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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Pestynol, an Antifungal Compound Discovered Using a Saccharomyces cerevisiae 12geneΔ0HSR-iERG6-Based Assay Katsuyuki Sakai,† Tomoyasu Hirose,†,‡ Masato Iwatsuki,†,‡ Takumi Chinen,§ Toru Kimura,† Takuya Suga,†,‡ Kenichi Nonaka,†,‡ Takuji Nakashima,‡ Toshiaki Sunazuka,†,‡ Takeo Usui,§ Yukihiro Asami,†,‡ Satoshi O̅ mura,*,‡ and Kazuro Shiomi*,†,‡ †

Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan § Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Ibaraki, Japan

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S Supporting Information *

ABSTRACT: The multidrug-sensitive budding yeast, Saccharomyces cerevisiae 12geneΔ0HSR-iERG6, is very useful in antifungal screens. A novel compound, named pestynol (1), was discovered from a culture of the fungus Pestalotiopsis humus FKI-7473 using the multidrug-sensitive yeast. The structure of 1 was elucidated by NMR studies and modified Mosher’s method as (1R,2R,3R,4R)-(E)-5-(7,11-dimethyl-3methylenedodeca-6,10-dien-1-yn-1-yl)cyclohex-5-ene-1,2,3,4tetraol. Compound 1 showed antimicrobial activity against the Gram-positive bacteria, Klebsiella pneumoniae, and S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus, but displayed only weak cytotoxicity against various human cancer cell lines. Compound 1 displayed antifungal activities against S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus at 10 μg/disc.

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icrobial secondary metabolites have been widely exploited as medicines, pesticides, and biochemical reagents. The budding yeast, Saccharomyces cerevisiae, is often used as a model eukaryote for biological screening. It is also useful for evaluation of bioactive compounds and target identification. However, the high level of drug resistance caused by drug efflux and membrane barrier systems of wildtype S. cerevisiae makes any evaluation difficult using this organism. One major factor involved in drug resistance in S. cerevisiae is drug efflux using ATP-binding cassette (ABC) transporters.1,2 If strains with fewer expressed ABC transporters were available, it should be possible to identify more bioactive compounds. Consequently, we used the multidrugsensitive budding yeast, S. cerevisiae 12geneΔ0HSR-iERG6 [MATa or MATα his3Δ1 leu2Δ0 metΔ15Δ0 ura3Δ0 pdr1Δ0 pdr3Δ0 pdr8Δ0 yrr1Δ0 aus1Δ0 pdr5Δ0 pdr10Δ0 pdr11Δ0 pdr12Δ0 pdr15Δ0 snq2Δ0 yor1Δ0 RME (ins-308A) KanMX6GAL1p-ERG6], for antifungal screening of the culture broths of various microorganisms.3,4 We devised a new paper disc screening method using S. cerevisiae 12geneΔ0HSR-iERG6 and S. cerevisiae BY4741 (wild type) to test and compare the growth inhibition of these culture extracts. As a result, a novel compound named pestynol (1) was detected and subsequently isolated from a culture of a fungus, Pestalotiopsis humus FKI-7473, and here we report taxonomic studies of the producing microorganism together with the fermentation, isolation, structure elucidation, and biological activity of pestynol. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Taxonomic Studies of the Pestynol Producing Strain. Fungal strain FKI-7473 (subsequently identified as Pestalotiopsis humus FKI-7473) was isolated from soil collected in Tokushima Prefecture, Japan. To determine the most closely related Pestalotiopsis species, the DNA sequence of the ITS region of FKI-7473 was compared to sequences in the GenBank database by BLASTN 2.5.0 analysis.5 The strain FKI-7473 had 100% similarity with Pestalotiopsis humus CBS 336.97 (ex-type, GenBank accession number KM199317). From the morphological characteristics and the BLAST search, strain FKI-7473 was identified as Pestalotiopsis humus (Figure 1).6 Production and Isolation of Pestynol (1). Cultures of Pestalotiopsis humus FKI-7473 were extracted with MeOH and EtOAc. The extract was fractionated on a silica gel column and Received: March 8, 2018

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DOI: 10.1021/acs.jnatprod.8b00200 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

70.8), C-4, and C-5, from H-4 to C-2, C-3, C-5, and C-6, and from H-5 to C-1 (δC 70.2), C-3, C-4, and C-6 proved the presence of a 1,2,3,4-tetrahydroxy-5-cyclohexene unit. Although the H-2 and H-3 signals overlapped, HMBC correlations from H-1 to C-3 and H-4 to C-2 revealed their connection (Figure 2a).

Figure 1. Pestalotiopsis humus FKI-7473. Microphotograph of conidia grown on poplar twigs on water agar. Scale bar = 20 μm.

ODS column and purified by HPLC and TLC. This gave 1 as a white powder. Structure Elucidation of Pestynol (1). The molecular formula of 1 was revealed as C21H30O4 by HR-ESIMS. The 1H and 13C NMR data of 1 are shown in Table 1. The 1H NMR Table 1. NMR Spectroscopic Data for 1 in CD3ODa position

δC, type

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′

70.2, CH 70.8, CH 70.4, CH 67.5, CH 136.2, CH 126.1, C 89.9, C 90.9, C 132.8, C 38.5, CH2 27.7, CH2 124.5, CH 136.8, C 40.8, CH2 27.7, CH2 125.4, CH 132.1, C 121.8, CH2

13′ 14′ 15′

16.2, CH3 25.9, CH3 17.8, CH3

a

δH (J in Hz)

HMBC

4.21, d (2.8) 3.91,b m 3.91,b m 4.31, dd (2.2, 4.1) 6.04, d (4.1)

3, 3, 2, 2, 1,

2.21, m 2.24, m 5.12, dt (1.3, 6.8 Hz)

2′, 3′, 5′, 6′, 12′ 3′, 4′, 6′, 7′ 4′, 5′, 8′, 13′

1.98, t (7.4) 2.08, dt (7.4, 7.1) 5.08, dt (1.3, 7.1)

6′, 7′, 9′, 10′, 13′ 7′, 8′, 10′, 11′ 8′, 9′, 14′

5.25, 5.31, 1.63, 1.67, 1.60,

2′, 3′, 4′

d d d d s

(1.8) (1.8) (1.3) (1.3)

5, 4, 4, 3, 3,

6, 1′ 6 5 5, 6 4, 6, 1′

The structure of the remaining part was elucidated by H−1H COSY and HMBC experiments. The cross-peaks from H2-4′ (δH 2.21) to H2-5′ (δH 2.24), from H2-5′ to H-6′ (δH 5.12), from H2-8′ (δH 1.98) to H2-9′ (δH 2.08), and from H29′ to H-10′ (δH 5.08) were observed in 1H−1H COSY. In addition, HMBC correlations from H2-12′ (δH 5.25, 5.31) to C-3′ (δC 132.8) and C-4′ (δC 38.5), from H2-4′ to C-3′, C-5′ (δC 27.7), C-6′ (δC 124.5), and C-12′ (δC 121.8), from H2-5′ to C-3′, C-4′, C-6′, and C-7′ (δC 136.8), from H-6′ to C-4′, C5′, C-8′ (δC 40.8), and C-13′ (δC 16.2), from H3-13′ (δH 1.63) to C-6′, C-7′, and C-8′, from H2-8′ to C-6′, C-7′, C-9′ (δC 27.7), C-10′ (δC 125.4), and C-13′, from H2-9′ (δH 2.08) to C7′, C-8′, C-10′, and C-11′ (δC 132.1), from H-10′ to C-8′, C9′, and C-14′ (δC 25.9), from H3-14′ (δH 1.67) to C-10′, C11′, and C-15′ (δC 17.8), and from H3-15′ (δH 1.60) to C-10′, C-11′, and C-14′ indicated the partial structure of an alkenyl chain with an exomethylene unit (Figure 2a). The ring and the alkenyl chain are connected to two carbons, C-1′ (δC 89.9) and C-2′ (δC 90.9), respectively, as evidenced by HMBC correlations from H-1 and H-5 to C-1′ and from H-4′ and H-12′ to C-2′.7−10 These two carbons were indicated as composing an internal acetylene group by the unsaturation degree of 1 and their 13C chemical shifts. The geometry of the double bond at C-6′ was elucidated as having an E-configuration by the NOESY cross-peaks between H-6′ and H-8′ (Figure 2b). Thus, the planar structure of 1 was elucidated as (E)-5-(7,11-dimethyl-3-methylenedodeca-6,10dien-1-yn-1-yl)cyclohex-5-ene-1,2,3,4-tetraol. 1

6′, 7′, 8′ 10′, 11′, 15′ 10′, 11′, 14′

Data were collected at 400 MHz for 1H and 100 MHz for Overlapped.

b

Figure 2. (a) 1H−1H COSY (bold lines) and HMBC correlations (arrows) of pestynol. (b) Key NOESY correlation (arrow) of pestynol.

13

C.

data indicated the existence of four oxygenated sp3 methines, three sp2 methines, five methylenes, including one sp 2 methylene, and three methyl groups. The 13C NMR and HSQC spectra indicated 21 carbons, which were classified into eight olefinic carbons, including one sp2 methylene, four oxygenated sp3 methine carbons, four sp3 methylene carbons, three sp3 methyl carbons, and two sp carbons. The 1H−1H COSY of 1 showed connections from H-1 (δH 4.21) to H-2 (δH 3.91), from H-3 (δH 3.91) to H-4 (δH 4.31), and from H-4 to H-5 (δH 6.04). The HMBC correlations from H-1 to C-3 (δC 70.4), C-5 (δC 136.2), and C-6 (δC 126.1), from H-2 to C-3, C-4 (δC 67.5), and C-6, from H-3 to C-2 (δC B



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Figure 3. Modified Mosher’s method and Δδ = δS − δR values (ppm) of 1a and 1b.

Application of the modified Mosher’s method 11−13 elucidated the relative and absolute configurations of the stereogenic centers C-1, C-2, C-3, and C-4 in the cyclohexene unit of 1. First, (R)-(+)- and (S)-(−)-α-methoxy-α(trifluoromethyl)phenylacetic acid (MTPA-OH) esters (1a and 1b) were obtained by reacting 1 with (R)- and (S)-MTPAOH, respectively, in the presence of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine (Figure 3). Compounds 1a and 1b were shown to be mono-MTPA esters by HRESIMS analysis. The change of the 1H chemical shift value of 1a and 1b in the H-4 position compared with that of 1 indicated structures of 1a and 1b were mono-MTPA esters at the 4-hydroxy group. The relative configuration of the cyclohexene unit was elucidated by analyzing the vicinal 1H−1H coupling constants (3JH,H) of 1a, since the proton signals at H-2 and H-3 of 1 were overlapped. A large coupling constant (9.7 Hz) between H-2 (δH 3.94) and H-3 (δH 4.19) indicated that these protons are in a diaxial orientation. Small couplings between the vicinal H1 (δH 4.35) and H-2, H-3 and H-4 (δH 5.76), and H-4 and H-5 (δH 6.10) established that H-1 and H-4 are equatorial (Figure 4). Therefore, the relative configuration of the cyclohexene

Compound 1 showed weak growth inhibition against all Gram-positive bacteria and one Gram-negative bacteria (Klebsiella pneumonia), but was inactive against other Gramnegative bacteria tested, mycoplasma, and Aspergillus niger. Although 1 did not show any inhibition against wild-type S. cerevisiae KF237 or BY4741 and Candida albicans KF1, it showed weak inhibition against S. cerevisiae BY25929. However, it displayed potent inhibition of S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus. Cytotoxic Evaluation of Pestynol (1). The cytotoxic activity of 1 using three floating cell lines (Jurkat, HL60, and THP-1 cells) and five adherent cell lines (HeLa S3, HT29, A549, H1299, and Panc1 cells) was examined. Compound 1 was weakly active against Jurkat, HL60, THP-1, HT29, and A549 cells, with IC50 values of 84, 19, 61, 83, and 92 μM, respectively. Other cells were not affected at 100 μM of 1. To our knowledge, the structure of 1 has not previously been published. It has been reported that Phomopsis sp. produces geranylcyclohexenetriol with sp carbons, such as the phomentrioloxins,6−9 which might have biosynthetic pathways similar to 1, which contains a farnesylcyclohexenetetraol with sp carbons. Although the phomentrioloxins are structurally similar to 1, they exhibit phytotoxic activity but are not antimicrobial or fungicidal,7−10 whereas 1 is weakly active against Gram-positive bacteria and inhibited S. cerevisiae 12geneΔ0HSR-iERG6 (compared to wild-type S. cerevisiae BY4741) in a dose-dependent manner (Table 2). Interestingly, 1 also inhibited the growth of Mucor racemosus IFO 4581 at 10 μg/disc, but did not have any drastic impact on mammalian cells. In addition to discovering pestynol, a promising new antimicrobial compound, we have demonstrated that S. cerevisiae 12geneΔ0HSR-iERG6 is a useful tool in antifungal discovery.

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Figure 4. 1H−1H coupling constants (Hz) of the cyclohexene unit of the mono-(R)-MTPA ester.

EXPERIMENTAL SECTION

General Experimental Procedures. High- and low-resolution mass data were recorded using an AB Sciex QSTAR Hybrid LC/MS/ MS System (AB Sciex, Framingham, MA, USA) and JEOL JMST100LP (JEOL, Tokyo, Japan). NMR spectra were measured by a Varian XL-400 spectrometer (Agilent Technologies, CA, USA) with 1 H NMR at 400 MHz and 13C NMR at 100 MHz and a JEOL JNMECA-500 (JEOL, Tokyo, Japan) with 1H NMR at 500 MHz in CD3OD and CDCl3. The chemical shifts are expressed in ppm and are referenced to CD3OD (3.31 ppm) or CDCl3 (7.26 ppm) in the 1H NMR spectra and CD3OD (49.0 ppm) in the 13C NMR. IR spectra (ATR) were measured with an FT-210 Fourier transform IR spectrometer (Horiba Ltd., Kyoto, Japan). UV spectra were observed using a Hitachi U-2801 spectrophotometer (Hitachi Ltd., Tokyo, Japan). Optical rotation was measured with a JASCO P2200 polarimeter (JASCO Corporation, Tokyo, Japan). Taxonomic Studies. For determination of morphological characteristics, the isolate was inoculated on oatmeal agar (OA, Difco Laboratories, Detroit, MI, USA) and cultured at 25 °C for 7

unit was elucidated as 1R*, 2R*, 3R*, and 4R*. The absolute configuration of 1 was deduced by the chemical shift differences of neighboring signals of H-4 in the diastereomeric MTPA esters 1a and 1b. The observed chemical shift differences (Δδ = δS − δR) clearly defined the R configuration at C-4. As a result, the overall absolute configuration of 1 was elucidated to be 1R, 2R, 3R, and 4R. Evaluation of Antimicrobial Activity of Pestynol (1). Antimicrobial activity of 1 against various Gram-positive and Gram-negative bacteria, mycoplasma, yeasts (including wildtype and multidrug-sensitive budding S. cerevisiae), and fungi was evaluated, and the results are shown in Table 2. All evaluation was carried out by the paper disc method using 6 mm discs. C



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Table 2. Antimicrobial Activity of 1 Using the Paper Disc Methoda 1 (μg/disc) organism Gram-Positive Bacteria Staphylococcus aureus KB210 (ATCC6538p) Bacillus subtilis KB211 (ATCC6633) Kocuria rhizophila KB212 (ATCC 9341) Mycobacterium smegmatis KB42 Gram-Negative Bacteria Escherichia coli KB213 (NIHJ) Klebsiella pneumoniae KB214 (ATCC10031) Pseudomonas aeruginosa KB359 (NBRC12582) Xanthomonas canpestris pv oryzae KB88 Proteus vulgaris KB127 (NBRC 3167) Mycoplasma Acoleplasma laidlawii KB174 (PG8) Yeast Candida albicans KF1 (ATCC 64548) Saccharomyces cerevisiae KF237 (ATCC9763) Saccharomyces cerevisiae BY4741 Saccharomyces cerevisiae BY25929 Saccharomyces cerevisiae 12geneΔ0HSR-iERG6 Filamentous Fungi Aspergillus niger KF103 (ATCC6275) Mucor racemosus KF223

MEPMb

EMc

AMPHd

100

30

10

3

1

1

10

8.2 16.2 14.7 9.4

6.6  7.5 7.3

   

   

35.2 24.1 38.0 9.5

N.T. N.T. N.T. N.T.

N.T. N.T. N.T. N.T.

 7.9   

    

    

    

31.0 27.0 47.3 31.6 37.8

N.T. N.T. N.T. N.T. N.T.










N.T.

28.0

N.T.

   8.0 12.1

    10.6

    7.9

    



13.0 14.5 8.2 11.7 9.8

 31.2

 28.1

 15.6

 

N.T. N.T.

N.T. N.T.

16.1 15.1

a Inhibition zone (mm); , no inhibition; N.T., not tested. bMPEM, meropenem trihydrate. cEM, erythromycin. dAMPH, amphotericin B; 6 mm paper discs were used.

extract, 0.05% MgSO4·7H2O, 0.5% Polypepton (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 0.1% KH2PO4, and 0.1% agar at pH 6.0] in a 500 mL Erlenmeyer flask and incubated for 3 days (210 rpm at 27 °C). After 3 days, 25 mL of the seed culture was inoculated into each of 20 Ulpack 47 (culture bags) (Hokken Co. Ltd., Tochigi, Japan) containing rice medium (500 g of water-sodden rice). Fermentation was maintained in static condition at 27 °C for 14 days. Isolation. MeOH (8 L) was added to the culture, and the mixture was centrifuged at 3000 rpm for 10 min. The supernatant was collected and evaporated under reduced pressure to remove MeOH. The remaining aqueous solution (1 L) was extracted three times with an equal volume of EtOAc. The organic layer was concentrated to dryness to afford a crude extract (21 g). The extract was fractionated on a silica gel column (65 i.d. × 200 mm) and eluted stepwise with a mixture of CHCl3−MeOH (100:0, 100:2, 100:20, 1:1, and 0:100). The 100:20 fraction (5 g) was applied to an ODS column and eluted stepwise with a mixture of MeOH−H2O (20:80, 30:70, 50:50, 70:30, 80:20, 90:10, and 100:0). The 80:20 fraction was concentrated in vacuo to remove the MeOH. The dried material (573 mg) was dissolved in a small amount of MeOH and applied to the preparative HPLC on a Pegasil ODS SP100 column (20 i.d. × 250 mm; Senshu Scientific Co. Ltd., Tokyo, Japan) with a linear gradient solvent system of CH3CN−H2O (from 30:70 to 100:0 for 40 min) at a flow rate of 10 mL/min to give a brown oil (403 mg). It was applied to the preparative HPLC using the same column with an isocratic solvent system of 50% CH3CN with 0.1% trifluoroacetic acid at a flow rate of 10 mL/min, and the peak, from 30 to 33 min, was collected to give 200 mg of yellow oil. This was then applied to preparative TLC (plate size, 200 × 200 mm; thickness, 1 mm) using two plates developed with CHCl3−MeOH (5:1, Rf value 0.5, 75.0 mg). Finally, it was purified by HPLC on a Pegasil ODS SP100 column (20 i.d. × 250 mm) with an isocratic solvent system of 45% CH3CN with 0.1% trifluoroacetic acid at a flow rate of 10 mL/min, and the peak of the retention time of 50−60 min was collected to give 33.0 mg of 1 as a white powder. Physicochemical Properties of Pestynol (1). Compound 1 was soluble in MeOH; [α]27D −49.4 (c 0.1, MeOH); UV (MeOH) λmax (ε) 202 (20 500), 261 (13 200), 274 (10 500); IR νmax (ATR) cm−1

days. For determination of the micromorphological characteristics of FKI-7473, it was grown on autoclaved poplar twigs in 2% water agar and incubated at room temperature, where it received diffused daylight to induce sporulation. Samples were examined with a VanoxS AH-2 microscope (Olympus, Tokyo, Japan), and digital photomicrographs were taken with a DP25 digital camera (Olympus). The pycnidial conidiomata culture on poplar twigs on water agar was globose, semi-immersed, aggregated, or scattered; exuding dark brown to black, globose conidial masses. Conidia fusoid to fusiform, 4septate, constricted at septum, 21−25(−27.5) × 5.0−6.0 (−7.5) μm; basal cell obconic to conic with a truncate base, hyaline, minutely verruculose and thin-walled; three median cells subcylindrical, wall rugose, concolourous, brown, septa darker than the rest of the cell; apical cell hyaline, subcylindrical; with 2−3 tubular apical appendages, arising from an apical crest, unbranched, filiform, flexuous, 10−16.5 μm; basal appendage single, tubular, unbranched, centric, (2.5−)5.0− 9.0 μm long. Culture characteristics: Colonies on OA grew fast at 25 °C, with smooth edge, pale yellow, with sparse aerial mycelium on the surface with black, gregarious conidiomata; reverse pale yellowish-brown. From the above morphological characteristics, strain FKI-7473 was classified into the genus Pestalotiopsis. DNA extraction and polymerase chain reaction amplification of the internal transcribed spacer (ITS) region, including the 5.8S rRNA gene, were conducted under the conditions detailed in a previous study,14 and annealing of the ITS region was carried out at 50 °C. Sequencing products were purified using a BigDye XTerminator purification kit (Applied Biosystems, Foster City, CA, USA), and samples were analyzed on an ABI PRISM 3130 genetic analyzer (Applied Biosystems). Contigs were assembled using the forward and reverse sequences, with the SeqMan Pro program from the Lasergene 10 package (DNASTAR Inc., Madison, WI, USA). The ITS sequence of the strain FKI-7473 was deposited at the DDBJ with accession number KM199317. Fermentation. Pestalotiopsis humus FKI-7473 grown on an LcA slant (0.1% glycerol, 0.08% KH2PO4, 0.02% K2HPO4, 0.02% MgSO4· 7H2O, 0.02% KCl, 0.2% NaNO3, and 1.5% agar at pH 6.0) was inoculated into 100 mL of seed medium [2.0% glucose, 0.2% yeast D



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drug resistance genes (Δyrr1, Δyrs1, Δpdr1, Δpdr3) deleted, was provided by the National Bio-Resource Project, Japan. This yeast, along with S. cerevisiae 12geneΔ0HSR-iERG6, was cultured under the same conditions as S. cerevisiae BY4741. All positive controls, meropenem trihydrate, erythromycin, and amphotericin B were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Cytotoxic Evaluation. Cytotoxic evaluation of 1 used three floating cell lines: Jurkat (human acute lymphocytic leukemia cell line), HL60 (human promyelocytic leukemia cell line), and THP-1 (human acute monocytic leukemia cell line), along with five adherent cell lines, HeLa S3 (human cervical cancer cell line), HT29 (human colorectal adenocarcinoma cell line), A549 (human adenocarcinoma cell line derived from lung cancer), H1299 (human non-small-cell lung carcinoma cell line derived from lymph nodes), and Panc1 (human cell line derived from pancreatic cancer). Culture conditions were as follows: floating cell lines [RPMI medium (Nacarai Tesque, Inc., Kyoto, Japan) added to 10% fetal bovine serum (FBS), 1% sodium pyruvate, and 1% penicillin− streptomycin, 37 °C, under 5% CO2] and adherent cell lines [DMEM medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) added to 10% FBS and 1% penicillin−streptomycin, 37 °C, under 5% CO2]. The cultivated floating (3 × 105 cells per well) and adherent (5 × 104 cells per well) cell lines were seeded in 96-well plates. After culturing overnight, a MeOH solution of 1 was added into each well. After 2 days of incubation at 37 °C, a WST-8 solution was added to each well and kept for 4 h at 37 °C. The absorbance of each well was measured using a Corona SH-9000 grating microplate reader (Corona Electric, Ibaraki, Japan) at 460 nm.

3375, 2358, 1398; HR-ESIMS found m/z 369.2049 [M + Na]+, calcd for C21H30NaO4, m/z 369.2042 [M + Na]+. Preparation Procedure of (R)-(+)-MTPA Ester of 1 (1a). A solution of 1 (3.2 mg, 9.24 μmol) in dry tetrahydrofuran (0.9 mL) at room temperature was treated with (R)-(+)-α-methoxy-α(trifluoromethyl)phenylacetic acid (2.6 mg, 11.1 μmol), dicyclohexylcarbodiimide (3.8 mg, 18.5 μmol), and a catalytic amount of 4dimethylaminopyridine (one crystal). The reaction mixture was warmed to 60 °C, stirred for 19 h, and cooled to room temperature. The mixture was then quenched with H2O (3 mL), stirred again for a further 10 min, and then extracted with dichloromethane (3 × 5 mL). The combined extracts were dried over Na2SO4, filtered, and concentrated. Preparative TLC (250 μm 20 × 20 cm; CHCl3− MeOH, 10:1) gave 1a (0.3 mg, 6% yield; 19% yield based on recovered starting material) as a colorless material, and unreacted 1 (1.9 mg, 59%) was recovered. HR-ESIMS m/z 580.2882 [M + NH4]+ (calcd m/z 580.2886 [M + NH4]+, molecular formula C31H41F3NO6); 1 H NMR (CDCl3, 500 MHz) δH 6.10 (1H, d, J = 4.9 Hz, H-5), 5.76 (1H, dd, J = 4.5, 4.9 Hz, H-4), 5.40 (1H, brs, H-12′), 5.33 (1H, brs, H-12′), 5.11 (1H, m, H-6′), 5.09 (1H, m, H-10′), 4.35 (1H, d, J = 4.3 Hz, H-1), 4.19 (1H, dd, J = 4.5, 9.7 Hz, H-3), 3.94 (1H, dd, J = 4.3, 9.7 Hz, H-2), 2.23 (2H, m, H-4′), 2.23 (2H, m, H-5′), 2.06 (2H, m, H-9′), 1.98 (2H, t, J = 7.2 Hz, H-8′), 1.68 (3H, brs, H-14′), 1.61 (3H, brs, H-13′), 1.60 (3H, brs, H-15′). Preparation Procedure of (S)-(−)-MTPA Ester of 1 (1b). Following the procedure described above for the preparation of 1a, 1 (2.8 mg, 8.08 μmol) was acylated with (S)-(−)-α-methoxy-α(trifluoromethyl)phenylacetic acid (2.3 mg, 9.70 μmol) to afford 1b (0.3 mg, 7% yield; 12% yield based on recovered starting material) as a colorless material, and unreacted 1 (1.3 mg, 46%) was recovered. HR-ESIMS m/z 580.28858 [M + NH4]+ (calcd m/z 580.28860 [M + NH4]+, molecular formula C31H41F3NO6); 1H NMR (CDCl3, 500 MHz) δH 6.15 (1H, d, J = 4.6 Hz, H-5), 5.77 (1H, dd, J = 4.5, 4.6 Hz, H-4), 5.42 (1H, brs, H-12′), 5.34 (1H, brs, H-12′), 5.10 (1H, m, H6′), 5.08 (1H, m, H-10′), 4.38 (1H, d, J = 4.3 Hz, H-1), 4.16 (1H, dd, J = 4.5, 9.2 Hz, H-3), 3.92 (1H, dd, J = 4.0, 9.2 Hz, H-2), 2.23 (2H, m, H-4′), 2.23 (2H, m, H-5′), 2.06 (2H, m, H-9′), 1.98 (2H, t, J = 7.7 Hz, H-8′), 1.68 (3H, brs, H-14′), 1.61 (3H, brs, H-13′), 1.60 (3H, brs, H-15′). Antimicrobial Activity. Antimicrobial activity against a variety of organisms was measured using the paper disc method (6 mm discs from Advantec Co. Ltd., Tokyo, Japan). Sterile filter discs impregnated with compound solution (10 μL) were placed on agar plates, and the plates were incubated at 27 or 37 °C for 24−48 h. After incubation, inhibition zones were measured. Culture conditions were as follows: Bacillus subtilis ATCC 6633 [nutrient agar (0.5% peptone, 0.5% meat extract, and 0.8% agar adjusted to pH 7.0), 0.5% inoculation, 37 °C, 24 h], Escherichia coli NIHJ [nutrient agar, 0.5% inoculation, 37 °C, 24 h], Klebsiella pneumoniae ATCC 10031 [nutrient agar, 0.5% inoculation, 37 °C, 24 h], Kocuria rhizophila ATCC 9341 [nutrient agar, 0.2% inoculation, 37 °C, 24 h], Proteus vulgaris NBRC 3167 [nutrient agar, 0.5% inoculation, 37 °C, 24 h], Pseudomonas aeruginosa NBRC 12582 [nutrient agar, 1.0% inoculation, 37 °C, 24 h], Staphylococcus aureus ATCC 6538p [nutrient agar, 0.2% inoculation, 37 °C, 24 h], Xanthomonas camestris pv oryzae KB 88 [nutrient agar, 1.0% inoculation, 27 °C, 24 h], Mycobacterium smegmatis KB 42 [Waksman agar (0.5% peptone, 0.5% meat extract, 0.3% NaCl, 1.0% glucose, and 0.8% agar adjusted to pH 7.0), 1.0% inoculation, 37 °C, 48 h], Acoleplasma laidlawii PG 8 [PPLO agar (3% PPLO broth (Becton Dickinson, Franklin Lakes, NJ, USA), 0.2% phenol red (5 mg/mL), 0.1% glucose, 1.5% agar, 1% penicillin G, and 15% horse serum), 7.5% inoculation, 37 °C, 48 h], Aspergillus niger ATCC 6275 [GY agar (1.0% glucose, 0.5% yeast extract, and 0.8% agar adjusted to pH 6.0), 0.5% inoculation, 27 °C, 48 h], Candida albicans ATCC 64548 [GY agar, 0.2% inoculation, 27 °C, 48 h], Mucor racemosus IFO 4581 [GY agar, 0.3% inoculation, 27 °C, 48 h], Saccharomyces cerevisiae ATCC 9763 [GY agar, 0.3% inoculation, 27 °C, 24 h], and S. cerevisiae BY4741 [YPD agar (1% yeast extract, 2% peptone, 2% glucose, and 1.5% agar), 1% inoculation, 27 °C, 48 h]. S. cerevisiae BY25929, with

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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.8b00200. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Toshiaki Sunazuka: 0000-0003-3452-6070 Kazuro Shiomi: 0000-0003-1264-5116 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study was supported by The Public Foundation of Elizabeth Arnold−Fuji, Japan. We thank Dr. K. Nagai and Ms. N. Sato, School of Pharmacy, Kitasato University, for measurement of MS and NMR spectra.

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REFERENCES

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