Aloperine Derivatives Research Articles

Design, synthesis and biological evaluation of aloperine derivatives as potential anticancer agents

Modifications at different positions on the aloperine molecule were performed to improve its anticancer activity and develop anticancer drugs. The in vitro anticancer activities of 44 synthesized compounds were evaluated. The effect of modification positions on anticancer activity was discussed and a structure–activity relationship analysis was established. A novel series of compounds with modifications at the N12 position showed much higher cytotoxicity than aloperine. Among them, compound 22 displayed promising in vitro anticancer activity against PC9 cells with a median inhibitory concentration (IC50) of 1.43 μM. The mechanism studies indicated that compound 22 induced cell apoptosis and cell cycle arrest in PC9 cells. These results demonstrate the potential of aloperine thiourea derivatives in anticancer activity.

Discovery and development of novel 10,12-disubstituted aloperine derivatives against HCoV-OC43 by targeting allosteric site of host TMPRSS2

By inducing steric activation of the 10CH bond with a 12-acyl group to form a key imine oxime intermediate, 20 novel (10S)-10,12-disubstituted aloperine derivatives were successfully synthesized and assessed for their antiviral efficacy against HCoV-OC43. Of them, compound 3i exhibited the moderate activities against HCoV-OC43, as well as against the SARS-CoV-2 variant EG.5.1 with the comparable EC50 values of 4.7 and 4.1 μM. A mechanism study revealed that it inhibited the protease activity of host TMPRSS2 by binding to an allosteric site, rather than the known catalytic center, different from that of camostat. Also, the combination of compound 3i and molnupiravir, as an RdRp inhibitor, showed an additive antiviral effect against HCoV-OC43. The results provide a new binding mode and lead compound for targeting TMPRSS2, with an advantage in combating broad-spectrum coronavirus.

Chemical construction and anti-HCoV-OC43 evaluation of novel 10,12-disubstituted aloperine derivatives as dual cofactor inhibitors of TMPRSS2 and SR-B1

Thirty-one new 10,12-disubstituted aloperine derivatives were subtly constructed through a selective oxidation on the 10-α-C–H induced by sulfonyl and a nucleophilic substitution with the stereoselectivity and scalability. Of them, compound 6b displayed a moderate anti-human coronavirus OC43 (HCoV-OC43) potency and blocked the viral entry stage through a host mechanism of action. Using chemoproteomic techniques, both transmembrane serine protease 2 (TMPRSS2) and scavenger receptor class B type 1 (SR-B1) proteins, which act as host cofactors of viral entry, were identified to be the direct targets of 6b against HCoV-OC43. Furthermore, 6b may deactivate the TMPRSS2 by inducing a change in protein conformation, rather than binding to its catalytic center, thus suppressing the viral membrane fusion. Accordingly, our study provided key scientific data for the development of aloperine derivatives into a new class of antiviral candidates against human β-coronavirus, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Quinolizidines as Novel SARS-CoV-2 Entry Inhibitors.

COVID-19, caused by the highly transmissible severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has rapidly spread and become a pandemic since its outbreak in 2019. We have previously discovered that aloperine is a new privileged scaffold that can be modified to become a specific antiviral compound with markedly improved potency against different viruses, such as the influenza virus. In this study, we have identified a collection of aloperine derivatives that can inhibit the entry of SARS-CoV-2 into host cells. Compound 5 is the most potent tested aloperine derivative that inhibited the entry of SARS-CoV-2 (D614G variant) spike protein-pseudotyped virus with an IC50 of 0.5 µM. The compound was also active against several other SARS-CoV-2 variants including Delta and Omicron. Results of a confocal microscopy study suggest that compound 5 inhibited the viral entry before fusion to the cell or endosomal membrane. The results are consistent with the notion that aloperine is a privileged scaffold that can be used to develop potent anti-SARS-CoV-2 entry inhibitors.

Structure–activity relationship and biological evaluation of 12 N-substituted aloperine derivatives as PD-L1 down-regulatory agents through proteasome pathway

Twenty-nine 12 N-substituted aloperine derivatives were synthesized and screened for suppression on PD-L1 expression in H460 cells, as a continuation of our work. Systematic structural modifications led to the identification of compound 6b as the most active PD-L1 modulator. Compound 6b could significantly down-regulate both constitutive and inductive PD-L1 expression in NSCLC cells, and successively enhance the cytotoxicity of co-cultured T cells against tumor cells at the concentration of 20 μM. Also, it exhibited a moderate in vivo anticancer efficacy against Lewis tumor xenograft with a stable PK and safety profile. The mechanism study indicated that 6b mediated the degradation of PD-L1 through a proteasome pathway, rather than a lysosome route. These results provided the powerful information for cancer immunotherapy of aloperine derivatives with unique endocyclic skeleton by targeting PD-L1 to activate immune cells to kill cancer cells.

Discovery and evolution of 12N-substituted aloperine derivatives as anti-SARS-CoV-2 agents through targeting late entry stage

So far, there is still no specific drug against COVID-19. Taking compound 1 with anti-EBOV activity as the lead, fifty-four 12N-substituted aloperine derivatives were synthesized and evaluated for the anti-SARS-CoV-2 activities using pseudotyped virus model. Among them, 8a exhibited the most potential effects against both pseudotyped and authentic SARS-CoV-2, as well as SARS-CoV and MERS-CoV, indicating a broad-spectrum anti-coronavirus profile. The mechanism study disclosed that 8a might block a late stage of viral entry, mainly via inhibiting host cathepsin B activity rather than directly targeting cathepsin B protein. Also, 8a could significantly reduce the release of multiple inflammatory cytokines in a time- and dose-dependent manner, such as IL-6, IL-1β, IL-8 and MCP-1, the major contributors to cytokine storm. Therefore, 8a is a promising agent with the advantages of broad-spectrum anti-coronavirus and anti-cytokine effects, thus worthy of further investigation.

Design and Synthesis of Quinolizidine Derivatives as Influenza Virus and HIV-1 Inhibitors.

We have previously reported that a quinolizidine natural product, aloperine, and its analogs can inhibit influenza virus and/or HIV-1 at low μM concentrations. The main goal of this study was to further optimize aloperine for improved anti-influenza virus activity. Structural modifications have been focused on the N12 position of aloperine scaffold. Conventional chemical synthesis was used to obtain derivatives with improved antiviral activities. The anti-HIV and anti-influenza virus activities of the synthesized compounds were determined using an MT4 cell-based HIV-1 replication assay and an anti- influenza virus infection of MDCK cell assay, respectively. Aloperine derivatives can be classified into three activity groups: those that exhibit anti-HIV activity only, anti-influenza virus only, or activity against both viruses. Aloperine optimized for potent anti-influenza activity often lost anti-HIV-1 activity, and vice versa. Compound 19 inhibited influenza virus PR8 replication with an IC50 of 0.091 μM, which is approximately 160- and 60-fold more potent than aloperine and the previously reported aloperine derivative compound 3, respectively. The data suggest that aloperine is a privileged scaffold that can be modified to become a selective antiviral compound with markedly improved potency against influenza virus or HIV-1.

Structure-Activity Relationship of Aloperine Derivatives as New Anti-Liver Fibrogenic Agents.

Twenty-seven novel 12N-substituted aloperine derivatives were synthesized and investigated for their inhibitory effects on collagen α1 (I) (COL1A1) promotor in human hepatic stellate LX-2 cells, taking aloperine (1) as the hit. A structure-activity relationship (SAR) study disclosed that the introduction of suitable substituents on the 12N atom might enhance the activity. Compound 4p exhibited a good promise on down-regulating COL1A1 expression with the IC50 value of 16.5 μM. Its inhibitory activity against COL1A1 was further confirmed on both mRNA and protein levels. Meanwhile, it effectively inhibited the expression of other fibrogenic proteins, such as transforming growth factor β1 (TGF-β1) and smooth muscle actin (α-SMA). It also exhibited good in vivo safety profile with the oral LD50 value of 400 mg kg−1 in mice. The results initiated the anti-liver fibrogenic study of aloperine derivatives, and the key compound 4p was selected as a novel lead for further investigation against liver fibrogenesis.

SA-49, a novel aloperine derivative, induces MITF-dependent lysosomal degradation of PD-L1.

BackgroundProgrammed death-ligand 1 (PD-L1) is a T-cell inhibitory checkpoint molecule that suppresses antitumor immunity. Anti-PD-L1 antibodies have shown remarkable promise in treating tumors, but the patient response rate is low. Therefore, small-molecule checkpoint inhibitors blocking PD-L1 function are urgently needed.MethodsChanges of protein expression and phosphorylation levels were determined by immunoblotting. The level of Membrane PD-L1 was examined by flow cytometer. Cytotoxicity of T cells and NK cells toward tumor cells were detected using LDH and cell index assays. Lysosome function was investigated by NAG assay. Changes in lysosomal-related genes were measured by RT-PCR. In vivo anti-NSCLC cancer effects were assessed using C57BL/6 mice bearing Lewis tumor xenografts.FindingsWe identified SA-49 as a new regulator of PD-L1 expression from a series of novel aloperine derivatives. SA-49 decreased the expression of PD-L1 in NSCLC cells and enhanced the cytotoxicity of co-cultured T and NK cells toward tumor cells. Importantly, lysosomal pathway contributed to SA-49-mediated down-regulation of PD-L1. SA-49 increased the biogenesis of lysosome and promoted translocation of PD-L1 to lysosome for proteolysis, which was associated with nuclear translocation of MITF. SA-49-induced MITF translocation acted through activation of PKCα and subsequently suppression of GSK3β activity. Furthermore, SA-49 suppressed Lewis tumor xenograft growth by activating immune microenvironment in C57BL/6 mice.InterpretationOur data demonstrate that SA-49 can be used to regulate PD-L1 in cancer cells and trigger its degradation by activating lysosome function.

SA-49, a Novel Aloperine Derivative, Induces MITF-Dependent Lysosomal Degradation of PD-L1

Programmed death-ligand 1 (PD-L1) is a T-cell inhibitory checkpoint molecule that suppresses antitumor immunity. Anti-PD-L1 antibodies have shown remarkable promise in treating tumors, but the patient response rate is low. Small-molecule checkpoint inhibitors blocking PD-L1 function are urgently needed. Here we reported that SA-49, a novel aloperine derivative, decreased the expression of PD-L1 in NSCLC cells and enhanced the cytotoxicity of co-cultured T and NK cells. Importantly, we found lysosomal pathway contributed to SA-49-mediated down-regulation of PD-L1. SA-49 increased the biogenesis of lysosome and promoted translocation of PD-L1 to lysosome for proteolysis, which was associated with nuclear translocation of MITF. SA-49-induced MITF translocation acted through activation of PKCα and subsequently suppression of GSK3β activity. Furthermore, SA-49 suppressed Lewis tumor xenograft growth by activating immune microenvironment in C57BL/6 mice. Our data demonstrate that SA-49 can be used to regulate PD-L1 in cancer cells and trigger its degradation by activating lysosome function. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (81773782, 81473248 and 81621064) and CAMS Major Collaborative Innovation Project (2016-12M-1-011). Declaration of Interests: The authors have declared no conflicts of interest. Ethics Approval Statement: The animal procedures were carried out with the approval of the Animal Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

Discovery and evolution of aloperine derivatives as novel anti-filovirus agents through targeting entry stage

Preventing filoviruses in the entry stage is an attractive antiviral strategy. Taking aloperine, a Chinese natural herb with an endocyclic skeleton, as the lead, 23 new aloperine derivatives were synthesized and evaluated for their anti-filovirus activities including ebola virus (EBOV) and marburg virus (MARV) using pseudotyped virus model. Structure-activity relationship (SAR) analysis indicated that the introduction of a 12N-dichlorobenzyl group was beneficial for the potency. Compound 2e exhibited the most potent anti-EBOV and anti-MARV effects both in vitro and in vivo. It also displayed a good pharmacokinetic and safety profile in vivo, indicating an ideal druglike feature. The primary mechanism study showed that 2e could block a late stage of viral entry, mainly through inhibiting cysteine cathepsin B activity of host components. We consider compound 2e to be a promising broad-spectrum anti-filovirus agent with the advantages of a unique chemical scaffold and a specific biological mechanism.

Discovery and evolution of aloperine derivatives as a new family of HCV inhibitors with novel mechanism

Aloperine (1), a Chinese natural product with a unique endocyclic scaffold, was first identified to be a potent hepatitis C virus (HCV) inhibitor in our laboratory. Thirty-four new aloperine derivatives were designed, synthesized and evaluated for their anti-HCV activities taking 1 as the lead. Among them, compound 7f exhibited the potential potency with EC50 values in a micromolar range against both wild-type and direct-acting antiviral agents (DAAs)-resistant variants, and synergistically inhibited HCV replication with approved DAAs. Furthermore, it also owned a good oral pharmacokinetic and safety profile, suggesting a highly druglike nature. The primary mechanism showed that 7f might target host components, distinctly different from the DAAs currently used in clinic. Therefore, we consider aloperine derivatives to be a novel class of anti-HCV agents, and compound 7f has been selected as a promising antiviral candidate for further investigation.

Structure Optimization of Aloperine Derivatives as HIV-1 Entry Inhibitors.

As a step toward developing novel anti-HIV agents, we have identified a class of quinolizidines, including aloperine, that inhibit HIV at 1-5 μM by blocking viral entry. In this study, we have optimized the structure of aloperine and derived compounds with markedly improved activity. Our structural optimization has yielded an aloperine derivative 19 with approximately a 15-fold increase in anti-HIV-1 activity. Our mechanism of action study reveals that compound 19 does not inhibit binding of HIV-1 to receptors but arrests the virus from fusion with the membrane. Binding of the compound to HIV-1gp120 might be responsible for its anti-HIV-1 entry activity.

P.8.b.002 - Prevalence and clinical correlates of intermittent explosive disorder in psychiatric outpatients

Aloperine (1), a Chinese natural product with a unique endocyclic scaffold, was first identified to be a potent hepatitis C virus (HCV) inhibitor in our laboratory. Thirty-four new aloperine derivatives were designed, synthesized and evaluated for their anti-HCV activities taking 1 as the lead. Among them, compound 7f exhibited the potential potency with EC50 values in a micromolar range against both wild-type and direct-acting antiviral agents (DAAs)-resistant variants, and synergistically inhibited HCV replication with approved DAAs. Furthermore, it also owned a good oral pharmacokinetic and safety profile, suggesting a highly druglike nature. The primary mechanism showed that 7f might target host components, distinctly different from the DAAs currently used in clinic. Therefore, we consider aloperine derivatives to be a novel class of anti-HCV agents, and compound 7f has been selected as a promising antiviral candidate for further investigation.

Chemical Modification of Plant Alkaloids. 7. T-Reaction of an Aloperine Derivative

N-(2-Carboxy-5-nitrophenyl)aloperine was prepared from aloperine and 2-fluoro-5-nitrobenzaldehyde and condensed with 1,3-dimethylbarbituric acid via a T-reaction with opening of the piperidine ring and formation of 1,3-dimethyl-5-{5-nitro-2-[3-(10-oxo-7-azatricyclo[7.3.1.02,7]trideca-11-en-12-yl)propylamino]} benzylhexahydro-2,4,6-pyrimidinetrione. The structure of the prepared compound was studied by an X-ray crystal structure analysis.

Aloperine and Its Derivatives as a New Class of HIV-1 Entry Inhibitors.

A quinolizidine-type alkaloid aloperine was found to inhibit HIV-1 infection by blocking HIV-1 entry. Aloperine inhibited HIV-1 envelope-mediated cell-cell fusion at low micromolar concentrations. To further improve the antiviral potency, more than 30 aloperine derivatives with a variety of N12-substitutions were synthesized. Among them, 12d with an N-(1-butyl)-4-trifluoromethoxy-benzamide side chain showed the most potent anti-HIV-1 activity with EC50 at 0.69 μM. Aloperine derivatives inhibited both X4 and R5 HIV-1 Env-mediated cell-cell fusions. In addition, both BMS-806, a compound representing a class of HIV-1 gp120-targeting small molecules in clinical trials, and resistant and sensitive HIV-1 Env-mediated cell-cell fusions were equally sensitive to aloperine derivatives. These results suggest that aloperine and its derivatives are a new class of anti-HIV-1 entry inhibitors.