The marine environment has recently been described as a source of novel chemical diversity for drug discovery, as many bioactive substances are isolated from marine organisms such as phytoplankton, algae, sponges, tunicates and mollusks.1, 2 Microorganisms, especially fungi, from marine habitats also constitute a promising untapped resource of novel compounds and are receiving special attention.3, 4, 5 We have recently discovered novel compounds, namely sesquiterpenes JBIR-27 and JBIR-28,6 aspochracin derivative JBIR-157 and glycosyl benzenediols JBIR-37 and JBIR-38,8 from marine-derived fungi. Therefore, we attempted to isolate fungi from a marine sponge, Demospongiae, to obtain novel substances from the fungal culture. In the course of our screening program for discovering novel compounds from marine-derived fungi, we isolated a new compound termed JBIR-59 (1, Figure 1a) together with six known compounds, that is, redoxcitrinin,9 bisorbibutenolide,10 bisvertinolone,11 trichodimerol12 and sclerotinin A and B,13 from the mycelial extract of Penicillium citrinum SpI080624G1f01. We report herein the fermentation, isolation and, in brief, the biological activity of 1.

Figure 1
figure 1

(a) Structure of 1. (b) Key correlations of 1H-1H double-quantum filtered COSY (bold line) and HMBC (arrow, proton to carbon) of 1. (c) NOE observation for 1 (dashed arrow).

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Penicillium citrinum SpI080624G1f01 was isolated from the marine sponge, Demospongiae, collected offshore of Ishigaki island, Okinawa Prefecture, Japan. SpI080624G1f01 was cultivated in 50 ml test tubes containing 15 ml of potato dextrose broth (24 g l−1 potato dextrose; BD Biosciences, San Jose, CA, USA). The test tubes were shaken on a reciprocal shaker (355 r.p.m.) at 27 °C for 3 days. Aliquots (5 ml) of the culture were transferred to 500-ml Erlenmeyer flasks containing 15 g of brown rice (Hitomebore, Miyagi, Japan), 30 mg of Bacto-yeast extract (BD Biosciences), 15 mg of sodium tartrate, 15 mg of potassium hydrogenphosphate and 45 ml of water; further, the aliquots were incubated in static culture at 27 °C for 14 days.

The mycelium (gathered from 10 flasks) was extracted with 80% aq. Me2CO. After concentration in vacuo, the aqueous concentrate was extracted with EtOAc. The obtained organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The dried residue was washed with n-hexane–CHCl3 (6:1), and its insoluble fraction was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI 60 μm, Moritex, Tokyo, Japan) and eluted with a stepwise solvent system of CHCl3–MeOH (100:0, 99:1, 98:2, 95:5, 90:10 and 50:50, successively). The CHCl3-eluate afforded redoxcitrinin, whereas the CHCl3–MeOH (99:1–98:2)-eluted fraction was chromatographed on reversed-phase medium-pressure liquid chromatography (Purif-Pack ODS 100 μm, Moritex) to yield bisorbibutenolide, bisvertinolone, trichodimerol and sclerotinin A and B. A portion (20 mg) of the CHCl3–MeOH (98:2)-eluate (134 mg) was purified by preparative reversed-phase HPLC using an XBridge Prep C18 column (5.0 μm OBD, 19 i.d. × 150 mm; Waters, Milford, MA, USA) developed with 55% MeOH–H2O (flow rate: 10 ml min−1) to yield 1 (12.3 mg, retention time: 17.2 min).

Compound 1 was obtained as a yellow amorphous solid ([α]27D –350°, c 0.1, in MeOH; UV λmax 221, 263 and 387 nm, in MeOH), and its molecular formula was determined to be C23H28O7 by HR-ESI-MS (m/z 417.1910, (M+H)+; calcd for C23H29O7, 417.1913). The IR spectra (νmax 1658 cm−1) of 1 showed the presence of a conjugated ketone. The direct connectivity between each proton and carbon was established by the HSQC spectrum, and the 13C and 1H NMR spectral data for 1 are listed in Table 1. A planar structure was established by double-quantum filtered COSY spectrum together with constant time HMBC14 spectrum as follows.

Table 1 13C- and 1H-NMR data for 1
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An allylic coupling between the methyl protons 10-H3 (δH 2.01) and an olefinic proton 2-H (δH 5.64) was observed in the double-quantum filtered COSY spectrum, as shown in Figure 1b. In the HMBC spectrum, the 1H–13C long-range correlations from 2-H to an olefinic carbon C-3 (δC 161.6) and two quaternary carbons C-4 (δC 76.9) and C-9b (δC 59.6); from 10-H3 to C-2 (δC 125.6), C-3 and C-4; from the methyl protons 11-H3 (δH 1.36) to C-3, C-4 and an acetal carbon C-4a (δC 106.7); and from the methyl protons 20-H3 (δH 1.28) to a carbonyl carbons C-1 (δC 194.9), C-4a and C-9b revealed a trimethylcyclohexenone ring (Figure 1b).

The long-range couplings from the methyl protons 12-H3 (δH 1.45) to an oxygenated quaternary carbon C-5a (δC 80.3), an oxygenated olefinic carbon C-6 (δC 162.5) and a methine carbon C-9a (δC 54.1); from the methyl protons 13-H3 (δH 1.63) to C-6, an olefinic carbon C-7 (δC 111.9) and a carbonyl carbon C-8 (δC 190.9); and from 9a-H (δH 3.65) to C-5a, C-6, C-8 and an olefinic carbon C-9 (δC 100.4) established dimethylcyclohexenone ring. In addition, a hydrogen-bonded hydroxyl proton 14-OH (δH 16.43) long-range coupled with three olefinic carbons C-9, C-14 (δC 170.8) and C-15 (δC 120.2) and a sequence from an olefinic proton 15-H (δH 6.38) to the methyl protons 19-H3 (δH 1.87) through three olefinic protons 16-H (δH 7.30), 17-H (δH 6.28) and 18-H (δH 6.11) determined the hydroxyhexatriene side chain to connect at C-9 of the dimethylcyclohexenone ring. The stereochemistry of the olefins at C-15 and C-17 was determined as trans by their coupling constants (J=15.0 Hz). Finally, long-range couplings from the methine protons 9a-H to C-1, C-9b and C-20 (δC 18.80) and from 20-H3 to C-9a elucidated the connectivity between the two cyclohexenone rings. Thus, the planar structure of 1 was determined as shown in Figure 1a.

The relative configuration was assigned on the basis of the analysis of NOE spectra. The NOE (Figure 1c) between 2-H and 13-H3, between 9a-H and 12-H3, between 9a-H and 20-H3 and between 12-H3 and 20-H3 indicated that the hydroxyl groups at C-4a, the hydrogen atom 9a-H and the two methyl groups C-12 and C-20 showed the same direction on the furan ring as shown in Figure 1c.

We herein isolated a new sorbicillin derivative together with three known dimeric sorbicillin derivatives. Some homodimeric analogs of sorbicillin, such as bisorbibutenolide,10 bisvertinolone11 and trichodimerol,12 which were isolated from fungi Penicillium spp., Trichoderma spp. or Verticillium spp., have been reported. On the other hand, only two sorbicillin derivatives, that is, sorbicillactones A and B, have been reported as heterodimeric compounds that consist of sorbicillin and alanine fumaramide units as secondary metabolites of marine sponge-derived fungus P. chrysogenum.15 The new sorbicillinoid, 1, may be produced by cyclization between sorbicillin and the trihydroxyquinol antibiotic, KS 506p,16 as precursors.

It has been reported that sorbicillin derivatives are radical scavengers.16 Therefore, to evaluate the activity of 1 as a radical scavenger, we examined its inhibitory effect on L-glutamate toxicity in neuronal hybridoma N18-RE-105 cells, which can assess the radical scavenging activity.17, 18, 19, 20 The protective activity of 1, bisorbibutenolide, bisvertinolone and trichodimerol against L-glutamate toxicity in N18-RE-105 cells was tested. Compound 1, bisorbibutenolide and bisvertinolone reduced L-glutamate toxicity in N18-RE-105 cells with EC50 values of 71, 61 and 49 μM, respectively; however, trichodimerol showed weak activity (EC50 >100 μM).