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A series of novel thienopyrimidine-based receptor tyrosine kinase inhibitors has been discovered. Investigation of structure−activity relationships at the 5- and 6-positions of the thienopyrimidine nucleus led to a series of N,N‘-diaryl ureas that potently inhibit all of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases. A kinase insert domain-containing receptor (KDR) homology model suggests that these compounds bind to the “inactive conformation” of the enzyme with the urea portion extending into the back hydrophobic pocket adjacent to the adenosine 5‘-triphosphate (ATP) binding site. A number of compounds have been identified as displaying excellent in vivo potency. In particular, compounds 28 and 76 possess favorable pharmacokinetic (PK) profiles and demonstrate potent antitumor efficacy against the HT1080 human fibrosarcoma xenograft tumor growth model (tumor growth inhibition (TGI) = 75% at 25 mg/kg·day, per os (po)).

A novel series of sulfone N-formylhydroxylamines (retrohydroxamates) have been investigated as matrix metalloproteinases (MMP) inhibitors. The substitution of the ether linkage of ABT-770 (5) with a sulfone group 13a led to a substantial increase in activity against MMP-9 but was accompanied by a loss of selectivity for inhibition of MMP-2 and -9 over MMP-1 and diminished oral exposure. Replacement of the biphenyl P1‘ substituent with a phenoxyphenyl group provided compounds that are highly selective for inhibition of MMP-2 and -9 over MMP-1. Optimization of the substituent adjacent to the retrohydroxamate center in this series led to the clinical candidate ABT-518 (6), a highly potent, selective, orally bioavailable MMP inhibitor that has been shown to significantly inhibit tumor growth in animal cancer models.

Symmetrical bis(quinolylmethoxyphenyl)alkylcarboxylic acids were investigated as inhibitors of leukotriene biosynthesis and 4,4-bis(4-(2-quinolylmethoxy)phenyl)pentanoic acid sodium salt (47·Na) met our design parameters for a drug candidate (ABT-080). This compound was readily synthesized in three steps from commercially available diphenolic acid. Against intact human neutrophils, 47·Na inhibited ionophore-stimulated LTB4 formation with an IC50 = 20 nM. In zymosan-stimulated mouse peritoneal macrophages producing both LTC4 and PGE2, 47·Na showed 9000-fold selectivity for inhibition of LTC4 (IC50 = 0.16 nM) over PGE2 (IC50 = 1500 nM). Preliminary pharmacokinetic evaluation in rat and cynomolgus monkey demonstrated good oral bioavailability and elimination half-lives of 9 and 5 h, respectively. Pharmacological evaluation of leukotriene inhibition with oral dosing was demonstrated in a rat pleural inflammation model (ED50 = 3 mg/kg) and a rat peritoneal passive anaphylaxis model (LTB4, ED50 = 2.5 mg/kg; LTE4, ED50 = 1.0 mg/kg). In a model of airway constriction induced by antigen challenge in actively sensitized guinea pigs, 47·Na dosed orally blocked bronchoconstriction with an ED50 = 0.4 mg/kg, the most potent activity we have observed for any leukotriene inhibitor in this model. The mode of inhibitory action of 47·Na occurs at the stage of 5-lipoxygenase biosynthesis as it blocks both leukotriene pathways leading to LTB4 and LTC4 but not PGH2 biosynthesis. However, 47·Na does not inhibit 5-lipoxygenase catalysis in a broken cell enzyme assay; therefore it is likely that 47·Na acts as a FLAP inhibitor.

A novel series of heteroarylmethoxyphenylalkoxyiminoalkylcarboxylic acids was studied as leukotriene biosynthesis inhibitors. A hypothesis of structure−activity optimization by insertion of an oxime moiety was investigated using REV-5901 as a starting point. A systematic structure−activity optimization showed that the spatial arrangement and stereochemistry of the oxime insertion unit proved to be important for inhibitory activity. The promising lead, S-(E)-11, inhibited LTB4 biosynthesis in the intact human neutrophil with IC50 of 8 nM and had superior oral activity in vivo, in a rat pleurisy model (ED50 = 0.14 mg/kg) and rat anaphylaxis model (ED50 = 0.13 mg/kg). In a model of lung inflammation, S-(E)-11 blocked LTE4 biosynthesis (ED50 of 0.1 mg/kg) and eosinophil influx (ED50 of 0.2 mg/kg). S-(E)-11 (A-93178) was selected for further preclinical evaluation.

As in vitro glucuronidation assay and several biochemical assays were utilized to discover potent new N-hydroxyurea-containing 5-lipoxygenase inhibitors with long durations of action. The best of these, A-78773, is a racemic mixture of two enantiomers. These enantiomers were purified and the R(+)-enantiomer A-79175 was found to be superior to the S(-)-enantiomer with respect to in vitro metabolism and duration of action in the monkey. A-79175 was a potent selective inhibitor of 5-hydroxyeicosatetraenoic acid formation in rat basophilic leukemic homogenates (IC50 = 54 nM) and of calcium ionophore-induced leukotriene B4 (LTB4) formation in purified human polymorphonuclear leukocytes (IC50 = 25 nM) and human whole blood (IC50 = 80 nM). The compound inhibited LT formation in the rat with oral ED50s of 1 to 2 mg/kg. It also was a potent inhibitor of edema and inflammatory cell influx in rats and mice. A-79175 was resistant to glucuronidation and had an elimination half-life of nearly 9 hr in cynomolgus monkeys. A-79175 inhibited ex vivo LTB4 formation by monkeys for extended periods. A single 0.5-mg/kg oral dose gave > 50% inhibition of calcium ionophore-induced LTB4 formation ex vivo for 12 hr. A good correlation was found between the elimination half-life for A-78773 and its enantiomers in cynomolgus monkeys and humans. These data indicate that A-79175 is a promising long-acting agent that should be useful to delineate the importance of LTs in animal and human studies.

Synthetic routes were developed to access a variety of novel 1-aryl-2H,4H-tetrahydro-1,2,4-triazin-3-one analogs which were evaluated as 5-lipoxygenase (5-LO) inhibitors. The parent structure, 1-phenylperhydro-1,2,4-triazin-3-one (4), was found to be a selective inhibitor of 5-LO in broken cell, intact cell, and human blood assays with IC50 values of 5−21 μM. In a rat anaphylaxis model, 4 blocked leukotriene formation with an ED50 = 7 mg/kg when administered orally. Compound 4 exhibited selectivity for inhibition of 5-LO with little activity against related enzymes:  12-LO from human platelets, 15-LO from soybean, and cyclooxygenase (COX) from sheep seminal vesicle. In pilot subacute toxicity testing, 4 did not produce methemoglobinemia in rats (400 mg/kg po daily for 9 days) or in dogs (200 mg/kg po daily for 28 days). These results indicated that the triazinone structure provided a 5-LO inhibitor template devoid of the toxicity problems observed in the related phenidone (1) and pyridazinone (3) classes of 5-LO inhibitors. The parent compound 4 is a selective, orally bioavailable 5-LO inhibitor which can serve as a useful reference standard for in vivo pharmacological studies involving leukotriene-mediated phenonmena.

The discovery of second generation N-hydroxyurea 5-lipoxygenase inhibitors was accomplished through the development of a broad structure−activity relationship (SAR) study. This study identified requirements for improving potency and also extending duration by limiting metabolism. Potency could be maintained by the incorporation of heterocyclic templates substituted with selected lipophilic substituents. Duration of inhibition after oral administration was optimized by identification of structural features in the proximity of the N-hydroxyurea which correlated to low in vitro glucuronidation rates. Furthermore, the rate of in vitro glucuronidation was shown to be stereoselective for certain analogs. (R)-N-[3-[5-(4-Fluorophenoxy)-2-furyl]-1-methyl-2-propynyl]-N-hydroxyurea (17c) was identified and selected for clinical development.

Representative nonsteroidal anti-inflammatory drug (NSAID) cyclooxygenase inhibitors such as ibuprofen, naproxen, and indomethacin were used as orally bioavailable scaffolds to design selective 5-lipoxygenase (5-LO) inhibitors. Replacement of the NSAID carboxylic acid group with a N-hydroxyurea group provided congeners with selective 5-LO inhibitory activity.

Zileuton [N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyure] inhibited 5-hydroxyeicosatetraenoic acid synthesis by rat basophilic leukemia cell 20,000 x g supernatant and rat polymorphonuclear leukocytes (PMNL) (IC50 = 0.5 and 0.3 microM) respectively. It also inhibited leukotriene (LT)B4 biosynthesis by rat PMNL (IC50 = 0.4 microM), human PMNL (IC50 = 0.4 microM) and human whole blood (IC50 = 0.9 microM). Inhibition of human PMNL LTB4 biosynthesis was removed readily by a simple wash procedure. At concentrations up to 100 microM, the compound produced little or no inhibition of several related enzymes, such as platelet 12-lipoxygenase, soybean and rabbit reticulocyte 15-lipoxygenase and sheep seminal vesicle cyclooxygenase. At p.o. doses from 0.5 to 5 mg/kg in the dog, zileuton produced a rapid and sustained inhibition of ex vivo blood LTB4 biosynthesis which correlated with the pharmacokinetic behavior of the compound. In a similar ex vivo study in the rat, the compound displayed an p.o. ED50 of 2 mg/kg. Zileuton was highly effective in preventing 6-sulfidopeptide LT formation in the rat peritoneal cavity triggered by an antigen-antibody reaction with an ED50 of 3 mg/kg. In experimental models of inflammation, zileuton significantly reduced arachidonic-acid induced mouse ear edema (ED50 = 31 mg/kg) and also attenuated inflammatory cell accumulation in the rat pleural Arthus reaction. The effectiveness of this compound for preventing LT formation in vitro, ex vivo and in vivo suggests its utility for preventing the pathophysiological effects of the LTs and other 5-lipoxygenase products in animals and in humans.

Our primary goal has been to discover leukotriene biosynthesis inhibitors with characteristics that are appropriate for use as clinical agents. The success of the use of zileuton in the treatment of asthma led us to explore further the use of the N-hydroxyurea class of 5-lipoxygenase inhibitors as longer-acting compounds with good lung penetration. A variety of in vitro and in vivo methods were used to evaluate a large number of compounds, from which ABT-761 [(R)-N-(3-(5-(4-fluorophenylmethyl)thien-2-yl)-1-methyl-2-pr opynyl)-N-hydroxyurea] was selected for study. ABT-761 exhibited potent and selective inhibition of leukotriene formation both in vitro and in vivo. More importantly, the compound potently inhibited antigen-induced bronchospasm in guinea pigs when given either prophylactically or therapeutically. In addition, ABT-761 was a potent inhibitor of eosinophil influx into the lungs of Brown Norway rats. These data provide added support for the role of leukotrienes in both bronchospasm and eosinophilic inflammation and characterize ABT-761 as a particularly potent inhibitor of leukotrienes formed in pulmonary tissues. These data combined with the excellent pharmacokinetic characteristics of the compound indicate its potential use in the treatment of leukotriene-dependent human disease.

An in vitro glucuronidation assay was used to optimize a series of N-hydroxyurea-containing 5-lipoxygenase inhibitors for metabolic stability. The glucuronidation of these compounds in cynomolgus monkey microsomes followed Michaelis-Menten kinetics allowing calculation of V(max) and K(M). The V(max) values ranged from 0.02 to 7.9 nmol/min/mg microsomal protein, a 400-fold difference, whereas K(M) ranged from 204 to 2500 microM, only a 12-fold difference. In vitro intrinsic clearance values (CL(int) were calculated for 18 compounds tested in the kinetic assay and compared with the in vivo plasma clearance (CL(p)) calculated from intravenous studies done in cynomolgus monkeys. These initial results suggested a relationship between the in vitro CL(int) and in vivo duration as defined by CL(p). A more rapid in vitro assay was developed in a 96-well format using a single concentration of substrate (100 microM) from which a glucuronidation rate was calculated. The results from this assay for 40 compounds correlated with in vivo plasma clearance (r = 0.57). This more efficient assay was used to test more than 100 compounds and develop structure-metabolism relationships based on metabolic stability and improved duration. The culmination of this effort contributed to the discovery of ABT-761, a 5-lipoxygenase inhibitor with in vivo duration in monkey improved 40-fold over thefirst generation inhibitor. Further studies performed in human liver microsomes demonstrated a similar trend that was corroborated by the 8-fold increase in duration after oral dosing in humans observed with ABT-761.