Penicilazaphilones D and E: two new azaphilones from a sponge-derived strain of the fungus Penicillium sclerotiorum †

Two new azaphilones, penicilazaphilones D ( 1 ) and E ( 2 ), along with four known analogs ( 3 – 6 ), were obtained from the sponge-derived fungus Penicillium sclerotiorum . Their structures were elucidated on the basis of comprehensive spectroscopic analyses, ECD calculation, single-crystal X-ray di ﬀ raction and comparison to the biosynthetically related compound 5 . Compound 1 represents the second reported azaphilone with a C-4 aliphatic side chain, and 2 is the ﬁ rst azaphilone with a tetrahydrofuran ring at C-3. Compound 3 displayed strong antiviral activity against HSV (IC 50 ¼ 19.5 m M) and EV71 (IC 50 ¼ 132 m M), 16 and 3 fold more potent than the positive control ribavirin, respectively.


Results and discussion
The sponge-derived fungal strain, Penicillium sclertiorum GDST-2013-0415, was cultured with rice medium in y Erlenmeyer asks at room temperature for 40 days. The fermented rice medium was extracted three times with EtOAc. The combined EtOAc layers were evaporated under reduced pressure to give the EtOAc extract (65 g). By repeated column chromatography (CC) over silica gel, octadecylsilyl (ODS), Sephadex LH-20, and semi-preparative HPLC, six compounds, 1-6, were obtained ( Fig. 1).
Penicilazaphilone D (1) was isolated as a yellow gum with the molecular formula of C 14 H 15 ClO 5 based on HRESIMS, for which seven degrees of unsaturation were required. The IR spectrum of 1 exhibited the presence of hydroxyl (3633 cm À1 ), and conjugated carbonyl (1701 cm À1 ) groups. The 1 H NMR spectroscopic data (Table 1) exhibited signals for three olenic protons at d H 7.20 (d, J ¼ 15.9 Hz), 6.61 (d, J ¼ 15.9 Hz) and 6.50 (s), two methyl groups at d H 2.33 (s) and 1.35 (s), two methine protons at d H 4.18 (d, J ¼ 3.1 Hz) and 3.29 (m), and one methylene group at d H 4.66 (dd, J ¼ 11.0, 4.9 Hz) and 4.21 (dd, J ¼ 13.0, 11.0 Hz). The 13 C NMR spectroscopic data (Table 1) showed eight sp 2 -hybridized carbons in 1, including two carbonyl carbons at d C 197.9 and 194.5, six olenic carbons at d C 159.5, 143.7, 136.1, 131.5, 120.5 and 108.7, and six sp 3 -hybridized carbons including two methyl groups at d C 27.7 and 23.4, two methine groups at d C 75.1 and 37.9, one methylene group at d C 69.5, and one quaternary carbon at d C 79.0. These spectroscopic features suggested that 1 shares the same bicyclic core of azaphilone as WB (CAS no. 1701443-52-8) (5), an azaphilone derivative isolated from a soil fungus and determined only the plane structure, 17 then from an entomopathogenic fungus Hypocrella sp. reported the absolute conguration. 18 The main differences were the presence of a 3-buten-2-one group [-CH] CHC(O)CH 3 ] at C-3 in 1 instead of the [-CH]CHCOH(CH 3 ) CH(OH)CH(CH 3 )CH 2 CH 3 ] fragment in 5, which was conrmed by the COSY cross-peaks and HMBC correlations (Fig. 2). In the 1 H-1 H COSY spectrum, the cross peak of H-1 0 /H-2 0 , together with HMBC correlations from H-1 0 to C-4 and C-3 0 , from H-2 0 to C-3, and from H-4 0 to C-2 0 established a 3-buten-2-one unit linked with C-3. The relative conguration of 1 was determined by 1 H-1 H coupling constants and NOESY correlations. The cisrelationship of H-8/H-8a was deduced from the eq/ax coupling constant (J 8,8a ¼ 3.1 Hz). In the NOESY spectrum (Fig. 3), NOESY correlations were observed between H 3 -9 and H-8, indicating the cofacial orientation relative to these protons. On the basis of these results, the complete planar assignment for compound 1 was established.
The absolute conguration of 1 was determined by comparing the theoretical calculation ECD with the experimental ECD. Conformational searches were carried out using Merck Molecular Force Field (MMFF) by means of the Spartan's 10 soware to acquire meaningful conformers for 7R,8R,8aS-1, followed by DFT optimizations at the B3LYP/6-31+G(d,p) level in MeOH by CPCM polarizable conductor calculation model. The ECD spectrum was calculated by the TDDFT methodology with a larger basis set at the B3LYP/6-311+G(d,p) level and generated using SpecDis 1.6 according to Boltzmann distributions. The theoretical ECD spectrum for 7S,8S,8aR-1 was obtained by directly reversing the spectrum of 7R,8R,8aS-1. For compound 1 the experimental positive Cotton effect at 250 nm (D3 +14.32), and negative Cotton effects at 218 nm (D3 À6.14) and 328 nm (D3 À3.83) compared well with the calculated ECD curve for 7R,8R,8aS-1, which showed three corresponding Cotton effects around 257 nm (D3 +15.21), 223 nm (D3 À17.65), and 325 nm (D3 À2.90), respectively (Fig. 4). Therefore, qualitative analysis of the results allowed the assignment of the absolute conguration of 1 as 7R,8R,8aS.
To conrm the absolute conguration, we undertook a single X-ray diffraction study. Ultimately, by slow crystallization from the mixture of CH 2 Cl 2 and CH 3 OH, single crystals of 1 suitable for X-ray diffraction were obtained. The Mo Ka radiation used for the X-ray diffraction allowed the assignment of the absolute conguration of all of the stereogenic centers in 1, which contains heavy atom chlorine, as 7R, 8R and 8aS through renement of Flack's parameter À0.07(3) (Fig. 5).  Penicilazaphilone E (2) was also obtained as a yellow gum, possessing a molecular formula of C 19 H 25 ClO 6 with seven degrees of unsaturation. The IR spectrum indicated the presence of hydroxyl (3752 cm À1 ) and conjugated carbonyl (1703 cm À1 ) groups. The 13 C NMR spectroscopic data (Table 1) displayed the presence of 19 carbon signals including one carbonyl carbon (d C 193.9) and six olenic carbons, three methyl groups, three methylene groups, four methine groups, and two quaternary carbons. In the 1 H NMR spectrum, three olenic protons at d H 6.77 (d, J ¼ 15.5 Hz), 6.36 (d, J ¼ 15.5 Hz) and 6.06 (s), three methyl groups, three methylene groups, and four methine groups were observed. Detailed analysis of the 1 H and 13 C NMR spectroscopic data (Table 1) revealed that 2 is an azaphilone derivative, also closely related to WB (CAS no. 1701443-52-8) (5). 17,18 Comparision of the 1 H and 13 C NMR data revealed that 2 differed from 5 only at the side chains. The terminal methyl of the side chain at d H 0.91, d C 11.8 in 5 were absent in 2. Instead, the signals for a methylene at d H 3.60, d C 60.1 were observed in 2. Furthermore, the above structural moiety accounts for six unsaturation degrees (one carbonyl, six olenic carbons, and the bicyclic core of azaphilone), indicated that 2 must contain another ring. The contiguous sequence of correlations from H-4 0 to H-7 0 in the COSY spectrum and the HMBC correlations from H-5 0 to C-7 0 and H-4 0 to C-6 0 indicated the presence of a tetrahydrofuran ring (Fig. 2).
The relative conguration of 2 was determined also by 1 H-1 H coupling constants and NOESY data revealing a bicyclic core with the same relative congurations as 1 and 5 (Fig. 3). Specically, the cis-relationship of H-4 0 /H-5 0 in the side chain was deduced from the coupling constant (J 4 0 , 5 0 ¼ 2.0 Hz). The NOESY correlations of H 3 -9 0 /H-4 0 indicated that these protons located on the same side of the moiety. Thus, the relative congurations of 2 were determined. The absolute conguration of 2 could not be assigned only by ECD method, while the absolute conguration of the side chain in 2 could not be determined by Mosher's method as 5 (ref. 18) because of the presence of tetrahydrofuran ring in the side chain. Unfortunately, the cultivation of single crystal of 2 was failed, even cultivated by the 4-bromobenzoate derivative of 2. And due to the limitation of compound amount, the absolute conguration of 2 was tentatively determined by biosynthetic pathways. Compound 2 was proposed to be a cyclized form of 5 between 7 0methyl and 4 0 -OH, that is, 5 was the precursor of 2. The plausible biosynthetic pathways from 5 to 2 were proposed. As depicted in Scheme 1, 5 is initially oxidized at side-chain by a cytochrome P450 hydroxylase to form a C-terminal hydroxylated product (a). Then the intermediate (a) undergoes a cyclic reaction between 7 0 -methyl and 4 0 -OH. It appears conceivable that a have two different cyclization pathways. Path 1: the dehydration of 7 0 -OH leads to the nucleophilic attack from 4 0 -O À to 7 0 -C + , which directly gives 2. Path 2: the dehydration of 4 0 -OH result in the nucleophilic attack from 7 0 -O À to 4 0 -C + , which could yield two possible products, 2 and b, on account of the rotation of the side chain. Owing to the fact that the relative conguration of 2 is identical with that of 5, the absolute conguration of 2 was proposed to be identical to that of the precursor 5 tentatively.
Azaphilones are a class of structurally variable fungal polyketide metabolites isolated mainly from the ascomyceteous fungi, such as genera Penicillium, Aspergillus, Chaetomium, Talaromyces and Xylariaceae. 8,19 The azaphilone derivatives could be dened as polyketides possessing a highly oxygenated pyranoquinone bicyclic core and a quaternary carbon center. 8 In the present study, the new compounds 1 and 2 belong to the substructure type of sclerotiorin (3)-like azaphilones, which usually possess a g-lactone, a conjugated ketone, a chlorine atom at C-5, and a branched C 7 side chain at C-3. 8 Their differences compared with sclerotiorin (3) lay on the imparity of the side chains at C-3 and substituent groups at C-7. Compound 1 represents the second reported azaphilone with a C4 aliphatic side chain, 20 a 3-buten-2-one moiety. Besides, compound 2 is a rstly reported azaphilone with a tetrahydrofuran ring at C-3.
The known compounds 3-6 belong to sclerotiorin structural skeleton with a branched C 7 side chain. As reported in the literature, the absolute congurations of the side chains [-CH] CHCOH(CH 3 )CH(OH)CH(CH 3 )CH 2 CH 3 ] in such azaphilones were determined by Mosher's method. 16,18 Fortunately, by slow crystallization from the mixture of EtOAc and hexane, single crystals of 6 suitable for X-ray diffraction were obtained. By detailed analysis of the crystal structure, the absolute conguration of 6 was established unambiguously as 7R,8R,8aR,3 0 R,4 0 R,5 0 S (Fig. 5) through renement of Flack's parameter 0.07(3). It was the rst time to conrm the absolute conguration of 6 by single-crystal X-ray diffraction.

Experimental section
General experimental procedure Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. ECD spectra were recorded on a Jasco J-815-150S circular dichroism spectrometer. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets. NMR spectra were recorded on an Agilent DD2 500 MHz NMR spectrometer (500 MHz for 1 H and 125 MHz for 13 C), using TMS as an internal standard. The HRESIMS and ESIMS spectra were obtained from a Micromass Q-TOF spectrometer. The crystallographic data were collected on a Bruker SMART APEX-II CCD diffractometer equipped with graphite monochromatized Mo Ka radiation (l ¼ 0.71073 A). Semi-preparative HPLC was performed on a Hitachi L-2000 HPLC system coupled with a Hitachi L-2455 photodiode array detector. A Kromasil C 18 semi-preparative HPLC column (250 Â 10 mm, 5 mm) was used. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200-300 mesh), Sephadex LH-20 (Amersham Biosciences) and octadecylsilyl silica gel (Unicorn; 45-60 mm) were used for column chromatography. Precoated silica gel GF 254 plates (Yantai Zifu Chemical Group Co., Yantai, People's Republic of China) were used for analytical TLC.

Fungal material
The fungal strain P. sclerotiorum GDST-2013-0415 was isolated from the inner part of an unidentied sponge GDST-2013-04 collected from the coral reef at a depth of 10 m in the sea area of Shantou, Guangdong Province of China, in May 2013. The fungal identication was performed by analysis of its ITS region of the rDNA as described previously. 21 The sequence data was submitted to the Genbank, with accession number MF467908. The strain was deposited in the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, PR China.

Extraction and isolation
Fiy Erlenmeyer asks of the fungal strain were cultivated in rice medium (3.6 g of natural sea salt from Yangkou saltern, China; 70 g of rice; 100 mL of H 2 O) for 40 days at room temperature. The fermented rice substrate was extracted repeatedly with EtOAc (400 mL for each ask), and the solvent was combined and concentrated in vacuo to afford a residue. The EtOAc residue (65 g) was subjected to silica gel CC using a step gradient elution with EtOAc-petroleum ether (0-100%) and then with MeOH-EtOAc (0-100%) to afford seven fractions (Fr.1-Fr.7). Fr.4 was rst subjected to Sephadex LH-20 CC eluting with a mixture of CHCl 3 -MeOH (1 : 1, v/v) and then was further puried by using semi-preparative HPLC eluting with Scheme 1 Proposed biosynthetic pathway of 2 from 5.

Antiviral activity assay
The antiviral activities against HSV, EV71, RSV1 and CoxB3 were determined by the CPE inhibition assay according to established procedures. 22 Ribavirin was used as a positive control.

Antibacterial activity assay
The antibacterial activity was evaluated by the conventional broth dilution assay. 23 Six pathogenic bacterial strains, M. lysodeikticus, B. subtilis, B. cereus, M. luteus, B. megaterium, and S. dysenteriae, were used, and ciprooxacin was used as a positive control.

Cytotoxicity assay
The cytotoxicity against human erythroleukemia K562 cell lines was evaluated using MTT method. 24 The cytotoxicity against human cervical carcinoma HeLa, human cancer of colon HCT-116, and human lung carcinoma A549 cell lines were evaluated using SRB method. 25 The four human cancer cell lines above were purchased from Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Chinese Academy of Sciences (CAS). Adriamycin was used as a positive control.

Conclusion
In conclusion, two new azaphilones, penicilazaphilones D and E (1 and 2), along with four known analogs (3)(4)(5)(6) were obtained from the sponge-derived fungus P. sclerotiorum. The absolute congurations of 1 and 2 were determined by single-crystal Xray diffraction analysis and comparison to the biosynthetically related known compound 5, respectively. It was the rst time to conrm the absolute congurations of known compound, geumsanol G (6) by single-crystal X-ray diffraction. Compound 1 represents the second reported azaphilone with a C-4 aliphatic side chain, while compound 2 is the rst reported azaphilone with a tetrahydrofuran ring at C-3 side chain. Compound 3 showed potent antiviral activity against HSV and EV71 with the IC 50 values of 19.5 and 132 mM, respectively, more stronger than the positive control ribavirin.

Conflicts of interest
The authors declare that they have no conict of interest.