Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase (JAK) inhibitors

A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g–4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3–JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3–JAK1 pathway versus JAK2, and active in a human whole blood assay.     

The discovery of 2,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 2 (JAK2) inhibitors versus JAK1 and JAK3

Members of the Janus kinase (JAK) family are potential therapeutic targets. Abnormal signaling by mutant JAK2 is related to hematological malignancy, such as myeloproliferative neoplasms (MPNs), and tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC). We discovered a potent and highly selective inhibitor of JAK2 over JAK1 and -3 based on the structure of 4-(2,5-triazole)-pyrrolopyrimidine. Among all triazole compounds tested, 2,5-triazole regioisomers more effectively inhibited JAK2 kinase activity than isomers with substitutions of various alkyl groups at the R₂ position, except for methyl-substituted 1,5-triazole, which was more potent than the corresponding 1,4- and 2,5-triazoles. None of the synthesized 1,4-isomers inhibited all three JAK family members. Compounds with phenyl or tolyl group substituents at the R₁ position were completely inactive compared with the corresponding analogues with a methyl substituted at the R₁ position. As a result of this structure–activity relationship, 54, which is substituted with a cyclopropylmethyl moiety, exhibited significant inhibitory activity and selectivity (IC₅₀ = 41.9 nM, fold selectivity JAK1/2 10.6 and JAK3/2 58.1). Compound 54 also exhibited an equivalent inhibition of wild type JAK2 and the V617F mutant. Moreover, 54 inhibited the proliferation of HEL 92.1.7 cells, which carry JAK2 V617F, and gefitinib-resistant HCC827 cells. Compound 54 also suppressed STAT3 phosphorylation at Y705.     

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.     

Identification of 4-(2-furanyl)pyrimidin-2-amines as Janus kinase 2 inhibitors

Janus kinases inhibitor is considered to have therapeutic potential for the treatment of oncology and immune-inflammatory diseases. Two series of 4-(2-benzofuranyl)pyrimidin-2-amine and 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl)pyrimidin-2-amine derivatives have been designed and synthesized. Primary SAR studies resulted in the discovery of a novel class of 4,5,6,7-tetrahydrofuro[3,2-c]pyridine based JAK2 inhibitors with higher potency (IC₅₀ of 0.7 nM) and selectivity (>30 fold) to JAK3 kinase than tofacitinib.     

Scaffold hopping towards potent and selective JAK3 inhibitors: Discovery of novel C-5 substituted pyrrolopyrazines

The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.     

Discovery of potent anti-inflammatory 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl) pyrimidin-2-amines for use as Janus kinase inhibitors

The Janus kinase (JAK) family of tyrosine kinases has been proven to provide targeted immune modulation. Orally available JAK inhibitors have been used for the treatment of immune-mediated inflammatory diseases, such as rheumatoid arthritis (RA). Here, we report the design, synthesis and biological evaluation of 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl) pyrimidin-2-amino derivatives as JAK inhibitors. Systematic structure–activity relationship studies led to the discovery of compound 7j, which strongly inhibited the four isoforms of JAK kinases. Molecular modeling rationalized the importance of cyanoacetyl and phenylmorpholine moieties. The in vivo investigation indicated that compound 7j possessed favorable pharmacokinetic properties and displayed slightly better anti-inflammatory efficacy than tofacitinib at the same dosage. Accordingly, compound 7j was advanced into preclinical development.     

Discovery of 3,6-diaryl-1H-pyrazolo[3,4-b]pyridines as potent anaplastic lymphoma kinase (ALK) inhibitors

A new series of 3,6-diaryl-1H-pyrazolo[3,4-b]pyridine compounds have been discovered as potent anaplastic lymphoma kinase (ALK) inhibitors. The 4-hydroxyphenyl in the 6-position of 1H-pyrazolo[3,4-b]pyridine were crucial and a fluorine atom substitution could give promising inhibitory activity. The IC₅₀ of compound 9v against ALK was up to 1.58?nM and a binding mechanism was proposed.     

Discovery of novel c-Met kinase inhibitors bearing a thieno[2,3-d]pyrimidine or furo[2,3-d]pyrimidine scaffold

A series of thieno[2,3-d]pyrimidines and furo[2,3-d]pyrimidines were synthesized and evaluated for the c-Met inhibition. Thieno[2,3-d]pyrimidine 6b stood out as the most potent showing an IC(50) of 35.7 nM. This compound displayed high inhibitory effect on cell proliferation in BaF3-TPR-Met cells and showed high selectivity for c-Met family against other 14 tested kinases. However, compound 6b was found ineffective in the c-Met-dependent U-87MG human gliobastoma xenograft model that may be relevant to its poor PK profile.     

Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors

Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs. Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine derivatives were designed and synthesized by various strategies. The pharmacological results identified the optimized compounds J-4 and J-6, which exerted high potency against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-6 effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4 and J-6 might serve as promising JAK1/2 inhibitors for further investigation.     

The impact of binding site waters on the activity/selectivity trade-off of Janus kinase 2 (JAK2) inhibitors

Structure based optimization of B39, an indazole-based low micromolar JAK2 virtual screening hit is reported. Analysing the effect of certain modifications on the activity and selectivity of the analogues suggested that these parameters are influenced by water molecules available in the binding site. Simulation of water networks in combination with docking enabled us to identify the key waters and to optimize our primary hit into a low nanomolar JAK2 lead with promising selectivity over JAK1.     

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.     

Design and optimization of a series of 4-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-2-amines: Dual inhibitors of TYK2 and JAK1

Herein, we disclose a new series of TYK2/ JAK1 inhibitors based upon a 3.1.0 azabicyclic substituted pyrimidine scaffold. We illustrate the use of structure-based drug design for the initial design and subsequent optimization of this series of compounds. One advanced example 19 met program objectives for potency, selectivity and ADME, and demonstrated oral activity in the adjuvant-induced arthritis rat model.     

Novel 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines as EGFR and erbB2 receptor tyrosine kinase inhibitors

Novel 4-anilino-1H-pyrazolo[3,4-d]pyrimidines have been synthesized and evaluated in vitro for erbB2 and EGFR kinase inhibition. A representative compound displaying oral bioavailability in rat and dog illustrates the potential of this series to provide orally active erbB2 inhibitors.     

Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine inhibitors of Erk2

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.     

Novel 1,5-diphenyl-6-substituted 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones induced apoptosis in RKO colon cancer cells

Novel 1,5-diphenyl-6-substituted-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones were synthesized and characterized. All compounds were screened for their anti-proliferative activities in five different cancer cell lines. The results showed that compounds 7a and 7b comprising aminoguanidino or guanidino moiety at position 6 inhibited proliferation of RKO colon cancer cells with IC₅₀ of 8 and 4?μM, respectively. Compounds 7a and 7b induced apoptosis in RKO cells, which was confirmed by TUNEL and annexin V-FITC assays. Flow cytometric analysis indicated that compounds 7a and 7b arrested RKO cells in the G1 phase and the most active compound 7b increased levels of p53, p21, Bax, ERK1/2 and reduced levels of Bcl2 and Akt. Compound 7b also activates release of cytochrome c, which is consistent with activation of caspase-9. Additionally, compound 7b increased caspase-3 activity and cleaved PARP-1 in RKO cells. Collectively, these findings could establish a molecular basis for the development of new anti-cancer agents.     

Synthesis and biological evaluation of novel pyrazolo[1,5-a]pyrimidines: Discovery of a selective inhibitor of JAK1 JH2 pseudokinase and VPS34

A series of novel 3,6-di-substituted or 3-substituted pyrazolo[1,5-a]pyrimidines were prepared via a microwave-assisted approach that generated a broad array of derivatives in good yields (20–93%, ave. = 59%). The straightforward synthesis involved sequential treatment of commercially-available acetonitrile derivatives with DMF-dimethylacetal (120 °C, 20 min), followed by treatment with NH₂NH₂·HBr (120 °C, 20 min), and 1,1,3,3-tetramethoxypropane or 2-aryl-substituted malondialdehdyes (120 °C, 20 min). Compounds were screened for antimitotic activities against MCF7 breast cancer and/or A2780 ovarian cancer cell lines in vitro. The most active compounds exhibited EC₅₀ values ranging from 0.5 to 4.3 μM, with the 3-(4-(trifluoromethyl)phenyl)-6-[4-(2-(piperidin-1-yl)ethoxy]phenyl analogue (34e) and the 3-(2-fluorophenyl)-6-[4-(2-(4-methylpiperizin-1-yl)ethoxy]phenyl analogue (35a) being two to three fold more active than Compound C (Dorsomorphin) in A2780 and MCF7 assays, respectively. Importantly, a monosubstituted 3-(benzothiazol-2-yl) derivative (13) was equipotent with the more synthetically challenging 3,6-disubstituted derivatives (34a–e and 35a–e), and exhibited a promising and unique selectivity profile when screened against a panel consisting of 403 protein kinases (Kinomescan™ selectivity score = 0.005, K_d = 0.55 ± 0.055 μM and 0.410 ± 0.20 μM for JAK1 JH2 pseudokinase and VPS34, respectively).     

Discovery of 3,6-dihydro-2H-pyran as a morpholine replacement in 6-aryl-1H-pyrazolo[3,4-d]pyrimidines and 2-arylthieno[3,2-d]pyrimidines: ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

The morpholine hinge-region binding group on a series of pyrazolopyrimidine and thienopyrimidine mammalian target of rapamycin (mTOR) inhibitors was replaced with 3,6-dihydro-2H-pyran (DHP), giving compounds of equivalent potency and selectivity versus PI3K. These results establish the DHP group as a hinge-region binding motif for the preparation of highly potent and selective mTOR inhibitors.     

Discovery of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors

Herein, we report the discovery of a series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies of these compounds led to the identification of the most potent compound, 3-(3-methoxybenzyl)-6-(1H-pyrrolo[2,3-b]pyridin-4-yl)thieno[2,3-d]pyrimidin-4(3H)-one (8k), which showed IC₅₀ values of 0.004 μM and 0.001 μM against ROCK Ⅰ and ROCK Ⅱ, respectively. In vitro, 8k significantly reduced the phosphorylation level of ROCK downstream signaling protein and induce changes in cell morphology and migration. Overall, this study provides a promising lead compound for drug discovery targeting ROCKs.     

Discovery of 2-((1H-benzo[d]imidazol-1-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-4-ones as novel PKM2 activators

The M2 isoform of pyruvate kinase is an emerging target for antitumor therapy. In this letter, we describe the discovery of 2-((1H-benzo[d]imidazol-1-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-4-ones as potent and selective PKM2 activators which were found to have a novel binding mode. The original lead identified from high throughput screening was optimized into an efficient series via computer-aided structure-based drug design. Both a representative compound from this series and an activator described in the literature were used as molecular tools to probe the biological effects of PKM2 activation on cancer cells. Our results suggested that PKM2 activation alone is not sufficient to alter cancer cell metabolism.