Four New Chromone Derivatives from Mangrove Endophytic Fungus Phomopsis Sp

marine drugs

Article Phomopsichin A–D; Four New Chromone Derivatives from Mangrove Endophytic Fungus Phomopsis sp. 33#

Meixiang Huang 1,†, Jing Li 2,3,†, Lan Liu 2,4,*, Sheng Yin 1, Jun Wang 1,* and Yongcheng Lin 3,4,5 1 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China; [email protected] (M.H.); [email protected] (S.Y.) 2 School of Marine Sciences, Sun Yat-sen University, Guangzhou 510006, China; [email protected] 3 Key Laboratory of Functional Molecules from Oceanic Microorganisms (Sun Yat-sen University), Department of Education of Guangdong Province, Guangzhou 510080, China 4 South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510006, China 5 School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China; [email protected] * Correspondence: [email protected] (L.L); [email protected] (J.W.); Tel.: +86-208-411-4834 (L.L.); +86-203-994-3090 (J.W.) † These authors contributed equally to this work.

Academic Editor: Russell Kerr Received: 9 September 2016; Accepted: 8 November 2016; Published: 22 November 2016 Abstract: Four new chromone derivatives, phomopsichins A–D (1–4), along with a known compound, phomoxanthone A (5), were isolated from the fermentation products of mangrove endophytic fungus Phomopsis sp. 33#. Their structures were elucidated based on comprehensive spectroscopic analysis coupled with single-crystal X-ray diffraction or theoretical calculations of electronic circular dichroism (ECD). They feature a tricyclic framework, in which a dihydropyran ring is fused with the chromone ring. Compounds 1–5 showed weak inhibitory activities on acetylcholinesterase as well as α-glucosidase, weak radical scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) as well as OH, and weak antimicrobial activities. Compounds 1–4 showed no cytotoxic activity against MDA-MB-435 breast cancer cells. Their other bioactivities are worthy of further study, considering their unique molecular structures.

Keywords: mangrove endophytic fungi; Phomopsis sp.; secondary metabolites; chromone derivatives

1. Introduction The chromone family of natural products exhibit a range of biological activities including anticancer, anti-inﬂammatory, antibacterial, antiviral, atypical antipsychotic, and anti-platelet properties [1–9]. In our continuous investigation of new bioactive secondary metabolites from the mangrove endophytic fungi in the South China Sea, four new chromone derivatives, phomopsichin A–D (1–4), along with a known compound, phomoxanthone A (5), were isolated from the metabolic products of endophytic fungus Phomopsis sp. 33# from the bark of the mangrove plant Rhizophora stylosa. Compounds 1–3 (Figure1) featured a tricyclic framework in which a dihydropyran ring is fused at C-3 and C-4 of the chromone ring. To our knowledge, the compounds with this type skeleton number approximately 10, which were reported to exhibit the activity attenuating resistin-induced adhesion of HCT-116 colorectal cancer cells to endothelial cells [10], the activity interrupting the dimer formation of Aβ17–42 peptide associated to Alzheimer’s disease [11], inhibitory activity against metallo-β-lactamases [12], moderate antibacterial activity and weak cytotoxic activity [13–15]. In this study, we report the isolation, structural elucidation, and exploration on the biological activities of compounds 1–5.

Mar. Drugs 2016, 14, 215; doi:10.3390/md14110215 www.mdpi.com/journal/marinedrugs Mar. Drugs 2016, 14, 215 2 of 11

InMar. this Drugs study,2016, 14 we, 215 report the isolation, structural elucidation, and exploration on the biological2 of 11 activities of compounds 1–5.

Figure 1. TheThe chemical structures of compounds 1–5.

2. Results Results

2.1. Structure Elucidation Phomopsichin A A ( 1,, Figure 11)) waswas obtainedobtained asas aa whitewhite solidsolid andand hadhad aa molecularmolecular formulaformula ofof C16HH1616OO7 7asas determined determined by by its its datum datum of of high high resolution resolution electrospray electrospray ionization mass spectroscopy (HRESIMS) (observed(observedm /mz 319.08184/z 319.08184 M− , calculatedM−, calculated 319.08233), 319.08233), requiring requiring nine degrees nine of unsaturation.degrees of unsaturation.The 13C-NMR The and 13C distortionless‐NMR and distortionless enhancement enhancement by polarization by polarization transfer (DEPT) transfer spectra (DEPT) (Table spectra1) 3 (Tableindicated 1) indicated the presence the ofpresence two carbonyl of two groups carbonyl (δc 169.5groups and (δc 173.2), 169.5 and eight 173.2), olefinic eight carbons, olefinic two carbons, sp CH 3 3 3 1 1 1 1 twogroups, sp CH one groups, sp CH 2onegroup, sp CH two2 methoxygroup, two groups, methoxy and groups, one methyl and one group. methyl The group.H-NMR The and H‐NMRH- H andcorrelation 1H‐1H correlation spectroscopy spectroscopy (COSY) (Table (COSY)1 and (Table Figure 1 2and) showed Figure the 2) showed signals ofthe two signalsm-hydrogens of two m‐ hydrogensof phenol of (δ Hphenol6.93 ( dδHJ 6.93= 2.4d J = Hz; 2.4 Hz; 6.85 6.85 d Jd =J = 2.4 2.4 Hz), two two methoxy methoxy groups groups (δH ( 3.85/3.42),δH 3.85/3.42), and oneand 2 one‐oxo 2-oxo-propyl‐propyl group group (δH 1.34 (δ dH J 1.34= 6.0 dHz;J =4.34 6.0 m; Hz; 2.67 4.34 dd J m; = 18.0, 2.67 4.0 dd Hz;J = 2.58 18.0, dd 4.0 J = 18.0, Hz; 2.584.0 Hz). dd TheJ = 18.0, remaining 4.0 Hz). Thetwo remaining degrees twoof degreesunsaturation of unsaturation supported supported a tricyclic a tricyclic carbon carbon framework framework of dihydropyrano[4,3of dihydropyrano[4,3-b]chromen-10(1H)-one‐b]chromen‐10(1H)‐one in 1 in, which1, which was was confirmed confirmed by the by the correlations correlations between between H‐ 13H-13 and and C‐ C-2/C-112/C‐11 in inheteronuclear heteronuclear multiple multiple-bond‐bond correlation correlation (HMBC) (HMBC) spectroscopy. spectroscopy. In In the the HMBC spectrum (Figure 22),), richrich correlationcorrelation datadata allowedallowed usus toto unambiguouslyunambiguously establishestablish thethe locationslocations ofof substituents on the carbon skeleton. The HMBC correlation between H H-8‐8 and C C-14‐14 revealed that the carbonyl group was located at the C-9C‐9 position; the correlation between H3‐-11 and C C-3‐3 demonstrated that the CH33‐-11 was locatedlocated atat thethe C-2 C‐2 position; position; and and the the correlations correlations between between H 3H-153‐15 and and C-14 C‐14 as as well well as asbetween between H 3H-163‐16 and and C-13 C‐13 indicated indicated that that the the two two methoxy methoxy groups groups were were located located atat thethe C-14C‐14 and C C-13‐13 positions, respectively.respectively. One One hydroxyl hydroxyl group group was was identified identified at theat the C-7 C position‐7 position based based on the on lower the lower field fieldchemical chemical shift (shiftδc 162.6, (δc 162.6, C-7). C‐7).

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Table 1. 1H-NMR and 13C-NMR data of compounds 1–4 (400/100 MHz, J in Hz).

1 (in C3D6O) 2 (in CDCl3) 3 (in CDCl3) 4 (in CD3OD)

δC δH δC δH δC δH δC δH 1 21.0 q 1.34 d 6.0 21.0 q 1.39 d 6.4 21.3 q 1.38 d 6.0 23.8 q 1.31 d 6.0 2 62.8 d 4.34 m 62.1 d 4.41 m 70.0 d 3.83 m 66.7 t 4.23 m 3 34.6 t 2.67 dd 18.0, 4.0 34.4 t 2.63 m 34.6 t 2.64 m 42.1 t 2.94 m 2.58 dd 18.0,10.8 4 164.1 s 163.6 s 160.7 s 167.7 s 5 158.4 s 144.4 s 144.8 s 159.3 s 6 104.3 d 6.93 d 2.4 134.7 s 134.9 s 104.4 d 6.87 d 2.4 7 162.6 s 149.2 s 149.0 s 164.2 s 8 113.8 d 6.85 d 2.4 108.0 d 6.89 s 107.9 d 6.93 s 114.8 d 6.77 d 2.4 9 114.6 s 124.5 s 124.0 s 113.7 s 10 136.4 s 116.0 s 115.6 s 136.2 s 11 173.2 s 173.5 s 174.1 s 177.2 s 12 117.4 s 116.6 s 116.5 s 122.1 s 13 95.2 d 5.40 s 94.5 d 5.57 s 62.5 t 4.82 d 15.2 55.0 t 4.55 s 4.48 d 15.2 14 169.5 s 169.8 s 170.0 s 171.6 s 15 52.8 q 3.85 s 53.2 q 3.95 s 53.2 q 3.96 s 53.3 q 3.91 s 16 55.6 q 3.42 s 55.9 q 3.55 s Mar. Drugs17 2016, 14, 215 56.8 q 3.98 s 56.9 q 3.99 s 3 of 11

Figure 2. The key 1HH-‐11HH COSY COSY and and HMBC HMBC correlations correlations of of compounds compounds 11––44..

Table 1. 1H‐NMR and 13C‐NMR data of compounds 1–4 (400/100 MHz, J in Hz). The relative stereochemistry of 1 was established by its nuclear Overhauser effect spectroscopy (NOESY). The NOE1 (in correlation C3D6O) between2 H-2(in CDCl and H3) -16 indicated3 (in CDCl the relative3) stereochemistry4 (in CD3OD) of 1 as 3 shown in FigureδC 3. δH δC δH δC δH δC δH The1 complete21.0 q structure1.34 d 6.0 and stereochemistry21.0 q 1.39 d of6.41 were21.3 q further1.38 confirmed d 6.0 23.8 by q X-ray1.31 diffractiond 6.0 analysis2 (Figure62.8 d4 ). The4.34 final m refinement 62.1 d of the4.41 Cu m K α data70.0 d resulted3.83 in m a small66.7 Flack t 4.23 parameter m of 0.02(3),3 allowing34.6 t an2.67 unambiguous dd 18.0, 4.0 assignment34.4 t of2.63 the m absolute34.6 t configuration2.64 m of42.11 as t 2S, 132.94R (Figure m 1). 2.58 dd 18.0,10.8 Phomopsichin B (2, Figure1) was obtained as a white solid and had a molecular formula of 4 164.1 s 163.6 s 160.7 s 167.7 s m z − C17H185O 8 based158.4 s on HRESIMS data (observed144.4 s / 349.09241144.8 M s , calculated 349.09289),159.3 s with one more 1 13 CH3O6 group 104.3 than d compound6.93 d 2.41 . The134.7H-NMR, s C-NMR, 134.9 and s HMBC spectra104.4 of 2 dwere 6.87 very d 2.4 similar to those7 of162.61 (Table s 1), except for the149.2 absence s of the H-6149.0 signal, s and an added164.2 CHs 3O-17 signals (δH/C 3.98/56.8).8 113.8 d The added6.85 d 2.4 CH 3O-17108.0 was d located6.89 s at the107.9 C-7 d position6.93 based s on114.8 the d NOE6.77 correlationd 2.4 9 114.6 s 124.5 s 124.0 s 113.7 s 10 136.4 s 116.0 s 115.6 s 136.2 s 11 173.2 s 173.5 s 174.1 s 177.2 s 12 117.4 s 116.6 s 116.5 s 122.1 s 13 95.2 d 5.40 s 94.5 d 5.57 s 62.5 t 4.82 d 15.2 55.0 t 4.55 s 4.48 d 15.2 14 169.5 s 169.8 s 170.0 s 171.6 s 15 52.8 q 3.85 s 53.2 q 3.95 s 53.2 q 3.96 s 53.3 q 3.91 s 16 55.6 q 3.42 s 55.9 q 3.55 s 17 56.8 q 3.98 s 56.9 q 3.99 s

The relative stereochemistry of 1 was established by its nuclear Overhauser effect spectroscopy (NOESY). The NOE correlation between H‐2 and H3‐16 indicated the relative stereochemistry of 1 as shown in Figure 3. The complete structure and stereochemistry of 1 were further confirmed by X‐ray diffraction analysis (Figure 4). The final refinement of the Cu Kα data resulted in a small Flack parameter of 0.02(3), allowing an unambiguous assignment of the absolute configuration of 1 as 2S, 13R (Figure 1). Phomopsichin B (2, Figure 1) was obtained as a white solid and had a molecular formula of C17H18O8 based on HRESIMS data (observed m/z 349.09241 M−, calculated 349.09289), with one more CH3O group than compound 1. The 1H‐NMR, 13C‐NMR, and HMBC spectra of 2 were very similar to those of 1 (Table 1), except for the absence of the H‐6 signal, and an added CH3O‐17 signals (δH/C 3.98/56.8). The added CH3O‐17 was located at the C‐7 position based on the NOE correlation between

Mar. Drugs 2016, 14, 215 4 of 11

Mar. Drugs 2016, 14, 215 4 of 11 betweenMar. H-17Drugs 2016 and, 14 H-8., 215 One hydroxyl group was identiﬁed at the C-6 position based on the4 of chemical 11 H‐17 and H‐8. One hydroxyl group was identified at the C‐6 position based on the chemical shift of shiftMar. of C-6 Drugs (δ 2016c 134.7), 14, 215 as well as the HMBC correlation between H-8 and C-6. 4 of 11 H‐17 and H‐8. One hydroxyl group was identified at the C‐6 position based on the chemical shift of C‐6 (δc 134.7) as well as the HMBC correlation between H‐8 and C‐6. HC‐‐176 ( δandc 134.7) H‐8. as One well hydroxyl as the HMBC group correlation was identified between at the H‐ C8 ‐and6 position C‐6. based on the chemical shift of C‐6 (δc 134.7) as well as the HMBC correlation between H‐8 and C‐6.

FigureFigureFigure 3. 3. 3.The The The key key key correlations correlationscorrelations ofof compounds compounds 1 and11 andand 2 in2 2 inNOESY.in NOESY. NOESY. Figure 3. The key correlations of compounds 1 and 2 in NOESY.

Figure 4. The X‐ray single‐crystal structure of 1. Figure 4. The X-ray single-crystal structure of 1. Figure 4. The X‐ray single‐crystal structure of 1. The relative stereochemistryFigure of 4. 2 The was X ‐establishedray single‐crystal by its structure NOESY. of The 1. NOE correlation between H‐2 and H3‐16, similar to those of 1, indicated the relative stereochemistry of 2 as shown in Figure 3. TheThe relative relative stereochemistry stereochemistry of of 22 waswas establishedestablished by by its its NOESY. NOESY. The The NOE NOE correlation correlation between between H-2 and HTheCompounds-16, relative similar stereochemistry 2 and to those 1 have of identical1 ,of indicated 2 was chiral established the spheres, relative by just its stereochemistry NOESY.opposite Thein the NOE signs of 2correlationas of shown their specificbetween in Figure 3. H‐2 and 3H3‐16, similar to those of 1, indicated the relative stereochemistry of 2 as shown in Figure 3. Hrotation‐2 and Hdata;3‐16, their similar ECD to spectra those of were 1, indicated symmetric the (Figure relative 5). stereochemistry The ECD spectrum of 2 of as 2 shown showed in negative Figure 3. Compounds 2 and 1 have identical chiral spheres, just opposite in the signs of their speciﬁc CottonCompoundsCompounds effect at 3182 2and and(Δε 1 − 10.48)have have nm identicalidentical as well chiralaschiral positive spheres, one at justjust 291 oppositeopposite (Δε +0.53) in in nm.the the Meanwhile,signs signs of oftheir their the specific ECD specific rotation data; their ECD spectra were symmetric (Figure5). The ECD spectrum of 2 showed negative rotationrotationspectrum data; data; of their 1 their displayed ECD ECD spectra spectraopposite were were Cotton symmetricsymmetric effects at (Figure the same 5).5). Thewavelengths. The ECD ECD spectrum spectrum For the of aboveof 2 showed2 showed reasons, negative negative the CottonCottonCottonabsolute effect effect effect configuration at at 318 at 318 318 (∆ (εΔε (Δε− − 0.48)of −0.48)0.48) 2 was nm nm nm suggested asas as well wellwell as asas positivepositive2R, 13S. oneone one atat at 291 291 291 ( Δε ( (Δε ∆+0.53)ε+0.53)+0.53) nm. nm. nm. Meanwhile, Meanwhile, Meanwhile, the the ECD the ECD ECD spectrumspectrumspectrum of of1 of 1displayed displayed1 displayed oppositeopposite opposite Cotton Cotton effectseffects effects at at thethe the samesame same wavelengths. wavelengths. wavelengths. For For the the For above above the reasons, above reasons, the reasons, the the absolute conﬁguration of4 2 was suggested as 2R, 13S. absoluteabsolute configuration configuration of of 2 2 was was suggested suggested as 2R, 13S.. ECD 1 ECD 2 4 4 ECD ECD 1 1

) ECDECD 2 2

-1 2 cm -1 ) )

-1 2

-1 2 (M cm  cm -1

-1 0

(M 250 300 350 400 (M   0 0 250 300 350 400 250 300Wavelength 350 (nm) 400 -2 Figure 5. The ECD spectra of 1 andWavelength 2. (nm) -2 Wavelength (nm) -2 FigureFigure 5. 5.The The ECD spectra spectra of of 1 1andand 2. 2. Figure 5. The ECD spectra of 1 and 2.

Mar. Drugs 2016, 14, 215 5 of 11

Mar. Drugs 2016, 14, 215 5 of 11 Mar. Drugs 2016, 14, 215 5 of 11 Phomopsichin C (3, Figure1) was obtained as a white solid and had a molecular formula of C 16 Phomopsichin C (3, Figure 1) was obtained as a white solid− and had a molecular formula of1 C16 H16O7 based on HRESIMS data (observed m/z 319.08200− M , calculated 319.08233).1 The H-NMR,13 16 H16OPhomopsichin7 based on HRESIMS C (3, Figure data (observed 1) was obtained m/z 319.08200 as a white M , solidcalculated and had 319.08233). a molecular The formulaH‐NMR, of C‐ 13 1 1 C-NMR,HNMR,16O7 based 1HH-‐1H on COSY,H HRESIMS COSY, and and HMBC data HMBC (observed spectra spectra of 3m were/z of 319.082003 verywere similar veryM−, calculated to similar those of to 319.08233). compound those of compound The2 (Table 1H‐NMR, 1, Figure2 (Table 13C ‐ 1, 1 1 FigureNMR,2), except2), exceptH‐ forH COSY,the for changes the and changes HMBC of CH of‐ spectra13 CH-13 signals of signals 3(δ wereH/C 5.57/94.5) very ( δH/ similarC 5.57/94.5)in 2 to to CH those2‐ in13 of 2signals tocompound CH (2δ-13H/C 4.82 signals2 (Table d; 4.48 ( 1,δ H d/62.5)Figure/C 4.82 d; 4.482),in d/62.5) 3except. These for in results 3the. These changes suggested results of CH that suggested‐13 compound signals ( thatδH /3C 5.57/94.5)is compound lacking ina methoxy23 tois CH lacking2‐ 13group signals a methoxyat the (δH /CC ‐4.8213 group position. d; 4.48 at d/62.5) theThe C-13 position.inabsolute 3. These The configuration results absolute suggested conﬁguration of compound that compound 3 of was compound determined 3 is lacking3 aswas 2aR methoxy by determined the result group that as at 2 thetheR byexperimental C‐13 the position. result ECD that The the experimentalabsoluteand calculated configuration ECD ECD and spectrum calculated of compound for ECD 2R 3isomer was spectrum determined matched for 2exactlyR asisomer 2R by(Figure the matched result 6). that exactly the experimental (Figure6). ECD and calculated ECD spectrum for 2R isomer matched exactly (Figure 6). )

-1 Calcd 3 ) 6 -1 cm Exptl Calcd 3 3 6 -1

cm Exptl 3 -1 (M

 4 (M

 4

2 2

0 250 300 350 0 250 300 350

-2 Wavelength (nm)

-2 Wavelength (nm) FigureFigure 6. 6.The The calculated calculated and experimental experimental ECD ECD spectra spectra of 3 of. 3. Figure 6. The calculated and experimental ECD spectra of 3. Phomopsichin D (4, Figure 1) had a molecular formula of C15H16O7 based on HRESIMS data Phomopsichin D (4, Figure1) had a molecular formula of C H O based on HRESIMS − 15 16 7 1 (observedPhomopsichin m/z 307.08194 D (4 ,M Figure, calculated 1) had 307.08233), a molecular requiring formula eight of C degrees15H16O7 basedof unsaturation. on HRESIMS The dataH‐ data (observed m/z 307.08194 M−, calculated 307.08233), requiring eight degrees of unsaturation. (observedNMR, 13C‐ m/zNMR, 307.08194 1H‐1H COSY, M−, calculated and HMBC 307.08233), spectra ofrequiring 4 were veryeight similar degrees to of those unsaturation. of 1 (Table The 1 and 1H ‐ The 1H-NMR, 13C-NMR, 1H-1H COSY, and HMBC spectra of 4 were very similar to those of 1 (Table1 NMR,Figure 132),C‐ NMR,except 1 Hfor‐1 Hthe COSY, change and of HMBC CH‐13 spectra signals of (δ 4H /Cwere 5.40/95.2) very similar in 1 to to CH those2OH of‐13 1 signals(Table 1( δandH/C and Figure2), except for the change of CH-13 signals ( δ 5.40/95.2) in 1 to CH OH-13 signals Figure4.55/55.0) 2), andexcept the forabsence the changeof a methoxy of CH group‐13 signals signal ( δinH/ C4 .5.40/95.2) AH /dicyclicC in 4 H1‐ chromento CH2OH‐4‐‐one13 2 signalssegment (δ ofH/C (δH/4.55/55.0)4C was4.55/55.0) decided and andthe based absence the on absence its of eight a ofmethoxy adegrees methoxy group of unsaturation, group signal signal in 4. A inwhich dicyclic4. A was dicyclic 4 supportedH‐chromen 4H-chromen-4-one by‐4‐ onethe absencesegment segment of of of 44HMBCwas was decideddecided correlation based between on its eightH eight‐13 anddegrees degrees C‐2. ofThe of unsaturation, unsaturation, hydroxymethyl which which group was was wassupported supported located byat C bythe‐12 theabsence based absence on of of HMBCHMBCthe HMBC correlation correlation correlation between between between H-13 H‐13 andH ‐and13 C-2. and C‐2. The C The‐11. hydroxymethyl hydroxymethylA 2‐hydroxypropyl group group group was was locatedwas located located atat C-12C at‐12 C based‐based4 based on the HMBCtheon theHMBC correlation HMBC correlation correlations between between H-13 between andH‐13 H C-11. ‐and1 and C A ‐C11. 2-hydroxypropyl‐4, Aas 2 well‐hydroxypropyl as, between group Hgroup‐ was3 and was located C‐12. located at C-4 at C based‐4 based on the HMBCon the correlationsThe HMBC absolute correlations configuration between between H-1 of and 4 was H C-4,‐1 confirmed and as C well‐4, asas as, well2S between based as, between on H-3 the result and H‐3 C-12. andthat Cthe‐12. experimental data andThe calculatedThe absolute absolute ECD conﬁguration configuration spectrum for of of 4the 4waswas 2S confirmed isomer conﬁrmed matched as 2 asS based 2exactlyS based on (Figurethe on result the 7). resultthat the that experimental the experimental data dataand and calculated calculated ECD ECD spectrum spectrum for the for 2 theS isomer 2S isomer matched matched exactly exactly (Figure (Figure 7). 7). 7 Calcd 4 7

6 Exptl Calcd 4 4

6 Exptl 4 5 ) 5 -1 4 ) cm -1

-1 4

cm 3 (M -1

 3

(M 2  2 1

1 0 250 300 350 0 Wavelength (nm) 250 300 350 Wavelength (nm) Figure 7. The calculated and experimental ECD spectra of 4.

FigureFigure 7. 7.The The calculated calculated and experimental ECD ECD spectra spectra of of4. 4.

Mar. Drugs 2016, 14, 215 6 of 11

Compound 5 was identiﬁed as phomoxanthone A (5, Figure1) by comparison of its spectral data with that of the literature [16,17]; both compound 5 and phomoxanthone A had the same NMR, MS, ECD data (Figure S27, in Supplementary Materials) and speciﬁc rotation data.

2.2. Biological Evaluation The various bioactivities of compounds 1–5 were evaluated in vitro. The ﬁve compounds displayed low inhibitory activities on acetylcholinesterase (AchE) as well as α-glucosidase, weak radical scavenging effects on DPPH as well as OH, and low antimicrobial activity against 13 pathogenic bacteria strains (Tables S8 and S9, in Supplementary Materials). Compounds 1–4 showed no cytotoxic activity against MDA-MB-435 breast cancer cells. It was reported that phomoxanthone A(5) has strong pro-apoptotic activity and immunostimulatory activity [17], so we did not consider its cytotoxicity assays in the study.

3. Materials and Methods

3.1. General Experimental Procedures Acetylcholinesterase (AchE), S-acetylthiocholine iodide, 5,50-dithio-bis-(2-nitrobenzoic acid), huperzine A, α-glucosidase and p-nitrophenyl-α-D-glucopyranoside were purchased from Sigma (St. Louis, MO, USA); 1,1-diphenyl-2-picrylhydrazyl (DPPH), H2O2, 1,10-phenanthroline, FeSO4, and other reagents were of analytical grade and commercially available; methanol was of chromatographic grade; potato dextrose agar (PDA) medium was purchased from Beijing L and Bridge Technology Co. Ltd. (Beijing, China). Optical rotation measurements were carried out using a Bellingham-Stanley 37–440 polarimeter (Bellingham Stanley Ltd., Kent, UK). UV spectra were determined using a UV-240 spectrophotometer (Shimadzu, Tokyo, Japan). ECD spectra were measured using a Chirascan Circular Dichroism Spectrometer (Applied Photophysics, London, UK). IR spectra were measured on a TENSOR37 spectrometer (Bruker Optics, Ettlingen, Germany). The 1H-NMR and 13C-NMR data were acquired using a Bruker Avance 400 spectrometer at 400 MHz for 1H nuclei and 100 MHz for 13C nuclei (Bruker Biospin, Rheinstetten, Germany). Tetramethylsilane (TMS) was used as an internal standard, and the chemical shifts (δ) were expressed in ppm. The HRESIMS were obtained using a LTQ-Orbitrap LC-MS (Thermo Fisher, Frankfurt, Germany). Single-crystal data were carried out on an Agilent Technologies Gemini A Ultra system (Agilent Tech, Santa Clara, CA, USA). HPLC was performed using a 515 pump with a UV 2487 detector (Waters, Milford, CT, USA) and an Ultimate XB-C-18 column (250 mm × 10 mm, 5 µm; Welch, Maryland, USA). Normal pressure preparative column chromatography was carried out on RP-18 gel (25–40 µm, Daiso Inc., Osaka, Japan), silica gel (200–400 mesh, Qingdao Marine Chemical Inc., Qingdao, China), or a Sephadex-LH-20 (GE Healthcare, Stockholm, Sweden) for reverse and direct phase elution modes, respectively. The thin-layer chromatography was performed over F254 glass plates (Qingdao Marine Chemical Inc.) and analyzed under UV light (254 and 366 nm).

3.2. Fungal Material Endophytic fungus Phomopsis sp. 33# was isolated with PDA medium from the bark of the mangrove plant Rhizophora stylosa, collected in the intertidal region of Zhanjiang, in Guangdong Province, China, and identiﬁed according to its morphological characteristics and internal transcribed spacer (ITS) region [18]. A voucher specimen is deposited in our laboratory at −20 ◦C.

3.3. Fermentation, Extraction, and Isolation Small agar slices bearing mycelia were placed in 1000 mL Erlenmeyer ﬂasks containing rice medium (composed of 60 g rice, 80 mL distilled water, and 0.24 g sea salt) and incubated for 30 days at 28 ◦C. In total, 140 ﬂasks of culture were obtained. Cultures were extracted with EtOAc. In total, Mar. Drugs 2016, 14, 215 7 of 11

250 g crude extract was obtained by evaporation of EtOAc. The crude extract was suspended in H2O (3 L) and partitioned with n-hexane (5 L × 2) and EtOAc (5 L × 2) to give n-hexane (90 g) and EtOAc (110 g) extracts, respectively. The EtOAc extract was subjected to a silica gel column, eluted with a n-hexane-EtOAc gradient (from 100:0 to 0:100) to obtain six fractions (Fractions 1–6). Fraction 2 (15 g) was extracted with 300 mL of chloroform to give dark yellow liquid phases and solid. The chloroform-soluble fraction was evaporated to dryness and washed with methanol (50 mL × 6) to give compound 5 (a light yellow solid, 5.2 g). Fraction 4 (15 g) was chromatographed over a column of RP-18 gel (2.5 cm × 30 cm, MeOH-H2O gradient from: 100:0 to 40:60) to obtain ﬁve fractions (Fractions 4.1–4.5). Fraction 4.1 and 4.3–4.5 were separated by HPLC (MeOH-H2O, 20:80, 2 mL/min, 254 nm), respectively, and then were puriﬁed separately using a Sephadex LH-20 column (MeOH) until pure compound 1 (17 mg), compound 2 (17mg), compound 3 (10 mg), and compound 4 (22 mg) were obtained.

3.4. Spectral Data

25 Phomopsichin A (1): white solid; [a]D +12.5 (c 0.48, MeOH); UV (MeOH) λmax (logε) 290 (4.0), 218 (4.4) nm; −1 ECD (MeOH) λmax (∆ε) 312 (+2.4), 281 (−1.5), 231 (−1.0) nm; IR (KBr) νmax 3410, 2925, 1655 cm ; for 1H-NMR and 13C-NMR data, see Table1; ESIMS m/z 319.0 [M−]; HRESIMS m/z 319.08184 [M−] (calculated for C16H15O7, 319.08233).

Crystal structure determination of phomopsichin A (1): crystal data for C16H16O7 (M = 320.29 g/mol): monoclinic, space group P21 (no. 4), a = 7.6234 (9) Å, b = 15.5426 (17) Å, c = 12.4889 (13) Å, β = 96.241 (10)◦, V = 1471.0 (3) Å3, Z = 4, T = 150 (2) K, µ (CuKα) = 0.973 mm−1, Dcalc = 1.446 g/cm3, ◦ ◦ 15,309 reﬂections measured (7.12 ≤ 2Θ ≤ 133.92 ), 5018 unique (Rint = 0.0877, Rsigma = N/A) which were used in all calculations. The ﬁnal R1 was 0.0849 (>2sigma(I)) and wR2 was 0.2334 (all data). The crystallographic data of 1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC), CCDC Depository Request CRM: 0001000638303.

25 Phomopsichin B (2): white solid; [a]D −8.0 (c 0.89, MeOH); UV (MeOH) λmax (logε) 299 (3.8), 238 (4.4) nm; −1 1 ECD (MeOH) λmax (∆ε) 318 (−0.48), 291 (+0.53) nm; IR (KBr) νmax 3284, 2922, 1649 cm ; for H-NMR and 13C-NMR data, see Table1; ESIMS m/z 349.0 [M−]; HRESIMS m/z 349.09241 [M−] (calculated for C17H17O8, 349.09289). 25 Phomopsichin C (3): white solid; [a]D −72 (c 0.05, MeOH); UV (MeOH) λmax (logε) 303 (3.9), 239 (4.5) nm; −1 ECD (MeOH) λmax (∆ε) 328 (−0.27), 278 (+0.61), 221 (+2.2) nm; IR (KBr) νmax 3325, 2922, 1665 cm ; for 1H-NMR and 13C-NMR data, see Table1; ESIMS m/z 319.1 [M−], 206, 175; HRESIMS m/z 319.08200 − [M ] (calculated for C16H15O7, 319.08233). 25 Phomopsichin D (4): white solid; [a]D +102 (c 0.05, MeOH); UV (MeOH) λmax (logε) 293 (3.8), 221 (4.2) nm; −1 1 ECD (MeOH) λmax (∆ε) 284 (+0.83), 226 (+2.4) nm; IR (KBr) νmax 3265, 2912, 1707 cm ; for H-NMR and 13C-NMR data, see Table1; ESIMS m/z 307.0 [M−]; HRESIMS m/z 307.08194 [M−] (calculated for C15H15O7, 307.08233).

3.5. Computational Analyses All of the theoretical methods and the basis set used for optimization and spectrum calculation were recommended in previous studies [19,20]. All of the theoretical calculations, including geometry optimization, frequency analysis, and ECD spectrum prediction, were carried out with the density functional theory (DFT) and time-dependent density functional theory (TDDFT) methods in the Gaussian 09 software package (Gaussian Inc., Wallingford, CT, USA) [21]. The geometry optimizations were performed at the B3LYP/6-31+G (d) level in the gas phase. Based on the ﬁnal optimized structure, the ECD spectra were calculated at the PBE1PBE-SCRF/6-311++g (d, p) level using the Polarized Continuum Model (PCM) with methanol as a solvent. The theoretical predicted ECD spectra were ﬁtted in the SpecDis 1.6 software package (University of Würzburg, Würzburg, Germany) [22]. Mar. Drugs 2016, 14, 215 8 of 11

3.6. X-ray Crystallographic Analysis of Compound 1

Single crystals of compound 1 were obtained from CH3OH-EtOAc. A suitable crystal was selected and all crystallographic data were collected at 150 K with Cu/Kα radiation (λ = 1.54178 Å). Using Olex2 (OlexSys Ltd., Durham University, Durham, UK), the structure was solved with the SIR2004 structure solution program using direct methods and reﬁned with the XL reﬁnement package using least squares minimization [23–25].

3.7. AchE Inhibitory Assay The inhibitory activities against AchE of compounds 1–5 were investigated in vitro using the modiﬁed Ellman method [26]. The substrates were S-acetylthiocholine iodide and 5,50-dithio-bis- (2-nitrobenzoic acid). Huperzine A was used as a positive control.

3.8. DPPH Radical Scavenging Assay The radical scavenging effect on DPPH of compounds 1–5 were determined according to previously reported methods [27,28], and 2,6-ditertbutyl-4-methylphenol was used as a positive control.

3.9. OH-Radical-Scavenging Assay Radical scavenging effect on OH of compounds 1–5 were carried out according to previously reported methods [29,30]. The indicator used was 1,10-phenanthroline-Fe2+; vitamin C was used as a positive control.

3.10. α-Glucosidase Inhibitory Assay The inhibitory activities against α-glucosidase of compounds 1–5 were investigated in vitro using the modiﬁed method described by Moradi-Afrapoli et al. [31]; p-nitrophenyl-α-D-glucopyranoside was used as the substrates, and trans-resveratrol was used as a positive control.

3.11. Antibacterial Experiment The antibacterial activity of compounds 1–5 were investigated in vitro using the modiﬁed 96 well microtiter-based method described by Pierce et al. [32].

4. Conclusions Mangrove endophytic fungi from the South China Sea provide rich fungal diversity, and are promising sources of structurally-unprecedented bioactive natural products [33–37]. Five chromone derivatives were isolated from the mangrove endophytic fungus Phomopsis sp. 33#, four of them are new compounds (1–4). Compounds 1–5 showed weak inhibitory activity of AchE as well as α-glucosidase, radical scavenging effects on DPPH as well as OH, and low antimicrobial activity. The compounds (1–4) showed no cytotoxic activity against MDA-MB-435 breast cancer cells. Their other bioactivities are worthy of further study, considering their unique tricyclic molecular structures, in which a dihydropyran ring is fused with the chromone ring.

Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/14/11/215/s1. Figure S1: 1H-NMR for phomopsichin A (1). Figure S2: 13C-NMR for phomopsichin A (1). Figure S3: 1H-1H COSY for phomopsichin A (1). Figure S4: heteronuclear single quantum coherence (HSQC) spectroscopy for phomopsichin A (1). Figure S5: HMBC for phomopsichin A (1). Figure S6: NOESY for phomopsichin A (1). Figure S7: HR mass spectrometry for phomopsichin A (1). Figure S8: 1H-NMR for phomopsichin B (2). Figure S9: 13C-NMR for phomopsichin B (2). Figure S10: 1H-1H COSY for phomopsichin B (2). Figure S11: HSQC for phomopsichin B (2). Figure S12: HMBC for phomopsichin B (2). Figure S13: NOESY for phomopsichin B (2). Figure S14: HR mass spectrometry for phomopsichin B (2). Figure S15: 1H-NMR for phomopsichin C (3). Figure S16: 13C-NMR for phomopsichin C (3). Figure S17: 1H-1H COSY for phomopsichin C (3). Figure S18: HSQC for phomopsichin C (3). Figure S19: HMBC for phomopsichin C (3). Figure S20: HR mass spectrometry for phomopsichin C (3). Figure S21: 1H-NMR for phomopsichin D (4). Figure S22: 13C-NMR for phomopsichin Mar. Drugs 2016, 14, 215 9 of 11

D(4). Figure S23: 1H-1H COSY for phomopsichin D (4). Figure S24: HSQC for phomopsichin D (4). Figure S25: HMBC for phomopsichin D (4). Figure S26: HR mass spectrometry for phomopsichin D (4). Figure S27: ECD spectrometry for phomoxanthone A (5). Table S1: Crystal data and structure refinement for phomopsichin A (1). Table S2: Fractional atomic coordinates (× 104) and equivalent isotropic displacement parameters (Å2 × 103) for phomopsichin A (1). Table S3: Anisotropic displacement parameters (Å2 × 103) for phomopsichin A (1). Table S4: Bond lengths for phomopsichin A (1). Table S5: Bond angles for phomopsichin A (1). Table S6: Torsion angles for phomopsichin A (1). Table S7: Hydrogen atom coordinates (Å × 104) and isotropic displacement parameters (Å2 × 103) for phomopsichin A (1). Table S8: Inhibitory activities against AchE as well as α-glucosidase, and the radical scavenging effects on DPPH as well as OH of compounds 1–5. Table S9: Antimicrobial activity of compounds 1–5. Acknowledgments: We are grateful for the financial support from the National Natural Science Foundation of China (21272286), China’s Marine Commonweal Research Project (201305017), Department of Science and Technology of Guangdong Province (No. 2014A020221006 and No. 2015A030313119), Science and Technology Planning Project of Guangdong Province (No. 2013B021100009), innovative development of marine economic demonstration project, GD2012-D01-001 and Traditional Chinese Medicine Bureau of Guangdong Province (20141052). Author Contributions: Jun Wang and Lan Liu took charge throughout the research, structural elucidation and writing. Meixiang Huang mainly took part in the extraction and isolation. Jing Li mainly took part in the computational analyses. Sheng Yin mainly took part in biological activity assay. Yongcheng Lin mainly checked the error about structures elucidation. Conflicts of Interest: The authors declare no conflict of interest.

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