A New Megastigmane Sesquiterpenoid from Zanthoxylum Schinifolium Sieb. et Zucc

Zanthoxylum schinifolium Sieb. et Zucc. (Rutaceae), a dioecious shrub with hooked prickly branches, has been used as folk medicine for the treatment of the common cold, stomach ache, diarrhea, and jaundice in China, Korea, and Japan. In our phytochemical investigations on this genus, a new megastigmane sesquiterpenoid, which is referred to as schinifolenol A (1), was isolated from Z. schinifolium. The stereochemistry was characterized via the analyses of extensive spectra. The absolute configuration was established by the application of a modified Mosher’s experiment and assisted by a time-dependent density functional theory (TD-DFT) on calculated electronic circular dichroism (ECD). Bioactivity screenings showed that compound 1 exhibited a safe hypotoxicity and a better selectivity on anti-Kaposi’s sarcoma associated herpes virus (KSHV).


Introduction
Zanthoxylum schinifolium Sieb. et Zucc. is a dioecious shrub with hooked prickly branches from the genus Zanthoxylum (family Rutaceae), which was termed Qinghuajiao, Yajiao, Tianjiao, and Xiaohuajiao, etc., and prosperously distributed from the south of the Yangtze River to the southwest provinces in China [1]. Clinical application in folk medicines of Z. schinifolium mainly included cures for the common cold, stomach ache, diarrhea, and jaundice in China, Korea, and Japan [2]. Recently, phytochemical studies on Zanthoxylum have resulted in the isolation of diverse chemical constituents such as alkaloids, amides, lignans, coumarins, essential oils and aliphatic acids [1,[3][4][5]. In our continuous research for structurally unique and biologically active metabolites from traditional Chinese medicine [6][7][8], a megastigmane sesquiterpenoid termed schinifolenol A (1) (Figure 1) was obtained from the dried rhizomes of Z. schinifolium. Herein, we elucidated the isolation procedures and the stereochemistry structure establishment of compound 1, as well as its inhibitory effect on Kaposi's sarcoma associated herpes virus (KSHV) infection. Schinifolenol A (1), a violet oil, has the elemental composition of C14H22O4, which corresponded to the (+)-HRESIMS peak (m/z 255.1596 [M + H] + , calcd as 255.1592). IR (KBr) spectrum showed the characteristic absorption bands for hydroxyl (3695 cm −1 ) and carbonyl (1724 and 1667 cm −1 ) functionalities along with UV (CH3OH) spectrum at λmax 244 nm, which are closely similar with those data of megastigmane sesquiterpenoids such as blumenol C in reported literatures [9][10][11]. According to the NMR data of the literature [11], the difference between compound 1 and blumenol C is that a methoxycarbonyl group at C-5 of 1 was replaced by a methyl function in blumenol C. Spectral analyses of 1D NMR (Table 1) and HSQC correlations indicated the presence of two carbonyls (δC 202.3 and 169.3), one oxygenated methyl (δH 3.84, s and δC 53.4), three methyls (δH 1.01, s; 1.12, d, J = 6.2 Hz; and 1.15, s), three methylenes, three methines (including an olefinic methine (δH 6.55, s and δC 131.4) and a hydroxylated carbon (δH 3.65, m and δC 68.8)), and two quaternary carbons (including one olefinic carbon (δC 155.2)). Referring to the literature data [9][10][11], the aforementioned analyses illustrate that compound 1 belongs to a class chemicals of megastigmane sesquiterpenoid. Combining with the signals of HSQC, iterative analyses of HMBC and 1 H-1 H COSY experiments could elucidate the planar constitution of 1. HMBC correlations from Me-12 and Me-13 to C-1, C-2, and C-6, from H-2 to C-3 and C-4, and from H-4 to C-5 and C-6 indicated the presence of a 5,5-dimethyl-cyclohex-3-one entity. Meanwhile, the HMBC cross peak of H-4 to the ester carbonyl C-13 implied that the ester carbonyl function was located at C-5. Moreover, HMBC correlations from Me-10 to C-8 and C-9, and from H-6 to C-7 and C-8, as well as the 1 H-1 H COSY spin systems of H-6/H-7/H-8/H-9/H-10 revealed that a 3-hydroxybutyl functionality was connected to the 3,3-dimethyl-cyclohexanone at C-6 ( Figure 2). No diagnostic NOESY could be applied to determine the relative configurations of C-6 and C-9, since both carbons were located on a rotational aliphatic

Results
The dried rhizomes of Z. schinifolium (30 kg) were exhaustively extracted with 95% EtOH to furnish a syrup (1.5 kg), which was successively partitioned by petroleum ether, CH 2 Cl 2 , and EtOAc against water. The petroleum ether fraction (300 g) was repeatedly subjected to silica gel column chromatography (silica gel CC), Sephadex LH-20, and semi-preparative High Performance Liquid Chromatography (HPLC) to afford a new megastigmane sesquiterpenoid, viz., compound 1, which is named schinifolenol A ( Figure 1).
Schinifolenol A (1), a violet oil, has the elemental composition of C 14 H 22 O 4 , which corresponded to the (+)-HRESIMS peak (m/z 255.1596 [M + H] + , calcd as 255.1592). IR (KBr) spectrum showed the characteristic absorption bands for hydroxyl (3695 cm´1) and carbonyl (1724 and 1667 cm´1) functionalities along with UV (CH 3 OH) spectrum at λ max 244 nm, which are closely similar with those data of megastigmane sesquiterpenoids such as blumenol C in reported literatures [9][10][11]. According to the NMR data of the literature [11], the difference between compound 1 and blumenol C is that a methoxycarbonyl group at C-5 of 1 was replaced by a methyl function in blumenol C. Spectral analyses of 1D NMR (Table 1) and HSQC correlations indicated the presence of two carbonyls (δ C 202.3 and 169.3), one oxygenated methyl (δ H 3.84, s and δ C 53.4), three methyls (δ H 1.01, s; 1.12, d, J = 6.2 Hz; and 1.15, s), three methylenes, three methines (including an olefinic methine (δ H 6.55, s and δ C 131.4) and a hydroxylated carbon (δ H 3.65, m and δ C 68.8)), and two quaternary carbons (including one olefinic carbon (δ C 155.2)). Referring to the literature data [9][10][11], the aforementioned analyses illustrate that compound 1 belongs to a class chemicals of megastigmane sesquiterpenoid. Combining with the signals of HSQC, iterative analyses of HMBC and 1 H-1 H COSY experiments could elucidate the planar constitution of 1. HMBC correlations from Me-12 and Me-13 to C-1, C-2, and C-6, from H-2 to C-3 and C-4, and from H-4 to C-5 and C-6 indicated the presence of a 5,5-dimethyl-cyclohex-3-one entity. Meanwhile, the HMBC cross peak of H-4 to the ester carbonyl C-13 implied that the ester carbonyl function was located at C-5. Moreover, HMBC correlations from Me-10 to C-8 and C-9, and from H-6 to C-7 and C-8, as well as the 1 H-1 H COSY spin systems of H-6/H-7/H-8/H-9/H-10 revealed that a 3-hydroxybutyl functionality was connected to the 3,3-dimethyl-cyclohexanone at C-6 ( Figure 2). No diagnostic NOESY could be applied to determine the relative configurations of C-6 and C-9, since both carbons were located on a rotational aliphatic chain. Thus, the aforementioned spectral analyses allowed us to assign compound 1 as a member of megastigmane sesquiterpenoid.
Molecules 2016, 21, 383 3 of 7 chain. Thus, the aforementioned spectral analyses allowed us to assign compound 1 as a member of megastigmane sesquiterpenoid. In order to determine the absolute configuration of compound 1, a modified Mosher's method was carried out to establish the stereochemistry characteristic of secondary alcohol carbon C-9. (S)-and (R)-MTPA esters of 1 were prepared as previously reported [12,13], then analyzed via 1 H-NMR chemical shifts study. The distinguishable values (∆δ = δS-MTPA-ester − δR-MTPA-ester) were calculated for proton chemical shifts adjacent to C-9, as shown in Figure 3. Correspondingly, the absolute configuration at C-9 was confirmed to be S. Furthermore, a time-dependent density functional theory (TD-DFT) method on ECD calculation was performed to determine the absolute configuration of the other chiral carbon, viz., C-6. Based on the ascertained absolute configuration of C-9, the calculated ECD curve of (6S, 9S)-1 showed a good consistency with the experimental ECD curve (Figure 4), which unequivocally established the absolute configuration of 1 as 6S,9S.  Since previous literatures reported that dictamnine (one type of alkaloids was isolated from Z. schinifolium) exhibited activity towards anti-Epstein-Barr virus (EBV) [14] and sesquiterpenes/ sesquiiterpene lactones exhibited moderately activities towards anti-HIV [15], these two assays were also assessed for compound 1 and neither of the results showed obvious activities (1 towards both assays with CC50 > 300 μM and EC50 > 300 μM). Besides the aforementioned assays of anti-EBV and anti-HIV, some other bioactive screenings for 1 were performed, which included inhibitory activities on β-site amyloid precursor protein cleaving enzyme 1 (BACE1) (1 towards this assay with IC50 > 40 μM), inhibitory activities on NO production (1 towards this assay with IC50 > 25 μM), and cytotoxic activities against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) (1 towards all these cytotoxicity assays with IC50 > 40 μM). Disappointingly, compound 1 did not show any activities in the above assays. In order to determine the absolute configuration of compound 1, a modified Mosher's method was carried out to establish the stereochemistry characteristic of secondary alcohol carbon C-9. (S)-and (R)-MTPA esters of 1 were prepared as previously reported [12,13], then analyzed via 1 H-NMR chemical shifts study. The distinguishable values (∆δ = δ S´MTPA´ester´δR´MTPA´ester ) were calculated for proton chemical shifts adjacent to C-9, as shown in Figure 3. Correspondingly, the absolute configuration at C-9 was confirmed to be S. Furthermore, a time-dependent density functional theory (TD-DFT) method on ECD calculation was performed to determine the absolute configuration of the other chiral carbon, viz., C-6. Based on the ascertained absolute configuration of C-9, the calculated ECD curve of (6S,9S)-1 showed a good consistency with the experimental ECD curve (Figure 4), which unequivocally established the absolute configuration of 1 as 6S,9S. chain. Thus, the aforementioned spectral analyses allowed us to assign compound 1 as a member of megastigmane sesquiterpenoid. In order to determine the absolute configuration of compound 1, a modified Mosher's method was carried out to establish the stereochemistry characteristic of secondary alcohol carbon C-9. (S)-and (R)-MTPA esters of 1 were prepared as previously reported [12,13], then analyzed via 1 H-NMR chemical shifts study. The distinguishable values (∆δ = δS-MTPA-ester − δR-MTPA-ester) were calculated for proton chemical shifts adjacent to C-9, as shown in Figure 3. Correspondingly, the absolute configuration at C-9 was confirmed to be S. Furthermore, a time-dependent density functional theory (TD-DFT) method on ECD calculation was performed to determine the absolute configuration of the other chiral carbon, viz., C-6. Based on the ascertained absolute configuration of C-9, the calculated ECD curve of (6S, 9S)-1 showed a good consistency with the experimental ECD curve (Figure 4), which unequivocally established the absolute configuration of 1 as 6S,9S.  Since previous literatures reported that dictamnine (one type of alkaloids was isolated from Z. schinifolium) exhibited activity towards anti-Epstein-Barr virus (EBV) [14] and sesquiterpenes/ sesquiiterpene lactones exhibited moderately activities towards anti-HIV [15], these two assays were also assessed for compound 1 and neither of the results showed obvious activities (1 towards both assays with CC50 > 300 μM and EC50 > 300 μM). Besides the aforementioned assays of anti-EBV and anti-HIV, some other bioactive screenings for 1 were performed, which included inhibitory activities on β-site amyloid precursor protein cleaving enzyme 1 (BACE1) (1 towards this assay with IC50 > 40 μM), inhibitory activities on NO production (1 towards this assay with IC50 > 25 μM), and cytotoxic activities against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) (1 towards all these cytotoxicity assays with IC50 > 40 μM). Disappointingly, compound 1 did not show any activities in the above assays. chain. Thus, the aforementioned spectral analyses allowed us to assign compound 1 as a member of megastigmane sesquiterpenoid. In order to determine the absolute configuration of compound 1, a modified Mosher's method was carried out to establish the stereochemistry characteristic of secondary alcohol carbon C-9. (S)-and (R)-MTPA esters of 1 were prepared as previously reported [12,13], then analyzed via 1 H-NMR chemical shifts study. The distinguishable values (∆δ = δS-MTPA-ester − δR-MTPA-ester) were calculated for proton chemical shifts adjacent to C-9, as shown in Figure 3. Correspondingly, the absolute configuration at C-9 was confirmed to be S. Furthermore, a time-dependent density functional theory (TD-DFT) method on ECD calculation was performed to determine the absolute configuration of the other chiral carbon, viz., C-6. Based on the ascertained absolute configuration of C-9, the calculated ECD curve of (6S, 9S)-1 showed a good consistency with the experimental ECD curve (Figure 4), which unequivocally established the absolute configuration of 1 as 6S,9S.  Since previous literatures reported that dictamnine (one type of alkaloids was isolated from Z. schinifolium) exhibited activity towards anti-Epstein-Barr virus (EBV) [14] and sesquiterpenes/ sesquiiterpene lactones exhibited moderately activities towards anti-HIV [15], these two assays were also assessed for compound 1 and neither of the results showed obvious activities (1 towards both assays with CC50 > 300 μM and EC50 > 300 μM). Besides the aforementioned assays of anti-EBV and anti-HIV, some other bioactive screenings for 1 were performed, which included inhibitory activities on β-site amyloid precursor protein cleaving enzyme 1 (BACE1) (1 towards this assay with IC50 > 40 μM), inhibitory activities on NO production (1 towards this assay with IC50 > 25 μM), and cytotoxic activities against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) (1 towards all these cytotoxicity assays with IC50 > 40 μM). Disappointingly, compound 1 did not show any activities in the above assays. Since previous literatures reported that dictamnine (one type of alkaloids was isolated from Z. schinifolium) exhibited activity towards anti-Epstein-Barr virus (EBV) [14] and sesquiterpenes/ sesquiiterpene lactones exhibited moderately activities towards anti-HIV [15], these two assays were also assessed for compound 1 and neither of the results showed obvious activities (1 towards both assays with CC 50 > 300 µM and EC 50 > 300 µM). Besides the aforementioned assays of anti-EBV and anti-HIV, some other bioactive screenings for 1 were performed, which included inhibitory activities on β-site amyloid precursor protein cleaving enzyme 1 (BACE1) (1 towards this assay with IC 50 > 40 µM), inhibitory activities on NO production (1 towards this assay with IC 50 > 25 µM), and cytotoxic activities against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) (1 towards all these cytotoxicity assays with IC 50 > 40 µM). Disappointingly, compound 1 did not show any activities in the above assays.
Kaposi's sarcoma associated herpes virus (KSHV) belongs to the gamma 2-herpesvirus subfamily, which is the etiological agent of all types of Kaposi's sarcoma, like primary effusion lymphoma, multicentric Castleman's disease, and posttransplant [16]. Currently, typical antiviral drugs, like acyclovir or ganciclovir, do not show a satisfactory potency to extinguish gamma herpes viruses in human bodies [17]. In this case, it is a challenge to discover more effective drugs or lead compounds against KSHV. Compound 1 was used to carry out an anti-KSHV assay referring to the procedure of previous literature [18]. The inhibitory activity of compound 1 against KSHV lytic replication was measured. The results indicated that 1 exhibited potency with a safe hypotoxicity and a definite selectivity (i.e., EC 50 of 501.3 µM and selectivity index of 1.99, respectively).

General Experiments
TLC was carried out by silica gel 60 F254 (Shanghai Beinuo Biological Technology Co. Ltd., Shanghai, China). Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), Silica gel (200-300 mesh; Shanghai Xibao Biological Technology Co. Ltd., Shanghai, China), and RP-18 (50 µm, Merck Co. Ltd., Darmstadt, Germany) were applied in column chromatography. Pseudomolecular ion peak was recorded by analyzing HRESIMS data through a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). A Perkin-Elmer 341 polarimeter (Perkin Elmer Inc., Waltham, MA, USA) was performed to measure optical rotation. The UV and IR spectra were obtained by a Varian Cary 50 (Varian Medical Systems, Salt Lake City, UT, USA) and Bruker Vertex 70 instruments (Brucker Corporation, Karlsruhe, Germany), respectively. The NMR spectra were acquired using a Bruker AM-600/400 spectrometer (Brucker Corporation). The chemical shifts of 1 H-and 13 C-NMR were referenced to the solvent peaks for methanol-d 4 at δ H 3.31 and δ C 49.2. HPLC procedures were carried out on a Dionex Ultimate 3000 (Thermo Fisher Scientific Inc.) applied with a UV detector and a semi-preparative column (5 µm, 10ˆ250 mm, Welch Ultimate ® XB-C 18 ).

Plant Material
The dried rhizomes of Z. schinifolium were collected in September 2013 at Da-Bie Mountain area of Hubei Province, China and authenticated by Changgong Zhang. A voucher specimen (ID 20131011) has been preserved in Herbarium of Material Medicine, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Anti-KSHV Assay
Anti-KSHV assay was evaluated via cytotoxicity assessments and anti-KSHV infectivity assays. Human iSLK.219 cells were adopted to assess the bioactivity of compound 1 towards anti-KSHV. The human iSLK.219 cells were embedded with the rKSHV.219 virus, which was a harbored green fluorescent protein (GFP), through control of the elongation factor 1α (EF-1α) promoter. The lytic replication of KSHV was activated by the addition with 1.2 mM sodium butyrate (NaB) (Sigma, Shanghai, China) and 1 µg/mL doxycycline (Dox) (Beyotime, Jiangsu, China) [19,20]. After cells grew to 70% confluence in 96-well culture plates, compound 1 in presence of Dox and NaB with assigned concentrations was instilled to the wells. According to AlamarBlue ® Cell Viability Assay (Invitrogen, Shanghai, China), the cell viability was evaluated after 48 h post drugs-induced. The luminescent expression was measured by the Envison 2102 Multilabel Reader (Perkin Elmer). The 50% cytotoxic concentration (CC 50 ) of compound 1 was obtained through mathematical statistics with Graphpad5.0 Prism. The result was shown in Figure S1, Supplementary Materials. An infectivity assay, like previous reports [18], was performed to determine the anti-KSHV activity of compound 1. The supernatants were harvested from iSLK.219-treated or untreated with compound 1 containing Dox and NaB at 48 h. Then, the supernatants were added to infect the Vero cells, which were seeded in a 96-well plate. Next, the Vero cells were centrifuged by a SORVALL ® Pico apparatus at 1500ˆg for 60 min [21]. The supernatants were replaced by fresh DMEM medium to remove superfluous viruses. At 48 h, the expression of GFP per well in Vero cells were detected via an Operetta High-Content Screening System (HCS) (Perkin Elmer). Image fields (9/well) were observed by the automated microscope based HCS. The GFP intensity of each well was afforded using the Harmony 3.5 software (Perkin Elmer). The DMSO control was used as normalized group. The 50% effective concentration (EC 50 ) of compound 1 towards anti-KSHV infectivity was calculated by reduced quantitative expression of the intensity of GFP by 50%. The result is exhibited in Figure S1, Supplementary Materials.

Conclusions
A new megastigmane sesquiterpenoid, methyl (S)-6-((S)-3-hydroxybutyl)-5,5-dimethyl-3oxocyclohex-1-ene-1-carboxylate (1), termed schinifolenol A, was discovered from the rhizomes of Zanthoxylum schinifolium. The absolute configuration was established by the analyses of the extensive spectra including HRESIMS, NMR, UV, and IR spectra, the application of the modified Mosher's method, and the method of calculated ECD spectra using the time-dependent density functional theory. Bioactivity screenings suggested that compound 1 had potential activity on anti-KSHV infection with a safe hypotoxicity and a definite selectivity.