New Antibacterial Phenone Derivatives Asperphenone A–C from Mangrove-Derived Fungus Aspergillus Sp

marine drugs

Article New Antibacterial Phenone Derivatives Asperphenone A–C from Mangrove-Derived Fungus Aspergillus sp. YHZ-1

Zhi-Kai Guo 1,2,†, Yi-Qin Zhou 1,†, Hao Han 1, Wen Wang 1, Lang Xiang 1, Xin-Zhao Deng 1, Hui-Ming Ge 1,* and Rui-Hua Jiao 1,*

1 State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210023, China; [email protected] (Y.-Q.Z.); [email protected] (H.H.); [email protected] (W.W.); [email protected] (L.X.); [email protected] (X.-Z.D.) 2 Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China; [email protected] * Correspondence: [email protected] (H.-M.G.); [email protected] (R.-H.J.) † These authors contributed equally to this work.

Received: 29 December 2017; Accepted: 25 January 2018; Published: 30 January 2018

Abstract: Marine fungi are a promising source of novel bioactive natural products with diverse structure. In our search for new bioactive natural products from marine fungi, three new phenone derivatives, asperphenone A–C (1–3), have been isolated from the ethyl acetate extract of the fermentation broth of the mangrove-derived fungus, Aspergillus sp. YHZ-1. The chemical structures of these natural products were elucidated on the basis of mass spectrometry, one- and two-dimensional NMR spectroscopic analysis and asperphenone A and B were conﬁrmed by single-crystal X-ray crystallography. Compounds 1 and 2 exhibited weak antibacterial activity against four Gram-positive bacteria, Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus, with the MIC values higher than 32.0 µM.

Keywords: mangrove endophyte; Aspergillus sp.; marine natural product; asperphenone; antibacterial activity

1. Introduction Marine ﬁlamentous fungi are a rich source of antimicrobial compounds, anti-inﬂammatory, anticancer and antiviral agents [1–5]. Among all the marine-sourced fungi, the species in the genus Aspergillus (Trichocomaceae) are an important source for novel pharmacological metabolites such as polyketides, alkaloids and terpenoids [6–9]. The mangrove plants inhabit the intertidal zones in the tropics and subtropics, providing a very unique habitat for animals and microbes. Thus, the mangrove-associated fungi, with the majority coming from endophytic species, have attracted much attention to discover structurally diverse and bioactive secondary metabolites. In the past several years, our research group has explored many novel secondary metabolites from mangrove-derived endophytic fungi, such as sesquiterpenoids diaporols A-I, diaporine with regulation activity of macrophage differentiation and other polyketides [10–12]. As part of our ongoing search for novel biologically active natural products from microbe from special environment [13,14], we chemically investigated an endophytic fungus, Aspergillus sp. YHZ-1, from mangrove plant from Hainan Island, China. Fractionation of the ethyl acetate extract of its liquid fermentation broth led to the discovery of three new phenone derivatives, which we have named asperphenone A–C (1–3).

Mar. Drugs 2018, 16, 45; doi:10.3390/md16020045 www.mdpi.com/journal/marinedrugs Mar.Mar. Drugs Drugs2018 2018, 16, 16, 45, x FOR PEER REVIEW 2 of2 7 of 7

of three new phenone derivatives, which we have named asperphenone A–C (1–3). Compounds 1 Compoundsand 2 were1 testedand 2 wereand displayed tested and weak displayed antibacterial weak antibacterial activity against activity fouragainst Gram-positive four Gram-positive bacteria. bacteria.Herein, Herein,we report we reportthe isolation the isolation and structure and structure elucidation elucidation of these of these new new compounds compounds and and their their antibacterialantibacterial activity. activity.

2.2. Results Results AA large-scale large-scale fermentation fermentation broth broth (50(50 L) of Aspergillus sp.sp. YHZ-1 YHZ-1 was was collected collected and and extracted extracted with with ethylethyl acetate acetate three three times times to to produce produce aa crude extract. The The subsequent subsequent fractionation fractionation by by repeated repeated column column chromatographychromatography over over silica silica gel, gel, octadecylsilyloctadecylsilyl silic silicaa gel gel (ODS), (ODS), Sephadex Sephadex LH-20 LH-20 and and semi-preparative semi-preparative reversed-phasereversed-phase highhigh performanceperformance liquidliquid chroma chromatographytography (HPLC) (HPLC) yielded yielded three three new new phenone phenone derivatives,derivatives, asperphenone asperphenone A–C A–C ((11––33)) (Figure(Figure1 1).).

FigureFigure 1. 1.The The structures structures ofof asperphenoneasperphenone A–C A–C ( (11––33) )isolated isolated from from AspergillusAspergillus sp.sp. YHZ-1. YHZ-1.

AsperphenoneAsperphenone AA ((11)) waswas isolated as as a areddish-brown reddish-brown needle needle crystal. crystal. The Themolecular molecular formula formula of 1 was determined to be C17H16O7 on the basis of the high-resolution (HR) ESI-MS (m/z 355.2716 [M + of 1 was determined to be C17H16O7 on the basis of the high-resolution (HR) ESI-MS (m/z 355.2716 + 1 13 [MNa] + Na, calcd.]+, calcd. 355.2706) 355.2706) along along with with the theH and1H andC 13NMRC NMR data data (Table (Table 1), 1indicating), indicating ten ten degrees degrees of of unsaturation. The1 1H,13 13C and HSQC NMR spectra (in supplementary materials) in DMSO-d6 revealed unsaturation. The H, C and HSQC NMR spectra (in supplementary materials) in DMSO-d6 revealed signals of two methyls (δC/δH 11.6/1.73, C-14/H3-14; δC/δH 26.6/2.52, C-16/H3-16), one oxygenated signals of two methyls (δC/δH 11.6/1.73, C-14/H3-14; δC/δH 26.6/2.52, C-16/H3-16), one oxygenated methyl (δC/δH 60.3/3.78, 10-OMe), one methylene (δC/δH 19.4/3.70, C-7/H2-7), one ketone carbon (δC methyl (δC/δH 60.3/3.78, 10-OMe), one methylene (δC/δH 19.4/3.70, C-7/H2-7), one ketone carbon 203.8, C-15), two α,β-unsaturated carbonyl carbons (δC 182.7, C-9; δC 185.1, C-12), three phenolic (δC 203.8, C-15), two α,β-unsaturated carbonyl carbons (δC 182.7, C-9; δC 185.1, C-12), three phenolic hydroxyl protons (δH 10.25, 1-OH; δH 13.21, 5-OH; δH 10.85, 11-OH) and ten aromatic/olefinic carbons, hydroxyl protons (δH 10.25, 1-OH; δH 13.21, 5-OH; δH 10.85, 11-OH) and ten aromatic/olefinic whose chemical shift values indicated the presence of one 1,2,3,4-tetrasubstituted benzene ring (δC carbons, whose chemical shift values indicated the presence of one 1,2,3,4-tetrasubstituted benzene 162.7, C-1; δC/δH 107.8/6.42 (d, J = 8.8 Hz), C-2/H-2; δC/δH 131.8/7.67 (d, J = 8.8 Hz), C-3/H-3; δC 112.8, ring (δ 162.7, C-1; δ /δ 107.8/6.42 (d, J = 8.8 Hz), C-2/H-2; δ /δ 131.8/7.67 (d, J = 8.8 Hz), C-4; δCC 162.4, C-5; δC C112.0,H C-6) and four quaternary carbons (δC 136.0,C C-8;H δC 138.8, C-10; δC 143.3, C-3/H-3;C-11; δC δ144.5,C 112.8, C-13). C-4; TheδC 162.4,tetrasubstituted C-5; δC 112.0, benzene C-6) ring and was four determined quaternary as carbons a 1,3-dihydroxy-4- (δC 136.0, C-8; δCacetophenone138.8, C-10; δ moiety,C 143.3, which C-11; δwasC 144.5, deduced C-13). by Theanalysis tetrasubstituted of the 1H-1H COSY benzene correlation ring was from determined H-2 to H- as a 1 1 1,3-dihydroxy-4-acetophenone3 and the HMBC correlations from moiety, H-2 which to C-4 was and deduced C-6, from by H-3 analysis to C-1, of C-5 the andH- C-15,H COSY from correlationH3-16 to fromC-4 H-2and C-15 to H-3 and and from the 5-OH HMBC to C-4, correlations C-5 and C-6 from (Figure H-2 2). to HMBC C-4 and correlations C-6, from from H-3 H to2-7 C-1, to C-1, C-5 C- and C-15,5 and from C-6 H permitted3-16 to C-4 the and linkage C-15 andof C-7 from to 5-OHC-6 of to the C-4, acetophenone C-5 and C-6 moiety. (Figure 2The). HMBCpresence correlations of a p- frombenzoquinone H2-7 to C-1, moiety C-5 could and C-6be deduced permitted from the the linkage chemical of shifts C-7 toof C8-C13 C-6 of in the 1, acetophenonewhich was partially moiety. Theconfirmed presence by of the a p observation-benzoquinone of HMBC moiety correlations could be deducedfrom H3-14 from to C-8, the C-12 chemical and C-13 shifts and of from C8-C13 H2-7 in 1, whichto C-8, was C-9 partially and C13. confirmed Meanwhile, by the the connectivity observation of of the HMBC p-benzoquinone correlations and from acetophenone H3-14 to C-8, moietiesC-12 and C-13via andthe methylene from H2-7 C-7 to was C-8, established C-9 and C13. by the Meanwhile, relevant HMBC the connectivity correlations. ofHowever, the p-benzoquinone the positions of and acetophenonethe hydroxyl moietiesgroup and via methoxyl the methylene group on C-7 the was p-benzoquinone established by moiety the relevant cannot HMBCbe determined correlations. as However,their 2D correlations the positions cannot of the be hydroxyl observed. group Finally, and a high-quality methoxyl group single oncrystal the pof-benzoquinone 1 was obtained moietyand cannotthe full be structure determined of 1 as was their determined 2D correlations by the cannotX-ray crys betallographic observed. Finally, analysis a high-qualityin a Cu Kα radiation single crystal in oflow1 was temperature obtained and(Figure the 3), full which structure also ofconfirmed1 was determined the proposed by structure the X-ray of crystallographic 1. analysis in a Cu Kα radiation in low temperature (Figure3), which also confirmed the proposed structure of 1.

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Table 1. 1H and 13C NMR data for compounds 1–3.

1 a 2 a 3 b No. δ , Mult. δ , Mult. δ , Mult. H δ , Type H δ , Type H δ , Type (J in Hz) C (J in Hz) C (J in Hz) C 1 162.7, C 162.3, C 163.3, C 1-OH 10.25 c, brs 10.75, brs 2 6.42, d (8.8) 107.8, CH 6.52, d (8.8) 107.4, CH 6.55, d (8.6) 108.3, CH 3 7.67, d (8.8) 131.8, CH 7.77, d (8.8) 132.1, CH 7.71, d (8.6) 132.8, CH 4 112.8, C 112.5, C 113.8, C 5 162.4, C 162.7, C 164.4, C 5-OH 13.21, s 13.09, s 13.73, s 6 112.0, C 109.5, C 111.4, C 7.07, dd 7 3.70, s 19.4, CH 3.61, d (2.0) 22.0, CH 124.1, CH 2 2 (16.2, 0.8) 6.78, dd 8 136.0 d,C 148.5, C 128.6, CH (16.2, 7.0) 4.42, dd 9 182.7, C 187.2, C 83.3, CH (7.0, 0.8) 9-OMe 3.37, s 57.0, CH3 10 138.8, C 116.9, C 171.7, C 10-OMe 3.78, s 60.3, CH3 3.70, s 52.0, CH3 11 143.3, C 153.8, C 204.3, C 11-OH 10.85 c, br s 10.75, brs 12 185.1, C 183.0, C 2.56, s 26.3, CH3 13 144.5 d,C 5.76, t (2.0) 127.1, CH 14 1.73, s 11.6, CH3 1.82, s 8.15, CH3 15 203.8, C 203.4, C Mar. Drugs16 2018, 16, x 2.52, FOR sPEER REVIEW 26.6, CH 3 2.55, s 26.2, CH3 3 of 7 Mar. Drugsa 2018, 16, x FOR PEER1 REVIEW 13 b 1 3 of 7 Acquired at 400 MHz for H NMR and 100 MHz for C NMR in DMSO-d6; Acquired at 600 MHz for H NMR 13 c,d and 150 MHz for C NMR in acetone-d6; Interchangeable signals.

Figure 2. 1 3 Figure 2. TheThe key key 2D 2D NMR NMR correlations correlations of asperphenone A–C ( 1–3). Figure 2. The key 2D NMR correlations of asperphenone A–C (1–3).

Figure 3. X-rayX-ray crystal crystal structure structure of 1.. Figure 3. X-ray crystal structure of 1.

Table 1. 1H and 13C NMR data for compounds 1–3. Asperphenone B (2) wasTable obtained 1. 1H and as a 13 yellowC NMR needle data for crystal, compounds with the 1–3 molecular. formula C16H14O6 + (ten degrees of unsaturation)1 a as derived from the HRESIMS2 a data (m/z 325. 13263 [M b + Na] , calcd. 13 1 a 2 a 3 b 325.0683).No. TheδH,C Mult. NMR spectrum showedδH, sixteen Mult. carbon signals for twoδH, methylsMult. (C-14 and C-16), No. δH, Mult. δC, Type δH, Mult. δC, Type δH, Mult. δC, Type one methylene( (C-7),J in Hz) one ketoneδC, Type carbon (C-15),(J in Hz) two α,β-unsaturatedδC, Type carbonyl(J in Hz) carbons (C-9δC, and Type C-12) (J in Hz) (J in Hz) (J in Hz) 1 162.7, C 162.3, C 163.3, C 1 162.7, C 162.3, C 163.3, C 1-OH 10.25 c, brs 10.75, brs 1-OH 10.25 c, brs 10.75, brs 2 6.42, d (8.8) 107.8, CH 6.52, d (8.8) 107.4, CH 6.55, d (8.6) 108.3, CH 2 6.42, d (8.8) 107.8, CH 6.52, d (8.8) 107.4, CH 6.55, d (8.6) 108.3, CH 3 7.67, d (8.8) 131.8, CH 7.77, d (8.8) 132.1, CH 7.71, d (8.6) 132.8, CH 3 7.67, d (8.8) 131.8, CH 7.77, d (8.8) 132.1, CH 7.71, d (8.6) 132.8, CH 4 112.8, C 112.5, C 113.8, C 4 112.8, C 112.5, C 113.8, C 5 162.4, C 162.7, C 164.4, C 5 162.4, C 162.7, C 164.4, C 5-OH 13.21, s 13.09, s 13.73, s 5-OH 13.21, s 13.09, s 13.73, s 6 112.0, C 109.5, C 111.4, C 6 112.0, C 109.5, C 111.4, C 7.07, dd 7 3.70, s 19.4, CH2 3.61, d (2.0) 22.0, CH2 7.07, dd 124.1, CH 7 3.70, s 19.4, CH2 3.61, d (2.0) 22.0, CH2 (16.2, 0.8) 124.1, CH (16.2, 0.8) 6.78, dd 8 136.0 d, C 148.5, C 6.78, dd 128.6, CH 8 136.0 d, C 148.5, C (16.2, 7.0) 128.6, CH (16.2, 7.0) 4.42, dd 9 182.7, C 187.2, C 4.42, dd 83.3, CH 9 182.7, C 187.2, C (7.0, 0.8) 83.3, CH (7.0, 0.8) 9-OMe 3.37, s 57.0, CH3 9-OMe 3.37, s 57.0, CH3 10 138.8, C 116.9, C 171.7, C 10 138.8, C 116.9, C 171.7, C 10-OMe 3.78, s 60.3, CH3 3.70, s 52.0, CH3 10-OMe 3.78, s 60.3, CH3 3.70, s 52.0, CH3 11 143.3, C 153.8, C 204.3, C 11 143.3, C 153.8, C 204.3, C 11-OH 10.85 c, br s 10.75, brs 11-OH 10.85 c, br s 10.75, brs 12 185.1, C 183.0, C 2.56, s 26.3, CH3 12 185.1, C 183.0, C 2.56, s 26.3, CH3 13 144.5 d, C 5.76, t (2.0) 127.1, CH 13 144.5 d, C 5.76, t (2.0) 127.1, CH 14 1.73, s 11.6, CH3 1.82, s 8.15, CH3 14 1.73, s 11.6, CH3 1.82, s 8.15, CH3 15 203.8, C 203.4, C 15 203.8, C 203.4, C 16 2.52, s 26.6, CH3 2.55, s 26.2, CH3 16 2.52, s 26.6, CH3 2.55, s 26.2, CH3 a Acquired at 400 MHz for 1H NMR and 100 MHz for 13C NMR in DMSO-d6; b Acquired at 600 MHz a Acquired at 400 MHz for 1H NMR and 100 MHz for 13C NMR in DMSO-d6; b Acquired at 600 MHz for 1H NMR and 150 MHz for 13C NMR in acetone-d6; c,d Interchangeable signals. for 1H NMR and 150 MHz for 13C NMR in acetone-d6; c,d Interchangeable signals.

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Asperphenone B (2) was obtained as a yellow needle crystal, with the molecular formula C16H14O6 (ten degrees of unsaturation) as derived from the HRESIMS data (m/z 325. 1326 [M + Na]+, calcd. 325.0683). The 13C NMR spectrum showed sixteen carbon signals for two methyls (C-14 and C- 16),Mar. Drugsone methylene2018, 16, 45 (C-7), one ketone carbon (C-15), two α,β-unsaturated carbonyl carbons (C-9 4and of 7 C-12) and ten aromatic/olefinic carbons. Comparison of the MS, 1H and 13C NMR data of 2 with those ofand 1 tenrevealed aromatic/olefinic the presence carbons. of the same Comparison 1,3-dihydroxy-4-acetophenone of the MS, 1H and 13C NMR moiety data as of 1 2andwith a thosedifferent of 1 substitutedrevealed the p presence-benzoquinone of the same moiety. 1,3-dihydroxy-4-acetophenone Further HMBC correlations moiety (Figure as 2)1 andfrom a H different3-14 (δHsubstituted 1.82) to C- 9 (δC 187.2), C-10 (δC 116.9) and C-11(δC 153.8), from H-13 (δH 5.76) to C-7 (δC 22.0), C-8 (δC 148.5), C-9 p-benzoquinone moiety. Further HMBC correlations (Figure2) from H 3-14 (δH 1.82) to C-9 (δC 187.2), and C-11 and from H2-7 (δH 3.61) to C-8, C-9 and C-13 (δC 127.1) along with the unsaturation C-10 (δC 116.9) and C-11(δC 153.8), from H-13 (δH 5.76) to C-7 (δC 22.0), C-8 (δC 148.5), C-9 and C-11 requirement of 2 confirmed the substituted pattern of the p-benzoquinone moiety. Finally, the and from H2-7 (δH 3.61) to C-8, C-9 and C-13 (δC 127.1) along with the unsaturation requirement of 2 connectionconfirmed thebetween substituted the two pattern moieties of the mentionedp-benzoquinone above moiety.was through Finally, C-7 the by connection interpretation between of the relevanttwo moieties HMBC mentioned correlations above from was H2 through-7. Detailed C-7 NMR by interpretation analysis allowed of the the relevant assignment HMBC of correlations 2 as shown in Figure 1, which was also confirmed by a low-temperature single-crystal X-ray diffraction from H2-7. Detailed NMR analysis allowed the assignment of 2 as shown in Figure1, which was also experimentconfirmed by (Figure a low-temperature 4). single-crystal X-ray diffraction experiment (Figure4).

Figure 4. X-ray crystal structure of 2. Figure 4. X-ray crystal structure of 2.

Asperphenone C C ( 33)) was isolated as as a colorless needle crystal that analyzed for the molecular + formula C14HH1616OO6 6(seven(seven degrees degrees of of unsaturation) unsaturation) by by HRESIMS (m/zz 303.303. 1215 1215 [M [M + Na] +, ,calcd. calcd. 13 13 303.0839) in combination with 13CC NMR NMR data (Table 11).). TheThe 13CC and and DEPT135 DEPT135 NMR NMR spectra spectra of of 3 displayed signals for fourteen carbon carbons,s, including one methyl carbon (δC 26.3, C-12), two oxygenated methyl carbons (δC 57.0,57.0, 9-OMe; 9-OMe; δC 52.0,52.0, 10-OMe), 10-OMe), one one oxygenated oxygenated methine ( δC 83.3,83.3, C-9), C-9), one one ketone ketone carbon ( (δδCC 204.3,204.3, C-11), C-11), one one carboxylic carboxylic carbon carbon (δ (Cδ C171.7,171.7, C-10) C-10) and and eight eight aromatic/olefinic aromatic/oleﬁnic carbons. carbons. In thisIn this molecule, molecule, the the same same moiety, moiety, 1,3-dihydroxy-4-acetophenone 1,3-dihydroxy-4-acetophenone as as found found in in 11, ,could could be be deduced 1 1 through analysis analysis of of the the 1H-H-1HH COSY COSY correlation correlation from from H-2 H-2 (δ (Hδ H6.55,6.55, d, d,J =J 8.6= 8.6 Hz) Hz) to H-3 to H-3 (δH( 7.71,δH 7.71, d, J d,= 8.6J = 8.6Hz) Hz and) and HMBC HMBC correlations correlations from from H-2 H-2 to C-4 to C-4 (δC (113.8)δC 113.8) and and C-6 C-6(δC (111.4),δC 111.4), from from H-3 H-3to C-1 to C-1(δC 163.3),(δC 163.3), C-5 C-5(δC 164.4) (δC 164.4) andand C-11, C-11, from from 5-OH 5-OH (δH (13.73)δH 13.73) to C-4, to C-4, C-5 C-5 and and C-6 C-6 and and from from H3 H-123-12 (δH ( δ2.56)H 2.56) to 1 1 C-4to C-4 and and C-11. C-11. 1H-1HH- COSYH COSY correlations correlations from fromH-7 ( H-7δH 7.07, (δH dd,7.07, J = dd, 16.2,J = 0.8 16.2, Hz) 0.8 to Hz)H-8 to(δH H-8 6.78, (δ dd,H 6.78, J = 16.2,dd, J 7.0= 16.2, Hz) and 7.0 Hz) from and H-8 from to H-9 H-8 (δ toH 4.42, H-9 (dd,δH J4.42, = 7.0, dd, 0.8J Hz)= 7.0, and 0.8 HMBC Hz) and cross-peaks HMBC cross-peaks from H-8 to from the carboxylicH-8 to the carboxyliccarbon C-10 carbon established C-10 established the fragment the fragmentC-7-C-8-C-9-C-10, C-7-C-8-C-9-C-10, which was which attached was attached to the acetophenoneto the acetophenone moiety moietythrough through C-6-C-7 C-6-C-7linkage on linkage the basis on theof the basis HMBC of the correlations HMBC correlations from H-8 to from C- 3 6H-8 and to from C-6 andH-7 fromto C-1 H-7 and to C-5. C-1 3 andJC-H diagnostic C-5. JC-H diagnosticcorrelations correlations from the oxygenated from the oxygenated methyl protons methyl at δprotonsH 3.37 to at C-9δH 3.37 and tofrom C-9 the and oxygenated from the oxygenated methyl protons methyl at protons δH 3.70 at toδ HC-103.70 demonstrated to C-10 demonstrated that the methoxylthat the methoxyl groups should groups be should located be at locatedC-9 and atC- C-910, respectively. and C-10, respectively. Thus, the structure Thus, theof compound structure of3 wascompound elucidated3 was as elucidated shown in as Figure shown 1. in However, Figure1. However, the absolute the absolute configuration conﬁguration of C-9 ofhas C-9 not has been not determined.been determined. In the the primary primary screen screen for for antibacterial antibacterial compounds, compounds, compounds compounds 1 and1 and 2 were2 were tested tested against against four Gram-positivefour Gram-positive bacteria, bacteria, StaphylococcusStaphylococcus aureus aureus CMCC(B)CMCC(B) 26003, 26003, StreptococcusStreptococcus pyogenes pyogenes ATCC19615,ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus . As As summarized summarized in in Table Table 22,, BothBoth ofof thesethese twotwo compounds showed weak weak activity activity against against the the tested tested bacteria, bacteria, StaphylococcusStaphylococcus aureus aureus CMCC(B)CMCC(B) 26003, 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus ,, with with the MIC values higher than 32.0 µµM.M.

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Table 2. Antibacterial activities of compounds 1 and 2 (MIC, µM).

Compounds S. aureus B. subtilis S. pyogenes M. luteus 1 64.0 64.0 64.0 32.0 2 32.0 64.0 32.0 32.0 Ampicillin 4.0 8.0 2.0 1.0

3. Materials and Methods

3.1. General Experimental Procedures The HR-ESI-MS data were obtained on an Agilent 6210 time of ﬂight LC-MS instrument (Agilent Technologies Inc., Palo Alto, CA, USA). NMR experiments were conducted on a Bruker DPX-400 NMR spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR) or Bruker DRX-600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR) (Bruker Corporation, Karlsruhe, Germany). The chemical shifts were given in δ (ppm) and referenced to the solvent signal (DMSO-d6, δH 2.50, δC 39.5; acetone-d6, δH 2.05, δC 29.8). Column chromatography (CC) was accomplished on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS (40–70 µm, Merck Company, Darmstadt, Germany) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Semi-preparative reverse-phase (RP) HPLC was performed on a Hitachi HPLC system with a L-7110 pump, a L-7420 UV/vis detector and an Hypersil RP-C18 column (5 µm, 250 × 10.0 mm, Thermo Fisher Scientiﬁc, Waltham, MA, USA). Thin-layer chromatography (TLC) was conducted on silica gel GF254 (10–20 µm, Qingdao Marine Chemical Inc., Qingdao, China). All chemicals used were of HPLC grade or analytical grade.

3.2. Strain Isolation and Cultivation The fungus Aspergillus sp. YHZ-1 was isolated by one of the authors (Y.-Q.Z.) from an unidentified mangrove plant from Hainan Island, China, in October 2015. The isolate was identified as Aspergillus sp. by its morphological characteristics and the voucher specimen (IFB-YHZ-1) was deposited in the Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University. The strain was cultivated on MEA agar plate (consisting of 20 g/L malt extract, 20 g/L sucrose, 1 g/L peptone, 20 g/L agar and deionized water) at 30 ◦C for 5 days. Then small agar plugs with mycelia were inoculated into five 1 L-Erlenmeyer flasks, each containing 400 mL MEA liquid medium, which were cultivated at 28 ◦C with 108 rpm/min. After 3 days of fermentation, 10 mL of the seed cultures were inoculated into 200 flasks with 250 mL ME liquid medium and fermented on a rotary shaker with 120 rpm/min at 28 ◦C for 14 days.

3.3. Extraction and Isolation The entire ﬁltrate of the fermented broth (50 L) was harvested and extracted three times with an equivalent volume of ethyl acetate at room temperature. The organic solvent was evaporated in vacuo to yield 25 g of crude extract. Then the crude extract was fractionated by silica gel (200–300 mesh) CC eluting with a gradient of CH2Cl2-MeOH mixtures (v/v, 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, 0:100) to give 8 fractions (Fr.1–8). Fr.3 (CH2Cl2-MeOH, v/v, 100:2) was subsequently subjected to ODS CC with MeOH-H2O(v/v, 30:70, 40:60, 50:50, 60:40, 70:30, 100:0) to afford 6 sub fractions (Fr.3.1–Fr.3.6). Finally, compounds 1 (12.5 mg), 2 (11.2 mg), 3 (6.3 mg) were puriﬁed from Fr.3.4 and Fr.3.5 by semi-preparative reverse-phase HPLC (2 mL/min, detector UV λmax 254 nm, CH3CN-H2O 55:45).

3.4. Crystal Data of 1 The single crystal X-ray diffraction data of compound 1 was collected on a Bruker APEX-II diffractometer at 130 K with Cu Kα radiation (λ = 1.54178 Å). The structure was solved using the program SHELXS-97 and refined by full-matrix least-squares on F2. Crystal data of compound 1 have Mar. Drugs 2018, 16, 45 6 of 7 been deposited with the Cambridge Crystallographic Data Centre (deposition No. CCDC 1584390), which can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif. Crystal data for 1: molecular formula C17H16O7, Mr = 332.30, monoclinic crystals, a = 13.2620 (3) Å, b = 16.2202 (4) Å, c = 7.0447 (2) Å, α = 90.00◦, β = 97.7510◦ (10), γ = 90.00◦, Z = 4, µ = 0.977 mm−1, F (000) = 696 and T = 130 K; Crystal dimensions: 0.15 × 0.1 × 0.05 mm3, Volume = 1501.56 (7) Å3, 10,794 reflections measured, 2783 independent reflections (Rint = 0.0472), the final R indices [I > 2σ(I)] 2 R1 = 0.0469, wR2 = 0.1286, R indices (all data) R1 = 0.0500, wR2 = 0.1330. The goodness of fit on F was 1.026.

3.5. Crystal Data of 2 The single crystal data of compound 2 was collected on a Bruker APEX-II diffractometer at 130 K with Cu Kα radiation (λ = 1.54178 Å). The structure was solved using the program SHELXS-97 and refined by full-matrix least-squares on F2. Crystal data of compound 2 have been deposited with the Cambridge Crystallographic Data Centre (deposition No. CCDC 1584387), which can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif. Crystal data for 2: molecular formula C16H14O6, Mr = 302.27, monoclinic crystals, a = 11.4611 (4) Å, b = 4.9487 (2) Å, c = 13.6864 (5) Å, α = 90.00◦, β = 111.933◦ (2), γ = 90.00◦, Z = 2, µ = 0.909 mm−1, F (000) = 316 and T = 130 K; Crystal dimensions: 0.22 × 0.2 × 0.15 mm3, Volume = 720.07 (5) Å3, 4697 reflections measured, 1843 independent reflections (Rint = 0.0350), the final R indices [I > 2σ(I)] 2 R1 = 0.0385, wR2 = 0.1115, R indices (all data) R1 = 0.0389, wR2 = 0.1119. The goodness of fit on F was 1.095.

3.6. Antibacterial Activity Assay The in vitro antibacterial activity of compounds 1 and 2 were evaluated against four bacteria including Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus in accordance with previously reported methods [14,15]. In the assays, the medium used in the test was Müller-Hinton (MH) broth. All test compounds and the positive control ampicillin were dissolved in dimethyl sulfoxide (DMSO). The minimum inhibitory concentration (MIC) values were determined in the 96-well plates (triplicate) and determined as the lowest sample concentration exhibiting no bacterial growth.

4. Conclusions Three new phenone derivatives, asperphenone A–C (1–3), were isolated from the ethyl acetate extract of the fermentation broth of the mangrove-derived fungus Aspergillus sp. YHZ-1. The chemical structures of these compounds were elucidated on the basis of HR-ESI-MS, 1D and 2D NMR spectroscopic analysis, as well as X-ray crystallographic data. Both of the tested compounds 1 and 2 displayed weak antibacterial activity against four Gram-positive bacteria, Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus, indicating that the mangrove-associated fungi are still a rich source for discovering diverse new bioactive natural products which could be used as lead compounds in drug development.

Supplementary Materials: The 1D and 2D NMR spectra for compounds 1–3 are available online at www.mdpi. com/1660-3397/16/2/45/s1. Acknowledgments: This work was financially supported by the National Natural Science Fund of China (Nos. 81673333, 81522042, 21572100, 81421091, 81500059, 41406083, 21672101 and 21661140001), SBK2015022152 and Jiangsu Provincial Key Medical Discipline (laboratory) (No. ZDXKA2016020). Author Contributions: Rui-Hua Jiao and Hui-Ming Ge conceived and designed the experiments; Yi-Qin Zhou and Zhi-Kai Guo cultured, isolated and identified the compounds; Xin-Zhao Deng performed the biological tests; Zhi-Kai Guo, Yi-Qin Zhou, Hao Han, Wen Wang and Lang Xiang analyzed the data; Zhi-Kai Guo, Hui-Ming Ge and Rui-Hua Jiao wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. Mar. Drugs 2018, 16, 45 7 of 7

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