Identification of quinazoline based inhibitors of IRAK4 for the treatment of inflammation.

Interleukin-1 receptor associated kinase 4 (IRAK4) has been implicated in IL-1R and TLR based signaling. Therefore selective inhibition of the kinase activity of this protein represents an attractive target for the treatment of inflammatory diseases. Medicinal chemistry optimization of high throughput screening (HTS) hits with the help of structure based drug design led to the identification of orally-bioavailable quinazoline based IRAK4 inhibitors with excellent pharmacokinetic profile and kinase selectivity. These highly selective IRAK4 compounds show activity in vivo via oral dosing in a TLR7 driven model of inflammation.

A Pharmacokinetic Modeling Approach to Predict the Contribution of Active Metabolites to Human Efficacious Dose.

A preclinical drug candidate, MRK-1 (Merck candidate drug parent compound), was found to elicit tumor regression in a mouse xenograft model. Analysis of samples from these studies revealed significant levels of two circulating metabolites, whose identities were confirmed by comparison with authentic standards using liquid chromatography-tandem mass spectrometry. These metabolites were found to have an in vitro potency similar to that of MRK-1 against the pharmacological target and were therefore thought to contribute to the observed efficacy. To predict this contribution in humans, a pharmacokinetic (PK) modeling approach was developed. At the mouse efficacious dose, the areas under the plasma concentration time curves (AUCs) of the active metabolites were normalized by their in vitro potency compared with MRK-1. These normalized metabolite AUCs were added to that of MRK-1 to yield a composite efficacious unbound AUC, expressed as "parent drug equivalents," which was used as the target AUC for predictions of the human efficacious dose. In vitro and preclinical PK studies afforded predictions of the PK of MRK-1 and the two active metabolites in human as well as the relative pathway flux to each metabolite. These were used to construct a PK model (Berkeley Madonna, version 8.3.18; Berkeley Madonna Inc., University of California, Berkeley, CA) and to predict the human dose required to achieve the target parent equivalent exposure. These predictions were used to inform on the feasibility of the human dose in terms of size, frequency, formulation, and likely safety margins, as well as to aid in the design of preclinical safety studies.

Identification of N-(1H-pyrazol-4-yl)carboxamide inhibitors of interleukin-1 receptor associated kinase 4: Bicyclic core modifications.

IRAK4 plays a critical role in the IL-1R and TLR signalling, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of permeable N-(1H-pyrazol-4-yl)carboxamides was developed by introducing lipophilic bicyclic cores in place of the polar pyrazolopyrimidine core of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides. Replacement of the pyrazolo[1,5-a]pyrimidine core with the pyrrolo[2,1-f][1,2,4]triazine, the pyrrolo[1,2-b]pyridazine, and thieno[2,3-b]pyrazine cores guided by cLogD led to the identification of highly permeable IRAK4 inhibitors with excellent potency and kinase selectivity.

Applying the pro-drug approach to afford highly bioavailable antagonists of P2Y(14).

Our series of competitive antagonists against the G-protein coupled receptor P2Y(14) were found to be highly shifted in the presence of serum (>99% protein bound). A binding assay using 2% human serum albumin (HSA) was developed to guide further SAR studies and led to the identification of the zwitterion 2, which is substantially less shifted (18-fold) than our previous lead compound 1 (323-fold). However, as the bioavailability of 2 was low, a library of ester pro-drugs was prepared (7a-7j) and assessed in vitro. The most interesting candidates were then profiled in vivo and led to the identification of the pro-drug 7j, which possesses a substantially improved pharmacokinetic profile.

The identification of 4,7-disubstituted naphthoic acid derivatives as UDP-competitive antagonists of P2Y14.

A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY(14) with a naphthoic acid core was identified through high-throughput screening. Optimization provided compounds with improved potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuronidation and improved the pharmacokinetic profile. Additional optimization led to the identification of compound 38 which is an 8 nM UDP-competitive antagonist of P2Y(14) with a good pharmacokinetic profile.

lfnA from Pseudomonas aeruginosa O12 and wbuX from Escherichia coli O145 encode membrane-associated proteins and are required for expression of 2,6-dideoxy-2-acetamidino-L-galactose in lipopolysaccharide O antigen.

The rare sugar 2,6-dideoxy-2-acetamidino-L-galactose (L-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of Pseudomonas aeruginosa serotype O12 and Escherichia coli O145. P. aeruginosa is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic E. coli. The acetamidino (NAm) moiety of L-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a pseA homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in Campylobacter jejuni. In P. aeruginosa O12 and E. coli O145, the pseA homologues are lfnA and wbuX, respectively, and we hypothesized that these genes function in L-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the lfnA mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-L-galactose (L-FucNAc) in place of L-FucNAm. The mutation could be complemented by expression of either His(6)-tagged lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His(6) tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.

Discovery of 5-Amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Inhibitors of IRAK4.

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing.

Biosynthesis of UDP-N-acetyl-L-fucosamine, a precursor to the biosynthesis of lipopolysaccharide in Pseudomonas aeruginosa serotype O11.

UDP-N-acetyl-L-fucosamine is a precursor to l-fucosamine in the lipopolysaccharide of Pseudomonas aeruginosa serotype O11 and the capsule of Staphylococcus aureus type 5. We have demonstrated previously the involvement of three enzymes, WbjB, WbjC, and WbjD, in the biosynthesis of UDP-2-acetamido-2,6-dideoxy-L-galactose or UDP-N-acetyl-L-fucosamine (UDP-l-FucNAc). An intermediate compound from the coupled-reaction of WbjB-WbjC with the initial substrate UDP-2-acetamido-2-deoxy-alpha-D-glucose or UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) was purified, and the structure was determined by NMR spectroscopy to be UDP-2-acetamido-2,6-dideoxy-L-talose (UDP-L-PneNAc). WbjD could then convert this intermediate into a new product with the same mass, consistent with a C-2 epimerization reaction. Those results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-l-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.