Ostalactones A–C, β- and ε-Lactones with Lipase Inhibitory Activity

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Ostalactones A−C, β- and ε‑Lactones with Lipase Inhibitory Activity from the Cultured Basidiomycete Stereum ostrea Hahk-Soo Kang† and Jong-Pyung Kim*,‡ †

Department of Bio-industrial Technologies, Konkuk University, Seoul 05029, Korea Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk 28116, Korea

‡

S Supporting Information *

ABSTRACT: Ostalactones A−C (1−3), three new β- and ε-lactone natural products, were isolated from the culture broth of the basidiomycete Stereum ostrea. The structures were elucidated by interpretation of HRFABMS and 1D and 2D NMR data. The structures of 1 and 2 are characterized by the presence of a β-lactone containing a fused 4/5 bicyclic core structure. Compound 3 possesses a 2-oxepinone ring system, which is likely to be a biosynthetic precursor of compounds 1 and 2. Ostalactones A (1) and B (2) displayed potent inhibitory activity against human pancreatic lipase.

B

Compound 1 was obtained as a colorless oil. HRFABMS analysis displayed a pseudomolecular ion peak at m/z 225.1130 ([M + H]+), suggesting the molecular formula C12H16O4. The 1 H NMR spectrum of 1 showed signals for two olefinic (δH 5.60 and 5.45), one oxymethine (δH 4.91), two oxymethylene (δH 4.15 and 3.94), two diastreotopic methylene (δH 2.81 and 2.67; δH 2.67 and 2.51), and one methyl (δH 1.68) proton. Analysis of the 13C NMR and HMQC spectra suggested the presence of 12 carbons including signals for one carbonyl (δC 175.1), four olefinic (δC 149.2, 140.3, 123.1, and 119.3), one methine (δC 80.4), one quaternary (δC 76.2), two oxymethylene (δC 68.4 and 61.5), two methylene (δC 38.4 and 28.3), and one methyl (δC 14.1) carbon. The planar structure of 1 was determined by combined analysis of 2D NMR data including COSY, HMBC, and ROSEY. A COSY correlation between H2-6 (δH 2.68 and 2.81) and H-7 (δH 4.91) together with HMBC correlations from H2-13 (δH 4.15) to C-4 (δC 123.1), C-5 (δC 149.2), and C-6 (δC 38.4), from H2-6 to C-4 and C-5, from H-7 to C-2 (δC 175.1), C-4, C-5, and C-6, and from H-4 (δH 5.6) to C-3 (δC 76.2), C-5, C-6, C-7 (δC 80.4), and C-13 (δC 61.5) determined the structure of a fused 4/5 bicyclic core functionalized with a hydroxymethyl group at C-5 (substructure A). The second substructure (substructure B) was suggested to be a dimethylallyl moiety with a hydroxy group attached at C-12 due to a COSY correlation between H28 (δH 2.51 and 2.67) and H-9 (δH 5.45) as well as HMBC correlations from H3-11 (δH 1.68) to C-9 (δC 119.3), C-10 (δC 140.3), and C-12 (δC 68.4), from H2-12 (δH 3.94) to C-9, C-10, and C-11 (δC 14.1), and from H2-8 to C-9 and C-10. The geometric configuration of a double bond between C-9 and C10 was assigned an E configuration by an ROE correlation observed between H-9 and H2-12. Substructures A and B were

asidiomycetes are known producers of products with unique fused multicyclic ring systems. The examples include trefolane A, a sesquiterpene with a 5/6/4 tricyclic core, from Tremella foliacea1 and hirsutenols A−C, sesquiterpenes with a 5/5/5 tricyclic core, from Stereum hirsutum.2 As a part of our ongoing efforts to find structurally unique natural products, we screened the culture broth extracts of basidiomycetes isolated from samples collected across Korea for strain-specific metabolites using LC-MS equipped with a photodiode array detector. During our screening campaign, the culture broth extract of Stereum ostrea showed strain-specific LC peaks with molecular weights of 224 and 252 Da. Dereplication of these peaks using 1H NMR in combination with HRMS indicated that these are likely to be new natural products. Therefore, we decided to conduct a full chemical investigation of the culture broth extract of S. ostrea. Here, we report the isolation and structure determination of three new lactones, named ostalctones A (1), B (2), and C (3). Ostalactones A (1) and B (2) displayed potent inhibitory activity against human pancreatic lipase. For the structure determination and bioassay, a culture of S. ostrea was prepared using a 4 L stirred batch fermenter. The culture broth was separated from the mycelium and then extracted with ethyl acetate. The ethyl acetate extract was fractionated using a silica column chromatography followed by a Sephadex LH-20 size exclusion chromatography. Final purification was achieved by reversed-phase HPLC to yield pure compounds 1 (4.5 mg), 2 (2.8 mg), and 3 (12.0 mg).

Received: July 12, 2016 © XXXX American Chemical Society and American Society of Pharmacognosy

A

DOI: 10.1021/acs.jnatprod.6b00647 J. Nat. Prod. XXXX, XXX, XXX−XXX

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connected at C-3 via HMBC correlations from H2-8 to C-2, C3, C-4, and C-7. The planar structure of 1 resembles that of a previously reported metabolite, vibralactone, from the basidiomycete Boreostereum vibrans.3 The difference was hydroxylation of C12 in ostalactone A (1). This allowed us to determine the absolute configurations of two chiral carbons, C-3 and C-7, simply by comparing the optical rotation of 1 with that of vibralactone with the aid of ROE analysis. First, an ROE correlation observed between H-7 and H2-8 indicated the relative configuration between H-7 and C-8 to be cis. In measurement of an optical rotation, compound 1 showed a negative [α]D value (−93.0) that is similar to that of vibralactone (−135.1). Therefore, the same absolute configurations 2R,5S were assigned, completing the structure determination of 1. Compound 2 was isolated as a colorless oil. The HRFABMS spectrum of 2 displayed a pseudomolecular ion peak at 253.1442 [M + H]+, suggesting the molecular formula of 2 to be C14H20O4. The 1H and 13C NMR spectra of 2 were similar to those of 1 with few differences. The major differences were the appearance of new signals corresponding to two methoxy protons, H3-14 (δH 3.29) and H3-15 (δH 3.31), and methoxy carbons C-14 (δC 53.0) and C-15 (δC 53.2). In addition, the chemical shifts of C-13 and H2-13 were downfield shifted to δH 4.87 and δC 100.5, respectively. The chemical shift of C-13 and two methoxy groups both correlating to C-13 in the HMBC spectrum indicated that C-13 is functionalized with two methoxy groups in 2. Another difference was found in the hydroxylated dimethylallyl moiety in which signals of H2-12 and C-12 were upfield shifted to δH 1.71 and δC 18.2, respectively, suggesting that C-12 is dehydroxylated in 2. Therefore, the structure of 2 was determined to be the dehydroxylated, dimethoxylated analogue of 1. Compound 2 showed a nearly identical [α]D value (−109) to that (−93.0) of 1; thus the same absolute configurations 2R,5S were assigned. Compound 3 was isolated as a white, amorphous powder. The molecular formula of 3 was suggested as C12H16O4 due to the pseudomolecular ion peak observed at 225.1126 [M + H]+ in the HRFABMS spectrum. The 1H and 13C NMR spectra of 3 were quite distinct from those of 1 and 2, suggesting that compound 3 possesses a different core structure. The 1H NMR spectrum of 3 displayed signals of four olefinic (δH 6.68, 5.97, 5.51, and 5.48), two oxymethylene (δH 4.13 and 3.93), one diastereotopic methylene (δH 2.74 and 2.62), one methine (δH 2.64), and one methyl (δH 1.72) proton. In the 13C NMR spectra, 12 signals were observed including signals of one carbonyl (δC 168.3), six olefinic (δC 140.5, 139.0, 137.5, 122.9, 121.4, and 112.6), two oxymethylene (δC 67.4 and 62.9), one methylene (δC 27.4), one methine (δC 45.2), and one methyl (δC 12.8) carbon. The structure of 3 was determined by combined analysis of the COSY, HMBC, and ROESY spectra in the same manner as described for 1. Sequential COSY correlations between H2-8 (δH 2.62 and 2.74) and H-9 (δH 5.48) together with HMBC correlations from H3-11 (δH 1.72) to C-9 (δC 121.4) and C-12 (δC 67.4) and from H2-12 (δH 3.93) to C-9 and C-11 (δC 12.8) determined the structure of a dimethylally moiety with a hydroxy group substituted at C-12 (substructure B in Figure 1). An ROE correlation observed between H-9 and H2-12 assigned an E configuration to the double bond between C-9 and C-10. The hydroxylated dimethylally substructure was further extended by connecting an oxepinone ring to C-8 using COSY correlations between H2-

Figure 1. 2D NMR correlations used for the structure determination of ostalactone A (1).

8/H-3 (δH 2.64)/H-4 (δH 5.51) and between H-6 (δH 5.97) and H-7 (δH 6.68) together with HMBC correlations from H-6 to C-4 (δC 122.9), from H-7 to C-2 (δC 45.2) and C-5 (δC 139.0), and from H2-8 to C-2 and C-4. HMBC correlations from H2-13 (δH 4.13) to C-4, C-5, and C-6 (δC 112.6) linked a methyl alcohol moiety to C-5, completing the structure determination of 3. Previous reports proposed that a fused βlactone containing a 4/5 bicyclic core is derived from an oxepinone structure.4,5 Referring to this, compound 3 is likely to be a biosynthetic precursor of 1 and 2 (Figure 3).

Figure 2. 2D NMR correlations used for the structure determination of ostalactone C (3).

Figure 3. Proposed biosynthetic origin of the fused β-lactone structure of ostalactone A (1). B

DOI: 10.1021/acs.jnatprod.6b00647 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Spectroscopic Data (600 MHz) for Ostalactones A (1), B (2), and C (3) ostalactone A (1) position

δ Ca

2 3 4 5 6

175.1 76.2 123.1 149.2 38.4

7 8

80.4 28.3

9 10 11 12 13 14 15

119.3 140.3 14.1 68.4 61.5

δHa (J in Hz)

5.60 s 2.81 2.68 4.91 2.67 2.51 5.45

dd (20.0, 5.0) d (20.0) d (5.5) dd (15.0, 7.6) dd (15.0, 7.6) t (7.6)

1.68 s 3.94 s 4.15 s

ostalactone B (2) δ Ca

δHa (J in Hz)

172.7 75.5 126.8 143.6 36.7

5.78 s

78.5 27.8 117.4 136.3 26.0 18.2 100.5 53.0 53.2

4.79 2.62 2.45 5.11

d (4.8) dd (15.2, 7.2) dd (15.2, 7.2) t (7.6)

1.63 1.71 4.87 3.29 3.31

s s s s s

ostalactone C (3) δ Ca 168.3 45.2 122.9 139.0 112.6 140.5 27.4 121.4 137.5 12.8 67.4 62.9

δHa (J in Hz) 2.64 m 5.51 d (3.2) 5.97 d (6.8) 6.68 2.74 2.62 5.48

d (6.8) m m t (7.6)

1.72 s 3.93 s 4.13 s

a1 H and 13C NMR were measured at 600 and 100 MHz, respectively, in MeOH-d4 for 1 and 3 and in chloroform-d for 2, and solvent signals were used as reference.

Ostalactone A (1): colorless oil; [α]25D −93.0 (c 0.12, MeOH); IR (KBr) 3413, 2924, 2860, 1811, 1635, 1627, 1571 cm−1; 1H and 13C NMR data (Table 1); ESIMS (negative ion mode) m/z 223 [M − H]−; HRFABMS m/z 225.1130 [M + H]+ (calcd for C12H17O4, 225.1127). Ostalactone B (2): colorless oil; [α]25D −109.3 (c 0.09, MeOH); IR (KBr) 3434, 2927, 2861, 1821, 1649 cm−1; 1H and 13C NMR data (Table 1); ESIMS (negative ion mode) m/z 251 [M − H]−; HRFABMS m/z 253.1442 [M + H]+ (calcd. for C14H21O4, 253.1440). Ostalactone C (3): white, amorphous powder; [α]25D +18.5 (c 0.34, MeOH); IR (KBr) 3417, 2924, 2867, 1751, 1649, 1609 cm−1; UV (MeOH) λmax (log ε) 205.5 (4.01), 222.5 (3.38), 272.0 (3.38) nm; 1H and 13C NMR data (Table 1); ESIMS (negative ion mode) m/z 223 [M − H]−; HRFABMS m/z 225.1126 [M + H]+ (calcd. for C12H17O4, 225.1127). Lipase Inhibitory Activity Assay. The lipase inhibitory activity of compounds 1−3 was measured using a previously published protocol.3 Twenty five microliters of each compound in DMSO and 50 μL of a 0.1 mM 4-methylumbelliferyl oleate solution in Tris-HCl buffer (13 mM Tris-HCl, 150 mM NaCl, and 1.3 mM CaCl2; pH 8.0) were mixed in a 96-well microtiter plate. To this mixture was added 25 μL of the lipase solution (50 U/mL) in the same buffer to start the reaction. After incubation at 25 °C for 30 min, the reaction was halted by adding 100 μL of 0.1 M sodium citrate solution (pH 4.0). The amount of 4-methylumbelliferone generated by the reaction was monitored using a fluorometrical microplate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. IC50 values of compounds 1−3 were calculated using the least-squares regression line of the plots of the logarithm of the sample concentration (log) versus the pancreatic lipase activity (%). The known lipase inhibitor orlistat was used as a positive control (IC50 = 0.5 μM).

β-Lactone-containing natural products have been known to inhibit lipase by blocking hydrolysis of triglycerides via covalent modification of the serine residues in the active site.6,7 Therefore, compounds 1 and 2 were tested for their inhibitory activity against a human pancreatic lipase using the method previously described.3 As expected, compounds 1 and 2 strongly inhibited the lipase activity with IC50 values of 9.0 and 3.2 μM, respectively, whereas no inhibitory activity was observed for compound 3.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter. UV and IR spectra were recorded on a Pharmacia Biotech Ultrospec 3000 UV/visible spectrometer and a Shimazu 8400S FT-IR spectrometer, respectively. 1D and 2D NMR spectra including 1H NMR, COSY, TOCSY, HMQC, HMBC, and ROESY spectra were obtained on a Bruker Avance DRX 600 MHz NMR. 1H and 13C NMR chemical shifts were referenced to the MeOH-d4 solvent signals (δH 3.31 and δC 49.15, respectively). The HMBC spectrum was recorded with an average 3JCH of 8 Hz, and the HMQC spectrum was measured with an average 1JCH of 145 Hz. Fermentation and Isolation. The S. ostrea strain (IUM00326) was acquired from the Korean culture collection of mushrooms at Incheon University and maintained on potato dextrose agar. For a metabolite production, a seed culture was prepared by culturing in a 500 mL volume flask containing 100 mL of YPS medium (20 g of glucose, 2 g of yeast extract, 5 g of bactopeptone, 0.5 g of MgSO4, and 1 g of KH2-PO4 per liter of dH2O) for 1 week at 26 °C. For scale-up fermentation, 10 mL of the seed culture was inoculated into 4 L fermenters each containing 3 L of YPS medium. Fermentation was carried out at 28 °C with aeration and agitation. After 4 days of fermentation, a total of 12 L of fermentation product was harvested, and the broth was separated from the mycelium by filtration. The fermentation broth was extracted three times with EtOAc. The EtOAc extract was concentrated in vacuo and fractionated using a silica column and stepwise gradient of chloroform/methanol from 50:1 to 3:1. Fractions containing compounds 1−3 were further separated using a Sephadex LH-20 size exclusion column and methanol as an elution solvent. Final purification of LH-20 fractions by reversed-phase HPLC using an ODS C18 column and aqueous acetonitrile yielded pure compounds 1 (4.5 mg), 2 (2.8 mg), and 3 (12.0 mg).

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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.6b00647. NMR spectroscopic data of 1, 2, and 3 (PDF) C

DOI: 10.1021/acs.jnatprod.6b00647 J. Nat. Prod. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Author

*E-mail (J.-P. Kim): [email protected]. ORCID

Hahk-Soo Kang: 0000-0001-9602-6042 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the KRIBB Research Initiative Program (KGM1221622). REFERENCES

(1) Ding, J. H.; Feng, T.; Li, Z. H.; Yang, X. Y.; Guo, H.; Yin, X.; Wang, G. Q.; Liu, J. K. Org. Lett. 2012, 14, 4976−8. (2) Yun, B.-S.; Lee, I.-K.; Cho, Y.; Cho, S.-M.; Yoo, I.-D. J. Nat. Prod. 2002, 65, 786−788. (3) Liu, D.-Z.; Wang, F.; Liao, T.-G.; Tang, J.-G.; Steglich, W.; Zhu, H.-J.; Liu, J.-K. Org. Lett. 2006, 8, 5749−5752. (4) Schwenk, D.; Brandt, P.; Blanchette, R. A.; Nett, M.; Hoffmeister, D. J. Nat. Prod. 2016, 79, 1407−1414. (5) Zhao, P.-J.; Yang, Y.-L.; Du, L.; Liu, J.-K.; Zeng, Y. Angew. Chem., Int. Ed. 2013, 52, 2298−2302. (6) Borgstrom, B. Biochim. Biophys. Acta, Lipids Lipid Metab. 1988, 962, 308−16. (7) Hadváry, P.; Lengsfeld, H.; Wolfer, H. Biochem. J. 1988, 256, 357−361.

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DOI: 10.1021/acs.jnatprod.6b00647 J. Nat. Prod. XXXX, XXX, XXX−XXX