Anticancer Evaluation of Novel Benzofuran-Indole Hybrids as Epidermal Growth Factor Receptor Inhibitors against Non-Small-Cell Lung Cancer Cells.

The epidermal growth factor receptor (EGFR), also known as ErbB1 and HER1, belongs to the receptor tyrosine kinase family. EGFR serves as the primary driver in non-small-cell lung cancer (NSCLC) and is a promising therapeutic target for NSCLC. In this study, we synthesized a novel chemical library based on a benzofuran-indole hybrid scaffold and identified 8aa as a potent and selective EGFR inhibitor. Interestingly, 8aa not only showed selective anticancer effects against NSCLC cell lines, PC9, and A549, but it also showed significant inhibitory effects against the double mutant L858R/T790M EGFR, which frequently occurs in NSCLC. In addition, in PC9 and A549 cells, 8aa potently blocked the EGFR signaling pathway, cell viability, and cell migration. These findings suggest that 8aa, a benzofuran-indole hybrid derivative, is a novel EGFR inhibitor that may be a potential candidate for the treatment of NSCLC patients with EGFR mutations.

Catalyst-free assembly of a polyfunctionalized 1,2,4-triazole-fused N-heterocycle, 6-acylated pyrrolo[1,2-a][1,2,4]triazolo[5,1-c]pyrazine.

A catalyst-free synthetic route to a new 1,2,4-triazole-pyrrolo[1,2-a]pyrazine hybrid system, 6-acylpyrrolo[1,2-a][1,2,4]triazolo[5,1-c]pyrazine, was realized upon sequential exposure of pyrrole-2-carbonitrile-derived substrates to DMF-DMA and acyl hydrazide through which acylated pyrazine and 1,2,4-triazole rings were consecutively formed with the formation of multiple bonds (one C-C bond and three C-N bonds). This annulative functionalization approach towards N-fused polycycles enabled us to install various substituents at specific positions on the core skeleton.

Regioselective Access to 1,2,4-Triazole-Fused N-Heterocycle, Pyrrolo[1,2-a][1,2,4]triazolo[5,1-c]pyrazine, via Double Dehydrative Cyclizations.

A highly efficient and regioselective approach to a novel 1,2,4-triazole-fused N-heterocyclic scaffold, pyrrolo[1,2-a][1,2,4]triazolo[3,4-c]pyrazine, was established by base-promoted reaction of pyrrole-2-carbonitrile-derived substrate with acyl hydrazide where domino double ring closures comprised of enamine formation, attack on nitrile, and cyclodehydration enabled sequential construction of pyrazine and 1,2,4-triazole ring systems with formation of three C-N bonds.

Benzo[d]imidazole-pyrrolo[1,2-a]pyrazine Hybrids Ameliorate Amyloid Aggregates in the Brain of Alzheimer Transgenic Mice.

Abnormal assembly of amyloid ß (Aß) in the brain is implicated in Alzheimer's disease (AD) and is associated with cognitive impairments. Since Aß accumulation occurs in advance of the onset of clinical symptoms, identifying preventable drug candidates regulating Aß accumulation is regarded as a promising approach in AD therapeutic. Herein, we synthesized eight Yonsei Institute of pharmaceutical sciences Alzheimer's Drug (YIAD) compounds based on 5-benzyl-6-phenylbenzo[4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazine structures. Subsequently, YIAD-0203 and YIAD-0205 were selected as effective candidates via thioflavin T assays and gel electrophoresis. The potential therapeutic effect of YIAD-0203 and YIAD-0205 on Aß aggregates was investigated through an AD transgenic mouse model with five familial AD mutations (5XFAD) by oral gavage. Significant amounts of Aß plaque and oligomer reduction were observed in the hippocampus region of both 4.3-month-old (early stage of AD) and 6.0-month-old (mid stage of AD) YIAD-0205-treated 5XFAD mice brains when compared to the nontreated brains. The ability of YIAD-0205 to ameliorate Aß aggregates in the early and mid stages of AD progression supports the notion that YIAD-0205 could be utilized as a reliable scaffold for the development of preventive AD drug candidates.

4-Acyl-3,4-dihydropyrrolo[1,2-a]pyrazine Derivative Rescued the Hippocampal-Dependent Cognitive Decline of 5XFAD Transgenic Mice by Dissociating Soluble and Insoluble Aß Aggregates.

Cerebral amyloid-ß (Aß) deposition is a representative hallmark of Alzheimer's disease (AD). Development of Aß-clearing small molecules could be an advantageous therapeutic strategy for Aß clearance considering the advantages in terms of side effects, cost-effectiveness, stability, and oral bioavailability. Here, we report an Aß-dissociating small molecule, YIAD-0121, a derivative of 4-acyl-3,4-dihydropyrrolo[1,2-a]pyrazine. Through a series of anti-Aß screening assays, YIAD-0121 was identified to inhibit Aß aggregation and dissociate preformed Aß fibrils in vitro. Furthermore, the administration of YIAD-0121 in 5XFAD transgenic AD mice inhibited the increase of cerebral Aß aggregation and progression of hippocampus-dependent cognitive decline, with ameliorated neuroinflammation.

C3 Functionalization of Indolizines via HFIP-Promoted Friedel-Crafts Reactions with (Hetero)arylglyoxals.

A highly efficient Friedel-Crafts type hydroxyalkylation at the C3 position of indolizines with (hetero)arylglyoxals has been achieved by the action of hexafluoroisopropanol (HFIP) under mild reaction conditions, leading to direct access to a variety of polyfunctionalized indolizines in excellent yields. Installation of more diverse functional groups at the C3 site of indolizine scaffold was realized via further elaboration of the resulting α-hydroxyketone moiety, allowing for expansion of indolizine chemical space.

Divergent synthesis of two types of indolizines from pyridine-2-acetonitrile, (hetero)arylglyoxal, and TMSCN.

Herein we describe the divergent synthesis of two types of indolizines via construction of the pyrrole moiety from pyridine-2-acetonitriles, arylglyoxals, and TMSCN. While one-pot three-component coupling provided 2-aryl-3-aminoindolizines via an unusual fragmentation process, a sequential two-step assembly protocol with these starting materials allowed efficient access to a wide range of new 2-acyl-3-aminoindolizines through an aldol condensation-Michael addition-cycloisomerization process. The subsequent manipulation of 2-acyl-3-aminoindolizines enabled direct access to novel polycyclic N-fused heteroaromatic skeletons.

Cu-Catalyzed Ullmann-Type Double C-N Coupling Approach to 5-Aryl-5H-indolizino[3,2-b]indoles.

A modular approach to a polyfunctionalized 5H-indolizino[3,2-b]indole, an indolizine-indole fused system, was achieved from readily available pyridine-2-acetonitrile, 2-bromobenzaldehyde, and TMSCN via the strategic combination of a one-pot three-component assembly and Cu-catalyzed Ullmann-type double C-N coupling reactions through which five new bonds (two C-C and three C-N) were formed in two steps.

Aryloxypropanolamine targets amyloid aggregates and reverses Alzheimer-like phenotypes in Alzheimer mouse models.

BACKGROUND: Aggregated amyloid-ß (Aß) is considered a pathogenic initiator of Alzheimer's disease (AD), in strong association with tau hyperphosphorylation, neuroinflammation, synaptic dysfunction, and cognitive decline. As the removal of amyloid burden from AD patient brains by antibodies has shown therapeutic potential, the development of small molecule drugs inducing chemical dissociation and clearance of Aß is compelling as a therapeutic strategy. In this study, we synthesized and screened aryloxypropanolamine derivatives and identified 1-(3-(2,4-di-tert-pentylphenoxy)-2-hydroxypropyl)pyrrolidin-1-ium chloride, YIAD002, as a strong dissociator of Aß aggregates. METHODS: The dissociative activity of aryloxypropanolamine derivatives against Aß aggregates were evaluated through in vitro assays. Immunohistochemical staining, immunoblot assays, and the Morris water maze were used to assess the anti-Alzheimer potential in YIAD002-treated 5XFAD and transgenic APP/PS1 mice. Target-ligand interaction mechanism was characterized via a combination of peptide mapping, fluorescence dissociation assays, and constrained docking simulations. RESULTS: Among 11 aryloxypropanolamine derivatives, YIAD002 exerted strongest dissociative activity against ß-sheet-rich Aß aggregates. Upon oral administration, YIAD002 substantially reduced amyloid burden and accordingly, improved cognitive performance in the Morris water maze and attenuated major pathological hallmarks of AD including tauopathy, neuroinflammation, and synaptic protein loss. Mechanism studies suggest that YIAD002 interferes with intermolecular ß-sheet fibrillation by directly interacting with KLVFFA and IGLMVG domains of Aß. In addition, YIAD002 was found to possess dissociative activity against aggregates of pyroglutamate-modified Aß and tau. CONCLUSIONS: Collectively, our results evince the potential of chemical-driven dissociation of Aß aggregates by aryloxypropanolamines as a therapeutic modality of the amyloid clearance approach.

Synthesis and Biological Evaluation of a Fused Structure of Indolizine and Pyrrolo[1,2-c]pyrimidine: Identification of Its Potent Anticancer Activity against Liver Cancer Cells.

A highly efficient approach to a new indolizine scaffold fused with pyrrolo[1,2-c]pyrimidine was achieved via one-pot three-component coupling followed by an oxidative cyclization reaction. The simple two-step sequence allowed rapid access to various tetracyclic compounds from commercially available starting materials with the formation of five new bonds. Here, we observed the effects of these compounds on cell viability in HepG2, H1299, HT29, AGS, and A549 cancer cell lines. Interestingly, this fused scaffold had more potent anticancer activity in hepatocellular carcinoma HepG2 and Huh7 cells than other cancer cells. In particular, 5r strongly decreased cell viability in HepG2 and Huh7 cells with an IC50 value of 0.22 ± 0.08 and 0.10 ± 0.11 µM, respectively, but had a very weak inhibitory effect on the cell viability of other cancer cell lines. In addition, 5r significantly inhibited cell migration and induced apoptosis in HepG2 and Huh7 cells via the activation of caspase-3 and cleavage of PARP in a dose-dependent manner. Notably, the co-treatment of 5r with gemcitabine resulted in the significant additional inhibition of cell viability in HepG2 and Huh7 cells. Our results suggest that 5r could be used to develop new chemotype anticancer agents against liver cancers.

VI-116, A Novel Potent Inhibitor of VRAC with Minimal Effect on ANO1.

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3'-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I- quenching with an IC50 of 1.27 ± 0.18 µM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 µM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K+ channel activity up to 10 µM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.

Generation of a poly-functionalized indolizine scaffold and its anticancer activity in pancreatic cancer cells.

A highly efficient domino [4 + 2] annulation process was employed to construct a novel indolizine chemical scaffold. Biological investigation led us to identify 6w as a potent anticancer agent. 6w significantly inhibited cell viability in BxPC3 pancreatic cancer, MCF7 breast cancer, and PC3 prostate cancer cell lines with IC50 values of 0.47 ± 0.04, 1.82 ± 0.08 and 2.68 ± 0.08 µM, respectively. Remarkably, 6w showed a weak effect on cell viability of nontumorigenic human keratinocyte cell line HaCaT compared to the above three types of cancer cells. 6w most potently inhibited cell viability of BxPC3 cells, and 6w also potently reduced cell migration and induced apoptosis in BxPC3 cells through activation of caspase-3 and cleavage of PARP in a dose-dependent manner. These results suggest that 6w can be used for the development of potential anticancer drugs for the treatment of pancreatic cancer.

Anti-amyloidogenic indolizino[3,2-c]quinolines as imaging probes differentiating dense-core, diffuse, and coronal plaques of amyloid-ß.

Abnormal deposition of amyloid-ß (Aß) is a major biomarker that is often used to diagnose Alzheimer's disease (AD). The Aß plaque levels in the cortex and hippocampus are measured by either brain histology or positron emission tomography. Although cerebral plaques are found in several phenotypes, such as dense-core, diffuse, and coronal, imaging probes differentiating these plaques are currently unavailable. Here, we report that fluorescent indolizino[3,2-c]quinoline derivatives (YIQ) distinguish Aß plaque phenotypes in brains of 5XFAD Alzheimer transgenic mice. We synthesized and screened 64 YIQ compounds through a series of in vitro and ex vivo Aß staining assays. We found 20 compounds that could stain the Aß phenotypes, 10 for dense-core plaques, eight for both dense-core and diffuse plaques, and two for solely visualizing only the coronal plaques while leaving the centric core unstained. Among the 20 imaging candidates, five YIQs displaying anti-Aß aggregation efficacy were confirmed by thioflavin T assays and electrophoretic analyses.

Development of a DABCO-Succinic Acid Based Catalytic System for the Aza-Michael Addition and Aza-Michael/Knoevenagel Tandem Reaction of Thiazolidine-2,4-dione to Electron Deficient Alkenes.

A DABCO catalyzed aza-Michael addition of thiazolidine-2,4-dione to a variety of electron deficient alkenes has been developed. Additionally, a DABCO/succinic acid salt system has been designed that allows for the one pot tandem aza-Michael/Knoevenagel reaction of thiazolidine-2,4-dione to give difunctionalized thiazolidine-2,4-dione products. To the best of our knowledge, this is the first example of a one-pot tandem aza-Michael/Knoevenagel reaction involving thiazolidine-2,4-dione.

Diastereoselective Synthesis of Densely Functionalized 3a,8a-Dihydro-8H-furo[3,2-a]pyrrolizines through One-Pot Three-Component Assembly.

A new domino mode of assembly was discovered from the one-pot three-component reactions of pyrrole derivatives, active methylene compounds (malononitrile, methyl cyanoacetate, or ethyl cyanoacetate), and sodium cyanide in the presence of piperidinium acetate in EtOH at room temperature, leading to a novel tricyclic skeleton in excellent yield under mild and eco-friendly conditions. This well-choreographed domino process enabled formation of multiple bonds (three C-C and one C-O) for consecutive construction of two rings (pyrrolidine and dihydrofuran) in a diastereoselective manner.

Michael-Aldol Double Elimination Cascade to Make Pyridines: Use of Chromone for the Synthesis of Indolizines.

A domino Michael-aldol double elimination route to indolizines having two different acyl groups at the C5 and C7 positions is described where chromone is employed as a two-carbon unit for the synthesis of a pyridine moiety for the first time. Various analogues were readily accessed in good yields under metal-free and eco-friendly conditions. Further manipulation of the resulting products allowed entry to novel indolizine-heterocycle adducts, which are difficult to access by other methods.

One-pot three-component coupling access to 1,2-dihydropyrrolo[1,2-a]pyrazine-1-phosphonates: multi-functionalization of a pyrazine unit.

A new pyrrolo[1,2-a]pyrazine chemical space with a poly-substituted pyrazine unit was readily accessed by Sc(OTf)3-catalyzed one-pot three-component coupling of a pyrrole derivative, amine, and trialkylphosphite under environment-friendly conditions. The formation of multiple bonds (two C-N and one C-P) via a domino process consisting of the chemoselective Kabachnik-Fields reaction and intramolecular cyclodehydration allowed for the construction of highly functionalized pyrazines.

A regioselective [4 + 2] annulation approach to 5-acylindolizine-7-carbonitriles: generation of poly-substituted pyridines.

The reaction of ß-ketonitrile and N-substituted pyrrole-2-carboxaldehyde in the presence of piperidinium acetate in CH3CN permitted regioselective access to 5-acylindolizine-7-carbonitrile through a domino Knoevenagel condensation-intramolecular aldol cyclization sequence. A wide range of new indolizine derivatives with a poly-functionalized pyridine moiety were readily synthesized in an atom-economical fashion, thereby expanding the indolizine chemical space for further applications.

X-ray Crystal Structure-Guided Design and Optimization of 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile Scaffold as a Potent and Orally Active Monopolar Spindle 1 Inhibitor.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile scaffold. Our lead optimization was guided by key X-ray crystal structure analysis. In vivo evaluation of candidate (9) is shown to effectively mitigate human TNBC cell proliferation.