Article pubs.acs.org/jnp
Synthesis of C‑4-Substituted Steviol Derivatives and Their Inhibitory Effects against Hepatitis B Virus Shwu-Jiuan Lin,†,‡ Ta-Chi Su,† Chin-Nan Chu,§,⊥ Yi-Chih Chang,∥ Li-Ming Yang,†,# Yu-Cheng Kuo,*,¶ and Tsurng-Juhn Huang*,□ †
School of Pharmacy and ‡Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan § Department of Radiation Oncology and College of Medicine, China Medical University Hospital, and ⊥Graduate Institute of Clinical Medical Science, China Medical University, Taichung 404, Taiwan ∥ Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan # Division of Chinese Medicinal Chemistry, National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan ¶ Department of Radiation Oncology, Show Chwan Memorial Hospital, Changhua 500, Taiwan □ School of Medicine, China Medical University, Taichung 404, Taiwan S Supporting Information *
ABSTRACT: ent-13-Hydroxykaur-16-ene-19-N-butylureide (6) was one of 33 synthesized C-4-substituted steviol derivatives that were evaluated for their effects on hepatitis B virus (HBV) surface antigen (HBsAg) secretion. The IC50 (16.9 μM) and SI (57.7) values for inhibiting HBV DNA replication of compound 6 were greater than those of the reference compound, lamivudine (3-TC; IC50: 107.5 μM; SI: 22.0). Thus, the anti-HBV mechanism of 6 was investigated, and it specifically inhibited viral gene expression and reduced viral DNA levels, as well as potently attenuated all of the viral promoter activity of HBV-expressing Huh7 cells. Examination of cellular signaling pathways found that 6 inhibited the activities of the nuclear factor (NF)-κB- and activator protein (AP)-1 elementcontaining promoters, but had no effects on AP-2 or interferon-stimulated response element (ISRE)-containing promoters in HBV-expressing cells. Meanwhile, it significantly eliminated NF-κB and extracellular signal-regulated kinase (ERK)/mitogenactivated protein kinase (MAPK) signaling-related protein levels and inhibited their phosphorylation in HBV-transfected Huh7 cells. The inhibitory potency of 6 against HBV DNA replication was reversed by cotransfecting the NF-κB p65 expression plasmid. Using the MAPK-specific activator anisomycin also reversed the inhibitory effect of 6 on viral DNA replication. The present findings suggest that the anti-HBV mechanism of 6 is partly mediated through the NF-κB and MAPK signaling pathways.
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classes of small-molecule inhibitors of HBV with novel action mechanisms distinct from currently available drugs.10 Steviol (ent-13-hydroxykaur-16-en-19-oic acid) (1) is an aglycone of the natural sweetener stevioside, a constituent of Stevia rebaudiana (Bertoni) Bertoni (Compositae).11 Stevioside can be completely converted to steviol (1) after incubation with human colon microflora in vitro.12,13 Pharmacological research demonstrated that steviol (1) inhibits glucose absorption,14a acts as a diuretic agent without hypotensive activity,14b has antihyperglycemic,14c anti-inflammatory, and immunomodulatory activities,14d and can reduce Madin-Darby canine kidney cyst formation and growth.14e It has been reported that stevioside was not carcinogenic in rats.15 In contrast, steviol (1) was reported to be a toxic substance with mutagenic and bactericidal activities against Salmonella typhimurium TM 677.16 Inspection of the molecular structure of steviol (1) reveals that
epatitis B virus (HBV) infection commonly leads to acute and chronic infections in humans and is associated with a high risk of developing cirrhosis and hepatocellular carcinoma.1,2 More than 400 million people worldwide are chronically infected with HBV.3,4 Current therapies for treating patients with chronic HBV infection involve the use of interferons (INFs) or nucleoside analogues, which reduce HBV replication in order to control its progression.5,6 However, clinical treatment with INF-based analogues is limited due to their low efficacy, high cost, and significant adverse effects.7 Longterm therapy with approved nucleoside analogues eventually leads to the development of drug resistance and undesirable side effects.8 Recent studies have been performed to determine how the viral gene is regulated and controlled to ameliorate HBV. This is a crucial issue for antiviral strategies9 because the focus is not on viral genome replication but on cell signaling pathways that are regulated during HBV infection. To date, there is no effective treatment that completely eliminates HBV infection in patients. Thus, it is critical to explore other novel © XXXX American Chemical Society and American Society of Pharmacognosy
Received: July 19, 2016
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DOI: 10.1021/acs.jnatprod.6b00671 J. Nat. Prod. XXXX, XXX, XXX−XXX
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procedures are depicted in Schemes 1 and 2. The starting material of 1 was prepared by reacting stevioside with NaIO4
modifications of its reactive sites include the carboxylic acid group, the allylic double bond, and the tertiary alcohol at C-13. Thus, steviol (1) is an interesting scaffold for synthesizing analogues to develop new compounds with unknown bioactivities as a result of its characteristic backbone and biological properties.17 According to the literature, the exocyclic double bond is responsible for some biological activities, and thus, the most often used modification of 1 is the esterification of its carboxylic acid group to maintain the ent-kaurene backbone.17,18 In addition, isosteviol and steviol (1) were originally derived from stevioside.11,19 Although they both have different skeletons, the carboxylic acid group is present on both at the C-4 position. In general, several potentially therapeutic agents have been found in the parent skeletons of naturally occurring functional products.20−22 From the point of view of chemical properties, urea and amide are valuable functional moieties in biological and pharmaceutical molecules.23,24 Thus, they were introduced into the C-4 position of the isosteviol molecule to produce novel compounds with anti-HBV activities.9,25 As a result of steviol (1) inhibiting Epstein−Barr virus early antigen (EBV-EA) activation26 and to continue a research program of discovering novel anti-HBV agents from naturally occurring lead compounds,9,25 chemical modification of steviol was performed to find new analogues with anti-HBV properties and to demonstrate their possible mechanisms and molecular targets. Herein, the synthesis of C-4-substituted steviol derivatives and evaluation of their anti-HBV activities, as well as mechanistic information, are described.
Scheme 2. Synthesis of Target Compounds 21−36a
a Reagents and conditions; (i) KOH, dioxane, reflux; (ii) RCOCl, DMAP, Et3N, DMF, rt.
and then refluxing the mixture with KOH as previously reported.27 The structure of 1 was characterized using various spectroscopic techniques.28 The target compounds 3−19 and 21−36 were synthesized from steviol (1) through the respective intermediates 2 and 20. Initially, isocyanate 2 was obtained by reacting 1 with diphenylphosphoryl azide (DPPA) in benzene. Thereafter, hydrolysis of 2 with sodium hydroxide in dioxane afforded amine 20. The C-4 ureido compounds 3− 19 were synthesized by reacting 2 with various amines in dimethylformamide (DMF) in the presence of triethylamine at room temperature (Scheme 1). In addition, C-4 amidesubstituted derivatives were synthesized by treating 20 with acyl chlorides/benzoyl chlorides in the presence of 4dimethylaminopyridine (DMAP) at room temperature to afford the desired compounds 21−36 (Scheme 2). All of the synthesized derivatives were determined by NMR and HRESIMS data analysis. Subsequently, the anti-HBV activities of the synthesized C-4 ureido and amide derivatives with substituents of different electronic, steric, and lipophilic moieties were evaluated in HepG2.2.15 cells using lamivudine as a reference compound. Levels of HBV DNA, HBsAg, and HBeAg in culture supernatants were detected to determine the inhibitory effect of each compound. The bioactivity of each compound was evaluated by combining the IC50 and SI values, and results are summarized in Tables 1 and S1 (Supporting Information). As the results show, parent compound 1 exhibited moderate inhibitory potency against the secretion of
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RESULTS AND DISCUSSION A series of compounds of C-4-substituted steviol (1) bearing ureido and amide moieties was synthesized. The synthesis Scheme 1. Synthesis of Target Compounds 3−19a
a
Reagents and conditions; (i) NaIO4, rt; (ii) KOH, reflux; (iii) DPPA, benzene, reflux; (iv) RNH2 or R1R2NH, Et3N, DMF, rt. B
DOI: 10.1021/acs.jnatprod.6b00671 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. Anti-Hepatitis B Virus (HBV) Activities of Steviol and Its Derivativesa HBsAgc b
e
HBeAgd f
compound
TC50 (μM)
IC50 (μM)
SI
1 (steviol) 2 6 20 30 34 35 5-FUg lamivudineh
474.5 337.1 972.4 168.9 412.8 56.3 124.9 227.9 2360.9
20.8 19.1 23.5 30.7 14.2 2.7 8.3
22.8 17.6 41.4 5.5 29.1 20.9 15.1
e
DNA replication
IC50 (μM)
SIf
IC50e (μM)
SIf
16.6 28.5 20.4 31.2 23.9 3.4 13.6
28.6 11.8 47.7 5.4 17.3 16.6 9.2
19.9 30.0 16.9 35.8 21.9 4.0 9.0
23.8 11.2 57.7 4.7 18.8 14.1 13.9
107.5
22.0
a
Data of other synthesized compounds are in Table S1 (Supporting Information). bTC50, 50% cytotoxic concentration in HepG2.2.15 cells. cHBsAg, HBV surface antigen. dHBeAg, HBV e antigen. eIC50, 50% inhibitory concentration. fSI (selectivity index) = TC50/IC50. g5-FU (fluorouracil), positive control of cytotoxicity. hLamivudine (3TC) as the antiviral positive control.
inhibitory mechanism of compound 6 against HBV in HBVtransfected Huh7 cells was further investigated. Effect of Compound 6 on HBV-Transfected Huh7 Cell Viability. To exclude the possibility that HBV production was suppressed by 6 due to its cytotoxicity, the effect of compound 6 on HBV-transfected Huh7 cell viability was tested using an MTS assay. The results indicated that 6 caused cytotoxic effects at doses of >80 μg/mL in HBV-transfected Huh7 cells (Figure 1). Thus, a noncytotoxic dosage range (2.5−10 μg/mL) was used for the antiviral treatment of HBV-transfected Huh7 cells.
HBsAg and HBeAg (Table 1). Among 17 synthesized C-4 ureido analogues, the methyl group at the 4-position of the phenyl ring (15) showed weak activity (IC50: 59.6 μM) against HBsAg secretion and was less potent than the unsubstituted phenyl derivative 10 (IC50: 33.5 μM) (Table S1, Supporting Information). However, the electron-donating methoxy group substituted at the 4-, 3,4-, and 3,4,5-positions of the phenyl ring (16−18) had good inhibitory activities against secretion of HBsAg (IC50: 11.7−14.0 μM) and HBeAg (IC50: 7.7−13.6 μM). Their IC50 values against HBV DNA replication were 14.7, 14.3, and 6.3 μM, respectively, which were superior to that of lamivudine (IC50: 107.5 μM). However, their SI values were lower than that of lamivudine (Table S1, Supporting Information). Introduction of the electron-withdrawing halogen at the C-4 position of the phenyl ring revealed that the chloride-substituted derivative 13 inhibited the secretion of HBsAg (IC50: 8.0 μM) and HBeAg (IC50: 5.0 μM) and was more potent than the other halide-substituted analogues (12 and 14). The IC50 value of 13 (4.5 μM) against HBV DNA replication was more potent than that of lamivudine (107.5 μM), but its SI value (6.6) was lower than that of the latter compound (22.0) (Table S1, Supporting Information). To identify the effects of the carbon chain length, compound 6, with four carbon atoms (a butyl group) attached to the nitrogen atom of urea, exhibited significant inhibition of the secretion of HBsAg (IC50: 23.5 μM; SI: 41.4) and HBeAg (IC50: 20.4 μM; SI: 47.7). IC50 and SI values of the inhibition of HBV DNA replication were 16.9 μM and 57.7, respectively, which were more potent than those of lamivudine (Table 1). Moreover, replacement of the C-4-carboxylic acid with an amino group (20) showed weak anti-HBV activity (Table 1). Subsequently, to investigate the effect of the amide moiety at the C-4 position, 16 analogues (21−36) were prepared from 20 with various substituted acyl chlorides. As the assay results show in Tables 1 and S1 (Supporting Information), these compounds exhibited weak to moderate inhibitory effects against secretion of HBsAg and HBeAg. In addition, their IC50 values of inhibiting HBV DNA replication were more potent than that of lamivudine, with the exception of 22 and 23. However, none of these C-4 amide derivatives possessed SI values superior to that of lamivudine. Overall, although modification of the steviol scaffold at the C-4 position did not provide clear structure−activity relationship information for anti-HBV activity, compound 6 had the most potent anti-HBV activity among all of the tested compounds. Hence, the
Figure 1. Cytotoxicity of compound 6 on hepatitis B virus (HBV)expressing Huh7 cells. To determine the cytotoxicity, cells were plated in 96-well plates for 24 h and treated with serial dilutions (2.5−160 μg/mL) of 6 for 3 days. After treatment, cells were subjected to a cytotoxic assay. Data are expressed as the mean and the SD of the mean (n = 4) (*p < 0.05 vs untreated cells).
Effects of Compound 6 on HBV Virions and Antigen Secretion in Huh7 Cells. The effects of 6 on the expression of viral antigens were examined by measuring secretion rates of HBsAg and HBeAg from Huh7 cells using an enzyme immunoassay (EIA). Cells were treated with various concentrations (2.5, 5, and 10 μg/mL) of 6, and HBsAg and HBeAg secretions were significantly suppressed in dose-dependent manners compared to the controls (Figure 2A). The function of 6 against virion secretion was further examined by isolating viral DNA from a conditioned medium. Real-time PCR was performed to detect extracellular viral DNA levels. Results showed that treatment with compound 6 significantly reduced secreted viral DNA levels in a dose-dependent manner compared to the controls (Figure 2B). Effect of Compound 6 on HBV Gene Expression. The molecular regulatory effect of compound 6 on HBV gene C
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Figure 2. Effects of compound 6 on (A) the secretion of hepatitis B virus (HBV) antigens and (B) replicated viral DNA levels in HBVexpressing Huh7 cells. Cells were treated with various concentrations (2.5, 5, and 10 μg/mL) of 6 for 3 days, and conditioned media were collected for the EIA of hepatitis B surface antigen (HBsAg) and e antigen (HBeAg). In addition, HBV DNA was isolated from virion particles and subjected to a real-time PCR analysis. Data are expressed as the mean and the SD of the mean (n = 3) (*p < 0.05 vs untreated cells).
expression was determined by transiently transfecting Huh7 cells with the pHBV1.2 HBV genome-containing plasmid29 and treating cells with three doses (2.5, 5, and 10 μg/mL) of 6 for 2 days. Northern blotting was performed to analyze HBV viral RNA expression levels. Treatment with 6 significantly reduced major S/preS RNA; the inhibitory potential of 6 was higher against surface RNA than against precore/pregenomic RNA (Figure 3A,B). In addition, an immunoblot analysis was used to detect the intracellular levels of small antigen of hepatitis B surfaces (SHBs), Core (HBcAg), and HBx proteins, and results indicated that compound 6 potently reduced SHBs, HBcAg, and HBx protein levels compared to the vehicle controls (Figure 3C). Effects of Compound 6 on the HBV Gene and Cellular Signaling Pathways Related to the Responsive Element That Contains Promoter Activity. The regulatory effect of 6 on HBV gene expression was examined by isolating and cloning four promoters that correspond to the viral gene into a pGL4.17 luciferase-reporter vector,30 and the effect of 6 on the activity level of the promoter was examined. In this study, pCore-Luc, pS-Luc (S promoter), pPreS-Luc (preS promoter), or pX-Luc constructs were cotransfected with pHBV1.2 into Huh7 cells for 24 h, and then cells were treated with the maximum inhibitory dose of 6 prior to performing the luciferase assay. Results showed that treatment with 6 significantly reduced Core and S promoter activities, whereas it had a slight inhibitory effect on the X and preS promoters (Figure 4A). Four luciferase reporters that contained the NF-
Figure 3. Effects of compound 6 on hepatitis B virus (HBV) gene expressions and viral DNA replication in Huh7 cells. Huh7 cells were transfected with the pHBV1.2 plasmid for 2 days and treated with three concentrations (2.5, 5, and 10 μg/mL) of 6 for another 2 days. Treated cells were harvested and subjected to total RNA and protein isolation. (A) Total RNA from the HBV genome-transfected and -treated Huh7 cells was subjected to a Northern blot analysis using HBV whole-genome DNA as the probe as described in the Experimental Section. GAPDH was used as an RNA loading control, and β-gal was used to detect the efficiency of each transfection. (B) The intensity of each RNA band from three sets of experiments was quantitated with a densitometer, and the relative amount was normalized with GAPDH loading control (*p < 0.05 vs untreated control). (C) Total cellular proteins were extracted, and immunoblotting was used to detect small antigen of hepatitis B surfaces (SHBs), hepatitis B core antigen (HBcAg), hepatitis B virus X (HBx), and αtubulin for comparison. Data shown in (A) and (B) are representative of three sets of experiments.
κB-, AP-1-, AP-2-, or interferon-sensitive response element (ISRE)-binding elements were cotransfected with pHBV1.2 into Huh7 cells to determine whether 6 inhibits HBV gene expression in a host’s mediated cellular signaling pathways. After treatment with 6 (10 μg/mL), cells were harvested and subjected to a promoter activity assay. Figure 4B shows that 6 decreased NF-κB- and AP-1-containing promoter activities, but did not affect AP-2- or ISRE-containing promoter activities (p < 0.05). D
DOI: 10.1021/acs.jnatprod.6b00671 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 4. Effects of compound 6 on hepatitis B virus (HBV) gene promoter- and cellular signaling pathway-responsive promoter activities. Huh7 cells were seeded in 24-well plates for 24 h and transfected with either the (A) pCore-Luc, pS-Luc, pPreS-Luc, or pXLuc viral gene promoter reporter construct and the (B) pAP1-Luc, pAP2-Luc, pNF-κB-Luc, or pISRE-Luc cellular signaling pathway responsive to the promoter reporter construct together with pRLSV40 for 24 h and then treated with 6 (10 μg/mL) for another 24 h. Cellular lysates were prepared for the luciferase assay as described in the Experimental Section. Data are expressed as the mean and the SD of the mean (n = 3) (*p < 0.05 vs untreated cells).
Figure 5. Effects of compound 6 on (A) cellular nuclear factor (NF)κB p65/p50, phospho-p65, inhibitor NF-κB kinase (IKK)α/β, and IκBα/phosphor-IκBα protein expressions and (B) cellular extracellular signal-regulated kinase (ERK)1/2, phosphor-Erk1/2, SAPK/c-Jun Nterminal kinase (JNK), phosphor-SAPK/JNK, and p38 mitogenactivated protein kinase (MAPK) in Huh7 cells that expressed the hepatitis B virus (HBV) genome. Huh7 cells were transfected with pHBV1.2 for 2 days and treated with three concentrations (2.5, 5, and 10 μg/mL) of 6 for another 2 days. Cellular proteins were extracted, and immunoblotting was used to detect NF-κB and MAPK signalingrelated protein levels. Data shown are representative of the three sets of experiments.
Effects of Compound 6 on NF-κB and MAPK Signaling Pathway-Related Protein Expressions in Huh7 Cells. During compound 6 treatment, NF-κB and MAPK had regulatory roles in HBV gene expression; thus, Huh7 cells were treated with this substance, and Western blotting was used to determine the effects of this compound on NF-κB and MAPK signaling pathway-related protein expression. Results showed that treatment with 6 reduced the inhibitor of NF-κB kinase (IKK)α/β and p65/p50 NF-κB protein levels and reduced phosphorylated NF-κB p65. However, the cytoplasmic IκBα level and its phosphorylation also decreased (Figure 5A). For another signaling pathway, compound 6 treatment potently decreased ERK-1/2 and stress-activated protein kinase/c-JunN-terminal (SAPK/JNK) levels and attenuated their phosphorylation. In addition, p38 MAPK expression decreased during treatment with 6 (Figure 5B). NF-κB and MAPK Are Partly Involved in the Inhibitory Effects of Compound 6 against HBV. To further examine whether the regulatory effect of 6 against HBV is mediated through inhibition of the NF-κB signaling pathway, the p65 expression plasmid was cotransfected with pHBV1.2 in Huh7 cells and treated with this compound (10 μg/mL). HBV DNA from the culture medium was extracted for a real-time PCR analysis, and results showed that an increase in the p65 plasmid transfected dose (0.5 and 2.5 μg) significantly reversed the inhibitory effect of 6 on the HBV DNA level compared to empty vector transfection (Figure 6A). Furthermore, to examine whether the MAPK signaling pathway is involved in
the action of 6 against HBV inhibition, a specific activator of MAPK, anisomycin, was treated with or without this compound in HBV-transfected Huh7 cells for 2 days. Results indicated that anisomycin significantly restored the inhibitory effect of 6 against the HBV DNA level, but had no effect if it was treated alone (Figure 6B). Compound 6, a synthesized steviol derivative with anti-HBV activity, was used to evaluate the potential inhibitory effects against HBV. Results demonstrated that 6 inhibited viral HBsAg and HBeAg secretion and supernatant viral DNA levels in HBV-transfected Huh7 cells (Figure 2A,B). As to viral gene expression, compound 6 significantly downregulated viral S/ preS relative to precore/pregenomic RNA levels (Figure 3B). Treatment with 6 significantly inhibited the synthesized viral protein expression level of a small form of viral surface protein (SHBs), HBcAg, and HBx (Figure 3C). The results demonstrated that this derivative regulates the HBV gene at the transcription level. Viral promoter activity was further E
DOI: 10.1021/acs.jnatprod.6b00671 J. Nat. Prod. XXXX, XXX, XXX−XXX
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To determine whether the effects of 6 on HBV gene expression and viral propagation are correlated with NF-κB, the pCMV-p65 NF-κB expression plasmid was cotransfected with pHBV1.2 into Huh7 cells. Secreted HBV viral particles were isolated, and a real-time PCR detected viral DNA levels. As seen in Figure 6A, when cells were treated with a maximum dose (10 μg/mL) of 6, secretion of the HBV viral particles was inhibited by about 38% compared to the vehicle-treated control. However, the inhibitory effect of 6 on HBV viral particle secretion was significantly reversed when cells were cotransfected with an increasing dose (0.5 and 2.5 μg) of the pCMV-p65 plasmid under the same treatment. There was no difference in secreted levels of HBV DNA at the maximum transfection dose of the pCMV-p65 plasmid combined treatment with 6 compared to the untreated controls. Furthermore, the specific activator of MAPK, anisomycin, was used concomitantly with or without 6. Results indicated that anisomycin reversed the inhibitory effect of compound 6 on HBV DNA replication but had no effect when used alone (Figure 6B). These results clearly suggest that the effect of 6 on HBV gene expression and viral DNA replication might be partly mediated through the NF-κB and MAPK signaling pathways. For medicinal applications, controlling the HBV well enough to ameliorate the disease is the most crucial issue for antiviral therapeutic strategies, in addition to preventing viral resistance commonly caused by viral reverse transcriptase inhibitors. The present findings indicate that compound 6 regulated the HBV gene expression and viral DNA replication by interfering with the host cell’s NF-κB and MAPK signaling pathways. In addition, previous studies proposed that NF-κB activation in cells infected with HBV31−34 might be a possible treatment for HBV and that activation of the NF-κB pathway is mediated by viral proteins.9 Further study is required to determine whether compound 6 is directly involved in regulating viral promoter activities or just disrupts the feedback activation of viral proteins involved in NF-κB or MAPK gene expression and activation. In summary, 33 C-4 ureido- and amide-substituted steviol derivatives were synthesized, and their inhibitory activities against HBV were evaluated in vitro. Among them, compound 6 showed more effective inhibition of HBV DNA replication than did lamivudine. The mechanism underlying the anti-HBV effects of 6 was elucidated. This work demonstrates that modification of the steviol (1) parent structure can potentially produce new derivatives with anti-HBV activity. More significantly, this is the first report to synthesize steviol derivatives as anti-HBV compounds.
Figure 6. Effect of the (A) nuclear factor (NF)-κB signaling pathway and (B) mitogen-activated protein kinase (MAPK) signaling pathway on compound 6-mediated hepatitis B virus (HBV) inhibition. Huh7 cells were cotransfected with pHBV1.2 and with two doses (0.5 and 2.5 μg) of the pCMV-p65 plasmid for 2 days and then treated with 6 (10 μg/mL) for another 2 days. In another set of experiments, HBVexpressing Huh7 cells were treated with 6 (10 μg/mL) or concomitantly with anisomycin (10 μM) for 2 days. Secreted viral particles from cells were isolated with a kit and subjected to a real-time PCR analysis of HBV DNA (n = 3) (*p < 0.05 vs untreated cells).
examined, and the four promoters of HBV were attenuated by 6 in HBV-expressing Huh7 cells (Figure 4A). These observations suggest that compound 6 can potentially inhibit HBV gene expression by regulating its promoter activity. To examine the mechanisms involved in the ability of 6 to affect a host’s cellular signaling pathways, transcriptional activities of four major signaling pathways, AP-1, AP-2, NFκB, and ISRE, were analyzed using a promoter-reporter assay. After transfection and treatment, compound 6 specifically decreased NF-κB- and AP-1 element-containing promoter activities, whereas there were no effects on AP-2- or ISRE element-containing promoters in HBV-transfected Huh7 cells. These results indicate that 6 specifically regulates the NF-κB and MAPK signaling pathways in HBV-infected host cells (Figure 4B). Expressions of NF-κB p65/p50 and IKKα/β proteins and their phosphorylated p65 forms decreased after compound 6 treatment. However, the IκBα protein level decreased after the same treatment in a dose-dependent manner (Figure 5A). These results suggest that 6 can potentially downregulate overall NF-κB-related protein expression, phosphorylation, and subsequent activation. In addition, compound 6 attenuated the level of MAPK signaling-related protein expressions and inhibited their phosphorylation (Figure 5B).
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EXPERIMENTAL SECTION
General Experimental Procedures. All chemicals were of reagent grade and were used as received. Reagent-grade solvents were distilled prior to use. Melting points were determined using a Yanagimoto micromelting point apparatus and are uncorrected. Optical rotations were determined on a JASCO DIP-1020 digital polarimeter. 1H NMR, 13C NMR, and DEPT spectra were recorded on Varian Unity INOVA-500 and -600 spectrometers in C5D5N, CDCl3, or CD3OD. Chemical shifts are reported in parts per million (ppm) with respect to the corresponding solvent as an internal standard, and coupling constants (J) are in hertz (Hz). High-resolution mass spectra were recorded on a Shimadzu LCMS-TOF mass spectrometer and SYNAPT G2 (Waters MS Technologies, Manchester, UK) orthogonal acceleration QTOF MS. Spots for all compounds were detected by F
DOI: 10.1021/acs.jnatprod.6b00671 J. Nat. Prod. XXXX, XXX, XXX−XXX
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ent-13-Hydroxykaur-16-ene-19-N-butylureide (6). Following the general procedure described for method A, compound 6 was prepared from 2 (60 mg, 0.19 mmol) using butylamine (220 mg, 3 mmol). White crystals (45 mg, 60.9%): mp 144−146 °C (H2O− CH3OH); [α]28D −77.1 (c 1.0, CHCl3); Rf 0.50 [CH2Cl2−CH3OH (30:1)]; 1H NMR (C5D5N, 500 MHz) δ 0.77 (1H, m), 0.78 (3H, t, J = 7.5 Hz,), 0.86 (1H, d, J = 12.0 Hz), 0.93 (1H, d, J = 8.0 Hz), 1.13 (1H, m), 1.16 (3H, s), 1.25−1.32 (3H, m), 1.37−1.51 (6H, m), 1.60 (3H, s), 1.62−1.86 (6H, m), 1.99 (1H, m), 2.11−2.22 (3H, m), 3.33− 3.43 (3H, m), 4.85 (1H, s), 4.99 (1H, s), 5.41 (1H, s), 6.77 (1H, br s, OH); 13C NMR (C5D5N, 125 MHz) δ 158.5, 157.5, 103.1, 80.0, 56.9, 55.0, 54.6, 48.2, 47.7, 41.6, 41.5, 40.8, 40.5, 40.0, 39.4, 37.4, 33.1, 28.4, 20.5, 20.4, 19.9, 18.6, 17.9, 14.0; HRESIMS m/z 389.3170 [M + H]+ (calcd for C24H41N2O2, 389.3168). Synthesis of 4α-Amino-19-nor-ent-13-hydroxykaur-16-ene (20). To a solution of 2 (60 mg, 0.19 mmol) in dioxane (15 mL) was added KOH (8.4 g, 0.15 mol). The reaction mixture was refluxed for 2 h. After completion of the reaction as monitored by thin-layer chromatography (TLC), water was added to terminate the reaction. The resulting solution was evaporated in vacuo and then filtered and washed with a large amount of water to remove excess KOH, which afforded a white powder. After recrystallization from CH3OH−H2O, compound 20 was obtained as white needles (40 mg, 70%): mp 134− 136 °C; [α]26D −48.8 (c 1.05, CH3OH); Rf 0.14 [CH2Cl2−CH3OH (10:1)]; 1H NMR (CD3OD, 500 MHz) δ 0.89 (1H, td, J = 13.0, 3.5 Hz,), 1.07−1.10 (2H, m), 1.14 (3H, s), 1.17 (3H, s), 1.28−1.31 (2H, m), 1.40−1.82 (11H, m), 1.89 (1H, m), 2.05−2.12 (2H, m), 2.21 (1H, dt, J = 17.0, 2.5 Hz), 4.79 (1H, s), 4.96 (1H, s); 13C NMR (CD3OD, 125 MHz) δ 156.7, 103.7, 80.8, 55.7, 55.6, 54.5, 48.5, 47.4, 42.5, 41.9, 40.7, 40.4, 41.0, 39.8, 30.4, 21.0, 20.4, 18.5, 17.4; HRESIMS m/z 290.2483 [M + H]+ (calcd for C19H32NO, 290.2484). General Procedure for the Preparation of C-4 Amide Derivatives 21−36. To a solution of 20 (60 mg, 0.19 mmol) in DMF (4 mL) were added DMAP (0.52 mmol) and triethylamine (400 μL), and the mixture was stirred at room temperature for 10 min. Various acyl chlorides/benzoyl chlorides (0.8 mmol) were then added to the mixture and stirred for 2 h at room temperature. After completion of the reaction as monitored by TLC, water was added to the reaction mixture, which was filtered and washed with water. The crude product was subjected to column chromatography using mixtures of n-hexane−EtOAc as the eluent to afford compounds 21−36. Structures of all compounds were characterized by NMR and HRESIMS analysis. Spectroscopic data of 21−36 are given in the Supporting Information. Cell Culture. Human Huh7 hepatoma cells (purchased from ATCC, Manassas, VA, USA) were maintained and grown as described previously.9,25 Cell Viability Assay. The cytotoxic effect of compound 6 toward Huh7 hepatoma cells was evaluated according to the procedures reported previously.9,25 Determination of HBsAg and HBeAg Levels. After treating HBV-transfected Huh7 cells, levels of HBsAg and HBeAg were determined by an EIA kit (Johnson and Johnson, Skillman, NJ, USA) according to the manufacturer’s instructions. Plasmid Construction. The production of all plasmid constructs was carried out as described previously.9,25,29 Briefly, the pHBV1.2 plasmid containing 1.2-fold of the HBV adw2 serotype genome (nt 2186-1986) was cloned into the EcoRI site of pGEM-7Zf (+) (Promega).30 The four viral promoter-reporter plasmids were constructed by amplifying HBV genomic fragments that corresponded to the Core (nt 1636-1851), S (nt 3114-220), PreS (nt 2438-2855), and X (nt 1071-1357) gene promoter regions with a polymerase chain reaction (PCR) that used the pHBV1.2 plasmid as a template and was subsequently inserted into SacI/XhoI sites of the pGL4.17 luciferasereporter expression vector (Promega) as described previously.29 All DNA sequences were verified with the appropriate restriction enzyme digestion and direct sequencing. Promoter-Reporter Activity Assay. Cells were plated in 24-well culture plates, transfected with promoter-reporter constructs (1 μg/ well) in serum-free DMEM for 24 h, washed with 1× phosphate-
spraying with Dragendorff’s reagent and 10% H2SO4 followed by heating. Anti-NF-κB p65/p50 (#8242, #13586), antiphospho-p65 (#3033), anti-IκBα/β (#4814, #8635), anti-IKKα/β (#11930, #8943), anti-p38 MAPK (#8690), anti-SAPK/JNK (#9252), antiphospho-SAPK/JNK (#9255), anti-Erk1/2 (#4695), and antiphospho-ERK 1/2 (#4370) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against the HBcAg (13A9 clone, sc-23946) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-HBsAg (GTX29193) and anti-HBx (X36C clone, GTX22741) antibodies were purchased from GeneTex (Irvine, CA, USA). The PCR DIG Probe synthesis kit and DIG luminescent detection kit were obtained from Roche (Mannheim, Germany), whereas the Power SYBR Green PCR master mix was purchased from Applied Biosystem (Foster City, CA, USA). The QIAquick Gel extraction kit was purchased from Qiagen (Valencia, CA, USA), and the Dual-Luciferase reporter assay kit was purchased from Promega (Madison, WI, USA). The NueloBond Xtra plasmid DNA purification kit was purchased from Macherey-Nagel KG (Düren, Germany). The Trizol total RNA isolation solution and Lipofectamine 2000 transfection reagent were from Invitrogen (Carlsbad, CA, USA). Lamivudine and an anti-α-tubulin antibody were purchased from Sigma (St. Louis, MO, USA). pNF-κB-Luc, p-AP-1-Luc, pAP2-Luc, and p-ISRE-Luc promoter-luciferase-reporter constructs from Stratagene (La Jolla, CA, USA) were provided by Dr. Cheng-Wen Lin (Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan). The pCMV-p65 expression plasmid was provided by Dr. Chen-Kung Chou (Department of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan). Synthesis of 4α-Isocyanato-19-nor-ent-13-hydroxykaur-16ene (2). Steviol (1) was prepared as described previously,27 and was characterized by 1D and 2D NMR and HRFABMS.28 Subsequently, DPPA (10 mL) and triethylamine (4 mL) were added to a solution of 1 (6.56 g, 0.02 mol) in benzene (60 mL), and the reaction mixture was refluxed for 3 h. After removing the solvent, the residue was purified by column chromatography over silica gel, using CH2Cl2 as an eluent, to give isocyanate 2. After recrystallization from n-hexane, compound 2 was obtained as white crystals (2.5 g, 39%): mp 102−104 °C; [α]28D −81.6 (c 1.05, CHCl3); Rf 0.70 [CH2Cl2−CH3OH (20:1)]; 1H NMR (C5D5N, 500 MHz) δ 0.65 (1H, m), 0.77 (1H, m), 0.84 (1H, d, J = 7.0 Hz), 1.09 (3H, s), 1.17 (1H, m), 1.20 (3H, s), 1.28−1.42 (4H, m), 1.49−1.71 (7H, m), 1.83 (1H, m), 2.02 (1H, m), 2.11 (1H, dd, J = 14.5, 2.0 Hz), 2.19 (1H, dt, J = 14.0, 2.0 Hz), 2.26 (1H, dd, J = 9.0, 2.0 Hz), 4.99 (1H, s), 5.46 (1H, s); 13C NMR (C5D5N, 125 MHz) δ 157.5, 122.5, 103.1, 79.8, 59.5, 54.9, 54.0, 48.2, 47.7, 41.5, 41.4, 40.8, 40.6, 39.2, 39.1, 31.6, 20.4, 20.1, 18.4, 16.7; HRESIMS m/z 316.2277 [M + H]+ (calcd for C20H29NO2, 316.2277). General Procedure for the Preparation of C-4 Ureido Derivatives 3−19. Method A. To a solution of 2 (60 mg, 0.19 mmol) in DMF (5 mL) were added triethylamine and various amines (3 mmol). The reaction mixture was stirred overnight at room temperature. Target compounds were obtained by adding 1 N HCl. The resulting white precipitate was collected and purified by recrystallization from H2O−CH3OH. Method B. To a solution of 2 (60 mg, 0.19 mmol) in DMF (5 mL) was added triethylamine and various amines (3 mmol). The reaction mixture was stirred overnight at room temperature. After completion of the reaction as monitored by TLC, the reaction mixture was diluted with water and extracted with EtOAc. The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated in vacuo to yield the crude product. The resulting product was subjected to column chromatography over silica gel using n-hexane−EtOAc or CH2Cl2− CH3OH as eluents to afford expected C-4 ureido derivatives. Method A was used to prepare compounds 3−6, 9, and 10. Method B was used to prepare compounds 7, 8, and 11−19. Structures of all compounds were characterized by NMR and HRESIMS analysis. The spectroscopic data of all compounds except 6 are given in the Supporting Information. G
DOI: 10.1021/acs.jnatprod.6b00671 J. Nat. Prod. XXXX, XXX, XXX−XXX
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buffered saline (PBS), incubated in DMEM supplemented with 2% FBS, and treated with or without 6 (10 μg/mL) for 2 days. Subsequent luciferase assays were carried out as described previously.9,25 Transfection. The experiment was carried out following the procedure described previously.25 Analysis of Intracellular HBV-RNA by Northern Blotting. The detailed procedures for analysis of intracellular HBV-RNA by Northern blotting were carried out as described previously.9,25 Real-Time PCR Analysis of Intracellular HBV DNA Synthesis. The detailed procedures for analysis of intracellular HBV DNA synthesis by real-time PCR were carried out as described previously.25 Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis and Western Blotting of Intracellular Viral Antigens and Cellular Signaling Proteins. Whole-cell proteins of Huh7 cells were extracted with a PRO-PREP protein extraction solution (iNTRON Biotechnology, Daejeon, Korea) with protease inhibitors. Subsequent assays were carried out as described previously.9,25 Statistical Analysis and Quantification of Data. Methods for statistical analysis and quantification of data were carried out as described previously.25
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(6) Aspinall, E. J.; Hawkins, G.; Fraser, A.; Hutchinson, S. J.; Goldberg, D. Occup. Med. 2011, 61, 531−540. (7) Wakui, Y.; Inoue, J.; Ueno, Y.; Fukushima, K.; Kondo, Y.; Kakazu, E.; Obara, N.; Kimura, O.; Shimosegawa, T. Biochem. Biophys. Res. Commun. 2010, 396, 508−514. (8) Leemans, W. F.; Ter Borg, M. J.; de Man, R. A. Aliment. Pharmacol. Ther. 2007, 26 (Suppl 2), 171, 182. (9) Huang, T. J.; Chou, B. H.; Lin, C. W.; Weng, J. H.; Chou, C. H.; Yang, L. M.; Lin, S. J. Phytochemistry 2014, 99, 107−114. (10) Zhang, F.; Wang, G. Eur. J. Med. Chem. 2014, 75, 267−281. (11) Geuns, J. M. C. Phytochemistry 2003, 64, 913−921. (12) Hutapea, A. M.; Toskulkao, C.; Buddhasukh, D.; Wilairat, P.; Glinsukon, T. J. Clin. Biochem. Nutr. 1997, 23, 177−186. (13) Gardana, C.; Simonetti, P.; Canzi, E.; Zanchi, R.; Pietta, P. J. Agric. Food Chem. 2003, 51, 6618−6622. (14) (a) Yamamoto, N. S.; Kelmer, A. M.; Bracht, E. L.; Ishii, F. S.; Kemmelmeier, F. S.; Alvarez, M.; Bracht, A. Experientia 1985, 41, 55− 57. (b) Melis, M. S. J. Ethnopharmacol. 1992, 36, 213−217. (c) Jeppesen, P. B.; Gregersen, S.; Poulsen, C. R.; Hermansen, K. Metab. Clin. Exp. 2000, 49, 208−214. (d) Boonkaewwan, C.; Toskulkao, C.; Vongsakul, M.; Boonkaewwan, C.; Toskulkao, C.; Vongsakul, M. J. Agric. Food Chem. 2006, 54, 785−789. (e) Yuajit, C.; Homvisasevongsa, S.; Chatsudthipong, L.; Soodvilai, S.; Muanprasat, C.; Chatsudthipong, V. PLoS One 2013, 8, e58871. (15) Toyoda, K.; Matsui, H.; Shoda, T.; Uneyama, C.; Takada, K.; Takahashi, M. Food Chem. Toxicol. 1997, 35, 597−603. (16) Pezzuto, J. M.; Compadre, C. M.; Swanson, S. M.; Nanayakkara, P. D.; Kinghorn, A. D. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 2478− 2482. (17) Moons, N.; De Borggraeve, W.; Dehaen, W. Curr. Org. Chem. 2012, 16, 1986−1995. (18) Khaibullin, R. N.; Strobykina, I. Y.; Kataev, V. E.; Musin, R. Z. Russ. J. Gen. Chem. 2009, 79, 2197−2200. (19) Avent, A. G.; Hanson, J. R.; de Oliveira, B. H. Phytochemistry 1990, 29, 2712−2714. (20) Lee, K. H. J. Nat. Prod. 2010, 73, 500−516. (21) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629−661. (22) Korochkina, M. G.; Nikitashina, A. D.; Khaybullin, R. N.; Petrov, K. A.; Strobykina, Y. I.; Zobov, V. V.; Kataev, V. E. MedChemComm 2012, 3, 1449−1454. (23) Flekhter, O. B.; Boreko, E. I.; Nigmatullina, L. R.; Tret’yakova, E. V.; Pavlova, N. I.; Baltina, L. A.; Nikolaeva, S. N.; Savinova, O. V.; Galin, F. Z.; Tolstikov, G. A. Russ. J. Bioorg. Chem. 2003, 29, 594−600. (24) Zhang, L.; Wang, X. J.; Wang, J.; Grinberg, N.; Krishnamurthy, D.; Senanayake, C. H. Tetrahedron Lett. 2009, 50, 2964−2966. (25) Huang, T. J.; Yang, C. L.; Kuo, Y. C.; Chang, Y. C.; Yang, L. M.; Chou, B. H.; Lin, S. J. Bioorg. Med. Chem. 2015, 23, 720−728. (26) Takasaki, M.; Konoshima, T.; Kozuka, M.; Tokuda, H.; Takayasu, J.; Nishino, H.; Miyakoshi, M.; Mizutani, K.; Lee, K. H. Bioorg. Med. Chem. 2009, 17, 600−605. (27) Ogawa, T.; Nozaki, M.; Matsui, M. Tetrahedron 1980, 36, 2641−2648. (28) Yang, L. M.; Hsu, F. L.; Cheng, J. T.; Chang, S. F.; Hsu, R. Y.; Liu, P. C.; Lin, S. J. Phytochemistry 2007, 68, 562−570. (29) Huang, T. J.; Liu, S. H.; Kuo, Y. C.; Chen, C. W.; Chou, S. C. Antiviral Res. 2014, 101, 97−104. (30) Blum, H. E.; Galun, E.; Liang, T. J.; von Weizsacker, F.; Wands, J. R. J. Virol. 1991, 65, 1836−1842. (31) Doria, M.; Klein, N.; Lucito, R.; Schneider, R. J. EMBO J. 1995, 14, 4747−4757. (32) Hildt, E.; Urban, S.; Eckerskorn, C.; Hofschneider, P. H. Hepatology 1996, 24, 502−507. (33) Kwon, J. A.; Rho, H. M. Biochem. Cell Biol. 2002, 80, 445−455. (34) Meyer, M.; Caselmann, W. H.; Schluter, V.; Schreck, R.; Hofschneider, P. H.; Baeuerle, P. A. EMBO J. 1992, 11, 2991−3001.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00671. Spectroscopic data for the synthesized compounds (3−5, 7−19, and 21−36) and NMR spectra of 2, 6, and 20, as well as data of anti-HBV activities (Table S1) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +886 4 7256166, ext 66022. Fax: +886 4 7233190. Email:
[email protected] (Y.-C. Kuo). *Tel: +886 4 22053366, ext 2159. Fax: +886 2 22053764. Email:
[email protected] (T.-J. Huang). ORCID
Tsurng-Juhn Huang: 0000-0002-2189-5486 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by grants from the Committee on Chinese Medicine and Pharmacy, Department of Health, Executive Yuan, Taiwan (CCMP102-RD-116), China Medical University (CMU105-S-18), China Medical University Hospital (DMR-103-042 and DMR-105-044), Show Chwan Memorial Hospital Research Foundation (RD105020), and the University System of Taipei Joint Research Program (USTP-NTUTTMU-105-05). The authors express their heartfelt thanks to Dr. C.-C. Lu for the pHBV1.2 and pHBV2 plasmids, to Dr. H.-S. Liu for the pGAPDH plasmid, and to Dr. C.-W. Lin for the pAP1-Luc, pAP2-Luc, pNF-κB-Luc, and pISRE-Luc promoterreporter constructs.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.6b00671 J. Nat. Prod. XXXX, XXX, XXX−XXX
