Identification of a novel series of azabenzimidazole-derived inhibitors of spleen tyrosine kinase

Spleen Tyrosine Kinase (SYK) is a well-studied enzyme with therapeutic applications in oncology and autoimmune diseases. We identified an azabenzimidazole (ABI) series of SYK inhibitors by mining activity data of 86,000 compounds from legacy biochemical assays with SYK and other homologous kinases as target enzymes. A structure-based design and hybridization approach was then used to improve the potency and kinase selectivity of the hits. Lead compound 23 from this novel ABI series has a SYK IC₅₀ = 0.21 nM in a biochemical assay and inhibits growth of SUDHL-4 cells at a GI₅₀ = 210 nM.     

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC₅₀ = 160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC₅₀ = 47 nM) was a highly specific JNK inhibitor.     

Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): Optimization for JNK potency and physicochemical properties

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC₅₀ of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.     

In vivo optimization of 2,3-diaminopyrazine Rho Kinase inhibitors for the treatment of glaucoma

A series of 2,3,6-pyrazine Rho Kinase inhibitors were optimized for in vivo activity for topical ocular dosing. Modifications of the 2-(piperazin-1-yl)pyrazine derivatives produced compounds with improved solubility and physicochemical properties. Modifications of the 6-pyrazine substituent led to improvements in in vitro potency. Compound 9 had the best in vitro and in vivo potency of EC₅₀ = 260 nM with a 30% reduction of IOP in a non-human primate model at a dose of 0.33%.     

Structure-based optimization and biological evaluation of trisubstituted pyrazole as a core structure of potent ROS1 kinase inhibitors

Recently inhibition of ROS1 kinase has proven to be a promising strategy for several indications such as glioblastoma, non-small cell lung cancer (NSCLC), and cholangiocarcinoma. Our team reported trisubstituted pyrazole-based ROS1 inhibitors by which two inhibitors showed good IC₅₀ values in enzyme-based screening. To develop more advanced ROS1 inhibitors through SAR this trisubstituted pyrazole-based scaffold has been built. Consequently, 16 compounds have been designed, synthesized and shown potent IC₅₀ values in the enzymatic assay, which are from 13.6 to 283 nM. Molecular modeling studies explain how these ROS1 kinase inhibitors revealed effectively the key interactions with ROS1 ATP binding site. Among these compounds, compound 9a (IC₅₀ = 13.6 nM) has exerted 5 fold potency than crizotinib and exhibited high degree of selectivity (selectivity score value = 0.028) representing the number of non-mutant kinases with biological activity over 90% at 10 μM.     

Identification of 5H-chromeno[3,4-c]pyridine and 6H-isochromeno[3,4-c]pyridine derivatives as potent and selective dual ROCK inhibitors

A novel series of 5H-chromeno[3,4-c]pyridine, 6H-isochromeno[3,4-c]pyridine and 6H-isochromeno[4,3-d]pyrimidine derivatives as dual ROCK1 and ROCK2 inhibitors is described. Optimization led to compounds with sub-nanomolar inhibitory affinity for both kinases and excellent kinome selectivity. Compound 19 exhibited ROCK1 and ROCK2 IC₅₀ of 0.67 nM and 0.18 nM respectively.     

The optimization of aminooxadiazoles as orally active inhibitors of Cdc7

A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC₅₀ = 0.71 μM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL = 18 L/h/kg). Extensive optimization resulted in azaindole 26 (Cdc7 IC₅₀ = 1.1 nM, pMCM2 IC₅₀ = 32 nM) that demonstrated robust lowering of pMCM2 in a mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.     

The development of benzimidazoles as selective rho kinase inhibitors

Rho Kinase (ROCK) is a serine/threonine kinase whose inhibition could prove beneficial in numerous therapeutic areas. We have developed a promising class of ATP-competitive inhibitors based upon a benzimidazole scaffold, which show excellent potency toward ROCK (IC₅₀ <10 nM). This report details the optimization of selectivity for ROCK over other related kinases such as Protein kinase A (PKA).     

Development and characterization of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold

A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl)methanones and of (1H-benzo[d][1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)methanones has been prepared and tested on human fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). In the benzylpiperazinyl series, compound 29 (ML30) exhibited an IC₅₀ value of 0.54 nM on MAGL, combined with a 1000-fold selectivity versus FAAH, while compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors (IC₅₀ <10 nM). In the phenylpiperazinyl series, compounds 37, 38, 42, and 43 displayed IC₅₀ values against MAGL in the nanomolar range, whilst being between one and two orders of magnitude less potent on the FAAH, while compounds 31 and 32 were potent FAAH inhibitors (IC₅₀ <20 nM) and over 12-fold selective versus MAGL. The key structural determinants driving the structure–activity relationships were explored by the minimization of the inhibitors inside the active site of both enzymes.     

Development of amino-pyrimidine inhibitors of c-Jun N-terminal kinase (JNK): Kinase profiling guided optimization of a 1,2,3-benzotriazole lead

A series of amino-pyrimidines was developed based upon an initial kinase cross-screening hit from a CDK2 program. Kinase profiling and structure-based drug design guided the optimization from the initial 1,2,3-benzotriazole hit to a potent and selective JNK inhibitor, compound 24f (JNK1 and 2 IC₅₀ = 16 and 66 nM, respectively), with bioavailability in rats and suitable for further in vivo pharmacological evaluation.     

Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.     

Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors

Structure–activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC₅₀ 0.7 nM, FMS cell IC₅₀ 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.     

Oxindole derivatives as inhibitors of TAK1 kinase

Several series of oxindole analogues were synthesized and screened for inhibitory activity against transforming growth factor-β-activating kinase 1 (TAK1). Modifications around several regions of the lead molecules were made, with a distal hydroxyl group in the D region being critical for activity. The most potent compound 10 shows an IC₅₀ of 8.9 nM against TAK1 in a biochemical enzyme assay, with compounds 3 and 6 showing low micromolar cellular inhibition.     

Structure–activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors

Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase. An optimization study of the acridine series revealed that the structure–activity relationship (SAR) of the acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC₅₀ <60 nM) with 180-fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC₅₀ <400 nM) with a 5.4-fold selectivity over haspin was also identified.     

The discovery of thienopyridine analogues as potent IκB kinase β inhibitors. Part II

An SAR study that identified a series of thienopyridine-based potent IκB Kinase β (IKKβ) inhibitors is described. With focuses on the structural optimization at C₄ and C₆ of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C₄, whereas polar groups with proper orientation at C₆ efficiently enhance compound potency. The most potent analogues inhibit IKKβ with IC₅₀s as low as 40 nM, suppress LPS-induced TNF-α production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-κB reporter gene assay, demonstrating that they directly interfere with the NF-κB signaling pathway.     

Novel quinazoline derivatives bearing various 6-benzamide moieties as highly selective and potent EGFR inhibitors

A series of novel quinazoline derivatives bearing various C-6 benzamide substituents were synthesized and evaluated as EGFR inhibitors, and most showed significant inhibitory potency against EGFR kinase. In particular, compound 6g possessed potent inhibitory activity against EGFR wild-type (IC₅₀?=?5?nM), and strong antiproliferative activity against HCC827 and Ba/F3 (L858R) cell lines. Kinase profiling against a panel of 365 kinases showed that 6g was highly selective for EGFR. Furthermore, 6g showed desirable properties in assays of liver microsome metabolic stability and cytochromes P450 inhibition and preliminary pharmacokinetic study. The overall attractive profile of 6g made it an interesting compound for further development.     

3-[2-(Aminomethyl)-5-[(pyridin-4-yl)carbamoyl]phenyl] benzoates as soft ROCK inhibitors

Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC₅₀ = 2.5 nM) with rapid inactivation in plasma (t_1/2 <5′). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects.     

7-Phenyl-imidazoquinolin-4(5H)-one derivatives as selective and orally available mPGES-1 inhibitors

To identify compounds with strong mPGES-1 inhibitory activity and clear in vitro ADME profile, we optimized the lead compound 1 by carrying our substitutions at the C(7)- and C(8)-positions. Replacement of the bromine atom of 1 with various substituents led to identification of the phenyl group as the best C(7)-substituent giving strong inhibitory activity with good in vitro ADME profile. Further SAR examination on both the C(2)- and the C(7)-phenyl groups provided compound 39 as the best candidate for further development. Compound 39 exhibited strong mPGES-1 inhibitory activity (IC₅₀ = 4.1 nM), potent cell-based functional activity (IC₅₀ = 33 nM) with good mPGES-1 selectivity (over 700-fold), excellent in vitro ADME profile, and good oral absorption in rat PK study.     

Design and synthesis of spirocyclic compounds as HCV replication inhibitors by targeting viral NS4B protein

Two novel series of spirocyclic piperidine analogs appended to a pyrazolo[1,5-a]pyridine core were designed, synthesized and evaluated for their anti-HCV activity. A series of piperidine ketals afforded dispiro 6p which showed excellent in vitro anti-HCV activities (EC₅₀ of 1.5 nM and 1.2 nM against genotype 1a and 1b replicons, respectively). A series of piperidine oxazolidinones afforded 27c which showed EC₅₀’s of 10.9 nM and 6.1 nM against 1a and 1b replicons, respectively. Both compounds 6p and 27c bound directly to non-structural NS4B protein in vitro (IC₅₀’s = 10.2 and 30.4 nM, respectively) and exhibited reduced potency in replicons containing resistance mutations encoding changes in the NS4B protein.     

Development of 2-aryl substituted quinazolin-4(3H)-one,pyrido[4,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4(3H)-one derivatives as microsomal prostaglandin E2 synthase-1 inhibitors

mPGES-1 is inducible terminal synthase acting downstream of COX enzymes in arachidonic acid pathway, regulates the biosynthesis of pro-inflammatory prostaglandin PGE₂. Cardiovascular side effect of coxibs and NSAIDs, selective for COX-2 inhibition, stimulated interest in mPGES-1, a therapeutic target with potential to deliver safe and effective anti-inflammatory drugs. The synthesis and structure activity relationship of a series of compounds from 2-aryl substituted quinazolin-4(3H)-one, pyrido[4,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds as mPGES-1 inhibitor are discussed. A set of analogs (28, 48, 49) were identified with <10 nM potencies in the recombinant human mPGES-1 enzyme and in the A549 cellular assays. These analogs were also found to be potent in the human whole blood assay (<400 nM). Furthermore, the representative compound 48 was shown to be selective with other prostanoid synthases and was able to effectively regulate PGE₂ biosynthesis in clinically relevant inflammatory settings, in comparison with celecoxib.