Steven Hansel

Dynamics of β-Amyloid Reductions in Brain, Cerebrospinal Fluid, and Plasma of β-Amyloid Precursor Protein Transgenic Mice Treated with a γ-Secretase Inhibitor

γ-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimers disease (AD). γ-Secretase inhibitors reduce brain β-amyloid peptide (Aβ), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human β-amyloid precursor protein (APP) are valuable models to examine the dynamics of Aβ changes with γ-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a γ-secretase inhibitor, showed dose- and time dependent reductions of Aβ in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Aβ levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Aβ in plaques showed reductions in CSF Aβ in the absence of measurable changes in plaque Aβ in the brain after up to 2 weeks of treatment. Hence, CSF Aβ levels were a valuable measure of γ-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8+ thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for γ-secretase inhibitors to reduce brain Aβ without causing Notch-mediated toxicity.

AN EFFICIENT METHOD FOR THE SYNTHESIS OF GUANIDINO PRODRUGS

Abstract A new class of guanidino prodrug is efficiently synthesized from an amine and an appropriate N,N′-bis(acyloxymethoxycarbonyl)-S-methylisothiourea. The N,N′-bis(acyloxymethoxycarbonyl)-S-methylisothiourea is readily prepared from S-methylisothiourea and the corresponding acyloxymethyl carbonochloridate in good yield. The N,N′-bis(acyloxymethyl carbamate) derivatives of the highly basic guanidino group serve as lipophilic, uncharged, esterase-activatable prodrugs of simple guanidine containing molecules.

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.

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.

Utilization of in vitro Caco-2 permeability and liver microsomal half-life screens in discovering BMS-488043, a novel HIV-1 attachment inhibitor with improved pharmacokinetic properties

Optimizing pharmacokinetic properties to improve oral exposure is a common theme in modern drug discovery. In the present work, in vitro Caco-2 permeability and microsomal half-life screens were utilized in an effort to guide the structure-activity relationship in order to improve the pharmacokinetic properties of novel HIV-1 attachment inhibitors. The relevance of the in vitro screens to in vivo pharmacokinetic properties was first demonstrated with a number of program compounds at the early stage of lead optimization. The Caco-2 permeability, tested at 200 microM, was quantitatively predictive of in vivo oral absorption, with complete absorption occurring at a Caco-2 permeability of 100 nm/s or higher. The liver microsomal half-life screen, conducted at 1 microM substrate concentration, can readily differentiate low-, intermediate-, and high-clearance compounds in rats, with a nearly 1:1 correlation in 12 out of 13 program compounds tested. Among the >100 compounds evaluated, BMS-488043 emerged as a lead, exhibiting a Caco-2 permeability of 178 nm/s and a microsomal half-life predictive of a low clearance (4 mL/min/kg) in humans. These in vitro characteristics translated well to the in vivo setting. The oral bioavailability of BMS-488043 in rats, dogs, and monkeys was 90%, 57%, and 60%, respectively. The clearance was low in all three species tested, with a terminal half-life ranging from 2.4 to 4.7 h. Furthermore, the oral exposure of BMS-488043 was significantly improved (6- to 12-fold in rats and monkeys) compared to the prototype compound BMS-378806 that had a suboptimal Caco-2 permeability (51 nm/s) and microsomal half-life. More importantly, the improvements in preclinical pharmacokinetics translated well to humans, leading to a >15-fold increase in the human oral exposure of BMS-488043 than BMS-378806 and enabling a clinical proof-of-concept for this novel class of anti-HIV agents. The current studies demonstrated the valuable role of in vitro ADME screens in improving oral pharmacokinetics at the lead optimization stage.

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.

Carbamate-appended N-alkylsulfonamides as inhibitors of γ-secretase

The synthesis and gamma-secretase inhibition data for a series of carbamate-appended N-alkylsulfonamides are described. Carbamate 54 was found to significantly reduce brain Abeta in transgenic mice. 54 was also found to possess markedly improved brain levels in transgenic mice compared to previously disclosed 1 and 2.