Hitoyol A and B, Two Norsesquiterpenoids from the Basidiomycete

Letter pubs.acs.org/OrgLett

Hitoyol A and B, Two Norsesquiterpenoids from the Basidiomycete Coprinopsis cinerea Junnosuke Otaka,† Daisuke Hashizume,‡ Yui Masumoto,§,⊥ Atsuya Muranaka,§ Masanobu Uchiyama,§,⊥ Hiroyuki Koshino,∥ Yushi Futamura,† and Hiroyuki Osada*,† †

Chemical Biology Research Group and ∥Molecular Structure Characterization Unit, RIKEN Center for Sustainable Resource Science (CSRS), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan § Elements Chemistry Laboratory, RIKEN, and Advanced Elements Chemistry Research Team, RIKEN CSRS, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan ‡ Materials Characterization Support Unit, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan ⊥ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan S Supporting Information *

ABSTRACT: Hitoyol A (1), an unprecedented norsesquiterpenoid with an exo-tricyclo[5.2.1.02,6]decane skeleton, was isolated from the culture broth of Basidiomycete Coprinopsis cinerea along with a novel skeletal hitoyol B (2) containing 4-cyclopentene-1,3dione. Their structures and absolute configurations were analyzed by single-crystal X-ray diffraction and electronic circular dichroism spectroscopic methods. Compound 1 is possibly biosynthesized through decarboxylation-induced cyclization of lagopodin B, a known cuparene-type sesquiterpenoid. Compound 2 showed weak antimalarial activity with an IC50 of 59 μM.

S

mall molecules are useful tools for exploring biological function in medicinal chemistry and chemical biology.1b,c Therefore, structurally diverse secondary metabolites (e.g., terpenoids, alkaloids, and polyketides) are suitable sources to expand the chemical space in drug discovery.1a,b Coprinopsis cinerea is an important model basidiomycete used to study fruiting body development, mating, and evolution.2 Whole-genome sequencing was completed in 2010.3 A characteristic ecological phenomenon of the Coprinopsis genus (Hitoyotake, in Japanese) is the formation of an inky fluid cap containing black spores immediately after its fruiting body has matured.4 Earlier studies of Coprinus species (synonym of Coprinopsis sp.) led to the isolation of cuparene-type sesquiterpenoid quinones, lagopodin A, B, and their derivatives.6f−h Although six sesquiterpene synthases and two terpene oxidizing cytochrome P450 monooxygenases, involved in the biosynthesis of lagopodin A, were identified from C. cinerea,5 few chemical investigations of Coprinopsis sp. have been carried out.6 In research for secondary metabolites of C. cinerea, we isolated two novel skeletal norsesquiterpenoids, hitoyol A (1) possessing an exo-tricyclo[5.2.1.02,6]decane core and hitoyol B (2), with a 4-cyclopentene-1,3-dione, together with lagopodin B as a main sesquiterpenoid (Figure 1).6g The absolute configurations of 1 and 2 were analyzed by single-crystal X-ray crystallography and electronic circular dichroism (ECD) spectroscopy, respectively. Herein, we describe the isolation, © 2017 American Chemical Society

Figure 1. Structures of hitoyol A (1) and B (2).

structural determination, plausible biosynthetic route, and bioactivities of 1 and 2. The ethyl acetate extract (1.0 g) from culture broth (2.7 L) of C. cinerea (NBRC 100011) was separated and purified by silica gel chromatography and reversed-phase HPLC to give 1 (9.0 mg), 2 (3.3 mg), and lagopodin B (128 mg) (see the Supporting Information for details). The molecular formula of hitoyol A (1), which was obtained as colorless crystals with a melting point of 178−180 °C, was determined to be C14H20O4 by HR-ESI-MS at m/z 253.1448 [M + H]+ (calcd 253.1440 for C14H21O4), requiring five degrees of unsaturation. The IR absorption peaks at 3421 and 3326 cm−1 showed the presence of hydroxyl groups. The UV absorption band at 225 nm (log ε = 3.90) and IR absorption peaks at 1685 (CO) and 1617 cm−1 (CC) were attributed to an α,β-unsaturated ketone in 1, which was confirmed by 13C and DEPT-135 spectra [δ 206.0 (C), 179.4 (C), and 131.6 Received: June 13, 2017 Published: July 20, 2017 4030

DOI: 10.1021/acs.orglett.7b01784 Org. Lett. 2017, 19, 4030−4033

Letter

Organic Letters Table 1. 1H and 13C NMR Data of Compounds 1 and 2a 1

a

no.

δC

1 2 3 4 5 6 7a 7b 8 9a 9b 10 11 12 13 14 4-OH 5-OH 8-OH

206.0 131.6 179.4 83.9 82.8 48.9 42.8 83.2 45.7 41.9 26.2 31.7 9.7 16.5

2

δH, mult (J in Hz) 6.01, q (1.0)

1.65, d (10.5) 1.03, dd (10.5, 3.0) 2.44, dd (11.5, 3.0) 1.30, d (11.5) 1.19, 0.98, 1.05, 2.24, 3.80, 4.45, 4.17,

one portion (C-1−C-5, C-14) was deduced by the HMBC correlations from H-2/C-1, C-3, C-4, C-5, and C-14; H3-14/C3 and C-4; and 5-OH/C-1 and C-4. In addition, the strongest fragment ion peak was observed at m/z 125 [M − C6H7O3]+ in EI-MS of 1 (obsd m/z 125.0959 in HR-EI-MS/calcd 125.0966 for C8H13O), indicating the presence of dual α-cleavages at C4−C8 and C5−C6 (Figure S24). Consequently, the planar structure of 1 with the tricyclo[5.2.1.02,6]decane skeleton was identified as shown in Figure 1. To elucidate the relative configuration of 1, 1D-NOE difference spectra were measured (Figure 2). Irradiation of H3-12 enhanced the signals at H-7a, H-9b, and H3-13, indicating H-9b/H3-12 for exo-orientation and H-7a for the syn-position of H3-12, respectively; however, we were not able to prove definitively the stereochemistry of 1, i.e., endo(4R*,5S*,6S*,8R*)-1 or exo-(4S*,5R*,6S*,8R*)-1, from NOE and NOESY data (Figures S6−S8). Thus, to resolve the stereochemical problem, including the absolute configuration of 1, X-ray crystal structure analysis was performed using a single-crystal crystallized from CDCl3/ acetone. The absolute stereochemistry of 1 was unambiguously demonstrated, affording exo-(4S,5R,6S,8R)-1 = (1S,2R,6S,7R)1,2,6-trihydroxy-3,7,8,8-tetramethyltricyclo[5.2.1.02,6]dec-3-en5-one with a Flack χ parameter of −0.02(6), as illustrated in Figure 3. The absolute configuration of 1 was also corroborated by UV and ECD spectra, coupled with theoretical calculations (Figure S34).

s s s d (1.0) brs brs brs

δC 203.8 146.4 161.2 205.1 78.6 49.1 47.0 215.6 54.7 43.1 27.7 24.0 22.1 11.2

δH, mult (J in Hz) 7.28, q (1.5)

3.31, d (19.0) 1.99, dd (19.0, 1.0) 2.33, d (19.0) 1.90, dd (19.0, 1.0) 1.37, 1.04, 0.99, 2.10,

s s s d (1.5)

5.05, brs

Measured at 500 MHz for 1H and 125 MHz for 13C in acetone-d6.

(CH)] (Table 1). In addition to three signals for an enone, the 1 H NMR, 13C NMR, DEPT-135, and HSQC spectra of 1 revealed 11 carbon signals that represent three tertiary methyls, one olefinic methyl, two methylenes, and five non-hydrogen attached sp3 carbons, including three oxygenated tertiary carbons, together with three exchangeable OH proton signals (Table 1). The 5/5/5 fused tricyclic scaffold, fulfilling three degrees of unsaturation, except for the conjugated system from C-1 to C-3, was established by detailed interpretation of 2DNMR data (Figure 2). The HMBC cross peaks were as follows: H2-7/C-4, C-5, C-6, C-8, and C-13; H2-9/C-4, C-8, and C-10; H3-11/C-6, C-9, C-10, and C-12; H3-13/C-5, C-6, and C-10; and 8-OH/C-7. This analysis yielded the 4-hydroxy-1,2,2trimethylbicyclo[2.2.1]heptane partial structure (C-4−C-13). Location of another 4,5-dihydroxy-3-methyl-2-cyclopenten-1-

Figure 3. ORTEP drawing of 1 at 50% probability levels.

Focusing on structural features of 1, the vestige of sesquiterpene was recognized within 1. On the other hand, because the third isoprene unit (C-2−C-4, C-14) is incomplete, and two juncture carbons at C-4 and C-5 are oxygenated, it is conceivable that the biosynthetic process of 1 may undergo unusual cyclization. Hitoyol B (2) was isolated as yellow oil with the molecular formula of C14H18O4 [HR-ESI-MS at m/z 251.1286 [M + H]+ (calcd 251.1283 for C 14 H 19 O 4 )] with six degrees of unsaturation, implying that 2 was a structural analogue of 1. In the IR spectrum of 2, a ketone CO stretching peak at 1739 cm−1 was observed, and a strong peak at 1697 cm−1 was assigned to an enone CO stretching peak. The presence of a hydroxyl group (3425 cm−1, δH 5.05) was suggested based on the IR and 1H NMR data. The combined 1H-, 13C-, DEPT-135, and HSQC spectroscopic analysis gave all 14 carbon signals: three tertiary methyls, one olefinic methyl, two methylene, one olefinic methine, two quaternary carbons, three carbonyl carbons, one olefinic carbon, and one oxygenated tertiary carbon. These carbon signals were assigned (Table 1). The planar structure of 2 was elucidated by HMBC spectra (Figure 2). The HMBC cross peaks from H-2/C-1, C-3, C-5; H3-14/C-

Figure 2. Selected key HMBC, NOE, and NOESY correlations of 1 and 2. 4031


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Organic Letters

a negative Cotton effect corresponding to the conjugated ketone n−π* transition at ∼380 nm and a positive Cotton effect for the nonconjugated ketone n−π* transition at 296 nm. We then calculated theoretical UV and ECD spectra for optimized structures of (5S,6S)-2 and (5S,6R)-2 at the B3LYP/ 6-31G** level. For each epimer, three conformers were obtained by geometry optimization, and the theoretical UV and ECD spectra were generated based on the Boltzmann factors at 297 K (see Figure S36 and Table S2). Since the overall calculated spectral patterns for (5S,6S)-2 reproduced the experimental patterns well, the absolute configuration for 2 was determined as 5S,6S. To the best of our knowledge, compound 2 is the first example of norsesquiterpenoid with a 4-cyclopentene-1,3-dione system. In nature, a unique tricyclic[5.2.1.02,6]decane carbon skeleton is biosynthesized. Hence, we propose a plausible biosynthetic pathway for 1 and related 2, as shown in Scheme 1. Both compounds 1 and 2 may share the same key precursor named “pre-hitoyol”, which is derived from lagopodin B.6f,g Indeed, as with prehitoyol, several secondary metabolites with a 4carboxy(or ester)-4-hydroxycyclopentenone instead of a quinone have been reported.8a−c Two possible mechanisms are envisaged for the biosynthesis of prehitoyol: (1) benzilic acid rearrangement of an o-quinone from lagopodin B in the hemiketal type and (2) initial epoxidation of lagopodin B in the hydroxy quinone type, followed by benzilic acid rearrangement.8d Following decarboxylation, prehitoyol may undergo a reaction that constructs a new linkage between C−4 and C−8 to yield 1. Meanwhile, the 4-cyclopentene-1,3-dione moiety in 2 was assumed to be formed via a decarboxylation from prehitoyol or oxidative cleavage of 1. The inhibitory activities of 1 and 2 against cancer cells (HeLa, HL-60, and srcts-NRK), the malarial parasite (Plasmodium falciparum), bacteria (Staphylococcus aureus and Escherichia coli), and fungi (Aspergillus f umigatus, Candida albicans, and Pyricularia oryzae) were investigated. As a result, compound 1 showed no activities (>100 μM, for each), but 2 displayed weak antimalarial activity against P. falciparum with an IC50 value of 59 μM (see the Supporting Information for details). Finally, we comment on the significance of the discovery of hitoyol A (1). Multifarious useful properties of synthetic compounds with the tricyclo[5.2.1.02,6]decane framework have been reported: D609 as the phosphatidylcholine-specific phospholipase C and sphingomyelin synthase inhibitor;9 JP10 as jet fuel;10 and tricyclodecane-4,8-dimethanol for chemical

2, C-3, and C-4 and 5-OH/C-1 and C-4 were observed, suggesting the presence of the 2-hydroxy-4-methyl-4-cyclopentene-1,3-dione part (C-1−C-5) (Figure 2). The conjugated ketone system (OC−CC−CO) was also suggested by the weak and broadened UV absorption band of the n−π* transition at ∼397 nm (log ε = 1.75) and the relatively strong band of the π−π* transition at 231 nm (log ε = 3.82) (Figure 4).7 In addition, assignments of key HMBC correlations from

Figure 4. Experimental and Boltzmann-averaged calculated ECD (top) and UV (bottom) spectra of 2. The experimental spectra were measured in CH3OH at room temperature. Gaussian bands with a half-bandwidth of 2500 cm−1 were used to produce the calculated spectra of (5S,6S)-2 and (5S,6R)-2. Inset shows chemical structures of two epimers.

H3-11/C-6, C-9, C-10, and C-12; H3-13/C-6, C-7, and C-10; H2-7/C-6 and C-8; and H2-9/C-8 and C-10 afforded the elucidation of the 3,3,4-trimethylcyclopentanone moiety (C-6− C-13) in 2. The HMBC correlations from H2-7/C-5, H3-13/C5 and C-6, and 5-OH/C-6 showed the connectivity between C5 and C-6, which suggested 2 adopts the planar structure depicted in Figure 1. The relative configuration in 2 was assigned by a combination of 1D-NOE and 2D-NOESY experiments (Figure 2). The NOE detection of H3-11/H-7a and H-9b; H3-13/H-7b, H-9a, and H-12 suggested that a set of protons (H-7a, H-9b, and H3-11) and (H-7b, H-9a, H3-12, and H3-13) were located on the same side, respectively. Both configurations of C-5 and C-6 were deduced as 5S*,6S* based on NOE from H-13/H-2 as well as NOESY correlation of 5-OH/H-11. To establish the absolute configuration of 2, the ECD spectrum of 2 was measured. As shown in Figure 4, 2 exhibited

Scheme 1. Plausible Biosynthetic Pathway for Hitoyol A (1) and Hitoyol B (2)

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Organic Letters materials.11 Given its applicability, hitoyol A (1) shows promise as a sustainable intermediate toward bioactive molecules.

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K.; Herald, D. L.; Cichacz, Z. A.; Doubek, D. L.; Richert, L. J. Nat. Prod. 2010, 73, 388−392. (d) Gonzalez Del Val, A.; Platas, G.; Arenal, F.; Orihuela, J. C.; Garcia, M.; Hernández, P.; Royo, I.; De Pedro, N.; Silver, L. L.; Young, K.; Vicente, M. F.; Pelaez, F. Mycol. Res. 2003, 107, 1201−1209. (e) Johansson, M.; Sterner, O.; Labischinski, H.; Anke, T. Z. Naturforsch., C: J. Biosci. 2001, 56, 31−34. (f) Bu’Lock, J. D.; Darbyshire, J. Phytochemistry 1976, 15, 2004−2004. (g) Bottom, C. B.; Siehr, D. J. Phytochemistry 1975, 14, 1433−1433. (h) Thomson, R. H. Naturally Occurring Quinones; Academic Press: London, 1971; pp 131−135. (7) Agosta, W. C.; Smith, A. B. J. Org. Chem. 1970, 35, 3856−3860. (8) (a) Fukuda, T.; Nagai, K.; Tomoda, H. J. Nat. Prod. 2012, 75, 2228−2231. (b) Zhao, A. H.; Li, S. H.; Zhao, Q. S.; Lin, Z. W.; Sun, H. D.; Lu, Y.; Zhang, L. L.; Zheng, Q. T. Helv. Chim. Acta 2003, 86, 3470−3475. (c) Yun, B. S.; Kang, H. C.; Koshino, H.; Yu, S. H.; Yoo, I. D. J. Nat. Prod. 2002, 64, 1230−1231. (d) Cunningham, I. D.; Danks, T. N.; O’Connell, K. T. A.; Scott, P. W. J. Org. Chem. 1999, 64, 7330−7337. (9) (a) Kato, M.; Hammam, M. A.; Taniguchi, T.; Suga, Y.; Monde, K. Org. Lett. 2016, 18, 768−771. (b) Li, Y.; Maher, P.; Schubert, D. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 7748−7753. (c) Wiegmann, K.; Schütze, S.; Machleidt, T.; Witte, D.; Krönke, M. Cell 1994, 78, 1005− 1015. (10) Hudzik, J. M.; Castillo, A.; Bozzelli, J. W. J. Phys. Chem. A 2015, 119, 9857−9878. (11) (a) Oesterreicher, A.; Wiener, J.; Roth, M.; Moser, A.; Gmeiner, R.; Edler, M.; Pinter, G.; Griesser, T. Polym. Chem. 2016, 7, 5169− 5180. (b) Suzuki, Y.; Higashihara, T.; Ando, S.; Ueda, M. Macromolecules 2012, 45, 3402−3408.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01784. X-ray data for compound 1 (CIF) Experimental procedures; full (HR)-MS, IR, UV, ECD, NMR spectra; computational details (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Junnosuke Otaka: 0000-0002-7947-1846 Hiroyuki Osada: 0000-0001-7400-8388 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. T. Nakamura from the RIKEN Center for Sustainable Resource Science for measurement of mass spectra. We also thank Ms. H. Aono, Ms. M. Tanaka, and Mr. K. Yamamoto at the RIKEN Center for Sustainable Resource Science for performing bioassays. This work was supported by the RIKEN SPDR Program (to J.O.). We are grateful to HOKUSAI−GreatWave for the computer resources used for the DFT calculations.

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REFERENCES

(1) (a) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629−661. (b) Harvey, A. L.; Edrada-Ebel, R.; Quinn, R. J. Nat. Rev. Drug Discovery 2015, 14, 111−129. (c) Leeson, P. D.; Springthorpe, B. U. Nat. Rev. Drug Discovery 2007, 6, 881−890. (2) (a) Muraguchi, H.; Umezawa, K.; Niikura, M.; Yoshida, M.; Kozaki, T.; Ishii, K.; Sakai, K.; Shimizu, M.; Nakahori, K.; Sakamoto, Y.; Choi, C.; Ngan, C. Y.; Lindquist, E.; Lipzen, A.; Tritt, A.; Haridas, S.; Barry, K.; Grigoriev, I. V.; Pukkila, P. J. PLoS One 2015, 10, e0141586. (b) Liu, Y.; Srivilai, P.; Loos, S.; Aebi, M.; Kües, U. Genetics 2006, 172, 873−884. (3) Stajich, J. E.; Wilke, S. K.; Ahrén, D.; Au, C. H.; Birren, B. W.; Borodovsky, M.; Burns, C.; Canbäck, B.; Casselton, L. A.; Cheng, C. K.; Deng, J.; Dietrich, F. S.; Fargo, D. C.; Farman, M. L.; Gathman, A. C.; Goldberg, J.; Guigó, R.; Hoegger, P. J.; Hooker, J. B.; Huggins, A.; James, T. Y.; Kamada, T.; Kilaru, S.; Kodira, C.; Kües, U.; Kupfer, D.; Kwan, H. S.; Lomsadze, A.; Li, W.; Lilly, W. W.; Ma, L. J.; Mackey, A. J.; Manning, G.; Martin, F.; Muraguchi, H.; Natvig, D. O.; Palmerini, H.; Ramesh, M. A.; Rehmeyer, C. J.; Roe, B. A.; Shenoy, N.; Stanke, M.; Ter-Hovhannisyan, V.; Tunlid, A.; Velagapudi, R.; Vision, T. J.; Zeng, Q.; Zolan, M. E.; Pukkila, P. J. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11889−11894. (4) Hopple, J. S., Jr.; Vilgalys, R. Mol. Phylogenet. Evol. 1999, 13, 1− 19. (5) (a) Lopez-Gallego, F.; Agger, S.; Abate-Pella, D.; Distefano, M. D.; Schmidt-Dannert, C. ChemBioChem 2010, 11, 1093−1106. (b) Agger, S.; Lopez-Gallego, F.; Schmidt-Dannert, C. Mol. Microbiol. 2009, 72, 1181−1195. (6) (a) Ki, D. W.; Kim, D. W.; Hwang, B. S.; Lee, S. W.; Seok, S. J.; Lee, I. K.; Yun, B. S. J. Antibiot. 2015, 68, 351−353. (b) Liu, Y. Z.; Li, Y. Y.; Sun, Y. F.; Zheng, Z. H.; Song, S. Y.; Su, W. J.; Shen, Y. M. Helv. Chim. Acta 2012, 95, 282−285. (c) Pettit, G. R.; Meng, Y.; Pettit, R. 4033


Hitoyol A and B, Two Norsesquiterpenoids from the Basidiomycete

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