Highly potent and selective 3-N-methylquinazoline-4(3H)-one based inhibitors of B-Raf(V600E) kinase.

Herein we describe the design of a novel series of ATP competitive B-Raf inhibitors via structure-based methods. These 3-N-methylquinazoline-4(3H)-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 16, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies. Our work also demonstrates that by replacing an aryl amide with an aryl sulfonamide, a multikinase inhibitor such as AZ-628, can be converted to a selective B-Raf inhibitor, a finding that should have broad application in kinase drug discovery.

Imidazo[4,5-b]pyridine inhibitors of B-Raf kinase.

This Letter details the synthesis and evaluation of imidazo[4,5-b]pyridines as inhibitors of B-Raf kinase. These compounds bind in a DFG-in, αC-helix out conformation of B-Raf, which is a binding mode associated with significant kinase selectivity. Structure-activity relationship studies involved optimization of the ATP-cleft binding region of these molecules, and led to compound 23, an inhibitor with excellent enzyme/cell potency, and kinase selectivity.

Pyrazolopyridine inhibitors of B-Raf(V600E). Part 3: an increase in aqueous solubility via the disruption of crystal packing.

A single crystal was obtained of a lead B-Raf(V600E) inhibitor with low aqueous solubility. The X-ray crystal structure revealed hydrogen-bonded head-to-tail dimers formed by the pyrazolopyridine and sulfonamide groups of a pair of molecules. This observation suggested a medicinal chemistry strategy to disrupt crystal packing and reduce the high crystal lattice energy of alternative inhibitors. Both a bulkier group at the interface of the dimer and an out-of-plane substituent were required to decrease the compound's melting point and increase aqueous solubility. These substituents were selected based on previously developed structure-activity relationships so as to concurrently maintain good enzymatic and cellular activity against B-Raf(V600E).

Pyrazolopyridine inhibitors of B-Raf(V600E). Part 4: rational design and kinase selectivity profile of cell potent type II inhibitors.

Cell potent inhibitors of B-Raf(V600E) that bind to the kinase in the DFG-out conformation are reported. These compounds utilize the hinge-binding group and lipophilic linker from a previously disclosed series of B-Raf(V600E) inhibitors that bind to the kinase in an atypical DFG-in, αC-helix-out conformation. This new series demonstrates that DFG-out kinase inhibitors can be rationally designed from related inhibitors which utilize an unconventional binding mode. Kinase selectivity profiles are compared. The pattern of kinase selectivity was found to be determined by the feature of the inhibitor which extends into the back pocket of the kinase and leads to the kinase conformation, rather than by the hinge-binding group or other minor modifications.

The discovery of potent and selective pyridopyrimidin-7-one based inhibitors of B-RafV600E kinase.

Herein we describe the discovery of a novel series of ATP competitive B-Raf inhibitors via structure based drug design (SBDD). These pyridopyrimidin-7-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 17, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies.

Non-oxime inhibitors of B-Raf(V600E) kinase.

The development of inhibitors of B-Raf(V600E) serine-threonine kinase is described. Various head-groups were examined to optimize inhibitor activity and ADME properties. Several of the head-groups explored, including naphthol, phenol and hydroxyamidine, possessed good activity but had poor pharmacokinetic exposure in mice. Exposure was improved by incorporating more metabolically stable groups such as indazole and tricyclic pyrazole, while indazole could also be optimized for good cellular activity.

The discovery of furo[2,3-c]pyridine-based indanone oximes as potent and selective B-Raf inhibitors.

Virtual and high-throughput screening identified imidazo[1,2-a]pyrazines as inhibitors of B-Raf. We describe the rationale, SAR, and evolution of the initial hits to a series of furo[2,3-c]pyridine indanone oximes as highly potent and selective inhibitors of B-Raf.

Pyrazolopyridine inhibitors of B-RafV600E. Part 2: structure-activity relationships.

Structure-activity relationships around a novel series of B-Raf(V600E) inhibitors are reported. The enzymatic and cellular potencies of inhibitors derived from two related hinge-binding groups were compared and3-methoxypyrazolopyridine proved to be superior. The 3-alkoxy group of lead B-Raf(V600E) inhibitor 1 was extended and minimally affected potency. The propyl sulfonamide tail of compound 1, which occupies the small lipophilic pocket formed by an outward shift of the αC-helix, was expanded to a series of arylsulfonamides. X-ray crystallography revealed that this lipophilic pocket unexpectedly enlarges to accommodate the bulkier aryl group.

Preclinical assessment of novel BRAF inhibitors: integrating pharmacokinetic-pharmacodynamic modelling in the drug discovery process.

The objective of these studies were to determine the preclinical disposition of the two BRAF inhibitors, G-F and G-C, followed by pharmacokinetic (PK)-pharmacodynamic (PD) modelling to characterize the concentration-efficacy relationship of these compounds in the Colo205 mouse xenograft model. With G-F, the relationship of pERK inhibition to concentration was also characterized. Compounds G-F and G-C were administered to mice, rats and dogs and the pharmacokinetics of G-F and G-C was determined. In addition, using indirect response models the concentration-efficacy relationship was described. The clearance of G-F was low; 0.625 and 4.65 mL/min/kg in rat and dog respectively. Similarly, the clearance of G-C was low in rat and dog, 0.490 and 4.43 mL/min/kg, respectively. Both compounds displayed low volumes of distribution (0.140-0.267 L/kg), resulting in moderate half-lives across species (~2.5 to 4 h). Bioavailability was formulation dependent and decreased with increasing dose. Using the indirect response models, the KC(50) (50% K(max); maximal response) value for tumor growth inhibition for G-F and G-C were 84.5 and 19.2 µM, respectively. The IC(50) for pERK inhibition in Colo205 tumors by G-F was estimated to be 29.2 µM. High exposures of G-F and G-C were required for efficacy. Despite good PK properties of low CL and moderate half-life, limitations in obtaining exposures adequate for safety testing in rat and dog resulted in development challenges.

Biomimetic macrocycle-forming Diels-Alder reaction of an iminium dienophile: synthetic studies directed toward gymnodimine.

The macrocyclic core of gymnodimine has been constructed via an intramolecular Diels-Alder reaction of an alpha,beta-unsaturated iminium dienophile in water. The cycloaddition furnished a single exo-product, along with two endo-products. Through X-ray analysis of a suitable derivative, the stereochemistry of the exo-product was established, thereby demonstrating that its stereochemistry matches that of gymnodimine. In contrast, macrocyclization of an analogous alpha,beta-unsaturated ketone dienophile gave only undesired endo-products. Interestingly, the imine dienophile shows remarkable stability in water. [reaction: see text]

Discovery of danoprevir (ITMN-191/R7227), a highly selective and potent inhibitor of hepatitis C virus (HCV) NS3/4A protease.

HCV serine protease NS3 represents an attractive drug target because it is not only essential for viral replication but also implicated in the viral evasion of the host immune response pathway through direct cleavage of key proteins in the human innate immune system. Through structure-based drug design and optimization, macrocyclic peptidomimetic molecules bearing both a lipophilic P2 isoindoline carbamate and a P1/P1' acylsulfonamide/acylsulfamide carboxylic acid bioisostere were prepared that possessed subnanomolar potency against the NS3 protease in a subgenomic replicon-based cellular assay (Huh-7). Danoprevir (compound 49) was selected as the clinical development candidate for its favorable potency profile across multiple HCV genotypes and key mutant strains and for its good in vitro ADME profiles and in vivo target tissue (liver) exposures across multiple animal species. X-ray crystallographic studies elucidated several key features in the binding of danoprevir to HCV NS3 protease and proved invaluable to our iterative structure-based design strategy.

Pyrazolo[3,4-b]pyridine kinase inhibitors: a patent review (2008--present).

INTRODUCTION: Numerous heterocycles occur as recurring motifs in the design of kinase inhibitors. Pyrazolo[3,4-b]pyridine has proved particularly versatile due to its ability to interact with kinases via multiple binding modes. As such, this scaffold has been claimed for kinase inhibition in many patents originating from several companies and universities, and these cover a broad range of kinase targets. AREAS COVERED: Patents from 2008 to the present in which pyrazolo[3,4-b] pyridine is utilized as a key element for inhibitor binding are included. This scaffold typically binds to the hinge region of the kinase, but examples in which other key interactions are formed are included in this review. Articles published in peer-reviewed journals that supplement information provided in the patent literature are highlighted. EXPERT OPINION: Several bicyclic heterocycles are capable of forming a hydrogen bond donor-acceptor pair. This interaction is common among kinase inhibitors, particularly at the hinge region. These heterocycles are elaborated to form additional interactions in the kinase pocket which provide potency and selectivity, and thus serve as key scaffolds in kinase inhibitor design. Pyrazolo[3,4-b]pyridine can be viewed as having elements of both pyrrolo[2,3-b]pyridine and indazole and can therefore achieve multiple kinase binding modes. This scaffold is often encountered in kinase inhibitors as a result. Examination of the patent literature suggests that in some cases this group is simply one of several hinge binders examined for a particular series. In other cases, the pyrazolo[3,4-b]pyridine appears to provide an advantage, either in terms of intellectual property, inhibitor activity, physical properties or synthetic flexibility.

Systemic in vitro and in vivo evaluation for determining the feasibility of making an amorphous solid dispersion of a B-Raf (rapidly accelerated fibrosarcoma) inhibitor.

It is well acknowledged that oral bioavailability of a drug candidate is often influenced by factors such as the permeability, physico-chemical properties, and metabolism of the drug. Among the physico-chemical properties, solubility and dissolution rate are considered the most critical factors affecting the oral bioavailability of a compound G-F is a potent and selective B-Raf inhibitor with poor solubility and adsorption is limited by solubility at high doses. In order to overcome this issue using a spray-dried amorphous dispersion (SDD) formulation was evaluated. A combination of theoretical solubility prediction and in vitro dissolution, were used to predict the in vivo exposure of G-F. The predicted value was found to have good agreement with the in vivo exposure from dosing the crystalline and amorphous form of G-F. In general, this combined approach demonstrated that the amorphous form of G-F offers an advantage over the crystalline form of G-F in terms of solubility; in vitro dissolution and in vivo absorption were predictable and consistent with the literature. This systemic approach provides a great value for compound development.

Potent and selective aminopyrimidine-based B-Raf inhibitors with favorable physicochemical and pharmacokinetic properties.

Recent clinical data provided proof-of-concept for selective B-Raf inhibitors in treatment of B-Raf(V600E) mutant melanoma. Pyrazolopyridine-type B-Raf inhibitors previously described by the authors are potent and selective but exhibit low solubility requiring the use of amorphous dispersion-based formulation for achieving efficacious drug exposures. Through structure-based design, we discovered a new class of highly potent aminopyrimidine-based B-Raf inhibitors with improved solubility and pharmacokinetic profiles. The hinge binding moiety possesses a basic center imparting high solubility at gastric pH, addressing the dissolution limitation observed with our previous series. In our search for an optimal linker-hinge binding moiety system, amide-linked thieno[3,2-d]pyrimidine analogues 32 and 35 (G945), molecules with desirable physicochemical properties, emerged as lead compounds with strong efficacy in a B-Raf(V600E) mutant mouse xenograft model. Synthesis, SAR, lead selection, and evaluation of key compounds in animal studies will be described.

Pyrazolopyridine Inhibitors of B-Raf(V600E). Part 1: The Development of Selective, Orally Bioavailable, and Efficacious Inhibitors.

The V600E mutation of B-Raf kinase results in constitutive activation of the MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-based design, a novel series of pyrazolopyridine inhibitors of B-Raf(V600E) was developed. Optimization led to the identification of 3-methoxy pyrazolopyridines 17 and 19, potent, selective, and orally bioavailable agents that inhibited tumor growth in a mouse xenograft model driven by B-Raf(V600E) with no effect on body weight. On the basis of their in vivo efficacy and preliminary safety profiles, 17 and 19 were selected for further preclinical evaluation.