Hepatitis B virus (HBV) infection remains a global health problem, which often leads to severe consequences such as liver failure, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).
There are about 2 billion people who have been infected with HBV, and more than 350 million people are lifelong patients [1e3] . Immunization therapy is the most effective measure for new infections, but millions of the HBV patients will eventually succumb to the infection sequence due to vaccine failure [4] . Interferona and polyethylene glycol interferon-a are used as anti-HBV agents clinically, however, their application is limited for low curing rate and serious side effects (influenza-like symptoms, fatigue, myalgia, nausea, headache, etc.) [5, 6] . Five nucleoside drugs, lamivudine (3TC), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT) and tenofovir-DF (TDF), have been approved by FDA for HBV treatment. The disadvantages of nucleosides, such as drug resistance and high recurrence, are becoming an important clinical issue because of the single target [6e8]. New strategies aiming at viral suppression, promoting virologic clearance and preventing drug resistance are fascinating topics [9] . Therefore, novel anti-HBV agents with unique antiviral target and mechanism are still needed.
Natural products and their derivatives by simple functionalgroup transformations offer many opportunities for finding novel anti-HBV leads or drugs with unique antiviral mechanisms [10e12]. For example, helioxanthin, initially isolated from the shrub of Taiwania cryptomerioides, had unique mechanisms by diminishing HBV promoter activity and blocking viral gene expression and replication. After chemical modification, its more active derivative 8e1 ( Fig. 1 ) was obtained [13e17] . Oxymatrine ( Fig. 1 ) and its derivatives showed anti-HBV activity through suppressing host heat-stress cognate 70 (Hsc70) expression [18] . In our previous study, alisol A from Alisma orientalis possessed potent anti-HBV activity, and a series of high-activity derivatives were obtained ( Fig. 1 ) after chemical modification [19e21] .
As an ongoing search for potential anti-HBV inhibitors [22e27], our latest study revealed that caudatin (1, Fig. 2 ) from Cynanchum auriculatum (Bai-Shou-Wu in Chinese) had activity inhibiting the secretion of HBsAg and HBV DNA replication with the IC 50 values of 142.67 mM (SI ¼ 1.7), 40.62 mM (SI ¼ 6.0), respectively. Furthermore, caudatin as a prospective anti-HCC drug with the mechanism of inhibiting cell proliferation and inducing cell apoptosis has been reported [28e32] . Consequently, it may be interesting for caudatin to be developed as a novel anti-HBV agent by chemical modification.
Cinnamic acid analogs (esters, amides and glycosides) have attracted much attention in biology and medicine because of their antiviral [33, 34] , antiatherogenic [35] , antitumor [36, 37] , antituberculosis [38, 39] , antioxidant [40, 41] , and antibacterial [42, 43] properties. For example, cinnamic acid (2, Fig. 2 ) could inhibit HIV/SARS-CoV S pseudovirus [33] , and the cholesteryl conjugated with the 3, 4-dimethoxy cimmamic acid (3, Fig. 2 ) could enhance the anti-poliovirus type 1 (PV1) activity [44] . Caffeic acid (3, 4dihydroxycinnamic acid, 4, Fig. 2 ) and chlorogenic acid (the ester of 3, 4-dihydroxycinnamic acid with quinic acid, 5, Fig. 2 ) showed potent anti-HBV activity in vivo and in vitro [45] , which suggested that the molecular with cinnamic acid moiety might show potent anti-HBV activity. Meanwhile, cinnamic acid has been widely applied as a food additive with good safety [46] . Molecular hybrids with two pharmacophores often lead to synergistic activity [47, 48] . Thus, the combination of caudatin with cinnamic acids as novel anti-HBV agents was designed, which was anticipated that the hybrids could enhance the anti-HBV activity and decrease the cytotoxicity. Herein, we reported the chemical modification and HBV inhibitory properties of caudatin derivatives designed by molecular hybridization. The most active compound 18 was further investigated for the mechanism of action on HBV promoters (preC⁄pregenomic, S1, S2 and X promoters) and enhancers (ENI and ENII), as well as the acute toxicity in mice.
Compounds 6e10 and 12e51 were synthesized by the reaction of corresponding cinnamic acids with caudatin in the presence of N 0 , N 0 -dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) (Scheme 1). An exception is that compound 11 was obtained starting from caudatin and 3, 4-dihydroxycinnamic acid in the presence of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine (TPP) (Scheme 1) [49] . Generally, the introduction of fluorine is useful to alter the physical properties, binding characteristics, and metabolic disposition in developing drug leads [50] , thus various fluorinated cinnamic acids were used to react with caudatin. The structures of the synthesized derivatives (Table 1) were identified by spectroscopic means ( 1 H, 13 C NMR and MS). The structures of 3, 17-O-dicinnamoyl caudatin derivatives (48e51) were further determined by comparison with caudatin and/or the mono-cinnamoyl analogs.
All the caudatin derivatives were tested for their anti-HBV activity, namely inhibiting the secretion of HBsAg, HBeAg, and HBV DNA replication in HepG 2.2.15 cells. The data of their anti-HBV activity and cytotoxicity were listed in Table 2 .
Derivative 6 was more effective on inhibiting HBsAg, HBeAg secretion and HBV DNA replication than that of caudatin with the IC 50 values of 48.33, 94.24, 5.91 mΜ, and SI values of more than 29.3, 15.0, 239.9, respectively. After methyl group introduced to the cinnamoyl moiety, the anti-HBV activity of compounds 7e9 slightly decreased comparing to compound 6 but still was greater than that of caudatin. The introduction of formyl group into cinnamoyl moiety of compound 10 could increase the anti-HBV activity against secretion of the HBsAg and HBeAg, and HBV DNA replication, but the cytotoxicity (CC 50 When hydroxyl group was introduced to cinnamoyl, the activity and cytotoxicity of compound 11 were increased. Compounds 12e21 exhibited greater activity and weaker cytotoxicity than those of compound 11 after the hydroxy on the cinnamoyl replaced by alkoxyl groups (MeOe, EtOe, eOCH 2 Oe). 3-O-(3, 4, 5-Trimethoxy) cinnamoyl caudatin (18) possessed the highest activity inhibiting not only the secretion of HBsAg (IC 50 ¼ 5.52 mΜ, SI > 330.0), HBeAg (IC 50 ¼ 5.52 mΜ, SI > 330.0), but also HBV DNA replication (IC 50 ¼ 2.44 mΜ, SI > 746.6). Both the anti-HBV activity and cytotoxicity of the derivatives 22e23 were increased comparing to caudatin when acetoxy group was incorporated into the cinnamic acid moiety.
The introduction of halogen atoms into caudatin derivatives (25e35) could significantly enhance their anti-HBV activity. But, more fluorine atoms in the derivatives led to cytotoxicity (36, CC 50 ¼ 396.82 mΜ and 37, CC 50 < 8.22 mΜ) increase.
Compounds 24, 40e42, 44 with one CF 3 group showed potent activity against HBsAg secretion and HBV-DNA replication but weaker against HBeAg secretion, while the co-existence of 3 00 -and 5 00 -CF 3 groups on cinnamoyl (43) remarkably decreased the anti-HBV activity. The anti-HBV activity of compounds 45 and 46 with nitro group substituted on the cinnamoyl group was increased, and the cytotoxicity was decreased.
The positions of substituents on the cinnamoyl moiety could impact the anti-HBV activity of the 3-O-cinnamoyl caudatin derivatives. When the substitutes were methyl (8 vs 7, 9) , alkoxy (13 vs 12, 14; 21 vs 20) or F (26 vs 25, 27) , Cl (29 vs 28, 30), Br (31 vs 32), the meta-substituted analogs showed greater activity than those of ortho-and para-substituted. In contrary, the activity of the metasubstituted was weaker than those of ortho-and para-substituted with the substituents of trifluoromethyl (41 vs 40, 42) and nitro (46 vs 45) group. From the above results, it is proposed that small substitutions could affect the steric clash, electron density, or hydrogen-bonding capacity, resulting different anti-HBV activity of caudatin derivatives.
Due to the good activity of the conjugated derivatives of caudatin with one cinnamoyl moiety, 3, 17-O-dicinnamoyl caudatin derivatives (48e51) were further synthesized. Disappointedly, the anti-HBV activity of compounds 48e51 obviously decreased. These results indicate that free hydroxyl group at C-17 of caudatin is essential to the anti-HBV activity, and the steroidal skeleton itself plays a crucial role in maintaining anti-HBV activity of the conjugated esters.
To elucidate the mechanism of action, a luciferase reporter gene assay was used to determine the effects of compound 18 on HBV promoters (preC⁄pregenomic, S1, S2 and X promoters) and enhancers (ENI and ENII). The result of compound 18 (10 mΜ) in the luciferase reporter assay was shown in Fig. 3 (Cells viability was up to 97.4% and 85.6% at 48 h and 72 h, respectively according to the MTT assay). Compound 18 mainly inhibited 5.6 times of the transcript activity of HBV X promoter (Xp) and increased 9.4 times of the HBV enhancer ENI compared with mock-treated control. HBV sequences are transcribed under the control of promoters and enhancers which are important for replication of HBV [51, 52] . The Xp regulates transcription of the small 0.9 kb mRNA sequence encoding HBV X protein (HBx), which plays an important role in stimulating HBV transcription and replication [53, 54] . The HBV ENI element takes a crucial effect in the overall liver-specific regulation of HBV gene expression [55] . Thus, compound 18 might exert anti-HBV activity by interfering promoters and enhancers to influence HBV transcriptions. Considering that there are about 20 nucleotides overlapped between the minimal Xp sequence and the 3 0 end of the ENI [56e58], compound 18 was presumed to simultaneously influence Xp and ENI on the overlapped region. Further studies are needed to clarify how compound 18 takes effect and which region responds exactly. Nucleoside analogs can suppress HBV replication by inhibiting HBV polymerase and terminating premature DNA chain, but do not directly affect the HBV gene expression [59] . Compound 18 exhibits anti-HBV activities by interfering HBV gene expression instead of inhibiting HBV DNA polymerase, which is different from those of the nucleoside analogs.
To evaluate the safety of compound 18, the acute toxicity was tested in mice. Groups of healthy Kunming mice of both genders were orally administrated compound 18 with a single dose at 50, 250, and 1250 mg/kg, respectively. The survival and abnormality of mice were monitored up to 14 days post administration, and no dies and abnormality were observed (including the body weight, Table 3 ) in the mice throughout the observation period. It is demonstrated that compound 18 has good safety in vivo with the LD 50 value of more than 1250 mg/kg in oral route.
In summary, our design and synthesis have led to a series of non-nucleoside anti-HBV agents by attaching of the cinnamic acids to caudatin. Most of the derivatives displayed potent anti-HBV activity especially inhibited the HBV DNA replication with the Preliminary mechanism study proposed that compound 18 exerted antivirus effects via interfering HBV promoters and enhancers to influence HBV transcriptions. The low cytotoxicity and acute toxicity in mice indicated that compound 18 may be potential novel non-nucleoside anti-HBV drug candidate with unique mechanisms to be further investigated.
Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AM 400 ( 1 H/ 13 C, 400 MHz) or DRX-500 ( 1 H/ 13 C, 500 MHz) spectrometers and chemical shifts were given in d (ppm) with TMS as the internal standard (Bruker, Bremerhaven, Germany); MS and HRMS spectra were determined on an AutoSpec Premier P776 (VG, Manchester, UK) or API QSTAR Pulsar (AB, Foster City, USA) mass spectrometers; column chromatography (CC): silica gel (200e300 mesh; Qingdao Makall Group Co., Ltd; Qingdao; China). All reactions were monitored using thin-layer chromatography (TLC) on silica gel plates. Caudatin was isolated from C. auriculatum (Bai-Shou-Wu) and had the purity of >95.0%. Substituted cinnamic acids were purchased from Alfa Aesar or J&K Scientific Ltd. Organic solvents were analytical reagent grade and purchased from Tianjin Chemical Reagent Co., Ltd.
A solution of 1 (0.2 mmol), DMAP (0.2 equiv), and the proper cinnamic acid (1.2 equiv) in anhydrous CH 2 Cl 2 (8 mL) was added 9 Hz, H-3 00 ); 13 
The promoter regions of HBV core [nucleotides (nt) 1501e1822], X (nt 831e1280), preS (nt 2461e2820), S (nt 2871e30), ENI (1070e1234) and the ENII (1627e1774) were cloned upstream of the luciferase gene of pGL3-Basic (Promega), respectively. HepG 2 was transiently transfected with the reporter vector in a 24-well dish by using LipofectamineÔ 2000 according to the manufacturer's instructions (Invitrogen). HepG 2 cells were transiently transfected with a constant amount of pGL3 Vector (expressing firefly luciferase) as Basic Control, phRL-CMV vector (expressing Renilla luciferase) as an internal control. Cells were split into 24well plates for 24 h after transfection and continuously cultured for 3 days. Compound was then added to the medium for 3 days (similar to an antiviral assay). Data represent the average AE standard deviation of triplicated samples. Transcriptional activity was determined by measuring luciferase activity in a multiwell plate luminometer (MDS Analytical Technologies, US) using the Luciferase Reporter Assay System (Promega).
Hep G 2.2.15 cells were cultured in RPMI-1640 medium (Gibco)
supplemented with 10% fetal calf serum (Gibco), 100 mg/mL G148 (Gibco), 100 IU/mL penicillin (Gibco), and 100 IU/mL streptomycin (Gibco), and maintained at 37 C in a moist atmosphere containing 5% CO 2 . Cells were subcultured once a week, and fresh medium was added every other day.
Male and female Kunming mice (21 AE 2 g) were purchased from Chengdu Dashuo Biotechnology Co., Ltd. (Chengdu, China) with animal certificate No. 0012039 and license No. SCXK (Chuan) 2008e24. They were randomly divided into 4 groups with 10 mice each (5 male plus 5 female). Groups of mice were orally administrated compound 18 at a single dose 0 (blank group, the same volume of physiological saline), 50, 250, and 1250 mg/kg, respectively. Mice were housed with free access to water and food in stainless cages in a room with controlled temperature (25 AE 1 C) and a 12-h light/dark cycle. The mouse survival and body weight were monitored and recorded up to 14 days posttreatment. The protocol complied with the guidelines of Kunming City Laboratory Animal Administration Committee of China for the care and use of laboratory animals.