Stereochemical Assignment and Biological Evaluation of BE-14106

Jun 22, 2016 - Heronamides are a class of potent antifungal metabolites produced by marine-derived actinomycetes. The number of hydroxy groups and the...
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Stereochemical Assignment and Biological Evaluation of BE-14106 Unveils the Importance of One Acetate Unit for the Antifungal Activity of Polyene Macrolactams Kohei Fujita,† Ryosuke Sugiyama,† Shinichi Nishimura,*,† Naoki Ishikawa,‡ Midori A. Arai,‡ Masami Ishibashi,‡ and Hideaki Kakeya*,† †

Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan ‡ Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan S Supporting Information *

ABSTRACT: Heronamides are a class of potent antifungal metabolites produced by marine-derived actinomycetes. The number of hydroxy groups and the stereochemistry of the two hydroxylated methine carbons are important for the activity of heronamide C, whereas the effect of the hydrocarbon chains is not known. In this study, the stereochemistry and the biological activity of BE-14106, another member of the heronamide class of antibiotics, isolated from an actinomycete Actinoalloteichus cyanogriseus IFM 11549 was investigated. Spectroscopic analysis coupled with photo- and O2-induced conversion revealed that BE-14106 and the heronamides had the same stereochemistry. BE-14106 showed potent growth inhibition against fission yeast cells with an MIC value of 0.50 μM (0.21 μg/mL), being 4 times less potent than heronamide C, which revealed the importance of the structure of the hydrocarbon tail for the activity.

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logical changes in mammalian cells. In particular, heronamide C and 8-deoxyheronamide C exhibit potent antifungal activity by targeting membrane phospholipids possessing saturated hydrocarbon chains.4 Although the binding mode at an atomic resolution remains to be revealed, a structure−activity relationship study using natural congeners and synthetic derivatives indicated that the presence of the hydroxy groups and stereochemistry of the oxygenated carbons C-8 and C-9 are important for their potent activities.4,14 To investigate the involvement of the hydrocarbon tail in the biological activity, we have compared the activities of BE-14106 and heronamide C. In this report, we describe the stereochemistry and biological activities of BE-14106. BE-14106 has been isolated from several actinomycetes, and the biosynthetic gene cluster has been identified,1,2,15 although the stereochemistry remained to be determined. We found that another actinomycete, Actinoalloteichus cyanogriseus IFM 11549, which was isolated from a soil sample collected in Chiba, Japan, produces BE-14106. From a culture broth (4 L), we purified 28.7 mg of BE-14106 by solvent partitioning and repeated chromatography. MS and NMR data in DMSO-d6 matched those reported (Table S1).1 As in the case of heronamide C, the 1H NMR spectra showed overlapping signals at δ 5.4−5.5 and 5.8−6.2 ppm, which hampered analysis of the stereochemistry by NOESY data.

growing number of polyene macrolactams have been reported from actinomycetes.1−13 Their chemical structures are based on a 20- to 26-membered lactam ring to which hydroxy groups (sometimes glycosylated), methyl groups, and a hydrocarbon tail are attached (Figure S1). Heronamides are 20membered macrolactams. So far, six actinomycete metabolites that share the macrolactam structure have been reported: BE14106/GT32-A,1,2 GT32-B,2 ML-449,6 heronamides C and F,3,5 and 8-deoxyheronamide C (Figure 1, Figure S1).4 Some of them were reported to exhibit significant biological activities, for example, antimicrobial activities and nontoxic morpho-

Figure 1. Chemical structures of BE-14106/GT-32A (1), the deoxy congener GT-32B (2), and heronamides. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 19, 2016

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DOI: 10.1021/acs.jnatprod.6b00250 J. Nat. Prod. XXXX, XXX, XXX−XXX

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shown in Figures 2a and S2, which was the same as heronamide B. Compounds 6 and 7 have the same relative stereochemistries at C-8, C-9, and C-19. Since these carbons are not directly involved in the photo- or O2-induced transformation, the relative stereochemistry of BE-14106 (1) must be 8R*, 9S*, 19R*. Finally, the absolute stereochemistry was determined by comparing CD spectra. BE-14106 showed a characteristic Cotton effect: 282.5 nm (−138.0 mdeg) and 323.5 nm (+265.2 mdeg) (Figure S3). Since heronamide C (3) and its 8-deoxy congener (5) showed similar CD spectra,4 we concluded the stereochemistry of BE-14106 (1) to be 8R, 9S, 19R. We previously reported that heronamide C (3) shows potent growth inhibition against fission yeast cells with an MIC value of 0.13−0.28 μM (0.056−0.13 μg/mL), which was comparable to that of the clinically used amphotericn B (MIC = 0.27 μM (0.25 μg/mL)).4,14 We examined the activity of BE-14106 (1) to find that the MIC value was 0.50 μM (0.21 μg/mL), which was 4 times less potent than heronamide C (3) (Figure 3a). To

We then investigated nonenzymatic conversion of BE-14106 to obtain compounds that may show less complicated NMR spectra; we previously showed that heronamide C can be converted to heronamide A and heronamide B by O2 and UV irradiation, respectively.4,14 As expected, BE-14106 was converted to a tetracyclic compound 6 after 14 days’ incubation in DMSO at 40 °C, although the yield was moderate (37% brsm). The NMR spectra closely resembled those of heronamide A except for the absence of two olefinic proton and carbon signals. 1D and 2D NMR analyses confirmed the planar structure of this tetracyclic compound as 6 (Figure 2a,

Figure 3. Biological activities of BE-14106 (1) and its derivatives. (a) Growth inhibitory activities of BE-14106 (1), compounds 6 and 7, and heronamide C (3) against fission yeast wild-type cells. Red diamonds: BE-14106 (1); black circles: heronamide C (3); orange squares: compound 6; blue triangles: compound 7. Data represent the mean ± SD (n = 3). Amphotericin B showed an MIC value of 0.27 μM (0.25 μg/mL). (b) Abnormal cell wall morphology induced by compound treatment. Cells were treated with DMSO (1%), BE-14106 (0.5 μM), or heronamide C (0.25 μM) for 2 h at 30 °C. Cells were fixed, stained with calcofluor white (Cfw), and observed under fluorescence microscopy. In control cells, an intense fluorescent signal was detected only at the cell division site. After compound treatment, bright fluorescent signals were detected not only at the division site but also at the cell tips. Scale bar: 10 μm.

Figure 2. Photo- and O2-induced conversion of BE-14106 (1). (a) Chemical structures of compounds 6 and 7 and the reaction scheme. (b) Key NOESY correlations in compound 6. NOESY correlations on the α-face are shown in red; those on the β-face are in blue.

Figure S14). NOESY correlations from H-7 to H-5 and H-13 and from H-15 to H-2 and H-5 placed H-2, H-5, H-7, H-13, and H-15 on the α-face of the 10-membered ring. NOESY correlations from H-19 to H-18a and from H-17 to H-2 and H18a indicated that H-17, H-18a, and H-19 are also placed on the α-face (Figure 2b). On the other hand, H-3, H-4, H-8, H-9, H-12, H-16, H-18b, H3-26, and H3-27 were placed on the βface based on the NOESY correlations (Figure 2b). Thus, the relative stereochemistry of compound 6 was revealed to be the same as heronamide A.16 Next, we examined photoinduced transformation of BE14106 (1). Heronamide C can be converted to heronamide B by UV irradiation.14 As expected, one major product appeared after irradiation of BE-14106 (1) at 365 nm for 5 min. The NMR analyses revealed the planar structure of this cycloadduct as 7, which possessed a shorter side chain when compared with heronamide B (Figure 2a, Figures S15, S21). Detailed analysis of the NMR spectra revealed the relative stereochemistry as

investigate whether BE-14106 (1) and heronamide C (3) share their mode of action, we examined the morphological changes of yeast cells after compound treatment (Figure 3b). We reported that heronamide C induced abnormal accumulation of cell wall material, probably through modulation of the function of the plasma membrane lipids.4 Fluorescence microscopic analysis revealed that BE-14106 induced the abnormal accumulation of the cell wall material, which can be visualized by calcofluor white, a fluorescent dye for detecting 1,3-β-glucan. Although a higher concentration of BE-14106 was required to induce the characteristic phenotype when compared to heronamide C, these results suggested that BE-14106 also B

DOI: 10.1021/acs.jnatprod.6b00250 J. Nat. Prod. XXXX, XXX, XXX−XXX

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HPLC (Cosmosil 5C8-MS, 20 × 250 mm, 7.56 mL/min, monitored at 220 nm) using an isocratic solvent system (80% MeOH(aq)) to obtain compound 7 (0.7 mg, 15.6%) as a white powder: 1H and 13C NMR data in Table S1; HRESIMS m/z 424.2850 [M + H]+ (calcd for C27H38NO3, 424.2846). The purity was assessed to be 96.6% by HPLC analysis. Biological Activities. Growth inhibition and morphological changes by compounds were tested as described previously using wild-type fission yeast JY1 (h−).18

targets the cell membrane. It is noted that the converted products 6 and 7 lost activity (not active at 20 μM, Figure 3a). This is not surprising since heronamides A and B (Figure S1) also significantly decreased their activities (not active at 100 and 50 μM, respectively).14 In this report, we unambiguously determined the absolute stereochemistry of BE-14106 (1) and unveiled the importance of the hydrocarbon chain for the potent activity. The absolute stereochemistry of BE-14106 (1) was the same as heronamides. This enabled the current structure−activity relationship study of heronamides to reveal that the presence of one more olefin in heronamide C enhanced the potency of the antiyeast activity by 4 times. A further SAR study using natural and synthetic heronamide analogues would contribute to the development of more effective and safer antifungal drugs.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00250. NMR and CD spectral data for BE-14106 and derivatives (PDF)

EXPERIMENTAL SECTION

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General Experimental Procedures. High-resolution ESIMS spectra were recorded on an LC-IT-TOF MS. NMR spectra were recorded on a 500 MHz instrument. 1H and 13C chemical shifts are shown relative to the residual solvent: δH 2.50 (DMSO-d6) or 3.31 (CD3OD) and δC 39.52 (DMSO-d6) or 49.00 (CD3OD). Chemical shifts (δ) are shown in parts per million (ppm), and coupling constants (J) are in hertz (Hz). CD spectra were recorded using a CD spectrometer with a 1 mm path length cell. Strain and Cultivation. Actinoalloteichus cyanogriseus IFM 11549 was isolated from a soil sample collected at the Sakazuki forest in Chiba city, Chiba prefecture, Japan. A voucher specimen is deposited at Medical Mycology Research Center, Chiba University (IFM 11549).17 Spores of A. cyanogriseus IFM 11549 grown on solid Waksman medium were inoculated into a 2 L Sakaguchi flask that contained 500 mL of liquid medium, and the flask was incubated at 28 °C for 3 days with reciprocal shaking at 120 rpm to produce seed culture. The seed culture (20 mL) was inoculated into 3 L flasks that contained 750 mL of liquid Waksman medium, and the flasks were incubated at 28 °C for 4 days with reciprocal shaking at 120 rpm to obtain 12 L of fermentation broths. Liquid Waksman medium was prepared using 2% glucose, 0.5% peptone, 0.5% meat extract, 0.3% yeast extract, 0.5% NaCl, and 0.3% CaCO3 in distilled water. Agar (1.5%) was added for solidification. Extraction and Isolation. After cultivation, an equal volume of acetone (12 L) was added to the culture broth and mixed at 28 °C for 3 h with reciprocal shaking at 120 rpm. The culture broth was filtered and evaporated in vacuo. The pH of the crude aqueous solution (ca. 12 L) was adjusted to 7.0 by HCl(aq). Part of the crude aqueous solution (4 L) was extracted with EtOAc three times, and the combined extracts were concentrated. The residue was partitioned between 60% MeOH(aq) and CHCl3. The CHCl3 layer (537 mg) was fractionated by SiO2 column chromatography (CHCl3−MeOH). Fractions containing BE-14106 (1) were subjected to RP-HPLC (Cosmosil 5C8-MS, 20 × 250 mm, 7.56 mL/min, monitored at 220 nm) using an isocratic solvent system (71% MeOH(aq)) to afford BE-14106 (1, 28.7 mg) as a white powder: 1H and 13C NMR data in Table S1; HRESIMS m/z 424.2847 [M + H]+ (calcd for C27H38NO3, 424.2846). The purity was assessed to be 98.7% by HPLC analysis. O2-Induced Conversion from BE-14106 (1) to Compound 6. BE-14106 (8.0 mg) was dissolved in DMSO (1 mM) and stirred at 40 °C for 14 days. The solution was lyophilized, dissolved in DMSO (800 μL), and subjected to RP-HPLC (Cosmosil 5C8-MS, 20 × 250 mm, 7.56 mL/min, monitored at 220 nm) using an isocratic solvent system (71% MeOH(aq)) to obtain compound 6 (1.2 mg, 37.1% brsm) as a white powder: 1H and 13C NMR data in Table S1; HRESIMS m/z 440.2796 [M + H]+ (calcd for C27H38NO4, 440.2796). The purity was assessed to be 90.1% by HPLC analysis. Photoinduced Conversion from BE-14106 (1) to Compound 7. BE-14106 (4.4 mg) was dissolved in DMSO (1 mM) and subjected to UV irradiation at 365 nm for 5 min. The solution was lyophilized, dissolved in DMSO−MeOH (200 μL/600 μL), and subjected to RP-

AUTHOR INFORMATION

Corresponding Authors

*Tel (S. Nishimura): +81-75-753-4534. E-mail: nshin@pharm. kyoto-u.ac.jp. *Tel (H. Kakeya): +81-75-753-4524. E-mail: scseigyo-hisyo@ pharm.kyoto-uac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. T. Gonoi (Medical Mycology Research Center, Chiba University) for identifying the actinomycete species. This work was supported in part by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).

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DOI: 10.1021/acs.jnatprod.6b00250 J. Nat. Prod. XXXX, XXX, XXX−XXX