Synthesis and biological evaluation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors

The synthesis and biological evaluation of a number of differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives are reported. From the inhibition results on a selection of disease-relevant protein kinases [IC₅₀ (μM) DYRK1A = 11; CDK5 = 0.41; GSK-3 = 1.5] we have observed that 3,6-diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4) constitutes a potential new and simple lead compound in the search of drugs for the treatment of Alzheimer’s disease.     

Methanesulfonamido-cyclohexylamine derivatives of 2,4-diaminopyrimidine as potent ALK inhibitors

The incorporation of R,R-1,2-diaminocyclohexane at C4 in a series of 2,4-diaminopyrimidines led to a number of ALK inhibitors in which optimized activity was achieved by conversion of the 2-amino group into a methanesulfonamide. Tumor growth inhibition was observed when an orally bioavailable analog was evaluated in a Karpas-299 tumor xenograft mouse model.     

Discovery of novel tetracyclic compounds as anaplastic lymphoma kinase inhibitors

Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is considered a promising therapeutic target for human cancers. We identified novel tetracyclic derivatives as potent ALK inhibitors. Among them, compound 27 showed strong cytotoxicity against KARPAS-299 with an IC₅₀ value of 21 nM and significant antitumor efficacy in ALK fusion-positive blood and solid cancer xenograft models in mice without body weight loss.     

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.     

Pyrazolone-based anaplastic lymphoma kinase (ALK) inhibitors: control of selectivity by a benzyloxy group

Anaplastic lymphoma kinase (ALK) is transmembrane receptor tyrosine kinase, with oncogenic variants that have been implicated in ALCL, NSCLC and other cancers. Screening of a VEGFR2-biased kinase library resulted in identification of 1 which showed cross-reactivity with ALK. SAR on the indole segment of 1 showed that a subtle structural modification (the ethoxy group of 1 changed to a benzyloxy to generate 5a) enhanced potency (ALK), selectivity for VEGFR2 and IR along with improvement in metabolic stability. From docking studies of ALK versus VEGFR2 kinase, we postulated that the loss of entropy of the VEGFR2 in the bound form with 5a might be the origin of the reduced activity against that protein. Modification of the heterocyclic segment showed that thiazole-bearing pyrazolones preserved enzyme potency, and enhanced inhibition of NPM-ALK autophosphorylation in ALK-positive ALCL cells (Karpas-299). SAR of the benzyloxy group resulted in compounds which demonstrated good cellular potency in Karpas-299 cells. Compound 8 showed best overall profile for the series with broad kinome selectivity and liver micorsome stability. Compound 8 showed reasonable iv PK in rat, but with little oral exposure.     

Discovery,synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.     

Identification of acidic heterocycle-substituted 1H-pyrazolo[3,4-b]pyridines as soluble guanylate cyclase stimulators

Novel guanylate cyclase stimulators are disclosed. Design, synthesis, SAR, and pharmacological profile of the compounds are discussed.     

Discovery of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines as potent inhibitors of the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase

Exploration of the SAR around a series of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines led to the discovery of novel pyrrolopyridine inhibitors of the IGF-1R tyrosine kinase. Several compounds demonstrated nanomolar potency in enzyme and cellular mechanistic assays.     

Structure-based design and synthesis of 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives as Janus kinase 3 inhibitors

Janus kinases (JAKs) regulate various inflammatory and immune responses and are targets for the treatment of inflammatory and immune diseases. Here we report the discovery and optimization of 1H-pyrazolo[3,4-d]pyrimidin-4-amino as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Our optimization study gave compound 12a, which exhibited potent JAK3 inhibitory activity (IC₅₀ of 6.2?nM) as well as excellent JAK kinase selectivity (>60-fold). In cellular assay, 12a exhibited potent immunomodulating effect on IL-2-stimulated T cell proliferation (IC₅₀ of 9.4?μM). Further, compound 12a showed efficacy in delayed hypersensitivity assay. The data supports the further investigation of these compounds as novel JAKs inhibitors.     

Discovery of potent,low-absorbable sodium-dependent glucose cotransporter 1 (SGLT1) inhibitor SGL5213 for type 2 diabetes treatment

A new series of C-phenyl d-glucitol derivatives was designed and synthesized, and their SGLT1 inhibitory potency and absorbability were evaluated. We also investigated whether kidney drug retention could be avoided by creating molecules with different excretion pathways. To achieve a class of molecules with low absorption and that were excreted in bile, optimized synthesis was performed to bring the ClogP value and the topological polar surface area to within the appropriate ranges. Compounds 34d and 34j were poorly absorbed, but the absorbed compounds were mainly excreted in bile. Thus, smaller amounts of persistent residue in the kidneys were observed. Since 34d exerted a glucose-lowering effect at a dose of 0.3?mg/kg (p.o.) in SD rats, this compound (SGL5213) could be a clinical candidate for the treatment of type 2 diabetes.     

Identification of amides derived from 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained.     

6-aryl-pyrazolo[3,4-b]pyridines: potent inhibitors of glycogen synthase kinase-3 (GSK-3)

A novel series of 6-aryl-pyrazolo[3,4-b]pyridines has been identified that are potent inhibitors of glycogen synthase kinase-3 (GSK-3).     

Identification of 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c]carbazoles as new Pim kinase inhibitors

New 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c]carbazoles were prepared and evaluated for their Pim kinase inhibitory potencies as well as their antiproliferative activities toward two prostatic cancer cell lines. Pyrazolocarbazole 15a was found to be a potent Pim kinase modulator with inhibitory potency toward the three isoforms. Compound 6c strongly inhibited Pim-3 with weaker effect toward Pim-1 and Pim-2, and thus could be used as an interesting molecular tool to study Pim-3 biological functions.     

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.     

5-aryl-pyrazolo[3,4-b]pyridines: potent inhibitors of glycogen synthase kinase-3 (GSK-3)

A novel series of pyrazolo[3,4-b]pyridines has been identified that are potent inhibitors of glycogen synthase kinase-3 (GSK-3).     

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 novel substituted octahydropyrrolo[3,4-c]pyrroles as dual orexin receptor antagonists for insomnia treatment

A series of octahydropyrrolo[3,4-c]pyrroles were synthesized and evaluated by orexin 1 and 2 receptor (OX_{1 & 2} R) antagonists assays. Compound 14l with potent OXR antagonist activity and suitable pharmacokinetic behavior was chosen to be investigated in an EEG study, which demonstrated effects of sleep promotion comparable to Suvorexant. Furthermore, the di-fluro substituted analogs exhibited reduced hERG inhibition while maintaining moderate potency.     

Discovery and structure-activity relationship of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a new series of potent apoptosis inducers

We report the discovery and SAR study of a series of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as potent inducers of apoptosis. N-(3-Acetylphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine (2) was discovered through our cell- and caspase-based HTS assays as an inducer of apoptosis. Compound 2 is active against cancer cells derived from several human solid tumors, with EC(50) values ranging from 400 to 700 nM. SAR study of hit 2 led to the discovery of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a novel series of potent apoptosis inducers, with 1,3-dimethyl-N-(4-propionylphenyl)-1H-pyrazolo[3,4-b]quinolin-amine (6b) having EC(50) values ranging from 30 to 70 nM in cancer cells. These compounds also demonstrated potent activity in the cell growth inhibition assay, with GI(50) values of 16-42 nM for compound 6b.     

4,5-Dihydro-1H-pyrazolo[4,3-h]quinazolines as potent and selective Polo-like kinase 1 (PLK1) inhibitors

A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1 inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of action and with good in vivo antitumor efficacy in two xenograft models after i.v. administration.     

Potency switch between CHK1 and MK2: Discovery of imidazo[1,2-a]pyrazine- and imidazo[1,2-c]pyrimidine-based kinase inhibitors

Chemistry has been developed to access both imidazo[1,2-a]pyrazines and imidazo[1,2-c]pyrimidines. Small structural modifications in both series led to a switch of potency between two kinases involved in mediating cell cycle checkpoint control, CHK1 and MK2.