Highly selective c-Jun N-terminal kinase (JNK) 2 and 3 inhibitors with in vitro CNS-like pharmacokinetic properties prevent neurodegeneration

In this Letter, we describe the discovery of selective JNK2 and JNK3 inhibitors, such as 10, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs, p38α and ERK2. Substitution of the naphthalene ring affords an isoform selective JNK3 inhibitor, 30, with approximately 10-fold selectivity over both JNK1 and JNK2. A naphthalene ring penetrates deep into the selectivity pocket accounting for the differentiation amongst the kinases. Interestingly, the gatekeeper Met146 sulfide interacts with the naphthalene ring in a sulfur-π stacking interaction. Compound 38 ameliorates neurotoxicity induced by amyloid-β in human cortical neurons. Lastly, we demonstrate how to install propitious in vitro CNS-like properties into these selective inhibitors.     

Design and synthesis of 1-aryl-5-anilinoindazoles as c-Jun N-terminal kinase inhibitors

Starting from pyrazole HTS hit (1), a series of 1-aryl-1H-indazoles have been synthesized as JNK3 inhibitors with moderate selectivity against JNK1. SAR studies led to the synthesis of 5r as double digital nanomolar JNK3 inhibitor with good in vivo exposure.     

Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-phenylurea derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor treatment

Based on the roles of Raf1 and JNK1 in hepatocarcinoma development, scaffold-based drug design was employed to produce a series of compounds, which subsequently were synthesized and explored as potential dual inhibitors Raf1 and JNK1 kinases for anti-tumor treatment. The compound 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chloro-phenyl)urea (3d) showed 66%, 67% and 13% inhibition rate at 50 μM against Raf1, JNK1 and p38-alpha, respectively, but no effect on ERK1 and ERK2, and inhibited the expression of pERK1/2 markedly and HepG2 cells proliferation with IC₅₀ at 8.3 μM. Furthermore, 3d showed lower toxicity against normal liver cell-lines QSG7701 and HL7702. Molecular docking study further showed that 3d can fit into binding domain of JNK1 and Raf1. Our data suggested the activities of 3d were associated with dual inhibition of JNK1 and Raf1 kinases.     

Synthesis and SAR of piperazine amides as novel c-jun N-terminal kinase (JNK) inhibitors

A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.     

Highly selective c-Jun N-terminal kinase (JNK) 3 inhibitors with in vitro CNS-like pharmacokinetic properties II. Central core replacement

In this Letter, we describe the evolution of selective JNK3 inhibitors from 1, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs. Strong SAR was found for substitution of the naphthalene ring, as well as for inhibitors adopting different central scaffolds. Significant potency gains were appreciated by inverting the polarity of the thione of the parent triazolothione 1, resulting in potent compounds with attractive pharmacokinetic profiles.     

Discovery of a novel series of 4-quinolone JNK inhibitors

A novel series of highly selective JNK inhibitors based on the 4-quinolone scaffold was designed and synthesized. Structure based drug design was utilized to guide the compound design as well as improvements in the physicochemical properties of the series. Compound (13c) has an IC₅₀ of 62/170 nM for JNK1/2, excellent kinase selectivity and impressive efficacy in a rodent asthma model.     

Peripheral but crucial: A hydrophobic pocket (Tyr706, Leu337, and Met336) for potent and selective inhibition of neuronal nitric oxide synthase

Selective inhibition of the neuronal isoform of nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) has become a promising strategy for the discovery of new therapeutic agents for neurodegenerative diseases. However, because of the high sequence homology of different isozymes in the substrate binding pocket, developing inhibitors with both potency and excellent isoform selectivity remains a challenging problem. Herein, we report the evaluation of a recently discovered peripheral hydrophobic pocket (Tyr⁷⁰⁶, Leu³³⁷, and Met³³⁶) that opens up upon inhibitor binding and its potential in designing potent and selective nNOS inhibitors using three compounds, 2a, 2b, and 3. Crystal structure results show that inhibitors 2a and 3 adopted the same binding mode as lead compound 1. We also found that hydrophobic interactions between the 4-methyl group of the aminopyridine ring of these compounds with the side chain of Met³³⁶, as well as the π–π stacking interaction between the pyridinyl motif and the side chain of Tyr⁷⁰⁶ are important for the high potency and selectivity of these nNOS inhibitors.     

Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors

The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c, whose kinase selectivity is reported.     

Discovery of new potent inhibitors for carbonic anhydrase IX by structure-based virtual screening

Through structure-based virtual screening, some dozen of benzene sulfonamides with novel scaffolds are identified as potent inhibitors against carbonic anhydrase (CA) IX with IC₅₀ values ranging from 2.86 to 588.34 nM. Among them, compounds 1 and 9 show high selectivity against tumor-target CA IX over CA II (the selectivity ratios are 21.3 and 136.6, respectively). The possible binding poses of hit compounds are also explored and the selectivity is elucidated by molecular docking simulations. The hit compounds discovered in this work would provide novel scaffolds for further hit-to-lead optimization.     

Benzimidazole design,synthesis, and docking to build selective carbonic anhydrase VA inhibitors

The similarity of human carbonic anhydrase (CA) active sites makes it difficult to design selective inhibitors for one or several CA isoforms that are drug targets. Here we synthesize a series of compounds that are based on 5-[2-(benzimidazol-1-yl)acetyl]-2-chloro-benzenesulfonamide (1a) which demonstrated picomolar binding affinity and significant selectivity for CA isoform five A (VA), and explain the structural influence of inhibitor functional groups to the binding affinity and selectivity. A series of chloro-substituted benzenesulfonamides bearing a heterocyclic tail, together with molecular docking, was used to build inhibitors that explore substituent influence on the binding affinity to the CA VA isoform.     

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors

The structure–activity and structure–kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11 Å channel leading to the Zn²⁺ catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.     

Design,synthesis and biological evaluation of phosphopeptides as Polo-like kinase 1 Polo-box domain inhibitors

Polo-like kinase 1 (Plk1) is an anti-cancer target due to its critical role in mitotic progression. A growing body of evidence has documented that Peptide-Plk1 inhibitors showed high Plk1 binding affinity. However, phosphopeptides-Plk1 inhibitors showed poor cell membranes permeability, which limits their clinical applications. In current study, nine candidate phosphopeptides consisting of non-natural amino acids were rationally designed and then successfully synthesized using an Fmoc-solid phase peptide synthesis (SPPS) strategy. Moreover, the binding affinities and selectivity were evaluated via fluorescence polarization (FP) assay. The results confirmed that the most promising phosphopeptide 6 bound to Plk1 PBD with the IC₅₀ of 38.99?nM, which was approximately 600-fold selectivity over Plk3 PBD (IC₅₀?=?25.44?μM) and nearly no binding to Plk2 PBD. Furthermore the intracellular activities and the cell membrane permeability of phosphopeptide 6 were evalutated. Phosphopeptide 6 demonstrated appropriate cell membrane permeability and arrested HeLa cells cycle in G2/M phase by regulating CyclinB1-CDK1. Further, phosphopeptide 6 showed typical apoptotic morphology and induced caspase-dependent apoptosis. In conclusion, we expect our discovery can provide new insights into the further optimization of Plk1 PBD inhibitors.     

Design and synthesis of highly selective pyruvate dehydrogenase complex E1 inhibitors as bactericides

In order to obtain PDHc-E1 inhibitors with high selectivity and efficacy, four series (7, 12, 15, and 19) of 35 novel 4-aminopyrimidine derivatives were rationally designed and synthesized based on the binding site of ThDP in E. coli PDHc-E1. 12, 15, and 19 were confirmed to be potent inhibitors against E. coli PDHc-E1. Selected compounds 12g, 12i, 15f, and 19a showed negligible inhibition against porcine PDHc-E1. To understand their selectivity, the interaction of inhibitor and E. coli PDHc-E1 or porcine PDHc-E1 was studied by molecular docking. The newly introduced acylhydrazone and N-phenylbenzamide moieties could form stronger interaction by hydrogen bond at the active site of E. coli PDHc-E1 compared with that of porcine PDHc-E1. A part of title compounds as potent PDHc-E1 inhibitors also exhibited notable antibacterial activity. In particular, 12e, 12f, 12g, 12o, and 19a exhibited 72–92% inhibition against Xanthomonas oryzae pv. Oryzae and Ralstonia solanacearum at 100?μg/mL, which was better than thiodiazole-copper (34 and 29%, respectively) and bismerthiazol (56 and 55%, respectively). The results proved that we could obtain effective bactericidal compounds as highly selective PDHc inhibitors by rational molecular design utilizing the binding model of active site of E. coli PDHc-E1.     

Discovery of (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide as potent and isoform selective ROCK2 inhibitors

ROCK1 and ROCK2 are highly homologous isoforms. Accumulated studies indicate that they have distinct different functions, and the development of isoform selective ROCK inhibitors will pave new roads for the treatment of various diseases. In this work, a series of amide-chroman derivatives were synthesized and biologically evaluated in order to develop potent and isoform selective ROCK2 inhibitors. Remarkably, (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide ((S)-7c) possessed ROCK2 inhibitory activity with an IC₅₀ value of 3?nM and 22.7-fold isoform selectivity (vs. ROCK1). Molecular docking indicated that hydrophobic interactions were the key element for the high potency and isoform selectivity of (S)-7c. The binding free energies predicted by MM/GBSA were in good agreement with the experimental bioactivities, and the analysis of individual energy terms suggested that residue Lys105 in ROCK1 or Lys121 in ROCK2 was the key residue for the isoform selectivity of (S)-7c.     

2,3,5-Trisubstituted pyridines as selective AKT inhibitors. Part II: Improved drug-like properties and kinase selectivity from azaindazoles

A novel series of AKT inhibitors containing 2,3,5-trisubstituted pyridines with novel azaindazoles as hinge binding elements are described. Among these, the 4,7-diazaindazole compound 2c has improved drug-like properties and kinase selectivity than those of indazole 1, and displays greater than 80% inhibition of GSK3β phosphorylation in a BT474 tumor xenograft model in mice.     

Identification of new dual FABP4/5 inhibitors based on a naphthalene-1-sulfonamide FABP4 inhibitor

Fatty acid binding protein 4 (FABP4) and fatty acid binding protein 5 (FABP5) are mainly expressed in adipocytes and/or macrophages and play essential roles in energy metabolism and inflammation. When FABP4 function is diminished, FABP5 expression is highly increased possibly as a functional compensation. Dual FABP4/5 inhibitors are expected to provide beneficial synergistic effect on treating diabetes, atherosclerosis, and inflammation-related diseases. Starting from our previously reported selective FABP4 inhibitor 8, structural biology information was used to modulate the selectivity profile and to design potent dual FABP4/5 inhibitors with good selectivity against FABP3. Two compounds A16 and B8 were identified to show inhibitory activities against both FABP4/5 and good selectivity over FABP3, which could also reduce the level of forskolin-stimulated lipolysis in mature 3T3-L1 adipocytes. Compared with compound 8, these two compounds exhibited better anti-inflammatory effects in lipopolysaccharide-stimulated RAW264.7 murine macrophages, with decreased levels of pro-inflammatory cytokines TNFα and MCP-1 and apparently inhibited IKK/NF-κB pathway.     

Identification of novel series of pyrazole and indole-urea based DFG-out PYK2 inhibitors

Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide–urea and a pyrazole–urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.     

Discovery of novel selective Janus kinase 2 (JAK2) inhibitors bearing a 1H-pyrazolo[3,4-d]pyrimidin-4-amino scaffold

Janus kinases (JAKs) regulate various cancers and immune responses and are targets for the treatment of cancers and immune diseases. A new series of 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives were synthesized and optimized by introducing a functional 3,5-disubstituted-1H-pyrazole moiety into the C-3 moiety of pyrazole template, and then were biologically evaluated as potent Janus kinase 2 (JAK2) inhibitors. Among these molecules, inhibitors 11f, 11g, 11h and 11k displayed strong activity and selectivity against the JAK2 kinase, with IC₅₀ values of 7.2?nM, 6.5?nM, 8.0?nM and 9.7?nM, respectively. In particular, the cellular inhibitory assay and western blot analysis further support the JAK2 selectivity of compound 11g also in cells. Furthermore, compound 11g also exhibited potent inhibitory activity in lymphocytes proliferation assay and delayed hypersensitivity assay. Taken together, the novel JAK2 selective inhibitors discovered in this study may be potential lead compounds for new drug discovery via further development of more potent and selective JAK2 inhibitors.     

Tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives as microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors: SAR and in vivo efficacy in hyperalgesia pain model

A series of substituted tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives have been synthesized and their mPGES-1 biological activity has been disclosed in detail. Structure-activity relationship (SAR) optimization provided inhibitors with excellent mPGES-1 potency and low to moderate PGE₂ release A549 cell potency. Among the mPGES-1 inhibitors studied, 7, 9 and 11l provided excellent selectivity over COX-2 (>200-fold) and >70-fold selectivity for COX-1 except 11l, which exhibited dual mPGES-1/COX-1 activity. Furthermore, the above tested mPGES-1 inhibitors demonstrated good metabolic stability in liver microsomes, high plasma protein binding (PPB) and no significant inhibition observed in clinically relevant CYP isoforms. Besides, selected mPGES-1 tool compounds 9 and 11l provided good in vivo pharmacokinetic profile and oral bioavailability (%F = 33 and 85). Additionally, the representative mPGES-1 tool compounds 9 and 11l revealed moderate in vivo efficacy in the LPS-induced thermal hyperalgesia guinea pig pain model.     

Monocyclic 4-amino-6-(phenylamino)-1,3,5-triazines as inhibitors of human DNA topoisomerase IIα

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Starting from the available information about the binding of the purine-based htIIα inhibitors in the ATP binding site we designed a virtual screening campaign combining structure-based and ligand-based pharmacophores with a molecular docking calculation searching for compounds that would contain a monocycle mimetic of the purine moiety. We discovered novel 4-amino-6-(phenylamino)-1,3,5-triazines 6, 7 and 11 as monocyclic htIIα inhibitors targeting the ATP binding site. Compound 6 from the 1,3,5-triazine series also displayed cytotoxicity properties in hepatocellular carcinoma (HepG2) cell lines and selectivity against human umbilical vein endothelial (HUVEC) cell lines.