3743, 3600, 2925, 1673, 1609, 1466, 1294, 1216, 1050 cm−1; ECD (CH3CN) λnm (Δε) 340 (+0.29), 281 (−7.09), 257 (+0.15), 240 (−3.08), 217 (+3.58); 1H and 13C NMR, see Table 1; HRMS (ESI) m/z 633.1595 [M + Na]+ (calcd for C37H26N2O7Na 633.1638).

4-Methoxy-8-oxodehydrodiscretamine (5): pale yellow solid; UV (MeOH) λmax (log ε) 372 (3.9), 336 (4.1), 223 (4.4); IR (ATR) νmax 3379, 2934, 1644, 1602, 1499, 1328 cm−1; 1H and 13C NMR, see Tables S1 and S3; HRMS (ESI) m/z 392.1124 [M + Na]+ (calcd for C20H19NO6Na 392.1110). 4-Hydroxy-3-O-methyl-8-oxodehydrodiscretamine (6): pale yellow solid; UV (MeOH) λmax (log ε) 537 (1.7), 365 (4.0), 333 (4.4), 225 (4.5); IR (ATR) νmax 3734, 3141, 2935, 1645, 1604, 1501, 1466, 1289, 1021 cm−1; 1H and 13C NMR, see Tables S1 and S3, Supporting Information; HRMS (ESI) m/z 392.1130 [M + Na]+ (calcd for C20H19NO6Na 392.1110). 4-Methoxy-8-oxodiscretamine (7): pale yellow solid; [α]23 D −794 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 371 (3.0), 310 (3.7), 232 (4.5); IR (ATR) νmax 3328, 2938, 1739, 1635, 1464, 1289, 1220, 1119, 1052, 686 cm−1; ECD (MeOH) λnm (Δε) 308 (−9.30), 275 (−0.94), 242 (−20.33), 228 (−13.17), 219 (−21.81), 217 (−20.68), 207 (−44.51); 1H and 13C NMR, see Tables S2 and S3, Supporting Information; HRMS (ESI) m/z 394.1285 [M + Na]+ (calcd for C20H21NO6Na 394.1267). 4-Hydroxy-3-O-methyl-8-oxodiscretamine (8): pale yellow solid; [α]24 D −609 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 310 (3.8), 229 (4.8); IR (ATR) νmax 3844, 3739, 3596, 1741, 1643, 1513, 1465, 1369, 1286, 1220, 676 cm−1; ECD (MeOH) λnm (Δε) 309 (−7.63), 274 (−1.24), 243 (−16.71), 230 (−8.82), 210 (−40.80); 1H and 13C NMR, see Tables S2 and S3, Supporting Information; HRMS (ESI) m/z 394.1287 [M + Na]+ (calcd for C20H21NO6Na 394.1267). 8-Oxodehydrodiscretamine (9): pale yellow solid; UV (MeOH) λmax (log ε) 537 (1.6), 372 (3.9), 337 (4.2), 224 (4.4); IR (ATR) νmax 3120, 1644, 1597, 1513, 1340, 1282, 1206, 1160, 1018 cm−1; 1H and 13 C NMR, see Tables S1 and S3, Supporting Information; HRMS (ESI) m/z 362.0992 [M + Na]+ (calcd for C19H17NO5Na 362.1004). 8-Oxodiscretamine (10): pale yellow solid; [α]23 D −549 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 370 (2.9), 309 (3.6), 281 (3.9), 234 (4.3); IR (ATR) νmax 3365, 2933, 1634, 1513, 1436, 1285 cm−1; ECD (MeOH) λnm (Δε) 310 (−6.68), 276 (−1.02), 242 (−14.89), 230 (−10.63), 222 (−14.55), 217 (−10.05), 205 (−35.79); 1H and 13 C NMR, see Tables S2 and S3, Supporting Information; HRMS (ESI) m/z 364.1177 [M + Na]+ (calcd for C19H19NO5Na 364.1161).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00409. Crystallographic data (CIF) Tables for 1H and 13C NMR spectroscopic data of new compounds, X-ray crystal analysis data, chiral HPLC analysis, ECD spectroscopic comparisons, inhibition data of TCF/β-catenin transcriptional activities of isolated compounds, and spectroscopic charts (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel and Fax: +81-43-226-2923. E-mail: [email protected] (M. Ishibashi). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by KAKENHI Grant Nos. 26305001 and 25870128 from the Japan Society for the Promotion of Science and KAKENHI Grant No. 23102008 on Innovative Areas “Chemical Biology of Natural Products” from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Takeda Science Foundation, and the Strategic E

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Priority Research Promotion Program of Chiba University. We are grateful to Prof. J. Nathans, John Hopkins University School of Medicine, for the STF/293 cells and Prof. R. T. Moon, University of Washington, for the SuperFOPFlash plasmid.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00409 J. Nat. Prod. XXXX, XXX, XXX−XXX