Design, synthesis and biological evaluation of novel aminothiazoles as antiviral compounds acting against human rhinovirus.

We describe here the design, synthesis and biological evaluation of antiviral compounds acting against human rhinovirus (HRV). A series of aminothiazoles demonstrated pan-activity against the HRV genotypes screened and productive structure-activity relationships. A comprehensive investigational library was designed and performed allowing the identification of potent compounds with lower molecular weight and improved ADME profile. 31d-1, 31d-2, 31f showed good exposures in CD-1 mice. The mechanism of action was discovered to be a host target: the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). The identification of the pan-HRV active compound 31f combined with a structurally distinct literature compound T-00127-HEV1 allowed the assessment of target related tolerability of inhibiting this kinase for a short period of time in order to prevent HRV replication.

Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors.

Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.

Investigation of the binding pocket of human hematopoietic prostaglandin (PG) D2 synthase (hH-PGDS): a tale of two waters.

The inhibition of hH-PGDS has been proposed as a potential target for the development of anti-allergic and anti-inflammatory drugs. Herein we describe our investigation of the binding pocket of this important enzyme and our observation that two water molecules bind to our inhibitors and the enzyme. A series of compounds were prepared to the probe the importance of the water molecules in determining the binding affinity of the inhibitors to the enzyme. The study provides insight into the binding requirements for the design of potent hH-PGDS inhibitors.

Potent and selective thiophene urea-templated inhibitors of S6K.

S6K1 (p70 S6 kinase-1) is thought to play a critical role in the development of obesity and insulin resistance, thus making it an attractive target in developing medicines for the treatment of these disorders. We describe a novel thiophene urea class of S6K inhibitors. The lead matter for the development of these inhibitors came from mining the literature for reports of weak off-target S6K activity. These optimized inhibitors exhibit good potency and excellent selectivity for S6K over a panel of 43 kinases.

Enhanced selectivity profile of pyrazole-urea based DFG-out p38alpha inhibitors.

By targeting an extended region of the conventional 'DFG-out' pocket of p38alpha, while minimizing interactions with the specificity pocket and eliminating interactions with the adenine binding site, we are able to design and synthesize a number of pyrazole-urea based DFG-out p38alpha inhibitors with good potencies, and excellent selectivity.

Selective cell adhesion inhibitors: Barbituric acid based alpha4beta7--MAdCAM inhibitors.

A novel series of barbituric acid derivatives were identified as selective inhibitors of alpha4beta7 MAdCAM (mucosal addressin cell adhesion molecule-1) interactions via a high throughput screening exercise. These inhibitors were optimized to submicromolar potencies in whole cell adhesion assays, retaining their selectivity over alpha4beta1 VCAM.

Discovery of Potent, Orally Bioavailable Inhibitors of Human Cytomegalovirus.

A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 µM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.

The design, synthesis and potential utility of fluorescence probes that target DFG-out conformation of p38alpha for high throughput screening binding assay.

The design, synthesis and utility of fluorescence probes that bind to the DFG-out conformation of p38alpha kinase are described. Probes that demonstrate good affinity for p38alpha, have been identified and one of the probes, PF-04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non-classical p38alpha inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non-classical kinase inhibitors to the unactive form of the enzyme.

The specificity of JAK3 kinase inhibitors.

PF-956980 is a selective inhibitor of JAK3, related in structure to CP-690550, a compound being evaluated in clinical trials for rheumatoid arthritis and prevention of allograft rejection. PF-956980 has been evaluated against a panel of 30 kinases, and found to have nanomolar potency against only JAK3. Cellular and whole blood activity of this compound parallels its potency and selectivity in enzyme assays. It was effective in vivo at inhibiting the delayed type hypersensivity reaction in mice. We compared 2 commercially available JAK3 inhibitors (WHI-P131 and WHI-P154) in the same panel of biochemical and cellular assays and found them to be neither potent nor selective for JAK3. Both were found to be nanomolar inhibitors of the EGF receptor family of kinases. As these compounds have been used in numerous publications in the transplant and autoimmune disease literature, their specificity should be considered when interpreting these results.

Bipiperidinyl carboxylic acid amides as potent, selective, and functionally active CCR4 antagonists.

A cell-based assay for the chemokine G-protein-coupled receptor CCR4 was developed, and used to screen a small-molecule compound collection in a multiplex format. A series of bipiperidinyl carboxylic acid amides amenable to parallel chemistry were derived that were potent and selective antagonists of CCR4. One prototype compound was shown to be active in a functional model of chemotaxis, making it a useful chemical tool to explore the role of CCR4 in asthma, allergy, diabetes, and cancer.

Selectivity-determining residues in Plk1.

Polo-like kinase 1 is an important regulator of cell cycle progression whose over-expression is often associated with oncogenesis. Polo-like kinase 1 hence represents an attractive target for cancer intervention. BI 2536 (Boehringer Ingelheim, Ingelheim, Germany), a Polo-like kinase 1 inhibitor currently in clinical trials, exhibits nanomolar potency against Polo-like kinase isoforms and high selectivity against other kinases. We have previously published the crystal structures of the Polo-like kinase 1 domain in complex with AMPPNP and an Aurora A inhibitor. In this work, we present the co-crystal structure of Polo-like kinase 1 with BI 2536. The structure, in combination with selectivity data for BI 2536 and related compounds, illustrates important features for potency and selectivity. In particular, we show that the methoxy group of BI 2536 is an important specificity determinant against non-Polo-like kinases by taking advantage of a small pocket generated by Leu 132 in the hinge region of Polo-like kinase 1. The work presented here provides a framework for structure-based drug design of Polo-like kinase 1-specific inhibitors.

Efficient use of the iron ortho-nitrophenylporphyrin chloride to mimic biological oxidations of dimethylaminoantipyrine.

Major metabolites of dimethylaminoantipyrine have been synthesized using iron ortho-nitrophenylporphyrin chloride as biomimetic catalyst. Reactivity of iron tetrakis-ortho-nitrophenylporphyrin chloride [Fe(TNO2PP)Cl] has been compared with iron tetrakis-pentafluorophenylporphyrin chloride and iron tetrakis-2,6-dichlorophenylporphyrin chloride using various oxidants such as hydrogen peroxide, iodosobenzene, and cumene hydroperoxide in either protic or aprotic solvent. Effect of imidazole has been showed on the reactivity of Fe(TNO2PP)Cl/cumene hydroperoxide system.

Structure of the catalytic domain of human polo-like kinase 1.

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.

Use of quinolinium salts in parallel synthesis for the preparation of 4-amino-2-alkyl-1,2,3,4-tetrahydroquinoline.

Compounds of pharmacological interest containing a 4-amino-2-alkyl-1,2,3,4-tetrahydroquinoline core structure were prepared starting from 4-chloroquinoline. This has been executed both in solution with a 1-benzyl-4-chloroquinolinium salt and on a solid support with a 1-(4-benzyloxybenzyl-PS)-4-chloroquinolinium resin as key intermediates. Diversification of such intermediates was accomplished through N-arylation of position 4 and subsequent nucleophilic addition of Grignard reagents of position 2 to deliver the expected 4-amino-2-alkyl-1,2,3,4-tetrahydroquinolines in 20-60% yields. The methods described within clearly demonstrate that the quinolinium salts are very efficient intermediates for parallel synthesis.

Synthesis and biological evaluation of new cross-conjugated dienone marine prostanoid analogues.

The synthesis of a series of brominated cross-conjugated dienones, marine prostanoid analogues, was considered using two cyclopentannelation processes, from enamine (by a domino 3-aza Claisen/Mannich reaction) and from dioxolane ester alkylation followed by intramolecular Wittig reaction. All the compounds synthesized featured the same cross-conjugated dienone system, with a vicinal syn or anti diol on the omega-chain. The replacement of the omega-side-chain of the natural prostanoids with a 1-hydroxyphenyl-butyl moiety gave new prostanoids (32-34) with good cytotoxicities. In a second series of products, the possibility of a shorter alpha-side-chain bearing a simple phenyl ester was investigated. The results indicated a dramatic increase in the cytotoxicity (39, 40, 43, 44). Finally, in a third series, the omega-1-hydroxyphenyl-butyl was replaced by a 1-hydroxymethyloxybenzyl chain. These simpler compounds (45, 46, 47, 48, 60) are still highly cytotoxic, in the medium range of 60 nM, close to the value of natural punaglandins.

Solid-phase enolate chemistry investigated using HR-MAS NMR spectroscopy.

Supported P4-t-Bu enolate chemistry of phenylacetyloxymethyl polystyrene (PS) resin was investigated using high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Direct analysis of the crude reaction suspensions through the use of a diffusion filter (DF) allowed a rapid selection of the optimal experimental conditions, but also the characterization of the enolate on the solid phase. Comparison with solution experiments and literature data allowed us to address partially the structure of the enolate. HR-MAS NMR spectra of the enolate revealed also a tight interaction of P4-t-Bu base with the polymer matrix.