1. Based on binding affinity, 2'-amino-N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-methylpropyl)[1,1'-biphenyl]-2-sulfonamide (2) was identified as an initial lead in a programme to identify selective endothelin (ET) receptor antagonists. However, the compound was extensively metabolized in preclinical animal species and human in vitro systems due to oxidative biotransformation. 2. To optimize this structural class, the site of metabolism of 2 was determined. This allowed for focussed structure-activity and structure-metabolism studies aimed at finding more metabolically stable analogues that maintained potency. New analogues were screened for their ET binding characteristics and their stability in rat and human liver microsomes. 3. The use of the microsomal stability screen was tested by the determination of the pharmacokinetic parameters of select analogues. A good correlation was found between reduced rates of rat microsomal metabolism and reduced clearance in the rat. 4. N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)[1,1'-biphenyl]-2-sulfonamide (3) was identified as an analogue with improved in vitro properties and further studies revealed that the compound had improved pharmacokinetic properties. 5. N-[[2'-[[(3,4-dimethyl-5-isoxazolyl)amino]sulfonyl]-4-(2-oxazolyl)[1,1'-biphenyl]-2-yl]methyl]acetamide (4) was subsequently identified as a compound with superior in vitro properties compared with compound 3, but when tested in vivo it had a substantially increased rate of clearance. Further studies demonstrated that the clearance of this closely related structural analogue was not dictated by metabolic processes, but was mediated by transport-mediated direct biliary excretion. 6. The utility of screening for in vitro liver microsomal stability as part of the lead optimization process for compounds with metabolic liabilities was shown. It was also shown that relatively small molecular changes can dramatically change the disposition of closely related analogues and care must be used when screening for a single property.

We have previously disclosed the selective ET(A) receptor antagonist N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)[1,1'-biphenyl]-2-sulfonamide (1, BMS-193884) as a clinical development candidate. Additional SAR studies at the 2'-position of 1 led to the identification of several analogues with improved binding affinity as well as selectivity for the ET(A) receptor. Following the discovery that a 3-amino-isoxazole group displays significantly improved metabolic stability in comparison to its 5-regioisomer, the 3-amino-isoxazole group was combined with the optimal 2'-substituent leading to 16a (BMS-207940). Compound 16a is an extremely potent (ET(A) K(i) = 10 pM) and selective (80,000-fold for ET(A) vs ET(B)) antagonist. It is also 150-fold more potent and >6-fold more selective than 1. The bioavailability of 16a was 100% in rats and the systemic clearance and volume of distribution are higher than that of 1. In rats, intravenous 16a blocks big ET pressor responses with 30-fold greater potency than 1. After oral dosing at 3 micromol/kg, 16a displays enhanced duration relative to 1.

The ET(A) receptor antagonist (2) (N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)-[1,1'-biphenyl]-2-sulfonamide, BMS-193884) shares the same biphenyl core as a large number of AT(1) receptor antagonists, including irbesartan (3). Thus, it was hypothesized that merging the structural elements of 2 with those of the biphenyl AT(1) antagonists (e.g., irbesartan) would yield a compound with dual activity for both receptors. This strategy led to the design, synthesis, and discovery of (15) (4'-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(3,4-dimethyl-5-isoxazolyl)-2'-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl]-[1,1'-biphenyl]-2-sulfonamide, BMS-248360) as a potent and orally active dual antagonist of both AT(1) and ET(A) receptors. Compound 15 represents a new approach to treating hypertension.

1. The disposition of acyl prodrugs was studied to improve the delivery of a guanidine-containing parent compound with poor membrane permeability and poor absorption.2. The prodrugs were evaluated in vitro and in vivo for conversion to drug. Prodrugs were evaluated for hydrolytic or oxidative bioactivation in intestinal homogenate and rat liver S9 or microsomes. The disposition of the prodrugs in vivo was monitored in bile duct-cannulated rats.3. Compounds with n-alkylacyl groups were efficiently bioactivated, but were hydrolysed before absorption.4. Hydrolytic bioactivation could be blocked in vitro by branching in the alkyl chain. These compounds showed modest improvements in absorption, despite favourable permeability. Experiments with liver microsomes demonstrated efficient NADPH-dependent oxidative bioactivation, which was proposed to occur through a CYP-mediated side chain oxidation followed by cyclization and release of parent compound. Ketoconazole co-administration yielded approximately a twofold increase in absorption.5. The hydrolytically stable prodrugs were successful in increasing absorption of parent drug and were efficiently bioactivated, but they did not yield increased systemic levels of drug.

This review article will focus on the various techniques that are currently employed by drug discovery scientists in evaluating permeability/absorption of drug candidates during the drug candidate selection process. Various preclinical methodologies are available; each having advantages and disadvantages, but it is the judicious use of these techniques that can help identify drug candidates that will be well absorbed in humans. It is well recognized that the human intestinal permeability cannot be accurately predicted based on a single methodology (in vitro: tissue/cell culture, in situ, or in vivo).

The synthesis and structure-activity relationship (SAR) studies of a series of 4'-oxazolyl-N-(3,4-dimethyl-5-isoxazolyl)[1,1'-biphenyl]-2-sulfonamide derivatives as endothelin-A (ETA) receptor antagonists are described. The data reveal a remarkable improvement in potency and metabolic stability when the 4'-position of the biphenylsulfonamide is substituted with an oxazole ring. Additional 2'-substitution of an acylaminomethyl group further increased the binding activity and provided one of the first subnanomolar ETA-selective antagonists in the biphenylsulfonamide series (17, ETA K-i = 0.2 nM). Among the compounds described, 3 (N(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)[1,1'-biphenyl]-2-sulfonamide BMS-193884) had the optimum pharmacological profile and was therefore selected as a clinical candidate for studies in congestive heart failure.

To improve the oral absorption of thrombin inhibitors, Argatroban analogs 1, 2, 3 and 4, with reduced guanidine basicity were prepared. The in vitro thrombin inhibitory potency and their permeability of the Caco-2 cell monolayers were studied as a model for intestinal permeability. While the neutral molecule 2 was quite permeable through the Caco-2 cell monolayers the other charged compounds 1, 3, 4 and 5 did not have appreciable permeability. Interestingly, the N-hydroxyguanidine compound 4 maintains potent intrinsic activity, which implies that the N-hydroxyguanidine can be used in this series of compounds as a guanidine surrogate with reduced basicity.

A series of HIV protease inhibitors containing a novel C2 symmetrical "aminodiol" core structure were prepared from amino acid starting materials. The ability of the aminodiols to inhibit HIV replication in cell culture is comparable to their ability to inhibit the isolated enzyme, a result compatible with good cell membrane penetration by this class of compounds. Optimization of the structure-activity in this series led to aminodiol 9a (Ki = 100 nM; ED50 (HIV-1) = 80 nM) containing P1/P1 benzyl and P2/P2 Boc substituents. Compound 9a is a selective inhibitor of HIV protease versus other aspartyl proteases such as human renin, human cathepsin D, and porcine pepsin. In addition, 9a is equipotent against HIV-1 and HIV-2 in cell culture and demonstrates similar activity in infected T-lymphocytes and PBMCs. After i.v. and oral administration in rats, 9a displayed significant oral bioavailability (ca. 40%) and a promising plasma elimination half-life (4 h).

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Pravastatin sodium, a competitive inhibitor of HMG-CoA reductase, is a new orally effective hypocholesterolaemic agent. In a two-way crossover study, eight healthy male subjects each received an intravenous and an oral dose of [14C]-pravastatin sodium. The oral absorption of [14C] activity from pravastatin sodium was about 34% and the oral bioavailability was about 18%, suggesting first-pass metabolism of pravastatin. After the intravenous dose, the recovery of radioactivity averaged 60% and 34% in urine and faeces, respectively. Corresponding values were 20% (urine) and 71% (faeces) for the oral dose. The estimated average plasma elimination half-life of pravastatin was 0.8 and 1.8 h for the intravenous and oral routes, respectively. The average values for total and renal clearances were 13.5 and 6.3 ml min-1 kg-1, respectively, and the steady-state volume of distribution averaged 0.51 kg-1. These results suggest that both kidney and liver are important sites of elimination for pravastatin.