Asperindoles A–D and a p-Terphenyl Derivative from the Ascidian-Derived Fungus Aspergillus sp. KMM 4676

Four new indole-diterpene alkaloids asperindoles A–D (1–4) and the known p-terphenyl derivative 3″-hydroxyterphenyllin (5) were isolated from the marine-derived strain of the fungus Aspergillus sp., associated with an unidentified colonial ascidian. The structures of 1–5 were established by 2D NMR and HRESIMS data. The absolute configurations of all stereocenters of 1–4 were determined by the combination of ROESY data, coupling constants analysis, and biogenetic considerations. Asperindoles C and D contain a 2-hydroxyisobutyric acid (2-HIBA) residue, rarely found in natural compounds. Asperindole A exhibits cytotoxic activity against hormone therapy-resistant PC-3 and 22Rv1, as well as hormone therapy-sensitive human prostate cancer cells, and induces apoptosis in these cells at low-micromolar concentrations.

Indole-diterpene alkaloids are widely represented among the fungal metabolites. These compounds have been isolated from fungi of the genera Claviceps, Acremonium, Eupenicillium, Penicillium, and Aspergillus (including Emericella striata). Most natural indole-diterpenes have an invariable framework ( Figure 1). Usually, C-7, C-13, and C-27 are oxidized. The oxygenation of C-27 is often followed by the formation of an ether bridge between C-27 and C-7 with inversion of the stereoconfiguration at C-7 [12]. Interestingly, some fungi produce metabolites with relatively

Results
The HRESIMS spectrum of 1 exhibited a pseudo-molecular peak at m/z 526.1980 [M + H] + , showing the characteristic isotope pattern with one chlorine atom, therefore establishing its molecular formula as C29H32NO6Cl, which was supported by the 13 C NMR spectrum.
The 1 H and 13 C NMR spectra of 1 ( Table 1) resembled those of paspalinine [16], suggesting that 1 has an indole-diterpene core similar to that of paspalinine. However, the differences in chemical shift values of C-19 (δC 123.3) and C-22 (δC 123.7) of 1 from the corresponding carbons in paspalinine [16]; the HMBC correlations ( Figure 2, Figure S5

Results
The HRESIMS spectrum of 1 exhibited a pseudo-molecular peak at m/z 526.1980 [M + H] + , showing the characteristic isotope pattern with one chlorine atom, therefore establishing its molecular formula as C 29 H 32 NO 6 Cl, which was supported by the 13 C NMR spectrum.
The effect of the asperindoles A (1) and C (3) on cell viability, cell cycle progression, and induction of apoptosis in human prostate cancer cell lines was investigated. MTT assays revealed that asperindole C (3) was noncytotoxic against human PC-3, LNCaP (androgen-sensitive human prostate adenocarcinoma cells), and 22Rv1 cell lines with an IC50 > 100 µ M. In contrast, asperindole A (1) showed cytotoxicity in all three cell lines, with IC50 values of 69.4 µ M, 47.8 µ M, and 4.86 µ M, respectively. Docetaxel, which was used as a reference substance, displayed IC50 values of 15.4 nM, 3.8 nM, and 12.7 nM, respectively. Asperindole A (1) was able to induce apoptosis in human cancer 22Rv1 cells at low-micromolar concentrations (Figure 8). Cell cycle progression analysis of 22Rv1 cells treated with asperindole A (1) for 48 h revealed a S-phase arrest (as well as a discrete G2/Mphase arrest, Figure 8). Thus, asperindole A (1) may be a promising candidate for further studies in human drug-resistant prostate cancer. In contrast, 22Rv1 cells treated with 100 µ M of asperindole C (3) for 48 h revealed only minimal induction of apoptosis (8.9 ± 0.6% vs 1.2 ± 0.1% in the control) and no significant changes in cell cycle progression.  The biosynthesis of related indole-diterpenes was previously proposed for paspalinine [29]. Apparently, the common biosynthetic precursor of asperindoles and 1,3-dioxolane indole-diterpenoids (including paspalinine) is 7α-hydroxypaxilline (Figure 7). Oxidation of the isopropyl substituent, followed by cyclization at C-7 and C-2 , generates a 1,3-dioxane ring. Asperindoles are then formed by acylation and halogenation.
The effect of the asperindoles A (1) and C (3) on cell viability, cell cycle progression, and induction of apoptosis in human prostate cancer cell lines was investigated. MTT assays revealed that asperindole C (3) was noncytotoxic against human PC-3, LNCaP (androgen-sensitive human prostate adenocarcinoma cells), and 22Rv1 cell lines with an IC 50 > 100 µM. In contrast, asperindole A (1) showed cytotoxicity in all three cell lines, with IC 50 values of 69.4 µM, 47.8 µM, and 4.86 µM, respectively. Docetaxel, which was used as a reference substance, displayed IC 50 values of 15.4 nM, 3.8 nM, and 12.7 nM, respectively. Asperindole A (1) was able to induce apoptosis in human cancer 22Rv1 cells at low-micromolar concentrations (Figure 8). Cell cycle progression analysis of 22Rv1 cells treated with asperindole A (1) for 48 h revealed a S-phase arrest (as well as a discrete G2/M-phase arrest, Figure 8). Thus, asperindole A (1) may be a promising candidate for further studies in human drug-resistant prostate cancer. In contrast, 22Rv1 cells treated with 100 µM of asperindole C (3) for 48 h revealed only minimal induction of apoptosis (8.9 ± 0.6% vs 1.2 ± 0.1% in the control) and no significant changes in cell cycle progression.

Fungal Strain
The strain was isolated from an unidentified colonial ascidian (Shikotan Island, Pacific Ocean) on malt extract agar, and identified on the basis of morphological and molecular features. For DNA extraction, the culture was grown on malt extract agar under 25 • C for 7 days. DNA extraction was performed with the HiPurATM Plant DNA Isolation kit (CTAB Method) (HiMedia Laboratories Pvt. Ltd., Mumbai, India) according to the manufacturer's instructions. Fragments containing the ITS (internal transcribed spacer) regions were amplified using ITS1 and ITS4 primers. The newly obtained sequences were checked visually and compared to available sequences in the GenBank database (www.mycobank.org). According to BLAST analysis of the ITS1-5.8S-ITS2 sequence, the strain KMM 4676 had 98% similarity with Aspergillus candidus. The sequences were deposited in the GenBank nucleotide sequence database under MG 241226. The strain is deposited in the Collection of Marine Microorganisms of G. B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS under the code KMM 4676.

Extraction and Isolation
The fungal mycelia with the medium were extracted for 24 h with 5.6 L of EtOAc. Evaporation of the solvent under reduced pressure gave a dark brown oil (6.25 g), to which 250 mL H 2 O-EtOH (4:1) was added, and the mixture was thoroughly stirred to yield a suspension. It was extracted successively with n-hexane (150 mL × 2), EtOAc (150 mL × 2), and n-BuOH (150 mL × 2). After evaporation of the EtOAc layer, the residual material (3.92 g) was passed over a silica gel column (35.0 cm × 2.5 cm, 75 g), which was eluted first with n-hexane (1.0 L), followed by a step gradient from 5% to 100% EtOAc in n-hexane (total volume 30 L). Fractions of 250 mL each were collected and combined on the basis of TLC (Si gel, toluene-2-propanol, 6:1 and 3:1, v/v).

Cell Culture
The human prostate cancer cells lines 22Rv1, PC-3, and LNCaP were purchased from ATCC. Cell lines were cultured in 10% FBS/RPMI media (Invitrogen Ltd., Paisley, UK) with (for LNCaP) or without (for 22Rv1 and PC-3) 1 mM sodium pyruvate (Invitrogen). Cells were continuously kept in culture for a maximum of 3 months, and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma. Cell line authentication was performed by DSMZ (Braunschweig, Germany) using highly polymorphic short tandem repeat loci [30].

Cell Cycle and Apoptosis Induction Analysis
The cell cycle distribution was analyzed by flow cytometry using PI (propidium iodide) staining as described before with slight modifications [32]. In brief, cells were preincubated overnight in 6-well plates (2 × 10 5 cells/well in 2 mL/well). The medium was changed to fresh medium containing different concentrations of the substances. After 48 h of treatment, cells were harvested with a trypsin-EDTA solution, fixed with 70% EtOH, stained, and analyzed by BD Bioscience FACS Calibur analyzer (BD Bioscience, Bedford, MA, USA). The results were quantitatively analyzed using BD Bioscience Cell Quest Pro v.5.2.1. software (San Jose, CA, USA). Cells detected in the sub-G1 peak were considered as apoptotic.

Conclusions
Four new metabolites, the indole-diterpene alkaloids asperindoles A-D (1-4), and the known p-terphenyl derivative 3 -hydroxyterphenyllin (5) were isolated from a marine-derived strain of the fungus A. candidus KMM 4676, associated with an unidentified colonial ascidian. To the best of our knowledge, 3 and 4 are the first examples of naturally occurring compounds containing a 2-hydroxyisobutiric acid (2-HIBA) residue. This is the first report of the spectral data and reliable assignment for 3 -hydoxyterphenyllin (5). Asperindole A (1) was proved to be highly cytotoxic in 22Rv1 human prostate cancer cells resistant to androgen receptor-targeted therapies. Therefore, this compound is a promising candidate for further evaluation in human drug-resistant prostate cancer cells.