Highly Modified Lanostane Triterpenes from Fruiting Bodies of the

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Highly Modified Lanostane Triterpenes from Fruiting Bodies of the Basidiomycete Tomophagus sp. Masahiko Isaka,* Panida Chinthanom, Tuksaporn Thummarukcharoen, Thitiya Boonpratuang, and Wilunda Choowong National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF SOUTHERN INDIANA on 04/15/19. For personal use only.

S Supporting Information *

ABSTRACT: Thirty-one highly modified lanostanes (1−31), together with 19 known compounds (32−50), were isolated from fruiting bodies of the wood-rot basidiomycete Tomophagus sp. The structures were elucidated by analyses of HRMS and NMR spectroscopic data. The present work demonstrates the high structural diversity of modified lanostane triterpenoids from Tomophagus. This paper also discusses structural revisions of several known derivatives. Some of the isolated compounds exhibited moderate antimalarial activity against Plasmodium falciparum K1 (IC50 5.1−19 μM).

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As part of our research on the utilization of fungal resources in Thailand, we have recently investigated several Ganoderma species that are taxonomically close to G. lucidum with respect to basidiome morphology and phylogeny. These studies have resulted in the isolation of lanostane triterpenoids from mycelial cultures, and many of them exhibited antimalarial and antitubercular activities.9 In continuation of this research, we have been expanding the targets into other species in the family Ganodermataceae. We report here the isolation of 31 new highly modified lanostane triterpenoids (1−31) together with 19 known compounds (32−50) from natural fruiting bodies of a Tomophagus sp., which were collected from a dead oil palm tree. Their antimalarial and antitubercular activities were evaluated.

racket fungi in the family Ganodermataceae have been investigated as sources of novel bioactive compounds.1 In particular, the chemical analysis of Ganoderma lucidum, which is a very popular medicinal mushroom known as lingzhi, has been comprehensively performed. Lanostane triterpenoids constitute one of the key metabolite groups of lingzhi, and a wide range of their biological activities have been evaluated.2,3 Several other species of Ganoderma have also been extensively chemically explored. To date, more than 300 lanostane-type triterpenoides, including highly oxygenated and modified derivatives, have been isolated from basidiocarps (natural or cultivated mushroom specimens) and/or mycelial cultures of this genus.1,2 In contrast, other genera of the family Ganodermataceae have been relatively rarely investigated. Tomophagus is a small genus within Ganodermataceae with only two described species, namely, T. colossus (current name: Ganoderma colossus) and T. cattienensis. Both species form soft and yellowish fruiting bodies, unlike G. lucidum and closely related species that commonly form harder and dark-brown fruiting bodies. Several highly modified lanostanes, colossolactones A−H4,5 and colossolactones I− VIII,6 have been isolated from G. colossus. Tomophagus cattienensis is a recently described species,7 with only one report of a chemical investigation, i.e., the isolation of cattienoids A−C and side-chain fragmented modified lanostanes.8 © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1, named tomophagusin A, was isolated as a colorless solid. Its molecular formula was determined to be C30H42O5 by HRESIMS. The IR spectrum exhibited intense carbonyl absorption bands at νmax 1737 and 1714 cm−1. The 1H and 13C NMR, DEPT, and HMQC data for 1 supported the presence of two ester carbonyl carbons (δH 171.0 and 166.6), two sp2 tetrasubstituted carbons (δC 139.9 and 128.2), two Received: October 18, 2018

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DOI: 10.1021/acs.jnatprod.8b00869 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. NMR Spectroscopic Data for Compounds 1−3 in CDCl3 tomophagusin A (1) no.

δC, mult.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 4-OCOCH3 4-OCOCH3

34.6, CH2 27.9,a CH2 171.0, C 76.1, C 38.1, CH 18.6, CH2 26.7, CH2 74.4, C 139.9, C 39.4, C 121.2, CH 37.4, CH2 43.9, C 49.8, C 26.7, CH2 26.7, CH2 46.7, CH 17.3, CH3 78.0, CH2 39.9, CH 12.9, CH3 80.1, CH 28.0,a CH2 139.7, CH 128.2, C 166.6, C 17.1, CH3 30.8, CH3 31.1, CH3 21.3, CH3

δH, mult. (J in Hz) 2.20, m; 1.87, m 2.71, dt (18.6, 5.9); 2.57, m

1.64, m 2.06, m; 1.71, m 1.89, m; 1.70, m

5.52, dd (6.0, 1.6) α 2.15, br d (17.7); β 2.06, m

2.07, m; 1.09, m 1.97, m; 1.30, m 2.25, m 0.93, s 4.67, dd (11.8, 2.4); 4.60, d (11.8) 1.51, m 0.98, d (6.7) 4.50, dd (13.4, 3.1) 2.55, m; 2.00, m 6.61, d (6.3)

1.91, s 1.37, s 1.29, s 0.82, s

tomophagusin B (2) δC, mult. 32.1, CH2 26.9,b CH2 170.4, C 86.5, C 47.8, CH 24.9, CH2 117.2, CH 141.5, C 133.2, C 39.4, C 122.4, CH 38.7, CH2 44.1, C 49.7, C 30.6, CH2 27.1,b CH2 46.4, CH 16.6, CH3 76.3, CH2 40.0, CH 13.2, CH3 80.2, CH 27.7, CH2 139.5, CH 128.3, C 166.4, C 17.1, CH3 28.0, CH3 23.6, CH3 24.3, CH3 170.2, C 23.0, CH3

δH, mult. (J in Hz) 1.97, m; 1.80, m 2.46, m; 2.41, m

2.46, m 2.53, m; 2.23, m 5.26, br s

5.66, br s α 2.35, m; β 2.21, m

1.64, m; 1.38, m 2.08, m; 1.35, m 2.18, m 0.63, s 4.83, dd (11.8, 2.5); 4.48, d (11.8) 1.55, m 1.02, d (6.8) 4.47, dd (13.2, 2.3) 2.56, m; 2.01, m 6.61, d (6.3)

1.91, s 1.54, s 1.43, s 0.90, s 2.00, s

tomophagusin C (3) δC, mult. 32.3, CH2 27.0,c CH2 170.3, C 86.6, C 47.9, CH 25.0, CH2 117.3, CH 141.7, C 133.5, C 39.5, C 122.4, CH 38.8, CH2 44.2, C 49.9, C 30.7, CH2 27.1,c CH2 46.1, CH 16.8, CH3 76.3, CH2 36.2 CH 12.5, CH3 84.3, CH 63.8, CH 143.7, CH 127.9, C 164.9, C 16.8 CH3 28.0, CH3 23.7, CH3 24.4, CH3 170.1, C 23.0, CH3


2.45, m 2.54, m; 2.24, m 5.27, br s

5.66, br s α 2.36, m; β 2.20, m

1.66, m; 1.39, m 2.05, m; 1.37, m 2.17, m 0.66, s 4.83, dd (11.8, 2.2); 4.48, d (11.8) 1.92, m 1.00, d (6.7) 4.20, d (10.5) 4.47, m 6.53, s

1.93, s 1.55, s 1.44, s 0.90, s 2.00, s

a−c

The assignments may be interchanged.

olefinic methines (δC 139.7, δH 6.61 and δC 121.2, δH 5.52), two oxygenated tetrasubstituted carbons (δC 76.1 and 74.4), an oxymethine (δC 80.1, δH 4.50), an oxymethylene (δC 78.0, δH 4.67 and 4.60), three sp3 quaternary carbons, three methines, eight methylenes, and six methyl groups (Table 1). The structural elucidation of 1 was accomplished by analysis of COSY and HMBC data (Figure 1). A spirocyclic ring system (AB-ring) was revealed by HMBC correlations from H2-1, H2-2, H-5, Ha-6 (δH 2.06), and Hb-19 (δH 4.60) to the quaternary carbon resonating at δC 39.4 (C-10). The presence of a δ-lactone was indicated by the HMBC correlations from H2-19, H2-1, and H2-2 to the carbonyl carbon at δC 171.0 (C-3). NMR spectroscopic data for the C-20−C-27 side-chain unit were similar to those of many other known co-metabolites such as 35−38. The ether linkage between C-5 and C-8 was a requirement, according to the molecular formula (HRMS). The relative configuration was deduced on the basis of the NOESY data (Figure 2). Intense NOESY correlations between Ha-19 (δH 4.67)/H3-28 (δH 1.37), Hb-19 (δH 4.60)/H3-28, Ha19/Ha-1 (δH 2.20), Ha-1/Ha-6 (δH 1.71), H3-29 (δH 1.29)/Hβ-6 (δH 2.06), and Hα-7 (δH 1.70)/H3-30 strongly suggested a 5R,8R,10R configuration. The similarity of the NMR spectroscopic data suggested that configurations for C-13, C-14, C-17, C-20, and C-22 should be the same as other known cometabolites. Thus, NOESY correlations between H3-30/Hα-12

(δH 2.15) and H3-30/H-17 indicated an α-orientation of these protons as well as the trans CD-ring junction. Intense NOESY correlations between H3-18/H-20, Hβ-12 (δH 2.06)/H3-21, H216/H-22, and H3-21/Ha-23 (δH 2.55, axial) have been commonly found for the compounds with the same D-ring and C-20−C-27 structure. Typical intense NOESY correlation patterns are H3-30/Hα-12 (δH 2.15), H3-30/H-17, H3-18/H-20, Hβ-12 (δH 2.06)/H3-21, H2-16/H-22, and H3-21/Ha-23 (δH 2.55, axial). Consequently, the relative configuration of 1 was determined. Although several hundred lanostane triterpenoids and modified lanostanes have been isolated from fungi of the family Ganodermataceae, ent-lanostanes have never been reported. Therefore, it is not unreasonable to propose the absolute configuration of 1 as shown. Compound 2, named tomophagusin B, was assigned the molecular formula C32H44O6 by HRESIMS. The 1H and 13C NMR spectra displayed similarity to those of 1, and the data suggested the presence of an acetoxy group. Interpretation of 2D NMR spectra (Figure 1) revealed the spiro-δ-lactone (C-1, C-2, C-3, C-10, and C-19), lanostane CD-ring, and the same C-20− C-27 unit as in 1. The significant structural difference with 1 was the presence of an additional olefin (C-7/C-8) conjugated with the C-9/C-11 double bond. The presence of the 4-acetoxy group was a requirement according to the molecular formula. The relative configuration was determined on the basis of NOESY B



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4.83)/Ha-1 (δH 1.97). The proposed absolute configuration (5R,10R) is based on the consideration of biosynthetic grounds, as discussed later in this paper. The molecular formula of compound 3, named tomophagusin C (3), was determined by HRESIMS to be C32H44O7, possessing one more oxygen atom than 2. The 1H and 13C NMR spectra were similar to those of 2. The only structural difference was the presence of an oxymethine (δC 63.8, δH 4.47) in 3, replacing the C-23 methylene carbon for 2. The large vicinal 1H−1H coupling constant for H-22 and H-23 (J = 10.5 Hz), the absence of the NOESY correlation for H-22/H-23, and the small J-value for H-23 and H-24 (H-24 resonated as a singlet) strongly suggested an anti-relation and pseudoaxial orientation of H-22 and H-23. NOESY correlations, H-22/H216, H-22/H-17, H-23/H3-21, and H-22/H-20, revealed a 22R,23S configuration, which is the same as some known compounds, such as 32 and 34. Compound 4, named tomophagusin D, had the molecular formula C30H42O5 as determined by HRESIMS. Interpretation of 1D and 2D NMR spectroscopic data suggested that it possesses the same CD-ring and C-20−C-27 unit as several cometabolites such as 1 and 2 and the presence of a 7,9(11)-diene (Table 2). The planar structure was elucidated by analysis of COSY and HMBC data (Figure 2). In particular, HMBC correlations from Ha-19 (δH 4.20) and Hb-19 (δH 3.58) to C-4 (δC 83.2) indicated an ether linkage between C-4 and C-10 to construct a tetrahydrofuran ring, which also suggested the relative configuration of C-5 and C-10 as 5R*,10R*. A NOESY correlation of Hb-1 (δH 1.68)/H-5 further supported the cis ring junction. The proposed absolute configuration (5R,10R) is based on consideration of biosynthetic grounds. NMR spectroscopic data of compound 5, indicating the presence of an acetal (δC 101.3, δH 4.82) and a ketal (δC 104.7) moiety, were similar to those of the co-metabolite ganoderma-

Figure 1. COSY and key HMBC correlations for 1, 2, 4, and 5.

correlations. Chemical shifts (1H and 13C) and NOESY data for the lanostane CD-ring and C-20−C-27 unit were similar to those of 1 and known compounds such as 38. Typical NOESY correlation patterns are H3-30/Hα-12 (δH 2.35), H3-30/H-17, Hα-12/H-17 H3-18/H-20, Hβ-12/H3-21, H2-16/H-22, and Hax24/H3-21. Relative configurations of C-5 and C-10 (5R*,10R*) were revealed by the NOESY correlations Hb-1 (δH 1.80)/H-5, Hb-1/Hb-19 (δH 4.48), Hβ-6 (δH 2.53)/Hb-19, and Ha-19 (δH

Figure 2. Selected NOESY correlations for 1, 2, 4, and 5. C



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Chart 1

ray crystallographic analysis.10 Its molecular formula C30H36O6, determined by HRESIMS, was the same as 46. The planar

lactone G (46), which was previously isolated from cultures of Ganoderma sp. KM01, and the structure was determined by XD



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Table 2. NMR Spectroscopic Data for Compounds 4−6 in CDCl3 tomophagusin D (4) no.

δC, mult.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

35.3, CH2 30.1, CH2 177.5, C 83.2, C 51.6, CH 23.0, CH2 117.4, CH 141.9, C 133.1, C 46.9, C 121.2, CH 38.4, CH2 44.1, C 50.3, C 31.1, CH2 27.3, CH2 46.5, CH 16.4, CH3 74.1, CH2 40.1, CH 13.1, CH3 80.2, CH 27.8, CH2 139.6, CH 128.3, C 166.5, C 17.1, CH3 29.6, CH3 23.2, CH3 24.0, CH3


1.88, d (8.5) 2.40, m; 2.07, m 5.30, br s

5.52, br s α 2.33, m; β 2.25, m

1.66, m; 1.37, m 2.07, m; 1.33, m 2.21, m 0.62, s 4.20, d (8.8); 3.58, d (8.8) 1.54, m 1.02, d (6.7) 4.48, dd (13.3, 2.9) 2.57, m; 2.00, m 6.61, d (6.3)

1.92, s 1.22, s 0.99, s 0.91, s

22-epi-ganodermalactone G (5) δC, mult. 143.0, CH 118.7, CH 166.7, C 80.1, C 49.3, CH 39.8, CH2 28.2, CH2 152.2, C 134.6, C 140.9, C 77.3, CH 30.1, CH2 54.5, C 52.3, C 31.1, CH2 22.2, CH2 39.7, CH 101.3, CH 140.6, CH 37.0, CH 14.3, CH3 104.7, C 29.0, CH2 134.8, CH 128.4, C 163.8, C 16.5, CH3 29.2, CH3 26.3, CH3 26.0, CH3

δH, mult. (J in Hz) 6.68, d (12.2) 5.84, d (12.2)

2.42, m 2.44, m; 2.34, m 2.08, m; 1.88, m

4.42, d (4.9) 2.52, dd (11.4, 4.9); 2.02, d (11.4)

1.71, m; 1.69, m 1.94, m; 1.64, m 2.60, m 4.82, s 6.37, s 2.37, m 1.11, d (7.2) 2.68, m; 2.47, m 6.36, d (7.5)

1.90, s 1.39, s 1.53, s 1.23, s

deacetylcolossolactone G (6) δC, mult. 148.1, CH 116.3, CH 164.3, C 77.8, C 92.8, C 44.0, CH2 27.1, CH2 151.0, C 127.7, C 132.6, C 28.3, CH2 31.4, CH2 43.8, C 56.6, C 76.4, CH 40.9, CH2 46.1, CH 17.3, CH3 139.6, CH 39.9, CH 13.4, CH3 80.0, CH 27.7, CH2 139.8, CH 128.3, C 166.4, C 17.1, CH3 24.8, CH3 24.6, CH3 24.5, CH3

δH, mult. (J in Hz) 6.94, d (9.8) 5.87, d (9.8)

2.48, m; 2.34, m 2.39, m; 2.15, m

2.26, m; 2.24, m α 1.89, m; β 1.73, m

4.06, d (7.0) 2.65, m; 1.44, dd (14.5, 9.5) 2.11, m 1.12, s 6.23, s 1.71, m 1.06, d (6.7) 4.47, dd (13.4, 2.8) 2.58, m; 2.03, m 6.62, d (6.3)

1.91, s 1.16, s 1.24, s 0.89, s

Chart 2

structure of 5 was elucidated to be the same as 46 on the basis of COSY, HMQC, and HMBC correlations (Figure 1). An ether linkage of C-11 and C-18 was revealed by the HMBC correlations from H-11 to C-18 and from H-18 to C-11. HMBC correlations from H-17, H-18, H-20, H3-21, Hb-23 (δH 2.47), and H-24 to the ketal carbon (C-22) indicated the location of the spiroketal as well as the ether linkage of C-18 and

C-22. The relative configuration was deduced from NOESY correlations (Figure 2). Thus, correlations of H3-30 with Hb-12 (δH 2.02), Hα-15, Hα-16 (δH 1.94), and H-17 and the intense correlation of H-17/H-20 revealed α-orientations of these protons. On the other hand, NOESY correlations of Hβ-15/H18, H-18/Ha-23 (δH 2.68), H-18/Hb-23 (δH 2.47), Ha-23/H321, and H3-21/Hβ-16 demonstrated the β-orientation of these E



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hydroxy-34. Accordingly, we revise herein the structure of colossolactone H to be that of 32. Modified lanostane triterpenoids, shown as formulas 7−13 (new compounds), 34 (colossolactone VIII),6b 35 (colossolactone D),4 36 (colossolactone E),4 37 (colossolactone F),4 and 38, constitute a major structural group in the present chemical investigation. The least oxygenated compound, 38, has been previously isolated from Ganoderma colossus,6,11 Ganoderma sp.,10 and Tomophagus cattienensis.8 However, it was wrongly reported as schisanlactone A, a constituent of the higher plant Schisandra sp., whose structure was determined by X-ray crystallographic analysis as the C-22 epimer (22R isomer) of 38.12 NMR data of compound 38 (in CDCl3), isolated in the present work, were different from those of schisanlactone A; however, the data for the C-20−C-27 side chains were very similar to those of several other known co-metabolites found in the present study (22S isomers), including colossolactone G (33).10 To avoid confusion regarding the trivial name, we propose here to newly name this compound (38), a metabolite of wood-rot mushrooms of the genera Tomophagus and Ganoderma (family, Ganodermataceae), as ganodermalactone H. Since its NMR spectroscopic data have not been previously reported, they are recorded in the Supporting Information (Table S10). An additional confusing issue in previous reports is the same configurational misassignment of C-22 for colossolactones I−VIII.6 Thus, the 22R configuration proposed for colossolactones I−VII (R1 = H) should be corrected to 22S. On the other hand, the configuration of colossolactone VIII, a 23hydroxy derivative (R1 = OH), should be corrected from 22S,23R to 22R,23S shown as 34. NMR spectroscopic data of compound 7 were similar to those of colossolactone E (36), possessing a 15β-acetoxy group (δC 77.6; δH 5.00, d, J = 6.9 Hz). The significant differences of the NMR data were the presence of a conjugated ketone (δC 197.0), which replaces one of the methylene groups in 36, and a significant downfield shift of C-8 (δC 168.9). Location of the ketone was revealed by the HMBC correlations from H2-12 and H-19 to C-11, from Hβ-12 (δH 2.65) and H2-7 to C-9, and from H2-7, H-19, and H3-30 to C-8. Consequently, compound 7 was identified as the 11-oxo derivative of 36, and it is named 11-oxocolossolactone E. Similarly, compound 8 was identified as the 11-oxo derivative of colossolactone VIII (34), and it is named 11-oxo-colossolactone VIII. NMR spectroscopic data of compound 9 showed a resemblance to colossolactone D (35); however, it possessed an additional secondary alcohol (oxygenated methine) replacing the C-11 methylene in 35. The location of the oxymethine was determined by the HMBC correlations from the downfield proton (δH 4.20, H-11) to C-8, C-9, C-13, and C-19 and from Hβ-12 (δH 2.00) to C-11 (δC 67.8). The α-orientation of H-11 was demonstrated by the intense NOESY correlations H-11/ Hα-12 (δH 2.29) and Hα-12/H3-30. Consequently, compound 9 was identified as the 11β-hydroxy derivative of 35, and it is named 11β-hydroxycolossolactone D. Similarly, compound 10 was identified as the 11β-hydroxy derivative of colossolactone VIII (34), and it is named 11β-hydroxycolossolactone VIII. The 1H and 13C NMR spectroscopic data of compound 11 suggested its structural similarity to colossolactone D (35), but indicated the presence of a 23-hydroxy group. The 22R,23S configuration was confirmed by comparison of the NMR data with other derivatives possessing the same C-20−C-27 unit. This compound (11) is named ganodermalactone I. Similarly, compound 12 was identified as the 23-hydroxy derivative of

protons. NMR spectroscopic data for the AB-ring were very similar to those of 46. The presence of weak NOESY cross-peaks of H3-29/H-1 and H3-29/H-2 were consistent with the axial-like orientation of CH3-29. These correlations were also observed in the NOESY spectra of 46 and other related compounds with the same AB-ring substructure such as 7−13. Consequently, compound 5 was identified as a stereoisomer of ganodermalactone G (46), which differs only in the C-22 configuration (22R) and is named 22-epi-ganodermalactone G. Both 5 and 46 may be produced from a hemiacetal/ hemiketal/carboxyl intermediate 51a, which is tautomeric with an aldehyde/ketone/carboxyl form, 51b (Figure 3). To confirm

Figure 3. Interconversion of 5 and 46 under acidic conditions.

the stereochemistry of the new compound 5, its chemical correlation to 46 was examined. To a solution of 5 (1.2 mg) in CDCl3 (0.5 mL) was added a CDCl3 solution of p-TsOH·H2O (catalytic amount), and the reaction was monitored by 1H NMR (Supporting Information, Figure S138). The 1H NMR spectrum after 10 min showed the presence of a ca. 1:2 mixture of 5 and 46. The 1H NMR spectrum after 4 h indicated the disappearance of 5 and its complete conversion into 46. Since the interconversion between 5 and 46, via intermediate 51a, should be a reversible process under the reaction conditions, the present result suggested that 46 is thermodynamically more stable than 5. The molecular formula of compound 6 was determined by HRESIMS to be C30H40O6. Its 1H and 13C NMR spectra were similar to those of the known co-metabolite colossolactone G (33).10 Major differences were the absence of the 15-O-acetyl group in 6 and an upfield shift of H-15 (δH 4.06 for 6; δH 4.81 for 33). Interpretation of the 2D NMR data of 6 further confirmed the same chemical skeleton as 33. Consequently, compound 6 was identified as the deacetyl derivative of 33 and named deacetylcolossolactone G. It should be noted that the originally reported structure of colossolactone G, 5-hydroxy-36,4 was later corrected to the δ-lactone 33 by X-ray crystallographic analysis performed by Kanokmedhakul and co-workers.10 In the present study, we isolated a related compound, which was identified as 32 on the basis of NMR data interpretation and comparison of the 1H and 13C NMR spectroscopic data with colossolactone G (33). NMR spectroscopic data of 32 were identical to those of colossolactone H,11 the reported structure of which is 5F



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skeleton as ganodermalactone B (39), which was previously isolated from cultures of Ganoderma sp. KM01.10 Compound 19, named ganodermalactone K, possessed a 9(11)-epoxy group (δC 62.0, C-9; δC 56.8, C-11; δH 3.50, d, J = 6.3 Hz, H-11). Location of the epoxide was assigned on the basis of the HMBC correlations from H-5, H-7, Hα-12 (δH 2.20), and H2-19 to C-9, from Hα-12 to C-11, and from H-11 to C-9 and C-13. The αepoxide configuration was revealed by the NOESY correlations of H3-18 with H-11 (weak), Hβ-12 (δH 1.94), Hβ-15 (δH 1.64), and H-20 and the intense correlation of H-11/Hβ-12, indicating their β-orientation. NOESY correlations of H3-30 with Hα-12, Hα-15 (δH 1.41), and H-17 demonstrated their α-orientation. The location of the acetoxy group was confirmed by the chemical shifts of H2-19 (δH 4.65 and 4.21) and the HMBC correlations from these protons to the carbonyl of the acetyl (δC 170.4). Compound 20, named ganodermalactone L, possessed a 23-hydroxy group and a 7,9(11)-diene. Compound 21, named ganodermalactone M, was identified as the 23-dehydroxy derivative of 20. Compound 22 was identified as the 23-hydroxy derivative of 39, and it is named ganodermalactone N. Structures of compounds 23 and 24, named ganodermalactones O and P, respectively, were elucidated by NMR analysis as closely related derivatives of the known ganodermalactone D (40).10 The only difference was that they lacked the 23-hydroxy group present in 40. Ganodermalactone O (23) was identified as a C-3 carboxylic acid derivative, while ganodermalactone P (24) was the corresponding methyl ester. Compounds 25, 26, and 27 possessed the classic lanostane carbon skeleton. Compound 25 was identified as the 23-hydroxy derivative of the known colossolactone B (41),4 and it is named ganodermalactone Q. Compound 26 is the 3-oxo variant of 41, and it is named ganodermalactone R. Compound 27 was identified as the 23-hydroxy derivative of colossolactone I (42),6a and it is named ganodermalactone S. The molecular formula of compound 28 was determined by HRESIMS and 13C NMR as C24H34O4, with six carbon atoms lacking from the C30 lanostane. NMR spectroscopic data of 28 were similar to those of tomophagusin D (4) with respect to the polycyclic main skeleton; however, it was lacking the δ-lactone unit (C-22−C-27). The presence of an acetyl group (δC 209.3, C-20; δC 30.9, C-21; δH 2.13, 3H, s, H-21) bonded to C-17 was revealed by the HMBC correlations from H3-21 and H-17 to the ketone carbon (C-20) and from H3-21 to C-17. The αorientation of H-17 was confirmed by its intense NOESY correlation with H3-24. This compound is named cattienoid D. NMR spectroscopic data of compound 29, named cattienoid E, were similar to those of cattienoid A (43), which was previously isolated from T. cattienensis.8 The most significant difference was the presence of an 11β-hydroxy group (δC 67.7, C-11; δH 4.30, m) in 29, in addition to the 15β-hydroxy group in 43. Configurations of these secondary alcohols (C-11 and C-15) were elucidated based on the NOESY data. NOESY correlations H3-24/H-15, H3-24/H-17, H3-24/Hα-12 (δH 2.49), and Hα-12/ H-11 indicated the α-orientation of these protons. Compound 30, named cattienoid F, is also structurally related to 43. On the basis of HRMS and 2D NMR spectroscopic data, it was identified as the 15-dehydroxy derivative of 43. Compound 31, named cattienoid G, had the molecular formula C22H28O4 as determined by HRESIMS. Its NMR spectroscopic data were similar to the known co-metabolite cattienoid B (44);8 however, it possessed a 15β-hydroxy group. The location of the ketone at C-17 was confirmed by the HMBC correlations from H-15, H2-16, and H3-18 to the carbonyl

ganodermalactone H (38), and it is named ganodermalactone J. NMR spectroscopic data of compound 13 suggested that it is also structurally related to ganodermalactone H (38); however, it possessed an aliphatic ketone (δC 213.7). The location of the ketone at the C-15 position was assigned by the HMBC correlations from H3-30 and Ha-16 (δH 2.75) to the carbonyl carbon. Consequently, this compound (13) is named 15-oxoganodermalactone H. The molecular formula of compound 14, named tomophagusin E, was determined by HRESIMS to be C32H42O8. Interpretation of the NMR spectroscopic data revealed that it contains the same carbon skeleton as the major compound group (7−13 and 34−38). It possessed an 11β-hydroxy group (δC 65.7; δH 4.41, d, J = 7.5 Hz) and 15β-acetoxy group (δC 77.4; δH 4.79, d, J = 6.7 Hz). This compound additionally possessed an epoxide (δC 59.4, C-10; δC 60.9, C-19; δH 3.82, s, H-19), replacing the C-10/C-19 trisubstituted olefin. Location of the epoxide was assigned by the HMBC correlations from the epoxy proton (H-19) to C-1, C-8, C-9, and C-10, from H-1 to C-19, and from H-2, H-5, and H2-6 to C-10. NOESY correlations of H3-28 (β-oriented) with H-1 and H-2 strongly suggested an αorientation of the epoxide oxygen atom (10S,19S). Intensities of these cross-peaks relative to other NOESY correlations in the same spectrum were higher when compared to those in the NOESY spectra of compounds 7−13 and 38. In addition, the correlation for H-19/H-11 was not observed in the NOESY spectrum. Compound 15, named tomophagusin F, was structurally related to 14, as suggested from its NMR spectra. The only structural difference was the replacement of the C-11 secondary alcohol of 14 by a ketone (δC 197.2) in 15. The location of this ketone was confirmed by the HMBC correlations from H2-12 to the carbonyl carbon, as well as the significant downfield shift of C-8 (δC 161.7; β-position of enone). NMR spectroscopic data of compound 16, named tomophagusin G, suggested its structural similarity to colossolactone E (36). It contained one less olefinic double bond but two additional methylene groups. The locations of these methylenes at C-2 and C-19 positions were revealed by the HMBC correlations from H2-2 to C-1, C-3, and C-10 and from H2-19 to C-1, C-5, C-8, C-9, C-10, and C-11. Compound 17, named tomophagusin H, is structurally related to ganodermalactone H (38), while the location of its double bonds was the same as 16, as confirmed by the analysis of COSY and HMBC data. This compound possessed an oxymethine (secondary alcohol), the location of which was assigned to the C-19 position by the HMBC correlations from the oxymethine proton (δH 4.50, H19) to C-1, C-5, C-8, and C-11. The β-orientation of H-19 was determined by NOESY data. Thus, correlations between H3-18/ Hβ-12 (δH 1.68), H3-18/Hβ-11 (δH 1.97), and Hβ-11/H-19 revealed the β-orientation of these protons. NOESY correlations of H3-30/Hα-12 (δH 1.83) and Hα-12/Hα-11 (δH 2.45) demonstrated their α-orientation. Compound 18, named tomophagusin I, retained the original cyclohexanone A-ring for lanostane, which was confirmed by the HMBC correlations from H3-28 and H3-29 to the ketone carbon (δC 216.1, C-3). The presence of a tertiary alcohol and its location at C-10 was established by the HMBC correlations from Ha-1 (δH 1.99), Ha-2 (δH 3.08), Ha-7 (δH 1.71), and H2-19 to the oxygenated carbon (δC 70.3). The trans AB-ring junction and β-orientation of 10-OH were revealed by the NOESY correlation of H-5 and Hα-19 (δH 2.37). Analysis of the NMR spectroscopic data of the new compounds 19−22 revealed that they possess the same carbon G



Article

Scheme 1. Plausible Biosynthetic Pathways for Highly Modified Lanostanes in Tomophagus sp.

lactone unit (C-22−C-27), suggesting that this is formed at a relatively early stage of post-lanostane biosynthesis. Several compounds bear 22-hydroxy or 22-acetoxy groups, although the timing of 22-hydroxylation is uncertain. Hydroxylation of 52 at C-11 and subsequent vinylogous dehydration will afford 7,9(11)-diene 53. Intramolecular translactonization of 53 will produce the spirocyclic δ-lactone 54, which could be further transformed to tomophagusins A (1) and B (2). On the other hand, ganodermalactone O (23) could be produced from 53 by acetylation of 19-OH and hydrolysis of the A-ring lactone. Compound 23 could be converted to tomophagusin D (4) through acid-promoted tetrahydropyran ring formation. In these transformations, 19-OH acetylation may not be a requirement. A main biosynthetic route in Tomophagus sp. would be the Bring expansion of 52 to create compound 55 via the 19-O-acetyl derivative ganodermalactone B (39) or directly (Wagner− Meerwein rearrangement). Hydroxylation of C-19 will generate tomophagusin H (17), which can then be converted into ganodermalactone H (38). Compound 51b, a possible precursor of the acetal/ketal derivatives 5 and 46 (discussed earlier), should be produced from 38 via 11β-hydroxylation, C18 oxidation (to aldehyde), and C-22 oxidation (to ketone). Dihydroxylation of 38 at C-5 and C-15 will synthesize compound 56, which would undergo trans-lactonization (probably in two steps) to produce a δ-lactone derivative,

carbon (δC 216.8). The configuration of the secondary alcohol (C-15) was confirmed by the intense NOESY correlation of H322 and H-15. The additional side-chain fragmented derivative 45 was also isolated, and its structure was elucidated by analysis of HRESIMS and 2D NMR spectroscopic data and by comparison of the NMR data with those of colossolactone G (33) and deacetylcolossolactone G (6). NMR spectroscopic data of 45 were consistent with those of cattienoid C,8 the originally reported structure of which was 5α-hydroxy-43. We conclude that the same structure elucidation error existed as in the cases of colossolactones G and H (discussed earlier in this paper) in the original report. Accordingly, the structure of cattienoid C is revised to be 45. In the present study, the following four additional known compounds were isolated and identified: colossolactone IV (47),6a colossolactone VII (48),6b ganodermalactone E (49),10 and colossolactone II (50).6a Plausible biosynthetic pathways for some of the highly modified lanostanes from the Tomophagus sp. are proposed in Scheme 1. All compounds may be derived from simple lanostane triterpenes such as lanosterol. Oxidation of C-3 (to ketone) and C-26 (to carboxylic acid), hydroxylation of C-19 and C-22, and Baeyer−Villiger-type oxidation reaction to insert an oxygen atom into the C-3/C-4 bond will provide a plausible common precursor, 52. Most of the isolated compounds contain a δH



Article

deacetylcolossolactone G (6). Ganodermalactone G (33), the 15-O-acetate of 6, was the most abundant lanostanoid in this fungus. Biosynthesis of a side-chain fragmented derivative, cattienoid F (30), from 38 can be explained by C-20 hydroxylation and C22 oxidation to afford 57 and its oxidative C-20/C-22 bond cleavage. Similarly, cattienoid B (44) could be produced from 38 by dihydroxylation at C-17 and C-20 (58) and subsequent oxidative cleavage of the C-17/C-20 bond. We previously reported antimalarial and antitubercular activities of lanostane triterpenoids isolated from cultures of Ganoderma species that are close in macro- and micromorphology to the medicinal mushroom Ganoderma lucidum.9 Compounds isolated in the present study were primarily more oxygenated and modified than those from the major group of Ganoderma species. Isolated compounds with sufficient sample quantities (37 compounds) were tested for antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain), antitubercular activity against Mycobacterium tuberculosis H37Ra, and cytotoxicity to nonmalignant Vero cells (African green monkey kidney fibroblasts) (Table 3). Fourteen compounds, 2, 4, 7, 16, 23−25, 34−36, 38, 40, 47, and 48, exhibited antimalarial activity, with IC50 values of 5.1−19 μM, whereas only ganodermalactone E (49) showed weak antitubercular activity (MIC 12.5 μg/mL). Several compounds also displayed weak cytotoxicity. Tomophagusin D (4) exhibited the most potent antimalarial activity (IC50 5.1 μM), while it was noncytotoxic to Vero cells at 104 μM (50 μg/mL). In conclusion, the present work demonstrates the richness and extensive structural diversity of the triterpene metabolites of the yellow wood-rot mushroom Tomophagus. Most are 3,4-secolanostanes with a rearranged (expanded) B-ring, and there are various modification patterns. Some are side-chain fragmented derivatives. Several of these compounds exhibit antimalarial activity.

■

Table 3. Antiplasmidial, Antimycobacterial, and Cytotoxic Activities of the Highly Modified Lanostanes

compound tomophagusin A (1) tomophagusin B (2) tomophagusin C (3) tomophagusin D (4) 22-epi-ganodermalactone G (5) deacetylcolossolactone G (6) 11-oxo-colossolactone E (7) 11-oxo-colossolactone VIII (8) 11β-hydroxycolossolactone D (9) 11β-hydroxycolossolactone VIII (10) ganodermalactone I (11) tomophagusin E (14) tomophagusin G (16) ganodermalactone K (19) ganodermalactone N (22) ganodermalactone O (23) ganodermalactone P (24) ganodermalactone Q (25) ganodermalactone R (26) ganodermalactone S (27) cattienoid E (29) colossolactone G (33) colossolactone VIII (34) colossolactone D (35) colossolactone E (36) colossolactone F (37) ganodermalactone H (38) ganodermalactone B (39) ganodermalactone D (40) colossolactone B (41) colossolactone I (42) cattienoid A (43) ganodermalactone G (46) colossolactone IV (47) colossolactone VII (48) ganodermalactone E (49) colossolactone II (50) dihydroartemisinina isoniazidb ellipticinec

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded on an Analytik-Jena SPEKOL 1200 spectrophotometer. FTIR spectra were acquired on a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. Preparative HPLC experiments were performed using reversed-phase columns: Dionex SunFire Prep C18 OBD, 19 × 250 mm, 10 μm; Phenomenex Luna 10 μ C18(2) 100A, 21.2 × 150 mm, 10 μm; Grace Grom-Sil 120 ODS-4 HE, 20 × 150 mm, 5 μm. Fungal Material. The mushroom specimens were collected from a dead oil palm (Elaeis guineensis) trunk in a private plantation area, Nuea Khlong District, Krabi Province, Thailand, on June 13, 2015. The voucher mushroom specimen was deposited in the BIOTEC Bangkok Herbarium as BBH 40430. Initial identification work by phylogenetic analysis using the voucher specimen had been previously unsuccessful. Fortunately, after 12 months, a large specimen of the same species, apparent from the characteristic basidiome morphology, was collected while regrowing on the same dead oil palm trunk. The quality of the ITS rDNA gene sequence data (GenBank accession number: MK007287) was sufficient for phylogenetic analysis and BLAST search to conclude that the fungus is assignable to the genus Tomophagus of the family Ganodermataceae. Extraction and Isolation. Natural mushroom specimens (523 g) were chopped into small pieces and extracted twice with CH2Cl2 (7 L, 7 days; 6 L, 7 days) to obtain a CH2Cl2 extract (23.0 g). The mushroom residue was extracted twice with MeOH (6 L, 7 days; 6 L, 7 days) to obtain a MeOH extract (6.94 g). The CH2Cl2 extract was subjected to fractionation using column chromatography (CC) on silica gel (8.5 ×

a c

antimalaria

antituberculosis

cytotoxicity

P. falciparum K1

M. tuberculosis H37Ra

Vero cells

IC50, μM

MIC, μg/mL

IC50, μM

>21 7.7 >18 5.1 >20

>50 50 >50 >50 >50

98 38 >92 >104 >102

>20 10 >18

>50 50 >50

>101 92 >90

>20

>50

>101

>18

>50

>90

>20 >18 12 >18 >18 5.5 8.1 6.6 >20 >21 >25 >19 7.0 12 18 >19 18 >19 6.3 >20 >22 >26 >20 19 15 >21 >21 0.0028

>50 >50 >50 >50 >50 >50 50 >50 >50 >50 >50 >50 50 50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 12.5 >50 >50

39 >90 94 >92 >92 88 32 34 >98 36 >125 33 22 42 11 34 >108 >95 32 >98 >110 >130 >102 45 11 >106 >106

0.047 3.1 b

Standard antimalarial drug. Standard antituberculosis drug. Standard compound for the cytotoxicity assay.

15 cm) eluting with a step gradient of acetone−hexane (0:100, 20:80, 40:60, 60:40, 80:20, then 100:0) to obtain 13 pooled fractions, Fr-1− Fr-13. Fr-3−Fr5 (total 1.40 g) contained significant compositions of ergosterol. Fr-6 (2.85 g) was fractionated by silica gel CC (4.8 × 14 cm) eluting with a step gradient of acetone−CH2Cl2, and the fractions were further separated by preparative HPLC using a reversed-phase column (mobile phase MeCN−H2O, 70:30 or 80:20) to furnish 18 (3.0 mg), 26 (32 mg), 30 (2.8 mg), 38 (207 mg), and 42 (101 mg). Fr-7 (3.48 g) was also fractionated by the combination of silica gel CC (4.8 × 14 cm, step gradient of acetone−CH2Cl2) and preparative HPLC (MeCN− I



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11-Oxo-colossolactone VIII (8): colorless solid; [α]24D +70 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 247 (4.14), 304 (4.14) nm; IR (ATR) νmax 3418, 1723, 1663, 1246 cm−1; for 1H NMR (500 MHz) and 13 C NMR (125 MHz) spectroscopic data in CDCl3, see Table S1; HRMS (ESI-TOF) m/z 575.2623 [M + Na]+ (calcd for C32H40O8Na, 575.2621). 11β-Hydroxycolossolactone D (9): colorless solid; [α]26D +106 (c 0.24, CHCl3); UV (MeOH) λmax (log ε) 219 (3.87), 318 (3.97) nm; IR (ATR) νmax 3448, 1700, 1662, 1389, 1296, 1242 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S1; HRMS (ESI-TOF) m/z 519.2711 [M + Na]+ (calcd for C30H40O6Na, 519.2717). 11β-Hydroxycolossolactone VIII (10): colorless solid; [α]24D +114 (c 0.15, CHCl3); UV (MeOH) λmax (log ε) 220 (4.03), 315 (4.07) nm; IR (ATR) νmax 3439, 1721, 1663, 1230 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S2; HRMS (ESI-TOF) m/z 577.2767 [M + Na]+ (calcd for C32H42O8Na, 577.2772). Ganodermalactone I (11): colorless solid; [α]22D +259 (c 0.15, CHCl3); UV (MeOH) λmax (log ε) 216 (4.09), 265 (4.01), 329 (4.24) nm; IR (ATR) νmax 3420, 1718, 1659, 1371 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S2; HRMS (ESI-TOF) m/z 519.2731 [M + Na]+ (calcd for C30H40O6Na, 519.2723). Ganodermalactone J (12): colorless solid; [α]25D +222 (c 0.15, CHCl3); UV (MeOH) λmax (log ε) 217 (3.83), 263 (3.71), 331 (3.93) nm; IR (ATR) νmax 3400, 1718, 1696, 1662, 1372, 1294 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S2; HRMS (ESI-TOF) m/z 503.2764 [M + Na]+ (calcd for C30H40O5Na, 503.2768). 15-Oxo-ganodermalactone H (13): colorless solid; [α]22D +93 (c 0.14, CHCl3); UV (MeOH) λmax (log ε) 219 (3.59), 322 (3.68) nm; IR (ATR) νmax 1731, 1711, 1678, 1292, 1239 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S3; HRMS (ESI-TOF) m/z 501.2616 [M + Na]+ (calcd for C30H38O5Na, 501.2611). Tomophagusin E (14): colorless solid; [α]24D −72 (c 0.09, CHCl3); UV (MeOH) λmax (log ε) 221 (4.05), 228 (4.05), 318 (3.56) nm; IR (ATR) νmax 3495, 1702, 1250 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S3; HRMS (ESI-TOF) m/z 577.2771 [M + Na]+ (calcd for C32H42O8Na, 577.2772). Tomophagusin F (15): colorless solid; [α]25D −32 (c 0.13, CHCl3); UV (MeOH) λmax (log ε) 232 (3.77) nm; IR (ATR) νmax 1710, 1666 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S3; HRMS (ESI-TOF) m/z 575.2618 [M + Na]+ (calcd for C32H40O8Na, 575.2615). Tomophagusin G (16): colorless solid; [α]25D −26 (c 0.34, CHCl3); UV (MeOH) λmax (log ε) 219 (3.72) nm; IR (ATR) νmax 1716, 1241, 1131 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S4; HRMS (ESI-TOF) m/z 547.3041 [M + Na]+ (calcd for C32H44O6Na, 547.3036). Tomophagusin H (17): colorless solid; [α]25D −61 (c 0.15, CHCl3); UV (MeOH) λmax (log ε) 217 (3.97), 331 (3.65) nm; IR (ATR) νmax 3450, 1704, 1131 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S4; HRMS (ESI-TOF) m/z 505.2928 [M + Na]+ (calcd for C30H42O5Na, 505.2924). Tomophagusin I (18): colorless solid; [α]22D +9 (c 0.16, CHCl3); UV (MeOH) λmax (log ε) 217 (3.97), 331 (3.65) nm; IR (ATR) νmax 3455, 1704, 1139 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S4; HRMS (ESI-TOF) m/z 491.3135 [M + Na]+ (calcd for C30H44O4Na, 491.3132). Ganodermalactone K (19): colorless solid; [α]24D −36 (c 0.28, CHCl3); UV (MeOH) λmax (log ε) 216 (3.57) nm; IR (ATR) νmax 1716, 1238, 1133, 1116, 1044 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S5; HRMS (ESI-TOF) m/z 563.2959 [M + Na]+ (calcd for C32H44O7Na, 563.2979). Ganodermalactone L (20): colorless solid; [α]26D +89 (c 0.18, CHCl3); UV (MeOH) λmax (log ε) 238 (3.96), 320 (3.23) nm; IR

H2O, 70:30) to obtain 1 (5.0 mg), 2 (75 mg), 7 (18 mg), 16 (19 mg), 20 (9.0 mg), 21 (2.2 mg), 24 (53 mg), 27 (12 mg), 36 (370 mg), 38 (70 mg), 39 (233 mg), 41 (164 mg), 44 (3.5 mg), and 47 (83 mg). Fr-8 (1.28 g) was also fractionated by the combination of silica gel CC (3.8 × 19 cm, step gradient of acetone−CH2Cl2) and preparative HPLC (MeCN−H2O, 60:40 or 70:30) to afford 12 (7.0 mg), 17 (5.8 mg), 24 (24 mg), 36 (329 mg), 46 (15 mg), and 47 (5.0 mg). Fr-9 (1.76 g) was fractionated by the combination of silica gel CC (3.8 × 16 cm, step gradient of acetone−CH2Cl2) and preparative HPLC (MeCN−H2O, 60:40, 65:35, or 70:30) to furnish 3 (32 mg), 5 (8.0 mg), 7 (35 mg), 13 (2.7 mg), 17 (2.8 mg), 19 (5.0 mg), 20 (14 mg), 22 (39 mg), 25 (8.7 mg), 31 (4.4 mg), 34 (117 mg), 35 (76 mg), 36 (11 mg), 40 (16 mg), 43 (8.6 mg), 46 (5.2 mg), 49 (5.0 mg), and 50 (43 mg). Fr-10 (1.41 g) was fractionated by the combination of silica gel CC (4.8 × 14 cm, step gradient of acetone−CH2Cl2) and preparative HPLC (MeCN−H2O, 52:48, 55:45, or 60:40) to obtain 3 (7.0 mg), 7 (71 mg), 15 (2.8 mg), 20 (4.0 mg), 22 (9.0 mg), 33 (68 mg), 34 (54 mg), 35 (5.8 mg), 37 (171 mg), 39 (28 mg), and 40 (14 mg). Fr-11 (6.56 g) was fractionated by the combination of silica gel CC (4.8 × 12 cm, step gradient of acetone−hexane) and preparative HPLC (MeCN−H2O, 45:55, 52:48, or 60:40) to obtain 4 (39 mg), 6 (12 mg), 7 (18 mg), 8 (49 mg), 9 (11 mg), 10 (47 mg), 11 (28 mg), 14 (14 mg), 23 (167 mg), 28 (21 mg), 29 (4.0 mg), 32 (50 mg), 33 (1.00 g), 34 (8.0 mg), 37 (516 mg), 41 (2.4 mg), and 45 (11 mg). Fr-12 (649 mg) and Fr-13 (920 mg) were subjected to preparative HPLC (MeCN−H2O, 65:35) to furnish 23 (242 mg). The MeOH extract was subjected to silica gel CC (4.8 × 15 cm, step gradient of acetone−hexane), and the fractions were further separated by preparative HPLC (MeCN−H2O, 65:35 or 70:30) to furnish 2 (7.4 mg), 4 (12 mg), 11 (3.0 mg), 24 (31 mg), 33 (65 mg), 36 (83 mg), 37 (103 mg), 38 (15 mg), 39 (9.0 mg), 41 (10 mg), 48 (9.0 mg), and 50 (2.6 mg). Tomophagusin A (1): colorless solid; [α]27D +70 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 217 (3.59), 311 (3.19) nm; IR (ATR) νmax 1737, 1714, 1379, 1139, 1050 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table 1; HRMS (ESI-TOF) m/z 505.2920 [M + Na]+ (calcd for C30H42O5Na, 505.2924). Tomophagusin B (2): colorless solid; [α]25D +70 (c 0.23, CHCl3); UV (MeOH) λmax (log ε) 237 (4.04) nm; IR (ATR) νmax 1717, 1370, 1247, 1138 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table 1; HRMS (ESI-TOF) m/z 547.3042 [M + Na]+ (calcd for C32H44O6Na, 547.3030). Tomophagusin C (3): colorless solid; [α]25D +119 (c 0.16, CHCl3); UV (MeOH) λmax (log ε) 237 (4.12) nm; IR (ATR) νmax 3439, 1722, 1368, 1205, 1137 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table 1; HRMS (ESI-TOF) m/z 563.2988 [M + Na]+ (calcd for C32H44O7Na, 563.2979). Tomophagusin D (4): colorless solid; [α]24D +61 (c 0.21, CHCl3); UV (MeOH) λmax (log ε) 237 (3.98) nm; IR (ATR) νmax 1711, 1383, 1140, 1047 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table 2; HRMS (ESI-TOF) m/z 505.2926 [M + Na]+ (calcd for C30H42O5Na, 505.2924). 22-epi-Ganodermalactone G (5): colorless solid; [α]27D +97 (c 0.48, CHCl3); UV (MeOH) λmax (log ε) 222 (3.91), 324 (4.02) nm; IR (ATR) νmax 1722, 1670, 1293 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table 2; HRMS (ESI-TOF) m/z 515.2403 [M + Na]+ (calcd for C30H36O6Na, 515.2404). Deacetylcolossolactone G (6): colorless solid; [α]26D +17 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 229 (3.91), 330 (3.73) nm; IR (ATR) νmax 3431, 1701, 1133 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table 2; HRMS (ESI-TOF) m/z 519.2731 [M + Na]+ (calcd for C30H40O6Na, 519.2717). 11-Oxo-colossolactone E (7): colorless solid; [α]24D +45 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 247 (4.14), 304 (4.15) nm; IR (ATR) νmax 1734, 1665, 1369, 1237 cm−1; for 1H NMR (400 MHz) and 13 C NMR (100 MHz) spectroscopic data in CDCl3, see Table S1; HRMS (ESI-TOF) m/z 559.2688 [M + Na]+ (calcd for C32H40O7Na, 559.2672). J



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(ATR) νmax 3425, 1720, 1374, 1233, 1138, 1041 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S5; HRMS (ESI-TOF) m/z 563.2948 [M + Na]+ (calcd for C32H44O7Na, 563.2979). Ganodermalactone M (21): colorless solid; [α]24D +71 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 238 (4.00) nm; IR (ATR) νmax 1717, 1376, 1237, 1137, 1044 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S5; HRMS (ESI-TOF) m/z 547.3027 [M + Na]+ (calcd for C32H44O6Na, 547.3030). Ganodermalactone N (22): colorless solid; [α]24D +240 (c 0.14, CHCl3); UV (MeOH) λmax (log ε) 215 (3.79) nm; IR (ATR) νmax 3446, 1722, 1374, 1232, 1137, 1032 cm−1; for 1H NMR (500 MHz) and 13 C NMR (125 MHz) spectroscopic data in CDCl3, see Table S6; HRMS (ESI-TOF) m/z 565.3143 [M + Na]+ (calcd for C32H46O7Na, 565.3136). Ganodermalactone O (23): colorless solid; [α]24D +69 (c 0.22, CHCl3); UV (MeOH) λmax (log ε) 237 (4.04) nm; IR (ATR) νmax 1717, 1373, 1141, 1045 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S6; HRMS (ESITOF) m/z 565.3150 [M + Na]+ (calcd for C32H46O7Na, 565.3136). Ganodermalactone P (24): colorless solid; [α]24D +78 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 237 (4.16) nm; IR (ATR) νmax 1736, 1719, 1373, 1233, 1140, 1045 cm−1; for 1H NMR (400 MHz) and 13 C NMR (100 MHz) spectroscopic data in CDCl3, see Table S6; HRMS (ESI-TOF) m/z 579.3307 [M + Na]+ (calcd for C33H48O7Na, 579.3292). Ganodermalactone Q (25): colorless solid; [α]27D +66 (c 0.34, CHCl3); UV (MeOH) λmax (log ε) 214 (3.67) nm; IR (ATR) νmax 3405, 1736, 1707, 1370, 1235, 1146, 1285 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S7; HRMS (ESI-TOF) m/z 551.3352 [M + Na]+ (calcd for C32H48O6Na, 551.3343). Ganodermalactone R (26): colorless solid; [α]24D +99 (c 0.16, CHCl3); UV (MeOH) λmax (log ε) 216 (3.74) nm; IR (ATR) νmax 1738, 1709, 1384, 1368, 1233, 1132, 1046 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S7; HRMS (ESI-TOF) m/z 533.3247 [M + Na]+ (calcd for C32H46O5Na, 533.3237). Ganodermalactone S (27): colorless solid; [α]26D +48 (c 0.15, CHCl3); UV (MeOH) λmax (log ε) 217 (3.58) nm; IR (ATR) νmax 3426, 1706, 1371, 1145, 1028 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S7; HRMS (ESI-TOF) m/z 493.3295 [M + Na]+ (calcd for C30H46O4Na, 493.3288). Cattienoid D (28): colorless solid; [α]27D +95 (c 0.43, CHCl3); UV (MeOH) λmax (log ε) 238 (3.89) nm; IR (ATR) νmax 1704, 1364, 1214, 1180 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S8; HRMS (ESI-TOF) m/z 409.2339 [M + Na]+ (calcd for C24H34O4Na, 409.2349). Cattienoid E (29): colorless solid; [α]23D +141 (c 0.21, CHCl3); UV (MeOH) λmax (log ε) 226 (3.66), 312 (3.93) nm; IR (ATR) νmax 3443, 1692, 1661, 1298, 1128, 987 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S8; HRMS (ESI-TOF) m/z 423.2131 [M + Na]+ (calcd for C24H32O5Na, 423.2142). Cattienoid F (30): colorless solid; [α]24D +265 (c 0.13, CHCl3); UV (MeOH) λmax (log ε) 218 (3.74), 262 (3.71), 328 (3.92) nm; IR (ATR) νmax 1684, 1292, 1130, 987 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S8; HRMS (ESI-TOF) m/z 391.2241 [M + Na]+ (calcd for C24H32O3Na, 391.2244). Cattienoid G (31): colorless solid; [α]25D +252 (c 0.15, CHCl3); UV (MeOH) λmax (log ε) 219 (3.74), 267 (3.77), 315 (3.98) nm; IR (ATR) νmax 3460, 1734, 1664, 1297, 1129, 986 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Table S9; HRMS (ESI-TOF) m/z 379.1876 [M + Na]+ (calcd for C22H28O4Na, 379.1880). Ganodermalactone H (38): colorless solid; [α]25D +228 (c 0.16, CHCl3); UV (MeOH) λmax (log ε) 221 (3.85), 264 (3.76), 332 (3.96)

nm; IR (ATR) νmax 1710, 1676, 1291, 1132, 987 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table S10; HRMS (ESI-TOF) m/z 487.2815 [M + Na]+ (calcd for C30H40O4Na, 487.2819). Biological Assays. An assay for activity against Plasmodium falciparum (K1, multidrug-resistant strain) was performed in duplicate using the microculture radioisotope technique.13 Anti-mycobacterial activity against Mycobacterium tuberculosis H37Ra and cytotoxicity to Vero cells were evaluated using the green fluorescent protein microplate assay.14,15

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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.8b00869. NMR spectra of compounds 1−32, 38, and 45 and tables of NMR spectroscopic data for 7−32, 38, and 45 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +66 25646700, ext 3554. E-mail: [email protected]. ORCID

Masahiko Isaka: 0000-0002-9229-3394 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Thailand Research Fund (Grant No. DBG5980002) is gratefully acknowledged. We thank Mr. W. Sanyapan for the permission for and technical support of the collection of the mushroom materials used in this study at his oil palm plantation.

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REFERENCES

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