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Aim: A reliable, sensitive, HPLC method was developed and validated to simultaneously quantify raloxifene (RLX) and cladrin (CLD). Method: The C18 column was used to analyze RLX and CLD at λmax 285 and 249 nm. The mobile phase was composed of acetonitrile and 35:65% v/v aqueous solution of 0.1% formic acid. Results: The method was linear over the linearity range of 0.078-20 µg/ml, and the limit of detection and limit of quantification for RLX and CLD were 0.191 and 0.228 and 0.581 and 0.69 µg/ml, respectively. Conclusion: In accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines, the developed method is precise and accurate for simultaneous estimation of RLX and CLD with applications in in vitro liver microsomal stability in mice, rabbits, dogs, monkeys and humans.

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Problems with drug ADME are responsible for many clinical failures. By understanding the ADME properties of marketed drugs and modeling how chemical structure contributes to these inherent properties, we can help new projects reduce their risk profiles. Kinetic aqueous solubility, the parallel artificial membrane permeability assay (PAMPA), and rat liver microsomal stability constitute the Tier I ADME assays at the National Center for Advancing Translational Sciences (NCATS). Using recent data generated from in-house lead optimization Tier I studies, we update quantitative structure-activity relationship (QSAR) models for these three endpoints and validate in silico performance against a set of marketed drugs (balanced accuracies range between 71% and 85%). Improved models and experimental datasets are of direct relevance to drug discovery projects and, together with the prediction services that have been made publicly available at the ADME@NCATS web portal (https://opendata.ncats.nih.gov/adme/), provide important tools for the drug discovery community. The results are discussed in light of our previously reported ADME models and state-of-the-art models from scientific literature.Graphical Abstract[Figure: see text].

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We synthesized and evaluated novel 5-[2-(thiophen-2-yl)propan-2-yl]-4H-1,2,4-triazole derivatives as 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors. Optimization of the thiophene ring and the substituents on the 1,2,4-triazole ring produced 3,4-dicyclopropyl-5-{2-[3-fluoro-5-(trifluoromethyl)thiophen-2-yl]propan-2-yl}-4H-1,2,4-triazole monohydrochloride (9a), which showed potent and selective inhibitory activity against human 11ß-HSD1. Compound 9a was also metabolically stable against human and mouse liver microsomes. Oral administration of 9a to diabetic ob/ob mice lowered corticosterone levels in adipose tissue, and thereby reduced plasma glucose and insulin levels in a dose-dependent manner.

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In our efforts to develop druggable diphenyl ethers as potential antitubercular agents, a series of novel diphenyl ether derivatives (5a-f, 6a-f) were designed and synthesized. The representative compounds showed promising in vitro activity against drug-susceptible, isoniazid-resistant, and multidrug-resistant strains of Mycobacterium tuberculosis with MIC values of 1.56 µg/ml (6b), 6.25 µg/ml (6a-d), and 3.125 µg/ml (6b-c), respectively. All the synthesized compounds exhibited satisfactory safety profile (CC50  > 300 µg/ml) against Vero and HepG2 cells. Reverse phase HPLC method was used to probe the physicochemical properties of the synthesized compounds. This series of compounds demonstrated comparatively low logP values. pKa values of representative compounds indicated that they were weak acids. Additionally, in vitro human liver microsomal stability assay confirmed that the synthesized compounds possessed acceptable stability under study conditions. The present study thus establishes compound 6b as the most promising antitubercular agent with acceptable drug-likeness.

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We have previously identified and reported several potent piperidine-derived amide inhibitors of the human soluble epoxide hydrolase (sEH) enzyme. The inhibition of this enzyme leads to elevated levels of epoxyeicosatrienoic acids (EETs), which are known to possess anti-inflammatory, vasodilatory, and anti-fibrotic effects. Herein, we report the synthesis of 9 analogs of the lead sEH inhibitor and the follow-up structure-activity relationship and liver microsome stability studies. Our findings show that isosteric modifications that lead to significant alterations in the steric and electronic properties at a specific position in the molecule can reduce the efficacy by up to 75-fold. On the other hand, substituting hydrogen with deuterium produces a notable increase (∼30%) in the molecules' half-lives in both rat and human microsomes, while maintaining sEH inhibition potency. These data highlight the utility of isosteric replacement for improving bioavailability, and the newly-synthesized inhibitor structures may thus, serve as a starting point for preclinical development. Our docking study reveals that in the catalytic pocket of sEH, these analogs are in proximity of the key amino acids involved in hydrolysis of EETs.

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LpxC inhibitors are new-type antibacterial agents developed in the last twenty years, mainly against Gram-negative bacteria infections. To develop novel LpxC inhibitors with good antibacterial activities and biological metabolism, we summarized the basic skeleton of reported LpxC inhibitors, designed and synthesized several series of compounds and tested their antibacterial activities against Escherichial coli and Pseudomonas aeruginosa in vitro. Structure-activity relationships have been discussed in this article. The metabolism stability of YDL-2, YDL-5, YDL-8, YDL-14, YDL-20-YDL-23 have been evaluated in liver microsomes, which indicated that the 2-amino isopropyl group may be a preferred structure than the 2-hydroxy ethyl group in the design of LpxC inhibitors.

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We present the first prospective application of our mouse liver microsomal (MLM) stability Bayesian model. CD117, an antitubercular thienopyrimidine tool compound that suffers from metabolic instability (MLM t1/2 < 1 min), was utilized to assess the predictive power of our new MLM stability model. The S-substituent was removed, a set of commercial reagents was utilized to construct a virtual library of 411 analogues, and our MLM stability model was applied to prioritize 13 analogues for synthesis and biological profiling. In MLM stability assays, all 13 analogues had superior metabolic stability to the parent compound, and six new analogues had acceptable MLM t1/2 values greater than or equal to 60 min. It is noteworthy that whole-cell efficacy and lack of relative mammalian cell cytotoxicity could not be predicted simultaneously. These results support the utility of our new MLM stability model in chemical tool and drug discovery optimization efforts.

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As a bioisosteric strategy to overcome the poor metabolic stability of lead compound KYS05090S, a series of new fluoro-substituted 3,4-dihydroquinazoline derivatives was prepared and evaluated for T-type calcium channel (Cav3.2) block, cytotoxic effects and liver microsomal stability. Among them, compound 8h (KCP10068F) containing 4-fluorobenzyl amide and 4-cyclohexylphenyl ring potently blocked Cav3.2 currents (>90% inhibition) at 10µM concentration and exhibited cytotoxic effect (IC50=5.9µM) in A549 non-small cell lung cancer cells that was comparable to KYS05090S. Furthermore, 8h showed approximately a 2-fold increase in liver metabolic stability in rat and human species compared to KYS05090S. Based on these overall results, 8h (KCP10068F) may therefore represent a good backup compound for KYS05090S for further biological investigations as novel cytotoxic agent. In addition, compound 8g (KCP10067F) was found to partially protect from inflammatory pain via a blockade of Cav3.2 channels.

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PURPOSE: Mouse efficacy studies are a critical hurdle to advance translational research of potential therapeutic compounds for many diseases. Although mouse liver microsomal (MLM) stability studies are not a perfect surrogate for in vivo studies of metabolic clearance, they are the initial model system used to assess metabolic stability. Consequently, we explored the development of machine learning models that can enhance the probability of identifying compounds possessing MLM stability. METHODS: Published assays on MLM half-life values were identified in PubChem, reformatted, and curated to create a training set with 894 unique small molecules. These data were used to construct machine learning models assessed with internal cross-validation, external tests with a published set of antitubercular compounds, and independent validation with an additional diverse set of 571 compounds (PubChem data on percent metabolism). RESULTS: "Pruning" out the moderately unstable / moderately stable compounds from the training set produced models with superior predictive power. Bayesian models displayed the best predictive power for identifying compounds with a half-life ≥1 h. CONCLUSIONS: Our results suggest the pruning strategy may be of general benefit to improve test set enrichment and provide machine learning models with enhanced predictive value for the MLM stability of small organic molecules. This study represents the most exhaustive study to date of using machine learning approaches with MLM data from public sources.

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Phosphoinositide 3-kinase (PI3K) is activated in various human cancer cells and well known as a cancer therapy target. We previously reported a dihydropyrrolopyrimidine derivative as a highly potent PI3K inhibitor that has strong tumor growth inhibition in a xenograft model. In this report, we describe further optimization to improve its bioavailability.

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A new series of indole analogues based on our earlier lead compound, 2-(1H-indol-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo[4,5-c]pyridine (42), was prepared as tubulin inhibitors in an effort to find a molecule with improved cytotoxic potency and metabolic stability. A series of indolyl-imidazopyridines (IIP) were synthesized and exhibited potent tubulin polymerization inhibitory activity with potent IC50 values ranging from 3 to 175 nM against a panel of human melanoma and prostate cancer cell lines. Among these compounds, the 6-indolyl compound 43 showed improved cytotoxic potency (average IC50 of 9.75 nM vs 55.75 nM) and metabolic stability in human liver microsomes (half-life time was 56.3 min vs. 45.4 min) as compared to previously reported 42. It was also shown to be effective against P-glycoprotein (P-gp) mediated multiple drug resistance (MDR) and taxol resistance.

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In our previous Letter, we reported the discovery of a novel benzoxazinyl-oxazolidinone antibacterial candidate 2. In order to identify a potential backup compound, extensive modifications on the B/C ring and C3 side chain were undertaken. A series of novel [6,6,5] tricyclic analogues were synthesized and their in vitro antibacterial activities were tested against a panel of susceptible and resistant Gram-positive pathogens. Among of them, benzothiazinyl-oxazolidinones with acetamide or thioamide as C3 side chains exhibited moderate to good antibacterial activity, such as compounds 54, 58, 59 and 63. In vitro liver microsomal stability was further evaluated and the results manifested that compounds 54 and 58 were both metabolically stable in rat and human liver microsomes. Additionally, insights gained from this investigation should provide directions for the further research of new oxazolidinone antibiotics.

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AIM: To discover novel [20(OH)D3] analogs as antiproliferative therapeutics. MATERIALS AND METHODS: We studied in vitro liver microsome stability, in vivo toxicity using mice, vitamin D receptor (VDR) translocation, in vitro antiproliferative effect, CYP enzyme metabolism. RESULTS: 20S- and 20R(OH)D3 had reasonable half-lives of 50 min and 30 min (average) respectively in liver microsomes. They were non-hypercalcemic at a high dose of 60 µg/kg. Three new 20(OH)D3 analogs were designed, synthesized and tested. They showed higher or comparable potency for inhibition of proliferation of normal keratinocytes and in the induction of VDR translocation from cytoplasm to nucleus, compared to 1,25(OH)2D3. These new analogs demonstrated different degrees of metabolism through a range of vitamin D-metabolizing CYP enzymes. CONCLUSION: Their lack of calcemic toxicity at high doses and their high biological activity suggest that this novel 20(OH)D3 scaffold may represent a promising platform for further development of therapeutically-useful agents.

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