English

• 1.   Introduction

  Hepatitis C virus (HCV) afflicts more than 170 million people total worldwide and leads to chronic liver disease [1, 2]. Direct antiviral agents such as boceprevir and telaprevir targeting the NS3-4A protease are approved for using in patients infected with HCV [3]. However,these direct acting approaches are associated with resistance and require concomitant use of ribavarin and interferon [4, 5, 6].

  Thus,a different way was taken by focusing on the discovery and development of small molecule HCV entry inhibitors. The viral entry is a critical step in the life cycle of HCV,and inhibitors of viral entry can add significantly to a drug cocktail targeting multi-steps of the viral life cycle [7, 8, 9]. Also,the entry inhibitors can protect cured cells from re-infection and prevent viral spread. Recently,we found that echinocystic acid (EA),displayed potential anti-HCV entry activity with IC₅₀ at 1.4 mmol/L. Previous studies elucidated the structure-activity relationships of EA as an HCV entry inhibitor, and we found that when its ring D is modified properly at the 28- COOH,the potency is significantly increased [8, 10, 11].

  As continue to our program for the discovery of novel HCV inhibitors targeting entry,we report herein the synthesis of EA 28-COOH derivatives and evaluation of their anti-HCV entry activities. This study establishes the importance of pentacyclic triterpene analogues as leads for the development of potential HCV entry inhibitors.

  2.   Experimental

  Routine ¹H NMR and ¹³C NMR spectra were recorded. Samples were dissolved in CDCl₃ or CD₃OD and tetramethylsilane (TMS) was used as reference. High Resolution Mass Spectra (HRMS) were obtained with an APEX IV FT_MS (7.0 T) spectrometer (Bruker) in positive ESI mode. NMR spectra were recorded on a Bruker DRX 400 spectrometer at ambient temperature. Analytical TLC was performed on Merck silica gel 60 F254. Compounds were visualized by staining with a yellow solution containing Ce(NH₄)₂(NO₃)₆ (0.5 g) and (NH₄)₆Mo₇O₂₄-4H₂O (24.0 g) in 6% H₂SO₄ (500 mL) followed by heating. Chemicals for the synthesis were analytical grade. Echinocystic acid was isolated from Gleditsia sinensis Lam,which was characterized by comparing their spectral data with those reported in the literature. The method employed in determining the purity of synthesized compounds was HPLC using a Agilent 1260 instrument and Agilent 1260 VWD detector (256 nm),with chromatography performed on a ZORBAX 250 × 4.6 mm,5 mm C18 column,and eluted with CH₃OH-H₂O (80:20 to 100:0) at a flow rate of 0.80 mL/min,the details see Supporting information.

  As shown in Scheme 1,the 28-COOH of EA was first activated by TBTU and then reacted with corresponding amines,giving the targeting EA derivatives (compounds 2-8) with high yield as previous report [8, 10]. However,it is difficult to obtain compounds 9-12 by the same strategy,because of the steric hindrance and decreasing nucleophilicity of the aryl amine. Thus another method was used for the preparation of compounds 9-12. Compared the reaction conditions and selectivity of carbodiimide and acylchloride condensation,the former was applied as a feasible plan.

  图 1

  

  图 1  Synthesis of EA derivatives 1–8. Reagents and conditions: (a) TBTU, DIEA, THF, r.t., 4 h, 89%; (b) corresponding amines, Na₂CO₃, DMF, r.t., 20 min,72%–91%.

  Figure 1.  Synthesis of EA derivatives 1–8. Reagents and conditions: (a) TBTU, DIEA, THF, r.t., 4 h, 89%; (b) corresponding amines, Na₂CO₃, DMF, r.t., 20 min,72%–91%.

  下载: 全尺寸图片 幻灯片

  Interestingly,we employed 4-(N,N-dimethylamino) pyridine (DMAP) as catalyst for the condensation,and found that the reaction did not proceed completely and EA anhydride was formed as a main side product. Without DMAP,certain EA derivatives (9- 12) were obtained with corresponding amines at the presence of EDC in dry THF (Scheme 2).

  图 S2

  

  图 S2  Synthesis of EA derivatives 9–12. Reagents and conditions: (a) EDC,corresponding amines, THF, r.t., 8 h, 37%–62%.

  Figure S2.  Synthesis of EA derivatives 9–12. Reagents and conditions: (a) EDC,corresponding amines, THF, r.t., 8 h, 37%–62%.

  下载: 全尺寸图片 幻灯片

  3.   Results and discussion

  The synthesized EA 28-COOH derivatives were tested for their capacities to block HCV entry using an HCV pseudo-particle (HCVpp) entry assay. Vesicular stomatitis virus G protein pseudo particle (VSVpp) was also tested in parallel to determine the specificity and toxicity,as previously reported [11, 12, 13, 14]. Two concentrations,1 mmol/L and 5 mmol/L,were tested for each compound. DMSO is used as a negative control for monitoring the maximum HCVpp entry in the presence of 1%,and CD81 is a positive control to reflect the maximum blocking of HCVpp entry with the concentration of 5 nmol/L. As shown in Fig. 1,introducing 2-phenylethanamine (2) or 2-(4-methoxyphenyl) ethanamine (3) group at C-28 significantly decreased or even eliminated the anti-HCV entry activity. Compound 4,with one more methoxy group than 3,showed moderate anti-HCV activity. While,other EA derivatives bearing a phenyl ring (5,6),a hydrophobic group, decreased their potency on blocking HCV entry. Pentacyclic triterpenes,such as EA,are hydrophobic,which may affect their potential pharmacokinetic properties. To increase the solubility of EA,some water soluble moieties,including morpholine (7,10), piperazine (8),sulfonamide (9) and azole groups (11,12),were conjugated to EA via 28-COOH. Besides,these functional groups are commonly used in drug design and show many biological activity. The above facts provide a biologic basis for the design and synthesis novel EA derivatives (7-12) to investigate the introduction of such moieties affecting the anti-HCV activity of EA.

  图 1

  

  图 1  Anti-HCV entry activity of EA and its derivatives. CD81 antibody, an entry inhibitor targeting host cell membrane, was utilized as a positive control, and the concentration of CD81 antibody was 5 nmol/L. 0.5% DMSO (final concentration) was used for normalization in each condition.

  Figure 1.  Anti-HCV entry activity of EA and its derivatives. CD81 antibody, an entry inhibitor targeting host cell membrane, was utilized as a positive control, and the concentration of CD81 antibody was 5 nmol/L. 0.5% DMSO (final concentration) was used for normalization in each condition.

  下载: 全尺寸图片 幻灯片

  By comparing the potencies of blocking HCV entry of this series of EA derivatives,we found that most of these compounds (7-12) displayed higher antiviral activity than compounds bearing a phenyl ring (2-6),especially compound 12,showed increasing anti-HCV entry activity than EA. In addition,VSVpp assay is used to monitor the toxicities of target compounds since VSVpp,unlike HCVpp,has a broad host range and can infect almost all cell lines. Most of these compounds showed no toxicity in VSVpp assay, however,compounds 4,8 and 11 decreased the report readings in VSVpp infected cells,which reflected cellular toxicity rather than potency enhancement.

  A series of studies demonstrated that the aglycon of triterpenoids have crucial influence on the hemolytic properties [15]. Here, we also found that EA gains substantial hemolytic side effect with CC₅₀ at 15 mmol/L [8]. Such hemolytic property may restrict EA used as potential anti-HCV entry inhibitor. Thus,the hemolytic properties of all these EA derivatives were tested with the concentration gradient of 0,6.25,12.5,25 and 50 mmol/L. The results showed that all the EA derivatives here depleted the hemolytic activity by modifying C-28 carboxyl group (Fig. 2).

  图 2

  

  图 2  Hemolytic properties of EA and its derivatives.

  Figure 2.  Hemolytic properties of EA and its derivatives.

  下载: 全尺寸图片 幻灯片

  4.   Conclusion

  In conclusion,we introduced different chemical groups at EA C- 28 to expand the structural diversity for SAR exploration and evaluated their anti-HCV entry activity for the first time. These novel compounds showed moderate anti-HCV entry activity, especial compound 12 showed higher anti-HCV entry activity than EA without significant cytotoxicity,and removed the undesired hemolytic effect. This study establishes the importance of EA as a new lead for the development of potential HCV entry inhibitors.

  Appendix A. Supplementary data

  Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2016.01.050.

• 1. [1]

     Sy T., Jamal M.M.. Epidemiology of hepatitis C virus (HCV) infection[J]. Int. J. Med. Sci., 2006, 3:  41-46.

  2. [2]

     Shepard C.W., Finelli L., Alter M.J.. Global epidemiology of hepatitis C virus infection[J]. Lancet Infect. Dis., 2005, 5:  558-567.

  3. [3]

     Sheridan C.. New Merck and Vertex drugs raise standard of care in hepatitis C[J]. Nat. Biotechnol., 2011, 29:  553-554.

  4. [4]

     Fried M., Shiffman M., Reddy K.. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection[J]. New Engl. J. Med., 2002, 347:  975-982.

  5. [5]

     Di Bisceglie A.M., McHutchison J., Rice C.M.. New therapeutic strategies for hepatitis C[J]. Hepatology, 2002, 35:  224-231.

  6. [6]

     Thompson A., McHutchison J.. Antiviral resistance and specifically targeted therapy for HCV (STAT-C)[J]. J. Viral Hepatitis, 2009, 16:  377-387.

  7. [7]

     Wong-Staal F., Syder A.J., McKelvy J.F.. Targeting HCV entry for development of therapeutics[J]. Viruses, 2010, 2:  1718-1733.

  8. [8]

     Yu F., Wang Q., Zhang Z.. Development of oleanane-type triterpenes as a new class of HCV nntry inhibitors[J]. J. Med. Chem., 2013, 56:  4300-4319.

  9. [9]

     Mittapalli G.K., Jackson A., Zhao F.. Discovery of highly potent small molecule hepatitis C virus entry inhibitors[J]. Bioorg. Med. Chem. Lett., 2011, 21:  6852-6855.

  10. [10]

      Yu F., Peng Y., Wang Q.. Development of bivalent oleanane-type triterpenes as potent HCV entry inhibitors[J]. Eur. J. Med. Chem., 2014, 77:  258-268.

  11. [11]

      Wang H., Wang Q., Xiao S.. Elucidation of the pharmacophore of echinocystic acid, a new lead for blocking HCV entry[J]. Eur. J. Med. Chem., 2013, 64:  160-168.

  12. [12]

      Yang J., Zhou D., Wong-Staal F.. Screening of small-molecule compounds as inhibitors of HCV entry[J]. Meth. Mol. Biol., 2009, 510:  295-304.

  13. [13]

      Bartosch B., Dubuisson J., Cosset F.. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes[J]. J. Exp. Med., 2003, 197:  633-642.

  14. [14]

      Hsu M., Zhang J., Flint M.. Hepatitis C virus glycoproteins mediate pHdependent cell entry of pseudotyped retroviral particles[J]. Proc. Natl. Acad. Sci. U.S.A., 2003, 100:  7271-7276.

  15. [15]

      Francis G., Kerem Z., Makkar H.P.. The biological action of saponins in animal systems:a review[J]. Br. J. Nutr., 2002, 88:  587-605.

• 

  Figure 1  Synthesis of EA derivatives 1–8. Reagents and conditions: (a) TBTU, DIEA, THF, r.t., 4 h, 89%; (b) corresponding amines, Na₂CO₃, DMF, r.t., 20 min,72%–91%.

  下载: 全尺寸图片 幻灯片
  

  Figure S2  Synthesis of EA derivatives 9–12. Reagents and conditions: (a) EDC,corresponding amines, THF, r.t., 8 h, 37%–62%.

  下载: 全尺寸图片 幻灯片
  

  Figure 1  Anti-HCV entry activity of EA and its derivatives. CD81 antibody, an entry inhibitor targeting host cell membrane, was utilized as a positive control, and the concentration of CD81 antibody was 5 nmol/L. 0.5% DMSO (final concentration) was used for normalization in each condition.

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Hemolytic properties of EA and its derivatives.

  下载: 全尺寸图片 幻灯片
•