Discovery and optimization of N-acyl and N-aroylpyrazolines as B-Raf kinase inhibitors

A high throughput screen identified N-aroylpyrazoline 1 as a selective inhibitor of the V600E mutant of B-Raf kinase. Parallel synthesis of acyl, aroyl, and sulfonyl derivatives led to the identification of several potent inhibitors in both enzymatic and cellular (pERK) assays such as compound 42.     

Discovery of novel pyrazolo[1,5-a]pyrimidines as potent pan-Pim inhibitors by structure- and property-based drug design

Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms.     

Synthesis,Pim kinase inhibitory potencies and in vitro antiproliferative activities of diversely substituted pyrrolo[2,3-a]carbazoles

The synthesis of new pyrrolo[2,3-a]carbazole derivatives diversely substituted at the C-6 to C-9 positions is described. These compounds were tested for their kinase inhibitory potencies toward three kinases (Pim-1, Pim-2, Pim-3) as well as for their in vitro antiproliferative activities toward a human fibroblast primary culture and three human solid cancer cell lines (PC3, DU145, and PA 1). Moreover, molecular docking studies were performed to explain the enhanced inhibitory activity of the most active compound 3d.     

Ethyl 2-(benzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate analogues as a new scaffold for protein kinase casein kinase 2 inhibitor

Protein kinase casein kinase 2 (PKCK2) is a constitutively active, growth factor-independent serine/threonine kinase, and changes in PKCK2 expression or its activity are reported in many cancer cells. To develop a novel PKCK2 inhibitor(s), we first performed cell-based phenotypic screening using 4000 chemicals purchased from ChemDiv chemical libraries (2000: randomly selected; 2000: kinase-biased) and performed in vitro kinase assay-based screening using hits found from the first screening. We identified compound 24 (C24)[(Z)-ethyl 5-(4-chlorophenyl)-2-(3,4-dihydroxybenzylidene)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a] pyrimidine-6-carboxylate] as a novel inhibitor of PKCK2 that is more potent and selective than 4,5,6,7-tetrabromobenzotriazole (TBB). In particular, compound 24 [half maximal inhibitory concentration (IC₅₀) = 0.56 μM] inhibited PKCK2 2.2-fold more efficiently than did TBB (IC₅₀ = 1.24 μM), which is quite specific toward PKCK2 with respect to ATP binding, in a panel of 31 human protein kinases. The K_i values of compound 24 and TBB for PKCK2 were 0.78 μM and 2.70 μM, respectively. Treatment of cells with compound 24 inhibited endogenous PKCK2 activity and showed anti-proliferative and pro-apoptotic effects against stomach and hepatocellular cancer cell lines more efficiently than did TBB. As expected, compound 24 also enabled tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-resistant cancer cells to be sensitive toward TRAIL. In comparing the molecular docking of compound 24 bound to PKCK2α versus previously reported complexes of PKCK2 with other inhibitors, our findings suggest a new scaffold for specific PKCK2α inhibitors. Thus, compound 24 appears to be a selective, cell-permeable, potent, and novel PKCK2 inhibitor worthy of further characterization.     

Triazolo and imidazo dihydropyrazolopyrimidine potassium channel antagonists

Previously disclosed C6 amido and benzimidazole dihydropyrazolopyrimidines were potent and selective blockers of I_Kur current. Syntheses and SAR for C6 triazolo and imidazo dihydropyrazolopyrimidines series are described. Trifluoromethylcyclohexyl N(1) triazole, compound 51, was identified as a potent and selective K_v1.5 inhibitor with an acceptable PK and liability profile.     

Identification of 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c]carbazoles as new Pim kinase inhibitors

New 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c]carbazoles were prepared and evaluated for their Pim kinase inhibitory potencies as well as their antiproliferative activities toward two prostatic cancer cell lines. Pyrazolocarbazole 15a was found to be a potent Pim kinase modulator with inhibitory potency toward the three isoforms. Compound 6c strongly inhibited Pim-3 with weaker effect toward Pim-1 and Pim-2, and thus could be used as an interesting molecular tool to study Pim-3 biological functions.     

Dialkylimidazole inhibitors of Trypanosoma cruzi sterol 14α-demethylase as anti-Chagas disease agents

New dialkylimidazole based sterol 14α-demethylase inhibitors were prepared and tested as potential anti-Trypanosoma cruzi agents. Previous studies had identified compound 2 as the most potent and selective inhibitor against parasite cultures. In addition, animal studies had demonstrated that compound 2 is highly efficacious in the acute model of the disease. However, compound 2 has a high molecular weight and high hydrophobicity, issues addressed here. Systematic modifications were carried out at four positions on the scaffold and several inhibitors were identified which are highly potent (EC₅₀ <1 nM) against T. cruzi in culture. The halogenated derivatives 36j, 36k, and 36p, display excellent activity against T. cruzi amastigotes, with reduced molecular weight and lipophilicity, and exhibit suitable physicochemical properties for an oral drug candidate.     

Docking- and pharmacophore-based virtual screening for the identification of novel Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) inhibitor with a thiobarbiturate scaffold

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an important virulence factor for Mtb that contributes to survival of the bacteria in macrophages. The absence of a human ortholog makes MptpB an attractive target for new therapeutics to treat tuberculosis. MptpB inhibitors could be an effective treatment to overcome emerging TB drug resistance. Adopting a structure-based virtual screening strategy, we successfully identified thiobarbiturate-based drug-like MptpB inhibitor 15 with an IC₅₀ of 22.4 μM, and as a non-competitive inhibitor with a K_i of 24.7 μM. Importantly, not only did it exhibit moderate cell membrane permeability, compound 15 also displayed potent inhibition of intracellular TB growth in the macrophage, making it an excellent lead compound for anti-TB drug discovery. To the best of our knowledge, this novel thiobarbiturate is the first class of MptpB inhibitor reported so far that leveraged docking- and pharmacophore-based virtual screening approaches. The results of preliminary structure-activity relationship demonstrated that compound 15 identified herein was not a singleton and may inspire the design of novel selective and drug-like MptpB inhibitors.     

Cyclic acyl guanidines bearing carbamate moieties allow potent and dirigible cholinesterase inhibition of either acetyl- or butyrylcholinesterase

A series of cyclic acyl guanidine with carbamate moieties have been synthesized and evaluated in vitro for their AChE and BChE inhibitory activities. Structure−activity relationships identified compound 23 as a nanomolar and selective BChE inhibitor, while compound 32 exhibited nanomolar and selective AChE inhibition, selectivity depending on both the structure of the carbamate substituent as well as the position of guanidines-N substitution. The velocity of enzyme carbamoylation was analyzed and showed similar behavior to physostigmine. Phenolic compounds formed after carbamate transfer to the active site of cholinesterases showed additional neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced intracellular reactive oxygen species generation.     

Synthesis and biological evaluation of C-glucosides with azulene rings as selective SGLT2 inhibitors for the treatment of type 2 diabetes mellitus: Discovery of YM543

Here, a series of C-glucosides with azulene rings in the aglycon moiety was synthesized and the inhibitory activities toward hSGLT1 and hSGLT2 were evaluated. Starting from the azulene derivative 7 which had relatively good SGLT2 inhibitory activity, compound 8a which has a 3-[(azulen-2-yl)methyl]phenyl group was identified as a lead compound for further optimization. Introduction of a phenolic hydroxyl group onto the central benzene ring afforded a potent and selective SGLT2 inhibitor 8e, which reduced blood glucose levels in a dose-dependent manner in rodent diabetic models. A mono choline salt of 8e (YM543) was selected as a clinical candidate for use in treating type 2 diabetes mellitus.     

Discovery and optimization of 1,7-disubstituted-2,2-dimethyl-2,3-dihydroquinazolin-4(1H)-ones as potent and selective PKCθ inhibitors

A high-throughput screening campaign helped us to identify an initial lead compound (1) as a protein kinase C-θ (PKCθ) inhibitor. Using the docking model of compound 1 bound to PKCθ as a model, structure-based drug design was employed and two regions were identified that could be explored for further optimization, i.e., (a) a hydrophilic region around Thr442, unique to PKC family, in the inner part of the hinge region, and (b) a lipophilic region at the forefront of the ethyl moiety. Optimization of the hinge binder led us to find 1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one as a potent and selective hinge binder, which resulted in the discovery of compound 5. Filling the lipophilic region with a suitable lipophilic substituent boosted PKCθ inhibitory activity and led to the identification of compound 10. The co-crystal structure of compound 10 bound to PKCθ confirmed that both the hydrophilic and lipophilic regions were fully utilized. Further optimization of compound 10 led us to compound 14, which demonstrated an improved pharmacokinetic profile and inhibition of IL-2 production in a mouse.     

Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.     

The discovery of novel benzofuran-2-carboxylic acids as potent Pim-1 inhibitors

Novel benzofuran-2-carboxylic acids, exemplified by 29, 38 and 39, have been discovered as potent Pim-1 inhibitors using fragment based screening followed by X-ray structure guided medicinal chemistry optimization. The compounds demonstrate potent inhibition against Pim-1 and Pim-2 in enzyme assays. Compound 29 has been tested in the Ambit 442 kinase panel and demonstrates good selectivity for the Pim kinase family. X-ray structures of the inhibitor/Pim-1 binding complex reveal important salt-bridge and hydrogen bond interactions mediated by the compound’s carboxylic acid and amino groups.     

Unexpected small molecules as novel SIRT2 suicide inhibitors

A series of sirtuin inhibitor candidates were assembled based on an intermediate ester (1a) our accidently discovered. After screening and evaluation, several SIRT2 selective inhibitors were identified, which can inhibit all the deacetylation, defatty-acylation and debenzoylation of SIRT2. Among these inhibitors, compound 1e was the best SIRT2 selective inhibitors. The primary study on the inhibitory mechanism indicated that compound 1e may be a suicide inhibitor acting as an irreversible way. Given almost all reported sirtuin inhibitors are non-covalent, sirtuin covalent inhibitors are still need to be developed. These findings will facilitate for further development of SIRT2 selective and suicide inhibitors.     

Design,synthesis, and evaluation of novel inhibitors for wild-type human serine racemase

Most of the endogenous free d-serine (about 90%) in the brain is produced by serine racemase (SR). d-Serine in the brain is involved in neurodegenerative disorders and epileptic states as an endogenous co-agonist of the NMDA-type glutamate receptor. Thus, SR inhibitors are expected to be novel therapeutic candidates for the treatment of these disorders. In this study, we solved the crystal structure of wild-type SR, and tried to identify a new inhibitor of SR by in silico screening using the structural information. As a result, we identified two hit compounds by their in vitro evaluations using wild-type SR.Based on the structure of the more potent hit compound 1, we synthesized 15 derivatives and evaluated their inhibitory activities against wild-type SR. Among them, the compound 9C showed relatively high inhibitory potency for wild-type SR. Compound 9C was a more potent inhibitor than compound 24, which was synthesized by our group based upon the structural information of the mutant-type SR.     

Discovery of potent PTP1B inhibitors via structure-based drug design,synthesis and in vitro bioassay of Norathyriol derivatives

Protein tyrosine phosphatase 1B (PTP1B) has recently been identified as a potential target of Norathyriol. Unfortunately, Norathyriol is not a potent PTP1B inhibitor, which somewhat hinders its further application. Based on the fact that no study on the relationship of chemical structure and PTP1B inhibitory activity of Norathyriol has been reported so far, we attempted to perform structural optimization so as to improve the potency for PTP1B. Via structure-based drug design (SBDD), a rational strategy based on the binding mode of Norathyriol to PTP1B, we designed 26 derivatives with substitutions at the four phenolic hydroxyl groups of Norathyriol. By chemical synthesis and in vitro bioassay, we identified seven PTP1B inhibitors that were more potent than Norathyriol, of which XWJ24 showed the highest potency (IC₅₀: 0.6 μM). We also found out that XWJ24 was a competitive inhibitor and showed the 4.5-fold selectivity over its close homolog, TC-PTP. Through molecular docking of XWJ24 against PTP1B, we highlighted the essential role of its hydrogen bond with Asp181 for PTP1B inhibition and identified a potential halogen bond with Asp48 that was not observed for Norathyriol. The current data indicate that our SBDD strategy is effective to discover potent PTP1B-targeted Norathyriol derivatives, and XWJ24 is a promising lead compound for further development.     

Novel 1,2-dihydroquinazolin-2-ones: Design,synthesis, and biological evaluation against Trypanosoma brucei

In 2014, a published report of the high-throughput screen of >42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.     

Identification of novel peptidomimetics targeting the polo-box domain of polo-like kinase 1

A series of new peptidomimetics targeting the polo-box domain (PBD) of polo-like kinase 1 (Plk1) was identified based on the potent and selective pentapeptide Plk1 PBD inhibitor PLHSpT. Unnatural amino acid residues were introduced to the newly designed compound and the N-terminal substituent of the peptidomimetic was investigated. The optimized compound 9 inhibited the Plk1 PBD with IC₅₀ of 0.267 μM and showed almost no inhibition to Plk2 PBD or Plk3 PBD at 100 μM. Biolayer interferometry studies demonstrated that compound 9 showed potent binding affinity to Plk1 with a K_d value of 0.164 μM, while no K_d were detected against Plk2 and Plk3. Compound 9 showed improved stability in rat plasma compared to PLHSpT. Binding mode analysis was performed and in agreement with the observed experimental results. There are only two natural amino acids remained in the chemical structure of 9. This study may provide new information for further research on Plk1 PBD inhibitors.     

A bromopyrrole-containing diterpene alkaloid from the Okinawan marine sponge Agelas nakamurai activates the insulin pathway in Huh-7 human hepatoma cells by inhibiting protein tyrosine phosphatase 1B

Agelasine G (1), a known bromine-containing diterpene alkaloid, was isolated as a new type of protein tyrosine phosphatase (PTP) 1B inhibitor together with ageline B (2), an inactive debromo-derivative of 1, from the marine sponge Agelas nakamurai collected at Iriomote Island in Okinawa, Japan. Further biological evaluations revealed that compound 1 exhibited selective inhibitory activity against PTP1B over T-cell PTP and CD45 phosphatase. Compound 1 also enhanced the insulin-stimulated phosphorylation levels of Akt in Huh-7 cells more strongly than compound 2. The results obtained in this study suggest that compound 1 activates the insulin signaling pathway by inhibiting PTP1B activity.