Stability In Human Liver Microsomes Research Articles

Coumarin-based Aldo-Keto Reductase Family 1C (AKR1C) 2 and 3 Inhibitors.

A series of 7-substituted coumarin derivatives have been characterized as pan-aldo-keto reductase family 1C (AKR1C) inhibitors. The AKR1C family of enzymes are overexpressed in numerous cancers where they are involved in drug resistance development. 7-hydroxy coumarin ethyl esters and their corresponding amides have high potency for AKR1C3 and AKR1C2 inhibition. Coumarin amide 3a possessed IC50 values of 50 nM and 90 nM for AKR1C3 and AKR1C2, respectively, and exhibits 'drug-like' metabolic stability and half-life in human and mouse liver microsomes and plasma. Compound 3a was employed as a chemical tool to determine pan-AKR1C2/3 inhibition effects both as a radiation sensitizer and as a potentiator of chemotherapy cytotoxicity. In contrast to previously reported pan-AKR1C inhibitors, 3a demonstrated no radiation sensitization effect in a radiation-resistant prostate cancer cell line model. Pan-AKR1C inhibition also did not potentiate the in vitro cytotoxicity of ABT-737, daunorubicin or dexamethasone, in two patient-derived T-cell ALL and pre-B-cell ALL cell lines. In contrast, a highly selective AKR1C3 inhibitor, compound K90, enhanced the cytotoxicity of both ABT-737 and daunorubicin in the T-cell ALL cell line model. Thus, the inhibitory profile of the AKR1C family inhibitor required to effect enhancement of chemotherapeutic cytotoxicity may be chemotherapeutic agent-specific in leukemia.

Ultra-fast UPLC–MS/MS approach for estimating X-376 in human liver microsomes: Evaluation of metabolic stability via in silico software and in vitro analysis

X-376 is a novel anaplastic lymphoma kinase (ALK) inhibitor that is capable of penetrating the blood brain barrier. This makes it suitable for use in patients with ALK-positive non-small cell lung cancer (NSCLC) who have metastases in the central nervous system. This study developed a highly sensitive, fast, eco-friendly, and reliable UPLC-MS/MS approach to quantify X-376 in human liver microsomes (HLMs). This approach was used to evaluate X-376's metabolic stability in HLMs in vitro. The UPLC-MS/MS analytical technique validation followed US-FDA bio-analytical method validation guidelines. StarDrop software, containing P450 metabolic and DEREK modules, was utilized to scan X-376's chemical structure for metabolic lability and hazardous warnings. X-376 and Encorafenib (ENF as internal standard) were resoluted on the Eclipse Plus C18 column utilizing an isocratic mobile phase method. The X-376 calibration curve was linear from 1 to 3000 ng/mL. The precision and accuracy of this study's UPLC-MS/MS approach were tested for intra- and inter-day measurements. Inter-day accuracy was −1.32% to 9.36% while intra-day accuracy was −1.5% to 10.00%. The intrinsic clearance (Clint) and in vitro half-life (t1/2) of X-376 were 59.77 mL/min/kg and 13.56 min. The high extraction ratio of X-376 supports the 50 mg twice-daily dose for ALK-positive NSCLC and CNS metastases patients. In silico software suggests that simple structural changes to the piperazine ring or group substitution in drug design may improve metabolic stability and safety compared to X-376.

Pharmacodynamic and pharmacokinetic profiles of a novel GLP-1 receptor biased agonist-SAL0112

Background and purposeGLP-1 receptor agonists are clinically utilized for type 2 diabetes and obesity. In vitro and in vivo preclinical studies were performed to assess the druggability of a novel small molecule GLP-1 receptor biased agonist SAL0112. Experimental approachThe HTRF assay, FLIPR assay, TR-FRET assay, and PathHunter assay were utilized for in vitro studies. Liver transporter tests were conducted using the HEK293-OATP1B1 and HEK293-OATP1B3 cell lines. In vitro stability assessments of various species and in vivo PK studies in rodents were performed. A model of type 2 diabetes and obesity induced by a high-energy diet in transgenic C57BL/6 mice expressing the human GLP-1 receptor gene was conducted. Principal resultsSAL0112 demonstrated high potency and selectivity in activating the Gαs pathway of the GLP-1 receptor, with no observed desensitization. SAL0112 demonstrated greater stability in human and rat liver microsomes compared to Danuglipron. In vivo PK studies revealed higher absorption of SAL0112 in rats. SAL0112 displayed a significantly lower potential for DDI on liver transporters compared to Danuglipron. SAL0112 led to significant reductions in body weight (P<0.001), blood glucose levels in OGTT (P<0.001), HbA1c (P<0.05) and improved insulin resistance (P<0.01). Notably, it increased peripheral adipocyte density and resolved hepatic steatosis. The efficacy of SAL0112 was found to be comparable to that of Danuglipron and Liraglutide. ConclusionSAL0112 demonstrated potent and selective GLP-1 receptor biased agonism, effectively alleviating signs of type 2 diabetes in a mouse model. These promising findings pave the way for the advancement of SAL0112 into clinical trials.

Prospects for Prostate Cancer Chemotherapy: Cytotoxic Evaluation and Mechanistic Insights of Quinolinequinones with ADME/PK Profile.

The evaluation of in vitro biological activity of several previously reported quinolinequinones (AQQ1-5) against 60 human cancer cell lines (NCI-60) used by the National Cancer Institute's Developmental Therapeutics Program (DTP) contributed to our earlier research on possible anticancer and/or antibacterial agents. Of interest, NCI-60 screening revealed that two quinolinequinones (AQQ1 and AQQ2) significantly reduced the proliferation of several cancer genotypes. Following the administration of a single dose and five additional doses, all quinolinequinones demonstrated a significant inhibitory effect on the growth of leukemia and other cancer cell lines. Hence, a series of subsequent in vitro biological assessments were performed to further understand the mechanistic impact of the compounds. In MTT assays, it was found that AQQ1 and AQQ2 exhibited higher efficacy against DU-145 cells (IC50 4.18 µM and 4.17 µM, respectively) compared to MDA-MB-231 (IC50 8.27 and 13.33 µM, respectively) and HCT-116 cells (IC50 5.83 and 9.18 µM, respectively). Additionally, AQQ1 demonstrated greater activity in this context. Further investigations revealed that AQQ1 inhibited DU-145 cell growth and migration dose-dependently. Remarkably, arrest of the DU-145 cell cycle at G0/G1 phase and ROS elevation were observed. Pharmacokinetic (PK) studies revealed that AQQ1 has better PK parameters than AQQ2 with %F of 9.83 in rat. Considering the data obtained with human liver microsomal stability studies, AQQ1 should have a better PK profile in human subjects. In silico studies (molecular dynamics) with three kinases (CDK2, CDK4, and MAPK) leading to cell cycle arrest at G0/G1 identified MAPK as a probable target for AQQ1. Taken together, our results showed that AQQ1 could be a potential chemotherapeutic lead molecule for prostate cancer.

Development of novel pyrimidine nucleoside analogs as potential anticancer agents: Synthesis, characterization, and In-vitro evaluation against pancreatic cancer

The present study proposed modification of 5-FU by conjugation with an acyl chloride and a 5-membered heterocyclic ring to improve its in-vitro cytotoxicity and metabolic stability. XYZ-I-71 and XYZ-I-73 were synthesized by introducing a tetrahydrofuran ring on 5-fluorocytosine (a precursor of 5-FU) and conjugation with octanoyl chloride and lauroyl chloride, respectively. The structure of the synthesized compounds was validated using NMR and micro-elemental analysis. The antiproliferative activity of the analogs was determined against MiaPaCa-2, PANC-1, and BxPC-3 pancreatic cancer cells. The analog's stability in human liver microsomes was quantified by HPLC. We found that the XYZ-I-73 (IC50 3.6 ± 0.4 μM) analog was most effective against MiaPaCa-2 cells compared to XYZ-I-71(IC50 12.3 ± 1.7 μM), GemHCl (IC50 24.2 ± 1.3 μM), Irinotecan (IC50 10.1 ± 1.5 μM) and 5-FU (IC50 13.2 ± 1.1 μM). The antiproliferative effects of this analog in Miapaca-2 cells is evident based on it having a 7-fold,3-fold, and 4-fold increased cytotoxic effect over Gem-HCl, Irinotecan, and 5-FU, respectively. On the other hand, XYZ-I-71 exhibited a 2-fold increased cytotoxic effect over Gem-HCl but a comparable cytotoxic effect to 5-FU and Irinotecan in MiaPaCa-2 cells. A similar trend of higher XYZ-I-73 inhibition was observed in PANC-1 and BxPC-3 cultures. For 48-h MiaPaCa-2 cell migration studies, XYZ-I-73 (5 μM) significantly reduced migration (# of migrated cells, 168 ± 2.9), followed by XYZ-I-71(315±2.1), Gem-HCl (762±3.1) and 5-FU (710 ± 3.2). PARP absorbance studies demonstrated significant inhibition of PARP expression of XYZ-I-73 treated cells compared to 5-FU, GemHCl, and XYZ-I-71. Further, BAX and p53 expressions were significantly increased in cells treated with XYZ-I-73 compared to 5-FU, GemHCl, and XYZ-I-71. In-vitro, metabolic stability studies showed that 80 ± 5.9% of XYZ-I-71 and XYZ-I-73 remained intact after 2 h exposure in liver microsomal solution compared to 5-FU. The XYZ-I-73 analog demonstrated a remarkable cytotoxic effect and improved in-vitro metabolic stability over the selected standard drugs and may have potential anticancer activity against pancreatic cancer.

Discovery of 2,3-Dihydro[1,4]dioxino[2,3-g]benzofuran Derivatives as Protease Activated Receptor 4 (PAR4) Antagonists with Potent Antiplatelet Aggregation Activity and Low Bleeding Tendency.

Patients with arterial embolic disease have benefited greatly from antiplatelet therapy. However, hemorrhage risk of antiplatelet agents cannot be ignored. Herein, we describe the discovery of 2,3-dihydro[1,4]dioxino[2,3-g]benzofuran compounds as novel PAR4 antagonists. Notably, the isomers 36 and 37 with the chemotype of phenoxyl methylene substituted on the 2,3-dihydro-1,4-dioxine ring exhibited potent in vitro antiplatelet activity (IC50 = 26.13 nM for 36 and 14.26 nM for 37) and significantly improved metabolic stability in human liver microsomes (T1/2 = 97.6 min for 36 and 11.1 min for BMS-986120). 36 also displayed good oral PK profiles (mice: T1/2 = 7.32 h and F = 45.11%). Both of them showed overall potent ex vivo antiplatelet activity at concentrations of 6 and 12 mg/kg, with no impact on the coagulation system and low bleeding liability. Our work will facilitate development of novel PAR4 antagonists as a safer therapeutic option for arterial embolism.

Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

N-(Heteroaryl)thiophene sulfonamides as angiotensin AT2 receptor ligands

Two series of N-(heteroaryl)thiophene sulfonamides, encompassing either a methylene imidazole group or a tert-butylimidazolylacetyl group in the meta position of the benzene ring, have been synthesized. An AT2R selective ligand with a Ki of 42 nM was identified in the first series and in the second series, six AT2R selective ligands with significantly improved binding affinities and Ki values of <5 nM were discovered. The binding modes to AT2R were explored by docking calculations combined with molecular dynamics simulations. Although some of the high affinity ligands exhibited fair stability in human liver microsomes, comparable to that observed with C21 undergoing clinical trials, most ligands displayed a very low metabolic stability with t½ of less than 10 min in human liver microsomes. The most promising ligand, with an AT2R Ki value of 4.9 nM and with intermediate stability in human hepatocytes (t½ = 77 min) caused a concentration-dependent vasorelaxation of pre-contracted mouse aorta.

The validation of new CHD1L inhibitors as a therapeutic strategy for cancer

Chromodomain helicase DNA-binding protein 1 like (CHD1L) is an oncogene that promotes tumor progression, metastasis, and multidrug resistance. CHD1L expression is indicative of poor outcomes and low survival in cancer patients with various cancer types. Herein, we report a set of CHD1L inhibitors (CHD1Li) discovered from high-throughput screening and evaluated using enzyme inhibition, 3D tumor organoid cytotoxicity and mechanistic assays. The structurally distinct compounds 8-11 emerged as hits with promising bioactivity by targeting CHD1L. CHD1Li were further examined for their stability in human and mouse liver microsomes, which showed compounds 9 and 11 to be the most metabolically stable. Additionally, molecular modeling studies of CHD1Li with the target protein shed light on key pharmacophore features driving CHD1L binding. Taken together, these results expand the chemical space of CHD1Li as a potential targeted therapy for colorectal cancer and other cancers.

Design, synthesis, and lead optimization of piperazinyl-pyrimidine analogues as potent small molecules targeting the viral capping machinery of Chikungunya virus

The worldwide re-emerge of the Chikungunya virus (CHIKV), the high morbidity associated with it, and the lack of an available vaccine or antiviral treatment make the development of a potent CHIKV-inhibitor highly desirable. Therefore, an extensive lead optimization was performed based on the previously reported CHVB compound 1b and the reported synthesis route was optimized — improving the overall yield in remarkably shorter synthesis and work-up time. Hundred analogues were designed, synthesized, and investigated for their antiviral activity, physiochemistry, and toxicological profile. An extensive structure-activity relationship study (SAR) was performed, which focused mainly on the combination of scaffold changes and revealed the key chemical features for potent anti-CHIKV inhibition. Further, a thorough ADMET investigation of the compounds was carried out: the compounds were screened for their aqueous solubility, lipophilicity, their toxicity in CaCo-2 cells, and possible hERG channel interactions. Additionally, 55 analogues were assessed for their metabolic stability in human liver microsomes (HLMs), leading to a structure-metabolism relationship study (SMR). The compounds showed an excellent safety profile, favourable physicochemical characteristics, and the required metabolic stability. A cross-resistance study confirmed the viral capping machinery (nsP1) to be the viral target of these compounds. This study identified 31b and 34 as potent, safe, and stable lead compounds for further development as selective CHIKV inhibitors. Finally, the collected insight led to a successful scaffold hop (64b) for future antiviral research studies.

HDAC specificity and kinase off-targeting by purine-benzohydroxamate anti-hematological tumor agents

A series of hybrid inhibitors, combining pharmacophores of known kinase inhibitors bearing anilino-purines (ruxolitinib, ibrutinib) and benzohydroxamate HDAC inhibitors (nexturastat A), were generated in the present study. The compounds have been synthesized and tested against solid and hematological tumor cell lines. Compounds 4d-f were the most promising in cytotoxicity assays (IC50 ≤ 50 nM) vs. hematological cells and displayed moderate activity in solid tumor models (EC50 = 9.3–21.7 μM). Compound 4d potently inhibited multiple kinase targets of interest for anticancer effects, including JAK2, JAK3, HDAC1, and HDAC6. Molecular dynamics simulations showed that 4d has stable interactions with HDAC and members of the JAK family, with differences in the hinge binding energy conferring selectivity for JAK3 and JAK2 over JAK1. The kinase inhibition profile of compounds 4d-f allows selective cytotoxicity, with minimal effects on non-tumorigenic cells. Moreover, these compounds have favorable pharmacokinetic profiles, with high stability in human liver microsomes (e.g., see t1/2: >120 min for 4f), low intrinsic clearance, and lack of significant inhibition of four major CYP450 isoforms.

Multifunctional agents against Alzheimer’s disease based on oxidative stress: Polysubstituted pyrazine derivatives synthesized by multicomponent reactions

As Alzheimer’s disease (AD) is a neurodegenerative disease with a complex pathogenesis, the exploration of multi-target drugs may be an effective strategy for AD treatment. Multifunctional small molecular agents can be obtained by connecting two or more active drugs or privileged pharmacophores by multicomponent reactions (MCRs). In this paper, two series of polysubstituted pyrazine derivatives with multifunctional moieties were designed as anti-AD agents and synthesized by Passerini-3CR and Ugi-4CR. Since the oxidative stress plays an important role in the pathological process of AD, the antioxidant activities of the newly synthesized compounds were first evaluated. Subsequently, selected active compounds were further screened in a series of AD-related bioassays, including Aβ1-42 self-aggregation and deaggregation, BACE-1 inhibition, metal chelation, and protection of SH-SY5Y cells from H2O2-induced oxidative damage. Compound A3B3C1 represented the best one with multifunctional potencies. Mechanism study showed that A3B3C1 acted on Nrf2/ARE signaling pathway, thus increasing the expression of related antioxidant proteins NQO1 and HO-1 to normal cell level. Furthermore, A3B3C1 showed good in vitro human plasma and liver microsome stability, indicating a potential for further development as multifunctional anti-AD agent.

1-Sulfonyl-3-amino-1H-1,2,4-triazoles as Yellow Fever Virus Inhibitors: Synthesis and Structure-Activity Relationship.

Yellow fever virus (YFV) transmitted by infected mosquitoes causes an acute viral disease for which there are no approved small-molecule therapeutics. Our recently developed machine learning models for YFV inhibitors led to the selection of a new pyrazolesulfonamide derivative RCB16003 with acceptable in vitro activity. We report that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class, which was recently identified as active non-nucleoside reverse transcriptase inhibitors against HIV-1, can also be repositioned as inhibitors of yellow fever virus replication. As compared to other Flaviviridae or Togaviridae family viruses tested, both compounds RCB16003 and RCB16007 demonstrate selectivity for YFV over related viruses, with only RCB16007 showing some inhibition of the West Nile virus (EC50 7.9 μM, CC50 17 μM, SI 2.2). We also describe the absorption, distribution, metabolism, and excretion (ADME) in vitro and pharmacokinetics (PK) for RCB16007 in mice. This compound had previously been shown to not inhibit hERG, and we now describe that it has good metabolic stability in mouse and human liver microsomes, low levels of CYP inhibition, high protein binding, and no indication of efflux in Caco-2 cells. A single-dose oral PK study in mice has a T1/2 of 3.4 h and Cmax of 1190 ng/mL, suggesting good availability and stability. We now propose that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class may be prioritized for in vivo efficacy testing against YFV.

Preclinical pharmacokinetic and in vitro metabolic stability study of lysosomotropic autophagy inhibitor, IITZ-01 in mice by using UPLC-MS/MS

The present study evaluates the pharmacokinetics and metabolic stability of a novel lysosomotropic autophagy inhibitor, IITZ-01 using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS-MS). It is required as this lead molecule awaits pre-clinical studies for development because of significant therapeutic outcomes in triple-negative breast cancer and renal cancer. A bioanalytical method for the quantitative determination of IITZ-01 in the plasma of mice was developed using the UPLC-MS/MS technique. The UPLC-MS/MS method was validated according to US-FDA bioanalytical guidance and successfully applied to study the pharmacokinetics and metabolic stability. Separation of IITZ- 01 and ZSTK474 (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved using Waters Acquity BEH C-18 column (50 mm × 2.1 mm, 1.7 μm). A gradient mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile was applied at a flow rate of 0.2 mL/min. Electrospray ionization was employed in positive ion mode for detection, while quantification utilized the multiple reaction monitoring (MRM) mode. This involved using [M+H]+fragment ions at m/z 483.19 → 235.09 for IITZ-01 and m/z 418 → 138 for the internal standard (IS). The method was validated over the calibration range of 0.5–800 ng/mL. The LLOQ of IITZ-01 was 0.5 ng/mL in mice plasma. The method demonstrated good in terms of intra- and inter-day precision and accuracy. The matrix effect was found to be negligible, and the stability data were within acceptable limits. The validated technique supports suitability, reliability, reproducibility, and sensitivity for the pre-clinical investigation of IITZ-01 pharmacokinetics in mice and metabolic stability in human liver microsomes.

Covalent Stapling of the Cereblon Sensor Loop Histidine Using Sulfur-Heterocycle Exchange.

Site-specific modification of amino acid residues in protein binding pockets using sulfonyl exchange chemistry expands the druggable proteome by enabling the development of covalent modulators that target residues beyond cysteine. Sulfonyl fluoride and triazole electrophiles were incorporated previously into the cereblon (CRBN) molecular glue degrader EM12, to covalently engage His353 within the CRBN sensor loop, but these probes had poor human plasma stability. Attenuation of intrinsic reactivity through the development of sulfonyl pyrazoles, imidazoles, and nucleobases enhanced plasma stability, and several compounds retained efficient labeling of His353. For example, sulfonyl imidazole EM12-SO2Im covalently blocked the CRBN binding site and possessed excellent metabolic stability in human plasma, liver microsomes, and hepatocytes. These results highlight the potential suitability of sulfonyl imidazole and related sulfur(VI)-diazole exchange (SuDEx) warheads for covalent drug development and further exemplify the therapeutic promise of site-specific histidine targeting.

Anti-methicillin-resistant Staphylococcus aureus activity and safety evaluation of 14-O-[(5-ethoxycarbonyl-4,6-dimethylpyrimidine-2-yl) thioacetyl] mutilin (EDT)

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have threated the public health worldwide, which emphasizes the urgent need for new drugs with novel mechanism of actions. 14-O-[(5-ethoxycarbonyl-4,6-dimethylpyrimidine-2-yl) thioacetyl] mutilin (EDT) is a pleuromutilin compound with high activity against several Gram-positive bacteria in vitro and in vivo. This study aimed to verifying the potential anti-MRSA activity and evaluating the safety of EDT. In in vitro antibacterial activity assays, EDT exhibited potent antibacterial activity against MRSA isolated from clinic (minimum inhibitory concentration = 0.0313–0.125 μg/mL), increased post-antibiotic effect (PAE) values and limited potential for the development of resistance. Docking model and green fluorescent protein (GFP) inhibition assay further elucidated the higher antibacterial activities of EDT via mechanism of action. In safety evaluation, EDT exhibited low cytotoxic effect and acute oral toxicity in mice and avoided to significantly increase the number of revertant colonies of six tested strains in the Ames study. Furthermore, EDT displayed a moderate inhibitory effect on CYP3A4 and moderate stability in mouse and human liver microsomes, providing a promising agent for the development of new antimicrobial candidate.

A Sensitive, Green, and Fast LC–MS/MS Analytical Method for the Quantification of Ribociclib: Evaluation of the Metabolic Stability in HLMs

Ribociclib (Kisqali®) is a pharmacological agent that has great selectivity as a cyclin-dependent kinase 4/6 inhibitor. It has received regulatory approval for its application in the treatment of breast cancer. The objective of the current study was to develop a rapid, green, highly sensitive, validated, and specific LC–MS/MS approach for the quantification of RCB in human liver microsomes (HLMs) over the linear range of 1–3000 ng/mL (LLOQ: 0.98 ng/mL). The inter- and intraday precision and accuracy exhibited values ranging from −0.31% to 3.16% and −5.67% to 5.46% correspondingly. The eco-scale technique (AGREE program) was employed to examine the environmental impact of the existing LC–MS/MS technology. The in vitro half-life and intrinsic clearance of RCB were determined to be 23.58 min and 34.39 mL/min/kg, respectively, which indicated the intermediate extraction ratio of RCB. The in silico P450 software (version 6.6) was used to confirm and validate the practical results. The metabolism of RBC was previously studied by our research group, indicating that the piperazine ring and N-dimethyl group are responsible for the metabolic instability of RCB. Drug discovery studies can be conducted taking into account this concept, allowing the development of new drugs with an enhanced safety profile and good metabolic stability.

Design, synthesis and biological evaluation of chalcone derivatives as potent and orally active hCYP3A4 inhibitors

Human cytochrome P450 3A4 (hCYP3A4), one of the most important drug-metabolizing enzymes, catalyze the metabolic clearance of ∼50% therapeutic drugs. CYP3A4 inhibitors have been used for improving the in vivo efficacy of hCYP3A4-substrate drugs. However, most of existing hCYP3A4 inhibitors may trigger serious adverse effects or undesirable effects on endogenous metabolism. This study aimed to discover potent and orally active hCYP3A4 inhibitors from chalcone derivatives and to test their anti-hCYP3A4 effects both in vitro and in vivo. Following three rounds of screening and structural optimization, the isoquinoline chalcones were found with excellently anti-hCYP3A4 effects. SAR studies showed that introducing an isoquinoline ring on the A-ring significantly enhanced anti-CYP3A4 effect, generating A10 (IC50 = 102.10 nM) as a promising lead compound. The 2nd round of SAR studies showed that introducing a substituent group at the para position of the carbonyl group on B-ring strongly improved the anti-CYP3A4 effect. As a result, C6 was identified as the most potent hCYP3A4 inhibitor (IC50 = 43.93 nM) in human liver microsomes (HLMs). C6 also displayed potent anti-hCYP3A4 effect in living cells (IC50 = 153.00 nM), which was superior to the positive inhibitor ketoconazole (IC50 = 251.00 nM). Mechanistic studies revealed that C6 could potently inhibit CYP3A4-catalyzed N-ethyl-1,8-naphthalimide (NEN) hydroxylation in a competitive manner (Ki = 30.00 nM). Moreover, C6 exhibited suitable metabolic stability in HLMs and showed good safety profiles in mice. In vivo tests demonstrated that C6 (100 mg/kg, orally administration) significantly increased the AUC(0-inf) of midazolam by 3.63-fold, and strongly prolonged its half-life by 1.66-fold compared with the vehicle group in mice. Collectively, our findings revealed the SARs of chalcone derivatives as hCYP3A4 inhibitors and offered several potent chalcone-type hCYP3A4 inhibitors, while C6 could serve as a good lead compound for developing novel, orally active CYP3A4 inhibitors with improved druglikeness properties.

Potent, Selective, and Membrane Permeable 2-Amino-4-Substituted Pyridine-Based Neuronal Nitric Oxide Synthase Inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS), based on a difluorobenzene ring linked to a 2-aminopyridine scaffold with different functionalities at the 4-position, is reported. In our efforts to develop novel nNOS inhibitors for the treatment of neurodegenerative diseases, we discovered 17, which showed excellent potency toward both rat (Ki 15 nM) and human nNOS (Ki 19 nM), with 1075-fold selectivity over human eNOS and 115-fold selectivity over human iNOS. 17 also showed excellent permeability (Pe = 13.7 × 10-6 cm s-1), a low efflux ratio (ER 0.48), along with good metabolic stability in mouse and human liver microsomes, with half-lives of 29 and >60 min, respectively. X-ray cocrystal structures of inhibitors bound with three NOS enzymes, namely, rat nNOS, human nNOS, and human eNOS, revealed detailed structure-activity relationships for the observed potency, selectivity, and permeability properties of the inhibitors.

Picomolar inhibitor of reverse transcriptase featuring significantly improved metabolic stability.

Considering the undesirable metabolic stability of our recently identified NNRTI 5 (t1/2 =96min) in human liver microsomes, we directed our efforts to improve its metabolic stability by introducing a new favorable hydroxymethyl side chain to the C-5 position of pyrimidine. This strategy provided a series of novel methylol-biphenyl-diarylpyrimidines with excellent anti-HIV-1 activity. The best compound 9g was endowed with remarkably improved metabolic stability in human liver microsomes (t1/2=2754min), which was about 29-fold longer than that of 5 (t1/2=96min). This compound conferred picomolar inhibition of WT HIV-1 (EC50=0.9nmol/L) and low nanomolar activity against five clinically drug-resistant mutant strains. It maintained particularly low cytotoxicity (CC50=264μmol/L) and good selectivity (SI=256,438). Molecular docking studies revealed that compound 9g exhibited a more stable conformation than 5 due to the newly constructed hydrogen bond of the hydroxymethyl group with E138. Also, compound 9g was characterized by good safety profiles. It displayed no apparent inhibition of CYP enzymes and hERG. The acute toxicity assay did not cause death and pathological damage in mice at a single dose of 2g/kg. These findings paved the way for the discovery and development of new-generation anti-HIV-1 drugs.