Cytoglobosins H and I, New Antiproliferative Cytochalasans from Deep-Sea-Derived Fungus Chaetomium globosum

Cytoglobosins H (1) and I (2), together with seven known cytochalasan alkaloids (3–9), were isolated from the deep-sea-derived fungus Chaetomium globosum. The structures of new compounds 1 and 2 were elucidated by extensive 1D and 2D NMR and mass spectroscopic data. All the compounds were evaluated for their antiproliferative activities against MDA-MB-231 human breast cancer cells, LNCaP human prostate cancer cells, and B16F10 mouse melanoma cells. Compound 6 showed significant antiproliferative activity against LNCaP and B16F10 cell lines with IC50 values of 0.62 and 2.78 μM, respectively. Further testing confirmed that compound 6 inhibited the growth of LNCaP cells by inducing apoptosis.


Introduction
Marine microbes have been recognized as a rich source of pharmacologically active metabolites, and a growing number of marine fungi have been reported to produce metabolites with unique structures and interesting biological activities [1]. Furthermore, secondary metabolites from deep-sea fungus, with antimicrobial, antifungal, cytotoxic, antiviral, and antiprotozoal activities, have been well studied during recent decades [2,3]. The genus Chaetomium, which includes both terrestrial-and marine-derived species, is well-known for producing structurally complex natural products with antioxidant, antimicrobial, and cytotoxic bioactivities [4][5][6][7][8]. Cytochalasans are an important class of fungal alkaloids with a wide range of structural diversity and biological activities; they have been an important chemical tool in cell and molecular biology and also represent an ambitious target for total synthesis. Biogenetically, the fungal PKS-NRPS (polyketide synthase and non-ribosomal polypeptide synthase) hybrid synthase CheA plays an important role in chaetoglobosin formation [9,10]. Compound 1 was obtained as a white amorphous powder with molecular formula C 32 H 40 N 2 O 6 through an analysis of its HRESIMS (549.2951, calcd. 549.2959), requiring 14 degrees of unsaturation. In the 1 H NMR spectrum of 1 (Table 1), the characteristic protons for four methyl groups (δ H 1.79, s; 1.24, s; 1.05, d, J = 7.6 Hz; and 1.03, d, J = 6.6 Hz) were observed. The 1 H and 13 C NMR spectra (Table 1) Mar. Drugs 2016, 14, 233 3 of 9 revealed the presence of four methyl groups, four methylene groups, 15 methine groups (including two oxygenated methine carbons and eight olefinic carbons), and nine quaternary carbons, which were quite similar to those of chaetoglobosin R [4]. The positions of two carbonyl groups (δ C 205.9, C-19; 209.8, C-23) were determined by HMBC correlations from H-17 (δ H 6.24, d) and CH 3 (Figure 2). Additionally, the chemical shifts of C-19 (δ C 205.9) and C-20 (δ C 72.3) combined with the 1 H-1 H COSY correlation between H-20 (δ H 4.75) and H 2 -21 (δ H 1.65 and 2.38) and the HMBC correlation from C-18 to C-19 indicated that the position of the carbonyl and the oxygenated methine were reversed compared to that of chaetoglobosin R. Further analysis of combined 1D and 2D NMR spectra revealed the difference of relative stereochemistry on C-6 between 1 and chaetoglobosin R. In ROESY spectrum, strong correlations between H 3 -12 and H-4/H-8, between H-8 and H-5, and between H-3 and H 3 -11/H-7 indicated the β-orientation of H-4/H-5/H-8/CH 3 -12 and α-orientation of H-3/H-7 ( Figure 3). Furthermore, the β-orientation of 20-OH was determined by ROESY correlations between H-17 and H-20/H-15α, between H-13 and H-15α/H-7. Additional supporting evidences for the structure of compound 1 were provided by HMBC and ROESY spectra (Supplementary Materials), which allowed the confirmation of the relative stereochemistry of compound 1.   Compound 2 was obtained as a white powder, for which the molecular formula was assigned as C32H40N2O6 on the basis of HRESIMS, from a [M + H] + ion at m/z 549.2943 (calcd. 549.2959). On analysis of its 1 H and 13 C NMR spectra, similar features to 1 were evident. However, the oxygenated carbons, including one methine (δC73.6, C-7) and one quaternary carbon (δC74.1, C-6), were quite   Compound 2 was obtained as a white powder, for which the molecular formula was assigned as C32H40N2O6 on the basis of HRESIMS, from a [M + H] + ion at m/z 549.2943 (calcd. 549.2959). On analysis of its 1 H and 13 C NMR spectra, similar features to 1 were evident. However, the oxygenated carbons, including one methine (δC73.6, C-7) and one quaternary carbon (δC74.1, C-6), were quite Compound 2 was obtained as a white powder, for which the molecular formula was assigned as C 32 H 40 N 2 O 6 on the basis of HRESIMS, from a [M + H] + ion at m/z 549.2943 (calcd. 549.2959). On analysis of its 1 H and 13 C NMR spectra, similar features to 1 were evident. However, the oxygenated carbons, including one methine (δ C 73.6, C-7) and one quaternary carbon (δ C 74.1, C-6), were quite different to those in 1 (δ C 76.7, C-7; 77.4, C-6), indicating the differences of relative stereochemistry on C-6. Furthermore, correlations between H 3 -11/H 3 -12 and H 3 -12/H-7α in the ROESY spectrum confirmed the α-orientation of methyl group and β-orientation of hydroxyl group on C-6, which established that compound 2 was the C-6 epimer of compound 1. Known compounds 3-9 were identified as cytoglobosin B (3), chaetoglobosinF ex (4), chaetoglobosin F (5), chaetoglobosin E (6), cytoglobosin C (7), chaetoglobosin B (8), and isochaetoglobosin D (9), respectively, by comparison of their MS and NMR data with those reported in the literature.

Antiproliferative Assay of Compounds 1-9
The antiproliferative bioactivities of compounds 1-9 towards MDA-MB-231 human breast cancer cells, LNCaP human prostate cancer cells, and B16F10 mouse melanoma cells were evaluated, and the IC 50 values are shown in Table 2. MDA-MB-231 cells were resistant to tested compounds with no obvious cytotoxicity (IC 50 > 10 µM). Most of them showed potent antiproliferative activity on LNCaP and B16F10 cells (IC 50 < 10 µM) expect compound 2. In LNCaP cells, compounds 1 and 4-9 possessed considerable antiproliferative effects with IC 50 ranging from 0.62 µM to 7.78 µM. In B16F10 cells, compounds 3-8 possessed considerable antiproliferative effects, with IC 50 ranging from 2.10 µM to 7.15 µM. Compound 6 showed the highest cytotoxic activity and displayed a promising antitumor activity against the LNCaP and B16F10 cell lines, with IC 50 values of 0.62 and 2.78 µM, respectively, similar to the positive control cisplatin (Figure 4). possessed considerable antiproliferative effects with IC50 ranging from 0.62 μM to 7.78 μM. In B16F10 cells, compounds 3-8 possessed considerable antiproliferative effects, with IC50 ranging from 2.10 μM to 7.15 μM. Compound 6 showed the highest cytotoxic activity and displayed a promising antitumor activity against the LNCaP and B16F10 cell lines, with IC50 values of 0.62 and 2.78 μM, respectively, similar to the positive control cisplatin (Figure 4).  Further experiments were conducted to determine whether or not the antiproliferative effect of compound 6 on LNCaP cell viability is closely associated with apoptosis. Quantification by Alexa Fluor ® 488 Annexin V/PI double staining assay showed that 6 increased the percentage of apoptotic cells in a dose-dependent manner in LNCaP cells ( Figure 5). Treatment with 6 for 24 h significantly increased the number of apoptotic LNCaP cells at both early-and late-stage apoptosis. Statistical analysis indicated that the apoptotic rates were approximately 12.10%, 15.39%, and 39.36% after treatment with compound 6 at 1.0, 2.0, and 5.0 μM, respectively. Investigations into the mechanistic aspects of the antigrowth effect of 6 against prostatic tumors are currently in progress.

Fungal Materials
The fungal strain of C. globosum MCCC 3A00607 was isolated from deep-sea sediments collected from the Indian Ocean. The strain was identified by Dr. Zhongze Shao, and a voucher specimen (C. globosum MCCC 3A00607) has been deposited in the Marine Culture Collection of China.

Fermentation and Isolation
The fungal strain of C. globosum MCCC 3A00607 was cultured in a rice medium at 28 °C without shaking for 30 days. The fermented solid rice medium (2 kg) was extracted with 70% acetone/water,

Fungal Materials
The fungal strain of C. globosum MCCC 3A00607 was isolated from deep-sea sediments collected from the Indian Ocean. The strain was identified by Dr. Zhongze Shao, and a voucher specimen (C. globosum MCCC 3A00607) has been deposited in the Marine Culture Collection of China.

Antiproliferative Activity of Compounds 1-9
We examined the effect of compounds 1-9 on the proliferation of the MDA-MB-231, LNCaP and B16F10 cell lines in vitro. Each cell line was seeded in 96-well plates at 1 × 10 4 cells/well in 100 µL culture medium. After seeding for 24 h, the medium was removed and replaced with a fresh medium containing the same concentration of DMSO (1%) as a vehicle control or with a medium containing increased concentrations of the target compounds (from 0 to 20 µM) in a final volume of 100 µL. The culture was maintained in a CO 2 incubator for an additional 48 h. After 48 h, 10 µL/well Cell Proliferation Reagent CCK-8 (Dojindo Molecular Technologies, Kyushu, Japan) was added and incubated for 1 h at 37% and 5% CO 2 . The absorbance of the formed formazan product was detected at 450 nm in a 96-cell spectrophotometric plate reader following the manufacturer's instructions. The concentration required to inhibit cell growth by 50% (IC 50 ) was calculated from inhibition curves.

Apoptosis Evaluation of Compound 6
LNCaP cells were seeded in six-well plates and then cultured for 24 h. The cells were treated with different concentrations of 6 (0, 1, 2, 5, 10, and 20 µM) for 24 h, collected, and stained using Alexa Fluor ® 488 Annexin V/dead cell apoptosis kit according to the manufacturer's protocol. Green fluorescence from Alexa Fluor ® 488 Annexin V and red fluorescence from PI were detected using an Epics XL cytometer (E x = 488 nm; E m = 530 nm for Alexa Fluor ® 488 Annexin V and E m = 575 nm for PI). Data were quantitatively analyzed with EXPO32 ADC software (Beckman Coulter, Brea, CA, USA). The cell population in the lower left quadrant (Annexin V − /PI − ) represents live cells, the population in the lower right quadrant (Annexin V + /PI − ) represents early apoptotic cells, and the population in the upper right quadrant (Annexin V + /PI + ) represents late apoptotic or dead cells ( Figure 5).