(±)-Tylopilusins, Diphenolic Metabolites from the Fruiting Bodies of

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(±)-Tylopilusins, Diphenolic Metabolites from the Fruiting Bodies of Tylopilus eximius Takashi Fukuda, Kenichiro Nagai, and Hiroshi Tomoda* Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan S Supporting Information *

ABSTRACT: Racemates of the diphenolic metabolites (±)-tylopilusin A (1) and (±)-tylopilusin B (2) were isolated from the fruiting bodies of Tylopilus eximius. Their structures were elucidated on the basis of spectroscopic analyses (1D and 2D NMR data and ROESY correlations) and X-ray crystallography. Each racemate was separated into its individual enantiomers, and electronic circular dichroism calculations were used to assign the absolute configuration of (+)- and (−)-tylopilusin A (1) and (+)- and (−)-tylopilusin B (2).

I

(±)-Tylopilusin A (1) was isolated by repeated chromatographic separation from the EtOAc fraction. The molecular formula for 1 was established as C20H18O8 ([M + Na]+ m/z 409.0892, calcd [M + Na]+ 409.0899) on the basis of highresolution ESIMS measurements, indicating that 1 contained 12 degrees of unsaturation. The high degrees of unsaturation and intense UV absorption at 339 nm suggested the presence of an extended conjugated system in this molecule. The 1H and 13 C NMR data (in DMSO-d6) confirmed the molecular formula (Table 1). The 13C NMR spectrum showed 20 resolved signals, which were classified as two oxygenated methyl carbons, eight sp2 methine carbons, and 10 quaternary carbons including two carbonyl carbons (C-9 and C-12), two oxygenated sp3 carbons (C-10 and C-11), and three oxygenated sp2 carbons (C-4, C-8, and C-16) (Table 1). The 1H NMR spectrum of 1 (in DMSOd6) showed four types of 1,4-disubstituted aromatic methine signals, one ester methyl signal, and one O-methyl signal. The connectivity of all carbon atoms was established by interpretation of HMBC spectroscopic data (Table 1). Analysis of the 1H NMR data using decoupling methods suggested the presence of two 1,4-disubstituted benzene rings, C-1 to C-6 and C-13 to C-18 partial structures. Analysis of HMBC spectroscopic data gave further structural information virtually defining the structure of 1. HMBC cross-peaks from 4-OH to C-3, C-4, and C-5 and from 16-OH to C-15, C-16, and C-17 established two p-hydroxyphenyl rings. The cross-peaks from 8OH to C-7, C-8, and C-9, from 10-OCH3 to C-10, from 11-OH to C-7, C-10, C-11, and C-12, and from 12-OCH3 to C-12 established the central ring.14 Furthermore, correlations from H-2 and H-6 to C-7 and from H-14 and H-18 to C-10 showed that the benzene rings were conjugated to the central ring at C7 and C-10 positions (Figure 1).

n the course of our studies of the chemistry of microbial products, we have reported novel and biologically active compounds.1−4 Recently, we focused on yellow pigment production by methicillin-resistant Staphylococcus aureus (MRSA) as a target of new anti-infectious agents against MRSA. The yellow pigment, named staphyloxanthin, works as an antioxidant agent, modifying the effectiveness of the host's immune system.5−7 We screened natural extracts of mushrooms collected at Nagano in Japan for inhibitors of yellow pigment production by MRSA. One of the strains collected was found to produce a unique series of diphenolic metabolites possessing three-ring systems. These biologically active compounds, designated (+)- and (−)-tylopilusin A (1) and (+)- and (−)-tylopilusin B (2), belong to a class of aromatic pigments that were previously isolated from the mushrooms of the orders Boletales, Suillus, Theleohora, and more.8−12 The fruiting bodies of Tylopilus eximius were collected in Nagano in 2010. The strain was identified from morphological characteristics as T. eximius.13 The fruiting bodies (300 g) were extracted with methanol (500 mL) for two months. After filtration and concentration of the methanol extract, the resulting aqueous solution was extracted with ethyl acetate. The ethyl acetate layer was concentrated in vacuo to yield 1.1 g of solid material.

Received: June 18, 2012 Published: December 6, 2012 © 2012 American Chemical Society and American Society of Pharmacognosy

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OMe should be shifted to higher fields, at 3.05 and 2.99 ppm, than those in the usual cases. The difference between (+)-1 and (−)-1 was apparent via optical rotation mesurment and electronic circular dichroism (ECD) calculations. From these data, (−)-1 was determined as the enantiomer of (+)-1. To determine the absolute configuration of (+)-1 and (−)-1, the ECD spectra of (+)-1 and (−)-1 were measured in methanol at 25 °C, respectively. Interestingly, (+)-1 and (−)-1 exhibit mirror-like CD curves (Figure 3). The CD curve of

Table 1. NMR Spectroscopic Data for (±)-Tylopilusins A (1) and B (2) (400 MHz for 1H, 100 MHz for 13C (DMSOd6))a 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 10-OMe 12-OMe 4-OH 8-OH 10-OH 11-OH 16-OH

δC 123.3, 130.7, 115.1, 158.1, 115.1, 130.7, 135.7, 149.1, 194.9, 80.9, 84.2, 171.8, 123.4, 129.9, 114.3, 157.1, 114.3, 129.9, 52.4, 51.7,

C CH CH C CH CH C C C C C C C CH CH C CH CH CH3 CH3

2 δH mult (J in Hz) 7.82, d (9.0) 6.75, d (9.0) 6.75, d (9.0) 7.82, d (9.0)

7.12, d (9.0) 6.70, d (9.0) 6.70, d (9.0) 7.12, d (9.0) 3.05, s 2.99, s 9.85, s 10.37, s

δC 123.8, C 130.5, CH 115.1, CH 158.0, C 115.1, CH 130.5, CH 136.2, C 149.2, C 198.8, C 77.3, C 83.3, C 172.1, C 128.72, C 128.69, CH 113.8, CH 156.6, C 113.8, CH 128.69, CH 51.4, CH3

5.90, s 9.48, s

δH mult (J in Hz) 7.79, d (9.0) 6.75, d (9.0) 6.75, d (9.0) 7.79, d (9.0)

7.00, d (9.0) 6.63, d (9.0) 6.63, d (9.0) 7.00, d (9.0)

Figure 3. CD spectra of (+)-1 (red), (−)-1 (blue), and calculated (10R,11R)-1 (green).

2.93, s 9.84, s 10.38, s 6.51, brs 5.89, brs 9.36, s

(+)-1 exhibits a strong positive Cotton effect at 363 nm and a negative Cotton effect at 323 nm. On the other hand, (−)-1 exhibits a strong negative Cotton effect at 363 nm and a positive Cotton effect at 324 nm. However, due to the lack of proper model compounds for reference, the assignment of its absolute configuration was unreliable by direct analysis of the CD curve.15 Therefore, to solve their absolute configuration, we calculated ECD by using time-dependent density functional theory (TDDFT).16,17 Recently, this method was reported as a strong tool in determining the absolute configuration of several compounds.18−21 The conformational analysis was performed by means of the semiempirical PM3 method, as implemented in the program package Gaussian 03, starting from preoptimized geometries generated by the MM2 force field in Chem 3D software. The corresponding minimum geometries were further optimized by DFT calculations at the B3LYP/6-31G level. The calculated CD data of (10R,11R)-1 are shown in Figure 3. The calculated CD data of (10R,11R)-1 were comparable to those observed for (+)-1, but were opposite those of (−)-1. This result suggested that the absolute configuration of (+)-1 was 10R, 11R and (−)-1 was 10S, 11S. The structure of 2 was established by comparison of all spectroscopic data with those of 1. The molecular formula of 2 was established as C19H16O8 ([M + Na]+ m/z 395.0746, calcd [M + Na]+ 395.0742) on the basis of high-resolution ESIMS measurements, which was 14 mass units less than that for 1. The difference between 1 and 2 was clearly that the 10-OCH3 (δ 3.05) in 1 was missing in the 1H NMR spectrum of 2 (Table 1). This suggested that 2 was a demethylated derivative of 1 at the 10 position. The relative configurations of C-10 and C-11 were determined to be cis by ROESY experiments and X-ray crystallography of racemic 2 (Figure 4). Furthermore, the absolute configuration of (+)-2 and (−)-2 was also determined as 10R, 11R and 10S, 11S by comparing the optical rotation and

a

Assignments made by interpretation of COSY, HSQC, and HMBC NMR data.

Figure 1. Key HMBC correlations of (±)-tylopilusin A (1).

The planar structure satisfied the molecular formula and the degree of unsaturation. The relative configurations of C-10 and C-11 were deduced by ROESY experiments (Figure 2). The

Figure 2. Key ROESY correlations of (±)-1.

cross-peaks between 10-OCH3 and 11-OH and between 12OCH3 and 14-H and/or 18-H indicated that 10-OCH3 and the benzene ring were cis. On the basis of the available data, the total structure of tylopilusin A was established as 1. 10-OMe and 12-OMe in 1 are placed on/under the benzene ring (Figure 2). This suggested that the chemical shifts of 10-OMe and 122229

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separations were performed using a Senshu Pak preparative C18 Pegasil ODS column (250 × 20 mm) at a flow rate of 8 mL/min and a Chiralcel OJ-H column (150 × 4.6 mm) at a flow rate of 0.8 mL/min using a Shimadzu LS20AT pump and a Shimadzu LS20AS UV detector. Collection of the Fruiting Bodies of T. eximius. The fruiting bodies of T. eximius were collected in Nagano in 2010. The edible mushroom was dark purple, and the size of the pileus was 10 × 10 cm. The strain was assigned as T. eximius from its morphological characteristics.13 Isolation of Tylopilusins. The EtOAc extract was dissolved in a small volume of chloroform, applied on a silica gel column (22 g, 28 × 14 cm, 0.04−0.063 mm, Merck), and eluted stepwise with 100% chloroform, 20:1, 10:1, 5:1, 1:1 (v/v) of chloroform−methanol and 100% methanol (200 mL each). Compounds (±)-1 and (±)-2 were recovered in the 10:1 chloroform−methanol fraction. This fraction was further purified by reversed-phase C18 HPLC (20 × 250 mm, Pegasil ODS), using a 40 min linear gradient from 10% to 40% aquous acetonitrile in 0.05% TFA, at a flow rate of 8.0 mL/min with UV detection at 340 nm. Under these conditions, racemic (±)-1 and (±)-2 were eluted as peaks with retention times of 32.8 and 24.5 min, respectively. These peaks were collected and concentrated to yield 5.0 and 4.3 mg of pale yellow solid and pale yellow needles. Racemic (±)-1 (5.0 mg) was further purified by reversed-phase HPLC (4.6 × 150 mm, Chiralcel OJ-H), using isocratic conditions of 20% aqueous acetonitrile, at a flow rate of 0.8 mL/min with UV detection at 340 nm. Under these conditions, (+)-1 and (−)-1 were eluted as the peaks with retention times of 14.6 and 25.4 min. These peaks were collected and concentrated to yield (+)-1 (2.0 mg) and (−)-1 (2.0 mg). The sample of racemic (±)-2 was recrystallized to afford crystals, which were analyzed by X-ray crystallography. The residue from the mother liquor (3.0 mg) was further purified by reversed-phase HPLC (150 × 4.6 mm, Chiralcel OJ-H), using isocratic conditions of 13% aquous acetonitrile, at a flow rate 0.8 mL/min with UV detection at 340 nm. Under these conditions, (+)-2 and (−)-2 were eluted as peaks with retention times of 32.6 and 47.0 min. These peaks were collected and concentrated to yield (+)-2 (1.2 mg) and (−)-2 (1.2 mg). (+)-Tylopilusin A (1, 2.0 mg): pale yellow solid; [α]24.5D 221.6 (c 0.04 CH3OH); CD (MeOH) λex termum (Δε) 363 (+16.6), 323 (−9.4), 231 (+8.9); IR (KBr) νmax 3391, 1735, 1698, 1604, 1515 cm−1; UV (MeOH) λmax (log ε) 339 (3.79), 278 (3.38), 227 (3.90); 1H and 13C NMR, see Table 1; ESIMS [M +Na]+ m/z 409.0892 (C20H18O8Na, calcd 409.0899). (−)-Tylopilusin A (1, 2.0 mg): pale yellow solid; [α]24.5D −253.6 (c 0.04 CH3OH); CD (MeOH) λex termum (Δε) 363 (−17.3), 324 (+8.9), 230 (−8.6); 1H and 13C NMR, see Table 1. (+)-Tylopilusin B (2, 1.2 mg): colorless needle; mp 185−187 °C; [α]24.5D 145.6 (c 0.02 CH3OH); CD (MeOH) λex termum (Δε) 361 (+20.9), 310 (−8.0), 230 (+13.7); IR (KBr) νmax 3357, 1745, 1696, 1605, 1516 cm−1; UV (MeOH) λmax (log ε) 338 (4.20), 227 (4.11); 1 H and 13C NMR, see Table 1; ESIMS [M + Na]+ m/z 395.0746 (C19H16O8Na, calcd 395.0742). (−)-Tylopilusin B (2, 1.2 mg): colorless needle; [α]24.5D −146.4 (c 0.02 CH3OH); CD (MeOH) λex termum (Δε) 360 (−20.7), 320 (+6.6), 230 (−13.1); 1H and 13C NMR, see Table 1. Crystal Structure Determination of Racemic Tylopilusin B (2). A colorless needle crystal of racemic tylopilusin B (2) was obtained from chloroform. All measurements were made on a RigakuAXIS-Rapid II diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.54184 Å). The data were collected at −180 °C. The structure was solved by direct methods (SHELXS-97) and refined by full-matrix least-squares on F2. The crystal data were monoclinic, space group P21/n, a = 12.5351(3) Å, b = 6.7851(2) Å, β = 100.2492(8)o, c = 21.4299(4) Å, V = 1793.57(6) Å3, and Z = 4. Inhibitory Activity on the Yellow Pigment Production in MRSA. The paper disk method for measuring the effect of tylopilusins is described below. MRSA was cultured in Mueller-Hinton Broth (MHB) at 37 °C for 20 h and adjusted to 1 × 108 colony forming unit (cfu)/mL. The inoculum (100 μL) was spread on TYB agar (25 mL) on a plate (sterile no. 2, square scale, 100 × 140 mm; Eiken Chemical

Figure 4. ORTEP view of (±)-tylopilusin B (2).

the CD data with those of 1, respectively (see Supporting Information). These data suggested that 1 was O-methyl-2. During our purification precedure, methanol extraction was carried out. Therefore, 1 could be an artifact of 2. Tylopilusins share structural characteristics (diphenolics having three rings) with mushroom metabolites atromentin (5),8,22 grevillin (6),9,23 variegatic acid (7),23,24 and involutin (8).25,26 They are all produced by mushrooms and were isolated as pigments. In this study, tylopilusins were isolated as inhibitors of yellow pigment production by pathogenic MRSA. Briefly, the effect of tylopilusins was tested on yellow pigment production in MRSA by the paper disk method.27 Compounds (+)-1, (−)-1, (+)-2, and (−)-2 showed a white zone (8, 9, 7, and 7 mm, respectively) without any inhibition at 20 μg/6 mm paper disk, suggesting that tylopilusins selectively inhibited the production of the yellow pigments without affecting the growth of MRSA. Diphenolics having three rings were reported to show a variety of biological activity such as antibacterial,28 inhibition of cytochrome P450 activity,24 inducing apoptosis of cancer cells,29 and inhibition of enoyl-ACP reductase (FebK) of Streptococcus pneumoniae.30 It might be intriguing to determine whether tylopilusins show these biological activities.

Figure 5. Diphenolic compounds from mushrooms.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded with a DIP-1000 digital polarimeter (JASCO, Tokyo, Japan). Melting points were measured with a micro melting apparatus (Yanaco, Kyoto, Japan). ESIMS data were obtained on a JMST1000LP spectrometer (JEOL, Tokyo, Japan). UV and IR spectra were measured with a U-2800 spectrophotometer (Hitachi, Tokyo, Japan) and an FT/IR-460 plus spectrometer (JASCO, Tokyo, Japan), respectively. CD spectra were recorded with a J-720 spectropolarimeter (JASCO, Tokyo, Japan). The various NMR spectra were measured with an INOVA400 MHz spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). Reversed-phase HPLC 2230

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Co., Ltd., Tochigi, Japan). After incubation at 37 °C for 4 h, paper disks (6 mm i.d.; Toyo Roshi Kaisha Ltd., Tokyo, Japan) containing 20 μg of each sample were placed on the plate and incubated at 37 °C for 68 h. The white zone indicates that the production of yellow pigments is inhibited without affecting the growth of MRSA. The positive control zaragozic acid showed a 10 mm white zone by a 50 μg/6 mm disk.

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(15) Stonard, R. J.; Trainor, D. A.; Nakatani, M.; Nakanishi, K. J. Am. Chem. Soc. 1983, 105, 130−131. (16) Bringmann, G.; Muhlbacher, J.; Reichert, M.; Dreyer, M.; Kolz, J.; Speicher, A. J. Am. Chem. Soc. 2004, 126, 9283−9290. (17) Bracher, F.; Eisenreich, W. J.; Muhlbacher, J.; Dreyer, M.; Bringmann, G. J. Org. Chem. 2004, 69, 8602−8608. (18) Yuan, T.; Zhu, R. X.; Zhang, H.; Odeku, O. A.; Yang, S. P.; Liao, S. G.; Yue, J. M. Org. Lett. 2010, 15, 252−255. (19) Feling, R.; Polborn, K.; Steglich, W.; Mü hlbacher, J.; Bringmann., G. Tetrahedron 2001, 57, 7857−7863. (20) Zhang, F.; Wang, J. S.; Gu, Y. C.; Kong, L. Y. J. Nat. Prod. 2012, 75, 538−546. (21) Diedrich, C.; Grimme, S. J. Phys. Chem. A 2003, 107, 2524− 2539. (22) Sullivan, G.; Guess, W. L. Lloydia 1969, 32, 72−75. (23) Negishi, O.; Negishi, Y.; Aoyagi, Y.; Sugahara, T.; Ozawa, T. J. Agric. Food Chem. 2001, 49, 5509−5514. (24) Huang, Y. T.; Onose, J.; Abe, N.; Yoshikawa, K. Biosci. Biotechnol. Biochem. 2009, 73, 855−860. (25) Edwards, R. L.; Elsworthy, G. C.; Kale, N. J. Chem. Soc. C 1967, 405−409. (26) Feling, R.; Polborn, K.; Steglich, W.; Mühlbacher, J.; Bringmann, G. Tetrahedron 2001, 57, 7857−7863. (27) Sakai, K.; Koyama, N.; Fukuda, T.; Morii, Y.; Onaka, H.; Tomoda, H. Biol. Pharm. Bull. 2012, 35, 48−53. (28) Brewer, D.; Jen, W. C.; Jones, G. A.; Taylor, A. Can. J. Microbiol. 1984, 30, 1068−1072. (29) Kim, J. H.; Lee, C. H. J. Microbiol. Biotechnol. 2009, 19, 946− 950. (30) Zheng, C. J.; Sohn, M. J.; Kim, W. G. J. Antibiot. (Tokyo) 2006, 59, 808−812.

ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures: copies of 1D and 2D NMR spectra, IR, CD, and X-ray crystallographic data of 1 and 2. This material and a cif data file are available free of charge via the Internet at http://pubs.acs.org. Crystallographic data have been deposited in the Cambridge Crystallographic Data Center with the deposition number CCDC 887770. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (e-mail: [email protected]).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-3-57916241. Fax: +81-3-3444-6197. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to thank Prof. H. Shibata, Shinsyu University, for collecting mushroom samples. We wish to thank Ms. N. Sato and Dr. T. Shirahata (School of Pharmacy, Kitasato University) for measurements of NMR spectra and calculated CD data. This work was supported by Takeda Science Foundation and a Kitasato University Research Grant for Young Researchers (T.F.)

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