Discovery of Novel and Highly Selective Cyclopropane ALK Inhibitors through a Fragment-Assisted, Structure-Based Drug Design.

Fragment screening is frequently used for hit identification. However, there was no report starting from a small fragment for the development of an anaplastic lymphoma kinase (ALK) inhibitor, despite the number of ALK inhibitors reported. We began our research with the fragment hit F-1 and our subsequent linker design, and its docking analysis yielded novel cis-1,2,2-trisubstituted cyclopropane 1. The fragment information was integrated with a structure-based approach to improve upon the selectivity over tropomyosin receptor kinase A, leading to the potent and highly selective ALK inhibitor, 4-trifluoromethylphenoxy-cis-1,2,2-trisubstituted cyclopropane 12. This work shows that fragments become a powerful tool for both lead generation and optimization, such as the improvement of selectivity, by combining them with a structure-based drug design approach, resulting in the fast and efficient development of a novel, potent, and highly selective compound.

Discovery of Potent, Selective, and Brain-Penetrant 1 H-Pyrazol-5-yl-1 H-pyrrolo[2,3- b]pyridines as Anaplastic Lymphoma Kinase (ALK) Inhibitors.

Anaplastic lymphoma kinase (ALK), a member of the receptor tyrosine kinase family, is predominantly expressed in the brain and implicated in neuronal development and cognition. However, the detailed function of ALK in the central nervous system (CNS) is still unclear. To elucidate the role of ALK in the CNS, it was necessary to discover a potent, selective, and brain-penetrant ALK inhibitor. Scaffold hopping and lead optimization of N-(2,4-difluorobenzyl)-3-(1 H-pyrazol-5-yl)imidazo[1,2- b]pyridazin-6-amine 1 guided by a cocrystal structure of compound 1 bound to ALK resulted in the identification of (6-(1-(5-fluoropyridin-2-yl)ethoxy)-1-(5-methyl-1 H-pyrazol-3-yl)-1 H-pyrrolo[2,3- b]pyridin-3-yl)((2 S)-2-methylmorpholin-4-yl)methanone 13 as a highly potent, selective, and brain-penetrable compound. Intraperitoneal administration of compound 13 significantly decreased the phosphorylated-ALK (p-ALK) levels in the hippocampus and prefrontal cortex in the mouse brain. These results suggest that compound 13 could serve as a useful chemical probe to elucidate the mechanism of ALK-mediated brain functions and the therapeutic potential of ALK inhibition.

Discovery of a novel series of indoline carbamate and indolinylpyrimidine derivatives as potent GPR119 agonists.

GPR119 has emerged as an attractive target for anti-diabetic agents. We identified a structurally novel GPR119 agonist 22c that carries a 5-(methylsulfonyl)indoline motif as an early lead compound. To generate more potent compounds of this series, structural modifications were performed mainly to the central alkylene spacer. Installation of a carbonyl group and a methyl group on this spacer significantly enhanced agonistic activity, resulting in the identification of 2-[1-(5-ethylpyrimidin-2-yl)piperidin-4-yl]propyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-carboxylate (20). To further expand the chemical series of indoline-based GPR119 agonists, several heterocyclic core systems were introduced as surrogates of the carbamate spacer that mimic the presumed active conformation. This approach successfully produced an indolinylpyrimidine derivative 37, 5-(methylsulfonyl)-1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indole, which has potent GPR119 agonist activity. In rat oral glucose tolerance tests, these two indoline-based compounds effectively lowered plasma glucose excursion and glucose-dependent insulin secretion after oral administration.

Visualizing cellular interactions with a generalized proximity reporter.

Interactions among neighboring cells underpin many physiological processes ranging from early development to immune responses. When these interactions do not function properly, numerous pathologies, including infection and cancer, can result. Molecular imaging technologies, especially optical imaging, are uniquely suited to illuminate complex cellular interactions within the context of living tissues in the body. However, no tools yet exist that allow the detection of microscopic events, such as two cells coming into close proximity, on a global, whole-animal scale. We report here a broadly applicable, longitudinal strategy for probing interactions among cells in living subjects. This approach relies on the generation of bioluminescent light when two distinct cell populations come into close proximity, with the intensity of the optical signal correlating with relative cellular location. We demonstrate the ability of this reporter strategy to gauge cell-cell proximity in culture models in vitro and then evaluate this approach for imaging tumor-immune cell interactions using a murine breast cancer model. In these studies, our imaging strategy enabled the facile visualization of features that are otherwise difficult to observe with conventional imaging techniques, including detection of micrometastatic lesions and potential sites of tumor immunosurveillance. This proximity reporter will facilitate probing of numerous types of cell-cell interactions and will stimulate the development of similar techniques to detect rare events and pathological processes in live animals.

Length of stay and cost for surgical site infection after abdominal and cardiac surgery in Japanese hospitals: multi-center surveillance.

PURPOSE: This study evaluated the influence of surgical site infections (SSIs) after abdominal or cardiac surgery on the post-operative duration of hospitalization and cost. METHODS: A retrospective 1:1 matched case-control study of length of stay and healthcare expenditures for patients who were discharged from nine hospitals, between April 1, 2006 and March 31, 2008, after undergoing abdominal or cardiac surgery and who did and did not have a SSI. RESULTS: Information was obtained from 246 pairs of patients who had undergone abdominal surgery and 27 pairs of patients who had undergone cardiac surgery. Overall, the mean post-operative hospitalization was 20.7 days longer and the mean post-operative healthcare expenditure was $8,791 higher in the SSI group than for the SSI-free group. Among the patients who had undergone abdominal surgery, development of SSI extended the average hospitalization by 17.6 days and increased the average healthcare expenditure by $6,624. Among the patients who had undergone cardiac surgery, SSI extended the post-operative hospitalization by an average of 48.9 days and increased the post-operative healthcare expenditure by an average of $28,534. CONCLUSIONS: Under the current healthcare system in Japan, the development of SSI after abdominal surgery necessitates extension of hospitalization two-fold and increases the post-operative healthcare expenditure 2.5-fold. Development of SSI after cardiac surgery necessitates extension of hospitalization fourfold and increases the healthcare expenditure six-fold.

Structure-activity relationships and key structural feature of pyridyloxybenzene-acylsulfonamides as new, potent, and selective peroxisome proliferator-activated receptor (PPAR) γ Agonists.

In our search for a novel class of non-TZD, non-carboxylic acid peroxisome proliferator-activated receptor (PPAR) γ agonists, we explored alternative lipophilic templates to replace benzylpyrazole core of the previously reported agonist 1. Introduction of a pentylsulfonamide group into arylpropionic acids derived from previous in-house PPARγ ligands succeeded in the identification of 2-pyridyloxybenzene-acylsulfonamide 2 as a lead compound. Docking studies of compound 2 suggested that a substituent para to the central benzene ring should be incorporated to effectively fill the Y-shaped cavity of the PPARγ ligand-binding domain (LBD). This strategy led to significant improvement of PPARγ activity. Further optimization to balance in vitro activity and metabolic stability allowed the discovery of the potent, selective and orally efficacious PPARγ agonist 8f. Structure-activity relationship study as well as detailed analysis of the binding mode of 8f to the PPARγ-LBD revealed the essential structural features of this series of ligands.

Destabilizing domains derived from the human estrogen receptor.

Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an experimental strategy to regulate the intracellular concentration of any protein of interest by using an engineered destabilizing protein domain and a cell-permeable small molecule. Destabilizing domains have general utility to confer instability to a wide range of proteins including integral transmembrane proteins. This study reports a destabilizing domain system based on the ligand binding domain of the estrogen receptor that can be regulated by one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen.

A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: design and synthesis of benzylpyrazole acylsulfonamides.

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.

Orally active CCR5 antagonists as anti-HIV-1 agents 2: synthesis and biological activities of anilide derivatives containing a pyridine N-oxide moiety.

In order to develop orally active CCR5 antagonists, we investigated 1-benzoxepine derivatives containing new polar substituents, such as phosphonate, phosphine oxide or pyridine N-oxide moieties, as replacements for the previously reported quaternary ammonium moiety. Among these compounds, the 2-(alpha-hydroxybenzyl)pyridine N-oxide 5e exhibited moderate CCR5 antagonistic activity and had an acceptable pharmacokinetic profile in rats. Subsequent chemical modification was performed and compound (S)-5f possessing the (S)-configuration hydroxy group was found to be more active than the (R)-isomer. Replacement of the 1-benzoxepine ring with a 4-methylphenyl group by a 1-benzazepine ring with a 4-[2-(butoxy)ethoxy]phenyl group enhanced the activity in the binding assay. In addition, introduction of a 3-trifluoromethyl group on the phenyl group of the anilide moiety led to greatly increased activity in the HIV-1 envelope-mediated membrane fusion assay. In particular, compound (S)-5s showed the most potent CCR5 antagonistic activity (IC(50)=7.2 nM) and inhibitory effect (IC(50)=5.4 nM) in the fusion assay, together with good pharmacokinetic properties in rats.

A novel oxyiminoalkanoic acid derivative, TAK-559, activates human peroxisome proliferator-activated receptor subtypes.

A novel oxyiminoalkanoic acid derivative, TAK-559, (E)-4-[4-[(5-methyl-2-phenyl-1, 3-oxazol-4-yl)methoxy]benzyloxyimino]-4-phenylbutyric acid, was synthesized as a candidate of a new type of insulin-sensitizing agent. We report here activation of human peroxisome proliferator-activated receptor (hPPAR) subtypes by TAK-559. In a transient transactivation assay, TAK-559 was a potent hPPARgamma1 and hPPARalpha agonist with EC50 values of 31 and 67 nM, respectively. Furthermore, TAK-559 was a partial agonist for hPPARgamma1 with about 68% of maximal activation obtained with rosiglitazone (5-(4-(2-(methyl(2-pyridinyl)amino)ethoxy) benzyl)-1,3-thiazolidine-2,4-dione), a thiazolidinedione derivative, which is known as a PPARgamma agonist. PPARdelta was significantly activated at a high concentration (10 microM) of TAK-559. Competition-binding assays using radiolabeled ligand indicated that the transactivation of all hPPAR subtypes by TAK-559 was due to direct binding of TAK-559 to each subtype. We also demonstrated that TAK-559 acts to recruit the coactivator SRC-1 to each of hPPARgamma1 and hPPARalpha, and to dissociate the corepressor NCoR from each of hPPARgamma1 and hPPARalpha. Taken together, we conclude that TAK-559 is a dual agonist for hPPARgamma1 and hPPARalpha with nearly equal EC50 values, a partial agonist for hPPARgamma1, and has a rather slight agonist activity for hPPARdelta.

Studies on non-thiazolidinedione antidiabetic agents. 3. Preparation and biological activity of the metabolites of TAK-559.

Preparation and biological activity of the metabolites of the potent antihyperglycemic and antihyperlipidemic agent, (E)-4-(4-[(5-methyl-2-phenyl-1,3-oxazol-4-yl)methoxy]benzyloxyimino)-4-phenylbutyric acid (TAK-559) (1), were investigated. Metabolites M-I (2), M-II (3), M-III (4) and M-IV (5) were synthesized and their biological activities were evaluated by in vitro and in vivo experiments. Compounds 2-4 activate human peroxisome proliferator-activated receptor gamma one (hPPARgamma1) and hPPARalpha, but their activities are weaker than those of TAK-559 (1). Compound 5 only activates hPPARgamma1 weakly. TAK-559 (1) showed potent in vivo plasma glucose and triglyceride lowering activities in Wistar fatty rats after intraperitoneal administration, while its metabolites (2-5) showed comparatively weak activities.

Studies on non-thiazolidinedione antidiabetic agents. 2. Novel oxyiminoalkanoic acid derivatives as potent glucose and lipid lowering agents.

We previously reported that (Z)-2-(4-[(5-methyl-2-phenyl-1, 3-oxazol-4-yl)methoxy]benzyloxyimino)-2-(4-phenoxyphenyl)acetic acid (3) showed potent glucose and lipid lowering effects in genetically obese and diabetic mice, KKA(y). This compound also showed transcriptional activity for peroxisome proliferator-activated receptor (PPAR)-gamma. We expanded on the structure-activity relationships of oxyiminoalkanoic acid derivatives based on this transcriptional activity (in vitro). Insertion of a carbon chain between the imino carbon and the carboxyl moiety of (Z)-2-(4-[(5-methyl-2-phenyl-1, 3-oxazol-4-yl)methoxy]benzyloxyimino)-2-phenylacetic acid (2) resulted in a marked increase in transcriptional activity at PPARgamma. In vivo potencies of synthesized compounds, which showed strong functional activity at PPARgamma, were tested using KKA(y) mice. Among these compounds, (E)-4-(4-[(5-methyl-2-phenyl-1, 3-oxazol-4-yl)methoxy]benzyloxyimino)-4-phenylbutyric acid (27) exhibited marked glucose and lipid lowering activity while showing no significant body weight gain. Compound (27) (TAK-559) showed favorable pharmacokinetic properties with good absorption and duration, and was considered as an attractive candidate for further evaluation.

Studies on non-thiazolidinedione antidiabetic agents. 1. Discovery of novel oxyiminoacetic acid derivatives.

A novel series of oxyiminoacetic acid derivatives were synthesized in an effort to develop a potent antidiabetic agent, which does not contain the 2,4-thiazolidinedione moiety. These compounds were evaluated for glucose and lipid lowering effects in genetically obese and diabetic KKA(y) mice. Several of the compounds showed strong antidiabetic activity, including functional potency at peroxisome proliferator-activated receptor (PPAR)-gamma. (Z)-2-[4-[(5-Methyl-2-phenyl-1,3-oxazol-4-yl)methoxy]benzyloxyimino]-2-(4-phenoxyphenyl)acetic acid (25) significantly reduced plasma glucose (33%, p<0.01) and plasma triglycelide levels (43%, p<0.01) even at a dosage of 0.001% in diet. Pharmacokinetic analyses of 25 are also reported.