Rhizoaspergillin A and Rhizoaspergillinol A, including a Unique Orsellinic Acid–Ribose–Pyridazinone-N-Oxide Hybrid, from the Mangrove Endophytic Fungus Aspergillus sp. A1E3

Two new compounds, named rhizoaspergillin A (1) and rhizoaspergillinol A (2), were isolated from the mangrove endophytic fungus Aspergillus sp. A1E3, associated with the fruit of Rhizophora mucronata, together with averufanin (3). The planar structures and absolute configurations of rhizoaspergillinol A (2) and averufanin (3) were established by extensive NMR investigations and quantum-chemical electronic circular dichroism (ECD) calculations. Most notably, the constitution and absolute configuration of rhizoaspergillin A (1) were unambiguously determined by single-crystal X-ray diffraction analysis of its tri-pivaloyl derivative 4, conducted with Cu Kα radiation, whereas those of averufanin (3) were first clarified by quantum-chemical ECD calculations. Rhizoaspergillin A is the first orsellinic acid–ribose–pyridazinone-N-oxide hybrid containing a unique β-oxo-2,3-dihydropyridazine 1-oxide moiety, whereas rhizoaspergillinol A (2) and averufanin (3) are sterigmatocystin and anthraquinone derivatives, respectively. From the perspective of biosynthesis, rhizoaspergillin A (1) could be originated from the combined assembly of three building blocks, viz., orsellinic acid, β-D-ribofuranose, and L-glutamine. It is an unprecedented alkaloid-N-oxide involving biosynthetic pathways of polyketides, pentose, and amino acids. In addition, rhizoaspergillinol A (2) exhibited potent antiproliferative activity against four cancer cell lines. It could dose-dependently induce G2/M phase arrest in HepG2 cells.


Introduction
Pyridazines and pyridazinones are rare in nature but are common building blocks for heterocyclic organic synthesis [1][2][3][4].Maleic hydrazide, i.e., 1,2-dihydro-3,6-pyradizinedione, is a synthesized selective herbicide and temporary plant growth regulator commonly used to prevent sprouting of potato tubers, onions, garlic, and radishes, etc., during storage.It can also inhibit crop growth and extend flowering periods [5].Pyridaben, another example of a pyridazinone, is a broad-spectrum and contact killing acaricide.It is a mitochondrial electron transport inhibitor (METI) acaricide that promotes the formation of damaging oxygen and nitrogen radicals [6][7][8].Pyridazine N-oxides are photoactivatable O( 3 P) precursors for applications in organic synthesis and chemical biology [9], whereas pyridazinone N-oxides are relatively stable.To the best of our knowledge, all the pyridazinone N-oxides are synthetic compounds.To date, no natural products of pyridazinone N-oxides have been reported.

Results and Discussion
Rhizoaspergillin A (1) was obtained as an amorphous powder.The molecular formula C17H19N2O9 with ten degrees of unsaturation was determined by HR-ESIMS (m/z: calcd: 395.1085; found: 395.1084 [M + H] + ).The 13 C-NMR spectroscopic data and DEPT 135 experimental results for 1 revealed the presence of a methyl group, an oxygenated methylene group, eight methine groups (four oxygenated and four olefinic), and seven nonprotonated carbons (two carbonyl and five olefinic).According to the 1D and 2D NMR spectroscopic data for 1, nine degrees of unsaturation are due to a carbon-carbon double bond, a carbon-nitrogen double bond, a tetrahydrofuran ring, an amide group, and a benzoate group.Thus, a pyridazinone ring should exist in the molecule.

Results and Discussion
Rhizoaspergillin A (1) was obtained as an amorphous powder.The molecular formula C 17 H 19 N 2 O 9 with ten degrees of unsaturation was determined by HR-ESIMS (m/z: calcd: 395.1085; found: 395.1084 [M + H] + ).The 13 C-NMR spectroscopic data and DEPT 135 experimental results for 1 revealed the presence of a methyl group, an oxygenated methylene group, eight methine groups (four oxygenated and four olefinic), and seven nonprotonated carbons (two carbonyl and five olefinic).According to the 1D and 2D NMR spectroscopic data for 1, nine degrees of unsaturation are due to a carbon-carbon double bond, a carbonnitrogen double bond, a tetrahydrofuran ring, an amide group, and a benzoate group.Thus, a pyridazinone ring should exist in the molecule.
The relative configuration of 1 was determined by NOE interactions (Figure 2b).Those between H-2/H-6, H-2/H2-9, H2-9/H-3, and H-3/H-7 revealed their cofacial relationships and were arbitrarily assigned as the α-oriented H-6 and H-7, whereas the diagnostic NOE interaction between H-5 and H-8 assigned their cofacial β-orientation.In order to reconfirm the constitution of 1 and establish its absolute configuration, single-crystal X-ray diffraction analysis was taken into account.However, it is impossible to obtain suitable crystals of 1 due to its poor solubility.Thus, derivatization reaction products were considered.Compound 1 was acylated by pivaloyl chloride to afford 4 (a tri-pivaloyl derivative of 1, Figure 1).Suitable crystals of 4 were obtained in MeOH after considerable effort.Finally, the constitution and absolute configuration of 1 were established by single-crystal X-ray diffraction analysis of 4, conducted with Cu Kα radiation [Flack parameter-0.12(9)] (Figure 3, CCDC 2291154).The absolute configuration of 1, named rhizoaspergillin A, was unambiguously determined to be (5S,6S,7S,8R).To the best of our knowledge, rhizoaspergillin A is the first reported orsellinic acid-ribosepyridazinone-N-oxide hybrid.The relative configuration of 1 was determined by NOE interactions (Figure 2b).Those between H-2/H-6, H-2/H 2 -9, H 2 -9/H-3, and H-3/H-7 revealed their cofacial relationships and were arbitrarily assigned as the α-oriented H-6 and H-7, whereas the diagnostic NOE interaction between H-5 and H-8 assigned their cofacial β-orientation.In order to reconfirm the constitution of 1 and establish its absolute configuration, single-crystal X-ray diffraction analysis was taken into account.However, it is impossible to obtain suitable crystals of 1 due to its poor solubility.Thus, derivatization reaction products were considered.Compound 1 was acylated by pivaloyl chloride to afford 4 (a tri-pivaloyl derivative of 1, Figure 1).Suitable crystals of 4 were obtained in MeOH after considerable effort.Finally, the constitution and absolute configuration of 1 were established by single-crystal X-ray diffraction analysis of 4, conducted with Cu Kα radiation [Flack parameter-0.12(9)] (Figure 3, CCDC 2291154).The absolute configuration of 1, named rhizoaspergillin A, was unambiguously determined to be (5S,6S,7S,8R).To the best of our knowledge, rhizoaspergillin A is the first reported orsellinic acid-ribose-pyridazinone-N-oxide hybrid.1), was corroborated by the 1 H-1 H COSY cross-peak between H-2/H-3 and HMBC correlations between H-2/C-1, H-3/C-1, H-3/C-2, and H-3/C-4 (Figure 2a).The Key HMBC correlation from H-5 to C-4 connected the β-D-ribofuranose unit and the above pyridazinone ring through the C-4-C-5 bond.In addition, the existence of a nitrogen-oxygen bond could be inferred by the molecular formula of 1 to be loaded on N1'.Taken together, the planar structure of 1 was elucidated as shown (Figure 2a).
The most potent 2 was selected to evaluate its effects on the cell cycle of HepG2 cancer cells using flow cytometry.As shown in Figure 10A,B, after treatment with increasing concentrations of 2 (4.0, 8.0, and 16.0 µM) for 48h, the percentages of cells arrested at G2/M phase were increased from 14.83% to 25.91%, as compared to the control group (15.03%).Therefore, 2 could dose-dependently induce G2/M phase arrest in HepG2 cells.The antiproliferative activity of 1-3 against four cancer cell lines were evaluated by using the standard MTT assay, with MS-275 (Entinostat, Figure 8) as the positive control [36].MS-275 is a known histone deacetylase inhibitor as an anticancer agent.As summarized in Table 4 and Figure 9, 2 is the most potent one among the three compounds, with IC50 values of 8.83, 14.18, and 15.12 μΜ against HepG2, LLC, and B16-F10 cancer cell lines, respectively.Compounds 1 and 3 showed lower activities with IC50 values around or greater than 50.0 μΜ.The antiproliferative activity of 1-3 against four cancer cell lines were using the standard MTT assay, with MS-275 (Entinostat, Figure 8) as the po [36].MS-275 is a known histone deacetylase inhibitor as an anticancer agen rized in Table 4 and Figure 9, 2 is the most potent one among the three com IC50 values of 8.83, 14.18, and 15.12 μΜ against HepG2, LLC, and B16-F lines, respectively.Compounds 1 and 3 showed lower activities with IC50 v or greater than 50.0 μΜ.The most potent 2 was selected to evaluate its effects on the cell cycle of HepG2 cancer cells using flow cytometry.As shown in Figure 10A,B, after treatment with increasing concentrations of 2 (4.0, 8.0, and 16.0 μΜ) for 48h, the percentages of cells arrested at G2/M phase were increased from 14.83% to 25.91%, as compared to the control group (15.03%).Therefore, 2 could dose-dependently induce G2/M phase arrest in HepG2 cells.

General Experimental Procedures
HR-ESIMS spectra were obtained on a Bruker Daltonics Apex-Ultra 7.0 T (Bruker Corporation, Billerica, MA, USA).Optical rotations were recorded on an MCP500 modular circular polarimeter (Anton Paar GmbH, Graz, Austria) with a 0.5 cm cell at 25 °C and UV spectra were measured on a UV-2600 UV-Vis spectrophotometer (SHIMADZU) at room temperature.IR spectra were obtained on a SHIMADZU IRAffinity-1 Fourier transform infrared spectrometer and ECD spectra were recorded on a circular dichromatic spectrometer (Chirascan, Applied PhotoPhysics, Leatherhead, Surrey, UK).X-ray

Fungus Material
The fungal strain Aspergillus sp.A1E3 was isolated from the fruit of Rhizophora mucronata, collected from the Thai mangrove swamps of the Trang Province in February 2012.It was identified as Aspergillus sp. according to ITS rDNA sequence data.The strain was preserved in the School of Pharmaceutical Sciences, Southern Medical University.

Synthesis of the Derivative 4
Compound 1 (20.0 mg) was dissolved in pyridine (0.5 mL).DMAP (3.0 mg) was then added, along with 50.0 µL of pivaloyl chloride under ice bath and stirred for 10 min.After the completion of the reaction (monitored by thin-layer chromatography, i.e., TLC), the reaction solution was evaporated under reduced pressure.The resulting residue was dissolved in methanol, purified by semi-preparative HPLC, and eluted with the mixture of MeCN/H 2 O (v/v, 58:42, 3.0 mL/min) to afford 4 (5.0 mg).Suitable crystals of 4 were obtained in MeOH after considerable effort.

Cell Culture and Cytotoxicity (MTT) Assay
An MTT assay was used to determine the antiproliferative activities of compounds 1-3 and MS-275 (positive control) against four cancer cell lines, including a human lung cancer cell line (HepG2), mouse Lewis lung carcinoma (LLC) cells, a mouse skin melanoma cell line (B16-F10), and a human breast cancer cell line (MCF-7).Fetal bovine serum (FBS, 10%) in RPMI-1640 medium was used to culture MCF-7 cell lines and Fetal bovine serum (FBS, 10%) in DMEM medium was used to culture B16-F10, HepG2 and LLC cells.Cells were seeded into 96-well plates at a density of 5000 cells/well and incubated at 37 • C with 5% CO 2 overnight.The next day, the three compounds and MS-275 (InvivoChem) were dissolved in a complete medium to prepare different concentrations of solution, which were added to each well, followed by incubation at 37 • C for 48 h.Finally, 20 µL of MTT (5 mg/mL dissolved in PBS) was added to each well and incubated with cells at 37 • C for 4 h.Then, the complete medium was removed and the formazan crystals were dissolved in 100 µL of DMSO in each well.The absorbance was measured in a TECAN microplate

Figure 3 .
Figure 3. Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP) illustration of the X-ray structure of 4 (Ellipsoids are given at the 30% probability level, Cu Kα).

Figure 3 .
Figure 3. Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP) illustration of the X-ray structure of 4 (Ellipsoids are given at the 30% probability level, Cu Kα).

Figure 3 .
Figure 3. Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP) illustration of the X-ray structure of 4 (Ellipsoids are given at the 30% probability level, Cu Kα).Rhizoaspergillinol A (2) was isolated as a light white amorphous powder.The molecular formula C 32 H 26 O 9 was determined by the positive HR-ESIMS ion at m/z 555.1644 (calcd for [M + H] + , 555.1650), indicating twenty degrees of unsaturation.According to 1 H-and 13 C-NMR spectroscopic data of 2 (Table2), thirteen degrees of unsaturation are due to a keto-carbonyl function and twelve carbon-carbon double bonds.Therefore, the molecule has to be heptacyclic.The 13 C-NMR spectroscopic data and DEPT 135 experimental results for 2 revealed the presence of three methyl groups (a methoxy and two tertiary), a methylene group, thirteen methine groups (twelve olefinic and one oxygenated), and fifteen nonprotonated carbons (one carbonyl, five olefinic, and nine oxygenated).

Figure 5 .
Figure 5. Experimental and calculated ECD curves for 2. Compound 3 was obtained as amorphous powder.The molecular formula C 20 H 18 O 7 was established by the positive HR-ESIMS ion at m/z 371.1136 (calcd for [M + H] + , 371.1125), indicating twelve degrees of unsaturation.According to the NMR spectroscopic data for 3 (Table3), eight degrees of unsaturation are due to two keto-carbonyl function, six carboncarbon double bonds.Therefore, the molecule has to be tetracyclic.The presence of a

a 1 H
-and13 C-NMR data measured at 400 and 100 MHz, respectively.
a 1 H-and 13 C-NMR data measured at 400 and 100 MHz, respectively.

Table 4 .
In vitro antiproliferative activities of 1
a MS-275 was used as a positive control.