Panaxadiol and Panaxatriol Derivatives as Anti-Hepatitis B Virus Inhibitors

Abstract 28 Derivatives of panaxadiol (PD) and panaxatriol were synthesized and evaluated for their anti-HBV activity on HepG 2.2.15 cells, of which 17 derivatives inhibited HBV DNA replication. Compounds 4, 9, 10, 14, and 15 showed moderate activity against HBV DNA replication with IC50 values ranged from 7.27 to 28.21 μM compared with PD. In particular, 3-O-2′-thenoyl panaxadiol (4) inhibited not only HBV DNA replication (IC50 = 16.5 μM, SI > 115.7) but also HBsAg (IC50 = 30.8 μM, SI > 62.0) and HBeAg (IC50 = 18.2 μM, SI > 105.14) secretions. Their structure–activity relationships were discussed for guiding future research toward the discovery of new anti-HBV agents. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0018-2) contains supplementary material, which is available to authorized users.


Introduction
Hepatitis B virus (HBV) infection is a serious health problem all over the world. There are about 350 million chronically infected individuals with the risk of approaching liver cirrhosis and hepatocellular carcinoma [1]. The current therapies for HBV infection involve immunomodulators, interferon-a, polyethylene glycol interferon-a, and nucleoside drugs, and are unsatisfactory due to high recurrence, drug resistance and inevitable side effects including influenza-like illness, myalgia, headache, reduction of neutrophilic granulocyte and blood platelet, etc. [2][3][4][5]. Therefore, it is interesting to explore novel anti-HBV agents with novel antiviral targets and mechanisms.
Natural products offer many opportunities to find lead compounds for drug discovery [6][7][8][9][10]. Dammarane triterpenes and their derivatives have antiviral and hepatoprotective potencies, as well as antitumor, hemolytic, antiplatelet, immunomodulatory, antioxidant and neuroprotective activities [11]. For example, chikusetsusaponin III reduced yield of herpes simplex virus type I with ID 50 value of 29 lM [12]; panaxadiol (PD) derivatives incorporated with 2,2-dimethylsuccinyl group at C-3 and panaxatriol (PT) derivatives with same groups at C-3 and C-6, could inhibit HIV-1 protein proteases (IC 50 = 2.7 ± 4.3 to 5.4 ± 3.8 lM) and HCV protein proteases (IC 50 = 1.8 ± 2.6 to 30.4 ± 3.0 lM) [13]; furthermore, ginsenosides Rb 3 , Rc, Rd, XVII and notoginsenoside R 1 from the flower buds of Panax notoginseng showed hepatoprotective activity against liver injury induced by D-galactosamine and lipopolysaccharide in mice [14]. Although derivatives of PD and PT ( Fig. 1) exhibited antiviral and hepatoprotective effects, no report was concerned with their anti-HBV activity. As our ongoing study for searching anti-HBV inhibitors from natural resources, PD and PT were revealed to be active against HBV DNA replication with IC 50 values of 148.15 and 668.60 lM but low SI values of 6.2 and 3.6 in our random assay. In order to increase the activity and safety, PD and PT were hybridized with heteroaromatic rings based on our previous experience from the modification on caudatin and hemslecin A [15,16]. Consequently, 28 panaxadiol and panaxatriol analogues were synthesized by modifying on rings A, B and C. Herein, we described the synthesis, in vitro anti-HBV activity and structure-activity relationships (SARs) of these derivatives (Scheme 1).

Chemistry
The Steglich esterification condition was applied for synthesis of 3-O-substituted derivatives of PD and 3,6-Odisubstituted derivatives of PT in presence of 4-dimethylaminopyridine (DMAP), and N 0 ,N 0 -dicyclohexylcarbodiimide (DCC). Derivatives (2-13, 20-21) of PD and PT were also prepared with anhydrides under a catalytic amount of DMAP. There were no 12-O-substituted derivatives produced, of which the substituent position could be determined by the chemical shifts of derivatives at H-3 and H-12 in 1 H NMR spectrum. For example, chemical shifts of H-3 and H-12 of PD appearred at d H 3.21 and 3.50 but at d H 4.42 and 3.52 of compound 1. Furthermore, the hydroxyl group at C-12 of 3-O-substituted derivatives (1, 4 and 14) were transformed as ketones by Jones reagent in order to disclose effects of hydroxyl groups.

Anti-HBV Activity
PD, PT and their derivatives were evaluated for anti-HBV activities on HBsAg and HBeAg secretions, as well as HBV DNA replication on HepG 2.2.15 cells [9], and the results were summarized in Table 1. Accordingly, 4 active derivatives (4, 9, 10 and 11) 14) secretions, which is worth for further investigating.
Among the 3-O-substituted derivatives of PD, introduction of acetyl (1) and cyclopentanecarbonyl (2) into C-3 of PD reduced cytotoxicity and activities against HBV DNA replication. The 3-O-cyclopentanecarbonyl group of compound 2 was replaced by heteroatomic rings to generate 3-O-2 0 -furoyl (3), 3-O-2 0 -thenoyl (4) and 3-O-2 0 -nicotinoyl (11) derivatives providing better inhibitory activity with IC 50 values of 50.27, 16.51 and 117.21 lM than PD and 3-O-benzoyl analogue (8). From the above analysis, it is suggested that heteroatomic rings played important roles in enhancing activity. Analogue 4 possessed the most active inhibition on HBsAg and HBeAg    exhibited less activity than 3-O-2 0 -thenoyl (4) analogue, indicating that substituents at 2-thenoyl moiety were unfavorable for anti-HBV activity. (16) analogues with free carboxyl groups were further prepared and showed better activity against HBV DNA replication than PD, of which compound 16 appeared the IC 50 value of 51.93 lM and the SI value higher than 32.8, inferring that oxygen atom at side chain reduced cytotoxicity. Phenolic hydroxyl groups were introduced into the benzene ring of inactive compound 8 to offer derivatives 9 and 10 with 60-folds growth of inhibition on HBsAg secretion and HBV DNA replication, together with the increased cytotoxicity, indicating phenolic hydroxyl groups enhanced both activity and cytotoxicity.

3-O-Succinyl (14), 3-O-glutaryl (15) and 3-O-diglycolyl
Further modification on ring C of derivatives 1, 4 and 14 by transforming the hydroxyl group at C-12 into the ketone group provided three inactive products 26-28 with IC 50 values higher than 485.1 lM, demonstrating that hydroxyl group at C-12 is crucial for antiviral activity. Compared with PD, PT with one hydroxyl group at C-6 reduced activity, inferring that hydroxyl group at C-6 was detrimental to anti-HBV activity. This analysis was further supported by six 3,6-O-disubstituted derivatives (20-25) exhibited slight activity against HBV with IC 50 values higher than 320.03 lM in contrast with PD derivatives which had same substituents, such as 2-furoyl, 2-thenoyl and succinyl groups.

Conclusion
According to the results mentioned above, SARs were summarized as follows: (1) 2-thenoyl group at C-3 are favorable to enhance anti-HBV activity; (2) the hydroxyl group at C-12 is necessary for inhibitory activity; (3) hydroxyl group at C-6 was detrimental to anti-HBV activity. This study indicated that panaxadiol derivatives had moderate anti-HBV activity, and were worth further investigating for non-nucleoside anti-HBV drug candidates.

General Procedure for Preparation of Compounds 2-13
The DCC (1.2 equiv.) was added to the solution of PD (0.2 mmol), DMAP (0.2 equiv.), and appropriate carboxylic acid (1.2 equiv.) in anhydrous CH 2 Cl 2 (8 mL) at 0 8C. The resulting mixture was stirred at room temperature until the starting material was vanished by TLC check. The reaction mixture was filtered and washed with CH 2 Cl 2 (10 mL 9 2). Then, the CH 2 Cl 2 solution was washed with 5 % HCl (30 mL 9 3), saturated NaHCO 3 (30 mL 9 3) and saturated NaCl (30 mL 9 3), respectively. Subsequently, the organic layer was dried over anhydrous Na 2 SO 4 and concentrated to dryness under reduced pressure. The residue was purified by column chromatography over the silica gel to yield the target compound.  13

General Procedure for Preparation of Compounds 1 and 14-19
A solution of panaxadiol (0.5 mmol), the corresponding anhydride (3 equiv.) in anhydrous pyridine (6 mL) was added DMAP (0.3 equiv.) and stirred at 90 8C for 5 h. The cooling reaction mixture was diluted with ice water (30 mL), extracted with ethyl acetate (30 mL 9 3). The ethyl acetate mixture was washed with 5 % HCl (30 mL 9 3) and saturated NaCl (30 mL 9 3). The ethyl acetate layer was dried over anhydrous Na 2 SO 4 and concentrated to dryness under reduced pressure. The crude products were purified by silica gel column chromatography.