Discovery of highly potent small molecule kallikrein inhibitors.

Uncontrolled kallikrein activation is involved in diseases such as hereditary angioedema, bacterial septic shock and procedures such as cardiopulmonary bypass. Here we report a series of small molecule compounds that potently inhibit kallikrein activity in vitro. Kinetic studies indicate that some of these compounds are slow binding inhibitors of kallikrein with Ki final less than a nanomolar. The ability of these compounds to inhibit the activity of kallikrein was further confirmed in a plasma model by quantitating the release of bradykinin, an endogenous cleavage product of plasma kallikrein. To understand the inhibitory mechanism of the selected compounds toward kallikrein, the interactions between the selected compounds and kallikrein was explored using molecular modeling based on the information of crystal structures of TF/FVIIa and kallikrein. The information presented in the current study provides an initial approach to develop more selective and therapeutically useful small molecule inhibitors.

Systematic structure-based design and stereoselective synthesis of novel multisubstituted cyclopentane derivatives with potent antiinfluenza activity.

The design and synthesis of novel, orally active, potent, and selective inhibitors of influenza neuraminidase differing structurally from existing neuraminidase inhibitors are described. X-ray crystal structures of complexes of neuraminidase with known five- and six-membered ring inhibitors revealed that potent inhibition of the enzyme is determined by the relative positions of the interacting inhibitor substituents (carboxylate, glycerol, acetamido, hydroxyl) rather than by the absolute position of the central ring. This led us to design potential neuraminidase inhibitors in which the cyclopentane ring served as a scaffold for substituents (carboxylate, guanidino, acetamido, alkyl) that would interact with the four binding pockets of the neuraminidase active site at least as effectively as those of the established six-membered ring inhibitors such as DANA (2), zanamivir (3), and oseltamivir (4). A mixture of the isomers was prepared initially. Protein crystallography of inhibitor-enzyme complexes was used to screen mixtures of isomers in order to identify the most active stereoisomer. A synthetic route to the identified candidate 50 was developed, which featured (3 + 2) cycloaddition of 2-ethylbutyronitrile oxide to methyl (1S,4R)-4[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (43). Structures of the synthetic compounds were verified by NMR spectroscopy using nuclear Overhauser effect methodology. Two new neuraminidase inhibitors discovered in this work, 50 and 54, have IC(50) values vs neuraminidase from influenza A and B of <1 and <10 nM, respectively. These IC(50) values are comparable or superior to those for zanamivir and oseltamivir, agents recently approved by the FDA for treatment of influenza. The synthetic route used to prepare 50 and 54 was refined so that synthesis of pure active isomer 54, which has five chiral centers, required only seven steps from readily available intermediates. Further manipulation was required to prepare deoxy derivative 50. Because the activities of the two compounds are comparable and 54 [RWJ-270201 (BCX-1812)] is the easier to synthesize, it was selected for further clinical evaluation.

Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir.

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.

Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design.

A series of 94 benzoic acid derivatives was synthesized and tested for its ability to inhibit influenza neuraminidase. The enzyme-inhibitor complex structure was determined by X-ray crystallographic analysis for compounds which inhibited the enzyme. The most potent compound tested in vitro, 5 (4-acetylamino)-3-guanidinobenzoic acid), had an IC50 = 2.5 x 10(-6) M against N9 neuraminidase. Compound 5 was oriented in the active site of the neuraminidase in a manner that was not predicted from the reported active site binding of GANA (4) with neuraminidase. In a mouse model of influenza, 5 did not protect the mice from weight loss due to the influenza virus when dosed intranasally.

Synthesis, ligand binding, and quantitative structure-activity relationship study of 3 beta-(4'-substituted phenyl)-2 beta-heterocyclic tropanes: evidence for an electrostatic interaction at the 2 beta-position.

A set of 3 beta-(4'-substituted phenyl)-2 beta-heterocyclic tropanes was designed, synthesized, and characterized. We discovered that these compounds can function as bioisosteric replacements for the corresponding WIN 35,065-2 analogs which possess a 2 beta-carbomethoxy group. Several of the compounds showed high affinity and selectivity for the dopamine transporter (DAT) relative to the serotonin and norepinephrine transporters. From the structure-activity relationship study, the 3 beta-(4'-chlorophenyl)-2 beta-(3'-phenylisoxazol-5-yl)tropane (5d) emerged as the most potent and selective compound. The binding data for 2 beta-heterocyclic tropanes were found to show a high correlation with molecular electrostatic potential (MEP) minima near one of the heteroatoms in the 2 beta-substituents. In contrast, low correlations were found for other MEP minima near the 2 beta-substituent as well as for calculated log P or substituent volume. These quantitative structure-activity relationship studies are consistent with an electrostatic contribution to the binding potency of these WIN 35,065-2 analogs at the DAT.

Cocaine and 3 beta-(4'-substituted phenyl)tropane-2 beta-carboxylic acid ester and amide analogues. New high-affinity and selective compounds for the dopamine transporter.

Several 2 beta-carboxylic acid ester and amide analogues of cocaine and of 3 beta-(4'-substituted phenyl)tropane-2 beta-carboxylic acid were prepared. The binding affinities of these compounds, and of some previously prepared analogues, at the dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters were determined. The phenyl esters of 3 beta-(4'-methylphenyl)- and 3 beta-(4'-chlorophenyl)tropane-2 beta-carboxylic acid are highly potent and highly selective for the DA transporter. The isopropyl esters of 3 beta-(4'-chlorophenyl)- and 3 beta-(4'-iodophenyl)tropane-2 beta-carboxylic acid also possess high DA affinity and show significant DA transporter selectivity. Similarly, the phenyl and isopropyl ester analogues of cocaine are much more selective for the DA transporter than cocaine. Tertiary amide analogues of cocaine and of 3 beta-(4'-substituted phenyl)tropane-2 beta-carboxylic acids are more potent inhibitors of radioligand binding at the DA transporter than the primary and secondary amide analogues. In particular, 3 beta-(4'-chlorophenyl)tropane-2 beta-N-morpholinocarboxamide as well as the 3 beta-(4'-chlorophenyl)- and 3 beta-(4'-iodophenyl)tropane-2 beta-N- pyrrolidinocarboxamides possess high affinity and selectivity for the DA transporter. The N,N-dimethylamide cocaine analogue is the most selective cocaine amide derivative for the DA transporter. High correlation between the inhibition of radioligand binding and inhibition of uptake at the DA, NE, and 5-HT transporter was found for a selected group of analogues. Within this group, one compound, the isopropyl ester of 3 beta-(4'-iodophenyl)-tropane-2 beta-carboxylic acid, was found to be more potent in the inhibition of radioligand binding than in the inhibition of DA uptake. Taken together with its high potency and selectivity at the DA transporter, this suggests that this compound may be a lead in the development of a cocaine antagonist.