Part 1: Structure–Activity Relationship (SAR) investigations of fused pyrazoles as potent,selective and orally available inhibitors of p38α mitogen-activated protein kinase

A novel class of fused pyrazole-derived inhibitors of p38α mitogen-activated protein kinase (MAPK) is disclosed. These inhibitors were evaluated for their ability to inhibit the p38α enzyme, the secretion of TNFα in a LPS-challenged THP1 cell line and TNFα-induced production of IL-8 in 50% human whole blood. This series was optimized through a SAR investigation to provide inhibitors with IC₅₀ values in the low single-digit nanomolar range in whole blood. Further investigation of their pharmacokinetic profiles led to the identification of two potent and orally bioavailable p38 inhibitors 10m and 10q. Inhibitor 10m was found to be efficacious in vivo in the inhibition of TNFα production in LPS-stimulated Lewis rats with an ED₅₀ of 0.1 mg/kg while 10q was found to have an ED₅₀ of 0.05–0.07 mg/kg.     

Structure-based design,synthesis, and biological evaluation of imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors

We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal structure, Phe169 adopts two conformations, where one interacts with 1 and the other shows no interaction with 1. Our structure-based design strategy shows that these two conformations converge into one via enhanced protein-ligand hydrophobic interactions. According to the strategy, we focused on scaffold transformation to identify imidazo[1,2-b]pyridazine derivatives as potent inhibitors of p38 MAP kinase. Among the herein described and evaluated compounds, N-oxide 16 exhibited potent inhibition of p38 MAP kinase and LPS-induced TNF-α production in human monocytic THP-1 cells, and significant in vivo efficacy in rat collagen-induced arthritis models. In this article, we report the discovery of potent, selective and orally bioavailable imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors with pyridine N-oxide group.     

Discovery of PH-797804, a highly selective and potent inhibitor of p38 MAP kinase

The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (−)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (−)-4a are described.     

Part 2: Structure–activity relationship (SAR) investigations of fused pyrazoles as potent,selective and orally available inhibitors of p38α mitogen-activated protein kinase

A novel class of pyrazolopyridazine p38α mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38α enzyme, the secretion of TNFα in a LPS-challenged THP1 cell line and TNFα-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC₅₀ values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38α inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38α/β over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFα production in LPS-stimulated Lewis rats with an ED₅₀ of ca. 0.08 mg/kg.     

Utilization of a nitrogen–sulfur nonbonding interaction in the design of new 2-aminothiazol-5-yl-pyrimidines as p38α MAP kinase inhibitors

The design, synthesis, and structure–activity relationships (SAR) of a series of 2-aminothiazol-5-yl-pyrimidines as novel p38α MAP kinase inhibitors are described. These efforts led to the identification of 41 as a potent p38α inhibitor that utilizes a unique nitrogen–sulfur intramolecular nonbonding interaction to stabilize the conformation required for binding to the p38α active site. X-ray crystallographic studies that confirm the proposed binding mode of this class of inhibitors in p38α and provide evidence for the proposed intramolecular nitrogen–sulfur interaction are discussed.     

Substituted N-aryl-6-pyrimidinones: a new class of potent, selective, and orally active p38 MAP kinase inhibitors

A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-α in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.     

2,4-Diamino-8-quinazoline carboxamides as novel,potent inhibitors of the NAD hydrolyzing enzyme CD38: Exploration of the 2-position structure-activity relationships

Starting from 4-amino-8-quinoline carboxamide lead 1a and scaffold hopping to the chemically more tractable quinazoline, a systematic exploration of the 2-substituents of the quinazoline ring, utilizing structure activity relationships and conformational constraint, resulted in the identification of 39 novel CD38 inhibitors. Eight of these analogs were 10–100-fold more potent human CD38 inhibitors, including the single digit nanomolar inhibitor 1am. Several of these molecules also exhibited improved therapeutic indices relative to hERG activity. A representative analog 1r exhibited suitable pharmacokinetic parameters for in vivo animal studies, including moderate clearance and good oral bioavailability. These inhibitor compounds will aid in the exploration of the enzymatic functions of CD38, as well as furthering the study of the therapeutic implications of NAD enhancement in metabolic disease models.     

The discovery and evaluation of 3-amino-2(1H)-pyrazinones as a novel series of selective p38α MAP kinase inhibitors

The discovery and optimisation of a novel series of potent and selective p38α inhibitors is described. Evaluating the structure-activity relationship of an aminoalkyl substituent at the 3 position of the 2(1H)-pyrazinone core, p38α potency was increased 20000-fold. The most advanced compound (25) demonstrated excellent in vivo properties suitable for an inhaled route of administration.     

The identification of novel p38α isoform selective kinase inhibitors having an unprecedented p38α binding mode

A novel series of p38 MAP kinase inhibitors with high selectivity for the p38α isoform over the other family members including the highly homologous p38β isoform has been identified. X-ray co-crystallographic studies have revealed an unprecedented kinase binding mode in p38α for representative analogs, 5c and 9d, in which a Leu108/Met109 peptide flip occurs within the p38α hinge region. Based on these findings, a general strategy for the rational design of additional promising p38α isoform selective inhibitors by targeting this novel binding mode is proposed.     

Design, synthesis and activity of a potent, selective series of N-aryl pyridinone inhibitors of p38 kinase

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.     

Synthesis,antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-hydrazone derivatives

In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth inhibition and apoptosis induction characteristics are being strongly screened for their cancer chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to evaluate for their anti-proliferative and apoptosis induction potential against cervical (HeLa), breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293 cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further, mechanistic studies were conducted on compound 14a to understand the biochemical mechanisms and functional interactions with various signaling pathways triggered in HeLa and MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen species. In downstream signaling pathway, activated p53 protein interacted with MAPK pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38 on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125) prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing and were dependent on p53 activity. Based on the results, compound 14a was identified as a promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo experiments on anti-proliferative activity.     

Synthesis and biological evaluation of p38α kinase-targeting dialkynylimidazoles

Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical intermediates, a series of 1,2-dialkynylimidazoles were designed as potential irreversible p38 MAP kinase α-isoform (p38α) inhibitors. The synthesis of these dialkynylimidazoles and their kinase inhibition activity is reported. The 1-ethynyl-substituted dialkynylimidazole 14 is a potent (IC₅₀ = 200 nM) and selective inhibitor of p38α. Moreover, compound 14 covalently modifies p38α as determined by ESI-MS after 12 h incubation at 37 °C. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450 2D6 inhibition by compound 14 demonstrate that dialkynylimidazoles are a new, promising class of p38α inhibitors.     

S-Benzylisothiourea derivatives as small-molecule inhibitors of indoleamine-2,3-dioxygenase

S-Benzylisothiourea 3a was discovered by its ability to inhibit indoleamine-2,3-dioxygenase (IDO) in our screening program. Subsequent optimization of the initial hit 3a lead to the identification of sub-μM inhibitors 3r and 10h, both of which suppressed kynurenine production in A431 cells. Synthesis and structure–activity relationship of S-benzylisothiourea analogues as small-molecule inhibitors of IDO are described.     

Fluorescence polarization-based assays for detecting compounds binding to inactive c-Jun N-terminal kinase 3 and p38α mitogen-activated protein kinase

Two fluorescein-labeled pyridinylimidazoles were synthesized and evaluated as probes for the binding affinity determination of potential kinase inhibitors to the c-Jun N-terminal kinase 3 (JNK3) and p38α mitogen-activated protein kinase (MAPK). Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using 1-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-yl)-3-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)thiourea (5) as an FP probe (JNK3: K_d = 3.0 nM; p38α MAPK: K_d = 5.7 nM). The validation of the assays with known inhibitors of JNK3 and p38α MAPK revealed that both FP assays correlate very well with inhibition data received by the activity assays. This, in addition to the viability of both FP-based binding assays for the high-throughput screening procedure, makes the assays suitable as inexpensive prescreening protocols for JNK3 and p38α MAPK inhibitors.     

Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers,as potent JAK3 kinase inhibitors

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.     

Discovery of pyrazolo[1,5-a]pyrimidine-based Pim inhibitors: A template-based approach

The synthesis and hit-to-lead SAR development from a pyrazolo[1,5-a]pyrimidine-derived hit 5 to the identification of a series of potent, pan–Pim inhibitors such as 11j are described.     

Synthesis and molecular docking study of new benzofuran and furo[3,2-g]chromone-based cytotoxic agents against breast cancer and p38α MAP kinase inhibitors

This study deals with synthesis of a new set of benzofuran and 5H-furo[3,2-g]chromone linked various heterocyclic functionalities using concise synthetic approaches aiming to gain new antiproliferative candidates against MCF-7 breast cancer cells of p38α MAP kinase inhibiting activity. The biological data proved the significant sensitivity of breast cancer cell lines MCF-7 towards most of the prepared compounds in comparison with doxorubicin. In addition, compounds IIa,b, Va,b, VIa,b, VIIa,b, VIIIa,b, XIc showed significant in vitro p38α MAPK inhibiting potency comparable to the reference standard SB203580. Cell cycle analysis and apoptosis detection data demonstrated that compound VIa induced G2/M phase arrest and apoptosis in MCF-7 cancer cells, in addition to its activation of the caspases-9 and -3. Gold molecular docking studies rationalized the highly acceptable correlation between the calculated docking scores of fitness and the biological data of p38α MAP kinase inhibition. The newly prepared benzofuran and 5H-furo[3,2-g]chromone derivatives might be considered as new promising nuclei in anti-breast cancer chemotherapeutics for further functionalization, optimization and in-depth biological studies.     

Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes: 1. Identification of 1-amino-1-cycloalkyl carboxylic acid headgroups

Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.     

7-Phenyl-pyrido[2,3-d]pyrimidine-2,4-diamines: Novel and highly selective protein tyrosine phosphatase 1B inhibitors

High throughput screening of the Roche compound collection led to the identification of diaminopyrroloquinazoline series as a novel class of PTP1B inhibitors. Structural modification of diaminopyrroloquinazoline series resulted in pyrido[2,3-d]pyrimidine-2,4-diamine series which was further optimized to give compounds 5 and 24 as potent, selective (except T-cell phosphatase) PTP1B inhibitors with good mouse PK properties.