Structure-Based Design, Synthesis, and Biological Evaluation of Imidazo[4,5-b]Pyridin-2-one-Based p38 MAP Kinase Inhibitors: Part†2.

We identified novel potent inhibitors of p38 mitogen-activated protein (MAP) kinase using a structure-based design strategy, beginning with lead compound, 3-(butan-2-yl)-6-(2,4-difluoroanilino)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (1). To enhance the inhibitory activity of 1 against production of tumor necrosis factor-α (TNF-α) in human whole blood (hWB) cell assays, we designed and synthesized hybrid compounds in which the imidazo[4,5-b]pyridin-2-one core was successfully linked with the p-methylbenzamide fragment. Among the compounds evaluated, 3-(3-tert-butyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-4-methyl-N-(1-methyl-1H-pyrazol-3-yl)benzamide (25) exhibited potent p38 inhibition, superior suppression of TNF-α production in hWB cells, and also significant in vivo efficacy in a rat model of collagen-induced arthritis (CIA). In this paper, we report the discovery of potent, selective, and orally bioavailable imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors.

Structure-Based Design, Synthesis, and Biological Evaluation of Imidazo[4,5-b]pyridin-2-one-Based p38 MAP Kinase Inhibitors: Part 1.

We identified a lead series of p38 mitogen-activated protein kinase inhibitors using a structure-based design strategy from high-throughput screening of hit compound 1. X-ray crystallography of 1 with the kinase showed an infrequent flip of the peptide bond between Met109 and Gly110, which was considered to lead to high kinase selectivity. Our structure-based design strategy was to conduct scaffold transformation of 1 with maintenance of hydrogen bond interactions with the flipped hinge backbone of the enzyme. In accordance with this strategy, we focused on scaffold transformation to identify imidazo[4,5-b]pyridin-2-one derivatives as potent inhibitors of the p38 MAP kinase. Of the compounds evaluated, 21 was found to be a potent inhibitor of the p38 MAP kinase, lipopolysaccharide-induced tumor necrosis factor-α (TNF-α) production in human monocytic leukemia cells, and TNF-α-induced production of interleukin-8 in human whole blood cells. Herein we describe the discovery of potent and orally bioavailable imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors that suppressed cytokine production in a human whole blood cell-based assay.

Multiscale enhanced sampling of glucokinase: Regulation of the enzymatic reaction via a large scale domain motion.

Enhanced sampling yields a comprehensive structural ensemble or a free energy landscape, which is beyond the capability of a conventional molecular dynamics simulation. Our recently developed multiscale enhanced sampling (MSES) method employs a coarse-grained model coupled with the target physical system for the efficient acceleration of the dynamics. MSES has demonstrated applicability to large protein systems in solution, such as intrinsically disordered proteins and protein-protein and protein-ligand interactions. Here, we applied the MSES simulation to an important drug discovery target, glucokinase (GCK), to elucidate the structural basis of the positive cooperativity of the enzymatic reaction at an atomistic resolution. MSES enabled us to compare two sets of the free energy landscapes of GCK, for the glucose-bound and glucose-unbound forms, and thus demonstrated the drastic change of the free energy surface depending on the glucose concentration. In the glucose-bound form, we found two distinct basins separated by a high energy barrier originating from the domain motion and the folding/unfolding of the α13 helix. By contrast, in the glucose-unbound form, a single flat basin extended to the open and super-open states. These features illustrated the two distinct phases achieving the cooperativity, the fast reaction cycle staying in the closed state at a high glucose concentration and the slow cycle primarily in the open/super-open state at a low concentration. The weighted ensemble simulations revealed the kinetics of the structural changes in GCK with the synergetic use of the MSES results; the rate constant of the transition between the closed state and the open/super-open states, kC/O = 1.1 ms-1, is on the same order as the experimental catalytic rate, kcat = 0.22 ms-1. Finally, we discuss the pharmacological activities of GCK activators (small molecular drugs modulating the GCK activity) in terms of the slight changes in the domain motion, depending on their chemical structures as regulators. The present study demonstrated the capability of the enhanced sampling and the associated kinetic calculations for understanding the atomistic structural dynamics of protein systems in physiological environments.

Structure-based design, synthesis, and biological evaluation of imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors.

We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal structure, Phe169 adopts two conformations, where one interacts with 1 and the other shows no interaction with 1. Our structure-based design strategy shows that these two conformations converge into one via enhanced protein-ligand hydrophobic interactions. According to the strategy, we focused on scaffold transformation to identify imidazo[1,2-b]pyridazine derivatives as potent inhibitors of p38 MAP kinase. Among the herein described and evaluated compounds, N-oxide 16 exhibited potent inhibition of p38 MAP kinase and LPS-induced TNF-α production in human monocytic THP-1 cells, and significant in vivo efficacy in rat collagen-induced arthritis models. In this article, we report the discovery of potent, selective and orally bioavailable imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors with pyridine N-oxide group.

Novel approach of fragment-based lead discovery applied to renin inhibitors.

A novel approach was conducted for fragment-based lead discovery and applied to renin inhibitors. The biochemical screening of a fragment library against renin provided the hit fragment which showed a characteristic interaction pattern with the target protein. The hit fragment bound only to the S1, S3, and S3SP (S3 subpocket) sites without any interactions with the catalytic aspartate residues (Asp32 and Asp215 (pepsin numbering)). Prior to making chemical modifications to the hit fragment, we first identified its essential binding sites by utilizing the hit fragment's substructures. Second, we created a new and smaller scaffold, which better occupied the identified essential S3 and S3SP sites, by utilizing library synthesis with high-throughput chemistry. We then revisited the S1 site and efficiently explored a good building block attaching to the scaffold with library synthesis. In the library syntheses, the binding modes of each pivotal compound were determined and confirmed by X-ray crystallography and the library was strategically designed by structure-based computational approach not only to obtain a more active compound but also to obtain informative Structure Activity Relationship (SAR). As a result, we obtained a lead compound offering synthetic accessibility as well as the improved in vitro ADMET profiles. The fragments and compounds possessing a characteristic interaction pattern provided new structural insights into renin's active site and the potential to create a new generation of renin inhibitors. In addition, we demonstrated our FBDD strategy integrating highly sensitive biochemical assay, X-ray crystallography, and high-throughput synthesis and in silico library design aimed at fragment morphing at the initial stage was effective to elucidate a pocket profile and a promising lead compound.

Prediction of Ligand Binding Affinity by the Combination of Replica-Exchange Method and Double-Decoupling Method.

A prediction method for ligand binding affinities to proteins is proposed. We first predict the structures of protein-ligand complex by the replica-exchange umbrella sampling or its extension. We then calculate ligand binding affinities based on these predicted ligand-protein bound structures by the double-decoupling method. As a test of the effectiveness of the proposed method, we applied it to the system of the oncoprotein MDM2 and a ligand. The value of the predicted binding affinity turned out to be in good agreement with that from experiments.

Two-dimensional replica-exchange method for predicting protein-ligand binding structures.

We have developed a two-dimensional replica-exchange method for the prediction of protein-ligand binding structures. The first dimension is the umbrella sampling along the reaction coordinate, which is the distance between a protein binding pocket and a ligand. The second dimension is the solute tempering, in which the interaction between a ligand and a protein and water is weakened. The second dimension is introduced to make a ligand follow the umbrella potential more easily and enhance the binding events, which should improve the sampling efficiency. As test cases, we applied our method to two protein-ligand complex systems (MDM2 and HSP 90-alpha). Starting from the configuration in which the protein and the ligand are far away from each other in each system, our method predicted the ligand binding structures in excellent agreement with the experimental data from Protein Data Bank much faster with the improved sampling efficiency than the replica-exchange umbrella sampling method that we have previously developed.

Ligand docking simulations by generalized-ensemble algorithms.

In protein chemistry and structural biology, conventional simulations in physical statistical mechanical ensembles, such as the canonical ensemble with fixed temperature and isobaric-isothermal ensemble with fixed temperature and pressure, face a great difficulty. This is because there exist a huge number of local-minimum-energy states in the system and the conventional simulations tend to get trapped in these states, giving wrong results. Generalized-ensemble algorithms are based on artificial unphysical ensembles and overcome the above difficulty by performing random walks in potential energy, volume, and other physical quantities or their corresponding conjugate parameters such as temperature and pressure. The advantage of generalized-ensemble simulations lies in the fact that they not only avoid getting trapped in states of energy local minima but also allow the calculations of physical quantities as functions of temperature or other parameters from a single simulation run. In this chapter, we review the generalized-ensemble algorithms. Some of their specific examples such as replica-exchange molecular dynamics and replica-exchange umbrella sampling are described in detail. Examples of their applications to drug design are presented.

Synthetic studies of centromere-associated protein-E (CENP-E) inhibitors: 1.Exploration of fused bicyclic core scaffolds using electrostatic potential map.

Centromere-associated protein-E (CENP-E), a mitotic kinesin that plays an important role in mitotic progression, is an attractive target for cancer therapeutic drugs. For the purpose of developing novel CENP-E inhibitors as cancer therapeutics, we investigated a fused bicyclic compound identified by high throughput screening, 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide 1a. Based on this scaffold, we designed inhibitors for efficient binding at the L5 site in CENP-E utilizing homology modeling as well as electrostatic potential map (EPM) analysis to enhance CENP-E inhibitory activity. This resulted in a new lead, 5-bromoimidazo[1,2-a]pyridine 7, which showed potent CENP-E enzyme inhibition (IC50: 50nM) and cellular activity with accumulation of phosphorylated histone H3 in HeLa cells. Our homology model and EPM analysis proved to be useful tools for the rational design of CENP-E inhibitors.

Design and synthesis of novel pyrimido[4,5-b]azepine derivatives as HER2/EGFR dual inhibitors.

A novel 7,6 fused bicyclic scaffold, pyrimido[4,5-b]azepine was designed to fit into the ATP binding site of the HER2/EGFR proteins. The synthesis of this scaffold was accomplished by an intramolecular Claisen-type condensation. As the results of optimization lead us to 4-anilino and 6-functional groups, we discovered 6-substituted amide derivative 19b, which has a 1-benzothiophen-4-yloxy group attached to the 4-anilino group. An X-ray co-crystal structure of 19b with EGFR demonstrated that the N-1 and N-3 nitrogens of the pyrimido[4,5-b]azepine scaffold make hydrogen-bonding interactions with the main chain NH of Met793 and the side chain of Thr854 via a water-mediated hydrogen bond network, respectively. In addition, the NH proton at the 9-position makes an additional hydrogen bond with the carbonyl group of Met793, as we expected. Compound 19b revealed potent HER2/EGFR kinase (IC50: 24/36 nM) and BT474 cell growth (GI50: 18 nM) inhibitory activities based on its pseudo-irreversible (PI) profile.

Prediction of Protein-Ligand Binding Structures by Replica-Exchange Umbrella Sampling Simulations: Application to Kinase Systems.

We have applied our prediction method, which is based on the replica-exchange umbrella sampling for protein-ligand binding structures, to two kinase systems (p38 and JNK3) with two different ligand molecules for each kinase. Starting from configurations in which the protein and the ligand are far away from each other, our method predicted the ligand binding structures in excellent agreement with the experimental data from PDB in all four cases, which suggests the general applicability of our method to kinase systems. In addition, the protein flexibility was shown to be essential to predict the correct binding structure for one of the systems, where dihydroquinolinone was bound to p38 alpha kinase (PDB ID: 1OVE ).

Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy.

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC(50): 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI(50): 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C=0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).

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.

Design and synthesis of pyrrolo[3,2-d]pyrimidine human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) dual inhibitors: exploration of novel back-pocket binders.

To develop novel human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) kinase inhibitors, we explored pyrrolo[3,2-d]pyrimidine derivatives bearing bicyclic fused rings designed to fit the back pocket of the HER2/EGFR proteins. Among them, the 1,2-benzisothiazole (42m) ring was selected as a suitable back pocket binder because of its potent HER2/EGFR binding and cell growth inhibitory (GI) activities and pseudoirreversibility (PI) profile as well as good bioavailability (BA). Ultimately, we arrived at our preclinical candidate 51m by optimization of the N-5 side chain to improve CYP inhibition and metabolic stability profiles without a loss of potency (HER2/EGFR inhibitory activity, IC(50), 0.98/2.5 nM; and GI activity BT-474 cells, GI(50), 2.0 nM). Reflecting the strong in vitro activities, 51m exhibited potent tumor regressive efficacy against both HER2- and EGFR-overexpressing tumor (4-1ST and CAL27) xenograft models in mice at oral doses of 50 mg/kg and 100 mg/kg.

Discovery of phenylpropanoic acid derivatives containing polar functionalities as potent and orally bioavailable G protein-coupled receptor 40 agonists for the treatment of type 2 diabetes.

As part of a program to identify potent GPR40 agonists with drug-like properties suitable for clinical development, the incorporation of polar substituents was explored with the intention of decreasing the lipophilicity of our recently disclosed phenylpropanoic acid derivative 1. This incorporation would allow us to mitigate the cytotoxicity issues observed with compound 1 and enable us to move away from the multifunctional free fatty acid-like structure. Substitutions on the 2',6'-dimethylbiphenyl ring were initially undertaken, which revealed the feasibility of introducing polar functionalities at the biphenyl 4'-position. Further optimization of this position and the linker led to the discovery of several 4'-alkoxybiphenyl derivatives, which showed potent GPR40 agonist activities with the best balance in terms of improved cytotoxicity profiles and favorable pharmacokinetic properties. Among them, 3-{2-fluoro-4-[({4'-[(4-hydroxy-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy]-2',6'-dimethylbiphenyl-3-yl}methyl)amino]phenyl}propanoic acid (35) exhibited a robust plasma glucose-lowering effect and insulinotropic action during an oral glucose tolerance test in rats with impaired glucose tolerance.

Identification of fused-ring alkanoic acids with improved pharmacokinetic profiles that act as G protein-coupled receptor 40/free fatty acid receptor 1 agonists.

The G protein-coupled receptor 40 (GPR40)/free fatty acid receptor 1 (FFA1) has emerged as an attractive target for a novel insulin secretagogue with glucose dependency. We previously identified phenylpropanoic acid derivative 1 (3-{4-[(2',6'-dimethylbiphenyl-3-yl)methoxy]-2-fluorophenyl}propanoic acid) as a potent and orally available GPR40/FFA1 agonist; however, 1 exhibited high clearance and low oral bioavailability, which was likely due to its susceptibility to ß-oxidation at the phenylpropanoic acid moiety. To identify long-acting compounds, we attempted to block the metabolically labile sites at the phenylpropanoic acid moiety by introducing a fused-ring structure. Various fused-ring alkanoic acids with potent GPR40/FFA1 activities and good PK profiles were produced. Further optimizations of the lipophilic portion and the acidic moiety led to the discovery of dihydrobenzofuran derivative 53 ((6-{[4'-(2-ethoxyethoxy)-2',6'-dimethylbiphenyl-3-yl]methoxy}-2,3-dihydro-1-benzofuran-3-yl)acetic acid), which acted as a GPR40/FFA1 agonist with in vivo efficacy during an oral glucose tolerance test (OGTT) in rats with impaired glucose tolerance.

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.

Identification of benzoxazin-3-one derivatives as novel, potent, and selective nonsteroidal mineralocorticoid receptor antagonists.

Mineralocorticoid receptor (MR) blockade has come into focus as a promising approach for the treatment of cardiovascular diseases such as hypertension and congestive heart failure. In order to identify a novel class of nonsteroidal MR antagonists that exhibit significant potency and good selectivity over other steroidal hormone receptors, we designed a novel series of benzoxazin-3-one derivatives and synthesized them from 6-(7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-1,4-benzoxazin-3(4H)-one (1a), high-throughput screening (HTS) hit compound. Our design was based on a crystal structure of an MR/compound complex and a docking model. In the course of lead generation from 1a, a 1,2-diaryl framework was characterized as a key structure with high binding affinity. On the basis of scaffold hopping and optimization studies, benzoxazin-3-one derivatives possessing 1-phenyl-3-trifluoromethylpyrazol-5-yl moiety at the 6-position were identified as a novel series of potent and selective MR antagonists. Among these compounds, 6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (14n) showed highly potent activity and good selectivity and also exhibited a significant antihypertensive effect in deoxycorticosterone acetate-salt hypertensive rats. On the basis of these results, compound 14n was progressed for further pharmacological evaluation.

Design and synthesis of novel human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) dual inhibitors bearing a pyrrolo[3,2-d]pyrimidine scaffold.

Dual inhibitors of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) have been investigated for breast, lung, gastric, prostate, and other cancers; one, lapatinib, is currently approved for breast cancer. To develop novel HER2/EGFR dual kinase inhibitors, we designed and synthesized pyrrolo[3,2-d]pyrimidine derivatives capable of fitting into the receptors' ATP binding site. Among the prepared compounds, 34e showed potent HER2 and EGFR (HER1) inhibitory activities as well as tumor growth inhibitory activity. The X-ray cocrystal structures of 34e with both HER2 and EGFR demonstrated that 34e interacts with the expected residues in their respective ATP pockets. Furthermore, reflecting its good oral bioavailability, 34e exhibited potent in vivo efficacy in HER2-overexpressing tumor xenograft models. On the basis of these findings, we report 34e (TAK-285) as a promising candidate for clinical development as a novel HER2/EGFR dual kinase inhibitor.

Discovery of orteronel (TAK-700), a naphthylmethylimidazole derivative, as a highly selective 17,20-lyase inhibitor with potential utility in the treatment of prostate cancer.

A novel naphthylmethylimidazole derivative 1 and its related compounds were identified as 17,20-lyase inhibitors. Based on the structure-activity relationship around the naphthalene scaffold and the results of a docking study of 1a in the homology model of 17,20-lyase, the 6,7-dihydro-5H-pyrrolo[1,2-c]imidazole derivative (+)-3c was synthesized and identified as a potent and highly selective 17,20-lyase inhibitor. Biological evaluation of (+)-3c at a dose of 1mg/kg in a male monkey model revealed marked reductions in both serum testosterone and dehydroepiandrosterone concentrations. Therefore, (+)-3c (termed orteronel [TAK-700]) was selected as a candidate for clinical evaluation and is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer.