Naphtho-Gamma-Pyrones (NγPs) with Obvious Cholesterol Absorption Inhibitory Activity from the Marine-Derived Fungus Aspergillus niger S-48

Eight naphtho-gamma-pyrones (NγPs) (1–8), together with four known biosynthetically related coumarin derivatives (9–12), were isolated from the potato dextrose agar media of a marine-derived fungus Aspergillus niger S-48. Among them, natural compounds 1 and 2 were tentatively subjected to benzohydrazide reaction to evaluate the importance of pyran rings in NγPs. Their structures were elucidated by extensive 1D and 2D NMR spectroscopic data and MS spectra. Compounds 1–4 showed obvious activity for reducing cholesterol absorption verging on ezetimibe. This work highlighted the potential of natural NγPs as NPC1L1 inhibitors.

In our continuing isolation on marine-derived fungi [3], an Aspergillus species, A. niger S-48, was obtained, and was subjected to chemical investigation to pursue biologically active NγPs pigments. As expected, twelve secondary metabolites (Figure 1), including eight known NγPs, were finally isolated from A. niger S-48. Following the established bioassay approaches in our laboratory, these isolates were evaluated for antibacterial, antifungal, cytotoxic, and quorum-sensing inhibitory activity, and cholesterol absorption inhibition activities. In order to tentatively evaluate the importance of pyran ring in NγPs for bioactivity, two compounds with enough amounts were modified for bioassay. Herein, the detailed isolation, identification, and bioactivities of natural or semisynthetic compounds are described.

Strain and Culturing Conditions
The fungal strain A. niger S-48 was isolated from the root of the mangrove plant, K. candel (L.) Druce, collected from the Beibu Bay of Guangxi Province, China. The fungus was identified according to its morphological characteristics and 18S rRNA sequences ( Figure S1 from Supplementary Materials; GenBank No. MZ573243). The fungus was deposited at the School of Pharmacy, Qingdao University, China, and was maintained at −80 • C. For large-scale fermentation, the fresh mycelia of A. niger S-48 were cultured on potato dextrose agar (PDA) media at 28 • C for 4 days. The agar plugs were cut into small pieces under aseptic conditions and 100 pieces were used to inoculate 100 flasks (1L) of PDA media, each containing potato extract powder 0.9 g, glucose 3.0 g, sea salt 4.5 g, agar 3.0 g, and distilled water 0.15 L at pH 5.6. The cultures were grown under static conditions at 28 • C for 40 days.

Structural Modification
Hydrazine monohydrate (3.75 µL, 0.12 mmol) was joined in a solution of compound 1 (7 mg, 0.012 mmol) in absolute ethanol (5 mL), and then the mixture was stirred for eight hours under 90 • C reflux, with TLC analysis indicating the consumption of the starting material. After reaction, water (5 mL) was added to terminate the reaction. The solution was evaporated slowly in vacuo and was further extracted with ethyl acetate (3 × 5 mL). The organic phase was washed with brine and dried with sodium sulfate, filtered, and concentrated slowly using rotary evaporation. The mixture was purified by a semi-preparative HPLC system (CH 3 CN/H 2 O, 80/20, 2 mL/min) using a C18 YMC-Pack ODS-A column to obtain semisynthetic compound 13 (2.1 mg, 30.0% yield, ≥98%). A similar reaction approach was applied to compound 2 to obtain the derivative 14 ( Figure S2 from Supplementary Materials).

Molecular Docking Methods
The molecular docking was carried out according to the previously described approach [11]. In general, it was carried out by SYBYL-X 2.0. The ligand molecule was drawn using the standard parameters of SYBYL-X. Their geometric conformations were energyminimized further by employing the Tripos force field for 1000 steps, and Gasteiger-Hückel charges were calculated. The protein receptor was prepared using the standard method. PyMOL was used as a viewer for interaction between ligands and the protein receptor.

Inhibition of Cholesterol Absorption
Cholesterol uptake in Caco-2 cells was performed according to a previously reported method [12,13]. Caco-2 cells were received from the American Type Culture Collection (Rockvill, MD) (ATCC ® -HTB-37 TM ). Samples in DMSO were dissolved in cell-culture medium and diluted to a concentration of 100 µM. Ezetimibe (100 µM) was used as a positive control for this study. The fluorescence was measured at the excitation wavelength of 485 nm and emission wavelength of 535 nm. BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine protein concentration with bovine serum albumin as standard. The whole protein represented the total number of cells used for normalization. The effects were expressed as the percentage of cholesterol uptake corresponding to the control values.

Antimicrobial Activities
Bacterial and fungal pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Candida albicans, Fusarium foetens, F. solanum, F. mangiferae, F. oxysporum f. sp. cubense, F. graminearum, Colletotrichum musae (ACCC 31244), C. coccodes (ACCC 36067), C. asianum, Cucumber fusarium wilt, Cowpea wilt, Nectria sp., and Alternaria solani) were used to assess the antimicrobial activities of compounds. Antimicrobial activity was carried out by the paper-agar disk diffusion assay as in the previously reported method [14]. Samples were dissolved in MeOH and diluted to a concentration of 4 mg/mL. Next, 10 µL of the sample solutions was dropped into 6 mm sterile filter paper disks, and the filter paper disks were placed evenly on the solid media with test strains [14]. Streptomycin, actidione, and fluconazole were used as the positive controls, and MeOH was used as the blank control. Antimicrobial activity was evaluated by the diameter of inhibitory zones in the solid media.
The ECD spectra of compounds 1-4 (see Figures S53 and S54) indicated that the absolute configurations of compounds 1-4 were (R), which was consistent with the previously reported data [18]. Specifically, compounds 1-8 are NγPs compounds, which were previously isolated from Aspergillus spp. The structures of NγPs consist of both naphthalene and γ-pyrone moiety, and there are monomeric and dimeric forms [23]. Moreover, compounds 1 and 2 displayed antibacterial activities against Helicobacter pylori [16]. The biological activity of NγPs is inextricably linked to its structure. The radical scavenging potential of NγPs is related to the number of hydroxyl substitutes on the skeleton [8], and the phytotoxicity of NγPs is connected to the γ-pyrone ring [24]. Compounds 9-12 are coumarin derivatives, and they had shown antibacterial and antifungal activities [7].

Modification and Identification of Semisynthetic NγPs
In order to evaluate the importance of the pyran ring in NγPs on bioactivity, compounds 1 and 2 were tentatively modified based on a benzohydrazide reaction [25]. Nucleophilic attack of hydrazine at C-2 of the NγP, followed by ring opening, further nucleophilic attack of the second nitrogen atom at the carbonyl carbon, and subsequent dehydration led to the formation of the pyrazole ring ( Figure S3 from Supplementary Materials). Finally, compounds 13 and 14 were obtained as the corresponding semisynthetic products consisting of the partial NγP and the pyrazole ring ( Figure 1).

Biological Activities
Niemann-Pick C1-Like 1 (NPC1L1) is a key target involving cholesterol cellar uptake [13]. Both compounds 1-4 and 9-14 were tested for cholesterol absorption inhibition activity [26]. Compounds 1-4 showed similar inhibitory activity in 100 µM compared with ezetimibe, the only FDA-approved NPC1L1 inhibitor (Figure 2) [12]. The results showed that compound 4 had the highest inhibitory activity for reducing cholesterol absorption among all compounds, and deserves further evaluation. The reason for the function of NγPs may be related to the pyrones site. To elucidate the binding mechanism of compound 4 and NPC1L1, molecular docking was performed to predict the binding mode of compound 4. Interestingly, Lys1027 and Phe532 were involved in the formation of hydrogen bonds with NγPs ( Figure 3). In addition, compound 4 and Phe532 interacted by π-π stacking, which further increased the affinity of compound 4 with NPC1L1. Through decreasing cholesterol absorption with a NPC1L1 inhibitor, ezetimibe was the first and only inhibitor approved for the treatment of hypercholesterolemia for nearly 20 years [12]. Natural products from marine fungi could be assumed to obtain NPC1L1 potential inhibitors.  The antimicrobial activities of all fourteen compounds, including eight NγPs, four coumarins, and two semisynthetic compounds, were evaluated by the paper-agar diskdiffusion methods. However, none of them showed activities against test pathogens, including Gram-positive and Gram-negative bacteria.

Conclusions
In summary, this study describes the isolation, identification, cholesterol absorption inhibition activity, and antimicrobial activity of NγPs and coumarins from the marine-derived fungus A. niger S-48. Their structures were identified by 1D and 2D NMR spectroscopic data and MS analysis. In addition, the reaction of natural NγPs with hydrazine afforded two semisynthetic compounds, containing naphtho-gamma-pyrones and the pyrazole ring. It has to be noted that benzohydrazide reaction was rarely reported on NγPs. All isolated or semisynthetic compounds had no effects during the assays of antimicrobial activities. Notably, compound 4, as one of the isolated NγPs, showed activity for reducing cholesterol absorption comparable to the positive drug ezetimibe. As far as we know, this is the first report to curb cholesterol cellar uptake activity using NγPs. Therefore, we obtain NγPs as potential NPC1L1 inhibitors, which call for further research.