Discovery of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of adaptor protein 2-associated kinase 1 for the treatment of pain.

Adaptor protein 2-associated kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases and plays a role in modulating receptor endocytosis. AAK1 was identified as a potential therapeutic target for the treatment of neuropathic pain when it was shown that AAK1 knock out (KO) mice had a normal response to the acute pain phase of the mouse formalin model, but a reduced response to the persistent pain phase. Herein we report our early work investigating a series of pyrrolo[2,1-f][1,2,4]triazines as part of our efforts to recapitulate this KO phenotype with a potent, small molecule inhibitor of AAK1. The synthesis, structure-activity relationships (SAR), and in vivo evaluation of these AAK1 inhibitors is described.

Inhibitors of HIV-1 attachment. Part 9: an assessment of oral prodrug approaches to improve the plasma exposure of a tetrazole-containing derivative.

7-(2H-Tetrazol-5-yl)-1H-indole 3 was found to be a potent inhibitor of HIV-1 attachment but the compound lacked oral bioavailability in rats. The cause of the low exposure was believed to be poor absorption attributed to the acidic nature of the tetrazole moiety and, in an effort to address this liability, three more lipohilic tetrazole analogs, N-acetoxymethyl 4, N-pivaloyloxymethyl 5, and N-methyl 6, were evaluated as potential oral prodrugs in rats. Prodrug 5 was ineffective in improving the plasma concentration of 3 in vivo but compound 4 provided a 15-fold enhancement of the plasma concentration of 3. Most interestingly, oral dosing of analog 6 afforded a substantial increase in the plasma concentration of the parent in rats when compared to dosing of parent. This represents a novel example of a methyl tetrazole that acts as a prodrug for a free NH tetrazole-containing compound.

Inhibitors of HIV-1 attachment. Part 8: the effect of C7-heteroaryl substitution on the potency, and in vitro and in vivo profiles of indole-based inhibitors.

As part of the SAR profiling of the indole-oxoacetic piperazinyl benzamide class of HIV-1 attachment inhibitors, substitution at the C7 position of the lead 4-fluoroindole 2 with various 5- and 6-membered heteroaryl moieties was explored. Highly potent (picomolar) inhibitors of pseudotyped HIV-1 in a primary, cell-based assay were identified and select examples were shown to possess nanomolar inhibitory activity against M- and T-tropic viruses in cell culture. These C7-heteroaryl-indole analogs maintained the ligand efficiency (LE) of 2 and were also lipophilic efficient as measured by LLE and LELP. Pharmacokinetic studies of this class of inhibitor in rats showed that several possessed substantially improved IV clearance and half-lives compared to 2. Oral exposure in the rat correlated with membrane permeability as measured in a Caco-2 assay where the highly permeable 1,2,4-oxadiazole analog 13 exhibited the highest exposure.

Inhibitors of HIV-1 attachment. Part 7: indole-7-carboxamides as potent and orally bioavailable antiviral agents.

A series of substituted carboxamides at the indole C7 position of the previously described 4-fluoro-substituted indole HIV-1 attachment inhibitor 1 was synthesized and the SAR delineated. Heteroaryl carboxamide inhibitors that exhibited pM potency in the primary cell-based assay against a pseudotype virus expressing a JRFL envelope were identified. The simple methyl amide analog 4 displayed a promising in vitro profile, with its favorable HLM stability and membrane permeability translating into favorable pharmacokinetic properties in preclinical species.

Simultaneous bioanalysis of a phosphate prodrug and its parent compound using a multiplexed LC-MS method.

BACKGROUND: Bioanalytical support of drug-discovery efforts increasingly requires more complex multiple component analysis, including the bioanalysis of drugs, prodrugs and metabolites. Just as the physiochemical properties of these components may differ widely from each other, optimal LC and MS conditions, including polarity, can also vary greatly among the analytes of interest, thus presenting significant challenges during quantitative LC-MS-based bioanalysis. A single compromised method for the determination of all analytes may sacrifice sensitivity or chromatographic conditions for one analyte in order to achieve adequate results for another. Manually switching between assay conditions to analyze samples under separately optimized conditions for individual compounds can be time consuming. RESULTS: The method presented here addresses the problem of differential analyte optimization using a multiplexed approach for simultaneous quantitative bioanalysis of multiple analytes in the same sample, employing a mixed mode of both turbulent- and laminar-flow chromatography. CONCLUSION: The approach is illustrated with the quantitation of a lipophilic drug and its hydrophilic phosphate ester prodrug in a biological matrix under individually optimized LC-MS conditions.