N-Substituted Benzyl Matrinic Acid Derivatives Inhibit Hepatitis C Virus (HCV) Replication through Down-Regulating Host Heat-Stress Cognate 70 (Hsc70) Expression

Heat-stress cognate 70 (Hsc70) is a host factor that helps hepatitis C virus (HCV) to complete its life cycle in infected hepatocytes. Using Hsc70 as a target for HCV inhibition, a series of novel N-substituted benzyl matrinic/sophoridinic acid derivatives was synthesized and evaluated for their anti-HCV activity in vitro. Among these analogues, compound 7c possessing N-p-methylbenzyl afforded an appealing ability to inhibit HCV replication with SI value over 53. Furthermore, it showed a good oral pharmacokinetic profile with area-under-curve (AUC) of 13.4 µM·h, and a considerably good safety in oral administration in mice (LD50>1000 mg/kg). As 7c suppresses HCV replication via an action mode distinctly different from that of the marketed anti-HCV drugs, it has been selected as a new mechanism anti-HCV candidate for further investigation, with an advantage of no or decreased chance to induce drug-resistant mutations.


Introduction
Currently, hepatitis C virus (HCV) infection is a significant health problem worldwide. Standard therapy for HCV infection in clinic is the combination of pegylated-interferon with ribavirin [1]. However, this treatment regimen is only effective in about 40%250% of patients infected with HCV genotype-1, which accounts for the majority of infections in the USA, Europe and Asia [2,3]. Meanwhile, serious adverse effects such as depression and flu-like symptoms also limit its application [3,4]. Two small molecular inhibitors for HCV nonstructural protein 3/4A (NS3/ 4A) protease, telaprevir and boceprevir, were approved by the Food and Drug Administration (FDA) in 2011 [5][6][7]. The NS3/ 4A inhibitors provide more therapeutic options for clinicians. However, antiviral therapy targeting specific viral enzyme such as HCV protease causes the emergence of drug-resistant mutations [8][9][10][11]. New anti-HCV therapeutic drugs with novel mechanisms that causes no or decreased chance of inducing drug-resistance are highly desirable.
It was reported that host heat-stress cognate 70 (Hsc70) protein played an important role in the HCV replication cycle. We have demonstrated that host Hsc70 was a new drug target and mechanism against HCV [12]. Using Hsc70 as a target for HCV inhibition, we found that 12-N-p-methoxybenzyl matrinic acid (1, Figure 1) synthesized in our laboratory showed an anti-HCV activity [13]. It significantly down-regulates host Hsc70 expression at the post transcriptional level through destabilizing Hsc70 mRNA [13]. Its mode of action is distinctly different from that of the current anti-HCV drugs such as telaprevir and boceprevir [14,15]. As this target is not a viral enzyme, antiviral agents acting through this mechanism might inhibit viral replication with no or decreased chance of causing drug-resistant mutations. This unique action mode and special scaffold of compound 1 strongly provoked our curiosity to explore the structure-activity relationship (SAR), with a goal of discovering novel anti-HCV agents.
Our previous SAR results [16,17] indicated that (i) carboxyl group in 1 was considered of significant importance in downregulating Hsc70 expression; (ii) substituted benzyl might significantly enhance the activity. As the inhibition rate of intracellular HCV replication was basically consistent with activity in Hsc70 down-regulation [13,16,17], SAR analysis for the inhibiting HCV replication was conducted with 1 as the lead in the present study. We retained the butyric acid chain, and focused the SAR study on the influence of the substituents on the phenyl ring including electron-withdrawing and electron-donating, and the effect of (S)or (R)-configuration of the chiral carbon at the 5-position, respectively. On the basis of this strategy, a series of new N-substituted benzyl matrinic acid (2, 5S-configuration, Figure 1) and sophoridinic acid (3, 5R-configuration, Figure 1) derivatives [18,19] was designed and synthesized. Herein, we describe the synthesis, in vitro anti-HCV evaluation, SAR analysis, in vivo toxicity, as well as pharmacokinetics of this kind of compounds.

Chemical Synthesis
Twenty-one new target compounds were grouped into matrinic core and sophoridinic core and then synthesized as described in Figure 2 and Figure 3, respectively. The key intermediate 6 was prepared with commercially available matrine (4) as the starting material, using a three-step sequence including hydrolysis, carboxyl protection via diphenyldiazomethane [20,21] and 12-Nalkylation with the methods reported previously [17]. Using hydrochloric acid as a de-protective reagent [22], the desired products 7a-o were obtained in 6 M HCl at refluxing temperature for 0.5-1 h with yields of 50-60%.
The second synthetic route used commercially available sophoridine (8) as the starting material. The synthetic strategies shown in Figure 3 illustrated our efforts on obtaining the key intermediate 11 through the reduction reaction of acyl group on the 12-nitrogen atom, rather than 12-N-alkylation reaction. Intermediate 9 was synthesized with the methods similar to that of 5 [17], and then converted into the corresponding 12-N-acyl sophoridinic acids (10) in CH 2 Cl 2 in the presence of K 2 CO 3 . Intermediate 11 was acquired through a selective reduction of 10, in which borane dimethyl sulfide (BMS) [23,24] was used as the reductive agent and THF as the solvent. Finally, all of the final products in series 7 and 12 were purified with silica gel column chromatography using CH 2 Cl 2 /MeOH as gradient eluent.
In the 12-N-alkylation in 5 series, the N-alkyl matrinic acid 6 was obtained as expected, because the lone pair electron on the 12-nitrogen atom could easily attack at the carbonium ion in RX. As shown in Figure 4 (left), the conformation analysis of matrinic core [18,19] could be a reasonable explanation for the Nalkylation at the 12-position. However, in the 12-N-alkylation of sopharidinic core in 9 series, the N-alkylation of 9 with RX took place just on the 1-N-nitrogen atom (Figure 4), owing to the steric hindrance at the 12-position in sophoridinic series.

SAR Analysis for Anti-HCV Activity in vitro
All of the synthesized compounds were examined for their anti-HCV activity and cytotoxicity in Huh 7.5 cells using specific realtime RT-PCR assay, as described in our previous publication [13]. Anti-HCV activity was evaluated by measuring both EC 50 (for anti-HCV activity) and CC 50 (for cytotoxicity) values. As a key indication, the selectivity index (SI) was calculated as a ratio of CC 50 to EC 50 . Anti-HCV activity of the study compound was estimated by combining its EC 50 value with SI. Structures of 21 Nbenzyl matrinic/sophoridinic acid analogues and their anti-HCV effect were shown in Table 1.
SAR analysis was first focused on the influences of the substituents on the phenyl ring in 1. Replacement of p-methoxy with o-, mor p-methyl respectively gave compounds 7a-c. Compound 7c possessing p-methylbenzyl showed a 2-fold improvement in anti-HCV activity as compared to 1. Similarly, attachment of o-, mor p-fluoro at the phenyl ring resulted in compounds 7d-f. All of them exhibited a moderate activity with EC 50 between 87-176 mM, similar to or less than that of 1 (EC 50 = 118 mM). Mono or di-chloro atom(s) was added at the aromatic ring respectively, by which five compounds (7g-k) were generated and tested. Compound 7h bearing m-chlorobenzyl had slight improvement in comparison to the lead. Introduction of a pbromo led to analogue 7l, which showed a reasonable activity (EC 50 = 119 mM) similar to that of 1. The electron-withdrawing groups vinyl and OCF 3 were attached to the phenyl ring, with which compounds 7m-n were produced. The highest anti-HCV activity was seen in 7m in this series (EC 50 4.70 mM). Compound 7o possessing a naphthylmethylene lost the activity completely. The results suggested that introduction of a substituent, either  electron-withdrawing (7m) or electron-donating (7c), to the phenyl ring could significantly enhance the anti-HCV activity. The dose-response curves of compounds 7c and 7m for anti-HCV effect were shown in Figure 5.
Next, the SAR analysis was moved on the effect of (S)-or (R)configuration of the asymmetric center at the 5-position in 1, in which six new derivatives of N-benzyl sophoridinic acid (12a-f) were prepared and tested. The results showed that compounds 12b-d decreased their inhibition on HCV partially or completely, as compared to the corresponding matrinic acids (7b-d). Compounds 12a, 12e-f exhibited a moderate anti-HCV activity with SI values between 10.4 and 18.5, less than that of the corresponding matrinic acids (1, 7f, 7n) with SI ranges of 15.1 to 21.9. It appeared that the matrinic scaffold or 5S-configuration might play an important role in the antiviral activity against HCV.

Anti-HCV Effect and Mode of Action
Since compound 7c exhibited the most potent effect against HCV with SI of 53, it was selected to verify its anti-HCV effect at protein level in Huh7.5 cells. As shown in Figure 6A, compound 7c treatment (62.5 mg/mL) significantly reduced HCV NS3 level, and the strongest anti-HCV effect was seen at the concentration of 250 mg/mL. To further confirm the mode of action for the activity against HCV, down-regulation of Hsc70 expression by 7c was also examined by Western Blot. As shown in Figure 6B, compound 7c afforded activity in down-regulating Hsc70 protein expression. As the anti-HCV activity of the compounds appeared over their effect on Hsc70, other mechanisms might be involved. Furthermore, therapeutic efficacy of 7m before, at and after infection was measured as well and the results are shown in Figure 7. It appears that 7m was effective before, at and after HCV infection, supporting its host environment-related action mode. The anti-HCV effect of compound 7m was similar to that of the positive control Intron A (interferon a-2b), consistent with our previous report [13]. Compound 7c exhibited an anti-HCV pattern close to that of 7m.
In addition, HCV NS3/4A protease activity (Michaelis constant Km = 0.60 mM) in the lysates was not altered after the treatment with compound 7c or 7m at the concentration of 250 mg/mL respectively ( Figure 8A, 8B), while the positive control VX-950 showed a potent inhibitory activity on HCV RNA protease with IC 50 of 98.8 nM ( Figure 8C). Down-regulating stability of host Hsc70 mRNA seems to be the main mechanism of the compounds ( Figure 6B) [13]. Therefore, we deduced that host Hsc70 might be at least one of the key drug targets for 7c in its action against HCV. Compound 7c working through down-regulating host Hsc70 expression might inhibit HCV replication with an advantage of no or decreased chance of inducing drug-resistant mutations.

Pharmacokinetic and Safety Assessment of 7c and 7m
Among the aimed analogues, compounds 7c and 7m afforded an increased anti-HCV effect compared with 1, both of them were chosen to evaluate their in vivo mice pharmacokinetic behavior in mice model. The study compounds were given to male ICR mice via oral (ig, 25 mg/kg) route. As indicated in Table 2, the absorption was rapid for both derivatives, and the maximum concentration (C max ) in plasma after dosing was reached in 15 min and 30 min respectively. The C max of 7m was 23.2 mM, 1.8-fold of that of 7c (C max = 13.2 mM). The area under the curve (AUC) of 7m (AUC = 25.9 mM?h) from 0 to 24 h was approximately 2-fold (Table 2 and Figure 9) higher than that of 7c (AUC = 13.4 mM?h). The plasma concentration levels (C max = 23.2 mM) of 7m in mice were higher than its anti-HCV EC 50 value in vitro (4.70 mM).
Single dose toxicity tests for 7c and 7m were carried out in mice as well. After 7c or 7m was given by intragastric administration (i.g) at a dose of 250, 500 or 1000 mg/kg, the mice were closely monitored for 7 days. No mouse died in the experiment duration, indicating that the LD 50 value for 7c or 7m via oral route was over 1000 mg/kg. In addition, this treatment with 7c or 7m showed no effect on body weight of mice as well (data not shown). The results suggested that compounds 7c and 7m were considerably safe in vivo.

Conclusions
In searching for novel anti-HCV agents that work through down-regulating host Hsc70 expression, a novel series of derivatives of N-benzyl matrinic or sophoridinic acids was synthesized and evaluated for their anti-HCV activities in Huh7.5 cells with 1 as the lead. SAR revealed that matrinic acid core was considered to be the optimal core structure for anti-HCV activity. Among the newly synthesized derivatives, compound 7c exhibited a moderate inhibitory activity on HCV replication, with a novel mode of action distinctly different from the marketed anti-HCV drugs. In addition, compound 7c showed a good PK profile and high safety in mice, indicating a druggable nature of the structure. Therefore, it was selected as a new mechanism anti-HCV candidate for further development, with a potential advantage of decreasing drug-resistant mutations in virus. In addition, combination of compound 7c with currently used anti-HCV drugs might provide a new regimen to improve therapeutic efficacy and reduce adverse effects.

Chemical Methods
Reagents and apparatus. Melting point (mp) was obtained with YRT-3 melting point apparatus and uncorrected. 1 H-NMR and 13 C-NMR spectra were performed on a Varian Inova 400 MHz spectrometer (Varian, San Francisco, CA) or 500 MHz spectrometer (AV500-III, Brvker, Swiss) in CD 3 OD, with Me 4 Si as internal standard. ESI high-resolution mass spectra (HRMS) were recorded on an AutospecUItima-TOF mass spectrometer (Micromass UK Ltd, Manchester, UK). Flash chromatography was performed on CombiflashRf 200 (Teledyne, Nebraska, USA), particle size 0.038 mm. All test compounds were confirmed to be $95% pure by HPLC.
General procedure for compound 7 and 12. Compound 4 or 8 (1 equiv) was added to a solution of KOH (6 equiv) in water. The reaction mixture was refluxed for 9 h, and then stirred at room temperature overnight. The reaction solution was cooled in ice-water bath, and acidified with HCl (3 M). The solvent was removed in vacuo and the residue was sufficiently dissolved in methanol to give a corresponding solution of crude 2 or 3. A mixture of diphenylmethanonehydrazone (1.5 equiv) and electrolytic MnO 2 (1.5 equiv) in petroleum ether (boiling range 30-60uC) was refluxed for 4 h, to give a purple mixture of diphenyldiazomethane. The insoluble solid was filtered off and the clear filtrate was added into the solution of crude 2 or 3 (1 equiv) in methanol mentioned above. The reaction mixture was stirred at room temperature until the purple color disappeared, and then filtered. The resulting filtrate was evaporated under reduced pressure to dryness. The residue was washed with petroleum ether to afford crude compound 5 or 9 which was used for next step without further purification.
12-N-2-Methylbenzyl matrinic acid (7a). To the mixture of compound 5 and anhydrous K 2 CO 3 (3 equiv) in CH 2 Cl 2 was added 2-methylbenzyl bromide (1 equiv) dissolved in CH 2 Cl 2 . The reaction mixture was stirred at room temperature till the reaction was completed (checked by TLC), then filtered. The filtrate was evaporated in vacuo to give the crude product 6 as oily residue. Then compound 6 was dissolved in 6 M HCl, and the mixture was refluxed for 1 h, cooled, and 3 M KOH was added to neutralize the excessive HCl. The solution was extracted with ethyl acetate, and the aqueous layer was evaporated to dryness, and the residue was purified through flash chromatography over silica gel to give the title compound as a light brown solid. Yield: 35%, m.p 1112113uC. 1    12-N-2-Chlorobenzyl matrinic acid (7g). The title compound was obtained from 5 and 2-chlorobenzyl chloride with a procedure similar to that of 7a. Yield: 32%. Light yellow solid, m.p 1402143uC. 1   12-N-4-Methoxybenzyl sophoridinic acid (12a). To a stirred mixture of 9 and anhydrous K 2 CO 3 (3 equiv) in CH 2 Cl 2 was added -methoxyl benzoyl chloride (1 equiv) in CH 2 Cl 2. The reaction solution was stirred at room temperature till the reaction was completed (checked by TLC), and then filtered. The filtrate was evaporated in vacuo giving an oily residue 10. Then 10 was dissolved in anhydrous THF and BMS (2 M, 1.7 eq.) was added. The reaction was be stirred at room temperature for 6 h. After the solvent was evaporated, 6 M HCl was added and the reaction was refluxed for 1 h, cooled, and 3 M KOH was added to neutralize the excessive HCl. The solvent was removed in vacuo, and the residue was purified through flash chromatography over silica gel affording the title compound as a light brown solid. Yield: 16%, m.p 992101uC. 1