Bioactive Aromatic Sesquiterpenoids Produced by the Mushroom

Letter Cite This: Org. Lett. 2018, 20, 6294−6297

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Structures and Synthesis of Hitoyopodins: Bioactive Aromatic Sesquiterpenoids Produced by the Mushroom Coprinopsis cinerea Junnosuke Otaka,† Takeshi Shimizu,† Yushi Futamura,† Daisuke Hashizume,‡ and Hiroyuki Osada*,† †

Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, 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 Org. Lett. 2018.20:6294-6297. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/05/18. For personal use only.

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ABSTRACT: The structures of sesquiterpenoids hitoyopodin A (1) and its hydroxy derivatives 2 and 3 from the mushroom Coprinopsis cinerea are reported. Their absolute structures (1−3) with a benzoxabicyclo[3.2.1]octane core were determined by spectroscopy, X-ray crystallography, and total synthesis of 1. Compound 1 displays antiproliferative activity against HL-60 cancer cells and the malarial parasite Plasmodium falciparum. It is proposed that 1 acts as a crucial precursor in the biosynthesis of 2, 3, and lagopodins.

C

from Coprinopsis cinerea, which could be derivatives of 4 (Figure 1).6a In F. velutipes the metabolite enokipodin A (5), which is considered to be a precursor of 6, has been the subject of numerous synthetic studies because of its hemiketal-bridged tricyclic structure that contains a hydroquinone,7 and its potent antimicrobial activity (Figure 1).2b,c On the basis of these results, we predicted that Coprinopsis sp. could produce specific benzoxabicyclo[3.2.1]octane compound(s). Only one study has reported a relative stereostructure of a hydroxylated compound from Coprinopsis sp.6b Since Bollinger isolated lagopodin A (4) in 1965,6c,d a key structural parent compound for 4 has not been discovered, and this is crucial for broader biological evaluation and biosynthetic studies. In this study, we document the isolation, structural elucidation, and biological evaluation of aromatic sesquiterpenoids hitoyopodin A (1) and two hydroxy derivatives (2 and 3) from a culture medium of C. cinerea. Total synthesis of 1 was achieved to determine its absolute configuration and to carry out subsequent extensive biological research. In addition, we propose a metabolic pathway of 1 to its oxidative derivatives. The C. cinerea was cultivated in 2.7 L of dried yeast powder−glucose liquid medium for 21 days at 28 °C. After extraction of the fermented medium with acetone, the mixture was evaporated under a vacuum to remove acetone. Through successive partitioning of the aqueous solution in n-hexane, ethyl acetate (EtOAc), and n-butanol, we obtained three extracts. UV-guided screening with monitoring at 290 nm detected three prospective compounds in the n-hexane and EtOAc extracts. Then, we performed silica gel column chromatog-

uparene sesquiterpenoids that carry the p-tolylcyclopentane motif have been found in various organisms, including red algae (Rhodophyta),1 mushroom-forming fungi (Basidiomycota),2 liverworts (Marchantiophyta),3 conifers (Pinophyta),4 and sea hares (Gastropoda).5 Oxidative modification can endow these small molecules with unexpected skeletal diversity and new biological activities for drug design. We focused on two Basidiomycete fungi, Coprinopsis sp. (Psathyrellaceae) (Figure S1) and Flammulina velutipes (Physalacriaceae), because they can produce the related cuparene quinones lagopodin A (4) and enokipodin B (6), even though these species are distinct in terms of morphology and habitat (Figure 1).2c,6 We have previously characterized two highly oxygenated norsesquiterpenoids hitoyols A and B

Figure 1. Hitoyopodins 1−3 and structurally related sesquiterpenoids. © 2018 American Chemical Society

Received: August 31, 2018 Published: September 20, 2018 6294

DOI: 10.1021/acs.orglett.8b02788 Org. Lett. 2018, 20, 6294−6297

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pentane moiety was also established by the HMBC correlations from three methyls (H3-12/C-7, C-10, C-11, and C-13; H3-13/C-7, C-10, and C-11; and H3-14/C-7, C-8, and C-11) and two methylenes (H2-8/C-9, and H2-10/C-9 and C11). The HMBC correlations from H-1/C-7, H2-8/C-6, and H3-14/C-6 established the linkage between C-6 and C-7. Considering that 1 had one degree of unsaturation, except for a benzene and cyclopentane ring, we deduced from the downfield shift of C-9 (δC 105.5 ppm) as a hemiketal carbon that 1 had an ether bridge from C-5 to C-9. Consequently, the planar structure of 1 was elucidated, as depicted in Figure 2. The bicyclic ring system of 1 permits the molecule to have only 7S* and 9S* configurations, and this was supported by NOESY experiments (Figure 2). Next, we attempted the total synthesis of 1 to establish the absolute configuration and evaluate the detailed biological activities. Our retrosynthetic analysis of 1 is shown in Figure 3. Compound 1 should be obtained by hydrolysis followed by

raphy, preparative thin layer chromatography, and reversedphase HPLC to obtain pure 1 (9.0 mg) from the n-hexane extract (490 mg), and 2 (2.0 mg) and 3 (4.3 mg) from the EtOAc extract (1.0 g). Compound 1 had a molecular formula of C15H20O3 as established by HR-field ionization-MS ([M]+ calcd, m/z 248.1412; found, m/z 248.1411) with six degrees of unsaturation. The presence of hydroxyl groups was suggested by IR absorption bands at 3369 and 3305 cm−1. UV adsorption at λmax 298 and 206 nm and 13C NMR data (δ 147.0, 146.3, 129.9, 123.0, 117.6, and 112.7 ppm) indicated that 1 contained a hydroquinone (Table 1). In the 1H NMR spectrum of 1, we Table 1. 13C (125 MHz) and 1H (500 MHz) NMR Spectral Data of 1 in CDCl3 δC

position 1 2 3 4 5 6 7 8a 8b 9 10a 10b 11 12 13 14 15 2-OH 9-OH

δH, mult. (J in Hz)

112.7 147.0 123.0 117.6 146.3 129.9 47.5 44.1

CH C C CH C C C CH2

105.5 53.3

CH CH2

45.5 25.3 27.4 17.1 15.5

C CH3 CH3 CH3 CH3

6.52, s

6.56, s

2.36, d (12.0) 1.84, dd (12.0, 2.5) 2.00, dd (14.5, 2.5) 1.94, d (14.5) 0.68, 1.06, 1.23, 2.18, 4.34, 3.12,

s s s s brs brs

Figure 3. Retrosynthetic analysis of 1.

cyclization of 2,5-dimethoxytoluene derivative 7, which is substituted at the C-4 position of 3,3,4-trimethylcyclopentanone. The 3,3,4-triimethylcyclopentanone part in 7 could be synthesized by transformation of 4,5,5-trimethylcyclopentenone 9 via 4,5,5-triimethyl-2-hydroxycyclopentenone 8.8 Kuwahara and co-workers reported that cyclopentenone 9 is an important intermediate for the enantioselective total synthesis of enokipodin A (5; Figure 1).7d,e First, we used Kuwahara’s protocol and 2,5-dimethoxytoluene to enantioselectively synthesize the epoxide 10 in 13 steps via cyclopentenone 9 (Scheme 1),.7d,e Heating of 10 with

observed two para-oriented signals at δH 6.56 and 6.52 ppm, which indicated the presence of a tetrasubstituted benzene ring. The 1D-NMR (1H, 13C, and DEPT) and HSQC results for 1 attributed 15 carbon signals, as follows: three tertiary methyls, one olefinic methyl, two methylenes, two aromatic methines, four aromatic quaternary carbons, two non-hydrogen attached sp3 carbons, and one oxygenated tertiary carbon (Table 1). Additionally, the benzoxabicyclo[3.2.1]octane nature was revealed by HMBC analyses (Figure 2). The HMBC correlations were as follows: H-1/C-2, C-3, and C-5; H-4/C6; and H3-15/C-2, C-3, and C-4. These results suggested that a 1,4-dioxygenated-2-methylbenzene unit was introduced into 1. The presence of a 1,1,2-trimethyl-4,4-disubstituted cyclo-

Scheme 1. Synthesis of 1

Figure 2. Selected HMBC and NOESY correlations of 1. 6295

DOI: 10.1021/acs.orglett.8b02788 Org. Lett. 2018, 20, 6294−6297

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The configurations of the two hitoyopodins were elucidated by NOESY experiments as (7S*,9S*)-2 and (7S*,9R*,11S*)-3, respectively. To determine all absolute configurations for 2 and 3, their electronic circular dichroism (ECD) spectra were measured and compared with that of (7S,9S)-1. Overall, the experimental ECD curves for compounds 1−3 showed very similar positive Cotton effects at around 295 and 234 nm because of a hydroquinone (Figure 5). The absolute structures were determined as (7S,9S)-15-hydroxy-1 and (7S,9R,11S)-13hydroxy-1.

sodium methoxide regioselectively afforded 2-methoxycyclopentenone 11 in 83% yield. Then, 11 was converted to 2hydroxycyclopentenone 8 by heating with 2 M hydrochloric acid at 80 °C. We envisaged that the treatment of 8 with hydriodic acid in acetic acid would give 1.8 However, this resulted in the formation of lagopodin A (4). The 4,5,5trimethyl-2-hydroxycyclopentenone part in 8 was cleanly transformed to 3,3,4-trimethylcyclopentanone by reduction with hydriodic acid. The 1,4-dimethoxytoluene part in 8 was converted to benzoquinone through oxidation of preformed 2,5-dihydroxytoluene. This oxidation might occur before cyclization of the hydroxyl group to the carbonyl group with iodine, which was formed during the reaction (see Scheme S1). Finally, 4 was reduced with sodium hydrosulfite to afford 1. Conversely, 1 was also oxidized to afford 4 with ceric ammonium nitrate. The 1H and 13C NMR data of synthetic 1 were in accordance with those of natural 1. Furthermore, the specific optical rotation of synthetic 1 ([α]D25 +80 (c 0.10, CH3OH)) was identical to that of natural 1 ([α]D25 +80 (c 0.25, CH3OH)) isolated from C. cinerea. Hence, the absolute configuration of 1 was definitely determined as 7S and 9S, and we gave it the name hitoyopodin A ((2S,5S)-4,4,5,8tetramethyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepine2,7-diol). Furthermore, the absolute configuration of 1 was confirmed by X-ray crystallographic analysis of natural 1 [Flack χ parameter: − 0.11(11)] (Figure 4).

Figure 5. ECD spectra of compounds 1−3.

We then tested the inhibitory activities of 1−3 against mammalian cells, bacteria, fungi, and a malarial parasite (Table 2 and Table S3). Hitoyopodin A (1) showed growth inhibition Table 2. Bioactivity Data of Hitoyopodins 1−3 growth inhibitory activities (IC50, μM) compounds

HL-60 cells

P. falciparum

1 (natural) 1 (synthetic) 2 3

3.7 2.5 16 14

6.7 6.2 >100 25

against human leukemia cells HL-60 (IC50 = 3.7 μM) and the malarial parasite Plasmodium falciparum 3D7 (IC50 = 6.7 μM). By contrast, 2 and 3, which bear an additional hydroxy group, showed much weaker activities than 1. We also confirmed that the growth inhibitory activities of synthetic 1 reproduced those of natural 1. In parallel with the synthetic approach, to obtain sufficient quantities of 1, we cultivated C. cinerea. Unfortunately, hydroquinones 1−3 were not detected, but substantial quantities of benzoquinone lagopodin B were observed as a major metabolite (Figure 6). This phenomenon is presumably caused by activation of oxidase from C. cinerea. The key precursor 1 from cuparene could undergo rapid conversion to lagopodin B via 4 by phenol oxidase, of which laccase is an example.9 Hence, depending on the activity of phenol oxidase, the quantities of accumulated benzoxabicyclo[3.2.1]octane compounds may fluctuate. In conclusion, we isolated hitoyopodin A (1), a novel sesquiterpenoid featuring a unique benzoxabicyclo[3.2.1]octane core, from a culture medium of C. cinerea. We also isolated the oxidative analogs 2 and 3. A combination of total synthesis and X-ray diffraction analysis unequivocally established the absolute configuration of 1. Our synthetic study indirectly demonstrated that the oxidative position on the cyclopentane of 1,4-dihydroxy cuparene controls the structural

Figure 4. X-ray crystal structure of natural 1.

The structural determinations for 2 and 3 based on the chemical structure of 1 proceeded uneventfully. Both compounds gave the same molecular formula of C15H20O4 in HR-ESI-MS and differed from that of 1 by one oxygen. The high similarity of UV spectral patterns among compounds 1−3 indicated that 2 and 3 both had a benzoxabicyclo[3.2.1]octane scaffold. The 1H and 13C NMR spectra of 2 resembled those of 1, except for the signal of C-15 [δH 2.18 (s, 3H)/δC 15.5 for 1 → δH 4.57 (d, J = 2.5 Hz, 2H)/δC 60.8 for 2] (Table S1). HMBC correlations from H2-15 to C-2, C-3, and C-4 were observed. Thus, the structure of 2 was determined to be 15-hydroxy-1. A comparison of the NMR data of 3 with those of 1 suggested the presence of an additional hydroxy group at C-13 in 3 ([δH 3.50 (d, J = 11.5 Hz), 3.38 (d, J = 11.5 Hz)/δC 68.9] for 3 and [δH 1.06 (s, 3H)/δC 27.4] for 1) (Table S2). From the HMBC cross peaks for H2-13/C-10, C-11, and C-12, we determined that 3 was 13-hydroxy-1. 6296

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carrying out bioassays. This work was supported by the RIKEN SPDR Program (to J.O.).

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Figure 6. Hypothetical biosynthetic pathway from cuparene to hitoyopodins (1−3) and enokipodins (5 and 6). Biosynthesis of lagopodin B from 4 proceeds via phenol oxidation of 1 (red arrows). Compounds 2 and 3 are generated from a measurable accumulation of 1 through methyl oxidation (blue arrows). The biosyntheses of 1 and 5 hinge upon an oxidative position at C-9 or C-10 of cuparene.

features for formation of 1 or 5 (Figure 6). Compound 1 showed growth inhibition against HL-60 cancer cells and the malarial parasite Plasmodium falciparum. Further chemical biology research utilizing 1 is currently in progress.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b02788. Experimental procedures, (HR)-MS, IR, UV, ECD, NMR spectra, and crystal data for natural 1 (PDF) Accession Codes

CCDC 1842177 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (H.O.). ORCID

Junnosuke Otaka: 0000-0002-7947-1846 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. T. Nakamura of the Molecular Structure Characterization Unit at the RIKEN CSRS for measurement of mass spectra. We also thank Ms. H. Aono, Dr. M. Uchida, Dr. R. Uson, Ms. E. Sanada, and Mr. K. Yamamoto of the Chemical Biology Research Group at the RIKEN CSRS for 6297

DOI: 10.1021/acs.orglett.8b02788 Org. Lett. 2018, 20, 6294−6297