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The anaplastic lymphoma kinase (ALK) is abnormally expressed and hyperactivated in a number of tumors and represents an ideal therapeutic target. Despite excellent clinical responses to ALK inhibition, drug resistance still represents an issue and novel compounds that overcome drug-resistant mutants are needed. We designed, synthesized, and evaluated a large series of azacarbazole inhibitors. Several lead compounds endowed with submicromolar potency were identified. Compound 149 showed selective inhibition of native and mutant drug-refractory ALK kinase in vitro as well as in a Ba/F3 model and in human ALK+ lymphoma cells. The three-dimensional (3D) structure of a 149:ALK-KD cocrystal is reported, showing extensive interaction through the hinge region and the catalytic lysine 1150.

The Anaplastic Lymphoma Kinase (ALK) is a therapeutic target for personalized medicine in selected cancers. Despite excellent clinical responses to ALK inhibitors, most patients develop drug resistance and relapse. New compounds with alternative binding modes are needed to overcome resistant mutants. Here we describe a medicinal chemistry effort to the design and development of novel ALK inhibitors based on a 4,6-substituted α-carboline scaffold. Active compounds were able to inhibit the gatekeeper L1196M mutant, in several cases better than the wild-type enzyme. Compound 43 showed potent non-ATP-competitive inhibition of wild-type and mutant ALK, including G1202R, in biochemical and cellular assays, as well as in xenograft mouse models.

Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the two phosphatidylinositol kinases PI4KB and PIP4K2C, the latter being known for participating in pain induction. Docking revealed that phenazopyridine forms strong H-bonds with the hinge region of the ATP-binding pocket of these kinases. As previous studies suggested increased autophagy upon inhibition of the phosphatidyl-inositol/AKT pathway, we also investigated the impact of phenazopyridine on this pathway and found an upregulation. In conclusion, our study demonstrates for the first time that phenazopyridine is a kinase inhibitor, impacting notably phosphatidylinositol kinases involved in nociception.

Purpose: To develop an animal model of atrophic age-related macular degeneration (aAMD) in aged mice that more closely mimics the natural progression of the disease. Methods: 12- and 18-month-old CBl57/6JRj mice were immunized with murine serum albumin (MSA) conjugated with carboxyethyl pyrrole (CEP). The immunization, given into the hock, was followed by 3 booster injections into the neck over a 3-month period. Immunized mice and age-matched controls were trained for a visual discrimination and an optokinetic response task to determine the objective visual threshold (OVT) at arrival and at 3 months; funduscopy and ocular coherence tomography were performed. After sacrifice, the eyes were enucleated for histological, immunofluorescent and electron microscopy analyses. Results: Retinal imaging confirmed that all mice had normal retinas upon arrival. Three months after mice were immunized the normal retinal age-related alterations were significantly more pronounced in CEP-immunized than in control mice as evidenced by drusenoid alterations, increased retinal thickness, immune activation, signs of retinal degeneration, decreased OVT. Electron microscopy indicated degeneration of the retinal pigment epithelium (RPE). Conclusions: The retinal and behavioral changes in the aged CEP-immunized mice will be useful for the investigation of novel treatments of aAMD. Translational relevance: The enhanced Aged-CEP-Mouse model enables the generation of results highly transferable to human patients and promotes the development of efficient, safe AMD therapies.

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.

Here, we report on the design, synthesis, and biological evaluation of 4-thiazolidinone (rhodanine) derivatives targeting Mycobacterial tuberculosis (Mtb) trans-2-enoyl-acyl carrier protein reductase (InhA). Compounds having bulky aromatic substituents at position 5 and a tryptophan residue at position N-3 of the rhodanine ring were the most active against InhA, with IC50 values ranging from 2.7 to 30 μM. The experimental data showed consistent correlations with computational studies. Their antimicrobial activity was assessed against Mycobacterium marinum (Mm) (a model for Mtb), Pseudomonas aeruginosa (Pa), Legionella pneumophila (Lp), and Enterococcus faecalis (Ef) by using anti-infective, antivirulence, and antibiotic assays. Nineteen out of 34 compounds reduced Mm virulence at 10 μM. 33 exhibited promising antibiotic activity against Mm with a MIC of 0.21 μM and showed up to 89% reduction of Lp growth in an anti-infective assay at 30 μM. 32 showed high antibiotic activity against Ef, with a MIC of 0.57 μM.

Novel benzodioxine and benzoxazine derivatives are prepared via a Diels-Alder cycloaddition in order to get potential anticancer lead compounds.

1,3-Oxazoline- (OXT) and 1,3-oxazolidine-2-thiones (OZT) can undergo direct Stille and Suzuki cross-coupling reactions under microwave activation to produce 2-aryloxazoles and 2-aryloxazolines in reasonable to good yields.

A vinyl bis-sulfone Michael type approach towards heteroatom vinylation was applied on nitrogen derivatives. Cyclic thionocarbamates––mainly 1,3-oxazolidine-2-thiones––were converted into their N-vinyl counterparts; the procedure proved particularly efficient in the case of carbohydrate-derived complex structures.

International audience; Chromosomal translocations involving anaplastic lymphoma kinase (ALK) are the driving mutations for a range of cancers and ALK is thus considered an attractive therapeutic target. We synthesized a series of functionalized benzo[4,5]imidazo[1,2-c]pyrimidines and benzo[4,5]imidazo[1,2-a]pyrazines by an aza-Graebe–Ullman reaction, followed by palladium-catalyzed cross-coupling reactions. A sequential regioselective cross-coupling route is reported for the synthesis of unsymmetrically disubstituted benzo[4,5]imidazo[1,2-a]pyrazines. The inhibition of ALK was evaluated and compound 19 in particular showed good activity against both the wild type and crizotinib-resistant L1196M mutant in vitro and in ALK-transfected BaF3 cells.

Several N-vinyl-l,3-oxazolidine-2-thione (N-vinyl OZT) were conveniently used as new dienophiles in Eu(fod) 3 -catalyzed reverse hetero-Diels-Alder reactions involving benzylidene pyruvic acid methyl ester. Simple chiral N-vinyl OZT analogues homogeneously led to moderate endo and facial diastereoselectivities, when compared to those obtained with the corresponding N-vinyl-l,3-oxazolidin-2-ones. In contrast, high diastereocontrols were observed with a sugar-derived N-vinyl OZT.

Novel benzodioxine and benzoxazine derivatives are prepared via a Diels-Alder cycloaddition in order to get potential anticancer lead compounds.

1,3-Oxazoline- (OXT) and 1,3-oxazolidine-2-thiones (OZT) can undergo direct Stille and Suzuki cross-coupling reactions under microwave activation to produce 2-aryloxazoles and 2-aryloxazolines in reasonable to good yields.

The general preparation of enantiopure monoacid side-chains of several esters of cephalotaxine is described. The strategy, similar to Weinreb's approach to the synthesis of deoxyharringtonine, used as key intermediate the chiral nonracemic epoxide 11a prepared from the commercially available monomethyl itaconate (8). The key step of the strategy was the ring-opening of the epoxide 11a by using different organocuprate nucleophiles. Hydrogenolysis as the final step gave the monacid side-chains of the corresponding esters of cephalotaxine in moderate to good overall yields from epoxide 11a.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

A vinyl bis-sulfone Michael type approach towards heteroatom vinylation was applied on nitrogen derivatives. Cyclic thionocarbamates––mainly 1,3-oxazolidine-2-thiones––were converted into their N-vinyl counterparts; the procedure proved particularly efficient in the case of carbohydrate-derived complex structures.