Meroterpenoids from the Alga-Derived Fungi Penicillium thomii

May 22, 2014 - Far Eastern Federal University, Suhanova 8, Vladivostok 690950, Russian .... between 1 and 3 indicated the absence of a hydroxy group a...
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Meroterpenoids from the Alga-Derived Fungi Penicillium thomii Maire and Penicillium lividum Westling Olesya I. Zhuravleva,†,‡ Maria P. Sobolevskaya,† Elena V. Leshchenko,‡ Natalya N. Kirichuk,† Vladimir A. Denisenko,† Pavel S. Dmitrenok,† Sergey A. Dyshlovoy,†,‡ Alexander M. Zakharenko,†,‡ Natalya Yu. Kim,† and Shamil Sh. Afiyatullov*,† †

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku 159, Vladivostok 690022, Russian Federation ‡ Far Eastern Federal University, Suhanova 8, Vladivostok 690950, Russian Federation S Supporting Information *

ABSTRACT: Ten new austalide meroterpenoids (1−10) were isolated from the alga-derived fungi Penicillium thomii KMM 4645 and Penicillium lividum KMM 4663. Their structures were elucidated by extensive spectroscopic analysis and by comparison with related known compounds. The absolute configurations of some of the metabolites were assigned by the modified Mosher’s method and CD data. Compounds 1, 2, 8, and 9 were able to inhibit AP-1-dependent transcriptional activity in JB6 Cl41 cell lines at noncytotoxic concentrations. Austalides 1−5, 8, and 9 exhibited significant inhibitory activity against endo-1,3-β-D-glucanase from a crystalline stalk of the marine mollusk Pseudocardium sachalinensis.

T

he austalides are a family of related meroterpenoid metabolites that were isolated for the first time from maize cultures of Aspergillus ustus.1 It was shown that the austalide skeleton is biosynthesized after subsequent cyclization and oxidative modification of 6-[(2E,6E)farnesyl]-5,7-dihydroxy-4-methylphthalide.2−4 The absolute configurations of the austalides A to L were assigned by X-ray structure analysis, which was carried out for a single crystal of the dicamphanate ester derivative of austalide D and by synthesis of austalide B.4,5 Recently, five new austalides (M−Q) were isolated from a sponge-derived fungus Aspergillus sp. The absolute configurations of austalides M and P were elucidated by electronic circular dichroism calculations.6 In our search for fungal secondary metabolites with novel chemical structures and/or cytotoxic activity we have investigated the strains Penicillium thomii KMM 4645 and Penicillium lividum KMM 4663 associated with the marine brown alga Sargassum miyabei. We report herein the isolation, structure determination, and biological assay results of the new austalides (1−10) produced by these fungi.

with the substituted phthalide moiety1a in structure 1. A comparison of the 1H and 13C NMR data (Tables 1 and 2) with those for the meroterpenoid austalide H showed a close similarity.1a The key differences were the lack of a methyl ester in 1 and the presence of three methylenes and an extra aliphatic methyl compared to austalide H. The correlations observed in the COSY-45 spectrum and the HMBC correlations H3-32/C-30 (δC 30.7), H2-31/C-28 (δC 64.7), and H2-28/C-17 (δC 173.6) revealed the location of a butyl ester at C-17 in 1. Thus, compound 1 has a butyl ester at C-17 instead of a methyl ester in austalide H,1a and it was named austalide H acid butyl ester. The NOE correlations H-14/H-13, H-13/H-24, H-24/H-21, H-22b (δH 2.81), and H3-27/H-22a (δH 3.10) determined the relative configuration of 1. The absolute configuration of 1 was

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RESULTS AND DISCUSSION The fungi were cultured for 21 days on specially modified rice medium.7 The EtOAc extracts of the mycelia were purified by a combination of Si gel column chromatography and reversedphase HPLC to yield compounds 1−5, 8, and 9 from P. thomii and 2−10 from P. lividum as amorphous solids. The molecular formula of 1 was determined to be C29H42O6 by HRESIMS and was in accordance with the 13C NMR data. The UV spectrum showed λmax at 216 and 267 nm, consistent © 2014 American Chemical Society and American Society of Pharmacognosy

Received: February 17, 2014 Published: May 22, 2014 1390

dx.doi.org/10.1021/np500151b | J. Nat. Prod. 2014, 77, 1390−1395

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Table 1. 1H NMR Spectroscopic Data (δ, J in Hz) for Austalides 1, 5, 7, 8, and 10 position

1a

5b

7c

8a

10b

1 12

5.13, s a: 2.53, dd (2.3, 15.3) b: 1.81, dd (3.3, 15.3) 4.68, m

5.12, s a: 2.51, dd (15.5; 2.5) b: 1.93, dd (19.0; 4.1) 4.08, brs

5.20, s a:.31, dd (2.1, 15.6) b: 2.19, dd (4.4, 15.6) 4.09, m

5.13, s a: 2.55, dd (2.7, 15.7) b: 1.98, dd (3.8, 15.7) 4.41, brs

1.35, d (2.5)

2.04, brs 2.36, m

23 24 25

a: 2.34, m b: 2.25, m a: 2.29, m b: 1.96, m 1.68, d (8.0) a: 3.10, d (18.0) b: 2.81, dd (8.0, 18.0) 2.05, s 1.26, s 1.46, s

26 27 29 28 30 31 32

1.47, 0.98, 4.15, 4.10, 1.63, 1.40, 0.96,

13 14 17 18 19 21 22

s s s dt (1.2, 7.0) m m t (7.4)

a: 1.80, m b: 1.70, m 1.68, d (7.8) a: 2.96, d (18.8) b: 2.84, m 2.05, s 1.26, s a: 5.06, s b: 5.01, s 1.92, s 0.85, s 4.14, s

a: 1.87, d (8.6) b: 1.64, d (8.6) a: 1.93, d (7.4) b: 1.89, d (11.3) 2.46, d (7.9) a: 2.96, d (18.5) b: 2.90, dd (7.9, 18.5) 2.07, s 1.24, s 1.56, s

a: 2.82, dd (8.3, 16.5) b: 2.70, dd (12.3, 16.5) a: 2.06, m b: 1.50, m 1.61, d (8.1) a: 3.05, d (18.7) b: 2.88, dd (8.2, 18.7) 2.05, s 1.27, s 1.75, s

5.11, s a: 2.27, m b: 2.24, m α: 1.64, m β: 1.60, m 0.93, d (11.5) 3.24, dd (4.5, 11.7) α: 1.57, m β: 1.68, m a: 1.91, m b: 0.88, m 1.39, d (8.8) a: 2.91, d (18.6) b: 2.73, dd (8.3, 18.6) 2.03, s 1.16, s 0.78, s

1.43, s 0.99, s 4.04, s

1.59, s 1.04, s 4.16, s

1.03, s 0.61, s 4.10, s

1.66, d (1.8)

a Chemical shifts referenced to CDCl3 at 700 MHz. bChemical shifts referenced to CDCl3 at 500 MHz. cChemical shifts referenced to CD3OD at 700 MHz.

Compound 3 showed characteristic Cotton effects at λ269 +2.63, λ230 +4.69, and λ209 −4.59 in the CD spectrum in MeOH. The two strong CEs of the high-energy region reproduced well those for austalides P,6 which confirmed the absolute configuration of 3 as 11S, 14R, 20S, 21R, and 3 was named austalide P acid butyl ester. The molecular formula of compound 4 was determined to be C25H34O7 from HRESIMS and 13C NMR data. The 1H and 13C data observed for the tetracyclic core of 4 (Table 2, Supporting Information S25, S27, S73) matched those reported for compound 3. The structure and location of the butyl ester at C-17 were established as for 2. Compound 4 exhibited a nearly identical CD spectrum to that of austalide P,6 establishing its absolute configuration as 11S, 14R, 20S, 21R. Compound 4 was named austalide P acid. The molecular formula of compound 5 was determined to be C25H32O7 from HRESIMS and 13C NMR data. The 1H and 13C NMR data (Tables 1 and 2) observed for the substituted tetracyclic backbone of 5 closely resembled those of 1 with the exception of the proton and carbon signals at positions 13 and 14. The HMBC correlations from methylene proton H-25a (δH 5.06) to the olefinic quaternary carbon signal detected at δC 145.0 (C-15) and from H3-26 to C-15 and C-25 (δC 116.0) revealed the presence of the exomethylene moiety in 5. The COSY-45 data and HMBC correlations from H3-24 to C-12 (δC 44.6) and C-13 (δC 71.2), from H-14 to C-13, from H2-25 to C-14 (δC 52.9), and from H2-19 to C-14, C-20 (δC 39.6), C17 (δC 177.3), and C-18 (δC 28.6) indicated the location of the hydroxy group and propenyl and carboxyethyl units at C-13, C14, and C-20, respectively, and established the structure of 5.

established by the modified Mosher’s method.8 Esterification of 1 with (R)- and (S)-MTPA chloride occurred at the C-13 hydroxy group to give the (S)- and (R)-MTPA esters 1a and 1b, respectively. The observed chemical shift differences Δδ (δS − δR) (Supporting Information S73, 74) revealed the 13R configuration, and hence the absolute configuration of 1 was determined as 11S, 13R, 14S, 20R, 21R, which is consistent with the absolute configurations for known austalides G, H, and I. The molecular formula of 2 was determined to be C25H34O8 from HRESIMS and 13C NMR data (Table 2). The structure of the substituted tetracyclic moiety of 2 was found by extensive NMR spectroscopy (1H and 13C NMR, COSY, HSQC, and HMBC, Supporting Information S11−S17) as for compound 1. The key difference was that the structure had a free acid rather than a butyl ester. Compound 2 showed characteristic Cotton effects (CEs) at λ270 +0.08, λ230 +0.42, and λ211 −0.36 in the CD spectrum in MeOH. The two CEs of the high-energy region were in good agreement with those for 1, which allowed us to determine the absolute configuration of 2 as 11S, 13R, 14S, 20R, 21R. Compound 2 was named austalide H acid. The molecular formula of 3 was determined to be C29H42O7 from HRESIMS and was in accordance with the 13C NMR data. The general features of the 1H and 13C NMR spectra (Table 2, Supporting Information S18, S20, S73) of 3 resembled those of 1 with the exception of the C-11−C-14 and C-25 proton and carbon signals. The correlations observed in the COSY and HSQC spectra (Supporting Information S19, S22) of 3 together with the molecular mass difference of 16 mass units between 1 and 3 indicated the absence of a hydroxy group at C13 in 3. 1391

dx.doi.org/10.1021/np500151b | J. Nat. Prod. 2014, 77, 1390−1395

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Table 2. 13C NMR Spectroscopic Data (δ in ppm) for Austalides 1−10 pos.

1a

2b

3a

4c

5c

6a

7d

8a

9c

10c

1 3 4 5 6 7 8 9 11 12 13 14 15 17 18 19 20 21 22 23 24 25 26 27 29 28 30 31 32

68.2 169.3 108.7 155.5 116.3 156.5 114.4 146.0 78.7 43.9 70.1 49.3 75.7 173.6 29.8 34.9 40.9 40.3 18.5 10.7 27.5 33.6 31.5 21.5 62.2 64.7 30.7 19.2 13.7

70.4 172.3 109.1 157.2 118.0 160.3 116.5 148.0 79.0 46.5 70.7 52.3 77.5 178.6 30.8 36.6 42.7 42.1 19.2 11.3 28.7 32.7 33.3 22.3 62.8

68.2 169.4 107.2 155.4 115.5 158.7 114.2 145.4 76.6 39.5 21.8 50.5 75.4 174.8 29.5 33.6 41.8 40.4 17.9 10.6 27.5 27.8 34.1 18.9 61.9 64.5 30.6 19.2 13.7

68.2 169.4 107.4 155.4 115.4 158.6 114.2 145.5 76.4 39.8 21.8 50.6 76.1 176.8 29.2 33.7 41.7 40.7 17.8 10.7 27.5 27.8 34.6 18.9 62.0

68.2 169.1 108.3 155.6 115.5 157.0 114.2 145.7 78.5 44.6 71.2 52.9 145.0 177.3 28.6 34.6 39.6 40.1 18.3 10.8 27.3 116.0 26.0 20.2 61.2

68.2 169.5 107.3 155.5 115.3 158.6 114.4 145.5 76.5 39.1 23.8 50.2 146.7 178.2 28.3 33.0 40.1 39.6 18.1 10.7 27.4 114.2 23.5 18.1 62.0

70.5 172.5 108.9 157.1 118.3 160.5 116.7 148.0 78.4 43.9 70.7 88.9 86.9 119.3 32.2 32.9 41.5 38.2 19.6 11.3 28.8 26.7 30.1 19.5 62.8

68.2 169.1 108.3 155.4 115.7 156.9 114.2 145.9 77.8 45.3 68.7 57.1 86.6 174.9 31.5 37.7 40.7 46.7 18.8 10.7 27.3 27.0 34.2 17.0 62.2

68.3 169.7 107.6 155.5 115.3 158.4 114.2 145.9 76.1 39.5 22.3 54.0 85.9 175.2 31.8 37.4 40.6 47.5 18.7 10.7 27.3 25.8 32.5 16.8 62.2

68.3 169.9 107.1 155.4 115.8 158.9 114.1 145.4 76.5 40.4 17.9 54.2 38.8 78.9 27.1 38.2 38.1 48.1 18.0 10.6 27.3 15.7 28.5 14.3 61.8

a Chemical shifts referenced to CDCl3 at 176 MHz. bChemical shifts referenced to CD3OD at 176 MHz. cChemical shifts referenced to CDCl3 at 125 MHz. dChemical shifts referenced to CD3OD at 125 MHz.

and H2-22/C-5, C-6 (δC 118.3), C-7, C-20, and C-21 (δC 38.2) established the structure of the substituted pyranophthalide moiety and its linkage with ring C in 7. The main structural difference between the new austalide 7 and the previously reported austalide B is the presence of a hydroxy group at C-17 in 7 instead of a methoxy group in austalide B. Thus, compound 7 was named 17-O-demethylaustalide B. The relative configurations of the C-11, C-13, C-14, C-17, C-20, and C-21 stereogenic centers in 7 were the same as in austalide M6 based on NOE correlations. The molecular formula of compound 8 was determined to be C25H32O7 from HRESIMS and 13C NMR data. The general features of 1H and 13C NMR spectra (Tables 1 and 2) of the C, D, and E rings of 8 closely resembled those of austalide I.1a The structure of the substituted pyranophthalide moiety in 8 was determined based on HMBC correlations as for compound 7. The molecular mass difference of 42 mass units between austalide I and 8 and the upfield chemical shift of H-13 (δH 4.41) indicated replacement of an acetoxy group at C-13 by a hydroxy group. The relative configuration of 8 was elucidated based on NOE correlations as for 1. Compound 8 was named 13-O-deacetylaustalide I. The molecular formula of 9 was determined to be C25H32O6 by HRESIMS and was in accordance with the 13C NMR data. The 13C NMR spectrum (Table 2) of 9 showed a close similarity of carbon chemical shifts to those of 13-Odeacetylaustalide I (8) with the exception of the C-11−C-14 and C-25 carbon signals. The COSY-45 data and HSQC

The relative configuration of 5 was elucidated based on the observed NOE correlations. Compound 5 exhibited a nearly identical CD spectrum to that of austalide Q.6 Thus, the absolute configuration of 5 was established as 11S, 13R, 14S, 20S, 21R. Compound 5 was named austalide Q acid. The molecular formula of compound 6 was determined to be C25H32O6 by HRESIMS and was in accordance with the 13C NMR data (Table 2). The 13C NMR data for this compound were very similar to those obtained for austalide Q acid (5) with the exception of the C-11−C-15 and C-25 carbon signals. The correlations observed in the COSY and HSQC spectra (Supporting Information S38, S41) of 6 together with the molecular mass difference of 16 mass units between 5 and 6 indicated the absence of a hydroxy group at C-13 in 6. The CD spectrum of 6 showed characteristic Cotton effects at λ297 −0.10, λ267 −0.02, λ231 +1.36, and λ212 −2.51, which were in good agreement with those for austalide P.6 Thus, the absolute configuration of 6 was established as 11S, 14S, 20S, 21R. Compound 6 was named 13-deoxyaustalide Q acid. The molecular formula of compound 7 was determined to be C25H32O8 from HRESIMS and 13C NMR data. The general features of the 13C NMR spectrum (Table 2) of 7 resembled those of austalides M−O6 with the exception of the C-6 and C20−C-22 carbon signals. The key HMBC correlations (Supporting Information S48) confirmed the structure of rings A, B, and C. The long-range correlations H2-1/C-3 (δC 172.5), C-4 (δC 108.9), C-8 (δC 116.7), and C-9 (δC 148.0); H3-23/C-7 (δC 160.5), C-8, and C-9; H3-29/C-5 (δC 157.1); 1392

dx.doi.org/10.1021/np500151b | J. Nat. Prod. 2014, 77, 1390−1395

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spectra of 9 revealed the connectivity sequence of the protons (−CH2(12)−CH2(13)−CH(14)