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

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 Hypericum japonicum. 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.


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 plants of Guttiferae are mainly found to be phloroglucinol derivatives with complex architectures and appealing therapeutical properties [1][2][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][6][7][8][9][10][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][13][14][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.

Results and Discussion
A crude extract (300 g) produced from the dried herbs of H. japonicum (4 kg) was subjected to the silica gel column chromatography (silica gel CC) eluted successively with the gradient mobile phases of petroleum ether, chloroform, and ethyl acetate. The fraction of petroleum ether was sequentially chromatographed by MCI gel column, ODS Middle Pressure Liquid Chromatography (MPLC), Sephadex LH-20, and High Performance Liquid Chromatography (HPLC) to give a new filicinic acid-based meroterpenoid (1) as drawn in Figure 1, which was named as hyperjaponol H.

Results and Discussion
A crude extract (300 g) produced from the dried herbs of H. japonicum (4 kg) was subjected to the silica gel column chromatography (silica gel CC) eluted successively with the gradient mobile phases of petroleum ether, chloroform, and ethyl acetate. The fraction of petroleum ether was sequentially chromatographed by MCI gel column, ODS Middle Pressure Liquid Chromatography (MPLC), Sephadex LH-20, and High Performance Liquid Chromatography (HPLC) to give a new filicinic acid-based meroterpenoid (1) as drawn in Figure 1, which was named as hyperjaponol H.   Figure S1). The analyses of IR absorption bands (3455, 1654, and 1612 cm −1 , Supplementary Materials Figure S9) demonstrated the characteristic scaffold of an enolic 1,3-diketo system, viz. acylated filicinic acid parent core [10,11,17,18]. Comparison of NMR data between 1 with hyperjaponols A-G [16] indicated that 1 was constructed via the incorporation of the same sesquiterpene unit as hyperJaponol G to the same acylated filicinic acid entity as hyperjaponols A-F. Accomplished by meticulous examination of HSQC, HMBC, and 1 H-1 H COSY spectra (Supplementary Materials Figure S4-S6), all 1 H-and 13 C-NMR data of 1 were unequivocally assigned as shown in Table 1 Figure  S3) denoted 28 carbon resonances consisting of one quaternary carbon, one carbonyl, two oxygenated, and five enolic or olefinic carbons, five methylenes, six methines (four aliphatic, and two olefinic carbons), and eight methyls. Taking the aforementioned analyses and its eight indices of proton deficiency into consideration, compound 1 contains a tricyclic system.    Figure S1). The analyses of IR absorption bands (3455, 1654, and 1612 cm −1 , Supplementary Materials Figure S9) demonstrated the characteristic scaffold of an enolic 1,3-diketo system, viz. acylated filicinic acid parent core [10,11,17,18]. Comparison of NMR data between 1 with hyperjaponols A-G [16] indicated that 1 was constructed via the incorporation of the same sesquiterpene unit as hyperjaponol G to the same acylated filicinic acid entity as hyperjaponols A-F. Accomplished by meticulous examination of HSQC, HMBC, and 1 H-1 H COSY spectra (Supplementary Materials Figures S4-S6), all 1 H-and 13 C-NMR data of 1 were unequivocally assigned as shown in Table 1 Figure S3) denoted 28 carbon resonances consisting of one quaternary carbon, one carbonyl, two oxygenated, and five enolic or olefinic carbons, five methylenes, six methines (four aliphatic, and two olefinic carbons), and eight methyls. Taking the aforementioned analyses and its eight indices of proton deficiency into consideration, compound 1 contains a tricyclic system.
The planar construction of 1 was established according to the HMBC and 1 H-1 H COSY experiments ( Figure 2). In ring C, the 2-hydroxyisoisopropyl residue was connected at position 8 due to the HMBC 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 suggested Me-15 and Me-14 was located at positions 1 and 5, respectively. Meanwhile, the clear 1 H-1 H COSY spin systems of H-2/H-3/H-4/H-5/H-6/H-7/H-8/H-9/H-10 supported the structural profile of ring C, a germacrane unit. Regarding to the ring A, a filicinic acid core, was confirmed by the HMBC correlations of Me-12 /Me-13 with C-3 , C-4 , and C-5 , H-7 with C-1 , C-5 , and C-6 along with an unassigned olefinic carbon (δ C 104.6) referring to literatures [10,11,17,18]. In addition, the isobutyryl functionality positioned at C-2 was illustrated by HMBC correlations from Me-10 /Me-11 to C-8 and C-9 . Definitively, the combination of the filicinic acid (ring A) and the germacrane (ring C) via C-7 was established by the 1 H-1 H COSY spin system of H-2/H-7 , and ring B formed to fit the unsaturation degrees of 1. The planar construction of 1 was established according to the HMBC and 1 H-1 H COSY experiments ( Figure 2). In ring C, the 2-hydroxyisoisopropyl residue was connected at position 8 due to the HMBC 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 suggested Me-15 and Me-14 was located at positions 1 and 5, respectively. Meanwhile, the clear 1 H-1 H COSY spin systems of H-2/H-3/H-4/H-5/H-6/H-7/H-8/H-9/H-10 supported the structural profile of ring C, a germacrane unit. Regarding to the ring A, a filicinic acid core, was confirmed by the HMBC correlations of Me-12′/Me-13′ with C-3′, C-4′, and C-5′, H-7′ with C-1′, C-5′, and C-6′ along with an unassigned olefinic carbon (δC 104.6) referring to literatures [10,11,17,18]. In addition, the isobutyryl functionality positioned at C-2′ was illustrated by HMBC correlations from Me-10′/Me-11′ to C-8′ and C-9′. Definitively, the combination of the filicinic acid (ring A) and the germacrane (ring C) via C-7′ was established by the 1 H-1 H COSY spin system of H-2/H-7′, and ring B formed to fit the unsaturation degrees of 1.  Figure 2). Furthermore, the value of 3 JH-7′b−H-2 (J 11.3 Hz) suggested that a dihedral angle 180 between H-7′b and H-2 assigned these two protons as trans-stereochemistry. Thus, a 6/6/10 ring system was incorporated by the sesquiterpenoid germacrane entity trans-fused into the acylfilicinic acid motif, which possessed a 1R*,2S*,5S*,8R* relative configuration.
As confirmation, the absolute stereocenters of C-1, C-2, C-5, and C-8 in 1 were assigned by means of the cautious comparison of electronic circular dichroism (ECD) data between 1 and its homologues,   Figure 2). Furthermore, the value of 3 J H-7 b −H-2 (J 11.3 Hz) suggested that a dihedral angle 180 between H-7 b and H-2 assigned these two protons as trans-stereochemistry. Thus, a 6/6/10 ring system was incorporated by the sesquiterpenoid germacrane entity trans-fused into the acylfilicinic acid motif, which possessed a 1R*,2S*,5S*,8R* relative configuration.
As confirmation, the absolute stereocenters of C-1, C-2, C-5, and C-8 in 1 were assigned by means of the cautious comparison of electronic circular dichroism (ECD) data between 1 and its homologues, i.e., hyperjaponols D-G, with the identical sesquiterpenoid germacrane. The ECD spectra exhibited positive Cotton effects at 226-231 nm (ECD (CH 3   Epstein-Barr virus (EBV, Lymphocryptovirus), a large DNA virus of the γ-herpes virus family, preferentially infects human B cells of at least 90% of the worldwide population in a latent state [19]. EBV is generally linked to a group of autoimmune ailments, such as systemic lupus erythematosus [20], multiple sclerosis [21], and rheumatoid arthritis [22]. Currently, anti-EBV drugs like ganciclovir and aciclovir, have efficacy against EBV lytic infections, while the increasing emergence of drugrelated toxicity, cross-resistance, and side effects also limit their clinical application [23][24][25]. As a successive biochemical research on this herb, compound 1 was carried out an inhibition assay on lytic DNA replication of EBV in B95-8 cells in terms of our previous 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).  Epstein-Barr virus (EBV, Lymphocryptovirus), a large DNA virus of the γ-herpes virus family, preferentially infects human B cells of at least 90% of the worldwide population in a latent state [19]. EBV is generally linked to a group of autoimmune ailments, such as systemic lupus erythematosus [20], multiple sclerosis [21], and rheumatoid arthritis [22]. Currently, anti-EBV drugs like ganciclovir and aciclovir, have efficacy against EBV lytic infections, while the increasing emergence of drug-related toxicity, cross-resistance, and side effects also limit their clinical application [23][24][25]. As a successive biochemical research on this herb, compound 1 was carried out an inhibition assay on lytic DNA replication of EBV in B95-8 cells in terms of our previous procedure [16]. Comparing the results with the reported compounds (hyperjaponols A-G), 1 exhibited a moderate effect with EC 50 25.00 µM, and the value of a CC 50 higher than 50 µM (Figure 4 and Table 2). Epstein-Barr virus (EBV, Lymphocryptovirus), a large DNA virus of the γ-herpes virus family, preferentially infects human B cells of at least 90% of the worldwide population in a latent state [19]. EBV is generally linked to a group of autoimmune ailments, such as systemic lupus erythematosus [20], multiple sclerosis [21], and rheumatoid arthritis [22]. Currently, anti-EBV drugs like ganciclovir and aciclovir, have efficacy against EBV lytic infections, while the increasing emergence of drugrelated toxicity, cross-resistance, and side effects also limit their clinical application [23][24][25]. As a successive biochemical research on this herb, compound 1 was carried out an inhibition assay on lytic DNA replication of EBV in B95-8 cells in terms of our previous 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).

General Experiments
Optical rotation was recorded on a JASCO P-2200 digital polarimeter (JASCO, Tokyo, Japan). IR, UV, and ECD spectra were measured by Bruker

Plant 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.

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][27][28][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 manufacturer s 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].

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 EC 50 value of 25.00 µM.