HETEROCYCLES

Short Paper | Regular issue | Vol. 87, No. 2, 2013, pp. 417-422
Received, 22nd November, 2012, Accepted, 17th December, 2012, Published online, 26th December, 2012.
DOI: 10.3987/COM-12-12633

■ Melanogenesis-Modulating Diketopiperazine Derivatives from Hypocrea spp.

Tetsuro Kawabata,* Chiaki Uchida, Hikaru Kato, Tomoyuki Tsubata, Fumihide Takano, and Tomihisa Ohta

School of Pharmacy and Pharmaceutical Sciences, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan

Abstract

A new dioxopiperazine derivative, hypocreasin (1), and five known (2–6) compounds were isolated from the fungus Hypocrea spp. The structure of 1 was determined by 1D/2D NMR and MS spectroscopic analyses. Compound 2 increased melanogenesis but 3 inhibited it in cultured murine melanoma B16F1 cells.

Diketopiperazines are the secondary metabolites of cyclic peptides produced mainly by various fungi.1-5 Diketopiperazine analogs exhibit antitumor,6,7 antimicrobial,7 and anti-inflammatory activities.8,9
To further investigate the beneficial effects of diketopiperazines, we tested the effects of various microbial metabolites and their constituents on melanogenesis using B16F1 murine melanoma cells. We report here that a new dioxopiperazine derivative, hypocreasin (1), along with five known compounds, cis-bis (methylthio) silvatin (2),10 3-[(4-hydroxyphenyl)methyl]-1,4-dimethyl-3,6-bis(methylthio)-2,5-piperazinedione (3),11 trans-bis (methylthio) silvatin (4),11 3-[4-(3-methyl-2-butenyl)phenylmethyl]-6-methylthio-2,5-piperazinedione (5)12 and fusaperazine E (6)13 were isolated from Hypocrea spp.
We found that some extracts inhibited melanogenesis in cultured melanoma cells. Among them, the extract of Hypocrea spp from the shellfish inhibited melanogenesis without any significantly cytotoxicity. The fungus strain of Hypocrea spp. was isolated from shellfish. A MeOH extract of culture plates was partitioned between EtOAc and H2O. The EtOAc layer was partitioned between n-hexane and 90% MeOH–H2O. The 90% MeOH–H2O layer showed inhibitory activity against melanogenesis. The 90% MeOH–H2O layer was subjected to column chromatography on silica gel and ODS. Compounds 1–6 were obtained by HPLC.

Compound 1 was obtained as white amorphous powder, and the molecular formula C16H20O4N2 was deduced by HRFABMS at m/z 303.1343 [M-H]-. The 1H NMR spectrum of 1 showed two methyl proton signals [δH 1.67 and δH 1.72], four aromatic proton signals of the AA’XX’ spin system [δH 6.78 (2H, d, J = 8.6 Hz) and δH 7.13 (2H, d, J = 8.6 Hz)] assigned to a para-disubstituted aromatic ring. An olefinic proton signal δH 5.39 (1H, t, J = 6.8 Hz), six methylene proton signals [δH 2.68 (1H, d, J = 8.6 Hz), δH 3.06 (1H, d, J = 17.9 Hz), δH 3.20 (1H, d, J = 8.6 Hz), δH 3.62 (1H, d, J = 17.9 Hz) and δH 4.45 (2H, d, J = 6.8 Hz)], and three exchangeable proton signals [δH 6.60 (1H, s), δH 7.86 (1H, s) and δH 8.64 (1H, s)] were noted.
HMBC correlations were observed between: 1-NH and C-6; H-3 and C-2; 4-NH/C-3, C-5, H-7 and C-9; H-9 and C-11; H-10 and C-8; H-12 and C-13; H-15 and C-14; H-16 and C-13 as well as C-14. In addition, a NOE key correlation was also observed between H-10 and H-12 (Figure 2). C-6 was observed as a deshielded chemical shift compared with the sulfur-substituted carbons in 411 (1: C-6, δC 81.8, in DMSO-d6 4: C-6, δC 44.3 in DMSO-d6), suggesting a hydroxy group attachment at this position. Therefore, 1 was defined as a diketopiperazine ring including tyrosine moiety in the structure. Known compounds 2–6 were identified by comparison of 1D NMR data with those in the literature.10-13
After establishing their structures, the effect of isolated compounds (2–5) on melanogenesis in cultured murine melanoma cells was tested. We measured the melanin production in the culture medium of B16F1 cells after stimulation of α-melanocyte-stimulating hormone (α-MSH). Kojic acid was used as the positive control. Melanogenesis was inhibited by compounds 3–5 in a concentration-dependent manner (Figure 3A). Further, cytotoxic activity was observed if cells were treated with compounds 4 or 5 but not with compound 3 (Figure 3B). Compounds 3–5 did not suppress tyrosinase activity, suggesting that these compounds inhibit melanin synthesis by a tyrosinase-independent mechanism. Interestingly, compound 2 enhanced the production of melanin at concentrations of 25 and 50 μM, whereas melanogenesis was inhibited at higher concentrations of this compound (Figure 3A). This compound was also cytotoxic at higher concentration up to 100 μM. This indicates compound 2 is a stimulator for melanin synthesis within a no toxic concentration. Taken together, these compounds could be new cosmetic agents.

EXPERIMENTAL
Optical rotations were measured using a Horiba SEPA-3000 High-sensitivity Polarimete (Horiba, Kyoto, Japan). UV spectra were measured using a Shimadzu UV-1600 UV–visible Spectrometer. NMR spectra were obtained using a JEOL ECA-600 Spectrometer in CDCl3 and DMSO-d6. Chemical shifts were referenced to the residual solvent peaks (δH 7.24 and δC 77.0 for CDCl3, δH 2.49 and δC 39.5 for DMSO-d6). Mass spectra were measured on a JEOL JMS-700 Mass Spectrometer. Reverse-phase HPLC was carried out on RP-23 (5 μm, Waters Corp., Milford, MA, U.S.A.). Silica gel (63–210 μm, Kanto Kagaku, Tokyo, Japan) and ODS (63–212 µm, Wako Pure Chemical, Osaka, Japan) were used for open-column chromatography. TLC was carried out on silica gel 60 F254 (Merck) and RP-18 F254S (Merck). Agar was purchased from Wako Pure Chemical (Osaka, Japan). Fungal culture plate (90φ×15 mm) was from Sansei Medical (Kyoto, Japan).

Fungal strain
The fungus was isolated from shellfish collected at the shore of Tokumitsu (Hakusan, Ishikawa) in the Sea of Japan. The collected fungus was identified by Bex Co., Ltd (Tokyo, Japan).

Extraction and Isolation
The MeOH extracts from thriving colonies of marine fungi (approx. 300 species) were tested for their capacity to inhibit melanogenesis in cultured murine B16F1 melanoma cells assay system. The fungus was cultivated on agar medium (24 L; composed of 1.5% agar, 2.0% malt extract, 0.5% peptone in 100% seawater) in fungal culture plate (1,200 plates in total, 20 mL of medium/plate) for 14 days at 25 °C. After cultivation, agar media were collected and extracted thrice with MeOH (15 L × 3). The MeOH extract was then partitioned between EtOAc and H2O. The EtOAc layer was partitioned between n-hexane and 90% MeOH–H2O, which showed anti-melanogenic activity. A part of the MeOH–H2O layer (7.0 g/7.3 g) was subjected to column chromatography on silica gel with gradient mixtures of n-hexane/EtOAc (4/1, 2/1, 1/1) and CHCl3/MeOH (20/1, 10/1, 1/1, 0/1) to afford 13 fractions (A01–A13). Fraction A03 (20.9 mg) was purified by ODS HPLC with 70% MeOH/H2O to afford 1 (0.2 mg) and 5 (0.9 mg). The fractions mixed in A06, A07 and A08 were purified by ODS HPLC with 50% MeOH/H2O to afford 2 (23.0 mg) and one fraction (B01). Fraction B01 was purified further by ODS HPLC with 70% MeOH/H2O to afford 3 (1.4 mg) and 4 (2.5 mg). Fraction A09 was also purified by ODS HPLC with 30% MeCN/H2O to afford 6 (0.2 mg).

Hypocreasin (1)
Amorphous powder, [α]D23 +84.5° (c 0.01, MeOH); UV (MeOH) λmax (log ε): 276 (3.43), 228 (4.16), 202 (4.58) nm; 1H and 13C NMR spectral data are shown in Table 1; HRFABMS m/z 303.1343 [M-H]- (calcd. for C16H19O4N2, 303.1345).

Inhibitory effects on melanogenesis in cultured murine melanoma B16F1 cells
Cultured B16 mouse melanoma cells were trypsinized (0.05% trypsin/EDTA at 37 °C for 5–10 min). Cells (5×104 cells/mL in 400 μL Dulbecco’s modified Eagle’s medium (DMEM)) were seeded onto 24-well plates and incubated for 24 h at 37 °C in a CO2 incubator. After 24 h incubation, 50 μL of each sample (final concentration: sample media in 0.2% DMSO) solution and α-MSH (final concentration: 10 ng/mL) was added to each well, and the 24-well plate incubated for 48 h at 37 °C in a CO2 incubator. In the control group, DMSO solution diluted with DMEM to 0.2% of the final concentration of DMSO was used instead of the sample solution. The positive control group was kojic acid (final concentration: 400 μM in 0.2% DMSO). After incubation, the culture medium was removed by pipette. The remaining melanoma cells were washed with phosphate-buffered saline (PBS) and added to 100 μL of 0.05% trypsin/EDTA solution. Cells were dissolved by the addition of 200 μL 1N NaOH solution. After 2 h, the resultant cell lysates were removed from the 96-well plates (each solution, 280 μL). The optical density at 475 nm of the resulting solution was measured, and the amount of intracellular melanin calculated.

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