Roquefortine J, a novel roquefortine alkaloid, from the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475

Abstract

Chemical investigation on the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475 led to the isolation of a previously undescribed (roquefortine J, 1) and four known (2−5) roquefortine alkaloids, along with six ergosterol analogues (6−11). The planar structure of 1 was established mainly on the basis of extensive analysis of its 1D, 2D NMR, and HRESIMS spectra. The absolute configuration of 1 was determined by comparison of the calculated and experimental electronic circular dichroism spectra. Compounds 5, 6, and 7 exhibited potent anti-proliferative effects against HepG2 tumor cells with IC₅₀ values of 7.0, 8.6, and 8.2 μM, respectively.

Marine fungi have been recognized as a new source for discovery of structurally fascinating and pharmaceutically useful secondary metabolites in recent years [1,2,3,4]. The deep-sea-derived fungi, which inhabit extreme environments, are a relatively untapped source because of the limitations of sampling and culturing technologies [5, 6]. Therefore, fewer investigations have been conducted on the secondary metabolites from marine-derived fungi living below 1000 m [6]. In our current study to search for novel bioactive secondary metabolites from deep-sea sediment-derived microorganisms [7,8,9], the fungus Penicillium granulatum MCCC 3A00475 was chosen for chemical investigation because its fermentation extract exhibited significant anti-allergic and antitumor effects. Previous bio-guided isolation had provided three anti-allergic diterpenoids [10]. A further investigation on the strain resulted in the isolation of 11 compounds (1−11) with cytotoxic activity (Fig. 1). This paper reports the isolation, structure elucidation, and cytotoxic activity of these compounds.

Fig. 1

Chemical structures of 1−11 from Penicillium granulatum MCCC 3A00475

The fungus Penicillium granulatum MCCC 3A00475 was isolated from the deep-sea sediment at the depth of 2284 m. Its EtOAc extract of the fermented cultures was fractionated by column chromatography on Sephadex LH-20, silica gel, and ODS to yield a novel roquefortine alkaloid (1) and 10 known compounds (2−11). By comparison of the NMR data with those reported in the literature, 10 known compounds were identified as roquefortine C (2) [11], 16-hydroxyroquefortine C (3) [12], roquefortine F (4) [13], meleagrin (5) [14], isonuatigenin I (6) [15], penicisteroid A (7) [16], anicequol (8) [17], 24ε-ethylcholest-5-en-3β-ol (9) [18], ergosterol (10) [19], and 5α,8α-epidioxyergosta-6,22-dien-3β-ol (11) [20].

Compound 1 was obtained as a yellow powder. Its molecular formula was determined to be C₂₂H₂₁N₅O₂ on the basis of the HRESIMS at m/z 388.1765 [M+H]⁺ (calcd for C₂₂H₂₂N₅O₂, 388.1773), indicating 15 degrees of unsaturation. The ¹H NMR spectrum exhibited signals characteristic of ortho-substituted benzene moiety (δ_H 6.66, d, J = 7.6 Hz, H-7; 7.04, t, J = 7.6 Hz, H-8; 6.67, t, J = 7.6 Hz, H-9; and 7.19, d, J = 7.6 Hz, H-10), as well as five olefinic protons (δ_H 5.94, dd, J = 17.2, 10.7 Hz, H-19; 6.42, s, H-11; 6.43, br s, H-12; 7.35, br s, H-17; and 7.73, br s, H-15), one exomethylene (δ_H 5.10, d, J = 17.2 Hz and 5.14, d, J = 10.7 Hz, H₂-20), and two methyl singlets (δ_H 1.09 and 1.15). The ¹³C NMR spectrum showed 22 carbon signals including 17 sp² carbons with six for one phenyl unit (δ_C 110.6, 119.4, 126.3, 129.8, 130.1, and 151.1), eight for four double bonds (δ_C 110.6, 114.5, 121.7, 125.0, 128.2, 133.7, 124.8, and 145.0), two for carbonyl carbons (δ_C 156.6 and 157.5), and one for nitrogen-bearing carbon (δ_C 137.9), in addition to 5 sp³ carbons comprising two quaternary carbons (δ_C 43.0 and 69.1), one nitrogen-bearing methine (δ_C 81.7), and two methyls (δ_C 22.8 and 22.9) (Table 1).

Table 1 ¹H (400 MHz) and ¹³C (100 MHz) NMR spectroscopic data for 1 in CD₃OD

The HMBC correlations from H-10 to C-6a (δ_C 151.1)/C-10a (δ_C 130.1)/C-10b (δ_C 69.1) and from H-5a (δ_H 6.08) to C-6a/C-10a/C-10b, together with the COSY correlations of H-7/H-8/H-9/H-10 deduced the presence of an indoline moiety. The HMBC correlations from H₃-21 (δ_H 1.09) and H₃-22 (δ_H 1.15) to C-10b/C-18 (δ_C 43.0)/C-19 (δ_C 145.0), and COSY correlations between H-19 and H₂-20 suggested the presence of an isoprenyl group on C-10b. The HMBC correlations from H-5a to C-10b, C-11 (δ_C 121.7), and C-11a (δ_C 134.8) confirmed the presence of a dihydropyrrole ring, while those from H-12 to C-3 (δ_C 125.0)/C-4 (δ_C 157.7)/C-13 (δ_C 128.2), from H-15 to C-13 and C-17 (δ_C 133.7), and from H-17 to C-12 (δ_C 110.6)/C-13/C-15 assigned a dehydrohistidine unit (Fig. 2). On the basis of the above evidence, the gross structure of 1 was established as 11,11a-dehydrogenated derivative of roquefortine C (2) [11].

Fig. 2

Key COSY, HMBC and NOESY correlations of 1

The NOESY correlations from H-5a to H₃-21 and H₃-22 revealed the same orientation of H-5a and the isoprenyl group (Fig. 2). The absolute configuration of 1 was assigned by comparison of the calculated and experimental electronic circular dichroism (ECD) spectra. The model molecules of (5aS,10bR)-1 (1a) and its enantiomer (1b) were calculated by the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-311G (d,p) level in MeOH with the IEFPCM model using the B3LYP/6-311G (d,p)-optimized geometries after systematic conformational searches by Confab program at the MMFF94 force field. The experimental ECD spectrum of 1 matched well with the calculated curve of 1a, indicating the absolute configuration of 1 to be 5aS and 10bR (Fig. 3). Therefore, the structure of 1 was elucidated to be 11(11a)-en-roquefortine C, and named roquefortine J.

Fig. 3

Calculated and experimental ECD spectra of 1 in MeOH

All the isolated compounds were evaluated for their cytotoxic activities against HepG2 tumor cells using the MTT method [21]. Compound 1 showed weak growth inhibitory effect against HepG2 tumor cells with a IC₅₀ value of 19.5 μM, while 5, 6, and 7 exhibited potent growth inhibitory effects with IC₅₀ values of 7.0, 8.6, and 8.2 μM, respectively (Table 2), suggesting potential application of these compounds for further development as antitumor agents.

Table 2 Growth inhibitory effects of 1−11 against HepG2 tumor cells

The present work reports a novel roquefortine alkaloid, roquefortine J (1), from the deep-sea sediment-derived fungus Penicillium granulatum MCCC 3A00475, together with four known roquefortine alkaloids (2–5) and six known ergosterol analogues (6–11). Biogenetically, the roquefortine alkaloids were assembled by condensation of tryptophan and histidine, which was different to the previously reported diterpenoids from the same fungus [10], indicating this fungus has multiple biogenetic pathways to produce structurally diverse secondary metabolites. The absolute configuration of 1 was determined by the calculated ECD spectra. All compounds were evaluated for their cytotoxic activities against HepG2 tumor cells. Compounds 5, 6, and 7 exhibited potent inhibitory effects with IC₅₀ values of 7.0, 8.6, and 8.2 μM, respectively, indicating their potential applications for further development as antitumor agents.

Roquefortine J (1): Yellow powder; [α]_D²⁶ + 2 (c 0.26, MeOH), [α]_D²³ −78 (c 0.28, CHCl₃); UV (MeOH) λ_max (log ε) 241 (3.48), 359 (3.75) nm; ECD (MeOH) λ_max (Δε) 204 (−4.26), 213 (−33.40), 222 (−9.25), 240 (−59.30), 260 (−1.42), 289 (+4.78); ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 388.1765 [M+H]⁺ (calcd for C₂₂H₂₂N₅O₂, 388.1773), 410.1586 [M+Na]⁺ (calcd for C₂₂H₂₁N₅O₂Na, 410.1593), 386.1619 [M−H]^– (calcd for C₂₂H₂₀N₅O₂, 386.1617), 422.1385 [M+Cl]^– (calcd for C₂₂H₂₁N₅O₂Cl, 422.1384).