Total Synthesis of Covalent Cysteine Protease Inhibitor N-Desmethyl Thalassospiramide C and Crystallographic Evidence for Its Mode of Action.

A total synthesis of N-desmethyl thalassospiramide C, a unique strained macrocyclic proteobacterial depsipeptide, enabled a detailed crystallographic study of its covalent complex with cathepsin K, a member of a medicinally important family of cysteine proteases. The study provides support for the mechanism of action, and the insight gained can be used for structure-based drug design targeting these calpain proteases.

Discovery of selective urokinase plasminogen activator (uPA) inhibitors as a potential treatment for multiple sclerosis.

We report here the design and synthesis of a novel series of benzylamines that are potent and selective inhibitors of uPA with promising oral availability in rat. Further evaluation of one representative (ZK824859) of the new structural class showed that this compound lowered clinical scores when dosed in either acute or chronic mouse EAE models, suggesting that uPA inhibitors of this type could be useful for the treatment of multiple sclerosis.

Reversible, orally available ADP receptor (P2Y12) antagonists Part I: Hit to lead process.

A hit to lead process to identify reversible, orally available ADP receptor (P2Y12) antagonists lead compounds is described. High throughput screening afforded 1. Optimization of 1, using parallel synthesis methods, a methyl scan to identify promising regions for optimization, and exploratory SAR on these regions, provided 22 and 23. Compound 23 is an orally available, competitive reversible antagonist (KB = 94 nM for inhibition of ADP-induced platelet aggregation). It exhibits high metabolic stability in human, rat and dog liver microsomes and is orally absorbed. Although plasma level after oral dosing of 22 and 23 to rats is low, reasonable levels were achieved to merit extensive lead optimization of this structural class.

Recent advances in drug discovery of benzothiadiazine and related analogs as HCV NS5B polymerase inhibitors.

Hepatitis C virus (HCV) is a major health burden, with an estimated 170 million chronically infected individuals worldwide, and a leading cause of liver transplantation. Patients are at increased risk of developing liver cirrhosis, hepatocellular carcinoma and even liver failure. In the past two decades, several approaches have been adopted to inhibit non-structural viral proteins. The RNA-dependent RNA polymerase (NS5B) of HCV is one of the attractive validated targets for development of new drugs to block HCV infection. In this review, we report the recent progress made towards identifying and developing benzothiadiazines as HCV NS5B polymerase inhibitors. The substituted benzothiadiazine class was identified by HTS in 2002 as an NS5B inhibitor. Further optimization and modification of the core has improved the potency and pharmacokinetic properties of substituted benzothiadiazines. Research on palm site-binding benzothiadiazine analogs and related derivatives and analogs is discussed in this article.

HCV NS5B polymerase inhibitors 3: Synthesis and in vitro activity of 3-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives.

3-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

HCV NS5B polymerase inhibitors 1: Synthesis and in vitro activity of 2-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-1-hydroxynaphthalene derivatives.

2-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-1-hydroxynaphthalene derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition and replicon activity are discussed.

HCV NS5B polymerase inhibitors 2: Synthesis and in vitro activity of (1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives.

(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

A novel P2Y(12) adenosine diphosphate receptor antagonist that inhibits platelet aggregation and thrombus formation in rat and dog models.

Irreversible platelet inhibitors, such as aspirin and clopidogrel, have limited anti-thrombotic efficacy in the clinic due to their bleeding risk. We have developed an orally active reversible P2Y(12) receptor antagonist, BX 667. The aim of this study was to determine if the reversible antagonist BX 667 had a greater therapeutic index than the irreversible P2Y(12) receptor antagonist clopidogrel. Since BX 667 is rapidly converted to its active metabolite BX 048 in rats, we first injected BX 048 intravenously (iv) in a rat arterial venous (A-V) shunt model of thrombosis. BX 048 dose- and concentration-dependently attenuated thrombosis. When administered orally, BX 667 and clopidogrel had similar efficacy, but BX 667 caused less bleeding than clopidogrel. In a rat model of a platelet-rich thrombus induced by vessel injury with FeCl(2), both BX 667 and clopidogrel exhibited higher levels of thrombus inhibition after oral administration compared to their potency in the A-V shunt model. Again, BX 667 caused less bleeding than clopidogrel. In a dog cyclic flow model, iv injection of either BX 667 or clopidogrel dose-dependently reduced thrombus formation with lower bleeding for BX 667 than clopidogrel. Inhibition of thrombosis was highly correlated with inhibition of ADP-induced platelet aggregation in these animal models. In dogs pre-treated with aspirin, BX 667 maintained its wider therapeutic index, measured by inhibition of platelet aggregation over bleeding, compared to the aspirin-clopidogrel combination. These data demonstrate that the reversible P2Y(12) receptor antagonist, BX 667, has a wider therapeutic index than clopidogrel in experimental models of thrombosis.

A novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) - part I: pharmacological characterization.

We have discovered a novel small-molecule (3-phosphinoylpropionic acid) inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa), BX 528, which had an IC (50) of 2 nM in an enzymatic assay and 50 nM in an in-vitro clot lysis assay, with 3,500- to 35,000-fold selectivity against other carboxypeptidases, such as CPN, CPZ and CPD, and 5- and 12-fold selectivity against CPE (CPH) and CPB, respectively. At 10 micro M, BX 528 had no significant activity (<50% inhibition or antagonism) in a panel of 137 enzymes and receptors. It had no effects on blood coagulation and platelet aggregation up to 300 and 10 micro M, respectively. The plasma half-life following intravenous administration was 0.85 hours in rats and 4.5 hours in dogs. No significant metabolism was detected in human, dog or rabbit hepatic microsomes, and no significant inhibition of cytochrome P450 3A4 and 2D6 up to 30 micro M. No cytotoxic or cell proliferative effects were found in three hepatic and renal cell lines up to 300 micro M and no mutagenic activity was seen in the Ames II screen. There were no significant hemodynamic effects in rats and dogs up to 100 and 30 mg/kg with peak plasma drug concentrations of approximately 1,000 and 300 micro M, respectively. In an in-vivo complement activation model in guinea pigs, BX 528 showed minimal inhibition of plasma CPN activity up to 60 mg/kg with peak plasma concentrations up to 250 micro M. Thus, these data demonstrate that BX 528 is a novel, potent, selective and safe TAFIa inhibitor.

A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis.

We have discovered a novel small-molecule TAFIa inhibitor, BX 528, which is potent, highly selective against other carboxypeptidases and safe. The present study was to determine if BX 528 can enhance exogenous and endogenous thrombolysis in four different animal models. In the first three models, a thrombus was induced by FeCl (2) (dogs) or laser (rats) injury of the femoral artery, or formed ex vivo and implanted in the jugular vein in rabbits. A low dose of exogenous t-PA was given to induce a low-level thrombolysis on an established thrombus. Co-treatment with BX 528 further enhanced the thrombolytic effects induced by the exogenous t-PA and, thus, reduced thrombosis in all three animal models. In a second rat model, fibrin deposition in the lungs was induced by batroxobin, which was spontaneously resolved in 30 minutes due to the activation of endogenous fibrinolysis. Pre-treatment with lipopolysaccharide (LPS) attenuated this spontaneous fibrinolysis. Co-treatment with 10 mg/kg BX 528 prevented the LPS-induced attenuation of endogenous fibrinolysis. Thus, these studies demonstrated that inhibition of TAFIa by BX 528, our newly discovered small-molecule TAFIa inhibitor, enhanced both the exogenous (induced by a low dose of t-PA) and endogenous (LPS-induced resistance) thrombolysis without increasing the bleeding risk in four different animal models of thrombosis in different species (rat, dog and rabbit) employing different thrombogenic stimuli (FeCl (2) , laser, ex vivo and batroxobin) to induce thrombus formation in different tissues (artery, vein and lung microcirculation).

3-Mercaptopropionic acids as efficacious inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa).

A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (-)2a displays TAFIa IC50 = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.

Solid-phase synthesis of naphthylamidines as factor VIIa/tissue factor inhibitors.

Reductive amination followed by acylation of polymer-linked formyl aryl amidines generate combinatorial libraries of aryl amidines 8-13. Potent small molecule naphthylamidine inhibitors 12 (Ki<100 nM) of FVIIa/TF have been discovered and their activity against other serine proteases in the coagulation cascade is reported.

Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1.

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.

Synthesis and biological evaluation of menthol-based derivatives as inhibitors of plasminogen activator inhibitor-1 (PAI-1).

Compound 1 was identified by high throughput screening as a novel PAI-1 inhibitor. Optimization of the B and C-segments of 1 resulted in a series of structurally simplified compounds with improved potency. The synthesis and SAR data of these compounds are presented here.

Design, synthesis, and activity of a novel series of factor Xa inhibitors: optimization of arylamidine groups.

A novel series of diaryloxypyridines have been designed as selective nanomolar factor Xa (fXa) inhibitors for use as anticoagulants. In this paper, we describe our efforts to identify an additional interaction and a replacement for the distal amidine group that binds in the S3/S4 pocket of fXa. Introduction of a hydroxyl group para to the proximal amidine group increases the potency vs fXa by 1-2 orders of magnitude, which is the result of a hydrogen bond to Ser195 of the catalytic triad. A methyl imidazoline and a dimethylamide are good alternatives for the second amidine. These substitutions have increased the selectivity vs the related serine proteases trypsin and thrombin. The synthesis, in vitro activity, and hypothetical modes of binding to fXa based on trypsin crystallographic data are outlined.

Automated parallel solid-phase synthesis of non-peptide CCR1 receptor antagonists.

An automated, parallel, solid-phase synthesis and screening strategy using commercially available aryl acetic acids as starting materials has discovered novel, non-peptide CCR1 antagonists (K(i) < 100 nM).

Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines.

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.