Chlorinated Azaphilone Pigments with Antimicrobial and Cytotoxic Activities Isolated from the Deep Sea Derived Fungus Chaetomium sp. NA-S01-R1

Four novel compounds, chaephilone C (1), chaetoviridides A–C (2–4), were obtained from the culture of a deep sea derived fungus Chaetomium sp. NA-S01-R1, together with four known compounds—chaetoviridin A (5), chaetoviridine E (6), chaetomugilin D (7) and cochliodone A (8). Their structures, including absolute configurations, were assigned based on NMR, MS and time-dependent density functional theory (TD-DFT) ECD calculations. A plausible biogenetic pathway for compounds 1–3 was proposed. Compounds 2 and 3 exhibited antibacterial activities against Vibrio rotiferianus and Vibrio vulnificus. Compounds 1, 3 and 4 displayed similar anti-methicillin resistant Staphylococcus aureus (anti-MRSA) activities in comparison to chloramphenicol. Compound 2 showed the most potent cytotoxic activities towards the Hep G2 cell and compounds 1 and 3 demonstrated relatively stronger cytotoxic activities than the other compounds against the HeLa cell.


Introduction
Fungi are well-known as prolific producers of secondary metabolites with structural and biological diversity. Azaphilones are a family of fungal pigments characterized by a highly oxygenated pyrano-quinone bicyclic core. The colored azaphilone derivatives are produced by species of ascomyceteous and basidiomyceteous fungi, including the genera Penicillium, Aspergillus, Chaetomium, Talaomyces, Emericella, Epicoccum, Pestalotiopsis, Phomopsis, Monascus and Hypoxylon. Several azaphilones are unique to one species and regarded as chemotaxonomical indicators. Azaphilones have been reported to exhibit a wide range of biological activities, such as antimicrobial, cytotoxic, antiviral and anti-inflammatory activities, as well as inhibitors of heat shock proteins (Hsp 90), MDM2-p53 interaction and gp120-CD4 binding [1].
As part of our ongoing efforts to discover bioactive compounds from marine-derived microorganisms, a Chaetomium sp. strain NA-S01-R1, isolated from the seawater sample at a depth of 4050 m (20 • 25 11.0321 N, 155 • 51 22.1549 E) in the West Pacific Ocean in 2017, attracted our attention. Studies on bioactive constituents of its pigment fraction led to the isolation of four novel chlorinated compounds, chaephilone C (1) and chaetoviridides A-C (2)(3)(4), together with four known compounds, chaetoviridin A (5), chaetoviridine E (6), chaetomugilin D (7) and cochliodone A (8) (Figure 1). Compounds 5-7 were azaphilones bearing a five-membered lactone and a fused tetrahydrofuran/δ-lactone respectively and compound 8 was a dimeric bis-spiro-azaphilone derivative. Their structures were elucidated through spectroscopic methods and cytotoxic and antimicrobial activities were both evaluated.

Results and Discussion
Chaephilone C (1), chaetoviridides A-C (2-4) were characterized as azaphilone derivatives with a chlorine atom at C-5, a methyl unit at C-7 and a branched pentenyl side chain at C-3. Chromium trioxide oxidation of compounds 1-5 gave 2-methylbutanoic acid showing (+)-rotation. Thus, the stereochemistry of C-11 for compounds 1-4 in the pentenyl side chain was established as (S).

Results and Discussion
Chaephilone C (1), chaetoviridides A-C (2-4) were characterized as azaphilone derivatives with a chlorine atom at C-5, a methyl unit at C-7 and a branched pentenyl side chain at C-3. Chromium trioxide oxidation of compounds 1-5 gave 2-methylbutanoic acid showing (+)-rotation. Thus, the stereochemistry of C-11 for compounds 1-4 in the pentenyl side chain was established as (S).
Chaephilone C (1)  ), implying ten degrees of unsaturation. The 13 C/DEPT and HSQC spectrum revealed the presence of one primary methyl group (C-13), three secondary methyl groups (C-6 , C-7 and C-14), one tertiary methyl group (C-15), one methylene group (C-12), five sp 3 -hybridized methine groups (C-1, C-4 , C-5 , C-8 and C-11) including two oxygen-bearing carbons (C-1 and C-5 ), three sp 2 -hybridized methine groups (C-4, C-9 and C-10), two sp 3 -hybridized quaternary oxygen-bearing carbons (C-7 and C-8a), five sp 2 -hybridized quaternary carbons (C-3, C-4a, C-5, C-2 and C-3 ) including one oxygen-bearing carbon (C-3 ) and two carbonyl carbons (C-6 and C-1 ) ( Figures S3 and S6). The 1 H-1 H COSY spectrum allowed the elucidation of two partial units as shown by bold-faced lines in Figure 2. The geometrical configuration of the double bond (C-9-C-10) was deduced as trans from the coupling constant of the olefinic protons 3 J 9,10 (15.95) ( Table 1). The connection of these units and the remaining groups was established based on the HMBC correlations as shown in Figure 2. The 3-methyl-1-pentenyl chain was connected to C-3 by HMBC correlations of H-9 with C-3 and C-4 and of H-10 with C-3. The connection of a chlorine atom to C-5 was reasonable from its chemical shift (δ C 124.2). Therefore, the planar structure of 1 was assigned and named chaephilone C (1). sp 3 -hybridized quaternary oxygen-bearing carbons (C-7 and C-8a), five sp 2 -hybridized quaternary carbons (C-3, C-4a, C-5, C-2′ and C-3′) including one oxygen-bearing carbon (C-3′) and two carbonyl carbons (C-6 and C-1′) ( Figures S3 and S6). The 1 H-1 H COSY spectrum allowed the elucidation of two partial units as shown by bold-faced lines in Figure 2. The geometrical configuration of the double bond (C-9-C-10) was deduced as trans from the coupling constant of the olefinic protons 3 J9,10 (15.95) ( Table 1). The connection of these units and the remaining groups was established based on the HMBC correlations as shown in Figure 2. The 3-methyl-1-pentenyl chain was connected to C-3 by HMBC correlations of H-9 with C-3 and C-4 and of H-10 with C-3. The connection of a chlorine atom to C-5 was reasonable from its chemical shift (δC 124.2). Therefore, the planar structure of 1 was assigned and named chaephilone C (1).  and H-7 oriented to the opposite side. Therefore, two possible isomers of (1S, 7S, 8R, 8aS, 11S, 4 S, 5 R)-1 and (1R, 7R, 8S, 8aR, 11S, 4 R, 5 S)-1 were proposed and their ECD spectra were calculated by time-dependent density functional theory (TD-DFT). The experimental ECD spectrum of 1 was in good agreement with the calculated ECD spectrum of (1S, 7S, 8R, 8aS, 11S, 4 S, 5 R)-1 ( Figure 4). Thus, the absolute configuration at C-1, C-7, C-8, C-8a, C-4 and C-5 of 1 was established as S, S, R, S, S, S and R, respectively and the structure of 1 was shown in Figure 1. oriented to the opposite side. Therefore, two possible isomers of (1S, 7S, 8R, 8aS, 11S, 4′S, 5′R)-1 and (1R, 7R, 8S, 8aR, 11S, 4′R, 5′S)-1 were proposed and their ECD spectra were calculated by time-dependent density functional theory (TD-DFT). The experimental ECD spectrum of 1 was in good agreement with the calculated ECD spectrum of (1S, 7S, 8R, 8aS, 11S, 4′S, 5′R)-1 ( Figure 4). Thus, the absolute configuration at C-1, C-7, C-8, C-8a, C-4′ and C-5′ of 1 was established as S, S, R, S, S, S and R, respectively and the structure of 1 was shown in Figure 1.      Figure 2). Partial spectra data of 2 were similar to chaetoviridin A, except that C-1, C-3 and C-8a were shifted to upfield and C-4a to downfield (Table 1). Chemical shift differences and the detailed 2D NMR ( 1 H-1 H COSY, HSQC and HMBC) correlations of the unassigned carbons suggested the nitrogen at position of 2 bearing a p-hydroxybenzamide moiety which was confirmed by the NOE correlations from H-1′′ to  Figure 2). Partial spectra data of 2 were similar to chaetoviridin A, except that C-1, C-3 and C-8a were shifted to upfield and C-4a to downfield (Table 1) Figures S25-S30), 4 were characterized as a chaetoviridide B analog with an ethyl group attached to C-3 instead of the 2-butanol-3-yl moiety. Therefore, the planar structure of 4 was deduced and named chaetoviridide C (4).
The optical rotation values of 2-4 exhibited the same sign compared to that of chaetoviridin A (5) isolated here, [α] 20.0 D 85 (c 0.15, MeOH). In addition, in CD spectra, the negative cotton effects at wavelengths of 225 and 378 nm and positive of 256 and 305 nm for 2-4, which were also similar to those of chaetoviridin A (5). Furthermore, the NMR data of C-7, C-4 and C-5 for 2-3 and of C-7 for 4 were almost identical to those of chaetoviridin A (5). The above evidence sufficiently allowed the assignment of the remaining asymmetric centers (7S, 4 S, 5 R). Therefore, the absolute configuration of 2-4 was established and shown in Figure 1.
Scheme 1A showed the postulated biogenetic pathway for 1, which started with hydration of chaetoviridin A (5) to form intermediate a, followed by attack of 3 -OH on C-8a, hydrolytic opening of the γ-lactone [2] and post dehydration to obtain chaephilone C (1). Scheme 1B showed the plausible mechanism for 2 and 3 via a Schiff base formation and dehydration reaction which has been reported previously [3][4][5].
The optical rotation values of 2-4 exhibited the same sign compared to that of chaetoviridin A (5) isolated here, [ ] . 85 0.15, MeOH . In addition, in CD spectra, the negative cotton effects at wavelengths of 225 and 378 nm and positive of 256 and 305 nm for 2-4, which were also similar to those of chaetoviridin A (5). Furthermore, the NMR data of C-7, C-4′ and C-5′ for 2-3 and of C-7 for 4 were almost identical to those of chaetoviridin A (5). The above evidence sufficiently allowed the assignment of the remaining asymmetric centers (7S, 4′S, 5′R). Therefore, the absolute configuration of 2-4 was established and shown in Figure 1.

Fungal Material
Strain NA-S01-R1 of C. sp. was identified by ITS sequence homology (99% similarity with Chaetomium globosum strain GYY2(1) with Genbank Accession No. KM268652.1 (max score 974, e value 0.0, query cover 97%)). The fungal strain was inoculated into a 15 mL centrifuge tube containing 5 mL of potato dextrose (PD) medium and cultured at 28 • C at 120 rpm for 3 days. Total genomic DNA was extracted as described by Lai et al. [9]. The internal transcribed spacer (ITS) region of rDNA was amplified by PCR using primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 with 32 cycles of 30 s at 95 • C, 30 s at 55 • C and 40 s at 72 • C and a final extension at 72 • C for 7 min. The ITS1-5.8S-ITS2 rDNA sequence of the fungus has been submitted to GenBank with the accession number MG786198. A voucher specimen was deposited at the Third Institute of Oceanography, SOA, China. The working strain was prepared on potato dextrose agar (PDA) slants and stored at 4 • C.

ECD Calculation
Conformational analysis was initially performed using Confab [10] with systematic search at MMFF94 force field for undetermined relative configurations of compound 1. Room-temperature equilibrium populations were calculated according to the Boltzmann distribution law Equation (1). Dominative conformers with Boltzmann-based population over 1% were delivered to subsequent Quantum Mechanics (QM) calculations. The energies and populations of conformers were provided in Table S1.
Mar. Drugs 2018, 16, 61 9 of 11 N i is the number of conformer i with energy E i and degeneracy g i at temperature T and k B is the Boltzmann constant. The theoretical calculations were carried out using Gaussian 09. At first, conformers were optimized at PM6 using semi-empirical theory method. The conformers with Boltzmann-based population lower than 1% were again filtered out and the remaining chosen for further optimization at B3LYP/6-311G(d,p) in methanol using the integral equation formalism polarizable continuum model (IEFPCM) (Table S2). Vibrational frequency analysis confirmed the stable structures. Under the same condition, the ECD calculation was conducted using TD-DFT. Rotatory strengths for a total of 30 excited states were calculated. The ECD spectrum was simulated in SpecDis [11] by overlapping Gaussian functions for each transition.

Antibacterial Assay
Antibacterial activities against V. rotiferianus (MCCC E385), V. vulnificus (MCCC E1758), V. campbellii (MCCC E333) and MRSA (ATCC 43300, CGMCC 1.12409), were tested by continuous dilution in 96-well plates using resazurin as a surrogate indicator. Blue resazurin was reduced by metabolically active bacteria to pink resorufin. A mid-logarithmic-phase tested strain was added at a starting inoculum of 5 × 10 5 CFU/mL to the plate containing tested compound (final concentration ranging from 125 to 0.98 µg/mL in two-fold dilution) plus 10% resazurin solution (6.75 mg/mL in sterile water). The foil covered plate was incubated for 24 h with shaking at 37 • C. The MIC value was determined to be the lowest concentration that did not induce the color change [12][13][14] by observing the blue-to-pink color change.

Cytotoxicity Assay
A549 (adenocarcinomic human alveolar basal epithelial cell), Hela (cervical cancer cell) and Hep G2 (human liver cancer cell) cells were maintained in F-12K, DMEM and MEM medium respectively and supplied with 10% FBS, 100 U/mL of penicillin and 100 mg/mL of streptomycin [15]. Cells were grown in a humidified chamber with 5% CO 2 at 37 • C. For cytotoxicity assays, cells were seeded at a density of 5000 cells per well in 96-well plates, grown at 37 • C for 12 h and then treated with tested compound at five different concentrations (100 µL medium/well). The cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) (DOJINDO) at 48 h post-treatment, following the manufacturer's instructions.
In brief, 10 µL of CCK-8 solution was added to each well of the 96-well plates. After incubation at 37 • C for 2 h, the absorbance at 450 nm was measured using a SpectraMAX M5 microplate reader. Wells with only culture medium and CCK-8 solution were used to determine the background and cells treated with DMSO were included as the negative controls [15].

Conclusions
In current research, we have isolated four chlorinated novel compounds, chaephilone C (1), chaetoviridide A-C (2-4), together with four known azaphilone derivatives, chaetoviridin A (5), chaetoviridine E (6), chaetomugilin D (7) and cochliodone A (8). The absolute configuration of 1-4 was elucidated by TD-DFT ECD method and plausible biogenetic pathway for 1-3 was proposed. Remarkably, compound 2 and 3 showed antibacterial activities against VR and VV respectively. Compounds 1, 3 and 4 displayed similar anti-MRSA activities in comparison to chloramphenicol. Compounds 1 and 3 demonstrated relatively stronger cytotoxic activities than the other compounds against HeLa cell and compound 2 showed the most potent cytotoxic activities towards Hep G2 cell with IC 50 below 5 µM. Further studies should be conducted to elucidate the antibacterial and cytotoxic mechanism of the related compounds as well as their roles in the life cycle of the deep sea ecological system. Supplementary Materials: NMR spectra for compounds 1-4 as well as computational data for compound 1 are available online at www.mdpi.com/1660-3397/16/2/61/s1 in Figures S1-S31 and Tables S1 and S2.