Antitubercular Lanostane Triterpenes from Cultures of the

Article pubs.acs.org/jnp

Antitubercular Lanostane Triterpenes from Cultures of the Basidiomycete Ganoderma sp. BCC 16642 Masahiko Isaka,*,† Panida Chinthanom,† Malipan Sappan,† Kannawat Danwisetkanjana,† Thitiya Boonpratuang,† and Rattaket Choeyklin‡ †

National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand ‡ Biodiversity-Based Economy Development Office, The Government Complex, Chaeng Wattana Road, Bangkok 10210, Thailand S Supporting Information *

ABSTRACT: Sixteen new lanostane triterpenoids (1−16), together with 26 known compounds (17−42), were isolated from cultures of the basidiomycete Ganoderma sp. BCC 16642. Antitubercular activities of these Ganoderma lanostanoids against Mycobacterium tuberculosis H37Ra were evaluated, and structure−activity relationships are proposed.

T

small-scale culture of Ganoderma sp. BCC 16642 exhibited relatively high anti-TB activity with an MIC value of 6.25 μg/ mL, and its 1H NMR spectrum suggested the presence of many lanostanoids. This strain was selected for scale-up fermentation and further chemical analysis. We report here the isolation of 16 new lanostanoids (1−16) together with 26 known derivatives (17−42) (Figure 1) and evaluation of their antiTB activity.

uberculosis (TB) remains one of the world’s deadliest communicable diseases. In 2013, an estimated 9.0 million people developed TB and 1.5 million died of the disease.1 Problematic issues in TB treatment include the increased incidence of multidrug-resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) and comobidity with HIV-AIDS. There is currently a re-emerging interest in natural products being able to provide novel structures for drug discovery and particularly as antibacterial leads.2,3 Fungi in the genus Ganoderma have been known to be a prolific source of highly oxygenated lanostane triterpenoids, such as ganoderic acids, which have been isolated both from fruiting bodies and cell cultures.4−6 As part of our research on the utilization of fungal resources in Thailand, we recently reported the isolation of ganoderic acid derivatives from mycelial cultures of the relatively rare species G. orbiforme, strain BCC 22324, and their antitubercular, antimalarial, and cytotoxic activities.7 In particular, the known C-3 epimer of ganoderic acid T (originally isolated from G. lucidum) was shown to exhibit significant antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MIC 0.781 μg/mL). Although a variety of biological activities of Ganoderma lanostanoids have been evaluated, this is the first report of their anti-TB activity. To discover new ganoderic acid derivatives with more potent anti-TB activity, other species of Ganoderma have been investigated. A mycelial extract from a © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The fungus BCC 16642 was cultured in malt extract broth under static conditions. The cultures were filtered to obtain mycelial cakes, which were extracted with MeOH. The mycelial extract was first fractionated by Sephadex LH-20 column chromatography (CC), and the fractions were subjected to silica gel CC and further separated by reversed-phase preparative HPLC to furnish new compounds 1−5, 8, and 10−16 and the known compounds 17−22, 24, and 27−42. The 7α-methoxy derivatives (10−13 and 27−30) were shown to be isolation artifacts. Thus, refermentation of the fungus (batch 2) and a non-MeOH extraction/isolation procedure (see Experimental Section) led to the isolation of additional compounds, two new (6, 7) and three known (23, 25, and 26) Received: September 14, 2015

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DOI: 10.1021/acs.jnatprod.5b00826 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−42.

an aliphatic ketone (δC 214.8), an α,β-unsaturated ketone (δC 198.2), two carboxyl or ester groups (δH 171.9 and 170.8), three sp2 quaternary carbons (δC 162.8, 139.6, and 129.3), an sp2 methine (δC 139.6/δH 6.81), an oxymethine (δC 74.9/δH 5.09), four sp3 quaternary carbons, three methines, eight methylenes, and eight methyl groups (including an acetyl methyl, δH 2.05) (Tables 1 and 2). The structural elucidation of 1 was accomplished by analysis of COSY and HMBC data (Figure 2). Key HMBC data were the 2J correlations from the six methyl group singlets (H3-18, H3-19, H3-27, H3-28, H3-29, and H3-30) to their attached carbons C-13, C-10, C-25, C-4, C4, and C-14, respectively, and their 3J correlations. The C-3 ketone was assigned on the basis of the HMBC correlations from Hα-2, Hβ-2, H3-28, and H3-29 to this carbon. A tetrasubstituted olefin was assigned to C-8/C-9 by the HMBC correlations from H3-30 to C-8 (δC 139.6) and from H3-19 and Hβ-11 to C-9 (δC 162.8). The C-7 α,β-unsaturated ketone was evident from the HMBC correlations from Hα-6 and Hβ-6 to this carbonyl carbon (δC 198.2) and the downfield chemical shift of C-9 (δC 162.8). The chemical shifts of protons and carbons and the 1H−1H coupling constant values for the C-20−C-27 side-chain were very similar to those of known (22R,24E)-22-acetoxy derivatives. The 24E configuration was confirmed by the NOESY correlations of H2-23/H3-27. The same relative configuration as lanosterol (“normal” relative configuration) was suggested by the NOESY correlations. The correlations from the equatorial methyl group (H3-28, δC 25.6) to H-5 (axial) and Hα-6 (equatorial) suggested their being in α-orientations. NOESY correlations from Hβ-6 (axial) to H3-19

7α-hydroxy derivatives and a new 7α-acetoxy derivative (9). The 7α-methoxy derivatives were absent in the fermentation batch 2 mycelial extract, while the 7α-hydroxy derivatives were isolated in larger quantities than batch 1. Since the structural variations of the mycelial Ganoderma lanostanoids were the functional groups at C-3, C-7, C-15, and C-22, the isolated compounds were initially identified by 1H NMR spectroscopy (CDCl3) and ESIMS. Structures of the new compounds (1−16) were further confirmed by analysis of 2D NMR spectroscopic data. The known compounds were identified by comparison of the spectroscopic data with those reported in the literature; these are ganorbiformin C (17),7 astraodoric acid A (18),8 astraodoric acid B (19),8 20,9 astraodoric acid C (21),8 ganorbiformin E (22),7 ganoderic acid V (23),7,10 ganorbiformin D (24),7 25,11 ganoderic acid Mb (26),12 ganorbiformin F (27),7 ganoderic acid Md (28),12 7-Omethylganoderic acid O (29),13 ganoderic acid Mi (30),14 ganorbiformin G (31),7 32,15 ganoderic acid TR (33),16,17 34,15 35,18 ganoderic acid R (36),19 ganoderic acid T (37),19 38,20 ganoderic acid S (39),19 ganoderic acid X (40),10,21 41,22 and ganorbiformin A (42).7 Compound 1 was isolated as a colorless solid, and the molecular formula was determined to be C32H46O6 by HRESIMS. Its IR spectrum exhibited intense overlapped carbonyl absorption bands at νmax 1733 and 1725 (sh) cm−1. The 1H and 13C NMR spectroscopic data suggested that 1 was a triterpenoid bearing one acetoxy group, and the skeleton was similar to the known lanostane co-metabolites. The 1H and 13C NMR, DEPT, and HMQC data for 1 supported the presence of B



Article

Table 1. 13C NMR Spectroscopic Data for Compounds 1−12 in CDCl3 no.

1

2

3

4

5

6

7

8

9

10

11

12

13

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 3-OCOCH3 3-OCOCH3 7-OCOCH3 7-OCOCH3 7-OCH3 15-OCOCH3 15-OCOCH3 22-OCOCH3 22-OCOCH3

35.6 34.6 214.8 47.5 50.6 37.4 198.2 139.6a 162.8 39.6 24.0 30.4 45.1 48.1 32.0b 28.6 45.9 15.9 18.2 39.7 13.3 74.9 32.0b 139.6a 129.3 171.9 12.5 25.6 21.6 25.2

29.9 23.4h 76.8 36.7 45.2 36.4 198.7 138.9 165.0 39.7 23.0h 30.1 44.9 47.9 31.8i 28.5 45.7 15.6 18.3 39.6 13.1 74.8 32.0i 139.2 129.0 171.5 12.4 26.9 21.6 25.0 170.6c 21.3j

36.0k 34.5 217.4 47.4 51.0 19.3 26.3 133.8 134.3 37.0 20.8 31.4 44.5 51.2 75.5 36.2k 45.7 16.0 18.6 39.9 12.7 74.4 31.9 139.0 129.3 171.6 12.3 26.2 21.3l 18.3

35.4 24.3 81.0 38.0 50.5 18.3 26.5 133.1 135.6 37.2 21.0 31.3 44.8 51.3 75.8 36.4 45.9 16.1 19.4 40.1 12.9 74.7 32.1 138.9 129.5 171.4 12.6 28.1 16.7 18.5 171.2m 21.2n

35.5 27.8 78.9 38.9 50.2 18.2 26.4 132.8 135.6 37.1 20.8 31.2 44.5 51.1 75.6 36.2 45.7 15.9 19.1 39.9 12.7 75.5 31.9 139.0 129.2 171.4 12.3 28.0 15.4 18.2

34.9d 24.1 80.7 37.4 45.3e 28.9 67.4 136.2 141.0 38.2 21.1 31.1 45.3e 49.7 30.0 28.2 50.7 16.2 17.5 36.5 18.5 34.9d 25.9 145.4 126.5 171.4 12.0 27.9 16.8 26.3 170.8 21.2

34.6 24.0 80.5 37.3 44.8 27.3 66.7 134.1 141.4 38.4 20.7 31.1 45.4 51.1 76.4 36.5 49.2 16.5 17.6 36.2 18.2 34.6 25.9 144.9 126.7 172.1 12.1 27.9 16.8 20.1 170.9o 21.3p

34.6 24.0 80.5 37.3 44.8 27.4 66.6 134.0 141.4 38.4 20.7 31.1 45.2 51.2 76.0 36.2 45.8 16.3 17.6 39.9 12.6 74.3 31.9 138.8 129.3 171.4 12.4 27.8 16.8 20.2 170.9q 21.2r

35.2 34.3 216.8 45.6 45.3 26.8 69.5 132.0 143.2 38.0 21.2 31.2 46.5 52.0 72.4 39.6 48.8 16.4 17.7s 36.2 18.3 34.6 25.8 145.0 126.8 171.7 12.1 26.3 21.1 17.5s

35.3 34.3 217.5 46.7 45.0f 23.3 76.2 135.4 139.5 37.8 21.0 31.1 45.0f 49.9 30.2 28.2 50.6 16.2 17.3 36.4 18.4 34.8 25.9 145.6 126.4 171.5 12.0 26.5 21.3 25.3

34.7 24.0 80.6 37.4 45.1 22.2 76.6 134.7 140.8 38.0 20.9 31.1 44.9 49.9 30.0 27.6 47.2 15.9 17.6 39.7 12.8 74.8 31.8 139.3 129.2 171.7 12.3 27.6 16.9 25.5 170.7t 21.0u

34.5 24.0 80.3 37.3 44.2 21.4 76.6 133.1 142.3 38.7 21.1 30.8 45.6 51.6 75.3 37.1 45.2 16.2 17.7 39.9 12.7 74.5 31.9 138.6 129.2 172.0 12.4 27.8 17.1 19.0 171.2v 21.7w

34.7g 24.0 80.3 37.4 44.8 21.3 76.5 133.2 143.1 38.4 20.9 31.9 46.2 52.7 72.0 37.8 49.9 16.7x 17.3 36.2 18.2 34.7g 25.8 144.9 127.3 171.8 12.0 27.8 16.8x 18.4 170.8 21.2

55.8

55.6

55.2 170.7v 21.0w 170.8v 21.3w

54.4

a−g

171.8 21.6

170.8 21.2

Overlapping signals.

170.6c 21.0j h−x

170.6 21.0l

171.2m 21.6n 170.0m 21.5n

170.7o 21.2p

170.6 21.0 171.0 21.4

170.7q 21.1r 170.5q 21.3r

170.8t 21.3u

The carbon assignment may be interchanged.

and H3-29 (axial, δC 21.6) were indicative of their being in βorientations. The NOESY cross-peak for H3-30/H-17 revealed their axial (α) orientations. Intense NOESY correlations of H318/H-20 and H2-16/H-22 were consistent with the NOESY spectroscopic data of the known 22-acetoxy derivatives of the normal C-17/C-20/C-22 configuration. Consequently, compound 1 was identified as a new Ganoderma lanostanoid, (22S,24E)-22-acetoxy-3,7-dioxolanosta-8,24-dien-26-oic acid. Compound 2 was assigned the molecular formula C34H50O7 by HRESIMS, which suggested the presence of two acetoxy groups. The 1H and 13C NMR spectra displayed similarity to those of 1. The significant difference was the presence of an acetoxy-bearing methine (δH 4.70, δC 76.8) replacing the C-3 ketone in 1. Location of this oxymethine (C-3) was assigned on the basis of the HMBC correlations from H-3 to C-1 and C-5 and the correlations from the geminal dimethyl group (H3-28 and H3-29) to C-3. The equatorial (β) orientation of H-3 was evident from the narrow peak width of this broad multiplet proton signal. Therefore, compound 2 was identified as

(22S,24E)-3α,22-diacetoxy-7-oxolanosta-8,24-dien-26-oic acid, which is the C-3 epimer of ganorbiformin B.7 The molecular formula of compound 3 was determined by HRESIMS as C34H50O7, a diacetoxy Ganoderma lanostanoid. The 1H and 13C NMR spectra of 3 suggested the same sidechain structure as 1 and 2, with a 22S-acetoxy group and 24Egeometry of the α,β-unsaturated carboxylic acid. Other functional groups in the tetracyclic core were a tetrasubsituted olefin, a ketone (δC 217.4), and an oxygenated methine (δC 75.5, δH 5.06, d, J = 9.5, 5.4 Hz) bonded with an acetoxy group. The location of the tetrasubstituted olefin was assigned to the C-8/C-9 position by HMBC correlations from H3-30 and Hβ-6 to C-8 (δC 133.8) and the correlation from H3-19 to C-9 (δC 134.3). The C-3 ketone functional group was assigned by the HMBC correlations from H3-28 and H3-29 to the ketone carbon. The 15α-acetoxy group was revealed by the HMBC correlations from H3-30 and Hα-16 (δH 1.81, m) to the oxymethine carbon C-15. The NOESY correlations H-15/H318, H-15/Hβ-16 (δH 2.08, m), and H3-30/H-17 confirmed the C



Article

Table 2. 1H NMR Spectroscopic Data for Compounds 1−5 in CDCl3 no.

1

2

3

4

5

1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 3-OAc 15-OAc 22-OAc

α 2.09, m; β 1.80, m α 2.46, m; β 2.70, m

α 1.59, m; β 1.75, m α 1.74, m; β 1.94, m 4.70, m 2.07, m α 2.33, m; β 2.40, m

α 1.61, m; β 1.97, m α 2.40, m; β 2.58, m

α 1.31, m; β 1.71, m α 1.68, m; β 1.61, m 4.48, dd (11.6, 4.3) 1.10, br d (12.1) α 1.65, m; β 1.49, m α 2.08, m; β 1.88. m 2.06−2.03, m 1.85, m; 1.64, m 5.03, m 2.06, m; 1.81, m 1.74, m 0.77, s 0.99, s 1.50, m 0.95, m 5.01, m 2.55, m; 2.32, m 6.75, t (7.0) 1.84, br s 0.87, s 0.87, s 0.96, s 2.05,a s 2.05,b s 2.03,b s

α 1.22, m; β 1.73, m α 1.68, m; β 1.60, m 3.22, dd (11.7, 4.4) 1.01, m α 1.66, m; β 1.49, m α 2.02, m; β 1.88, m 2.07−2.04 (2H), m 1.82, m; 1.64, m 5.03, m 2.06, m; 1.80, m 1.73, m 0.77, s 0.97, s 1.51, m 0.96, m 5.02, m 2.55, m; 2.33, m 6.76, t (7.0) 1.85, s 0.99, s 0.80, s 0.97, s

2.14, m α 2.34, m; β 2.54, m 2.34−2.31 (2H), m 1.80−1.78 (2H), m 2.08, m; 1.69, m 2.07, m; 1.34, m 1.57, m 0.67, s 1.33, s 1.53, m 1.00, d (6.3) 5.09, t (7.0) 2.56, m; 2.35, m 6.81, t (7.0) 1.86, br s 1.09, s 1.11, s 0.91, s

2.36, m; 2.28, m 1.79−1.77 (2H), m 2.08, m; 1.73, m 2.03, m; 1.35, m 1.54, m 0.66, s 1.19, s 1.58, m 1.00, d (6.4) 5.10, t (7.0) 2.57, m; 2.36, m 6.80, t (7.0) 1.87, br s 0.87, s 0.99, s 0.94, s 2.08,a s

2.05, s

2.06,a s

1.58, m 1.61−1.59 (2H), m α 2.07, m; β 1.92, m 2.09−2.08 (2H), m 1.87, m; 1.67, m 5.06, dd (9.5, 5.4) 2.08, m; 1.81, m 1.74, m 0.99, s 1.11, s 1.52, m 0.97, d (6.8) 5.02, t (7.1) 2.57, m; 2.33, m 6.77, t (7.0) 1.86, br s 1.08, s 1.06, s 0.98, s 2.04, s 2.05, s

2.05, s 2.03, s

a,b

The assignments may be interchanged.

lanostanoid, (24E)-3β-acetoxy-7α-hydroxylanosta-8,24-dien-26oic acid. Compound 7 had the molecular formula C34H52O7 (HRESIMS), and its NMR spectroscopic data showed close similarity to those of 6. The only significant difference was the presence of a 15α-acetoxy group (δC 76.4; δH 5.13, dd, J = 9.5, 5.9 Hz). Compound 8 was assigned the molecular formula C36H54O9 by HRESIMS. It possessed an additional acetoxy group at C-22 (δC 74.3; δH 5.02, br t, J = 7.0 Hz) and was identified as (22S,24E)-7α-hydroxy-3β,15α,22-triacetoxylanosta-8,24-dien-26-oic acid. Compound 9 had the molecular formula C32H48O6 as determined by HRESIMS. The 1H and 13C NMR spectroscopic data revealed the presence of a ketone (δC 216.8), a hydroxy group (δC 72.4; δH 4.27, dd, J = 9.6, 5.8 Hz), and an acetoxy (δC 69.5; δH 5.47, br s) group in addition to the C-8/C-9 double bond and an E-configured α,β-unsaturated carboxylic acid. The C-3 ketone was confirmed by the HMBC correlations from Hβ-1, Hα-2, Hβ-2, H3-28, and H3-29 to the carbonyl carbon. The 7α-acetoxy group was revealed by the HMBC correlations from the oxymethine proton (H-7) to C-5, C-8, and C-9. The 15α-hydroxy group was evident from the HMBC correlations from the oxymethine proton (H-15) to C-8 and C30 and the correlations from Hα-16, Hβ-16, and H3-30 to the oxymethine carbon (C-15). The NOESY correlations from H15 to H-7, H3-18, and Hβ-16 indicated the β-orientations of H15 and H-7. Therefore, compound 9 was identified as (24E)7α-acetoxy-15α-hydroxy-3-oxolanosta-8,24-dien-26-oic acid. The molecular formula of compound 10 was assigned by HRESIMS as C31H48O4. The 1H and 13C NMR spectroscopic data suggested the presence of a C-3 ketone (δC 217.5, assigned by HMBC correlations) and a methoxy group (δH 3.32, 3H, s). An intense HMBC correlation from the methoxy protons to the oxymethine carbon (δC 76.2, C-7) confirmed the location of

Figure 2. COSY and key HMBC correlations for 1.

normal relative configuration of ring D. Consequently, compound 3 was identified as a new lanostanoid, (22S,24E)15α,22-diacetoxy-3-oxolanosta-8,24-dien-26-oic acid. Compound 4 was assigned the molecular formula C36H54O8 by HRESIMS. The NMR spectroscopic data were similar to those of 3. The significant difference was the presence of an additional acetoxy-bearing methine (δC 81.0, δH 4.48, J = 11.6, 4.3 Hz) replacing the C-3 ketone in 3. The axial (α) orientation of H-3 was evident from its coupling constant values. Compound 5 had the molecular formula C34H52O7 (HRESIMS) and was assigned as the 3-O-deacetyl analogue of 4. The coupling constant values of H-3 (δH 3.22, dd, J = 11.7, 4.4 Hz) indicated its axial orientation. The molecular formula of compound 6 was determined by HRESIMS as C34H52O7. The 1H and 13C NMR spectroscopic data demonstrated the presence of a 3β-acetoxy group, the absence of a 22-acetoxy group, and the presence of a 7αhydroxy group (δC 67.4; δH 4.20, br s, equatorial). The location of this hydroxy group (axial) was assigned by comparison of the NMR data with those of the known compounds (22−26) and was further confirmed by the HMBC correlations from H-7 to C-5 and C-8. Accordingly, compound 6 was identified as a new D



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isolated from the cultured mycelium of G. lucidum by methanol extraction, but it was not present in the benzene extract. New compounds, excluding those of limited availability (9, 10, 14, and 16), were subjected to our bioassay protocols to determine anti-TB activity against Mycobacterium tuberculosis H37Ra and cytotoxicity against nonmalignant Vero cells (Table

the methoxy group. Similar to the known 7α-methoxy derivatives (27−30), H-7 exhibited very small coupling constants (δH 3.67, br s), which confirmed its equatorial (β) orientation. Consequently, compound 10 was identified as a new Ganoderma lanostanoid, (24E)-7α-methoxy-3-oxolanosta8,24-dien-26-oic acid. Compounds 11−13 were also identified as new 7α-methoxy derivatives. Other key functional groups of 11 (molecular formula C35H54O7, HRESIMS) were 3β-acetoxy and 22-acetoxy groups. The structure of compound 12 (molecular formula C37H56O9, HRESIMS) was similar to 11 with only an additional 15α-acetoxy group. Other key functional groups of compound 13 (molecular formula C33H52O6, HRESIMS) were 3β-acetoxy and 15α-hydroxy groups. Compound 14 had the molecular formula C32H46O4 as determined by HRESIMS. The 1H and 13C NMR spectroscopic data were similar to those of the known 7,9(11)-diene-type lanostanes (31−41). Interpretation of the 2D NMR (COSY, HMQC, and HMBC) data indicated the presence of the 3βacetoxy group, and it was identified as (24E)-3β-acetoxylanosta7,9(11),24-trien-26-oic acid. The molecular formula of compound 15 was assigned by HRESIMS as C34H50O6. The NMR spectroscopic data were similar to those of 14, possessing a conjugated 7,9(11)-diene and a 3β-acetoxy functional group in addition to an E-configured α,β-unsaturated carboxylic acid. The key difference was that 15 additionally possessed a 22acetoxy group. Compound 16 had the molecular formula C33H52O5 (HRESIMS), and it was assigned as the 3-O-deacetyl analogue of 14. The axial (α) orientation of H-3 (δH 3.25, dd, J = 11.4, 4.3 Hz) was evident from its coupling constants and the intense NOESY correlation of H-3/H-5. In the present study and the previous chemical investigation of G. orbiforme BCC 22324,7 we observed occasional conversion of 7α-hydroxy derivatives to 7,9(11)-diene isomers in the separation and purification by silica gel column chromatography. The same vinylogous dehydration was also occasionally found in the NMR spectroscopic analysis using CDCl3, due to the trace contamination of acid. This rare event has been successfully avoided by passing the NMR solvent (CDCl3) through a short column on aluminum oxide (Merck, aluminum oxide 90 active basic) prior to sample preparation. Recently, Li and co-workers reported the isolation and stability of a 7α-hydroxy derivative, 25.11 Assessment of the stability of 25 in various solvents revealed that the degradation of 25 is acid-catalyzed. On the other hand, heating 25 in MeOH led to slow conversions into ganoderic acid Md (28) and ganoderic acid R (36).11 In the present study, we also confirmed the acid sensitivity of the 7α-hydroxy derivatives. Treatment of ganobiformin E (22) with TsOH·H2O in MeOH at room temperature for 1 h led to its complete degradation, giving ganorbiformin G (31) as the sole product. No degradation of 22 occurred when a solution in MeOH−CH2Cl2 (1:1) was stirred with silica gel for 2 days. Ganobiforbin F (27), a 7αmethoxy isomer, was also shown to be acid sensitive, giving 31 by treatment with catalytic TsOH·H2O in MeOH. As mentioned earlier in this report, the 7α-methoxy derivatives should be isolation artifacts. The 1H NMR spectrum of the crude methanolic mycelial extract (fermentation batch 1) exhibited several resonances of 7α-methoxy groups (δH 3.40− 3.10), which suggested the intracellular conversion of the 7αhydroxy derivatives to 3α-methoxy isomers. This transformation in the extraction step was previously reported by Shiro and co-workers.13 7-O-Methylganoderic acid O (29) was

Table 3. 1H NMR Spectroscopic Data for Compounds 6−9 in CDCl3 no. 1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 3-OAc 7-OAc 15OAc 22OAc a,b

6

7

8

α 1.32, m; β 1.77, m α 1.76, m; β 1.64, m 4.54, dd (11.9, 4.2) 1.42, m

α 1.34, m; β 1.71, m α 1.73, m; β 1.62, m 4.53, dd (11.8, 4.1) 1.55, m

α 1.75, m; β 1.70, m 4.20, br s 2.08−2.02 (2H), m 1.77, m; 1.65, m 1.73, m; 1.42, m 2.00, m; 1.36, m 1.55, m 0.61, s 0.97, s 1.40, m 0.93, d (6.5) 1.56, m; 1.18, m 2.25, m; 2.11, m 6.90, br t (6.9) 1.84, s 0.92, s 0.89, s 1.13, s 2.06, s

α 1.76, m; β 1.68, m 4.12, br s 2.10−2.05 (2H), m 1.87, m; 1.64, m 5.13, dd (9.5, 5.9) 2.17, m; 1.75, m 1.68, m 0.70, s 0.98, s 1.38, m 0.91, m 1.50, m; 1.15, m 2.21, m; 2.09, m 6.84, t (6.6) 1.82, s 0.92, s 0.89, s 1.15, s 2.09,a s

α 1.34, m; β 1.72, m α 1.75, m; β 1.63, m 4.53, dd (11.4, 4.3) 1.55, dd (11.8, 1.8) α 1.74, m; β 1.70, m 4.11, br s 2.11, m; 2.05, m 1.88, m; 1.61, m 5.09, dd (9.7, 6.0) 2.15, m; 1.87, m 1.77, m 0.70, s 0.97, s 1.51, m 0.97, d (6.9) 5.02, t (7.0) 2.56, m; 2.33, m 6.75, t (6.5) 1.86, s 0.92, s 0.89, s 1.12, s 2.08,b s

9 α 1.75, m; β 1.99, m α 2.50, m; β 2.57, m

2.04, m α 1.67, m; β 1.77, m 5.47, br s 2.14−2.08 (2H), m 1.89, m; 1.68, m 4.27, dd (9.6, 5.8) 1.90, m; 1.76, m 1.66, m 0.68, s 1.08, s 1.36, m 0.91, d (6.3) 1.50, m; 1.19, m 2.23, m; 2.10, m 6.85, t (7.5) 1.83, s 1.04, s 1.04, s 1.04, s 2.02, s

2.06,b s 2.05,a s

2.06,b s


5). Activities of some known compounds (18, 20, 33, and 35), which have not been previously tested in our research group, were also evaluated. For comparison of the activities of Ganoderma lanostanoids, our previously reported data for the compounds isolated from G. orbiforme BCC 22324 are also listed in Table 5.7 All the high anti-TB activity compounds that showed MIC values of 3.13 μg/mL or less (4, 7, 8, 20, and 38) possessed a 3β-acetoxy group. Among these compounds, 20 and 38 showed the lowest MIC value of 0.781 μg/mL. Therefore, this functional group (3β-OAc) appears to be crucial for the anti-TB activity of the lanostanoids evaluated. Comparison of the anti-TB MIC values among the 3β-acetoxy derivatives suggested further structure−activity relationships. The MIC values of the 3β,13α,22-triacetoxy derivatives, 4, 8, 12, and 38, which differ only in the functional groups at C-7, E



Article

Table 4. 1H NMR Spectroscopic Data for Compounds 10− 13 in CDCl3 no. 1 2

10 α 1.71, m; β 1.94, m α 2.48, m; β 2.51, m

3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24

1.99, dd (13.9, 1.9) α 1.86, m; β 1.48, m 3.67, br s 2.11−2.08 (2H), m 1.80, m; 1.69, m 1.69, m; 1.38, m 2.02, m; 1.37, m 1.58, m 0.63, s 1.04, s 1.40, m 0.93, d (6.4) 1.57, m; 1.18, m 2.26, m; 2.10, m 6.90, br t (6.9) 1.85, s 1.12, s 1.09, s 1.04, s

27 28 29 30 3-OAc 73.32, s OCH3 15-OAc 22-OAc a

11

12 α 1.33, m; β 1.71, m α 1.74, m; β 1.61, m 4.53, dd (11.9, 4.4) 1.58, d (12.3)

α 1.90, d (12.1); β 1.45, m 3.60, br s 2.06, m; 2.04, m 1.77, m; 1.63, m 1.68, m; 1.38, m 2.04, m; 1.36, m 1.68, m 0.59, s 0.97, s 1.51, m 0.98, d (6.4) 5.09, t (7.0)

α 1.96, br d (14.1); β 1.36, m 3.55, br s 2.08, m; 2.06, m

2.56, m; 2.37, m 6.80, t (7.0)

2.55, m; 2.32, m 6.74, t (6.8)

4.04, m 2.11, m; 2.05, m 1.85, m; 1.60, m 4.34, dd (8.8, 6.6) 1.87, m; 1.84, m 1.67, m 0.64, s 0.98, s 1.34, m 0.89, d (6.5) 1.53, m; 1.18, m 2.24, m; 2.11, m 6.87, t (7.0)

1.86, 0.90, 0.90, 1.00, 2.04, 3.27,

s s s s s s

1.84, s 0.89, s 0.89, s 1.15, s 2.09,a s 3.17, s

1.83, 0.92, 0.91, 1.00, 2.06, 3.30,

2.04, s

2.05,a s 2.04,a s

5.09, dd (10.3, 5.2) 2.13, m; 1.80, m 1.79, 0.69, 0.98, 1.49, 0.96, 5.00,

m s s m d (6.7) t (7.0)

■

13

α 1.33, m; β 1.72, m α 1.71, m; β 1.63, m 4.52, dd (11.6, 4.2) 1.48, m

1.85, m; 1.61, m

the MIC values suggested that the 3β-acetoxy and 7α-acetoxy groups are crucial for the anti-TB activity.

α 1.31, m; β 1.74, m α 1.74, m; β 1.61, m 4.53, dd (11.9, 4.0) 1.42, d (12.9) α 2.07, m; β 1.50, m

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured with an Electrothermal IA9100 digital melting point apparatus. Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded on a GBC Cintra 404 spectrophotometer. FTIR spectra were taken 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. Fungal Material. The fungus used in this study was isolated from an unidentified wood sample from Doi Suthep-pui National Park, Chang Mai Province, Thailand. The living culture was deposited in the BIOTEC Culture Collection on September 6, 2004, as BCC 16642. The ITS rDNA sequence data (GenBank accession number: KR709152) and the results from a BLAST search showed 98% identity to Ganoderma fornicatum and Ganoderma mastoporum. Therefore, the strain (BCC 16642) was assigned to the genus Ganoderma of the family Ganodermataceae. Fermentation, Extraction, and Isolation. Batch 1. The fungus BCC 16642 was maintained on potato dextrose agar at 25 °C. The agar was cut into small plugs and inoculated into 4 × 250 mL Erlenmeyer flasks containing 25 mL of malt extract broth (MEB; malt extract 6.0 g/L, yeast extract 1.2 g/L, maltose 1.8 g/L, dextrose 6.0 g/ L). After incubation at 25 °C for 8 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1000 mL Erlenmeyer flask containing 250 mL of the same liquid medium (MEB) and incubated at 25 °C for 8 days on a rotary shaker (200 rpm). The secondary cultures were pooled, each 25 mL portion was transferred into 40 × 1000 mL Erlenmeyer flasks containing 250 mL of MEB, and the final fermentation was carried out at 25 °C for 95 days under static conditions. The cultures were filtered, and the residual wet mycelia were macerated in MeOH (2 L, 25 °C, 2 days) and filtered. Hexanes (2.7 L) were added to the filtrate, and the layers were separated. The MeOH (bottom) layer was partially concentrated by evaporation, and the residue was extracted with EtOAc (2.1 L), which was concentrated under reduced pressure to obtain a brown gum (mycelial extract, 2.15 g). The mycelial extract was passed through a Sephadex LH-20 column (3.7 × 60 cm) eluted with MeOH. The lanostanoid-containing fractions were combined (1.89 g) and subjected to column chromatography on silica gel (3.8 × 20 cm, EtOAc−CH2Cl2, step gradient elution); the fractions were further fractionated and purified by preparative HPLC using reversed-phase columns (Phenomenex Luna 10u C18 100A, 21.2 × 250 mm, 10 μm, or Grace Grom-Sil 120 ODS-4 HE, 20 × 150 mm, 5 μm; mobile phase MeCN−H2O, proportions 55:45−80:20; flow rate 8 mL/min) to furnish pure compounds 1 (6.0 mg), 2 (7.8 mg), 3 (4.8 mg), 4 (7.1 mg), 5 (5.5 mg), 8 (5.0 mg), 10 (2.0 mg), 11 (15 mg), 12 (4.6 mg), 13 (7.0 mg), 14 (30 mg), 15 (26 mg), 16 (1.8 mg), 17 (3.0 mg), 18 (7.3 mg), 19 (3.5 mg), 20 (9.1 mg), 21 (1.1 mg), 22 (36 mg), 24 (9.6 mg), 27 (48 mg), 28 (7.7 mg), 29 (4.1 mg), 30 (3.9 mg), 31 (39 mg), 32 (55 mg), 33 (18 mg), 34 (28 mg), 35 (6.4 mg), 36 (27 mg), 37 (43 mg), 38 (22 mg), 39 (14 mg), 40 (2.9 mg), 41 (8.0 mg), and 42 (9.4 mg). Fermentation, Extraction, and Isolation. Batch 2 (NonMeOH Procedures). The fermentation was conducted at the same scale and conditions as batch 1. The cultures were filtered, and the residual wet mycelia were macerated in acetone (2.8 L, 25 °C, 2 days) and filtered. The filtrate was concentrated under reduced pressure, and the residue was extracted with EtOAc (3.7 L) and concentrated under reduced pressure to obtain a brown gum (mycelial extract, 2.18 g). The mycelial extraction was repeated once again (1.23 g). The combined mycelial extract was subjected to CC on silica gel (EtOAc− CH2Cl2, step gradient elution), and the fractions were further fractionated and purified by preparative HPLC (MeCN−H2O) to furnish pure compounds 1 (5.0 mg), 2 (5.2 mg), 3 (1.7 mg), 4 (0.6 mg), 5 (8.3 mg), 6 (4.0 mg), 7 (5.0 mg), 8 (26 mg), 9 (7.0 mg), 15 (21 mg), 16 (3.5 mg), 17 (6.0 mg), 18 (8.5 mg), 20 (15 mg), 22 (130

s s s s s s


revealed that 7,9(11)-diene isomer 38 showed higher antimycobacterial activity than the 8-ene congeners. In addition, the bulky 7α-methoxy group (12) reduces the activity. Comparison of the MIC values of 15 (12.5 μg/mL) and 38 (0.781 μg/mL) indicated that the 17α-acetoxy group also plays an important role in the mycobacterial growth inhibition. The MIC values of 4 (3.13 μg/mL) and 20 (0.781 μg/mL) suggested that a 22-acetoxy group reduces the activity. Cytotoxic activities of these compounds were relatively weaker when compared with their anti-TB activity, and there was no clear correlation in the inhibitory potential between these biological activities. In conclusion, the present results demonstrate that the unidentified Ganoderma species (strain BCC 16642) isolated in northern Thailand is a rich source of lanostanoids. There are many overlaps of the same compounds with those from the most popular medicinal species such as G. lucidum and other closely related species including G. orbiforme; however, the strain studied provided 16 new analogues. Several of these compounds possess significant anti-TB activity. Comparison of F



Article

Table 5. NMR Spectroscopic Data for Compounds 14−16 in CDCl3 14 no.

δC, mult.

1 2 3 4 5 6 7 8 9 10 11 12

35.6, 24.5, 81.1, 37.8, 49.5, 23.1, 120.2, 142.9, 145.9, 37.5, 116.7, 38.1,

CH2 CH2 CH qC CH CH2 CH qC qC qC CH CH2

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3-OCOCH3 3-OCOCH3 22-OCOCH3 22-OCOCH3

44.0, 50.6, 31.7, 28.1, 51.1, 15.9, 23.0, 36.4, 18.5, 35.0, 26.1, 145.7, 126.9, 172.7, 12.3, 29.3, 17.2, 25.7, 171.2, 21.5,

qC qC CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH2 CH qC qC CH3 CH3 CH3 CH3 qC CH3

15

δH, mult. (J in Hz) α 1.52, m; β 1.99, m α 1.72, m; β 1.68, m 4.59, m 1.18, m α 2.07, m; β 2.06, m 5.46, m

5.31, br d (5.8) α 2.21, br d (5.8) β 2.09, m

1.62, 1.97, 1.57, 0.56, 1.00, 1.42, 0.92, 1.56, 2.24, 6.88,

m; 1.39, m; 1.31, m s s m d (6.4) m; 1.17, m; 2.11, t (6.8)

1.83, 0.89, 0.95, 0.88,

s s s s

2.06, s

m m

m m

δC, mult. 35.4, 24.2, 80.8, 37.6, 49.3, 22.8, 120.3, 142.4, 145.8, 37.3, 116.2, 37.8,

CH2 CH2 CH qC CH CH2 CH qC qC qC CH CH2

43.7, qC 50.3, qC 31.4, CH2 27.6, CH2 47.4, CH 15.5, CH3 22.7, CH3 39.4, CH 12.7, CH3 74.7, CH 31.9, CH2 139.3, CH 129.2, qC 172.0, qC 12.3, CH3 28.1, CH3 16.9, CH3 25.6, CH3 170.7, qC 21.3,a CH3 171.0, qC 21.0,a CH3

16

δH, mult. (J in Hz)

δC, mult.

δH, mult. (J in Hz)

α 1.52, m; β 1.98, m α 1.72, m; β 1.68, m 4.51, dd (11.4, 4.5)

35.7, CH2 27.8,b CH2 78.9, CH 38.7, qC 49.1, CH 23.0, CH2 120.6, CH 142.3, qC 146.1, qC 37.4, qC 115.9, CH 37.8, CH2

α 1.43, m; β 1.98, m α 1.71, m; β 1.66, m 3.25, dd (11.4, 4.3)

1.18, dd (10.1, 5.4) α 2.07, m; β 2.05, m 5.47, m

5.31, d (5.9) α 2.05, m β 2.22, m

1.62, 2.05, 1.70, 0.55, 0.99, 1.53, 0.98, 5.09, 2.56, 6.80,

m; 1.40, m m; 1.30, m m s s m d (6.7) t (7.0) m; 2.37, m t (7.0)

1.86, 0.88, 0.95, 0.85,

s s s s

43.7, qC 50.3, qC 31.4, CH2 27.6,b CH2 47.4, CH 15.5, CH3 22.7, CH3 39.5, CH 12.7, CH3 74.8, CH 31.9, CH2 139.2, CH 129.1, qC 171.8, qC 12.4, CH3 28.1, CH3 15.8, CH3 25.6, CH3

1.09, m α 2.09, m; β 2.06, m 5.48, m

5.30, br d (6.0) α 2.21, br d (17.3) β 2.04, m

1.62, 2.05, 1.69, 0.55, 0.98, 1.54, 0.98, 5.09, 2.56, 6.79,

m; 1.41, m m; 1.32, m m s s m d (6.5) t (7.0) m; 2.35, m t (7.0)

1.86, 1.00, 0.88, 0.86,

s s s s

2.06, s 2.06, s

171.0, qC 21.0, CH3

2.06, s

a,b

The assignments may be interchanged. cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESITOF) m/z 637.3719 [M + Na]+ (calcd for C36H54O8Na, 637.3711). (22S,24E)-15α,22-Diacetoxy-3β-hydroxylanosta-8,24-dien-26-oic acid (5): colorless solid; [α]27D +47 (c 0.26, CHCl3); UV (MeOH) λmax (log ε) 216 (3.87) nm; IR (ATR) νmax 1736, 1730 sh, 1374, 1236, 1030, 755 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESI-TOF) m/z 595.3610 [M + Na]+ (calcd for C34H52O7Na, 595.3605). (24E)-3β-Acetoxy-7α-hydroxylanosta-8,24-dien-26-oic acid (6): colorless solid; [α]25D +54 (c 0.215, CHCl3); UV (MeOH) λmax (log ε) 218 (3.83) nm; IR (ATR) νmax 1734, 1715, 1687, 1374, 1246 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESITOF) m/z 537.3545 [M + Na]+ (calcd for C32H50O5Na, 537.3550). (24E)-3β,15α-Diacetoxy-7α-hydroxylanosta-8,24-dien-26-oic acid (7): colorless solid; [α]26D +67 (c 0.255, CHCl3); UV (MeOH) λmax (log ε) 219 (3.84) nm; IR (ATR) νmax 1730, 1688, 1375, 1247 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESITOF) m/z 595.3609 [M + Na]+ (calcd for C34H52O7Na, 595.3606). (22S,24E)-7α-Hydroxy-3β,15α,22-triacetoxylanosta-8,24-dien-26oic acid (8): colorless solid; [α]26D +49 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 215 (3.55) nm; IR (ATR) νmax 1732, 1375, 1244 cm−1; for 1 H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in

mg), 23 (20 mg), 24 (147 mg), 25 (3.0 mg), 26 (7.0 mg), 31 (17 mg), 32 (17 mg), 36 (35 mg), 37 (6.0 mg), 38 (13 mg), 39 (10 mg), and 41 (26 mg). (22S,24E)-22-Acetoxy-3,7-dioxolanosta-8,24-dien-26-oic acid (1): colorless solid; [α]27D +6 (c 0.32, CHCl3); UV (MeOH) λmax (log ε) 217 (3.84), 248 (3.69) nm; IR (ATR) νmax 1733, 1725 sh, 1366, 1229, 1217 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESI-TOF) m/z 527.3365 [M + H]+ (calcd for C32H47O6, 527.3367). (22S,24E)-3α,22-Diacetoxy-7-oxolanosta-8,24-dien-26-oic acid (2): colorless solid; [α]27D −25 (c 0.135, CHCl3); UV (MeOH) λmax (log ε) 216 (3.86), 249 (3.77) nm; IR (ATR) νmax 1739, 1729 sh, 1366, 1229, 1217 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESITOF) m/z 571.3634 [M + H]+ (calcd for C34H51O7, 571.3629). (22S,24E)-15a,22-Diacetoxy-3-oxolanosta-8,24-dien-26-oic acid (3): colorless solid; [α]28D +50 (c 0.35, CHCl3); UV (MeOH) λmax (log ε) 216 (3.81) nm; IR (ATR) νmax 1738, 1730 sh, 1367, 1229, 1217 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESI-TOF) m/z 571.3634 [M + H]+ (calcd for C34H51O7, 571.3629). (22S,24E)-3β,15α,22-Triacetoxylanosta-8,24-dien-26-oic acid (4): colorless solid; [α]28D +46 (c 0.195, CHCl3); UV (MeOH) λmax (log ε) 216 (3.85) nm; IR (ATR) νmax 1738, 1730 sh, 1366, 1229, 1217 G



Article

CDCl3, see Tables 1 and 3; HRMS (ESI-TOF) m/z 653.3664 [M + Na]+ (calcd for C36H54O9Na, 653.3660). (24E)-7α-Acetoxy-15α-hydroxy-3-oxolanosta-8,24-dien-26-oic acid (9): colorless solid; [α]25D +72 (c 0.155, CHCl3); UV (MeOH) λmax (log ε) 217 (3.91) nm; IR (ATR) νmax 1708, 1375, 1245 cm−1; for 1 H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESI-TOF) m/z 551.3347 [M + Na]+ (calcd for C32H48O6Na, 551.3343). (24E)-7α-Methoxy-3-oxolanosta-8,24-dien-26-oic acid (10): colorless solid; [α]28D +58 (c 0.115, CHCl3); UV (MeOH) λmax (log ε) 215 (3.80) nm; IR (ATR) νmax 1739, 1730 sh, 1366, 1229, 1217 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESI-TOF) m/z 507.3452 [M + Na]+ (calcd for C31H48O4Na, 507.3445). (22S,24E)-3β,22-Diacetoxy-7α-methoxylanosta-8,24-dien-26-oic acid (11): colorless solid; [α]28D +19 (c 0.275, CHCl3); UV (MeOH) λmax (log ε) 215 (3.89) nm; IR (ATR) νmax 1738, 1730 sh, 1367, 1230, 1217 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESI-TOF) m/z 587.3944 [M + H]+ (calcd for C35H55O7, 587.3942). (22S,24E)-7α-Methoxy-3β,15α,22-triacetoxylanosta-8,24-dien26-oic acid (12): colorless solid; [α]28D +14 (c 0.24, CHCl3); UV (MeOH) λmax (log ε) 215 (3.79) nm; IR (ATR) νmax 1738, 1729 sh, 1367, 1229, 1217 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESITOF) m/z 667.3813 [M + Na]+ (calcd for C37H56O9Na, 667.3817). (24E)-3β-Acetoxy-15α-hydroxy-7α-methoxylanosta-8,24-dien26-oic acid (13): colorless solid; [α]27D +36 (c 0.225, CHCl3); UV (MeOH) λmax (log ε) 216 (3.86) nm; IR (ATR) νmax 3457, 1739, 1730 sh, 1366, 1229, 1217 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESI-TOF) m/z 567.3653 [M + Na]+ (calcd for C33H52O6Na, 567.3656). (24E)-3β-Acetoxylanosta-7,9(11),24-trien-26-oic acid (14): colorless solid; [α]28D +72 (c 0.165, CHCl3); UV (MeOH) λmax (log ε) 235 (4.09), 240 (4.09) nm; IR (ATR) νmax 1738, 1731 sh, 1367, 1229, 1217 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Table 5; HRMS (ESI-TOF) m/z 519.3442 [M + Na]+ (calcd for C32H48O4Na, 519.3442). (22S,24E)-3β,22-Diacetoxylanosta-7,9(11),24-trien-26-oic acid (15): colorless solid; [α]26D +72 (c 0.115, CHCl3); UV (MeOH) λmax (log ε) 220 sh (4.10), 234 (4.17), 241 (4.17) nm; IR (ATR) νmax 1739, 1730 sh, 1367, 1229, 1217 cm−1; for 1H NMR (500 MHz) and 13 C NMR (125 MHz) spectroscopic data in CDCl3, see Table 5; HRMS (ESI-TOF) m/z 577.3493 [M + Na]+ (calcd for C34H50O6Na, 577.3500). (22S,24E)-22-Acetoxy-3β-hydroxylanosta-7,9(11),24-trien-26-oic acid (16): colorless solid; [α]28D +43 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 220 (3.94), 234 (3.98), 241 (3.99) nm; IR (ATR) νmax 1739, 1730 sh, 1367, 1229, 1217 cm−1; for 1H NMR (500 MHz) and 13 C NMR (125 MHz) spectroscopic data in CDCl3, see Table 5; HRMS (ESI-TOF) m/z 551.3709 [M + Na]+ (calcd for C33H52O5Na, 551.3707). Acidic Degradations of Ganorbiformin E (22) and Ganorbiformin F (27). To a solution of 22 (4.7 mg) in MeOH (0.5 mL) was added TsOH·H2O (ca. 4 mg), and the mixture was stirred at room temperature for 1 h. The reaction was quenched by addition of 1 drop of aqueous ammonia solution, and the solution was concentrated under reduced pressure. The residue was dissolved in EtOAc (3 mL) and washed with H2O (1 mL). The organic layer was concentrated in vacuo to obtain the crude reaction product (3.9 mg), which was identified to be ganobiformin G (31) (1H NMR and ESIMS). In the same procedure, 27 (3.7 mg) was converted into 31 (3.1 mg). Biological Assays. Anitmycobacterial activity against Mycobacterium tuberculosis H37Ra and cytotoxicity to Vero cells (African green monkey kidney fibroblasts) were evaluated using the green fluorescent protein microplate assay.23

Table 6. Biological Activities of the Lanostane Triterpenoids compound

anti-TB M. tuberculosis H37Ra (MIC, μg/mL)

1 2 3 4 5 6 7 8 11 12 13 15 18 20 22a 24a 27a 31a 33 34a 35 37a 38a 39a isoniazidb ellipticinec

50 >50 >50 3.13 50 25 1.56 3.13 25 12.5 50 12.5 > 50 0.781 >50 >50 50 >50 50 >50 >50 6.25 0.781 50 0.094

cytotoxicity Vero cells (IC50, μM) 91 33 86 15 30 72 32 28 28 26 32 32 93 22 >95 >85 36 35 >107 >95 89 28 16 >98 3.3

a

Previously reported data of the compounds isolated from G. orbiforme BCC 22324.7 bPositive control for the anti-TB assay. cPositive control for the cytotoxicity assay.

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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.5b00826. NMR spectra of compounds 1−16 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +66 25646700, ext 3554. Fax: +66 25646707. E-mail: [email protected] (M. Isaka). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Science and Technology Development Agency (Grant No. P-13-00856) is gratefully acknowledged.

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REFERENCES

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