Synthesis and Evaluation of Acylated Derivatives of Hederagenin as Inhibitors of HIV-1 and HCV NS3/4A Proteases

Viral infection imposes a major threat to human health. To develop new potent antiviral agents, Hederagenin (HE), a known inhibitor of HIV-1 and HCV NS3/4A proteases, was used as a starting material to synthesize 4 types of HE derivatives, HE-3,23-diacyl, HES-3,23-diacyl, HES-3-acyl, and HES-3-oxo-23-acyl. We evaluated the in vitro inhibitory activities of the derivatives against HIV-1 and HCV NS3/4A proteases. (3β,23)-Di-O-diglutaryl-hederagenin (1b) and (3β,23)-di-O-(3′,3′-dimethylsuccinyl-hederagenin ethyl ester (2b) exhibited potent inhibitory activities against the HIV-1 and/or HCV NS3/4A proteases with IC50 values < 10 μM, but did not appreciably inhibit general human proteases renin and trypsin. The SARs showed that dicarboxylic acid hemiesters of HE significantly enhance the antiviral activities when C3 or C23 are linked with 6 carbon acyl chains.


Introduction
HIV and hepatitis C infections continue to be a major threat to human health worldwide. According to the World Health Organization, there were an estimated 37.7 million people living with HIV at the end of 2020, and hepatitis C killed about 290 000 people in 2019. 1,2 Although antiviral therapy has significantly reduced the death rate for both HIV and hepatitis C, drug resistance has emerged to be a drawback for the therapy and new classes of antiviral drugs are needed to combat the drug-resistant strains. 3 A key strategy in developing new drugs is to identify and modify bioactive compounds from natural resources.

Isolation of HE
HE was purified from flower buds of L fulvotomentosa using our previously published protocol. 21

Enzyme Assay Kits
Inhibitory activities of the acylated derivatives of HE and HES were evaluated against HIV-1 PR (Lot# AS-72028-5) using an HIV PR Assay Kit (Lot#71147-1028) and against HCV NS3/ 4A PR (Lot# AS-61017-10) using an HCV PR Assay Kit (Lot# AK 71145-1035). Both assay kits were purchased from AnaSpec Inc. (Fremont, CA, USA). The detailed experimental protocols have been previously reported. 13

Molecular Docking
To
Inhibitory Activities of HE Derivatives Against HIV-1 and HCV NS3/4A PRs Inhibitory activities of HE derivatives against HIV-1 and HCV NS3/4A PRs were measured and summarized in Table 1, with pepstatin A (PC A) and Emberlin (PC B) as the respective positive control in the HIV-1 PR and HCV NS3/4A PR assays. We towards HIV-1 PR was increased nearly 2-fold in comparison with that of 1a. This indicated that the terminal COOH group in SA and GA confers the inhibitory effect for 1a and 1b and the longer linker chain is likely to be more favorable for the inhibition of HIV-1 PR. Further studies are warranted to optimize linker size (distance between COOH and C3) for the best inhibition of HIV-1 PR. Both 1a and 1b have similar IC 50 s towards HCV NS3/4A PR, suggesting that HCV NS3/4A PR is likely to have a volume rather than a specific interaction (such as ionic interaction) requirement at C3 of HE. We observed similar patterns for the HES-3,23-diaryl derivatives.  14,19 Furthermore, compared to 2a and 2c, 2b is a stronger inhibitor with moderate inhibition of HIV-1 PR (IC 50 = 17.2 μM) and potent inhibition of HCV NS3/4A PR (IC 50 = 8.6 μM), implying that branching of the linker between the COOH group and HES might provide better binding to both proteases. We further decided to investigate the effect of 23-substitution on the antiviral activity of HE and HES. We synthesized 4a and 4b, leaving 23-OH unsubstituted. For HIV-1 PR, 4a and 4b showed similar inhibition (IC 50 of 24.5 and 26.0 μM, respectively). Towards HCV NS3/4A PR, 4a (IC 50 = 20.7 μM) is more potent than 4b (IC 50 = 37.4 μM). These observations show that the substitution of dicarboxylic acid hemiesters (such as DMS) at C23 will strengthen the antiviral activities of HE and HES derivatives.
Next, we evaluated how the COOH moiety in the 23-substituted functional groups affects the antiviral activity. A series of 23-TBS derivatives of HES was synthesized. As shown in Table 1, 3a did not show any inhibitory activity towards HIV-1 and HCV NS3/4A PRs, but 3b, 3c, and 3d exhibited moderate inhibition of both HIV-1 PR and HCV NS3/4A PR. This study suggests that a dicarboxylic acid hemiester-containing group at C23 is likely to enhance the inhibitory activities of HE and HES derivatives against HIV-1 PR, but may exhibit different effects against HCV NS3/4A PR. Thus, further studies are needed to identify the structural requirement of 23-substitution on HE or HES for potent anti-HCV PR activity.
Finally, we synthesized a series of 3-oxo-23-acyl derivatives of HES. As shown in Table 1, 5a and 5b did not inhibit either HIV-1 PR or HCV NS3/4A PR. 5c, 5d, and 5e moderately inhibited HIV-1 and HCV NS3/4A PRs, except 5c, which exhibited weak inhibition of HIV-1 PR. Thus, a dicarboxylic acid hemiester-containing group at C23 is favorable for the antiviral activity of HE, HES, and their derivatives.  consistent with the protease activity assays that showed that 2b is a more potent inhibitor of HIV-1 and HCV NS3/4A PRs than 1b. Furthermore, the molecular docking revealed that the (3β,23)-di-O-(3 ′ ,3 ′ -dimethylsuccinyl fragments of compound 2b were well buried into the binding sites of HIV-1 and HCV NS3/4A PRs via extensive hydrophobic interactions. As for the 2 FDA approved positive controls, pepstatin A formed 2 hydrogen bonds and 1 salt bridge with HIV-1 PR ( Figure 3C), and boceprevir formed 5 hydrogen bonds with HCV NS3/4A PR ( Figure 4C). Specifically for HIV-1 PR, the binding contribution comes from residues 1 to 9 and 94 to 99 [N-and C-termini], 21 to 32 [active site core], 47 to 56 [flap region], and 78 to 88 [substrate-binding region]. Resistant mutations are usually evolved from these conserved regions, especially in the flap and substrate-binding region. 24 Thus, compounds 1b and 2b might be alternatives to the current anti-HIV protease inhibitors to circumvent cross-resistance.

Conclusion
In this study, we synthesized 16 3-acyl and 23-acyl derivatives of either HE or HES. Inhibitory activity assays against HIV-1 and HCV NS3/4A PRs showed that dicarboxylic acid hemiestercontaining groups with 6 carbons at C3 and/or C23 strengthen the antiviral activity. 3,23-di-GA-HE (1b) and 3,23-di-DMS-HES (2b) could be used as lead compounds in developing new potent HE-based antiviral agents.