N-dealkylation of amines during water disinfection - Revealing a new direction in the formation of disinfection by-products.

Among numerous disinfection by-products (DBP) forming during aqueous chlorination nitrogen containing species are of special concern due to their toxicological properties. Nevertheless, corresponding reaction products of these natural and anthropogenic compounds are not sufficiently studied so far. An interesting reaction involves dealkylation of the substituted amine moiety. Here we present the results of the comparative study of one-electron oxidation and aqueous chlorination of several aliphatic and aromatic amines. The reaction products were reliably identified with gas chromatography - high resolution mass spectrometry (GC-HRMS), high pressure liquid chromatography - electrospray ionization high resolution mass spectrometry HPLC-ESI/HRMS), and electrochemistry - electrospray ionization high resolution mass spectrometry (EC-ESI/HRMS). Certain similarities dealing with the formation of the corresponding aldehydes and substitution of alkyl groups at the nitrogen atom for hydrogen were shown for the studied processes. The mechanism of the substituted amines' aqueous chlorination involving one-electron oxidation is proposed and confirmed by the array of the observed reaction products. Alternative reactions taking place in conditions of aqueous chlorination, i.e. aromatic electrophilic substitution, may successfully compete with dealkylation and produce major products.

Mild and Functional Group-Tolerant Aerobic N-Dealkylation of Tertiary Amines Promoted by Photoredox Catalysis.

This article describes the development of a mild method for the N-dealkylation of tertiary amines via photoredox catalysis and its application in late-stage functionalization. Using the developed method, more than 30 diverse aliphatic, aniline-type, and complex substrates are shown to undergo N-dealkylation, providing a method with broader functional group tolerance compared to methods found in the literature. The scope also includes tertiary and secondary amine molecules with complex substructures and drug substrates. Interestingly, α-oxidation to imines was observed in several cyclic substructures instead of N-dealkylation, suggesting that imines are relevant reaction intermediates.

N-Dealkylation of Amines.

N-dealkylation, the removal of an N-alkyl group from an amine, is an important chemical transformation which provides routes for the synthesis of a wide range of pharmaceuticals, agrochemicals, bulk and fine chemicals. N-dealkylation of amines is also an important in vivo metabolic pathway in the metabolism of xenobiotics. Identification and synthesis of drug metabolites such as N-dealkylated metabolites are necessary throughout all phases of drug development studies. In this review, different approaches for the N-dealkylation of amines including chemical, catalytic, electrochemical, photochemical and enzymatic methods will be discussed.

Cysteine Dealkylation in Bacillus subtilis by a Novel Flavin-Dependent Monooxygenase.

In this paper, we describe the biochemical reconstitution of a cysteine salvage pathway and the biochemical characterization of each of the five enzymes involved. The salvage begins with amine acetylation of S-alkylcysteine, followed by thioether oxidation. The C-S bond of the resulting sulfoxide is cleaved using a new flavoenzyme catalytic motif to give N-acetylcysteine sulfenic acid. This is then reduced to the thiol and deacetylated to complete the salvage pathway. We propose that this pathway is important in the catabolism of alkylated cysteine generated by proteolysis of alkylated glutathione formed in the detoxification of a wide range of electrophiles.

Nanoporous Gold Catalyst for the Oxidative N-Dealkylation of Drug Molecules: A Method for Synthesis of N-Dealkylated Metabolites.

A novel method for the selective catalytic N-dealkylation of drug molecules on a nanoporous gold (NPG) catalyst producing valuable N-dealkylated metabolites and intermediates is described. Drug metabolites are important chemical entities at every stage of drug discovery and development, from exploratory discovery to clinical development, providing the safety profiles and the ADME (adsorption, distribution, metabolism, and elimination) of new drug candidates. Synthesis was carried out in aqueous solution at 80 °C using air (oxygen source) as oxidant, in single step with good isolated yields. Different examples examined in this study showed that aerobic catalytic N-dealkylation of drug molecules on NPG has a broad scope supporting N-deethylation, N-deisopropylation and N-demethylation, converting either 3° amines to 2° amines, or 2° amines to 1° amines.

Elucidation of Metabolic and Disposition Pathways for Maribavir in Nonhuman Primates through Mass Balance and Semi-Physiologically Based Modeling Approaches.

Maribavir is in phase 3 clinical development for treatment of cytomegalovirus infection/disease in transplant recipients. Previous research conducted using only intact cynomolgus monkeys indicated biliary secretion as the primary elimination pathway for maribavir and that maribavir undergoes enterohepatic recirculation (EHR). To clarify the exact mechanisms of maribavir's EHR behavior, we studied its clearance pathways using intravenously administered 14C-labeled maribavir in intact and bile duct-cannulated (BDC) monkeys and constructed a semi-physiologically based pharmacokinetic (PBPK) model. Total radioactivity metabolite profiles in plasma and excreta were quantitatively determined along with plasma maribavir concentrations. Intact animals showed significantly lower clearance and longer half-lives in both total radioactivity and parent concentration in plasma than BDC monkeys. The primary in vitro and in vivo metabolic pathway for maribavir in monkey is direct glucuronidation; N-dealkylation and renal clearance are minor pathways. In BDC monkeys, 73% of dose was recovered as maribavir glucuronides in bile, and 3% of dose was recovered as parent in bile and feces; in intact animals' feces, 58% of dose was recovered as parent, and no glucuronides were detected. Therefore, EHR of maribavir occurs through biliary secretion of maribavir glucuronides, and this is followed by hydrolysis of glucuronides in the gut lumen and subsequent reabsorption of parent. A semi-PBPK model constructed from physiologic, in vitro, and in vivo BDC monkey data is capable of projecting maribavir's pharmacokinetic and EHR behavior in intact animals after intravenous or oral dosing and could be applied to modeling other xenobiotics that are subject to similar EHR processes. SIGNIFICANCE STATEMENT: Through both mass balance and semi-physiologically based pharmacokinetic (semi-PBPK) modeling approaches, this study mechanistically and quantitatively elucidates maribavir's enterohepatic recirculation (EHR) behavior in monkeys, which occurs via extensive direct glucuronidation, biliary secretion of these glucuronides, luminal hydrolysis of glucuronides to parent, and subsequent reabsorption of the parent. The study also identifies important drug- and animal-specific parameters that determine the EHR kinetics, and the semi-PBPK model is readily applicable to other drugs that undergo similar metabolic and recirculation mechanisms.

Metabolic N-Dealkylation and N-Oxidation as Elucidators of the Role of Alkylamino Moieties in Drugs Acting at Various Receptors.

Metabolic reactions that occur at alkylamino moieties may provide insight into the roles of these moieties when they are parts of drug molecules that act at different receptors. N-dealkylation of N,N-dialkylamino moieties has been associated with retaining, attenuation or loss of pharmacologic activities of metabolites compared to their parent drugs. Further, N-dealkylation has resulted in clinically used drugs, activation of prodrugs, change of receptor selectivity, and providing potential for developing fully-fledged drugs. While both secondary and tertiary alkylamino moieties (open chain aliphatic or heterocyclic) are metabolized by CYP450 isozymes oxidative N-dealkylation, only tertiary alkylamino moieties are subject to metabolic N-oxidation by Flavin-containing monooxygenase (FMO) to give N-oxide products. In this review, two aspects will be examined after surveying the metabolism of representative alkylamino-moieties-containing drugs that act at various receptors (i) the pharmacologic activities and relevant physicochemical properties (basicity and polarity) of the metabolites with respect to their parent drugs and (ii) the role of alkylamino moieties on the molecular docking of drugs in receptors. Such information is illuminative in structure-based drug design considering that fully-fledged metabolite drugs and metabolite prodrugs have been, respectively, developed from N-desalkyl and N-oxide metabolites.

A bifunctional enzyme belonging to cytochrome P450 family involved in the O-dealkylation and N-dealkoxymethylation toward chloroacetanilide herbicides in Rhodococcus sp. B2.

BACKGROUND: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown. RESULTS: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1 °C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCDB2), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthRB2), a ferredoxin reductase (EthAB2), a cytochrome P450 monooxygenase (EthBB2), a ferredoxin (EthCB2) and a 10-kDa protein of unknown function (EthDB2). Complementation with EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCDB2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthDB2 acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. CONCLUSIONS: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

White-rot fungus Phanerochaete chrysosporium metabolizes chloropyridinyl-type neonicotinoid insecticides by an N-dealkylation reaction catalyzed by two cytochrome P450s.

We previously identified a cytochrome P450 (CYP) derived from the white-rot fungus Phanerochaete chrysosporium as involved in degradation of acetamiprid, a neonicotinoid (NEO) insecticide. In the present study, we investigated biodegradation of other NEOs by P. chrysosporium, and attempted to identify the CYP enzyme responsible for NEO degradation. P. chrysosporium was able to degrade some NEOs (acetamiprid, clothianidin, imidacloprid, and thiacloprid) in nutrient-rich medium. Two CYPs in P. chrysosporium (PcCYPs), CYP5037B3 and CYP5147A3, were identified as major isozymes involved in metabolism of three neonicotinoids that have in common a chloropyridinyl moiety (acetamiprid, imidacloprid, and thiacloprid) by screening yeast that heterologously express PcCYPs. Both PcCYPs catalyzed cleavage of the chloropyridinyl moiety and side chain of the three NEOs by N-dealkylation, resulting in 6-chloro-3-pyridinemethanol and respective side chain fragments. In a culture of P. chrysosporium, 97 % and 74 % of imidacloprid and thiacloprid were modified to form degradation products, and one of these, 6-chloro-3-pyridinemethanol, was further degraded. These two PcCYPs catalyzed almost the same reaction but their substrate specificity and expression pattern are slightly different. Altogether, we found that P. chrysosporium degrades NEOs via the activity of at least two different CYP isozymes.

In vitro assessment of the photo(geno)toxicity associated with Lapatinib, a Tyrosine Kinase inhibitor.

The epidermal growth factor receptors EGFR and HER2 are the main targets for tyrosine kinase inhibitors (TKIs). The quinazoline derivative lapatinib (LAP) is used since 2007 as dual TKI in the treatment of metastatic breast cancer and currently, it is used as an oral anticancer drug for the treatment of solid tumors such as breast and lung cancer. Although hepatotoxicity is its main side effect, it makes sense to investigate the ability of LAP to induce photosensitivity reactions bearing in mind that BRAF (serine/threonine-protein kinase B-Raf) inhibitors display a considerable phototoxic potential and that afloqualone, a quinazoline-marketed drug, causes photodermatosis. Metabolic bioactivation of LAP by CYP3A4 and CYP3A5 leads to chemically reactive N-dealkylated (N-LAP) and O-dealkylated (O-LAP) derivatives. In this context, the aim of the present work is to explore whether LAP and its N- and O-dealkylated metabolites can induce photosensitivity disorders by evaluating their photo(geno)toxicity through in vitro studies, including cell viability as well as photosensitized protein and DNA damage. As a matter of fact, our work has demonstrated that not only LAP, but also its metabolite N-LAP have a clear photosensitizing potential. They are both phototoxic and photogenotoxic to cells, as revealed by the 3T3 NRU assay and the comet assay, respectively. By contrast, the O-LAP does not display relevant photobiological properties. Remarkably, the parent drug LAP shows the highest activity in membrane phototoxicity and protein oxidation, whereas N-LAP is associated with the highest photogenotoxicity, through oxidation of purine bases, as revealed by detection of 8-Oxo-dG.

Toward a systems approach to cytochrome P450 ensemble: interactions of CYP2E1 with other P450 species and their impact on CYP1A2.

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3-0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.

Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast.

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.

Investigating the in vitro metabolism of the dental resin monomers BisGMA, BisPMA, TCD-DI-HEA and UDMA using human liver microsomes and quadrupole time of flight mass spectrometry.

Dental resin systems have been in use for several decades. (Meth)acrylic monomers are an important part of the matrix system and are either based on BPA while others lack the BPA core. The degree of conversion during restoration is in general between 50-70 % allowing leaching from unreacted monomers to the oral cavity where they can be taken up through the pulp or gastrointestinal tract after ingestion with subsequent hepatic metabolism. This study identified the in vitro Phase I and Phase II metabolism of the dental resin monomers BisGMA, UDMA, BisPMA and TCD-DI-HEA, using human liver microsomes (HLM) and human liver cytosols. During Phase I incubation with HLM, the (meth)acrylic acid in the monomers was rapidly removed followed by oxidative and hydroxylation pathways. For BisPMA an O-dealkylation pathway occurred resulting in the formation of BPA. The carbamates present in TCD-DI-HEA and UDMA were resistant to biotransformation reactions. Phase II biotransformation products were only observed for BisPMA and included conjugation reactions with sulphate and glucuronic acid. In total 4, 3, 12 and 3 biotransformation products were identified in this study for BisGMA, UDMA, BisPMA and TCD-DI-HEA respectively. Possible human health effects of these biotransformation products remain unclear due to limited data availability.

Characterization of in vitro metabolism of focal adhesion kinase inhibitors by LC/MS/MS.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is critically involved in cell migration, spreading and proliferation at the early step of various cancers. Small molecule inhibitors of FAK are effective to inhibit its activation in the process of tumor formation in cell. To better understand biotransformation of FAK inhibitors, this work has investigated in vitro phase I metabolism of inhibitors (namely PF-573228, PF-562271 and PF-03814735) by rat liver microsomes model. Using liquid chromatography - quadrupole time of flight mass spectrometry and tandem mass spectrometry (LC/Q-TOF/MS and MS/MS), three metabolites of PF-573228 and PF-562271 were observed and characterized, respectively. These in vitro metabolites were reported for the first time. The structures and fragmentation patterns of these metabolites were elucidated, and phase I metabolic pathways for FAK inhibitors were proposed. The main metabolic pathways of PF-573228 were hydroxylation, dehydrogenation and N-dealkylation. For PF-562271, they were hydroxylation and dehydrogenation. Hydroxylation was observed as the primary metabolism for PF-0381473.

Demethylation of the Protein Phosphatase PP2A Promotes Demethylation of Histones to Enable Their Function as a Methyl Group Sink.

Dysregulation of chromatin methylation is associated with defects in cellular differentiation as well as a variety of cancers. How cells regulate the opposing activities of histone methyltransferase and demethylase enzymes to set the methylation status of the epigenome for proper control of gene expression and metabolism remains poorly understood. Here, we show that loss of methylation of the major phosphatase PP2A in response to methionine starvation activates the demethylation of histones through hyperphosphorylation of specific demethylase enzymes. In parallel, this regulatory mechanism enables cells to preserve SAM by increasing SAH to limit SAM consumption by methyltransferase enzymes. Mutants lacking the PP2A methyltransferase or the effector H3K36 demethylase Rph1 exhibit elevated SAM levels and are dependent on cysteine due to reduced capacity to sink the methyl groups of SAM. Therefore, PP2A directs the methylation status of histones by regulating the phosphorylation status of histone demethylase enzymes in response to SAM levels.

Kinetics and Degradation Mechanism of Adrenaline Derivative CpQ in Diluted Aqueous Solutions.

The degradation kinetics of an adrenaline (epinephrine) derivative, CpQ, was studied in solution in the pH range of 1-12 at 40-80 °C by high-performance liquid chromatography and ultraviolet-visible spectroscopy. The pH-rate profile exhibits a bell-shaped curve with two sigmoidal regions in the specific acid-catalyzed and specific base-catalyzed regions. The pH range of maximum stability was 2.5-4.5 with the main degradation pathway being the oxidative N-dealkylation of the aliphatic amino moiety followed by fast interconversion of the resulting fragments to stable degradation products. The autoxidation reaction was slower than the reaction of the oxygen reactive species. The chiral center underwent R to S racemization by a polar reaction mechanism under acidic conditions with a rate minimum at pH 4. The rates of degradation of the R and S enantiomers were similar across all pHs. CpQ degradation in the presence of hydrogen peroxide at 40 °C was significantly faster, and the extent of increases with pH. Metal ions bind to CpQ and catalyze its hydrolysis in the order Fe3+ > Fe2+ > Mg2+ > Mn2+ > Ti3+ > Sr2+ > Zn2+, with a rate enhancement of ≤1 order of magnitude at the studied pH values of 1 and 5. There was no buffer catalysis observed in the hydrolysis of the studied compound for maleate and phosphate but significant buffer catalysis in the case of citrate and malate.

In vitro and in vivo metabolic investigation of the Palbociclib by UHPLC-Q-TOF/MS/MS and in silico toxicity studies of its metabolites.

Palbociclib (PAB) is a CDK4/6 inhibitor and U. S Food and Drug Administration (FDA) granted regular approval for the treatment of hormone receptor (HR) positive, metastatic breast cancer in combination with an aromatase inhibitor in postmenopausal women. Metabolite identification is a crucial aspect during drug discovery and development as the drug metabolites may be pharmacologically active or possess toxicological activity. As there are no reports on the metabolism studies of the PAB, the present study focused on investigation of the in vitro and in vivo metabolic fate of the drug. The in vitro metabolism studies were carried out by using microsomes (HLM and RLM) and S9 fractions (Human and rat). The in vivo metabolism of the drug was studied by administration of the PAB orally to the Sprague-Dawley rats followed by analysis of urine, faeces and plasma samples. The sample preparation includes simple protein precipitation (PP) followed by solid phase extraction (SPE). The extracted samples were analyzed by ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry (UHPLC/Q-TOF/MS/MS). A total of 14 metabolites were detected in in vivo matrices. The PAB was metabolized via hydroxylation, oxidation, sulphation, N-dealkylation, acetylation and carbonylation pathways. A few of the metabolites were also detected in in vitro samples. Metabolite identification and characterization were performed by using UHPLC/Q-TOF/MS/MS in combination with HRMS data. To identify the toxicity potential of these metabolites, in silico toxicity assessment was carried out using TOPKAT and DEREK softwares.

Computationally Assessing the Bioactivation of Drugs by N-Dealkylation.

Cytochromes P450 (CYPs) oxidize alkylated amines commonly found in drugs and other biologically active molecules, cleaving them into an amine and an aldehyde. Metabolic studies usually neglect to report or investigate aldehydes, even though they can be toxic. It is assumed that they are efficiently detoxified into carboxylic acids and alcohols. Nevertheless, some aldehydes are reactive and escape detoxification pathways to cause adverse events by forming DNA and protein adducts. Herein, we modeled N-dealkylations that produce both amine and aldehyde metabolites and then predicted the reactivity of the aldehyde. This model used a deep learning approach previously developed by our group to predict other types of drug metabolism. In this study, we trained the model to predict N-dealkylation by human liver microsomes (HLM), finding that including isozyme-specific metabolism data alongside HLM data significantly improved results. The final HLM model accurately predicted the site of N-dealkylation within metabolized substrates (97% top-two and 94% area under the ROC curve). Next, we combined the metabolism, metabolite structure prediction, and previously published reactivity models into a bioactivation model. This combined model predicted the structure of the most likely reactive metabolite of a small validation set of drug-like molecules known to be bioactivated by N-dealkylation. Applying this model to approved and withdrawn medicines, we found that aldehyde metabolites produced from N-dealkylation may explain the hepatotoxicity of several drugs: indinavir, piperacillin, verapamil, and ziprasidone. Our results suggest that N-dealkylation may be an under-appreciated bioactivation pathway, especially in clinical contexts where aldehyde detoxification pathways are inhibited. Moreover, this is the first report of a bioactivation model constructed by combining a metabolism and reactivity model. These results raise hope that more comprehensive models of bioactivation are possible. The model developed in this study is available at http://swami.wustl.edu/xenosite/ .

Estimation of Circulating Drug Metabolite Exposure in Human Using In Vitro Data and Physiologically Based Pharmacokinetic Modeling: Example of a High Metabolite/Parent Drug Ratio.

(R)-4-((4-(((4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (TBPT), a serotonin-4 receptor partial agonist, is metabolized to two metabolites: an N-dealkylation product [(R)-3-(piperidin-4-ylmethoxy)-4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazole (M1)] and a cyclized oxazolidine structure [7-(((4-(((R)-tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)octahydro-3H (M2)]. After administration of TBPT to humans the exposure to M1 was low and the exposure to M2 was high, relative to the parent drug, despite this being the opposite in vitro. In this study, projection of the plasma metabolite/parent (M/P) ratios for M1 and M2 was attempted using in vitro metabolism, binding, and permeability data in static and dynamic physiologically based pharmacokinetic (PBPK) models. In the static model, the fraction of parent clearance yielding the metabolite (which also required taking into account secondary metabolites of M1 and M2), the clearance of the metabolites and parent, and an estimate of the availability of the metabolites from the liver were combined to yield estimated parent/metabolite ratios of 0.32 and 23 for M1 and M2, respectively. PBPK modeling that used in vitro and physicochemical data input yielded estimates of 0.26 and 20, respectively. The actual values were 0.12 for M1/TBPT and 58 for M2/TBPT. Thus, the ratio for M1 was overpredicted, albeit at values less than unity. The ratio for M2/TBPT was underpredicted, and the high ratio of 58 may exceed a limiting ceiling of the approach. Nevertheless, when considered in the context of determining whether a potential circulating metabolite may be quantitatively important prior to administration of a drug for the first time to humans, the approaches succeeded in highlighting the importance of M2 (M/P ratio >> 1) relative to M1, despite M1 being much greater than M2 in vitro.

Synthesis and Profiling of a Novel Potent Selective Inhibitor of CHK1 Kinase Possessing Unusual N-trifluoromethylpyrazole Pharmacophore Resistant to Metabolic N-dealkylation.

Checkpoint-mediated dependency of tumor cells can be deployed to selectively kill them without substantial toxicity to normal cells. Specifically, loss of CHK1, a serine threonine kinase involved in the surveillance of the G2-M checkpoint in the presence of replication stress inflicted by DNA-damaging drugs, has been reported to dramatically influence the viability of tumor cells. CHK1's pivotal role in maintaining genomic stability offers attractive opportunity for increasing the selectivity, effectivity, and reduced toxicity of chemotherapy. Some recently identified CHK1 inhibitors entered clinical trials in combination with DNA antimetabolites. Herein, we report synthesis and profiling of MU380, a nontrivial analogue of clinically profiled compound SCH900776 possessing the highly unusual N-trifluoromethylpyrazole motif, which was envisioned not to undergo metabolic oxidative dealkylation and thereby provide greater robustness to the compound. MU380 is a selective and potent inhibitor of CHK1 which sensitizes a variety of tumor cell lines to hydroxyurea or gemcitabine up to 10 times. MU380 shows extended inhibitory effects in cells, and unlike SCH900776, does not undergo in vivo N-dealkylation to the significantly less selective metabolite. Compared with SCH900776, MU380 in combination with GEM causes higher accumulation of DNA damage in tumor cells and subsequent enhanced cell death, and is more efficacious in the A2780 xenograft mouse model. Overall, MU380 represents a novel state-of-the-art CHK1 inhibitor with high potency, selectivity, and improved metabolic robustness to oxidative N-dealkylation. Mol Cancer Ther; 16(9); 1831-42. ©2017 AACR.