molecules

Article Hyperjaponol H, A New Bioactive Filicinic Acid-Based Meroterpenoid from japonicum Thunb. ex Murray

Rongrong Wu 1,†, Zijun Le 2,†, Zhenzhen Wang 3, Shuying Tian 1, Yongbo Xue 3, Yong Chen 1, Linzhen Hu 1,* and Yonghui Zhang 3 ID 1 National & Local Joint Engineering Research Center of High-throughput Drug Screening Technology, Key Laboratory of Biotechnology of Chinese Traditional Medicine, School of Life Science, Hubei University, Wuhan 430062, ; [email protected] (R.W.); [email protected] (S.T.); [email protected] (Y.C.) 2 Wuhan Rayson School, Wuhan 430040, China; [email protected] 3 Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; [email protected] (Z.W.); [email protected] (Y.X.); [email protected] (Y.Z.) * Correspondence: [email protected]; Tel.: +86-27-88663882 † These authors contributed equally to this work.

Academic Editor: Isabel C. F. R. Ferreira Received: 18 February 2018; Accepted: 16 March 2018; Published: 18 March 2018

Abstract: Hyperjaponol H (1), a new filicinic acid-based meroterpenoid, with a 6/6/10 ring system trans-fused by hetero-Diels–Alder cycloaddition between a germacrane sesquiterpenoid and a filicinic acid moiety, was isolated from aerial parts of . The elucidation of its structure and absolute configuration were accomplished by the analyses of extensive spectroscopic data and the comparison of Cotton effects of electron circular dichroism (ECD) with previously reported ones. The bioactivity assay showed that hyperjaponol H exhibited a moderate inhibitory efficacy on lytic Epstein-Barr virus (EBV) DNA replication in B95-8 cells.

Keywords: Hypericum japonicum; meroterpenoid; Epstein-Barr virus

1. Introduction Natural products are widely known to be a considerable resource of biologically active compounds that involve manifold and unusual scaffolds. Most secondary metabolites from of Guttiferae are mainly found to be phloroglucinol derivatives with complex architectures and appealing therapeutical properties [1–3]. Hypericum japonicum Thunb. ex Murray, as a member of Guttiferae family, also termed as Tianjihuang in China, is widespread chiefly in temperate regions of North America, Oceania, and Asia [4,5]. Also as a type of traditional Chinese medicine, H. japonicum has been extensively utilized for the medical treatment of the hemostasis, detumescence, dysentery, and hepatitis [4]. In recent years, literatures reported that various ranges of chemical constituents such as aliphatic compounds, terpenoids, flavonoids, xanthonoids, lactones, and phloroglucinol derivatives had been discovered from this herb [5–11]. In our on-going research on the genus Hypericum for structurally fascinating and biologically appealing metabolites, we have reported some meroterpenoids of polycyclic prenylated acylphloroglucinols (PPAPs) from H. sampsonii, H. ascyron, H. attenuatum, and H. perforatum [12–15] as well as a series of filicinic acid-based meroterpenoids (Hyperjaponols A–G) from H. japonicum [16]. In the present study, the isolation, structural confirmation, and anti-EBV assay of compound 1, named hyperjaponol H, a metabolite of H. japonicum, are illustrated in detail.

Molecules 2018, 23, 683; doi:10.3390/molecules23030683 www.mdpi.com/journal/molecules Molecules 2018, 23, 683 2 of 8

Molecules2016, 21,x 2 of 8 2. Results and Discussion 2. Results and Discussion A crude extract (300 g) produced from the dried herbs of H. japonicum (4 kg) was subjected to the silicaA crude gel columnextract (300 chromatography g) produced from (silica the gel dried CC) elutedherbs of successively H. japonicum with (4 kg) the was gradient subjected mobile to phasesthe silica of gel petroleum column chromatography ether, chloroform, (silica and gel ethyl CC) acetate. eluted successively The fraction with of petroleum the gradient ether mobile was sequentiallyphases of petroleum chromatographed ether, chloroform, by MCI gel and column, ethyl ODSacetate. Middle The Pressurefraction Liquidof petroleum Chromatography ether was (MPLC),sequentially Sephadex chromatographed LH-20, and by High MCI Performance gel column, LiquidODS Middle Chromatography Pressure Liquid (HPLC) Chromatography to give a new filicinic(MPLC), acid-based Sephadex meroterpenoidLH-20, and High (1) asPerformance drawn in Figure Liquid1, whichChromatography was named (HPLC) as hyperjaponol to give a H. new filicinic acid-based meroterpenoid (1) as drawn in Figure 1, which was named as hyperjaponol H.

Figure 1. Structure of compound 1.

2020 Hyperjaponol H ( 1), white amorphous amorphous powder powder { {[[αα]]D +16.4 +16.4 ((cc 0.06,0.06, CHClCHCl33)}, with the molecular formula C28HH4242OO5,5 ,was was manifested manifested by by the the high-resolution high-resolution electrospray electrospray ionization ionization mass spectraspectra + (HRESIMS) quasi-molecularquasi-molecular peakpeak ionion atat mm/z/z 459.3119459.3119 [M[M + + H] H]+,, calculatedcalculated forfor C C2828HH4343O55 459.3110 −1 (Supplementary MaterialsMaterials FigureFigure S1).S1). TheThe analysesanalyses ofof IRIR absorptionabsorption bandsbands(3455, (3455, 1654, 1654, and and 1612 1612 cm cm−1,, Supplementary Materials Figure S9) demonstrated the characteristic scaffold of an enolic 1,3-diketo system, viz. viz. acylated acylated filicinic filicinic acid acid parent parent core core [10,11,17,18]. [10,11,17,18 Comparis]. Comparisonon of NMR of NMR data databetween between 1 with1 withhyperjaponols hyperjaponols A–G A–G[16] [indicated16] indicated that that1 was1 was constructed constructed via via the the incorporation incorporation of of the the same sesquiterpene unit as hyper hyperjaponolJaponol G to the same acylated fi filiciniclicinic acid acid entity entity as as hyperjaponols hyperjaponols A–F. A–F. 1 1 Accomplished byby meticulous meticulous examination examination of HSQC, of HMBC,HSQC, and HMBC,H- H COSYand spectra1H-1H (SupplementaryCOSY spectra 1 13 Materials(Supplementary Figures Materials S4–S6), Figure all H- S4–S6), and C-NMRall 1H- and data 13C-NMR of 1 were data unequivocally of 1 were unequivocally assigned as assigned shown 1 inas Tableshown1. in The TableH-NMR 1. The (6001H-NMR MHz) (600 spectrum MHz) (Supplementaryspectrum (Suppl Materialsementary FigureMaterials S2) Figure displayed S2) resonancesdisplayed resonances for eight methylsfor eight [δmethylsH, 1.29 [ (s),δH, 1.29 1.24 (s), (s), 1.24 1.20 (s), (s), 1.20 1.19 (s), (s), 1.19 1.02 (s), (s), 1.02 1.11 (s), ( d1.11, J = (d 6.7, J = Hz), 6.7 1.10Hz), (1.10d, J = (d 6.7, J = Hz 6.7), Hz), 1.00 (1.00d, J =(d 7.0, J = Hz)], 7.0 Hz)], and twoand olefinictwo olefinic methine methine protons protons [δH, 5.59[δH, (5.59dd, J (=dd 16.2,, J = 16.2, 4.7 Hz), 4.7 13 5.47Hz), (5.47dd, J (=dd 16.2,, J = 16.2, 8.0 Hz) 8.0]. Hz)]. The TheC-NMR 13C-NMR (150 (150 MHz) MHz) spectrum spectrum (Supplementary (Supplementary Materials Materials Figure Figure S3) denotedS3) denoted 28 carbon 28 carbon resonances resonances consisting consisting of one of quaternaryone quaternary carbon, carbon, one carbonyl,one carbonyl, two oxygenated,two oxygenated, and fiveand enolicfive enolic or olefinic or olefinic carbons, carbons, five methylenes, five methylenes, six methines six methines (four aliphatic, (four aliphatic, and two and olefinic two carbons), olefinic andcarbons), eight methyls.and eight Taking methyls. the aforementionedTaking the aforementi analysesoned and analyses its eight indicesand its ofeight proton indices deficiency of proton into consideration,deficiency into compoundconsideration,1 contains compound a tricyclic 1 contains system. a tricyclic system. The planar construction of 1 was established according to the HMBC and 1H-1H COSY experimentsTable 1. 1 (FigureH-NMR2 (600). In MHz) ring and C, the13C-NMR 2-hydroxyisoisopropyl (150 MHz) spectral data residue of compound was connected 1 in CDCl at3 position(δ in 8 dueppm, to the J in HMBC Hz). correlations from Me-12/Me-13 to C-11 and C-8, while the HMBC cross-peaks between Me-15 with C-1, C-2, and C-10 as well as the cross-peaks between Me-14 with C-4, C-5, and C-6 Position δH (J) δC Position δH (J) δC 1 1 suggested Me-151 and Me-14 was located at positions84.9 1 and14 5, respectively.1.00d (7.0) Meanwhile, 16.4 the clear H- H COSY spin systems2 of H-2/H-3/H-4/H-5/H-6/H-7/H-8/H-9/H-101.47 m 36.1 15 supported1.02 s the21.3 structural profile of ring C, a germacrane3 unit. Regarding0.91 m to the ring25.2 A, a filicinic1′ acid core, was confirmed188.7 by the HMBC correlations of Me-12 0/Me-130 1.32with m C-3 0, C-40, and C-50, H-72′ 0 with C-10, C-50, and C-6104.60 along with an unassigned olefinic4 carbon (δC1.58104.6) m referring31.4 to literatures3′ [10,11,17,18]. In addition,197.1 the isobutyryl functionality positioned at C-201.51was m illustrated by HMBC4 correlations′ from Me-100/Me-1148.5 0 to C-80 and C-90. Definitively,5 the combination2.65 m of the filicinic33.9 acid (ring5′ A) and the germacrane 173.1 (ring C) via C-70 6 5.59 dd (16.2, 4.7) 136.8 6′ 101.9 was established by the 1H-1H COSY spin system of H-2/H-70, and ring B formed to fit the unsaturation 7 5.47 d (16.2, 8.0) 127.5 7′ 2.74 dd (16.6, 5.2) 21.8 degrees of 1. 8 2.31 t (7.0) 50.0 1.69 dd (16.6, 11.3) 9 1.90 m 24.7 8′ 207.9 1.43 m 9′ 3.93 sept (13.5, 6.7) 35.5 10 1.79 ddd (14.7, 10.0, 4.5) 32.1 10′ 1.10 d (6.7) 19.15 2.03 dt (14.8, 3.9) 11′ 1.11 d (6.7) 19.24

Molecules 2018, 23, 683 3 of 8

1 13 Table 1. H-NMR (600 MHz) and C-NMR (150 MHz) spectral data of compound 1 in CDCl3 (δ in ppm, J in Hz). Molecules2016, 21,x 3 of 8

Position δH (J) δC Position δH (J) δC 11 73.0 12′ 1.24 s 24.1 112 1.19 s 84.927.9 13 14′ 1.29s 1.00 d (7.0)25.4 16.4 213 1.471.20 m s 36.128.7 15 1.02 s 21.3 3 0.91 m 25.2 10 188.7 0 The planar construction1.32 mof 1 was established according 2 to the HMBC and 1104.6H-1H COSY 0 experiments4 (Figure 2). In ring 1.58 C, m the 2-hydroxyiso 31.4isopropyl 3 residue was connected at position197.1 8 due 0 to the HMBC correlations from1.51 mMe-12/Me-13 to C-11 and 4C-8, while the HMBC cross-peaks48.5 between 5 2.65 m 33.9 50 173.1 Me-15 with6 C-1, C-2, 5.59anddd C-10(16.2, as 4.7) well as the 136.8 cross-peaks 60 between Me-14 with C-4, C-5,101.9 and C-6 suggested Me-157 and Me-14 5.47 d (16.2,was located 8.0) at positions 127.5 1 and 70 5, respectively.2.74 dd (16.6, Meanwhile, 5.2) the 21.8 clear 1H- 1H COSY spin8 systems of 2.31 H-2/H-3/ t (7.0)H-4/H-5/H-6/H-7/ 50.0H-8/H-9/H-10 1.69 supporteddd (16.6, 11.3)the structural profile of ring C, a9 germacrane unit. 1.90 Regarding m to the 24.7 ring A, a 8 0filicinic acid core, was confirmed207.9 by the 0 HMBC correlations of Me-121.43′/Me-13 m′ with C-3′, C-4′, and 9 C-5′, H-73.93′ with sept C-1 (13.5,′, C-5 6.7)′, and 35.5 C-6′ along 10 1.79 ddd (14.7, 10.0, 4.5) 32.1 100 1.10 d (6.7) 19.15 with an unassigned olefinic carbon (δC 104.6) referring to literatures [10,11,17,18]. In addition, the 2.03 dt (14.8, 3.9) 110 1.11 d (6.7) 19.24 isobutyryl functionality positioned at C-2′ was illustrated by HMBC correlations from Me-10′/Me-11′ 11 73.0 120 1.24 s 24.1 to C-8′ and12 C-9′. Definitively, 1.19 the s combination 27.9 of the filicinic 130 acid (ring A)1.29 and s the germacrane 25.4 (ring C) via C-7′13 was established 1.20by the s 1H-1H COSY 28.7 spin system of H-2/H-7′, and ring B formed to fit the unsaturation degrees of 1.

FigureFigure 2. 2.Key Key 2D2D NMRNMR correlations of of compound compound 1.1 .

1 1 AccordingAccording to to the the 2D 2D NOESY NOESY spectrum spectrum (Supplementary(Supplementary Materials Figure Figure S7) S7) and and H-1H-H1 Hcoupling coupling constant,constant, the the relative relative configurations configurations of theof the chiral chiral centers centers of 1 wereof 1 were revealed. revealed. In light In oflight a large of a coupling large coupling constant value of H-6/H-7 (J = 16.2 Hz), an E geometry of the olefinic bond (∆6,7) was constant value of H-6/H-7 (J = 16.2 Hz), an E geometry of the olefinic bond (∆6,7) was ascertained. ascertained. NOE correlations between Me-14/H-7, H-7/H-10b (δH 2.03), H-10b/Me-15, and Me-15/H-0 NOE correlations between Me-14/H-7, H-7/H-10b (δH 2.03), H-10b/Me-15, and Me-15/H-7 b (δH 1.69) 7′b (δH 1.69) suggested that these protons should be assigned as the same side named β orientation. suggested that these protons should be assigned as the same side named β orientation. Analogously, Analogously, the observed NOE cross-peaks between H-7′a (δH 2.74)/H-2, H-6/H-5, and H-6/H-8 as the observed NOE cross-peaks between H-70a (δ 2.74)/H-2, H-6/H-5, and H-6/H-8 as well as the well as the absence of a key NOESY correlation betweenH H-2/Me-15, indicated H-2, H-5, H-6, and H- absence of a key NOESY correlation between H-2/Me-15, indicated H-2, H-5, H-6, and H-8 should be 8 should be placed at α orientation (Figure 2). Furthermore, the value of 3JH-7′b−H-2 (J 11.3 Hz) suggested α 3 0 placedthat a at dihedralorientation angle 180 (Figure between2). Furthermore,H-7′b and H-2 assigned the value these of JtwoH-7 protb−H-2ons(J as11.3 trans-stereochemistry. Hz) suggested that a 0 dihedralThus, a angle 6/6/10 180 ring between system H-7wasb incorporated and H-2 assigned by the these sesquiterpenoid two protons germacrane as trans-stereochemistry. entity trans-fused Thus, a 6/6/10into the ring acylfilicinic system wasacid incorporatedmotif, which possessed by the sesquiterpenoid a 1R*,2S*,5S*,8 germacraneR* relative configuration. entity trans-fused into the acylfilicinicAs confirmation, acid motif, whichthe absolute possessed stereocenters a 1R*,2S of*,5 C-1,S*,8 C-2,R* relative C-5, and configuration. C-8 in 1 were assigned by means of theAs confirmation,cautious comparison the absolute of electronic stereocenters circular ofdichroism C-1, C-2, (ECD) C-5, anddataC-8 between in 1 were 1 and assigned its homologues, by means ofi.e., the cautioushyperjaponols comparison D–G, with of electronic the identical circular sesquiterpenoid dichroism (ECD) germacrane. data between The ECD1 and spectra its homologues, exhibited i.e.,positive hyperjaponols Cotton effects D–G, at with 226–231 the identical nm (ECD sesquiterpenoid (CH3OH) λ (Δε): germacrane. 1, 231 (+10.34) The (Figure ECD 3); spectra hyperjaponol exhibited positiveD, 227 Cotton(+3.23); effects hyperjaponol at 226–231 E, 227 nm (+5.28); (ECD (CHhyperjaponol3OH) λ (∆ F,ε): 2291, 231(+4.48); (+10.34) hyperjaponol (Figure3); G, hyperjaponol 226 (+13.74)] D, 227together (+3.23); with hyperjaponol the dextrorotatory E, 227 (+5.28); optical hyperjaponolactivities of compound F, 229 (+4.48); 1 and hyperjaponol hyperjaponols G, D–G, 226 (+13.74)]which togetherdesignated with the the stereochemistry dextrorotatory of optical 1 as 1R activities,2S,5S,8R. of compound 1 and hyperjaponols D–G, which designated the stereochemistry of 1 as 1R,2S,5S,8R.

Molecules2016, Molecules21,x 2018, 23, 683 4 of 8 4 of 8

Molecules2016, 21,x 4 of 8

FigureFigure 3. 3.ExperimentalExperimental ECD ECD spectrum of of1 1(in (in CH CHOH).3OH). Figure 3. Experimental ECD spectrum of 1 (in3 CH3OH).

Epstein-BarrEpstein-Barr virus virus (EBV, (EBV, Lymphocryp Lymphocryptovirus),tovirus), a large DNADNA virus virus of of the theγ-herpes γ-herpes virus virus family, family, Epstein-Barrpreferentiallypreferentially virus infects infects (EBV, human human Lymphocryp B cells B cells of of at at least leasttovirus), 90% 90% of of athe large worldwideworldwide DNA population populationvirus of inthe in a latent aγ latent-herpes state state [19 virus]. [19]. family, preferentiallyEBV EBVis generallyinfects is generally human linked linked to B to acells agroup group of ofof at autoimmuneautoimmune least 90% ailments, of ailments, the suchworldwide such as systemic as systemic population lupus erythematosuslupus erythematosusin a latent [20], state [19]. EBV is generally[20], multiple linked sclerosis sclerosis to [21 [21],a], group and and rheumatoid rheumatoid of autoimmune arthritis arthritis [22]. ailments, [22]. Currently, Currently, anti-EBVsuch anti-EBV as drugs systemic like drugs ganciclovir lupuslike ganciclovir erythematosus and [20], multipleand aciclovir, sclerosis have have [21], efficacy efficacy and against againstrheumatoid EBV EBV lytic lytic infections, arthritis infections, while [22]. thewhile Currently, increasing the increasing emergence anti-EBV emergence of drugs drug-related likeof drug- ganciclovir toxicity, cross-resistance, and side effects also limit their clinical application [23–25]. As a successive and aciclovir,related toxicity,have efficacy cross-resistance, against andEBV side lytic effects infections, also limit while their clinical the increasing application emergence [23–25]. As aof drug- successivebiochemical biochemical research research on this herb,on this compound herb, compound1 was carried 1 was out carried an inhibition out an inhibition assay onlytic assay DNA on lytic replication of EBV in B95-8 cells in terms of our previous procedure [16]. Comparing the results with related DNAtoxicity, replication cross-resistance, of EBV in B95-8 and cells side in terms effects of our also previous limit procedure their clinical [16]. Comparing application the results[23–25]. As a the reported compounds (hyperjaponols A–G), 1 exhibited a moderate effect with EC50 25.00 µM, and successivewith biochemical the reported compounds research on (hyperjaponols this herb, compound A–G), 1 exhibited 1 was a carriedmoderate out effect an with inhibition EC50 25.00 assay μM, on lytic the value of a CC50 higher than 50 µM (Figure4 and Table2). DNA replicationand the value of ofEBV a CC in50 B95-8 higher cellsthan 50in μtermsM (Figure of our 4 and previous Table 2). procedure [16]. Comparing the results with the reported compounds (hyperjaponols A–G), 1 exhibited a moderate effect with EC50 25.00 μM, and the value of a CC50 higher than 50 μM (Figure 4 and Table 2).

FigureFigure 4. Effects 4. Effects on B95-8 on B95-8 cells cells viabilities viabilities and and inhibition inhibition on onlytic lytic EBV EBV replication replication of of compound compound 11 was × 5 measuredwas measured using GCV using as GCV positive as positive control control in vitro.in vitro B95-8. B95-8 cells cells (5 (5× 10510/well)/well) were were cultivated cultivated with with designated concentrations of compounds in present of 12-O-tetradecanoyl-phorbol-13-acetate designated concentrations of compounds in present of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). (TPA). The 50% cytotoxic concentration (CC50) of 1 was calculated from the dose-response curve The 50% cytotoxic concentration (CC50) of 1 was calculated from the dose-response curve by by Graphpad5.0 Prism. The 50% effective concentration (EC50) value correspond to compound Graphpad5.0concentrations Prism. required The to50% reduce effective quantitative concentration expression (EC of the50) copyvalue number correspond of intracellular to compound viral Figure 4. Effects on B95-8 cells viabilities and inhibition on lytic EBV replication of compound 1 was concentrationsgenomic DNA required by 50%. to Bothreduce values quantitative of CC50 and expression EC50 were of obtained the copy as meannumber values of intracellular with standard viral 5 measuredgenomic usingdeviations DNA GCV (nby= as50%. 3). positive Both values control of CC in50 andvitro. EC 50B95-8 were obtainedcells (5 as× mean10 /well) values were with cultivatedstandard with designateddeviations concentrations (n = 3). of compounds in present of 12-O-tetradecanoyl-phorbol-13-acetate (TPA).

The 50% cytotoxic concentration (CC50) of 1 was calculated from the dose-response curve by Table 2. Anti-EBV activities of positive control ganciclovir (GCV), 1, and the reported compounds Graphpad5.0 Prism. The 50% effective concentration (EC50) value correspond to compound (hyperjaponols A–G) (μM). concentrations required to reduce quantitative expression of the copy number of intracellular viral Compounds CC50 a EC50 b Selectivity Index (CC50/EC50) genomic DNA by 50%. Both values of CC50 and EC50 were obtained as mean values with standard GCV >300 2.86 >104.50 deviations (n = 3). 1 >50 25.00 >2 (+)-hyperjaponol A >41.35 10.33 >4.00 Table 2. Anti-EBV activities(−)-hyperjaponol of positi A ve >300control119.4 ganciclovir (GCV),>2.50 1 , and the reported compounds (hyperjaponols A–G) (+)-hyperjaponol(μM). B >30 0.57 >52.63 (−)-hyperjaponol B >120 6.60 >18.18 (+)-hyperjaponol C 31.75 a − b − Compounds CC50 EC50 Selectivity Index (CC50/EC50) GCV >300 2.86 >104.50 1 >50 25.00 >2 (+)-hyperjaponol A >41.35 10.33 >4.00 (−)-hyperjaponol A >300 119.4 >2.50 (+)-hyperjaponol B >30 0.57 >52.63 (−)-hyperjaponol B >120 6.60 >18.18 (+)-hyperjaponol C 31.75 − −

Molecules 2018, 23, 683 5 of 8

Table 2. Anti-EBV activities of positive control ganciclovir (GCV), 1, and the reported compounds (hyperjaponols A–G)(µM).

a b Compounds CC50 EC50 Selectivity Index (CC50/EC50) GCV >300 2.86 >104.50 1 >50 25.00 >2 (+)-hyperjaponol A >41.35 10.33 >4.00 (−)-hyperjaponol A >300 119.4 >2.50 (+)-hyperjaponol B >30 0.57 >52.63 (−)-hyperjaponol B >120 6.60 >18.18 (+)-hyperjaponol C 31.75 − − (−)-hyperjaponol C 17.78 − − hyperjaponol D 48.05 0.49 106.78 hyperjaponol E 60.49 17.53 3.45 hyperjaponol F 41.62 14.47 2.87 hyperjaponol G >300 >300 − a: 50% cytotoxic concentration; b: 50% effective concentration.

3. Materials and Methods

3.1. General Experiments Optical rotation was recorded on a JASCO P-2200 digital polarimeter (JASCO, Tokyo, ). IR, UV, and ECD spectra were measured by Bruker Vertex 70 (Brucker Co., Karlsruhe, Germany), Varian Cary 50 (Varian Medical Systems, Salt Lake City, UT, USA), and JASCO J-1700 (JASCO, Tokyo, Japan) apparatuses, respectively. HRESIMS was carried out on Agilent 6530 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, California, CA, USA) employed with positive ion mode with nebulizer pressure at 2.0 bar, dry gas temperature at 593 K, and assembled with Agilent Extend-C18 column (50 mm × 2.1 mm, 1.8 µm) under the mobile phase (MeOH/H2O, 90/10 (V/V)) with a flow rate of 0.5 mL/min. NMR spectra were run on a Bruker AM-600 spectrometer (Brucker Co., Karlsruhe, Germany), 1H-NMR (600 MHz), 13C-NMR (150 MHz), using TMS as the internal standard. 1 13 Chemical shifts of H and C-NMR were reported in ppm relative to the solvent peaks of CDCl3 (δH 7.24 ppm; δC 77.23 ppm). DEPT 135, HSQC (acquired size 512, 256; spectral size 1024, 1024), HMBC (acquired size 1024, 128; spectral size 2048, 1024), 1H-1H COSY (acquired size 1024, 128; spectral size 1024, 1024), NOESY (acquired size 1024, 128; spectral size 1024, 1024) experiments were performed. Silica gel (0.12–0.2 and 0.2–0.3 mm, Yantai Chemical Co. Ltd., Yantai, China), MCI gel (Mitsubishi Chemical Co., Tokyo, Japan), ODS (YMC Co., Tokyo, Japan), and Sephadex LH-20 (Mitsubishi Chemical Co., Tokyo, Japan) were used for column chromatography. Thin-layer chromatography (GF 254, Yantai Chemical Co. Ltd., Yantai, China) was performed for monitoring isolates under an ultraviolet-visible detector with λ 254 nm. Semi-preparative high performance liquid chromatography (HPLC) was carried out by a LC 3050 analysis of HPLC system (CXTH, Beijing, ® China) with a RP-C18 column (5 µm, 10 × 250 mm, Welchrom , Shanghai, China).

3.2. Material The aerial parts of herbs (H. japonicum) were collected from Da-Bie Mountain area, Qichun County, Hubei Province, P. R. China, in October 2016, and were authenticated by Professor Jianping Wang, Huazhong University of Science and Technology. A voucher specimen (no. 2016-1011) was deposited at the Herbarium of Hubei Key Laboratory of Biotechnology of Chinese Traditional Medicine, School of Life Science, Hubei University, Wuhan, P. R. China.

3.3. Extraction and Isolation The air-dried aerial parts of herbs (H. japonicum) (4 kg) were percolated with 95% aqueous EtOH (10 L) at 40 ◦C for 72 h to produce a crude extract (300 g), which was subjected to silica gel column Molecules 2018, 23, 683 6 of 8

(silica gel, 0.12–0.2 mm, 1.5 kg; column, 10 × 75 cm) chromatography (silica gel CC) eluted successively with the gradient mobile phases of petroleum ether (12 L), chloroform (8 L), and ethyl acetate (8 L). The petroleum ether fraction was subjected to silica gel column chromatography (petroleum ether/acetone, 100:1 to 5:1) to produce seven fractions (Fractions 1–7). With the aid of TLC analyses, fraction 3 was chosen and subjected on silica gel column (petroleum ether/acetone, 50:1 to 5:1) to yield four subfractions (fractions 3.1–3.4). Fraction 3.2 was purified using an ODS column with a gradient elution (MeOH–H2O (50:50 to 100:0), to produce four subfractions (fractions 3.2.1–3.2.4). Fraction 3.2.3 was further repurified by semi-preparative HPLC (MeOH–H2O, 85:15; flow rate, 2.0 mL/min; tR, 37.5 min) to afford 1 (2.1 mg). 20 Hyperjaponol H (1): white amorphous powder; [α]D +16.4 (c 0.06, CHCl3); UV (CH3OH) λmax (log ε) 243 (3.71) (Supplementary Materials Figure S8), 329 (3.90) nm; IR (KBr) νmax 3455, 2966, 2932, −1 1 13 2874, 1654, 1612, 1523, 1464 cm ; ECD λmax (∆ε) 231 (+10.34), 352 (−0.79) nm; H and C-NMR data, + see Table1; HRESIMS: m/z 459.3119 [M + H] (calcd for C28H43O5 459.3110) (Supplementary Materials Figure S1).

3.4. Anti-EBV Assay Regarding the pathogenicity of EBV infection, viral replication plays a critical role, and the inhibition of viral replication is a crucial parameter used to assess anti-virus activity of drugs. Hence the inhibitory activity on EBV DNA replication of compound 1 was investigated using previous procedures [26–29]. The cytotoxicity of compound 1 towards B95-8 cells was assessed by the AlamarBlue® cell viability assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer0s protocol. Thereupon, the antiviral activity of compound 1 against the lytic replication of EBV in B95-8 cells was measured using a qPCR assay to assess the intracellular viral DNA copy number, an accurate and rapid assessment of the efficacy of EBV DNA inhibitors as reported [26]. Extraction of the EBV genomic DNA, determination of the viral DNA copy number, and evaluation of the intracellular viral genomic DNA were undertaken referring to our previously described method [16].

4. Conclusions Hyperjaponols H (1), a new filicinic acid-based meroterpenoid, with a 6/6/10 ring system trans-fused by hetero-Diels–Alder cycloaddition between a germacrane sesquiterpenoid and a filicinic acid moiety, was discovered from Hypericum japonicum. The structure and absolute stereocenters were attributed to the analyses of extensive spectroscopic data and the Cotton effect of ECD undergoing a comparison with previously reported ones. Primary bioactivity screening suggested that 1 had a moderate inhibitory effect on lytic EBV DNA replication with the EC50 value of 25.00 µM.

Supplementary Materials: HRESIMS, NMR, UV, and IR spectra of compound 1 are available online. Acknowledgments: This work was financially supported by the Program for New Century Excellent Talents in University, the State Education Ministry of China (2008-0224), and the National Natural Science Foundation of China (nos. 81573316, 31770379, 81641129, 21502057, and 31700298). Author Contributions: Linzhen Hu conceived and designed the experiments; Rongrong Wu and Zijun Le carried out the experiments, analyzed the data, and wrote the manuscript; Zhenzhen Wang and Shuying Tian performed the biological assay; Yong Chen contributed reagents, materials, and analysis tools; Yongbo Xue and Yonghui Zhang modified the manuscript. All authors reviewed the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Singh, I.P.; Sidana, J.; Bansal, P.; Foley, W.J. Phloroglucinol compounds of therapeutic interest: Global patent and technology status. Expert Opin. Ther. Pat. 2009, 19, 847–866. [CrossRef][PubMed] 2. Grenning, A.J.; Boyce, J.H.; Porco, J.A., Jr. Rapid synthesis of polyprenylated acylphloroglucinol analogs via dearomative conjunctive allylic annulation. J. Am. Chem. Soc. 2014, 136, 11799–11804. [CrossRef][PubMed] Molecules 2018, 23, 683 7 of 8

3. Singh, I.P.; Sidana, J.; Bharate, S.B.; Foley, W.J. Phloroglucinol compounds of natural origin: Synthetic aspects. Nat. Prod. Rep. 2010, 27, 393–416. [CrossRef][PubMed] 4. Pang, Q.; Tian, Y.; Mi, J.; Wang, J.; Xu, Y. Simultaneous determination and pharmacokinetic study of eight components in rat plasma by UHPLC-MS/MS after oral administration of Hypericum japonicum Thunb extract. J. Pharm. Biomed. Anal. 2016, 118, 228–234. [CrossRef][PubMed] 5. Liu, L.S.; Liu, M.H.; He, J.Y. Hypericum japonicum Thunb. ex Murray: phytochemistry, pharmacology, quality control and pharmacokinetics of an important herbal medicine. Molecules 2014, 19, 10733–10754. [CrossRef] [PubMed] 6. Wu, Q.L.; Wang, S.P.; Zhang, S.M.; Yang, J.S.; Xiao, P.G. Chromone glycosides and flavonoids from Hypericum japonicum. Phytochemistry 1998, 49, 1417–1420. [CrossRef] 7. Zhang, W.D.; Fu, P.; Liu, R.H.; Li, T.Z.; Li, H.L.; Zhang, W.; Chen, H.S. A new bisxanthone from Hypericum japonicum. Fitoterapia 2007, 78, 74–75. [CrossRef][PubMed] 8. Verma, R.S.; Padalia, R.C.; Chauhan, A.; Chanotiya, C.S.; Yadav, A. Chemical composition of the aliphatic compounds rich essential oil of Hypericum japonicum Thunb. ex Murray from . J. Essent. Oil. Res. 2012, 24, 501–505. [CrossRef] 9. Wang, X.W.; Mao, Y.; Wang, N.L.; Yao, X.S. A new phloroglucinol diglycoside derivative from Hypericum Japonicum Thunb. Molecules 2008, 13, 2796–2803. [CrossRef][PubMed] 10. Ishiguro, K.; Nagata, S.; Fukumoto, H.; Yamaki, M.; Isoi, K. Phloroglucinol derivatives from Hypericum Japonicum. Phytochemistry 1994, 35, 469–471. [CrossRef] 11. Hu, L.H.; Khoo, C.W.; Vittal, J.J.; Sim, K.Y. Phloroglucinol derivatives from Hypericum japonicum. Phytochemistry 2000, 53, 705–709. [CrossRef] 12. Zhu, H.; Chen, C.; Yang, J.; Li, X.N.; Liu, J.; Sun, B.; Huang, S.X.; Li, D.; Yao, G.; Luo, Z.; et al. Bioactive acylphloroglucinols with adamantyl skeleton from . Org. Lett. 2014, 16, 6322–6325. [CrossRef][PubMed] 13. Zhu, H.; Chen, C.; Liu, J.; Sun, B.; Wei, G.; Li, Y.; Zhang, J.; Yao, G.; Luo, Z.; Xue, Y.; et al. Hyperascyrones A–H, polyprenylated spirocyclic acylphloroglucinol derivatives from Linn. Phytochemistry 2015, 115, 222–230. [CrossRef][PubMed] 14. Li, D.; Xue, Y.; Zhu, H.; Li, Y.; Sun, B.; Liu, J.; Yao, G.; Zhang, J.; Du, G.; Zhang, Y. Hyperattenins A–I, bioactive polyprenylated acylphloroglucinols from Hypericum attenuatum Choisy. RSC. Adv. 2015, 5, 5277–5287. [CrossRef] 15. Guo, Y.; Zhang, N.; Chen, C.; Huang, J.; Li, X.N.; Liu, J.; Zhu, H.; Tong, Q.; Zhang, J.; Luo, Z.; et al. Tricyclic polyprenylated acylphloroglucinols from St John0s Wort, . J. Nat. Prod. 2017, 80, 1493–1504. [CrossRef][PubMed] 16. Hu, L.; Zhang, Y.; Zhu, H.; Liu, J.; Li, H.; Li, X.N.; Sun, W.; Zeng, J.; Xue, Y.; Zhang, Y. Filicinic acid-based meroterpenoids with anti-Epstein-Barr virus activities from Hypericum japonicum. Org. Lett. 2016, 18, 2272–2275. [CrossRef][PubMed] 17. Ishiguro, K.; Yamaki, M.; Takagi, S.; Yamaga, Y.; Tomita, K. X-ray crystal structure of sarothralin, a novel antibiotic compound from Hypericum japonicum. J. Chem. Soc. Chem. Commun. 1985, 26–27. [CrossRef] 18. Ishiguro, K.; Yamaki, M.; Kashihara, M.; Takagi, S. Sarothralen A and B from H. japomicum. Planta Med. 1986, 288–290. [CrossRef][PubMed] 19. Wang, H.B.; Zhang, H.; Zhang, J.P.; Li, Y.; Zhao, B.; Feng, G.K.; Du, Y.; Xiong, D.; Zhong, Q.; Liu, W.L.; Du, H.; Li, M.Z.; Huang, W.L.; Tsao, S.W.; Hutt Fletcher, L.; Zeng, Y.X.; Kieff, E.; Zeng, M.S. Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells. Nat. Commun. 2015, 6, 6240–6252. [CrossRef][PubMed] 20. Ribeiro, F.M.; Gomez, V.E.; Albuquerque, E.M.; Klumb, E.M.; Shoenfeld, Y. Lupus and leprosy: Beyond the coincidence. Immunol. Res. 2015, 61, 160–163. [CrossRef][PubMed] 21. Serafini, B.; Rosicarelli, B.; Franciotta, D.; Magliozzi, R.; Reynolds, R.; Cinque, P.; Andreoni, L.; Trivedi, P.; Salvetti, M.; Faggioni, A.; et al. Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J. Exp. Med. 2007, 204, 2899–2912. [CrossRef][PubMed] 22. Balandraud, N.; Roudier, J.; Roudier, C. Epstein-Barr virus and rheumatoid arthritis. Autoimmun. Rev. 2004, 3, 362–367. [CrossRef][PubMed] 23. Billaud, G.; Thouvenot, D.; Morfin, F. Drug targets in herpes simplex and Epstein Barr virus infections. Infect. Disord. Drug Targets 2009, 9, 117–125. [CrossRef][PubMed] Molecules 2018, 23, 683 8 of 8

24. Coen, D.M.; Schaffer, P.A. Antiherpesvirus drugs: a promising spectrum of new drugs and drug targets. Nat. Rev. Drug Discov. 2003, 2, 278–288. [CrossRef][PubMed] 25. Shi, W.J.; Sun, H.X.; Mo, X.M.; Li, S.Y.; Li, X.Y.; Zhang, G.; Liu, H.A. Development of a broad-spectrum antiviral agent with activity against herpesvirus replication and gene expression. Trop. J. Pharm. Res. 2013, 12, 541–547. [CrossRef] 26. Ballout, M.; Germi, R.; Fafi-Kremer, S.; Guimet, J.; Bargues, G.; Seigneurin, J.M.; Morand, P. Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression. J. Virol. Methods 2007, 143, 38–44. [CrossRef][PubMed] 27. Wiedmer, A.; Wang, P.; Zhou, J.; Rennekamp, A.J.; Tiranti, V.; Zeviani, M.; Lieberman, P.M. Epstein-Barr virus immediate-early protein Zta co-opts mitochondrial single-stranded DNA binding protein to promote viral and inhibit mitochondrial DNA replication. J. Virol. 2008, 82, 4647–4655. [CrossRef][PubMed] 28. Stevens, S.J.; Vervoort, M.B.; van den Brule, A.J.; Meenhorst, P.L.; Meijer, C.J.; Middeldorp, J.M. Monitoring of Epstein-Barr virus DNA load in peripheral blood by quantitative competitive PCR. J. Clin. Microbiol. 1999, 37, 2852–2857. [PubMed] 29. Asahi Ozaki, Y.; Sato, Y.; Kanno, T.; Sata, T.; Katano, H. Quantitative analysis of Kaposi Sarcoma–Associated Herpesvirus (KSHV) in KSHV-associated diseases. J. Infect. Dis. 2006, 193, 773–782. [CrossRef][PubMed]

Sample Availability: Samples of the compound 1 are available from the authors.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).