Discovery of Potent and Selective Inhibitors of Cdc2-Like Kinase 1 (CLK1) as a New Class of Autophagy Inducers.

Autophagy inducers represent new promising agents for the treatment of a wide range of medical illnesses. However, safe autophagy inducers for clinical applications are lacking. Inhibition of cdc2-like kinase 1 (CLK1) was recently found to efficiently induce autophagy. Unfortunately, most of the known CLK1 inhibitors have unsatisfactory selectivity. Herein, we report the discovery of a series of new CLK1 inhibitors containing the 1H-[1,2,3]triazolo[4,5-c]quinoline scaffold. Among them, compound 25 was the most potent and selective, with an IC50 value of 2 nM against CLK1. The crystal structure of CLK1 complexed with compound 25 was solved, and the potency and kinase selectivity of compound 25 were interpreted. Compound 25 was able to induce autophagy in in vitro assays and displayed significant hepatoprotective effects in the acetaminophen (APAP)-induced liver injury mouse model. Collectively, due to its potency and selectivity, compound 25 could be used as a chemical probe or agent in future mechanism-of-action or autophagy-related disease therapy studies.

Identification of a small molecule that downregulates MITF expression and mediates antimelanoma activity in vitro.

Melanoma is a type of cancer arising from the melanocytes, which are the cells that make up the pigment melanin and are derived from the neural crest. There is no particularly effective therapy once the disease is metastatic, highlighting the need for discovery of novel potent agents. In this investigation, we adopted a zebrafish embryonic pigmentation model to identify antimelanoma agents by screening an in-house small molecule library. With this assay, we found that a small molecule compound, SKLB226, blocked zebrafish pigmentation and pigment cell migration. Mechanism of action studies showed that SKLB226 downregulated MITF mRNA level in both zebrafish embryos and mammalian melanoma cells. Further studies showed that it could efficiently suppress the viability and migration of mammalian melanoma cells. In summary, SKLB226 can be used as a chemical tool to study melanocyte development as well as an antimelanoma lead compound that should be subjected to further structural optimization.

Structural modification of an EGFR inhibitor that showed weak off-target activity against RET leading to the discovery of a potent RET inhibitor.

Here, we describe the structural optimization of a known EGFR inhibitor (compound 1) that showed weak off-target activity against RET. Twenty-six analogs of 1 were synthesized. SAR analysis led to the discovery of several compounds that showed considerable potency against the RET-dependent thyroid cancer cell line TT. Kinase inhibitory potency was then measured for the most active compound (2u) in the cellular assay. The results showed that 2u is a potent RET inhibitor with an IC(50) value of 7 nM.

Structure-activity relationship studies of pyrazolo[3,4-d]pyrimidine derivatives leading to the discovery of a novel multikinase inhibitor that potently inhibits FLT3 and VEGFR2 and evaluation of its activity against acute myeloid leukemia in vitro and in vivo.

We describe the structural optimization of a hit compound, 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)-3-(3-methoxyphenyl)urea (1), which exhibits inhibitory activity but low potency against FLT3 and VEGFR2. A series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized, and structure-activity relationship analysis using cell- and transgenic-zebrafish-based assays led to the discovery of a number of compounds that exhibited both high potency against FLT3-driven human acute myeloid leukemia (AML) MV4-11 cells and a considerable antiangiogenic effect in transgenic-zebrafish-based assays. The compound 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (33), which exhibited the highest activity in preliminary in vivo anti-AML assays, was chosen for further anti-AML studies. The results demonstrated that compound 33 is a multikinase inhibitor that potently inhibits FLT3 and VEGFR2. In an MV4-11 xenograft mouse model, a once-daily dose of compound 33 at 10 mg/kg for 18 days led to complete tumor regression without obvious toxicity. Western blot and immunohistochemical analyses were performed to determine the mechanism of action of compound 33.

Discovery of N6-phenyl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamine derivatives as novel CK1 inhibitors using common-feature pharmacophore model based virtual screening and hit-to-lead optimization.

Aberrant activation of casein kinase 1 (CK1) has been demonstrated to be implicated in the pathogenesis of cancer and various central nervous system disorders. Discovery of CK1 inhibitors has thus attracted much attention in recent years. In this account, we describe the discovery of N6-phenyl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamine derivatives as novel CK1 inhibitors. An optimal common-feature pharmacophore hypothesis, termed Hypo2, was firstly generated, followed by virtual screening using Hypo2 against several chemical databases. One of the best hit compounds, N6-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamine, was chosen for the subsequent hit-to-lead optimization under the guide of Hypo2, which led to the discovery of a new lead compound (1-(3-(3-amino-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)phenyl)-3-(3-chloro-4-fluorophenyl)urea) that potently inhibits CK1 with an IC(50) value of 78 nM.

Discovery of the novel potent and selective FLT3 inhibitor 1-{5-[7-(3- morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]thiadiazol-2-yl}-3-p-tolylurea and its anti-acute myeloid leukemia (AML) activities in vitro and in vivo.

Structure-activity relationship (SAR) studies of 2-(quinazolin-4-ylthio)thiazole derivatives, which are for optimizing the in vitro and in vivo antiacute myeloid leukemia (AML) activity of a previously identified FLT3 inhibitor 2-(6,7-dimethoxyquinazolin-4-ylthio)thiazole (1), are described. SAR studies centering around the head (thiazole) and tails (6- and 7-positions) of the quinazoline moiety of 1 led to the discovery of a series of compounds that exhibited significantly increased potency against FLT3-driven AML MV4-11 cells. Preliminary in vivo assays were carried out on three highly active compounds, whose results showed that 1-{5-[7-(3-morpholinopropoxy)quinazolin-4-ylthio]-[1,3,4]thiadiazol-2-yl}-3-p-tolylurea (20c) had the highest in vivo activity. Further in vitro and in vivo anti-AML studies were then performed on 20c; in an MV4-11 xenograft mouse model, a once-daily dose of 20c at 100 mg/kg for 18 days led to complete tumor regression without obvious toxicity. Western blot and immunohistochemical analysis were carried out to illustrate the mechanism of action of 20c.

4-(4-Nitro-benz-yl)morpholine.

In the title compound, C(11)H(14)N(2)O(3), an inter-molecular inter-action between a nitro group O atom and a neighboring benzene ring helps to stabilize the crystal structure [N⋯centroid = 3.933 (2) Å]. No classical hydrogen bonds are observed in the crystal packing.