Michael J. Costanzo

Potent, nonpeptide inhibitors of human mast cell tryptase. Synthesis and biological evaluation of novel spirocyclic piperidine amide derivatives

We have explored a series of spirocyclic piperidine amide derivatives (5) as tryptase inhibitors. Thus, 4 (JNJ-27390467) was identified as a potent, selective tryptase inhibitor with oral efficacy in two animal models of airway inflammation (sheep and guinea pig asthma models). An X-ray co-crystal structure of 4 x tryptase revealed a hydrophobic pocket in the enzymes active site, which is induced by the phenylethynyl group and is comprised of amino acid residues from two different monomers of the tetrameric protein.

Crystal structure of human carbonic anhydrase II complexed with an anti-convulsant sugar sulphamate.

The fructose-based sugar sulphamate RWJ-37497, a potent analogue of the widely used anti-epileptic drug topiramate, possesses anti-convulsant and carbonic anhydrase-inhibitory activities. We have studied the binding interactions of RWJ-37497 in the active site of human carbonic anhydrase II by X-ray crystallography. The atomic positions of the enzyme inhibitor complex were refined at a resolution of 2.1 A (1 A=0.1 nm) to the final crystallographic R and R(free) values of 0.18 and 0.23, respectively. The inhibitor co-ordinates to the active-site zinc ion through its oxygen atom and the ionized nitrogen atom of the sulphamate group by replacing the metal-bound water molecules, although the sulphamoyl oxygen atom provides a rather lengthy co-ordination. The 4,5-cyclic sulphate group is positioned in a hydrophobic pocket of the active site, making contacts with the residues Phe-131, Leu-198, Pro-201 and Pro-202. Since the ligand was found to be intact, concerns about RWJ-37947 irreversibly alkylating the enzyme through its 4,5-cyclic sulphate group were dispelled.

Solid-phase parallel synthesis applied to lead optimization: Discovery of potent analogues of the GPIIb/IIIa antagonist RWJ-50042

Abstract A study of β-turn peptide mimetics, related to the C-terminal γ-chain of fibrinogen and containing a nipecotic acid scaffold, led to RWJ-50042 (1), an interesting fibrinogen receptor (GPIIb/IIIa) antagonist. To enhance potency, we employed solid-phase parallel synthesis for the preparation of over 200 analogues in a protocol of optimization cycles. This strategy produced several promising nipecotamide analogues, such as 25, which is 35 times more potent than 1 in vitro.

Exploration of Potential Prodrugs of RWJ-445167, an Oxyguanidine-based Dual Inhibitor of Thrombin and Factor Xa

Compound 2 (RWJ‐445167; 3DP‐10017), a dual inhibitor of thrombin and factor Xa, was advanced into human clinical studies. However, its oral bioavailability in humans proved to be below acceptable limits. To address this issue, we explored a prodrug approach involving numerous guanidine derivatives. Prodrug candidates of classes A (carbamate derivatives), B (imidate derivatives), and C (alkyl and acyl derivatives), compounds 3–6, were synthesized and evaluated for anticoagulant activity at 2h after oral administration to rats. In comparison to the parent drug (2), little worthwhile improvement was observed for the prodrug candidates.

Remote acyclic diastereocontol involving a bicyclic metal chelate. High 1,5 asymmetric induction in the hydride reduction of δ-hydroxy ketones

Abstract A variety of reducing agents was explored to effect stereoselective reduction of acyclic δ-hydroxy ketone 3a ; R -Alpine-Hydride provided high anti stereoselectivity ( anti : syn = 7:1). Reduction of 3b in CH 2 Cl 2 with R -Alpine-Hydride or Zn(BH 4 ) 2 afforded impressive anti stereoselectivity: 10:1 and 13:1, respectively. The stereochemical outcome is attributed to a bicyclic metal-chelate species (viz. 10 ).

Cooperative antithrombotic effect from the simultaneous inhibition of thrombin and factor Xa.

Whereas heparin functions as an antithrombotic agent by promoting antithrombin III-based inhibition of thrombin and factor Xa, there is less appreciation for the combination behavior with small-molecule, direct inhibitors of these proteases. We conducted a study in a high-shear arterial environment to explore the potential for a cooperative antithrombotic effect with a thrombin inhibitor (argatroban), a factor Xa inhibitor (YM-60828), and a dual thrombin/factor Xa inhibitor (RWJ-445167). We employed a platelet-dependent vascular injury model in which rats were subjected to an acute electrical injury to the carotid artery. Antithrombotic efficacy was measured for thrombin inhibitor argatroban and factor Xa inhibitor YM-60828 administered alone or in combination. The results indicate that there is a cooperative antithrombotic effect in vivo when both thrombin and factor Xa are inhibited simultaneously. The dual thrombin/factor Xa inhibitor RWJ-445167 was found to have potent antithrombotic activity in this high-shear environment. A comparison of results for RWJ-445167 and argatroban showed additional efficacy with RWJ-445167, suggestive of drug synergy.

Antithrombotic properties of RWJ-50353, a potent and novel thrombin inhibitor.

The antithrombotic, anticoagulant, and kinetic properties of RWJ-50353, a novel, reversible, active-site-directed thrombin inhibitor, were evaluated. RWJ-50353 inhibited the catalytic activity of human alpha-thrombin with a K(i) of 0.19+/-0.02 nM. It showed a 16-fold selectivity relative to inhibition of trypsin and at least 330-fold selectivity relative to inhibition of other biologically important serine proteases. In a gel-filtered platelet preparation, RWJ-50353 inhibited alpha-thrombin-induced aggregation with an IC(50) of 32+/-6 nM. In a canine arteriovenous shunt antithrombotic model, RWJ-50353 demonstrated a significant dose-related (0.1-1.0 mg/kg, i.v.) reduction in thrombus formation with 50% inhibition (ID(50)) obtained at 0.46+/-0.1 mg/kg. In a rabbit deep vein thrombosis model, RWJ-50353 dose-dependently (0.1-1. 0 mg/kg, i.v.) reduced thrombus formation with an ID(50) of 0.25+/-0. 03 mg/kg. The antithrombotic activity in both of these models was associated with only mild prolongations in bleeding time and coagulation parameters. These results demonstrate that RWJ-50353 is a potent, selective thrombin inhibitor that is an effective antithrombotic agent after intravenous administration in models of arterial and venous thrombosis and may be useful in the management of various thrombotic disorders.

ANTICONVULSANT SUGAR SULFAMATES. POTENT CYCLIC SULFATE AND CYCLIC SULFITE ANALOGUES OF TOPIRAMATE

Abstract Exploration structure-activity relationships surrounding the clinically effective antiepileptic drug topiramate (1) led to a series of potent anticonvulsants with a 4,5-cyclic sulfate or 4,5-cycli sulfite functionality. Key derivative 2 (RWJ-37947) is ca. 8 times more potent than topiramate in mice; it also features a long duration of action and a very favorable neurotoxicity index.

Structure of human α-thrombin complexed with RWJ-51438 at 1.7 Å: unusual perturbation of the 60A–60I insertion loop

The three-dimensional structure of the ternary complex consisting of human alpha-thrombin, hirugen and the active-site inhibitor RWJ-51438 has been determined at 1.7 A resolution. The crystals of the complex belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 62.98, b = 117.52, c = 47.99 A. The refined R and R(free) values are 0.196 and 0.232, respectively. The ketone carbonyl group of the inhibitor is covalently linked to the hydroxyl O atom of Ser195, forming a tetrahedral intermediate hemiketal structure; the benzothiazole ring N atom of RWJ-51438 forms a hydrogen bond with His57. Surprisingly, the carboxylate substituent on the benzothiazole group forms salt bridges with Lys60F NZ and the NZ of the symmetry-related residues Lys236 and Lys240, which introduces steric effects that perturb the 60A-60I insertion loop, especially at residues Trp60D and Phe60H.

Non-chair six-membered-ring conformations. Preference for a twist-boat (or skew) structure in α-L-sorbopyranose derivatives

Abstract The conformational preferences for 2,3- O -isopropylidene-α- L -sorbopyranose derivatives 3–6 were determined by using 1 H NMR data and empirical force field calculations. Proton NMR studies of 3–6 indicate that a twist-boat (or skew) conformation ( 3 S 0 ) prevails over possible chair forms for each compound. Force-field calculations (MM2, MNDO, AM1) on a model 2,3- O -isopropylidene-α- L -sorbopyranose system ( 18 ) indicate that the 3 S 0 conformation is among the low-energy structures. X-Ray crystallographic analysis of α- L -sorbopyranose sulfamate 3 , a compound with potent anticonvulsant activity, demonstrates that the 3 S 0 skew conformation is manifested in the solid state, as well.