Characterization of Divergent Metabolic Pathways in Elucidating an Unexpected, Slow-Forming, and Long Half-Life Major Metabolite of Iclepertin.

PURPOSE: After single oral dosing of the glycine reuptake transporter (GlyT1) inhibitor, iclepertin (BI 425809), a single major circulating metabolite, M530a, was identified. However, upon multiple dosing, a second major metabolite, M232, was observed with exposure levels ~ twofold higher than M530a. Studies were conducted to characterize the metabolic pathways and enzymes responsible for formation of both major human metabolites. METHODS: In vitro studies were conducted with human and recombinant enzyme sources and enzyme-selective inhibitors. The production of iclepertin metabolites was monitored by LC-MS/MS. RESULTS: Iclepertin undergoes rapid oxidation to a putative carbinolamide that spontaneously opens to an aldehyde, M528, which then undergoes reduction by carbonyl reductase to the primary alcohol, M530a. However, the carbinolamide can also undergo a much slower oxidation by CYP3A to form an unstable imide metabolite, M526, that is subsequently hydrolyzed by a plasma amidase to form M232. This difference in rate of metabolism of the carbinolamine explains why high levels of the M232 metabolite were not observed in vitro and in single dose studies in humans, but were observed in longer-term multiple dose studies. CONCLUSIONS: The long half-life iclepertin metabolite M232 is formed from a common carbinolamine intermediate, that is also a precursor of M530a. However, the formation of M232 occurs much more slowly, likely contributing to its extensive exposure in vivo. These results highlight the need to employ adequate clinical study sampling periods and rigorous characterization of unexpected metabolites, especially when such metabolites are categorized as major, thus requiring safety assessment.

Future of Biotransformation Science in the Pharmaceutical Industry.

Over the past decades, the number of scientists trained in departments dedicated to traditional medicinal chemistry, biotransformation and/or chemical toxicology have seemingly declined. Yet, there remains a strong demand for such specialized skills in the pharmaceutical industry, particularly within drug metabolism/pharmacokinetics (DMPK) departments. In this position paper, the members of the Biotransformation, Mechanisms, and Pathways Focus Group (BMPFG) steering committee reflect on the diverse roles and responsibilities of scientists trained in the biotransformation field in pharmaceutical companies and contract research organizations. The BMPFG is affiliated with the International Society for the Study of Xenobiotics (ISSX) and was specifically created to promote the exchange of ideas pertaining to topics of current and future interest involving the metabolism of xenobiotics (including drugs). The authors also delve into the relevant education and diverse training skills required to successfully nurture the future cohort of industry biotransformation scientists and guide them toward a rewarding career path. The ability of scientists with a background in biotransformation and organic chemistry to creatively solve complex drug metabolism problems encountered during research and development efforts on both small and large molecular modalities is exemplified in five relevant case studies. Finally, the authors stress the importance and continued commitment to training the next generation of biotransformation scientists who are not only experienced in the metabolism of conventional small molecule therapeutics, but are also equipped to tackle emerging challenges associated with new drug discovery modalities including peptides, protein degraders, and antibodies. SIGNIFICANCE STATEMENT: Biotransformation and mechanistic drug metabolism scientists are critical to advancing chemical entities through discovery and development, yet the number of scientists academically trained for this role is on the decline. This position paper highlights the continuing demand for biotransformation scientists and the necessity of nurturing creative ways to train them and guarantee the future growth of this field.

Novel advances in biotransformation and bioactivation research - 2020 year in review.

This annual review is the sixth of its kind since 2016 (see references). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation and bioactivation. These fields are constantly evolving with new molecular structures and discoveries of corresponding pathways for metabolism that impact relevant drug development with respect to efficacy and safety. Based on the selected articles, we created three sections: (1) drug design, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation and safety (Table 1). Unlike in years past, more biotransformation experts have joined and contributed to this effort while striving to maintain a balance of authors from academic and industry settings.[Table: see text].

A unique sequential C-S or N-S inductive cleavage and retro-Diels-Alder fragmentation mechanism for alkylsulfonyl piperidine- and piperazine-containing compounds.

RATIONALE: BI 605906 undergoes a collision-induced dissociation (CID) fragmentation resulting in the loss of methylsulfinic acid and butadiene to produce a corresponding imine. The fragmentation is hypothesized to occur via inductive cleavage of the C-S bond, generating a six-membered cyclic ene, followed by the retro-Diels-Alder (RDA) reaction. The aim of this study was to provide mechanistic evidence for the proposed fragmentation by investigating the CID spectra of BI 605906 and other alkylsulfonyl piperidine- and piperazine-containing compounds. METHODS: The positive electrospray ionization tandem mass spectrometric (ESI+ -MS/MS) fragmentations of BI 605906, D9 -BI 605906, GK02935, GK02942, ketoconazole, terazosin, and homopiperazine were investigated. Additionally, incubations of BI 605906 and GK02942 in human liver microsomes (HLM) preparations were conducted. Metabolite identification experiments were performed following these incubations to investigate corresponding in vitro metabolism. RESULTS: BI 605906, D9 -BI 605906, GK02935, and GK02942 demonstrated the same fragmentation pattern by generating a respective imine ion, supporting the hypothesized inductive cleavage and subsequent RDA mechanism. Ketoconazole and terazosin, which contain either an N-acetyl or tetrahydrofuranyl piperazine group, respectively, did not demonstrate this mechanism, notably because they do not have the alkylsulfonyl moiety as a good leaving group. Although homopiperazine contains an arylsulfonyl diazepane group, and the initial step produced an unsaturated diazepane ring, the subsequent RDA reaction was unable to proceed due to the absence of a six-membered cyclic ene intermediate. Additionally, we identified oxidative metabolites of BI 605906 and GK02942 in HLM incubations utilizing the proposed fragmentation pattern. CONCLUSIONS: In the mass spectrometer, compounds containing alkylsulfonyl piperidine or piperazine groups can undergo inductive cleavage, leading to a six-membered cyclic ene intermediate. This intermediate will then form a corresponding imine ion via the RDA reaction. A practical application of this work is to utilize this fragmentation for elucidating structures of metabolites arising from parent compounds containing alkylsulfonyl piperidine or piperazine moieties.

A new LC-MS assay for the quantitative analysis of vitamin K metabolites in human urine.

Vitamin K (VK), in both its phylloquinone and menaquinone forms, has been hypothesized to undergo ω- and ß-oxidation on its hydrophobic side chain in order to generate the observed urinary metabolites, K acid I and K acid II, which are excreted primarily as glucuronide conjugates. Synthetic standards of K acid I, K acid II, and a putative intermediate metabolite, menaquinone (MK)1 ω-COOH, were used to develop and optimize a new atmospheric pressure negative chemical ionization LC-MS/MS assay for the quantitation of these compounds in urine from untreated individuals and subjects treated with a high dose VK supplement. VK catabolites were extracted from urine, deconjugated, and converted to their methyl ester derivatives using previously reported methodology. The assay showed a high degree of sensitivity, with limits of detection below 10-50 fmol of metabolite per milliliter of urine, as well as an inter-assay precision of 8-12%. Metabolite standards provided unambiguous evidence for MK1 ω-COOH as a new human urinary metabolite of VK. This assay provides a minimally invasive, highly sensitive, and specific alternative for monitoring VK status in humans.

Influence of Stereochemistry on the Bioactivation and Glucuronidation of 4-Ipomeanol.

A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4-ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, (R)-IPO and (S)-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated (R)- and (S)-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. (R)-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of (S)-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO's bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.

N-Methylation of BI 187004 by Thiol S-Methyltransferase.

BI 187004, an 11ß-hydroxysteroid dehydrogenase 1 inhibitor, was administered once daily for 14 days to eight patients with type 2 diabetes mellitus. N-methylation was identified as a major biotransformation pathway. In four patients treated with BI 187004, the plasma exposure of an N-methylbenzimidazole metabolite [N-methylbenzimidazole regioisomer 1 (M1)] was 7-fold higher than the remaining four patients, indicating a substantial degree of metabolic variation. To identify the methyltransferase enzymes responsible for N-methylation, BI 187004 was incubated with human liver microsomes (HLM), human kidney microsomes (HKM), and their respective cytosolic preparations in the presence and absence of isoform-selective chemical inhibitors. Additionally, BI 187004 was incubated with several human recombinant methyltransferases: catechol O-methyltransferase (rhCOMT), histamine N-methyltransferase (rhHNMT), nicotinamide N-methyltransferase (rhNNMT), glycine N-methyltransferase (rhGNMT), and thiopurine S-methyltransferase (rhTPMT). M1 was principally observed in HLM and HKM incubations, minimally formed in liver and kidney cytosol, and not formed during incubations with recombinant methyltransferase enzymes. In all microsomal and cytosolic incubations, the formation of M1 was inhibited only by 2,3-dichloro-α-methylbenzylamine (DCMB), an inhibitor of thiol S-methyltransferase (TMT), providing evidence that TMT catalyzed the formation of M1. Interestingly, the N-methylbenzimidazole regioisomer (M14) was only observed in vitro, predominantly during incubations with human kidney cytosol and rhHNMT. The formation of M14 was inhibited by amodiaquine (an HNMT inhibitor) and DCMB, providing additional evidence that both HNMT and TMT catalyzed M14 formation. Overall, using BI 187004 as a substrate, this study demonstrates a novel TMT-mediated N-methylation biotransformation and an HNMT-mediated regioselective N-methylation.

Species Differences in Microsomal Oxidation and Glucuronidation of 4-Ipomeanol: Relationship to Target Organ Toxicity.

4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species.

Addressing the ADME Challenges of Compound Loss in a PDMS-Based Gut-on-Chip Microphysiological System.

Microphysiological systems (MPSs) are promising in vitro technologies for physiologically relevant predictions of the human absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates. However, polydimethylsiloxane (PDMS), a common material used in MPSs, can both adsorb and absorb small molecules, thereby compromising experimental results. This study aimed to evaluate the feasibility of using the PDMS-based Emulate gut-on-chip to determine the first-pass intestinal drug clearance. In cell-free PDMS organ-chips, we assessed the loss of 17 drugs, among which testosterone was selected as a model compound for further study based on its substantial ad- and absorptions to organ chips and its extensive first-pass intestinal metabolism with well-characterized metabolites. A gut-on-chip model consisting of epithelial Caco-2 cells and primary human umbilical vein endothelial cells (HUVECs) was established. The barrier integrity of the model was tested with reference compounds and inhibition of drug efflux. Concentration-time profiles of testosterone were measured in cell-free organ chips and in gut-on-chip models. A method to deduce the metabolic clearance was provided. Our results demonstrate that metabolic clearance can be determined with PDMS-based MPSs despite substantial compound loss to the chip. Overall, this study offers a practical protocol to experimentally assess ADME properties in PDMS-based MPSs.

Characterization of AusA: a dimodular nonribosomal peptide synthetase responsible for the production of aureusimine pyrazinones.

Aureusimines have been identified as potential virulence factors in Staphylococcus aureus. These pyrazinone secondary metabolites are produced by a nonribosomal peptide synthetase (NRPS) annotated as AusA. We report the overproduction of AusA as a 277 kDa soluble protein with A(1)-T(1)-C-A(2)-T(2)-R bimodular architecture. The substrate specificity of each adenylation (A) domain was initially probed using an ATP-pyrophosphate exchange assay with A-domain selective bisubstrate inhibitors to chemically knock out each companion A-domain. The activity of AusA was then reconstituted in vitro and shown to produce all naturally occurring aureusimines and non-natural pyrazinone products with k(cat) values ranging from 0.4 to 1.3 min(-1). Steady-state kinetic parameters were determined for all substrates and cofactors, providing the first comprehensive steady-state characterization of a NRPS employing a product formation assay. The K(M) values for the amino acids were up to 60-fold lower with the product formation assay than with the ATP-pyrophosphate exchange assay, most commonly used to assess A-domain substrate specificity. The C-terminal reductase (R) domain catalyzes reductive release of the dipeptidyl intermediate, leading to formation of an amino aldehyde that cyclizes to a dihydropyrazinone. We show oxidation to the final pyrazinone heterocycle is spontaneous. The activity and specificity of the R-domain was independently investigated using a NADPH consumption assay. AusA is a minimal autonomous two-module NRPS that represents an excellent model system for further kinetic and structural characterization.

Synthesis, pH-dependent, and plasma stability of meropenem prodrugs for potential use against drug-resistant tuberculosis.

Meropenem, a broad-spectrum parenteral ß-lactam antibiotic, in combination with clavulanate has recently shown efficacy in patients with extensively drug-resistant tuberculosis. As a result of meropenem's short half-life and lack of oral bioavailability, the development of an oral therapy is warranted for TB treatment in underserved countries where chronic parenteral therapy is impractical. To improve the oral absorption of meropenem, several alkyloxycarbonyloxyalkyl ester prodrugs with increased lipophilicity were synthesized and their stability in physiological aqueous solutions and guinea pig as well as human plasma was evaluated. The stability of prodrugs in aqueous solution at pH 6.0 and 7.4 was significantly dependent on the ester promoiety with the major degradation product identified as the parent compound meropenem. However, in simulated gastrointestinal fluid (pH 1.2) the major degradation product identified was ring-opened meropenem with the promoiety still intact, suggesting the gastrointestinal environment may reduce the absorption of meropenem prodrugs in vivo unless administered as an enteric-coated formulation. Additionally, the stability of the most aqueous stable prodrugs in guinea pig or human plasma was short, implying a rapid release of parent meropenem.

Discovery of a Novel Potent and Selective HSD17B13 Inhibitor, BI-3231, a Well-Characterized Chemical Probe Available for Open Science.

Genome-wide association studies in patients revealed HSD17B13 as a potential new target for the treatment of nonalcoholic steatohepatitis (NASH) and other liver diseases. However, the physiological function and the disease-relevant substrate of HSD17B13 remain unknown. In addition, no suitable chemical probe for HSD17B13 has been published yet. Herein, we report the identification of the novel potent and selective HSD17B13 inhibitor BI-3231. Through high-throughput screening (HTS), using estradiol as substrate, compound 1 was identified and selected for subsequent optimization resulting in compound 45 (BI-3231). In addition to the characterization of compound 45 for its functional, physicochemical, and drug metabolism and pharmacokinetic (DMPK) properties, NAD+ dependency was investigated. To support Open Science, the chemical HSD17B13 probe BI-3231 will be available to the scientific community for free via the opnMe platform, and thus can help to elucidate the pharmacology of HSD17B13.

The Absolute Bioavailability, Absorption, Distribution, Metabolism, and Excretion of BI 425809 Administered as an Oral Dose or an Oral Dose with an Intravenous Microtracer Dose of [14C]-BI 425809 in Healthy Males.

BACKGROUND AND OBJECTIVES: BI 425809, a novel glycine transporter-1 inhibitor, may ameliorate cognitive deficits in schizophrenia. The objectives of the studies were: to assess absolute bioavailability of oral BI 425809 compared with intravenous (IV) microtracer infusion (study 1), and to determine the mass balance, distribution, metabolism, and excretion of BI 425809 (study 2). METHODS: These were Phase I, open-label, non-randomized, single-period, single-arm studies in healthy males. Study 1 administered a single oral dose of unlabeled BI 425809 25 mg, then an IV microtracer infusion of [14C]-BI 425809 30 µg. In study 2, participants received an oral dose of [14C]-BI 425809 25 mg containing [14C]-labeled (dose: 3.7 megabecquerel (0.41 mSv)) and unlabeled drug. Safety was assessed. RESULTS: In study 1 (n = 6), the absolute bioavailability of a 25 mg tablet of BI 425809 in a fasted state was 71.64%. The geometric mean dose-normalized maximum plasma concentration was approximately 80% lower after oral administration versus IV dose. In study 2 (n = 6), the total recovery of [14C]-BI 425809 was 96.7%, with ~ 48% of [14C]-radioactivity excreted in urine and ~ 48% excreted in feces. Among the labeled drug in urine, ~ 45% of the amount excreted was composed of BI 425809 (17.4%) and two metabolites (BI 758790, 21.0%; BI 761036, 5.9%). In feces, < 1% of BI 425809 was excreted as unchanged drug. In both studies, BI 425809 was generally well tolerated. CONCLUSIONS: After normalization, the absolute bioavailability of tablet-form BI 425809 was 71.64%. The total recovery of [14C]-BI 425809 25 mg was high (96.7%), with low intraindividual variability and similar amounts excreted in urine and feces. CLINICALTRIALS. GOV IDENTIFIERS: NCT03783000 and NCT03654170.

Development, validation and application of a comprehensive stereoselective LC/MS-MS assay for bupropion and oxidative, reductive, and glucuronide metabolites in human urine.

A stereoselective assay was developed for the quantification of bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine. Initially, authentic glucuronide standards obtained from commercial sources were found to be incorrectly labeled with regard to stereochemistry; the correct stereochemistry was unequivocally reassigned. A trifurcated urine sample preparation and analysis procedure was employed for the stereoselective analysis of bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion enantiomers, and hydroxybupropion, erythrohydrobupropion and threohydrobupropion ß-d-glucuronide diastereomers in urine. Method 1 stereoselectively analyzed bupropion (R and S), and unconjugated free hydroxybupropion (R,R and S,S), erythrohydrobupropion (1R,2S and 1S,2R), and threohydrobupropion (1R,2R and 1S,2S) using chiral chromatography with an α1-acid glycoprotein column. Because no hydroxybupropion ß-d-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by ß-glucuronidase. Hydroxybupropion ß-d-glucuronide (R,R and S,S) urine concentrations were calculated as the difference between total and free hydroxybupropion (R,R and S,S) concentrations. Due to incomplete ß-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion ß-d-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion ß-d-glucuronide diastereomers using C18 column chromatography. All analytes were quantified by positive ion electrospray tandem mass spectrometry. The assay was fully validated over analyte-specific concentrations. Intra- and inter assay precision were within 15% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), erythrohydrobupropion (1R,2S and 1S,2R) were 10, 50, 100, and 100ng/mL, respectively. The limits of quantification for (1R,2R)-threohydrobupropion ß-d-glucuronide, (1S,2S)-threohydrobupropion ß-d-glucuronide, and (1R,2R)-erythrohydrobupropion ß-d-glucuronide were each 50ng/mL. Due to the abundance of bupropion and metabolites in human urine, no efforts were made to optimize sensitivity. All analytes were stable following freeze thaw cycles at -80°C. This assay was applicable to clinical pharmacokinetic investigations of bupropion in patients and to in vitro metabolism of the primary bupropion metabolites to their glucuronides.

Development and validation of a high-throughput stereoselective LC-MS/MS assay for bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.

A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing an α1-acid glycoprotein column within a 12-min run time. Chromatograph separation was significantly influenced by mobile phase pH and variability between columns. Analytes were quantified by positive ion electrospray tandem mass spectrometry following plasma protein precipitation with 20% trichloroacetic acid. Identification of erythrohydrobupropion enantiomer peaks and threohydrobupropion enantiomer peaks was achieved by sodium borohydride reduction of enantiopure (R)- and (S)-bupropion. Initial assay validation and sensitivity determination was on AB Sciex 3200, 4000 QTRAP, and 6500 mass spectrometers. Accuracy and precision were within 15% for each analyte. The assay was fully validated over analyte-specific concentrations using an AB Sciex 3200 mass spectrometer. Intra- and inter-assay precision and accuracy were within 12% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), and erythrohydrobupropion (1R,2S and 1S,2R) were 0.5, 2, 1, and 1ng/mL, respectively. All analytes were stable following freeze thaw cycles at -80°C and while stored at 4°C in the instrument autosampler. This method was applicable to clinical pharmacokinetic investigations of bupropion in patients. This is the first chromatographic method to resolve erythrohydrobupropion and threohydrobupropion enantiomers, and the first stereoselective LC-MS/MS assay to quantify bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.

Efficient Syntheses of Vitamin K Chain-Shortened Acid Metabolites.

Vitamin K sequentially undergoes ω-oxidation followed by successive rounds of ß-oxidation to ultimately produce two chain-shortened carboxylic acid metabolites, vitamin K acid 1 and vitamin K acid 2. Two facile syntheses of these acid metabolites are described, each starting from commercially available menadione-cyclopentadiene adduct 3. Vitamin K acid 1 was synthesized in five steps via alkylation with a geranyl halide followed by subsequent oxidation reactions, while fully retaining the trans configuration of the side chain 2',3'-double bond. Vitamin K acid 2 was synthesized in 5 steps from 3via alkylation with dimethylallyl chloride and subsequent oxidation reactions.

9-Amino acridine pharmacokinetics, brain distribution, and in vitro/in vivo efficacy against malignant glioma.

PURPOSE: The delivery of drugs to the brain is a major obstacle in the design and development of useful treatments for malignant glioma. Previous studies by our laboratory have identified a series of 9-amino acridine compounds that block the catalytic cycle of topoisomerase II resulting in apoptosis and cell death in a variety of cancer cell lines. METHODS: This study reports the in vitro and in vivo activity of two promising lead compounds, [{9-[2-(1H-Indol-3-yl)-ethylamino]-acridin-4-yl}-(4-methyl-piperazin-1-yl)-methanone (1) and [9-(1-Benzyl-piperidin-4-ylamino)-acridin-3-yl]-(4-methyl-piperazin-1-yl)-methanone] (2), using an orthotopic glioblastoma mouse model. In addition, the absorption, distribution, and metabolism properties are characterized by determining metabolic stability, MDCK accumulation, Pgp efflux transport, plasma protein binding, and brain distribution in mouse pharmacokinetic studies. RESULTS: The efficacy results indicate low micromolar ED(50) values against glioma cells and a significant increase in the survival of glioma-bearing mice dosed with (2) (p < 0.05). Pharmacokinetic data collected at time intervals following a 60 mg/kg oral dose of acridine 1 and 2 showed both compounds penetrate the blood-brain barrier yielding peak concentrations of 0.25 µM and 0.6 µM, respectively. Peak plasma concentrations were determined to be 2.25 µM (1) and 20.38 µM (2). The results were further compared with data collected using a 15 mg/kg intravenous dose of 2 which yielded a peak concentration in the brain of 1.7 µM at 2.0 h relative to a 2.04 µM peak plasma concentration. The bioavailability was calculated to be 83.8%. CONCLUSION: Taken overall, the results suggest compounds in this series may offer new strategies for the design of chemotherapeutics for treating brain cancers with high oral bioavailability and improved efficacy.

Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening.

A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure-activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition.