Design and biological activity of (S)-4-(5-([1-(3-chlorobenzyl)-2-oxopyrrolidin-3-ylamino]methyl)imidazol-1-ylmethyl)benzonitrile, a 3-aminopyrrolidinone farnesyltransferase inhibitor with excellent cell potency.

The synthesis, structure-activity relationships, and biological properties of a novel series of imidazole-containing inhibitors of farnesyltransferase are described. Starting from a 3-aminopyrrolidinone core, a systematic series of modifications provided 5h, a non-thiol, non-peptide farnesyltransferase inhibitor with excellent bioavailability in dogs. Compound 5h was found to have an unusually favorable ratio of cell potency to intrinsic potency, compared with other known FTIs. It exhibited excellent potency against a range of tumor cell lines in vitro and showed full efficacy in the K-rasB transgenic mouse model.

Synthesis of conformationally constrained 5,6,7, 8-Tetrahydroimidazo[1,5-a]pyridine inhibitors of farnesyltransferase.

[reaction: see text] Synthesis of the 8-amino-5,6,7,8-tetrahydroimidazo[1,5-a]pyridine ring system was accomplished by intramolecular cyclization of an iminium ion, derived from condensation of an amine and a substituted gamma-(1-imidazolyl)butyraldehyde. The reaction was used to produce conformationally restricted farnesyltransferase inhibitor analogues which exhibit improved in vivo metabolic stability.

Nonpeptide alpha(v)beta(3) antagonists. 1. Transformation of a potent, integrin-selective alpha(IIb)beta(3) antagonist into a potent alpha(v)beta(3) antagonist.

Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.

Identification of MK-944a: a second clinical candidate from the hydroxylaminepentanamide isostere series of HIV protease inhibitors.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

Design and synthesis of N-alkylated saccharins as selective alpha-1a adrenergic receptor antagonists.

Benign prostatic hyperplasia can be managed pharmacologically with alpha-1 adrenergic receptor antagonists. Agents that demonstrate selectivity for the alpha-1a receptor subtype may offer advantages in clinical applications with respect to hypotensive side effects. The N-alkylated saccharins reported here represent a new class of subtype selective alpha-1a adrenergic receptor antagonists which demonstrate potent effects on prostate function in vivo and are devoid of blood pressure side effects.

Nonpeptidal P2 ligands for HIV protease inhibitors: structure-based design, synthesis, and biological evaluation.

Design and synthesis of nonpeptidal bis-tetrahydrofuran ligands based upon the X-ray crystal structure of the HIV-1 protease-inhibitor complex 1 led to replacement of two amide bonds and a 10 pi-aromatic system of Ro 31-8959 class of HIV protease inhibitors. Detailed structure-activity studies have now established that the position of ring oxygens, ring size, and stereochemistry are all crucial to potency. Of particular interest, compound 49 with (3S,3aS,6aS)-bis-Thf is the most potent inhibitor (IC50 value 1.8 +/- 0.2 nM; CIC95 value 46 +/- 4 nM) in this series. The X-ray structure of protein-inhibitor complex 49 has provided insight into the ligand-binding site interactions. As it turned out, both oxygens in the bis-Thf ligands are involved in hydrogen-bonding interactions with Asp 29 and Asp 30 NH present in the S2 subsite of HIV-1 protease. Stereoselective routes have been developed to obtain these novel ligands in optically pure form.

L-743, 726 (DMP-266): a novel, highly potent nonnucleoside inhibitor of the human immunodeficiency virus type 1 reverse transcriptase.

The clinical benefit of the human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) is limited by the rapid selection of inhibitor-resistant viral variants. However, it may be possible to enhance the clinical utility of this inhibitor class by deriving compounds that express both high levels of antiviral activity and an augmented pharmacokinetic profile. Accordingly, we developed a new class of NNRTIs, the 1, 4-dihydro-2H-3, 1-benzoxazin-2-ones. L-743, 726 (DMP-266), a member of this class, was chosen for clinical evaluation because of its in vitro properties. The compound was a potent inhibitor of the wild-type HIV-1 RT (Ki = 2.93 nM) and exhibited a 95% inhibitory concentration of 1.5 nM for the inhibition of HIV-1 replicative spread in cell culture. In addition, L-7743, 7726 was found to be capable of inhibiting, with 95% inhibitory concentrations of < or = 1.5 microM, a panel of NNRTI-resistant mutant viruses, each of which expressed a single RT amino acid substitution. Derivation of virus with notably reduced susceptibility to the inhibitor required prolonged cell culture selection and was mediated by a combination of at least two RT amino acid substitutions. Studies of L-743, 726 in rats, monkeys, and a chimpanzee demonstrated the compound's potential for good oral bioavailability and pharmacokinetics in humans.

HIV-1 protease inhibitors: synthesis and biological evaluation of glycopeptidemimetics.

A series of glycopeptidemimetics based on the hydroxyethylene Phe-Phe isostere have been synthesized and evaluated for their ability to inhibit the enzyme HIV-1 protease. Incorporation of carbohydrate moieties at the P'2-position and elimination of P'3 amino acid in our lead compound 1, provided inhibitors with only nanomolar potencies (400-800 nM). However, incorporation of a carbohydrate moiety at the P'3-position with branched chain amino acid at the P'2-position, resulted in inhibitors with subnanomolar potencies. Within this series, compound 21 was the most potent inhibitor (IC50 value 0.17 nM). This compound has also shown to block the spread of HIV-1 in T-lymphoid cells at an inhibitor concentration of 200 nM.

[3H]L-657,743 (MK-912): a new, high affinity, selective radioligand for brain alpha 2-adrenoceptors.

L-657,743 (MK-912), a highly potent and selective alpha 2-adrenoceptor antagonist was tritiated to a high specific activity and its binding characteristics to brain tissue were determined. The specific binding of [3H]L-657,743 to rat cerebrocortex was saturable, reversible, and dependent on tissue concentration. In saturation studies, [3H]L-657,743 binding was resolved into two high affinity components exhibiting Kd values of 86 pM and 830 pM with densities of 82 fmol/mg protein and 660 fmol/mg protein, respectively. Based on the binding potencies of a variety of compounds with differing receptor selectivities, the sites labeled by [3H]L-657,743 were characteristic of alpha 2-adrenoceptors. In contrast to alpha 2-antagonists, alpha 2-agonists displayed shallow competition curves. In the presence of 100 microM GTP, Gpp(NH)p or 150 mM NaCl, the competition curve for epinephrine was shifted to the right, whereas that for yohimbine was unaffected. In studies utilizing human cerebrocortical tissue, [3H]L-657,743 also bound with high affinity to sites characteristic of alpha 2-adrenoceptors.

Metabolism of synthetic inositol trisphosphate analogs.

A series of synthetic analogs was employed to explore structure-activity relationships in the metabolism of the second messenger inositol trisphosphate (IP3) in vascular tissue. Cytosolic IP3-5-phosphatase activity was purified approximately 240-fold from bovine aorta. All synthetic analogs tested were apparent competitive inhibitors of the 5-phosphatase activity. The order of potency was DL-1,3,4,5-IP3 greater than D-1,4,5-IP3 greater than DL-1,3,4-IP3 greater than L-1,4,5-IP3 greater than 1,3,5-IP3 greater than DL-6-methoxy-1,4,5-IP3 greater than DL-2,4,5-IP3 greater than DL-1,2,4-cyclohexane-P3. The least potent analogs had Ki values only 11 times higher than the apparent Km of the substrate D-1,4,5-[3H]IP3. However, only three synthetic compounds, DL-1,3,4,5-IP4, D-1,4,5-IP3, and DL-2,4,5-IP3, could serve as substrates for the 5-phosphatase. IP3 kinase activity in the same tissue exhibited considerably more selectivity with respect to inhibition by IP3 analogs. D-1,4,5-IP3 was about 30 times more potent than DL-1,3,4,5-IP4 and 100-1000 times more potent than the other compounds tested. The function of the IP3 receptor was evaluated by measuring labeled calcium mobilization in permeabilized bovine aortic smooth muscle cells in culture. While all analogs tested were full agonists, vast differences in potency were observed. D-1,4,5-IP3 was about 30 times more potent than DL-2,4,5-IP3 and 100-2000 times more potent than the other analogs tested. The results suggest that IP3-5-phosphatase activity is relatively nonselective in the binding of inositol polyphosphates, while IP3 kinase activity and the IP3 receptor exhibit great selectivity in the recognition of these compounds.

[3H]L-654,284 as a probe of the central alpha 2 adrenoceptor.

L-654,284 [2R, 12bS)-N-(1,3,4,6,7,12b-hexahydro-2H-benzo[b]-furo[2,3-a] quinolizin-2-yl)-N-methyl-2-hydroxyethanesulfonamide], a potent and selective antagonist of the alpha 2 adrenoceptor, was tritiated to high specific activity. Saturation binding to cell membrane suspensions obtained from calf cerebral cortex revealed a high affinity binding site (0.63 nM). Kinetics of association and dissociation were well represented by single exponential processes, and the equilibrium dissociation constant obtained from the ratio of rate constants agreed well with that found by saturation binding. A direct comparison of saturation binding revealed that the antagonist [3H]L-654,284 had roughly the same affinity for the alpha 2 adrenoceptor as the agonist [3H]clonidine and eight times the affinity of the antagonist [3H]rauwolscine. The maximum receptor densities of these radioligands were not significantly different. Competition assays with a series of compounds of known receptor affinity revealed that [3H]L-654,284 selectively binds to a site with all of the characteristics expected of the alpha 2 adrenoceptor.

A peripherally acting alpha-2 adrenoceptor antagonist: L-659,066.

L-659,066 has been characterized as a potent and selective alpha-2 adrenoceptor antagonist. Both in vitro and in vivo, L-659,066 exhibited specificity (comparable to rauwolscine) for alpha-2 over alpha-1 adrenoceptors. Studies comparing L-659,066 with a previously described antagonist, L-657,743, demonstrate that the new compound penetrates the blood-brain barrier only poorly after systemic administration. With a pA2 of 8.44 at alpha-2 adrenoceptors in the isolated rat vas deferens and an IC50 of 3.0 nM against the binding of [3H]rauwolscine to rat cerebrocortical membranes, L-659,066 possessed, respectively, about one-eighth and one-third of the potency of L-657,743. Similar relative potencies were obtained in vivo in pithed rats with regard to blocking peripherally located postjunctional and prejunctional alpha-2 adrenoceptors (L-659,066 = one-seventh and one-fourth of L-657,743, respectively). In tests carried out in vivo with rats for ascertaining alpha-2 adrenoceptor antagonism in the central nervous system--namely, accumulation of cortical dopa and antagonism of mydriasis induced by the alpha-2 agonist, clonidine--L-659,066 had, respectively, less than 1/345th and about 1/5000th of the potency of L-657,743. In mice, L-659,066 had, respectively, approximately 1/29th and 1/1400th of the potency of L-657,743 as an antagonist in vivo of the predominately peripherally mediated inhibition of colonic propulsion caused by clonidine as compared with the mainly centrally mediated antinocisponsive action elicited by the alpha-2 agonist UK 14,304. The foregoing findings are consistent with poor penetration of the blood-brain barrier by L-659,066.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-affinity relationships of arylquinolizines at alpha-adrenoceptors.

Hexahydroaryl[a]quinolizines comprise a prominent structural element in several alpha 2-adrenoceptor antagonists. Eight hexahydroheteroarylquinolizines were prepared as minimal ligands to investigate the relationship between the nature of the aromatic ring and affinity of these molecules for alpha-adrenoceptors. Affinity for alpha 1-and alpha 2-adrenoceptors was assessed by displacement of [3H]prasozin and [3H]clonidine, respectively. Lipophilicity of the aryl portion of the molecules, reflected by their partition coefficient between octanol and pH 7.4 buffer, correlated well with affinity at both receptor subtypes. Although some compounds showed nanomolar affinity for alpha-adrenoceptors, no subtype selectivity was observed. These results suggest that the aromatic ring enhances binding at both receptors chiefly through hydrophobic interactions and contributes little to subtype selectivity.