RESUMEN
Bruton's tyrosine kinase (BTK) is an essential node on the BCR signaling in B cells, which are clinically validated to play a critical role in B-cell lymphomas and various auto-immune diseases such as Multiple Sclerosis (MS), Pemphigus, and rheumatoid arthritis (RA). Although non-selective irreversible BTK inhibitors have been approved for oncology, due to the emergence of drug resistance in B-cell lymphoma associated with covalent inhibitor, there an unmet medical need to identify reversible, selective, potent BTK inhibitor as viable therapeutics for patients. Herein, we describe the identification of Hits and subsequence optimization to improve the physicochemical properties, potency and kinome selectivity leading to the discovery of a novel class of BTK inhibitors. Utilizing Met ID and structure base design inhibitors were synthesized with increased in vivo metabolic stability and oral exposure in rodents suitable for advancing to lead optimization.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacocinética , Agammaglobulinemia Tirosina Quinasa/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Humanos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Hypoxia-selective cytotoxins (HSCs) seek to exploit the oxygen-poor nature of tumor tissue for therapeutic gain. Typically, HSCs require activation by one-electron bioreductive enzymes such as NADPH:cytochrome P450 reductase (CYPOR). Thus, successful clinical deployment of HSCs may be facilitated by the development and implementation of diagnostic probes that detect the presence of relevant bioreductive enzymes in tumor tissue. The work described here develops analogues of the well-studied HSC tirapazamine (3-amino-1,2,4-benzotriazine 1,4-di- N-oxide, TPZ) as profluorescent substrates of the one-electron reductases involved in bioactivation of HSCs. Hypoxic metabolism of TPZ or 7-fluoro-TPZ by one-electron reductases releases inherently fluorescent mono- N-oxide metabolites that may serve as indicators, probes, markers, or stains for the detection of the enzymes involved in the bioactivation of HSCs. In particular, profluorescent compounds of this type can provide a foundation for fluorescence-based bioassays that help identify tumors responsive to HSCs.
Asunto(s)
Colorantes Fluorescentes/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Profármacos/metabolismo , Triazinas/metabolismo , Triazinas/farmacología , Hipoxia Tumoral/efectos de los fármacos , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Oxidación-Reducción , TirapazaminaRESUMEN
The 1,2,4-benzotriazine 1,4-dioxides are an important class of potential anticancer drugs that selectively kill the low-oxygen (hypoxic) cells found in solid tumors. These compounds undergo intracellular one-electron enzymatic reduction to yield an oxygen-sensitive drug radical intermediate that partitions forward, under hypoxic conditions, to generate a highly reactive secondary radical that causes cell killing DNA damage. Here, we characterized bioreductively activated, hypoxia-selective DNA-strand cleavage by 1,2,4-benzotriazine 1,4-dioxide. We found that one-electron enzymatic activation of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions in the presence of the deuterium atom donor methanol-d4 produced nondeuterated mono-N-oxide metabolites. This and the results of other isotopic labeling studies provided evidence against the generation of atom-abstracting drug radical intermediates and are consistent with a DNA-damage mechanism involving the release of hydroxyl radical from enzymatically activated 1,2,4-benzotriazine 1,4-dioxides.
Asunto(s)
Antineoplásicos/farmacología , Óxidos N-Cíclicos/farmacología , División del ADN/efectos de los fármacos , Hipoxia/metabolismo , Triazinas/farmacología , Antineoplásicos/química , Óxidos N-Cíclicos/química , Marcaje Isotópico , Estructura Molecular , Triazinas/químicaRESUMEN
Heterocyclic N-oxides are an interesting class of antitumor agents that selectively kill the hypoxic cells found in solid tumors. The hypoxia-selective activity of the lead compound in this class, tirapazamine, stems from its ability to undergo intracellular one-electron reduction to an oxygen-sensitive drug radical intermediate. In the presence of molecular oxygen, the radical intermediate is back-oxidized to the parent molecule. Under hypoxic conditions, the extended lifetime of the drug radical intermediate enables its conversion to a highly cytotoxic DNA-damaging intermediate via a "deoxygenative" mechanism involving the loss of oxygen from one of its N-oxide groups. The natural product myxin is a phenazine di-N-oxide that displays potent antibiotic activity against a variety of organisms under aerobic conditions. In light of the current view of heterocyclic N-oxides as agents that selectively operate under hypoxic conditions, it is striking that myxin was identified from Sorangium extracts based upon its antibiotic properties under aerobic conditions. Therefore, we set out to examine the molecular mechanisms underlying the biological activity of myxin. We find that myxin causes bioreductively activated, radical-mediated DNA strand cleavage under both aerobic and anaerobic conditions. Our evidence indicates that strand cleavage occurs via a deoxygenative metabolism. We show that myxin displays potent cytotoxicity against the human colorectal cancer cell line HCT-116 under both aerobic and anaerobic conditions that is comparable to the cell-killing properties of tirapazamine under anaerobic conditions. This work sheds light on the processes by which the naturally occurring aromatic N-oxide myxin gains its potent antibiotic properties under aerobic conditions. Furthermore, these studies highlight the general potential for aromatic N-oxides to undergo highly cytotoxic deoxygenative metabolism following enzymatic one-electron reduction under aerobic conditions.
Asunto(s)
Productos Biológicos/toxicidad , División del ADN , Aerobiosis , Anaerobiosis , Antineoplásicos/toxicidad , Productos Biológicos/síntesis química , Línea Celular Tumoral , Humanos , Oxidación-Reducción , Fenazinas/síntesis química , Fenazinas/toxicidad , Tirapazamina , Triazinas/toxicidadRESUMEN
Compound 1 (SNS-314) is a potent and selective Aurora kinase inhibitor that is currently in clinical trials in patients with advanced solid tumors. This communication describes the synthesis of prodrug derivatives of 1 with improved aqueous solubility profiles. In particular, phosphonooxymethyl-derived prodrug 2g has significantly enhanced solubility and is converted to the biologically active parent (1) following iv as well as po administration to rodents.
Asunto(s)
Compuestos de Fenilurea/química , Profármacos/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Tiazoles/química , Agua/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Aurora Quinasas , Masculino , Ratones , Compuestos de Fenilurea/farmacocinética , Compuestos de Fenilurea/farmacología , Profármacos/farmacocinética , Profármacos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Solubilidad , Tiazoles/farmacocinética , Tiazoles/farmacologíaRESUMEN
Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.