Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
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Ca2+-Signal Transduction Inhibitors, Kujiol A and Kujigamberol B, Isolated from Kuji Amber Using a Mutant Yeast Takeshi Uchida,† Hiroyuki Koshino,‡ Shunya Takahashi,‡ Eisaku Shimizu,† Honoka Takahashi,§ Jun Yoshida,⊥ Hisao Shinden,∥ Maiko Tsujimura,# Hisayoshi Kofujita,†,§,# Shota Uesugi,# and Ken-ichi Kimura*,†,§,# †
Graduate School of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan RIKEN Center for Sustainable Resource Science, Wako, Saitama 351-0198, Japan § Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan ⊥ Center for Liberal Arts and Sciences, Iwate Medical University, Yahaba, Iwate 028-3694, Japan ∥ Kuji Kohaku Co. Ltd., Kuji, Iwate 028-0071, Japan # The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan ‡
S Supporting Information *
ABSTRACT: A podocarpatriene and a labdatriene derivative, named kujiol A [13-methyl-8,11,13-podocarpatrien-19-ol (1)] and kujigamberol B [15,20-dinor-5,7,9-labdatrien-13-ol (2)], respectively, were isolated from Kuji amber through detection with the aid of their growth-restoring activity against a mutant yeast strain (zds1Δ erg3Δ pdr1Δ pdr3Δ), which is known to be hypersensitive with respect to Ca2+-signal transduction. The structures were elucidated by spectroscopic data analysis. Compounds 1 and 2 are rare organic compounds from Late Cretaceous amber, and the mutant yeast used seems useful for elucidating a variety of new compounds from Kuji amber specimens, produced before the K−Pg boundary.
A
structures, but this issue still remains unresolved.10 Although ambers, especially Baltic amber, have been used as medicinal ingredients, biologically active compounds have not been isolated and identified until quite recently.15 Our group has focused on the alcohol-soluble fractions (about 5%) from amber samples as natural sources for potential drug screening and has been studying biologically active compounds in ambers, using detection by growth-restoring activity against a mutant yeast involving Ca2+-signal transduction.16−18 A new compound named kujigamberol (15,20-dinor-5,7,9-labdatrien-18-ol) (3) from Kuji amber (Japan) [90−86 million years ago (Ma) (Prof. Hisao Ando, Ibaraki University, personal communication)]19 has been reported using activity-guided fractionation for the first time.20,21 Although a pure compound has been reported as quesnoin that might be formed as a byproduct of the polymerization process from Oise amber (55 Ma) and contributes to the Earth’s climate history, there is no
mber is a polymerized and fossilized tree resin that formed under favorable circumstances in soil and sediments up to hundreds of millions years ago and is distributed all over the world.1−4 During fossilization, the original compounds (bioterpenoids: unaltered biosynthetic natural products) underwent diagenetic transformation to form different structures (geoterpenoids: diagenetic products), which are found in amber. Some diagenetic pathways for the terpenoid precursors from some ambers have been proposed.5−9 GC-MS and pyrolysis (Py) GC-MS are commonly used methods for elucidating the molecular composition of amber samples by comparison of their GC-MS spectra with those of standard compounds and with library data.10−14 However, there is no guarantee that the ancient plants represented have survived or even that closely related species still exist. Moreover, GC-MS is quite effective for the identification of known molecules, but not conclusive for new compound structural elucidation. A further complication is that some compounds are interpreted to be the pyrolysis products of degraded labdanoids that are derived from macromolecular © XXXX American Chemical Society and American Society of Pharmacognosy
Received: November 2, 2017
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DOI: 10.1021/acs.jnatprod.7b00922 J. Nat. Prod. XXXX, XXX, XXX−XXX
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information on its biological activity.22 Recently 2,5,8-trimethyl1-butyltetralin was directly isolated from an extract of Cretaceous Basque-Cantabrian Basin (Spain) and has been revised subsequently as 1,6-dimethyl-5-isopentyltetralin (amberene) by NMR spectroscopy.9 Biologically active compounds from Baltic amber (56−34 Ma, Poland and Russia) such as agathic acid 15-monomethyl ester, dehydroabietic acid, and pimaric acid were also isolated from modern Araucaria and Pinus leaves using the same mutant yeast.20 One new biologically active compound, 5(10)-halimen-15-oic acid, and two known biologically active compounds, 3-cleroden-15-oic acid and 8-labden-15-oic acid, which inhibit the degranulation of RBL-2H3 cells (antiallergy), were isolated from Dominican amber (45−30 Ma and 20−15 Ma, Dominican Republic) and were also isolated from a modern Himenaea species.23 A key question that arises is why new compounds are isolated only from Kuji amber, whereas only known compounds occur in Baltic and Dominican ambers.20,23 As the environment of Earth on the Cretaceous−Paleogene (K− Pg) (formerly Cretaceous−Tertiary, K−T) boundary (65 Ma) was altered so as to destroy most of plants and dinosaurs, the botanical origin of Kuji amber that is older than the K−Pg boundary died out around the time of the K−Pg boundary. To try to find new compounds, we have isolated biologically active compounds other than 3 from Kuji amber. Described herein are the isolation, structure elucidation, and biological activity of two new compounds (1 and 2) from Kuji amber.
Figure 1. COSY and HMBC correlations of 1.
value (65.3 ppm) and was confirmed by the 1H−13C HSQC spectrum (Figure S6, Supporting Information). The connectivity of each partial structure was established by analysis of the long-range coupling correlations in the HMBC spectrum (Figure S7, Supporting Information). Three singlet methyl signals were observed at 1.05, 1.17, and 2.26 ppm, and the latter methyl was assigned to an aromatic methyl group from its chemical shift value and the long-range coupling to the aromatic signals at 6.94 and 6.85 ppm. Long-range correlations from the hydroxylmethyl and nonaromatic singlet methyl at 1.05 ppm to an sp3 quaternary carbon (C-4) at 38.7 ppm and to a terminal methylene carbon (C-3) of the trimethylene unit, and to the methine carbon (C-5), suggested that both are attached at C-4. The other singlet methyl at 1.17 ppm showed correlations to the C-10 quaternary carbon at 37.4 ppm and the C-9 sp2 carbon at 146.9 ppm, together with C-1 of the other terminal methylene of the trimethylene and the methine C-5, indicating that it is attached at C-10. Benzylic methylene signals at 2.89 and 2.81 ppm were assigned to H-7 and showed longrange correlations to C-5, C-8, C-9, and C-14. The position of the methyl group at 2.26 ppm on the aromatic ring was determined to be C-13 from HMBC correlations to C-12, C13, and C-14. Assignment of the observed HMBC correlations revealed the whole planar structure of 1. The NOESY spectrum also supported the proposed structure, from sequential NOEs between H-7 and H-14, H-14 and Me-13, Me-13 and H-12, H11 and H-1, and H-11 and Me-10 (Figure S8, Supporting Information). The relative configuration of the A and B ring system was determined to be trans by the NOE interaction between Me-10 and the hydroxylmethyl groups, indicating both groups to be 1,3-diaxially oriented. Based on this evidence, the structure of 1 was determined to be a new compound, 13methyl-8,11,13-podocarpatrien-19-ol. The absolute configuration of 1 was deduced to be 4S, 5R, 10S on the basis of the electronic circular dichroism (ECD) spectrum (Figure S9, Supporting Information) in comparison with those of similar compounds such as chlorabietins J and K.24 In the 1H NMR spectrum of 2, two typical ortho-coupled (J = 8.0 Hz) doublet signals were observed at 7.14 and 6.98 ppm with 1H NMR integral values of each signal. This indicated that the benzene ring in 2 is 1,2,3,4-tetrasubstituted. Two types of two equivalent singlet methyl signals and one singlet methyl signal were observed at 1.28, 1.33, and 2.29 ppm, respectively, and the latter methyl could be assigned to an aromatic methyl group from its chemical shift value (Figure S10, Supporting Information). From the DQF-COSY spectrum, other partial structures were elucidated as a trimethylene group, with three sets of equivalent methylene signals, and a benzylic ethylene group (Figure S11, Supporting Information). In the 13C NMR
Kujiol A (1) and kujigamberol B (2) were both isolated as a colorless oil by HPLC (Figures S1 and S2, Supporting Information). The molecular formulas of 1 and 2 were determined to be C18H26O and C18H28O, respectively, by HREIMS, with m/z 258.1982 and 260.2152 as the molecular ions. The UV spectra of these compounds resembled that of dehydroabietic acid and of 3. The presence of an aromatic ring in 1 and 2 was suggested by the UV absorption (259 nm for 1 and 265 nm for 2) and six aromatic carbon signals in the 13C NMR spectrum (Figure S3, Supporting Information). In the 1H NMR spectrum of 1, two typical ortho-coupled (J = 8.0 Hz) doublet signals were observed at 7.15 and 6.94 ppm, and the latter was meta-coupled with a methine proton resonating at 6.85 ppm, indicating the presence of a 1,2,4-trisubstituted aromatic ring (Figure S4, Supporting Information). From the analysis of the DQF-COSY spectrum, other partial structures (bold lines in 1, Figure 1) were elucidated as a trimethylene group for the A ring and a 1-propanyl-3-ylidene group for the B ring with nonequivalent methylene signals, and a methine signal at 1.49 ppm (Figure S5, Supporting Information). Isolated methylene signals were observed at 3.86 and 3.56 ppm (each, d, J = 10.8 Hz), and the chemical shift values and the coupling pattern indicated a hydroxylmethyl group attached to an asymmetric quaternary carbon. Assignment of this hydroxylmethyl group was supported by its 13C NMR chemical shift B
DOI: 10.1021/acs.jnatprod.7b00922 J. Nat. Prod. XXXX, XXX, XXX−XXX
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dane skeleton of compounds 2 and 3 has not yet been found among contemporary plants. The diagenesis process probably occurred under aerobic conditions, leading to reductive reactions such as decarboxylation of resin acids, with concurrent oxidation reactions, to form aromatic derivatives.5 Compound 1 is a reduction product of 16,17-bisnorcallitrisic acid7 (Figure S18a, Supporting Information) at the carboxylic group of C-17. There was no known related compound of 2 and 3 in any amber until the structure of 2,5,8-trimethyl-1isopentyltetralin was revised to 1,6-dimethyltetralin (amberene) (Figure S18b, Supporting Information).9 Methylionene (Figure S18c, Supporting Information) has already been identified in many ambers.5,8,12,14 A hydroxylmethyl group attached to C-4 in 1 and 3 is a common partial structure and is found in dehydroabietol6 (Figure S18d, Supporting Information), communol10−12 (Figure S18e, Supporting Information), and ozol10,12 (Figure S18f, Supporting Information). A tertiary hydroxy group with a dimethyl unit is a structural unit also found in 15-hydroxydehydroabietic acid6 (Figure S18g, Supporting Information) and 2, and an isopentyl group attached to C-9 in 3 is found in 1,6-dimethyl-5-isopentyltetralin (amberene) (Figure S18b, Supporting Information). In the structures of the new compounds in Kuji amber, each has common partial structures of compounds previously identified in ambers by GC-MS, and they are different from all compounds that have been isolated from existing organisms, such as microorganisms and plants using the same general screening method.25−31 Compounds 1 and 2 showed a clear growth restored zone on a plate in a dose-dependent manner (from 0.5 to 0.13 μg/spot) similar to that of 3 (Figure S19, Supporting Information). The Ca2+-signal transduction pathway of Saccharomyces cerevisiae is involved in both GSK-3β and calcineurin. Each inhibitor also shows growth-restoring activity,18,20 and the inhibitory activities of 1 and 2 were measured, with 1 inhibiting GSK-3β and calcineurin in a dose-dependent manner, with IC50 values of 178 and 42.7 μM, respectively. These activities are comparable to those of dehydroabietic acid isolated from Baltic amber or modern pine leaves.20 However, 2 did not inhibit GSK-3β and calcineurin even at 200 μM. These activities are comparable to those of 3 isolated from Kuji amber.20 Compound 1 also showed more potent cytotoxicity against the HL60 promyelocytic leukemia cell line (IC50 7.1 μM) than 2 (IC50 41.7 μM), but 1−3 were not cytotoxic against the WI-38 normal human lung fibroblast cell line at IC50 50 μM. It may be speculated that the new compounds 1 and 2 in Kuji amber are the result of diagenetic modification of the parent compounds (low molecular weight compounds and/or macromolecular materials) and that the origin of Kuji amber is different from those of Baltic and Dominican ambers. Moreover, the GC-MS analysis patterns of Kuji, Baltic, and Dominican ambers are also different from each other. Significantly, the SI values of Kuji amber are smaller than those of Baltic or Dominican ambers that are produced after the K−Pg boundary, and 3 was detected in only the hexane extract of Kuji amber by GC-MS (Figure S17, Supporting Information). Another possibility is that the origin of Kuji amber was from a plant extinct at the K−Pg boundary. It is documented that the related nor-, dinor-, and trinor analogues of the parent compounds such as dehydroabietate and callitrisate have been detected in Raritan amber (Upper Cretaceous) or Carboniferous amber (320 Ma).9,12 The
Figure 2. COSY and HMBC correlations of 2.
spectrum, four aromatic sp2 nonprotonated carbons and two sp3 nonprotonated carbons at 34.0 and 71.1 ppm were observed. One of the nonprotonated sp3 carbons at 71.1 ppm indicated that oxygen is attached to it as a tertiary hydroxy group (Figures S12 and S13, Supporting Information). The connectivity of each partial structure was established by analysis of the long-range coupling correlations in the HMBC spectrum (Figure S14, Supporting Information). Long-range correlations from a methylene signal at 1.61 ppm (H-12) of the ethylene to the oxygenated tertiary carbon (C-13) at 71.1 ppm and from the equivalent dimethyl signal at 1.33 ppm also to C-13 and to the methylene carbon (C-12) of the ethylene unit suggested that both are attached at C-13. Another methylene proton at 2.67 ppm of the ethylene unit showed correlations to C-8, C-9, and C-10 of the aromatic ring. The other nonaromatic equivalent dimethyl groups at 1.28 ppm exhibited correlations to an aromatic carbon (C-5) at 143.9 ppm, to an sp3 quaternary carbon (C-4) at 34.0 ppm, and also to one terminal methylene carbon (C-3) of the trimethylene group, and this suggested that the singlet dimethyl group is attached at C-4. The other terminal methylene protons of the trimethylene group at 2.73 ppm (H-1) showed correlations to the aromatic C-5, C-9, and C-10 carbons, together with C-2 and C-3. Additionally typical three-bond correlations were also observed from the aromatic protons H-6 (7.14 ppm) and H-7 (6.98 ppm) to the nonprotonated aromatic carbons C-8 and C-10 and to C-5 and C-9, respectively. Correlations of the aromatic methyl at 2.29 ppm to C-7, C-8, and C-9 were also observed. The sequential assignments of HMBC correlations and NOESY correlations (Figure S15, Supporting Information) around the aromatic ring revealed the entire planar structure of 2. Based on this evidence, the structure of 2 was determined to be 15,20dinor-5,7,9-labdatrien-13-ol, and this new compound has been named kujigamberol B. To compare Kuji amber to other ambers, GC-MS analysis was performed. GC-MS chromatogram patterns of each amber were very distinctive (Figure S16, Supporting Information), and the Baltic and Dominican ambers investigated afforded no GC signal indicative of the presence of 3 with expanded spectra shown in Figure S17, Supporting Information. The average similarity index (SI) value (the similarity between the analyzed abundant 25 compounds in each amber and the library compounds) of the Kuji amber extract showed the smallest value (75) (Figures S16b and S17b, Supporting Information) in all examined commercial ambers such as Baltic amber (84) (Figures S16c and S17c, Supporting Information) and Dominican amber (82) (Figures S16d and S17d, Supporting Information), respectively. Podocarpane skeletons like 1 have been reported previously from many modern-day plants. However, the 15,20-dinorlabC
DOI: 10.1021/acs.jnatprod.7b00922 J. Nat. Prod. XXXX, XXX, XXX−XXX
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configulation of C-4 (S) in 1 and 3 suggested that Kuji amber may be class I amber.11
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Table 1. NMR Spectroscopic Data of Kujiol A (1)
EXPERIMENTAL SECTION
General Experimental Procedures. The optical rotation was measured on a JASCO DIP-1000 polarimeter in MeOH. UV spectra were measured on a UV mini 22 instrument (Shimadzu Co. Ltd., Kyoto, Japan) in MeOH. The ECD spectrum was measured on a JASCO J-720 CD spectrophotometer in MeOH. 1H and 13C NMR spectra were recorded on a JEOL ECA-600 NMR spectrometer. Chemical shifts were referenced to the residual solvent signal δH 7.26 ppm or the solvent signal δC 77.0 ppm for CDCl3. HREIMS spectra were measured on a JEOL JMS700 mass spectrometer. The analytical and preparative HPLC system comprised a PU-2080 HPLC pump and an MD-2018 photodiode array detector (JASCO, Tokyo, Japan). GCMS spectra were measured on a QP-2010 instrument (Shimadzu Co, Ltd., Kyoto, Japan). Source Material. Kuji amber was excavated from mines of Kuji Kohaku Co. Ltd. located in the upper part of the Tamagawa Formation of Kuji Group of Kuji, Iwate Prefecture, northeastern Japan (October 2010). Baltic and Dominican ambers were imported by Kuji Kohaku Co. Ltd. Extraction and Isolation. Powdered Kuji amber (1029 g) was extracted with MeOH for 3 days at room temperature, and the resultant extract [33.81 g (3.3%)] was diluted with water, followed by two extractions with one volume of EtOAc (1 L) used. After evaporation of the EtOAc, the organic layer [18.34 g (1.8%)] was subjected to silica gel column chromatography (7.5 × 19 cm) with hexane−EtOAc (3:1 and 2:1) as solvents. Two active fractions were collected and concentrated under reduced pressure to afford the crude materials A [1.01 g (0.098%)] and B [758 mg (0.074%)]. Finally, kujigamberol (3) was purified (52.1 mg from fraction A and 23.6 mg from fraction B) as a colorless oil using HPLC [mobile phase MeOH− H2O (85:15); flow rate 5 mL/min] and a Capcell Pak C18 column (20 mm i.d. × 250 mm; Shiseido, Tokyo, Japan). Compounds 1 and 2 were isolated from fraction B (total of 12.4 mg) using HPLC [mobile phase MeOH−H2O (85:15); flow rate 5 mL/min] and finally purified (2.2 mg and 1.7 mg, respectively) as colorless oils using HPLC [mobile phase MeOH−H2O (70:30); flow rate 5 mL/min] (Figures S1 and S2, Supporting Information). GC-MS analysis of the underivatized hexane extract (0.2 μg) of each amber was performed with a QP-2010 instrument (Shimadzu Co, Ltd., Kyoto, Japan). Separation was achieved with an HP-5 column (30 m × 0.25 mm i.d. × 0.25 μm, Agilent Technologies, Tokyo, Japan), and the GC oven operating conditions were 60 °C (held 2 min) to 300 °C (held 10 min) at 6 °C min−1. Helium (He) was used as carrier gas. Samples were injected in a splitless mode with the injector temperature at 290 °C. The mass spectrometer was operated in the full-scan mode from 50 to 500 Da and with electron impact ionization (70 eV). Wiley 7 was used as a library tool, and the SI value was used as the similarity between constituents in each amber and known compounds in the library. Kujiol A (1): colorless oil; [α]24D +28.2 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 203 (4.07), 259 (2.69) nm; ECD (c 3.87 × 10−4 M, MeOH) λmax (Δε) 221 (1.12), 213 (0.45) (Figure S9, Supporting Information); 1H NMR and 13C NMR, see Table 1; HREIMS m/z 258.1981 [M]+ (calcd for C18H26O, 258.1984). Kujigamberol B (2): colorless oil; UV λmax (MeOH) (log ε) 204 (4.12), 265 (2.57) nm; 1H NMR and 13C NMR, see Table 2; HREIMS m/z 260.2152 [M]+ (calcd for C18H28O, 260.2140). Biological Testing. All yeast strains were derivatives of strain W303-1A and were YNS17 (MATa zds1::TRP1 erg3::HIS3 pdr1::hisG URA3 hisG pdr3::hisG).18,20 Difco YPD broth and YPD agar were obtained from Becton Dickinson (Franklin Lakes, NJ, USA). FK506 was kindly provided by the Fujisawa Pharmaceutical Co., Ltd. (now Astellas Pharma Inc., Tokyo, Japan). Extracted samples of 10 mg/mL in MeOH were prepared for subsequent assays. Screening was carried out using YNS17 strain, and 5 μL of each sample was added to a plate as described previously.18,20 The inhibitory activity of the Ca2+-signal
position
δC (ppm)
1
39.0
2
19.0
3
35.2
4 5
38.7 51.4
6
19.2
7
30.9
8 9 10 11 12 13 14 15 16 17
134.7 146.9 37.4 124.5 126.6 134.7 129.6 20.8 26.8 65.3
18
25.7
δH (ppm, J in Hz) 2.31 1.41 1.70 1.62 1.88 1.02
ddd ddd m m ddd ddd
HMBC
(13.1, 4.3, 3.2) (13.1, 13.1, 3.4)
(13.8, 4.6, 3.4) (13.8, 13.8, 3.4)
1.49 dd (12.6, 1.6) 1.97 dddd (13.2, 6.9, 1.7, 1.7) 1.68 dddd (13.2, 12.6, 11.5, 6.0) 2.89 brdd (16.4, 6.0) 2.81 ddd (16.4, 11.5, 6.0)
C-4, C-6, C-7, C-10, C17, C-18 C-8, C-10 C-8, C-10 C-5, C-8, C-9, C-14 C-8
7.15 d (8.0) 6.94 brd (8.0)
C-8, C-13 C-9, C-14, C-15
6.85 2.26 1.05 3.86 3.56 1.17
C-9, C-12, C-15 C-12, C-13, C-14 C-3, C-4, C-5, C-17 C-16 C-3, C-16 C-1, C-5, C-9, C-10
brs s s d (10.8) d (10.8) s
Table 2. NMR Spectroscopic Data of Kujigamberol B (2) position
δC (ppm)
δH (ppm, J in Hz)
HMBC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
27.3 19.8 38.8 34.0 143.9 124.4 127.8 132.9 138.2 134.1 24.1 42.4 71.1 29.1 29.1 19.5 32.2 32.2
2.73 t (6.3) 1.82 m 1.62 m
C-2, C-3, C-5, C-9, C-10 C-1, C-17
7.14 d (8.0) 6.98 d (8.0)
C-4, C-8, C-10 C-5, C-9, C-16
2.67 m 1.61 m
C-8, C-9, C-10, C-12 C-11, C-13, C-14, C-15
1.33 1.33 2.29 1.28 1.28
C-12, C-13, C-15
s s s s s
C-7, C-8, C-9 C-3, C-4, C-5, C-18
transduction was determined to be represented by the size and the intensity of the yeast growth zone. An immunosuppressive drug, FK506 (tacrolimus; 2.5 ng/spot), was used as a positive control. The calcineurin activity was measured using a commercial kit (AK-804, BIOMOL International LP, Plymouth Meeting, PA, USA) with a minor modification, in which the free phosphate ion released from a substrate phosphopeptide (DLDVPIPGRFDRRVpSVAAE) was quantified by colorimetric analysis (650 nm) using the malachite green method. The calmodulin antagonist trifluoperazine was used as a positive control.20 The substrate peptide (20 μM) was mixed with human GSK-3β (0.02 units) at a total volume of 50 μL in buffer [8 mM MOPS (pH 7.0), 0.2 mM EDTA, 5 μM ATP, 10 mM MgCl2] in D
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the presence or absence of inhibitors in 2 μL of DMSO. GSK-3β assays were performed in a white 96-well plate, and a GSK-3β inhibitor, GSK-3β inhibitor I (TDZD-8, 4-benzyl-2-methyl-1,2,4thiadiazolidine-3,5-dione), was used as a positive control.20 HL60 cells (RCB0041, RIKEN BioResource Center, Tsukuba, Japan) were grown in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum (Bio West Co., Vancouver, Canada) and penicillin (50 units/mL)−streptomycin (50 μg/mL) (Gibco, Carlsbad, CA, USA) and incubated with samples for 48 h in a humidified atmosphere at 37 °C under 5% CO2. The cytotoxicity was examined by an MTT assay, as described previously.32 Human diploid lung fibroblast WI-38 cells (RCB0742, RIKEN BioResource Center, Tsukuba, Japan) were cultured on 5 × 103 cells/well in DMEM and were used for the cytotoxicity assay.
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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.7b00922. HPLC analysis and isolation procedure of 1, 2, and 3; 1D and 2D NMR spectra of 1 and 2; ECD spectrum of 1; GC-MS spectra of amber extracts; structures of the related compound; growth-restoring activity of 1, 2, and 3 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81 19 621 6124. Fax: +81 19 621 6124. E-mail:
[email protected]. ORCID
Shunya Takahashi: 0000-0001-9148-9814 Ken-ichi Kimura: 0000-0002-4344-2525 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Mrs. S. Nakajyo of the Center for Regional Collaboration in Research and Education of Iwate University for the HREIMS, to Prof. H. Ando, Ibaraki University, for helpful comments, to Emeritus Prof. D. R. Phillips of La Trobe University for critical reading of the manuscript, and to Emeritus Prof. T. Miyakawa of Hiroshima University for donating to us the mutant yeast strain. This work was partially supported by the Japan Science and Technology Agency (JST) and Sanriku Fund.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.7b00922 J. Nat. Prod. XXXX, XXX, XXX−XXX
