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.

Design, synthesis and triglyceride-lowering activity of tricyclic matrine derivatives for the intervention of non-alcoholic fatty liver disease.

Thirty new tricyclicmatrinic derivatives were successively synthesized and evaluated for their inhibitory activity on the accumulation of triglycerides (TG) in AML12 cells, using 12 N-m-trifluoromethylbenzenesulfonyl matrine (1) as the hit compound. Among the analogues, compound 7n possessing 11-trimethylbutylamine quaternary exerted the highest in vitro TG-lowering potency, as well as a good safety profile. 7n significantly attenuated the hepatic injury and steatosis, and ameliorated dyslipidemia and dysglycemia in the mice with non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet. Primary mechanism study revealed that upregulation of peroxisome proliferator-activated receptors α (PPARα)-carnitine palmitoyltransferase 1A (CPT1A) pathway mediated the efficacy of 7n. Our study provides powerful information for developing this kind of compound into a new class of anti-NAFLD candidates, and compound 7n is worthy of further investigation as an ideal lead compound.

Discovery of new riminophenazine analogues as antimycobacterial agents against drug-resistant Mycobacterium tuberculosis.

Twenty-three new riminophenazine and pyrido[3,2-b]quinoxaline derivatives were prepared and examined for their antimycobacterial activities against Mycobacterium marinum and Mycobacterium tuberculosis H37Rv, taking clofazimine (1) as the lead. Structure-activity relationship (SAR) analysis revealed that the introduction of a heterocycle or diethylamine substituted benzene moiety on the N-5 atom might be beneficial for activity. The most potent compound 7m also displayed enhanced activity against wild-type as well as multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB clinical isolates, with the MICs ranging from 0.08 to 1.25 µg/mL, especially effective toward strain M20A507, resistant to 1. Further mechanism study indicated that its anti-TB activity was independent of cell membrane disruption, but related to NDH-2 reduction and the resulting high ROS production. Our study provides instructive guidance for the further development of clofazimine derivatives into promising antimicrobial agents against MDR and XDR TB.

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.

Structure-activity relationship and biological evaluation of berberine derivatives as PCSK9 down-regulating agents.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein and its deficiency markedly enhanced the survival rate of patient with cardiovascular diseases (CVDs). Forty berberine (BBR) derivatives were synthesized and evaluated for their activities on down-regulating the transcription of PCSK9 in HepG2 cells, taking BBR as the lead. Structure-activity relationship (SAR) analysis revealed that 2,3-dimethoxy moiety might be beneficial for activity. Among them, 9k displayed the most potent activity with IC50 value of 9.5 ± 0.5 µM, better than that of BBR. Also, it significantly decreased PCSK9 protein level at cellular level, as well as in the liver and serum of mice in vivo. Furthermore, 9k markedly increased LDLR expression and LDL-C clearance via down-regulating PCSK9 protein. The mechanism of action of 9k is targeting HNF1α and/or Sp1 cluster modulation upstream of PCSK9, a different one from BBR. Therefore, 9k might have the potential to be a novel PCSK9 transcriptional inhibitor for the treatment of atherosclerosis, worthy for further investigation.

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-1ß, 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.

MPB, a novel berberine derivative, enhances lysosomal and bactericidal properties via TGF-ß-activated kinase 1-dependent activation of the transcription factor EB.

Lysosome has a crucial role in clearance of endocytosed pathogens from the cell. Small molecules that can boost lysosome function and bactericidal ability to cope with subsequent infection are urgently needed. Here, we report that MPB, a novel berberine derivative, induced lysosome-based degradation and clearance of methicillin-resistant Staphylococcus aureus and enteroinvasive Escherichia coli in macrophages. MPB caused nuclear translocation of transcription factor EB (TFEB), which boosted expression of lysosome genes. TFEB silencing repressed the MPB-mediated enhancements in degradation and bacterial eradication. MPB switched on TFEB nuclear translocation by coupling 2 parallel signaling pathways. MPB-triggered JNK activation led to 14-3-3δ being released from TFEB, which, in turn, caused TFEB nuclear translocation. In addition, MPB induced AMPK activation and subsequent inhibition of mechanistic target of rapamycin activity, which also contributed to TFEB nuclear translocation. Importantly, genetical or pharmaceutical inhibition of TGF-ß-activated kinase 1 (TAK1) reduced MPB action remarkably. MPB acted through TAK1 at lysine 158 to activate JNK and AMPK and, thus, induced TFEB-dependent bactericidal activity in macrophages. Therefore, our study reveals a novel mechanism by which MPB controls JNK and AMPK phosphorylation cascades to activate lysosomal function and bactericidal activity via TAK1 K158-dependent manner, which may offer insight into novel therapeutic strategies to control bacterial infection.-Liu, X., Zhang, N., Liu, Y., Liu, L., Zeng, Q., Yin, M., Wang, Y., Song, D., Deng, H. MPB, a novel berberine derivative, enhances lysosomal and bactericidal properties via TGF-ß-activated kinase 1-dependent activation of the transcription factor EB.

Synthesis and antibacterial evaluation against resistant Gram-negative bacteria of monobactams bearing various substituents on oxime residue.

Based on the structural characteristics of aztreonam (AZN) and its target PBP3, a series of new monobactam derivatives bearing various substituents on oxime residue were prepared and evaluated for their antibacterial activities against susceptible and resistant Gram-negative bacteria. Among them, compounds 8p and 8r displayed moderate potency with MIC values of 0.125-32 µg/mL against most tested Gram-negative strains, comparable to AZN. Meanwhile, the combination of 8p and 8r with avibactam as a ß-lactamases inhibitor, in a ratio of 1:16, showed a promising synergistic effect against both ESBLs- and NDM-1-producing K. pneumoniae, with significantly reduced MIC values up to 8-fold and >256-fold respectively. Furthermore, both of them demonstrated excellent safety profiles both in vitro and in vivo. The results provided powerful information for further structural optimization of monobactam antibiotics to fight ß-lactamase-producing resistant Gram-negative bacteria.

Synthesis and biological evaluation of berberine derivatives as a new class of broad-spectrum antiviral agents against Coxsackievirus B.

A series of novel berberine (BBR) analogues were prepared and tested for their antiviral potencies against six different genotype Coxsackievirus B (CVB1-6) strains, taking BBR core for structural modification. Structure-activity relationship (SAR) research revealed that introduction of a primary amine through a linker at position 3 might be beneficial for both antiviral activity and safety. Compound 14c displayed most promising inhibitory potency with IC50 values of 3.08-9.94 µM against tested CVBs 2-6 strains and satisfactory SI value of 34.3 on CVB3, better than that of BBR. Also, 14c could inhibit CVB3 replication through down-regulating the expression of VP1 protein and VP1 RNA. The mechanism revealed that 14c could suppress host components JNK-MAPK, ERK-MAPK and p38-MAPK activation. Therefore, BBR derivatives were considered to be a new class of anti-CVB agents with an advantage of broad-spectrum anti-CVB potency.

The novel quinolizidine derivate IMB-HDC inhibits STAT5a phosphorylation at 694 and 780 and promotes DNA breakage and cell apoptosis via blocking STAT5a nuclear translocation.

Sophoridine is a quinolizidine natural product and the exploration of its derivatives has been carried out, and the potent anticancer compound IMB-HDC was acquired. Although previous studies have revealed that some sophoridine derivatives could induce DNA breakage, the underlying mechanisms of inhibition of DNA damage repair (ATR inactivation) and the apoptosis independent of p53, have not been elucidated. Our research reveals a novel DNA response mechanism different from general DNA-damaging agents, and that sophoridine derivate inhibits the phosphorylation of Tyr694 and Ser780 of STAT5a to induce the lessened shuttle from the cytoplasm to the nucleus, and leads to the decreased nuclear STAT5a and subsequently inhibits the expression of STAT5a target gene RAD51 that contributes to the checkpoint activation, thus inhibiting ATR activation. Meanwhile, IMB-HDC that induced the diminished expression of STAT5a target gene contributes to proliferation and leads to apoptosis. More importantly, we give the first evidence that promoting the effect of Tyr694 phosphorylation on nuclear location and subsequent STAT5a target gene transcription depends on Ser780 increased or unchanged phosphorylation and was not correlated with Ser726 phosphorylation.

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 ß1 (TGF-ß1) 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.

Discovery of 9O-Substituted Palmatine Derivatives as a New Class of anti-COL1A1 Agents via Repressing TGF-ß1/Smads and JAK1/STAT3 Pathways.

Twenty 9O-substituted palmatine derivatives were prepared and tested for their biological effect against collagen α1 (I) (COL1A1) promotor in human hepatic stellate LX-2 cells. The structure-activity relationship (SAR) indicated that the introduction of a benzyl motif on the 9O atom was favorable for activity. Among them, compound 6c provided the highest inhibitory effect against COL1A1 with an IC50 value of 3.98 µM, and it also dose-dependently inhibited the expression of fibrogenic COL1A1, α-soomth muscle actin (α-SMA), matrix metalloprotein 2 (MMP2) in both mRNA and protein levels, indicating extensive inhibitory activity against fibrogenesis. A further primary mechanism study indicated that it might repress the hepatic fibrogenesis via inhibiting both canonical transforming growth factor-beta 1 (TGF-ß1)/Smads and non-canonical janus-activated kinase 1 (JAK1)/singal transducer and activator of transcription 3 (STAT3) signaling pathways. Additionally, 6c owned a high safety profile with the LD50 value of over 1000 mg·kg-1 in mice. These results identified palmatine derivatives as a novel class of anti-fibrogenic agents, and provided powerful information for further structure optimization.

Synthesis and Structure-Activity Relationship of Palmatine Derivatives as a Novel Class of Antibacterial Agents against Helicobacter pylori.

Taking palmatine (PMT) as the lead, 20 new PMT derivatives were synthesized and examined for their antibacterial activities against six tested metronidazole (MTZ)-resistant Helicobacter pylori (H. pylori) strains. The structure-activity relationship (SAR) indicated that the introduction of a suitable secondary amine substituent at the 9-position might be beneficial for potency. Among them, compound 1c exhibited the most potent activities against MTZ-resistant strains, with minimum inhibitory concentration (MIC) values of 4-16 µg/mL, better than that of the lead. It also exhibited a good safety profile with a half-lethal dose (LD50) of over 1000 mg/kg. Meanwhile, 1c might exert its antimicrobial activity through targeting H. pylori urease. These results suggested that PMT derivatives might be a new family of anti-H. pylori components.

Novel cytisine derivatives exert anti-liver fibrosis effect via PI3K/Akt/Smad pathway.

A series of new cytisine derivatives with a unique endocyclic scaffold were synthesized and evaluated for their inhibitory effect on collagen α1 (I) (COL1A1) promotor in human LX2 cells, taking cytisine as the lead. Structure-activity relationship (SAR) revealed that introducing a 12N-benzyl substitution might significantly enhance the activity. Compound 5f exhibited a promising inhibitory potency against COL1A1 with an IC50 value of 12.8 µM in human LX2 cells, and an inspiring inhibition activity against COL1A1 on both mRNA and protein levels. It also effectively inhibited the expression of α smooth muscle actin (α-SMA), connective tissue growth factor (CTGA), matrix metalloprotein 2 (MMP-2), and transforming growth factor ß1 (TGFß1), indicating an extensive inhibitory effect against fibrogenetic proteins. In addition, compound 5f displayed reasonable PK and safety profiles. The primary mechanism study indicated that it might repress the hepatic fibrogenesis via PI3K/Akt/Smad signaling pathway. The results provided powerful information for further structure optimization, and compound 5f was selected as a novel anti-liver fibrosis agent for further investigation.

Synthesis and Biological Evaluation of Fangchinoline Derivatives as Anti-Inflammatory Agents through Inactivation of Inflammasome.

Twenty eight 7-substitued fangchinoline analogues, of which twenty two were novel, were synthesized and evaluated for their effect to inhibit lipopolysaccharide/nigericin (LPS/NIG)-induced IL-1ß release at both cell and protein levels at the concentration of 5 µM. Among them, compound 6 exhibited promising inhibitory potency against IL-ß activation with an IC50 value of 3.7 µM. Preliminary mechanism study revealed that 6 might target NLRP3 protein, and then block ASC pyroptosome formation with-NLRP3, rather than acting on the activation of the NLRP3 inflammasome (NF-κB and MAPK pathways) or caspase-1 protein. Our current study supported the potential role of compound 6 against IL-ß activation, and provided powerful information for developing fangchinoline derivatives into a novel class of anti-inflammatory agents.

12N-Substituted Matrinol Derivatives Inhibited the Expression of Fibrogenic Genes via Repressing Integrin/FAK/PI3K/Akt Pathway in Hepatic Stellate Cells.

Twenty new 12N-substituted matrinol derivatives were synthesized and evaluated for their inhibitory effects on collagen α1 (I) (COL1A1) promotor in human hepatic stellate LX-2 cells. The structure-activity relationship (SAR) revealed that introducing a 12N-benzeneaminoacylmethyl substitution might significantly enhance the activity. Compound 8a exhibited the highest inhibitory potency against COL1A1, and its inhibition activity against COL1A1 was further confirmed on both the mRNA and protein levels. It also effectively inhibited the expression of α smooth muscle actin (α-SMA), fibronectin and transforming growth factor ß1 (TGFß1), indicating an extensive inhibitory effect on the expression of fibrogenic genes. The primary mechanism study indicated that it might take action via the Integrin/FAK/PI3K/Akt signaling pathway. The results provided powerful information for further structure optimization, and compound 8a was selected as a novel anti-fibrogenic lead for further investigation.

Evolution and Biological Evaluation of Matrinic Derivatives with Amantadine Fragments As New Anti-Influenza Virus Agents.

A series of novel tricyclic matrinic derivatives with 11-adamantyl substitution were designed, synthesized, and evaluated for their activities against Influenza A H3N2 virus, based on the privileged structure strategy. Structure-activity relationship (SAR) analysis indicated that the introduction of an 11-adamantyl might be helpful for the potency. Among them, compounds 9f and 9j exhibited the promising anti-H3N2 activities with IC50 values of 7.2 µM and 10.2 µM, respectively, better than that of lead 1. Their activities were further confirmed at the protein level. Moreover, compound 9f displayed a high pharmacokinetic (PK) stability profile in whole blood and a safety profile in vivo. In primary mechanism, compound 9f could inhibit the virus replication cycle at early stage by targeting M2 protein, consistent with that of the parent amantadine. This study provided powerful information for further strategic optimization to develop these compounds into a new class of anti-influenza agents.

Evolution and Antibacterial Evaluation of 8-Hydroxy-cycloberberine Derivatives as a Novel Family of Antibacterial Agents Against MRSA.

Twenty-five new derivatives of 8-hydroxycycloberberine (1) were synthesized and evaluated for their activities against Gram-positive bacteria, taking 1 as the lead. Part of them displayed satisfactory antibacterial activities against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-intermediate Staphylococcus aureus (VISA). Especially, compound 15a displayed an excellent anti-MRSA activity with MICs (minimum inhibitory concentrations) of 0.25⁻0.5 µg/mL, better than that of 1. It also displayed high stability in liver microsomes and whole blood, and the LD50 value of over 65.6 mg·kg-1 in mice via intravenous route, suggesting a good druglike feature. The mode of action showed that 15a could effectively suppress topo IV-mediated decatenation activity at the concentration of 7.5 µg/mL, through binding a different active pocket of bacterial topo IV from quinolones. Taken together, the derivatives of 1 constituted a promising kind of anti-MRSA agents with a unique chemical scaffold and a specific biological mechanism, and compound 15a has been chosen for the next investigation.

Synthesis and Biological Evaluation of Quinoline Derivatives as a Novel Class of Broad-Spectrum Antibacterial Agents.

Nineteen new quinoline derivatives were prepared via the Mannich reaction and evaluated for their antibacterial activities against both Gram-positive (G⁺) and Gram-negative (G-) bacteria, taking compound 1 as the lead. Among the target compounds, quinolone coupled hybrid 5d exerted the potential effect against most of the tested G⁺ and G- strains with MIC values of 0.125⁻8 µg/mL, much better than those of 1. Molecular-docking assay showed that compound 5d might target both bacterial LptA and Top IV proteins, thereby displaying a broad-spectrum antibacterial effect. This hybridization strategy was an efficient way to promote the antibacterial activity of this kind, and compound 5d was selected for the further investigation, with an advantage of a dual-target mechanism of action.

Construction and synthesis of tricyclic matrinic derivatives against influenza A virus by privileged structure strategy.

A series of new matrinic derivatives with an 11-adamantyl group were designed, synthesized and evaluated for their anti-influenza A H3N2 activities, based on the privileged structure strategy.SAR analysis indicated that introduction of an 11-adamantyl by ester linker might be helpful for the activity. Among them, compound 7b exhibited promising anti-H3N2 activities with IC50 value of 5.14 µM, slightly better than that of amantadine. Its activity was further confirmed at the protein level. In primary mechanism, compound 7b could inhibit virus replication cycle at early stage by targeting M2 protein, consistent with that of amantadine. This study represents a successful application of combined strategy of privileged amantadine segment for further structural optimization and development of a new class of anti-influenza agents.