Hikiamides A–C, Cyclic Pentadepsipeptides from Fusarium sp. TAMA

Article pubs.acs.org/jnp

Hikiamides A−C, Cyclic Pentadepsipeptides from Fusarium sp. TAMA 456 Takao Fukuda,† Yuri Sudoh,‡ Yuki Tsuchiya,‡ Toru Okuda,§ Nobuyasu Matsuura,⊥ Atsuko Motojima,∥ Tsutomu Oikawa,∥ and Yasuhiro Igarashi*,† †

Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan ‡ Hyphagenesis Inc., 6-2-37 Tamagawa Gakuen, Machida, Tokyo 194-0041, Japan § Botanical Gardens, The University of Tokyo, 3-7-1 Hakusan, Bunkyo, Tokyo 112-0001, Japan ⊥ Department of Life Science, Faculty of Science, Okayama University of Science, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-0005, Japan ∥ Department of Nutritional Biochemistry, School of Nutrition and Dietetics, Kanagawa University of Human Services, 1-10-1 Heisei-cho, Yokosuka, Kanagawa 238-8522, Japan S Supporting Information *

ABSTRACT: Three new cyclic pentadepsipeptides, hikiamides A− C (1−3), were isolated from the culture extract of Fusarium sp. TAMA 456. The structures were determined by spectroscopic analysis using NMR and MS, and the absolute configurations were established by using Marfey’s method and chiral HPLC analysis. Hikiamides induced the differentiation of murine ST-13 preadipocytes into mature adipocytes at 2 μM and adiponectin mRNA expression (5- to 13-fold higher than control). They also induced PPAR-γ-dependent gene expression at a concentration from 0.63 to 10 μM in a gene reporter assay.

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expression of adiponectin in murine ST-3 preadipocyte cells. Agonistic activity of these compounds against PPARγ was also demonstrated by a gene reporter assay. Herein, we describe the isolation, structure elucidation, and biological activities of 1, 2, and 3.

iabetes mellitus is a disease characterized by the lack of blood glucose control and is associated with serious complications including neuropathy, nephritis, retinopathy, and atherosclerosis. The number of incidents is annually increasing worldwide: as of 2013, almost 400 million people were suffering from this disease.1 Diabetes is classified as type 1 and type 2. More than 90% of patients have type 2 diabetes,2 which is caused by insensitivity of peripheral tissues to insulin, termed insulin resistance, or by relatively low production of insulin. Adiponectin, a 30 kDa polypeptide secreted from adipose tissue, ameliorates the insulin resistance in type 2 diabetic patients through lowering the blood glucose level and lipid storage by stimulating glucose uptake and fatty acid oxidation in skeletal muscle.3 Thiazolidinediones (TZDs), potent PPARγ (peroxisome proliferator-activated receptor γ) agonists, are used in the treatment of type 2 diabetes. They were introduced in the late 1990s, but several serious adverse effects restricted their continuous usage, resulting in withdrawal from the market.4,5 Alternative agents without such deleterious properties are urgently needed.6,7 In our continuing search for new lead scaffolds of antidiabetic agents,7−12 three new cyclic depsipeptides, hikiamides A (1), B (2), and C (3), were isolated from Fusarium sp. TAMA 456. These peptides promoted adipocyte differentiation and mRNA © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The fungal strain TAMA 456 was isolated from rotten wood collected in Hiki County, Saitama, Japan. On the basis of morphological characteristics and ITS sequence similarity, this strain was identified as a Fusarium sp. Strain TAMA 456 was cultured in RB medium under static conditions at 25 °C for 21 days, and the whole culture broth was extracted with 1-butanol. The crude extract (18 g from 0.96 L) was suspended in water and sequentially extracted with n-hexane and EtOAc. The dried EtOAc extract (12 g) was subjected to consecutive fractionation using silica gel and ODS column chromatographies, followed by reversed-phase HPLC purification, to yield hikiamides A (1, 418 mg), B (2, 4.7 mg), and C (3, 81 mg). Received: December 24, 2014

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Combined analysis of COSY, TOCSY, and HMBC data revealed that 1 comprised two phenylalanines (Phe-1 and Phe2), valine (Val), N-methylleucine (N-MeLeu), and one leucic acid (OLeu) (Figure 1). The Val residue was assigned by sequential COSY correlations from an amide doublet proton (δH 7.10) to two terminal methyl protons (δH 0.96 and 0.81). The presence of an N-MeLeu was deduced from TOCSY correlations among the α-proton H-13 (δH 3.46), H2-14 (δH 2.09, 1.73), H-15 (δH 1.52), and two doublet methyls, H3-16 (δH 0.91) and H3-17 (δH 0.89), and an HMBC correlation from N-methyl protons (δH 3.19) to C-13 (δC 67.0). Another spin system starting from the oxymethine H-2 (δH 4.89) to the isopropyl terminus (H3-5, H3-6) was deduced from COSY and TOCSY correlations. This fragment was expanded to include carbonyl carbon C-1 by HMBC correlations from H-2 and H2-3 to this carbon, establishing an OLeu residue. In addition to these aliphatic portions, the presence of 12 aromatic carbons was recognized in the 1D and 2D NMR spectra. Aromatic carbons including two quaternary carbons (δC 138.4, 139.2) and 10 proton-bearing carbons (δC 127.3, 127.4, 129.1 × 2, 129.3 × 2, 130.0 × 2, 130.2 × 2) were assigned to two monosubstituted benzene rings. Two isolated spin systems defined by COSY analysis, NH (δH 7.35)/H-19 (δH 4.52)/H220 (δH 3.17, 3.04) and NH (δH 8.07)/H-28 (δH 5.02)/H2-29 (δH 3.31, 3.10), were respectively connected to the benzene ring by the HMBC correlations from the methylene protons to the aromatic carbons, providing two phenylalanines. The four amino acids and one hydroxy acid were assembled into one molecule on the basis of NOESY and HMBC analysis (Figure 1). NOEs between H-3 (δH 1.84) in OLeu and Val-NH, H-8 and H-9 of Val and N-methyl protons in N-MeLeu, H2-14 of NMeLeu and NH of Phe-1, and H-19 of Phe-1 and NH of Phe-2 established the sequence OLeu/Val/N-MeLeu/Phe-1/Phe-2. Finally, an HMBC correlation from H-2 to C-27 (δC 170.3) completed the planar structure of 1. The absolute configuration of the amino acid components was determined by Marfey’s analysis. After the treatment of 1 in 6 M HCl at 110 °C for 16 h, the hydrolysate was derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and the retention times were compared with the FDLA derivatives of standard amino acids. The LC/MS analysis gave the peaks

Hikiamide A (1) was obtained as an optically active, white, amorphous solid. High-resolution ESITOFMS gave an [M + Na]+ at m/z 657.3622, corresponding to the molecular formula C36H50N4O6 (Δ −0.1 mmu, calcd for C36H50N4O6Na). 1H, 13C, and edited HSQC NMR data of 1 confirmed the presence of 36 carbons assignable to five ester/amide carbonyls, 12 aromatic carbons including two quaternary sp2 carbons, eight methines, four methylenes, and seven methyl groups including one nitrogen-bonded methyl.

Figure 1. COSY and key HMBC correlations for 1, 2, and 3. B

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for L-Phe and L-Val, whereas both enantiomers of N-MeLeu were detected in a ratio of L:D = 2.5:1 (Figure S1). As this result indicated the partial racemization of this amino acid during the hydrolysis, 1 was hydrolyzed in 6 M DCl/D2O and the hydrolysate was similarly derivatized with L-FDLA. The LC/MS analysis displayed only one peak for nonracemized FDLA-NMe-L-Leu (m/z 439), while two peaks were detected for racemized (deuterized) N-MeLeu (m/z 440) (Figure S22). The absolute configuration of OLeu was determined to be S by comparing the retention time of commercially available OLeu with the acid hydrolysate of 1 using a chiral HPLC column. Standard R- and S-OLeu gave peaks at 24.0 and 21.6 min, respectively, while the hydrolysate gave a peak at 21.8 min (Figure S25). Therefore, 1 was determined to consist of L-Phe, L-Phe, N-Me-L-Leu, L-Val, and S-OLeu. Hikiamide B (2) was also obtained as a white, amorphous solid. An [M + Na]+ peak was observed at m/z 643.3463 in the positive ion HR-ESITOFMS appropriate for a molecular formula of C 35 H 48 N 4 O 6 (Δ −0.3 mmu, calcd for C35H48N4O6Na), corresponding to the loss of a methylene (CH2) from 1. Comparing the 1H and 13C NMR spectra of 2 with those for 1, a nitrogen-bonded methyl singlet present in 1 was missing and an additional amido proton was observed at δH 8.08 in CDCl3. This exchangeable proton was coupled to the αmethine proton H-8 of the Leu residue, confirming that 2 was an N-demethyl congener of 1 (Figure 1). The absolute configuration of 2 was determined by Marfey’s method for amino acids and chiral HPLC analysis for OLeu as L-Phe, L-Phe, L-Leu, L-Val, and S-OLeu (Figures S23 and S25). Hikiamide C (3) was obtained as a white, amorphous solid with a molecular formula of C38H51N5O6 on the basis of HRESITOFMS data that gave an [M + Na]+ ion at m/z 696.3732 (Δ 0.0 mmu, calcd for C38H51N5O6Na). The 1H and 13C NMR spectra of 3 were highly similar to those for 1 except for the aromatic region (Table S2), lacking two intense signals for ortho- and meta-positioned carbons in a phenyl group. Instead, the COSY spectrum showed four contiguously coupled aromatic protons (H-23, H-24, H-25, H-26), which could be assigned to the 1,2-disubstituted benzene protons. Two quaternary sp2 carbons were confirmed by HMBC correlations from H-23, H-24, and H-26 to C-22 and from H-23 and H-25 to C-27. This monocyclic ring was expanded to an indole ring on the basis of HMBC correlations from NH (δH 8.23) to C21, C-22, C-27, and C-28. Further HMBC correlations from H20 to C-21, C-22, and C-28 connected the indole unit to the methylene carbon C-20. The protons bonded to this carbon were correlated to a methine H-19, which in turn showed a correlation to an NH proton at δH 7.40 (Figure 1). The location of the Trp residue was determined by HMBC correlations from Trp-NH to C-12 and from H-20 and H-30 to C-18. The absolute configuration of 3 was assigned again by using Marfey’s method and chiral HPLC analysis in the same manner as described for 1 (Figures S24 and S25). Hikiamides belong to a relatively minor group of depsipeptides containing a leucic acid residue in their cyclic structures. Structurally related peptides sansalvamide, zygosporamide, and alternaramide are known to be produced by filamentous fungi of the genera Fusarium,13 Zygosporium,14 and Alternaria,15 respectively (Figure 2). These peptides exhibit cytotoxicity13,14 or antimicrobial activity,15 but their effects on adipocytes have not been described. Treatment with 2−4 μM hikiamides induced significant accumulation of lipid droplets in the cytosol of murine ST-13 preadipocytes, indicating their

Figure 2. Structures of hikiamide A and related cyclic peptides.

differentiation into mature adipocytes (Figure S26).7 In addition, mRNA expression of adiponectin in adipocytes was enhanced by the treatment with 2 μM 1 (13.3-fold), 2 (5.2fold), and 3 (10.0-fold) compared to the control (Figure 3).

Figure 3. Expression of adiponectin mRNA in murine ST-13 cells.

Induction of adiponectin secretion by adipocytes was reported with a wide range of natural and synthetic small molecules including phenylpropanoids (e.g., nobiletin), triterpenes (e.g., isoastragaloside I), aromatic polyketides (e.g., norlichexanthone), TZDs (e.g., rosiglitazone), and sulfonylurea (glimepiride) (Figure 4). Many of these compounds enhance adiponectin secretion through activation of PPARγ, a transcription factor of the nuclear receptor superfamily. In a gene reporter assay,7 hikiamides activated PPARγ-mediated transcription at a concentration from 0.63 to 10 μM in a concentration-dependent manner (Figure 5). Hikiamides are the first example of cyclic peptides with adipogenic activity, thereby making them unique as new scaffolds for designing compounds to treat type 2 diabetes. C

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2003). A culture study was done by observation of colonies grown on 2% malt extract agar and oatmeal agar at 25 °C for 7 to 14 days. A morphological study was performed using cultures grown on 2% malt extract agar using an optical microscope (Nikon ECLIPSE E600) with a Nomarsky phase contrast lens system. For the phylogenetic study, the internal transcribed spacer (ITS) sequence (512 nucleotides; DDBJ accession number LC027468) obtained from TAMA 456 was compared with those from DDBJ/EMBL/GenBank. The phylogenetic tree was constructed using the neighbor-joining (NJ) method. Wet and pale yellow sporodochial conidial structures were produced in a concentric ring-like manner on 2% malt extract agar. Conidiophores were densely branched to form cylindrical phialides with a distinct collarette on the apex. Conidia were characteristically sickle shaped with three to five transverse septa. No microconidia were observed. According to the NJ tree constructed with the ITS sequences obtained from DDBJ/EMBL/GenBank by BLAST search, TAMA 456 was most closely related to Fusarium spp. and Nectria hematococca, one of the teleomorphs of Fusarium (Leslie and Summerell 2006; Crous et al. 2009). Fermentation. Strain TAMA 456 grown on a PDA slant was inoculated into 150 mL polypropylene flasks, each containing RB medium consisting of rolled barley 10 g, yeast extract 20 mg, sodium tartrate 10 mg, KH2PO4 10 mg, and 10 mL of distilled water. The inoculated flasks were incubated under static conditions at 25 °C for 21 days. Extraction and Isolation. At the end of the fermentation period, 20 mL of 1-butanol was added to each flask, and the flasks were shaken on a rotary shaker (225 rpm) for 30 min. The mixture was centrifuged at 3000 rpm for 5 min, and the organic layer was separated from the aqueous layer containing mycelia. Evaporation of the organic solvent provided approximately 18.0 g of extract from 0.96 L of culture. The crude extract was suspended in water (150 mL) and extracted with nhexane (3 × 75 mL), and the remaining water layer was extracted with EtOAc (3 × 75 mL). After concentration under reduced pressure, the EtOAc extract was chromatographed on a silica gel column with a step gradient of CHCl3−MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v). Fraction 4 (4:1) was evaporated, and the remaining solid was suspended in CHCl3. The CHCl3-soluble fraction was separated by centrifugation (3000 rpm, 10 min) and fractionated by ODS column chromatography with MeCN−0.1% HCO2H (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 1:0 v/v). Fraction 7 (8:2) was subjected to preparative HPLC purification (CAPCELL PAK C18 MGII S5, 20 × 150 mm) with a gradient of MeCN−0.1% HCO2H (MeCN concentration: 70% for 0−15 min; 70−90% for 15−20 min), to yield hikiamides A (1, 418 mg, tR 15.0 min), B (2, 4.7 mg, tR 10 min), and C (3, 81 mg, tR 13.5 min). Hikiamide A (1): white, amorphous solid; [α]23D −146 (c 0.25, MeOH); IR νmax (ATR) 3295, 1750, 1701, 1670, 1646, 1621, 1543, 1511 cm−1; UV (MeOH) λmax (log ε) 206 (4.45), 258 (2.61) nm; for 1 H and 13C NMR data, see Table 1; HR-ESITOFMS m/z 657.3622 [M + Na]+ (calcd for C36H50N4O6Na, 657.3623). Hikiamide B (2): white, amorphous solid, [α]23D −145 (c 0.25, MeOH); IR νmax (ATR) 3291, 1748, 1674, 1649, 1516, 1498 cm−1; UV (MeOH) λmax (log ε) 206 (4.43), 258 (2.60) nm; for 1H and 13C NMR data, see Table S1; HR-ESITOFMS m/z 643.3463 [M + Na]+ (calcd for C35H48N4O6Na, 643.3466). Hikiamide C (3): white, amorphous solid, [α]23D −139 (c 0.25, MeOH); IR νmax (ATR) 3310, 1743, 1666, 1627, 1516, 1496 cm−1; UV (MeOH) λmax (log ε) 218 (4.51), 281 (3.69) nm; for 1H and 13C NMR data, see Table S2; HR-ESITOFMS m/z 696.3732 [M + Na]+ (calcd for C38H51N5O6Na, 696.3732). Determination of Absolute Configuration: Preparation of Reference Samples. Commercially available L-amino acid standards (0.5 mg each) were dissolved in 0.1 M NaHCO3 (100 μL), and 1fluoro-2,4-dinitrophenyl-5-L-leucinamide or -D-leucinamide (1% LFDLA or D-FDLA solution in acetone, 50 μL) was added. The mixtures were heated at 55 °C for 30 min. After cooling, 0.2 M HCl (50 μL) was added to the reaction mixture to quench the reaction, which was then diluted with 50% MeOH−0.2% HCO2H (1:1, 100 μL).

Figure 4. Activators of adiponectin secretion.

Figure 5. PPARγ agonist activity of hikiamides A−C (1−3) (V = vehicle (DMSO), trog = troglitazone).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a JASCO P-1030 polarimeter. UV spectra were recorded on a Hitachi U-3210 spectrophotometer. IR spectra were measured on a PerkinElmer Spectrum 100. NMR spectra were obtained on a Bruker AVANCE 500 spectrometer or a Bruker AVANCE 400 spectrometer. The 1H and 13C chemical shifts were referenced to the solvent signals (δH 2.05 and δC 29.8 for acetone-d6; δH 3.31 and δC 49.0 for CD3OD) and to tetramethylsilane in CDCl3. HR-ESITOFMS were measured on a Bruker microTOF spectrometer. HPLC analysis was carried out on an Agilent HP1100 system using a CAPCELL PAK C18 MGII S5 (Shiseido Co., Ltd., 4.6 × 250 mm). HPLC purification was performed using a CAPCELL PAK C18 MGII S5 (Shiseido Co., Ltd., 20 × 150 mm). Microorganism. Strain TAMA 456 was isolated from a rotten wood sample collected in Hiki County, Saitama, Japan (November D

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Table 1. 1H and 13C NMR Data for Hikiamide A (1) in Acetone-d6 unit OLeu

Val

N-Me-Leu

Phe-1

Phe-2

position

δC, mult.a

δH (J in Hz)b

HMBCb,c

COSYb

NOESYb

d

1 2 3

171.2, qC 75.9, CH 41.6, CH2

4 5 6 7 8 9 10 11 NH 12 13 14

25.1, CH 23.4,e CH3 21.7, CH3 173.4, qC 55.0, CH 31.0, CH 20.2, CH3 17.0, CH3 171.2,d qC 67.0, CH 37.7, CH2

15 16 17 N-Me 18 19 20

25.9, CH 22.3, CH3 23.4,e CH3 40.1, CH3 171.5, qC 56.3, CH 37.2, CH2

21 22, 26 23, 25 24 NH 27 28 29

139.2, 130.0, 129.1, 127.3,

30 31, 35 32, 34 33 NH

138.4, 130.2, 129.3, 127.4,

qC CH CH CH

170.3, qC 54.0, CH 37.5, CH2 qC CH CH CH

4.89, dd (10.3, 4.0) 1.86, ddd (13.7, 10.3, 5.0) 1.62, ddd (13.7, 9.1, 4.0) 1.55,h m 0.92, d (6.5) 0.86, d (6.5)

1, 3, 4, 27 1, 2, 4, 5, 6 1, 2, 4, 5, 6 3, 4, 6 3, 4, 5

3h 4h 4h

3 2, NH (Val) 2, 4 2 3 2, 3

4.57, dd (8.5, 5.0) 2.15,j m 0.96, d (7.0) 0.81, d (7.0) 7.10, br d (8.5)

1, 8, 8, 8, 1,

9, NH (Val) 8, 10, 11 9 9 8

9, 8, 8, 8, 3,

3.46, dd (9.3, 6.2) 2.09,j m 1.73, ddd (13.9, 8.1, 6.2) 1.52,h m 0.91, d (6.0) 0.89, d (6.5) 3.19, s

7, 12, 14,15, N-Me 12, 13, 15, 16, 17 12, 13, 15, 16, 17 13 14, 15, 17 14, 15, 16 7, 13

14 13, 15

14, 15, 16, N-Me 13, 17, NH (Phe-1) 13, 16, NH (Phe-1) 13, NH (Phe-1), N-Me 13, 14 14 8, 9, 13

4.52, ddd (10.8, 8.9, 4.1) 3.17i 3.04, dd (14.0, 10.8)

18, 20, 21 18, 19, 21, 22 (26) 18, 19, 21, 22 (26)

20, NH 19

20, NH (Phe-2), NH (Phe-1) 19, NH (Phe-1) 19, NH (Phe-1)

7.25−7.30f 7.25−7.30f 7.23d−g,j 7.35, br d (8.5)

12, 19

19

14, 19, NH (Phe-2)

5.02, m 3.31, dd (14.3, 7.3) 3.10, dd (14.3, 7.7)

18, 27, 29 27, 28, 30, 31 (35) 27, 28, 30, 31 (35)

29, NH (Phe-2) 28

29, NH (Phe-2) 28, NH (Phe-2)

7.25−7.30f 7.25−7.30f 7.23d−g,j 8.07, br d (9.0)

18, 28

28

19, 28, 29, NH (Phe-1)

a

7, 9, 10, 11 10, 11 9, 11 9, 10 7

3 2, 4h

14h 15h 15h

10, 11, N-Me, NH (Val) 10, 11, N-Me 9, N-Me 9, NH (Val) 8, 11, 19

Recorded at 100 MHz. bRecorded at 500 MHz. cHMBC correlations are from proton(s) stated to the indicated carbon. h Assignment by TOCSY experiments. iOverlapping with N-Me. j1H shifts are assigned by HSQC experiments. Hydrolysis of Hikiamides A (1), B (2), and C (3) in HCl−H2O and FDLA Derivatization. A solution of hikiamide (0.5 mg each) in 6 M HCl (200 μL) was heated at 110 °C overnight. After cooling to room temparature, the reaction mixture was evaporated to dryness. The remaining hydrolysate was reacted with L-FDLA to give a sample for Marfey’s analysis in a similar manner to that described above. HPLC Analysis. The FDLA derivatives of the hydrolysates and standards were analyzed by LC-MS (Cadenza CD-C18, 3 μm, 2.0 × 150 mm) at 40 °C at a flow rate of 0.2 mL/min. MeCN−2% HCO2H was used as a mobile phase in a gradient mode (MeCN concentration: 25% for 0−4 min; 25−55% for 4−19 min; 55−100% for 19−25 min; 100% for 25−33 min). FDLA derivatives were monitored by UV absorption at 340 nm and by selective ion monitoring in the negative ionization mode. The retention times for the amino acid standards were 24.1 and 26.7 min for L-and D-Phe, 22.1 and 26.0 min for L- and D-Val, 25.7 and 26.9 min for L- and D-N-MeLeu, 23.8 and 25.6 min for L- and D-Trp, and 24.0 and 27.5 min for L- and D-Leu, respectively. The L-FDLA-derivativzed hydrolysate of 1 gave peaks at 24.1 min (L-Phe, m/z 458) and 22.1 min (L-Val, m/z 410) and two peaks for NMeLeu at 25.7 min (L-form, m/z) and 26.9 min (D-form, m/z) in a

d−g

Overlapping signals.

ratio of 2.5:1. The derivatized hydrolysate of 2 gave peaks at 24.1 min (L-Phe, m/z 458), 22.1 min (L-Val, m/z 410), and 24.0 min (L-Leu, m/ z 424). The L-FDLA derivatives from the hydrolysate of 3 gave peaks at 24.1 min (L-Phe, m/z 458), 23.8 min (L-Trp, m/z 497), and 22.1 min (L-Val, m/z 410). Similar to the analysis for the hydrolysate from 1, both L- and D-FDLA-derivatized N-Me-Leu showed peaks for both N-Me-L- and D-Leu in a ratio of 3:1. Hydrolysis of Hikiamides A (1) and C (3) in DCl−D2O. Hikiamide A or C (0.5 mg) in 6 M DCl (200 μL) was allowed to stand at 110 °C overnight. The FDLA derivatives were prepared and analyzed in the same manner as described above. The base ion peak with m/z 438, indicating that no α-proton of N-MeLeu replaced deuterium during hydrolysis, was detected in the peak for N-Me-L-Leu, while the base ion peak with m/z 439 generated by epimerization was found in peaks for both N-Me-L- and D-Leu. Chiral HPLC Analysis. 1 or 2 (0.5 mg) was hydrolyzed with 6 M HCl (200 μL) at 110 °C overnight and evaporated to dryness. Each residue was dissolved in 2 mM CuSO4 (200 μL) and subjected to HPLC analysis (Sumichiral OA-5000, 4.6 × 150 mm; 6% 2-propanol in 2 mM CuSO4; 1 mL/min; UV 254 nm). Retention times for the E

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standards were 21.6 min for (S)-leucic acid and 24.0 min for (R)-leucic acid, while the hydrolysate of 1 or 2 gave a peak at 21.8 min. Adipocyte Differentiation Assay. The effect of hikiamides on adipogenesis of ST-13 cells was performed according to the procedure previously described.12 Real-Time Quantitative PCR Analysis. Total RNA (1.0 μg) was reverse transcribed to cDNA using Super Script II RT (Invitrogen, Tokyo, Japan). Quantification of the mRNA levels of several genes was measured by a Light Cyclersystem (Roche Diagnostic Co., Tokyo, Japan). Real-time PCR amplification was performed in a total volume of 20 μL containing 500 nM each of gene specific primers, cDNA, and SYBR Premix Ex Tag (Takara, Kyoto, Japan). Expression was normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Thermal cycling conditions were as follows: 95 °C for 5 min, 45 cycles each of 5 s at 95 °C, 15 s at 60 °C, and 10 s at 72 °C with a final melting curve analysis from 65 to 95 °C with a 0.1 °C increment per second. The primer sequences were as follows: adiponectin, 5′-GAAGCCGCTTATATGTATCG-3′ (forward) and 5′-GCCGTCATAATGATTCTGT-3′ (reverse); GAPDH, 5′CCAGAACATCATCCCTGC-3′ (forward) and 5′-CCACGACGGACACATT-3′ (reverse); fatty acid-binding protein (aP2), 5′GAAATCACCGCAGACG-3′ (forward) and 5′-ACATTCCACCACCAGC-3′ (reverse); PPARγ2, 5′-CTGTTGACCCAGAGCA-3′ (forward) and 5′-GCGAGTGGTCTTCCAT-3′ (reverse). Oil Red O Staining. After pretreatment of ST-13 cells with hikiamides A−C for 11 days, the cells were washed with phosphatebuffered saline three times followed by fixing with 10% formalin neutral buffer solution at room temperature for 10 min, washed with distilled water, and then rinsed with 60% 2-propanol for 5 min. The prepared cells were stained with 0.24% Oil Red O at room temperature for 20 min and photographed under a phase contrast microscope (100× magnification) equipped with a CCD camera (Leica Microsystems Japan, Tokyo, Japan). Luciferase Reporter Assay. A chimeric plasmid containing human PPARγ-ligand binding domain and GAL4 DNA-binding domain (pPPARγ-GAL4) and the luciferase reporter plasmid, 17m2G TATA Lue (p17m2G), were kindly donated by Dr. S. Kato (University of Tokyo, Tokyo, Japan). COS-1 cells (6 × 105) were transiently transfected with pPPARγ-GAL4 (0.25 μg) and p17m2G (1 μg) using an Effectene transfection reagent kit (QIAGEN, Tokyo, Japan), which was performed in triplicate in 24-well plates according to the manufacturer’s recommendations. After transfection, the cells were cultured for 16 h at 37 °C in a 5% CO2 incubater followed by addition of hikiamides A−C, rosiglitazone, or vehicle at the indicated concentration. The cells were finally incubated for an additional 24 h. The prepared cells were lysed, and luciferase assays were carried out using a Steady-Glo luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

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REFERENCES

(1) International Diabetes Federation. IDF Diabetes Atlas, 6th ed.; International Diabetes Federation: Brussels, Belgium, 2013. http:// www.idf.org/diabetesatlas. (2) http://www.idf.org/types-diabetes. (3) Kadowaki, T.; Yamauchi, T.; Kubota, N.; Hara, K.; Ueki, K.; Tobe, K. J. Clin. Invest. 2006, 116, 1784−1792. (4) Mutalik, M. Int. J. Cur. Biomed. Pharm. Res. 2011, 1, 141−147. (5) (a) Inzucchi, S. E.; Bergenstal, R. M.; Buse, J. B.; Diamant, M.; Ferrannini, E.; Nauck, M.; Peters, A. L.; Tsapas, A.; Wender, R.; Matthews, D. R. Diabetologia 2012, 55, 1577−1596. (b) Schernthaner, G.; Currie, C. J.; Schernthaner, G.-H. Diabetes Care 2013, 36, 5155− 5161. (6) Hino, K.; Nagata, H. Vitamins Horm. 2012, 90, 125−141. (7) Kunimasa, K.; Kuranuki, S.; Matsuura, N.; Iwasaki, N.; Ikeda, M.; Ito, A.; Sashida, Y.; Mimaki, Y.; Yano, M.; Sato, M.; Igarashi, Y.; Oikawa, T. Bioorg. Med. Chem. Lett. 2009, 19, 2062−2064. (8) Igarashi, Y.; Tanaka, Y.; Ikeda, M.; Oikawa, T.; Kitani, S.; Nihira, T.; Mongkol, P.; Janhom, M.; Panbangred, W. J. Antibiot. 2012, 65, 157−159. (9) Igarashi, Y.; Yu, L.; Ikeda, M.; Oikawa, T.; Kitani, S.; Nihira, T.; Bayanmunkh, B.; Panbangred, W. J. Nat. Prod. 2012, 75, 986−990. (10) Yu, L.; Oikawa, T.; Kitani, S.; Nihira, T.; Bayanmunkh, B.; Panbangred, W.; Igarashi, Y. J. Antibiot. 2014, 67, 1−3. (11) Indananda, C.; Igarashi, Y.; Ikeda, M.; Oikawa, T.; Thamchaipenet, A. J. Antibiot. 2013, 66, 1−3. (12) Ikeda, M.; Kurotobi, Y.; Namikawa, A.; Kuranuki, S.; Matsuura, N.; Sato, M.; Igarashi, Y.; Nakamura, T.; Oikawa, T. Med. Chem. 2011, 7, 250−256. (13) (a) Belofsky, G. N.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 1999, 40, 2913−2916. (b) Cueto, M.; Jensen, P. R.; Fenical, W. Phytochemistry 2000, 55, 223−226. (c) Song, H. H.; Lee, H. S.; Lee, C. A. Food Chem. 2011, 126, 472−478. (d) Lee, H. S.; Lee, C. J. Agric. Food Chem. 2012, 60, 4342−4347. (14) Oh, D. C.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 2006, 47, 8625−8628. (15) Kim, M. Y.; Sohn, J. H.; Ahn, J. S.; Oh, H. J. Nat. Prod. 2009, 72, 2065−2068.

ASSOCIATED CONTENT

S Supporting Information *

NMR data, 1D/2D NMR spectra of 1, 2, and 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-766-56-7500. Fax: +81-766-56-2498. E-mail: yas@ pu-toyama.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research (A) (25252037) from JSPS to T. Oikawa. F

DOI: 10.1021/np501047a J. Nat. Prod. XXXX, XXX, XXX−XXX