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.

Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (I): in vitro effects on platelets.

ADP plays a key role in platelet aggregation which has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the effects of two novel P2Y12 receptor antagonists, BX 667 and its active metabolite BX 048, on platelets. BX 667 and BX 048 block the binding of 2MeSADP to platelets and antagonize ADP-induced platelet aggregation in human, dog and rat washed platelets. Both compounds were shown to be reversible inhibitors of platelet aggregation. BX 048 prevents the decrease in cAMP induced by treatment of platelets with ADP. The specificity of BX 667 and BX 048 was demonstrated against cell lines expressing P2Y1 and P2Y6 as well as against a panel of receptors and enzymes. Taken all together these data show that both BX 048 and BX 667 are potent P2Y12 antagonists with high specificity which, in the accompanying paper are demonstrated to behave predictably in vivo.

Discovery of novel and potent aryl diamines as leukotriene A4 hydrolase inhibitors.

The synthesis and biological evaluation of a series of aryl diamines as inhibitors of LTA(4)-h inhibitors are described. The optimization which led to the identification of the optimal para-substitution on the diphenyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent enzyme and cellular potency as well as desirable pharmacokinetic properties.

Synthesis of N-alkyl glycine amides as potent inhibitors of leukotriene A4 hydrolase.

The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA(4)-h inhibitors and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic parameters of our analogs are described. The lead compound in the series, 4q, has excellent potency and oral bioavailability.

Fasudil, a Rho-kinase inhibitor, attenuates angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice by inhibiting apoptosis and proteolysis.

BACKGROUND: Angiotensin II (Ang II) accelerates atherosclerosis and induces abdominal aortic aneurysm (AAA) in an experimental mouse model. Agonism of a G protein-coupled receptor by Ang II activates Rho-kinase and other signaling pathways and results in activation of proteolysis and apoptosis. Enhanced proteolysis and smooth muscle cell apoptosis are important mechanisms associated with AAA. In this study, we tested the hypothesis that fasudil, a Rho-kinase inhibitor, could attenuate Ang II-induced AAA formation by inhibiting vascular wall apoptosis and extracellular matrix proteolysis. METHODS AND RESULTS: Six-month-old apolipoprotein E-deficient mice were infused with Ang II (1.44 mg x kg(-1) x d(-1)) for 1 month. Animals were randomly assigned to treatment with fasudil (136 or 213 mg x kg(-1) x d(-1) in drinking water) or tap water. Ang II infusion induced AAA formation in 75% of the mice, which was accompanied by an increase in proteolysis detected by zymographic analysis and quantified by active matrix metalloproteinase-2 activity, as well as apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and quantified by both caspase-3 activity and histone-associated DNA fragmentation. The level of DNA fragmentation in the suprarenal aorta correlated with AAA diameter. Ang II also increased atherosclerotic lesion area and blood pressure. Fasudil treatment resulted in a dose-dependent reduction in both the incidence and severity of AAA. At the higher dose, fasudil decreased AAA by 45% while significantly inhibiting both apoptosis and proteolysis, without affecting atherosclerosis or blood pressure. CONCLUSIONS: These data demonstrate that inhibition of Rho-kinase by fasudil attenuated Ang II-induced AAA through inhibition of both apoptosis and proteolysis pathways.

Novel 3-oxa lipoxin A4 analogues with enhanced chemical and metabolic stability have anti-inflammatory activity in vivo.

Lipoxin A(4) (LXA(4)) is a structurally and functionally distinct natural product called an eicosanoid, which displays immunomodulatory and anti-inflammatory activity but is rapidly metabolized to inactive catabolites in vivo. A previously described analogue of LXA(4), methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), was shown to have a poor pharmacokinetic profile after both oral and intravenous administration, as well as sensitivity to acid and light. The chemical stability of the corresponding E,E,E-trien-11-yne analogue, 3, was improved over 2 without loss of efficacy in the mouse air pouch model of inflammation. Careful analysis of the plasma samples from the pharmacokinetic assays for both 2 and 3 identified a previously undetected metabolite, which is consistent with metabolism by beta-oxidation. The formation of the oxidative metabolites was eliminated with the corresponding 3-oxatetraene, 4, and the 3-oxatrien-11-yne, 5, analogues of 2. Evaluation of 3-oxa analogues 4 and 5 in calcium ionophore-induced acute skin inflammation model demonstrated similar topical potency and efficacy compared to 2. The 3-oxatrien-11-yne analogue, 5, is equipotent to 2 in an animal model of inflammation but has enhanced metabolic and chemical stability and a greatly improved pharmacokinetic profile.

Measurements of drug-protein binding by using immobilized human serum albumin liquid chromatography-mass spectrometry.

An HPLC/MS based method was used for fast and convenient determination of drug plasma-protein interactions in early drug discovery screening by employing a human serum albumin affinity column. Results from this methodology were compared with data from ultrafiltration or dialysis methods, and good agreement was observed. A compound not suitable for ultrafiltration due to the very high non-specific binding to artificial membrane of ultrafiltration device was also successfully analyzed by this method, and the protein binding determined by this chromatography method was very similar to data obtained by dialysis technique employing biological membranes. The immobilized HSA column LC/MS method also proved to be more reproducible and precise compared to ultrafiltration method in drug protein binding measurements.

Potent triazolothione inhibitor of heat-shock protein-90.

Heat-shock protein-90 is an attractive target for anticancer drugs, as heat-shock protein-90 blockers such as the ansamycin 17-(allylamino)-17-demethoxygeldanamycin greatly reduce the expression of many signaling molecules that are disregulated in cancer cells and are key drivers of tumor growth and metastasis. While 17-(allylamino)-17-demethoxygeldanamycin has shown promise in clinical trials, this compound class has significant template-related drawbacks. In this paper, we describe a new, potent non-ansamycin small-molecule inhibitor of heat-shock protein-90, BX-2819, containing resorcinol and triazolothione rings. Structural studies demonstrate binding of BX-2819 to the ADP/ATP-binding pocket of heat-shock protein-90. The compound blocked expression of heat-shock protein-90 client proteins in cancer cell lines and inhibited cell growth with a potency similar to 17-(allylamino)-17-demethoxygeldanamycin. In a panel of four cancer cell lines, BX-2819 blocked growth with an average IC(50) value of 32 nM (range of 7-72 nM). Efficacy studies demonstrated that treatment with BX-2819 significantly inhibited the growth of NCI-N87 and HT-29 tumors in nude mice, consistent with pharmacodynamic studies showing inhibition of heat-shock protein-90 client protein expression in tumors for greater than 16 h after dosing. These data support further studies to assess the potential of BX-2819 and related analogs for the treatment of cancer.

CCR5 receptor antagonists: discovery and SAR of novel 4-hydroxypiperidine derivatives.

The guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromobenzaldehyde, 1, with an IC(50) of 840 nM against the CCR5 receptor was identified using high-throughput screening. Optimization efforts led to the discovery of a novel piperidine series of CCR5 antagonists. In particular, the 4-hydroxypiperidine derivative, 6k, had improved potency against CCR5, and was a starting point for further optimization. SAR elaboration using parallel synthesis led to the identification of 10h, a potent CCR5 antagonist with an IC(50) of 11 nM.

1-(1,3-Benzodioxol-5-ylmethyl)-3-[4-(1H-imidazol-1-yl)phenoxy]-piperidine analogs as potent and selective inhibitors of nitric oxide formation.

A new series of 1-(1,3-benzodioxol-5-ylmethyl)-3-[4-(1H-Imidazol-1-yl)phenoxy]-piperidine analogs were designed and identified as potent and selective inhibitors of NO formation based both on the crystal structure of a murine iNOS Delta114 monomer domain/ inhibitor complex and inhibition of the NO formation in human A172 cell assays. Compound 12S showed high potency and high iNOS selectivity versus nNOS and eNOS.

Synergistic effect of angiotensin II and nitric oxide synthase inhibitor in increasing aortic stiffness in mice.

Although they are implicated on their own as risk factors for cardiovascular disease, the potential link between nitric oxide (NO) deficiency, ANG II, and vascular stiffening has not been tested before. We evaluated the role of chronic ANG II treatment and NO deficiency, alone and in combination, on aortic stiffness in mice and tested parameters contributing to increases in active or passive components of vascular stiffness, including blood pressure, vascular smooth muscle contractility, and extracellular matrix components. Untreated (control) mice and mice treated with a NO synthase (NOS) inhibitor [N(omega)-nitro-L-arginine methyl ester (L-NAME), 0.5 g/l] were implanted with osmotic minipumps delivering ANG II (500 ng.kg(-1).min(-1)) for 28 days. Aortic stiffness was then measured in vivo by pulse wave velocity (PWV) and ex vivo by load-strain analysis to obtain values of maximal passive stiffness (MPS). Blood pressure and aortic contractility ex vivo were measured. ANG II treatment or NOS inhibition with L-NAME did not independently increase vascular stiffness; however, the combined treatments worked synergistically to increase PWV and MPS. The combined treatments of ANG II + L-NAME also significantly increased aortic wall collagen content while decreasing elastin. These novel results suggest that NO deficiency and ANG II act synergistically to increase aortic stiffness in mice predominantly via changes in aortic wall collagen/elastin ratio.

Anti-tumor efficacy of the nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) in human pancreatic and ovarian tumor xenograft models.

1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) is a deoxycytidine analog that has been shown previously to have impressive anti-proliferative and cytotoxic effects in vitro and in vivo toward colorectal and gastric tumors. In our present studies, the pharmacokinetic behavior in nude mice and the effectiveness of 4'-thio-FAC against human pancreatic and ovarian tumor growth were assessed in comparison with standard chemotherapeutic agents. Potent in vitro anti-proliferative effects were observed against pancreatic (Capan-1, MIA-PaCa-2, BxPC-3) and ovarian (SK-OV-3, OVCAR-3, ES-2) cancer cell lines with IC(50) of 0.01-0.2 microM. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously (s.c.) implanted human pancreatic tumor xenografts or intraperitoneally (i.p.) disseminated human ovarian xenografted tumors. Oral daily administration of 4'-thio-FAC for 8-10 days significantly inhibited the growth of gemcitabine-resistant BxPC-3 pancreatic tumors and induced regression of gemcitabine-refractory Capan-1 tumors. 4'-Thio-FAC was also a highly effective inhibitor of ovarian peritoneal carcinomatosis. In the SK-OV-3 and ES-2 ovarian cancer models, 4'-thio-FAC prolonged survival to a greater extent than that observed with gemcitabine. Furthermore, the superiority of 4'-thio-FAC to carboplatin and paclitaxel was demonstrated in the ES-2 clear cell ovarian carcinoma model. Studies provide evidence that 4'-thio-FAC is a promising new alternative to gemcitabine and other chemotherapeutic drugs in the treatment of a variety of tumor indications, including pancreatic and ovarian carcinoma.

A beta-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction.

Lipoxins and aspirin-triggered 15-epi-lipoxins (ATL) are counter-regulatory eicosanoids with potent antiinflammatory actions. Oral efficacy and mechanism of action of ZK-192, a beta-oxidation-resistant 3-oxa-ATL analog, were examined in trinitrobenzenesulphonate (TNBS)-induced colitis. When dosed orally once daily, 300 and 1,000 mug/kg ZK-192 markedly attenuated TNBS colitis in rodents both in preventive and therapeutic regimens. ZK-192 attenuated weight loss, macroscopic and histologic colon injury, mucosal neutrophil infiltration, and colon wall thickening. ZK-192 was as effective as 3-10 mg/kg oral prednisolone. ZK-192 decreased mucosal mRNA levels for several inflammatory mediators: inducible nitric oxide synthase, cyclooxygenase 2, and macrophage inflammatory protein 2. ZK-192 also decreased mucosal mRNA and protein levels of T helper 1 effector cytokines: tumor necrosis factor alpha, IL-2, and IFN-gamma. Systemic levels of these cytokines were also dramatically attenuated. CD3/CD28-mediated costimulation of T helper 1 effector cytokine release in lamina propria mononuclear cells was markedly inhibited by ZK-192 ex vivo and in vitro. ZK-192 also prevented colitis in lymphocyte-deficient severe combined immunodeficient mice, with approximately 75% inhibition of mucosal tumor necrosis factor alpha and IL-2 levels. The results are further evidence that innate immune cells function as triggers for hapten-induced colitis. The combined antiinflammatory and immunomodulatory effects of ZK-192 in TNBS colitis suggest that ATL analogs may be an attractive oral treatment approach for inflammatory bowel diseases.

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

Compound 2 was identified by high throughput screening as a novel PAI-1 inhibitor. Systematic optimization of the A, B, and C segments of 2 resulted in the identification of a more potent compound 39 with good oral bioavailability. The synthesis and SAR data are presented in this report.