Evaluation of Drug Blood-Brain-Barrier Permeability Using a Microfluidic Chip.

The blood-brain-barrier (BBB) is made up of blood vessels whose permeability enables the passage of some compounds. A predictive model of BBB permeability is important in the early stages of drug development. The predicted BBB permeabilities of drugs have been confirmed using a variety of in vitro methods to reduce the quantities of drug candidates needed in preclinical and clinical trials. Most prior studies have relied on animal or cell-culture models, which do not fully recapitulate the human BBB. The development of microfluidic models of human-derived BBB cells could address this issue. We analyzed a model for predicting BBB permeability using the Emulate BBB-on-a-chip machine. Ten compounds were evaluated, and their permeabilities were estimated. Our study demonstrated that the permeability trends of ten compounds in our microfluidic-based system resembled those observed in previous animal and cell-based experiments. Furthermore, we established a general correlation between the partition coefficient (Kp) and the apparent permeability (Papp). In conclusion, we introduced a new paradigm for predicting BBB permeability using microfluidic-based systems.

Design, Synthesis, and Biological Evaluation of New 2,6,7-Substituted Purine Derivatives as Toll-like Receptor 7 Agonists for Intranasal Vaccine Adjuvants.

TLR7/8 agonists are versatile immune stimulators capable of treating various diseases such as viral infections, autoimmune, and cancer. Despite the structural similarity of TLR7/8, their immune stimulation mechanisms and time-course responses significantly differ. In this study, a new series of TLR7-selective agonists was synthesized utilizing the economical building block 2,6-dichloropurine. Compound 27b showed the most potent activity on hTLR7 with an EC50 of 17.53 nM and demonstrated high hTLR7 selectivity (224 folds against TLR8). 27b effectively stimulated the secretion of proinflammatory cytokines in mouse macrophages and enhanced intranasal vaccine efficacy against influenza A virus in vivo. Assessment of humoral and mucosal antibody titers confirmed that 27b elevates IgG and IgA levels, protecting against both homologous and heterologous influenza viral infections. These findings suggest that 27b is a promising candidate as a vaccine adjuvant to prevent viral infections or as a robust immunomodulator with prolonged activity for treating immune-suppressed diseases.

Discovery of a peripheral 5HT2A antagonist as a clinical candidate for metabolic dysfunction-associated steatohepatitis.

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is currently the leading cause of chronic liver disease worldwide. Metabolic Dysfunction-Associated Steatohepatitis (MASH), an advanced form of MASLD, can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Based on recent findings by our team that liver 5HT2A knockout male mice suppressed steatosis and reduced fibrosis-related gene expression, we developed a peripheral 5HT2A antagonist, compound 11c for MASH. It shows good in vitro activity, stability, and in vivo pharmacokinetics (PK) in rats and dogs. Compound 11c also shows good in vivo efficacy in a diet-induced obesity (DIO) male mice model and in a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) male mice model, effectively improving histologic features of MASH and fibrosis. According to the tissue distribution study using [14C]-labeled 11c, the compound was determined to be a peripheral 5HT2A antagonist. Collectively, first-in-class compound 11c shows promise as a therapeutic agent for the treatment of MASLD and MASH.

Quantitative and Qualitative Analysis of Neurotransmitter and Neurosteroid Production in Cerebral Organoids during Differentiation.

In the human brain, neurophysiological activity is modulated by the movement of neurotransmitters and neurosteroids. To date, the similarity between cerebral organoids and actual human brains has been evaluated using comprehensive multiomics approaches. However, a systematic analysis of both neurotransmitters and neurosteroids from cerebral organoids has not yet been reported. Here, we performed quantitative and qualitative assessments of neurotransmitters and neurosteroids over the course of cerebral organoid differentiation. Our multiomics approaches revealed that the expression levels of neurotransmitter-related proteins and RNA, including neurosteroids, increase as cerebral organoids mature. We also found that the electrophysiological activity of human cerebral organoids increases in tandem with the expression levels of both neurotransmitters and neurosteroids. Our study demonstrates that the expression levels of neurotransmitters and neurosteroids can serve as key factors in evaluating the maturity and functionality of human cerebral organoids.

Synthesis and biological evaluation of xanthine derivatives with phenacyl group as tryptophan hydroxylase 1 (TPH1) inhibitors for obesity and fatty liver disease.

Tryptophan hydroxylase 1 (TPH1) has emerged as a target for the treatment of metabolic diseases including obesity and fatty liver disease. A series of xanthine derivatives were synthesized and evaluated for their TPH1 inhibition. Among the synthesized compounds, compound 40 showed good in vitro activity and liver microsomal stability. Docking studies revealed that compound 40 showed better binding to TPH1 via key intermolecular interactions involving the xanthine scaffold, imidazo-thiazolyl ring, and hydroxyl-containing phenacyl moiety. In addition, compound 40 effectively suppressed the adipocyte differentiation of 3 T3-L1 cells.

Inhibition of pyruvate dehydrogenase kinase 4 ameliorates kidney ischemia-reperfusion injury by reducing succinate accumulation during ischemia and preserving mitochondrial function during reperfusion.

Ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury (AKI), is still without effective therapies. Succinate accumulation during ischemia followed by its oxidation during reperfusion leads to excessive reactive oxygen species (ROS) and severe kidney damage. Consequently, the targeting of succinate accumulation may represent a rational approach to the prevention of IR-induced kidney injury. Since ROS are generated primarily in mitochondria, which are abundant in the proximal tubule of the kidney, we explored the role of pyruvate dehydrogenase kinase 4 (PDK4), a mitochondrial enzyme, in IR-induced kidney injury using proximal tubule cell-specific Pdk4 knockout (Pdk4ptKO) mice. Knockout or pharmacological inhibition of PDK4 ameliorated IR-induced kidney damage. Succinate accumulation during ischemia, which is responsible for mitochondrial ROS production during reperfusion, was reduced by PDK4 inhibition. PDK4 deficiency established conditions prior to ischemia resulting in less succinate accumulation, possibly because of a reduction in electron flow reversal in complex II, which provides electrons for the reduction of fumarate to succinate by succinate dehydrogenase during ischemia. The administration of dimethyl succinate, a cell-permeable form of succinate, attenuated the beneficial effects of PDK4 deficiency, suggesting that the kidney-protective effect is succinate-dependent. Finally, genetic or pharmacological inhibition of PDK4 prevented IR-induced mitochondrial damage in mice and normalized mitochondrial function in an in vitro model of IR injury. Thus, inhibition of PDK4 represents a novel means of preventing IR-induced kidney injury, and involves the inhibition of ROS-induced kidney toxicity through reduction in succinate accumulation and mitochondrial dysfunction.

The effects of environmental Microplastic on wharf roach (Ligia exotica): A Multi-Omics approach.

This is the first report to evaluate the potential effects of microplastics (MPs) on wild wharf roaches (Ligia exotica) in a shoreline habitant. L. exotica is an important plastic detritus consumer in coastal area. A survey was conducted from May to June in the years 2019 and 2020 in two South Korean nearshore sites: Nae-do (as MPs-uncontaminated) and Maemul-do (as MPs-contaminated). MPs (>20 µm in size) were detected highly in gastrointestinal tracts of the L. exotica from Maemul-do, at an average level of 50.56 particles/individual. They were detected in much lower levels in the L. exotica from Nae-do. at an average rate of 1.00 particles/individual. The polymer type and shape were dominated by expanded polystyrene (EPS, 93%) and fragment (99.9%) in L. exotica from Maemul-do. Especially, Hexabromocyclododecanes, brominated flame retardants added to EPS, have been detected highly in L. exotica from Maemul-do (630.86 ± 587.21 ng/g l. w.) than those of Nae-do (detection limit: 10.5 ng/g l. w). Genome-wide transcriptome profiling revealed altered expression of genes associated with fatty acid metabolic processes, the innate-immune response-activating system and vesicle cytoskeletal trafficking in L. exotica from Maemul-do. The activation of the p53 signaling pathway (which is related to proteasome, ER regulation and cell morphogenesis) is likely to be involved in the EPS-uptake of wild L. exotica. Four neurosteroids were also detected in head tissue, and cortisol and progesterone concentrations differed significantly in L. exotica from Maemul-do. Our findings also suggest that resident plastic detritus consumer might be a useful indicator organism for evaluating pollution and potential effects of environmental microplastics.

Integrative multi-omics reveals analogous developmental neurotoxicity mechanisms between perfluorobutanesulfonic acid and perfluorooctanesulfonic acid in zebrafish.

The molecular mechanism of perfluorobutanesulfonic acid (PFBS), an alternative to legacy perfluorooctanesulfonic acid (PFOS), is not fully understood yet. Therefore, we conducted a developmental toxicity evaluation on zebrafish embryos exposed to PFBS and PFOS and assessed neurobehavioral changes at concentrations below each point of departure (POD) determined by embryonic mortality. Using transcriptomics, proteomics, and metabolomics, biomolecular perturbations in response to PFBS were profiled and then integrated for comparison with those for PFOS. Although PFBS (7525.47 µM POD) was approximately 700 times less toxic than PFOS (11.42 µM POD), altered neurobehavior patterns and affected kinds of endogenous neurochemicals were similar between PFBS and PFOS at the corresponding POD-based concentrations. Multi-omics analysis revealed that the PFBS neurotoxicity mechanism was associated with oxidative stress, lipid metabolism, and glycolysis/glucogenesis. The commonalities in developmental neurotoxicity-related mechanisms between PFBS and PFOS interconnected by knowledge-based integration of multi-omics included the calcium signaling pathway, lipid homeostasis, and primary bile acid biosynthesis. Despite being less toxic than PFOS, PFBS exhibited similar dysregulated molecular mechanisms, suggesting that chain length differences do not affect the intrinsic toxicity mechanism. Overall, carefully managing potential toxicity of PFBS can secure its status as an alternative to PFOS.

Mechanism of Bisphenol F Affecting Motor System and Motor Activity in Zebrafish.

Bisphenol F (BPF; 4,4'-dihydroxydiphenylmethane) is one of the most frequently used compounds in the manufacture of plastics and epoxy resins. Previous studies have demonstrated that BPF affects locomotor behavior, oxidative stress, and neurodevelopment in zebrafish. However, its neurotoxic effects are controversial, and the underlying mechanisms are unclear. In order to determine whether BPF affects the motor system, we exposed zebrafish embryos to BPF and assessed behavioral, histological, and neurochemical changes. Spontaneous locomotor behavior and startle response were significantly decreased in BPF-treated zebrafish larvae compared with control larvae. BPF induced motor degeneration and myelination defects in zebrafish larvae. In addition, embryonic exposure to BPF resulted in altered metabolic profiles of neurochemicals, including neurotransmitters and neurosteroids, which may impact locomotion and motor function. In conclusion, exposure to BPF has the potential to affect survival, motor axon length, locomotor activity, myelination, and neurochemical levels of zebrafish larvae.

Evaluation of Antiviral Activity of Gemcitabine Derivatives against Influenza Virus and Severe Acute Respiratory Syndrome Coronavirus 2.

Gemcitabine is a nucleoside analogue of deoxycytidine and has been reported to be a broad-spectrum antiviral agent against both DNA and RNA viruses. Screening of a nucleos(t)ide analogue-focused library identified gemcitabine and its derivatives (compounds 1, 2a, and 3a) blocking influenza virus infection. To improve their antiviral selectivity by reducing cytotoxicity, 14 additional derivatives were synthesized in which the pyridine rings of 2a and 3a were chemically modified. Structure-and-activity and structure-and-toxicity relationship studies demonstrated that compounds 2e and 2h were most potent against influenza A and B viruses but minimally cytotoxic. It is noteworthy that in contrast to cytotoxic gemcitabine, they inhibited viral infection with 90% effective concentrations of 14.5-34.3 and 11.4-15.9 µM, respectively, maintaining viability of mock-infected cells over 90% at 300 µM. Resulting antiviral selectivity was comparable to that of a clinically approved nucleoside analogue, favipiravir. The cell-based viral polymerase assay proved the mode-of-action of 2e and 2h targeting viral RNA replication and/or transcription. In a murine influenza A virus-infection model, intraperitoneal administration of 2h not only reduced viral RNA level in the lungs but also alleviated infection-mediated pulmonary infiltrates. In addition, it inhibited replication of severe acute respiratory syndrome virus 2 infection in human lung cells at subtoxic concentrations. The present study could provide a medicinal chemistry framework for the synthesis of a new class of viral polymerase inhibitors.

Application of convolutional neural network for analyzing hepatic fibrosis in mice.

Recently, with the development of computer vision using artificial intelligence (AI), clinical research on diagnosis and prediction using medical image data has increased. In this study, we applied AI methods to analyze hepatic fibrosis in mice to determine whether an AI algorithm can be used to analyze lesions. Whole slide image (WSI) Sirius Red staining was used to examine hepatic fibrosis. The Xception network, an AI algorithm, was used to train normal and fibrotic lesion identification. We compared the results from two analyses, that is, pathologists' grades and researchers' annotations, to observe whether the automated algorithm can support toxicological pathologists efficiently as a new apparatus. The accuracies of the trained model computed from the training and validation datasets were greater than 99%, and that obtained by testing the model was 100%. In the comparison between analyses, all analyses showed significant differences in the results for each group. Furthermore, both normalized fibrosis grades inferred from the trained model annotated the fibrosis area, and the grades assigned by the pathologists showed significant correlations. Notably, the deep learning algorithm derived the highest correlation with the pathologists' average grade. Owing to the correlation outcomes, we conclude that the trained model might produce results comparable to those of the pathologists' grading of the Sirius Red-stained WSI fibrosis. This study illustrates that the deep learning algorithm can potentially be used for analyzing fibrotic lesions in combination with Sirius Red-stained WSIs as a second opinion tool in non-clinical research.

Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin-p65 interaction.

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics. Thus, there is a significant demand for new chemical entities (NCEs) targeting neuroinflammation. Herein, we rediscovered benzopyran-embedded tubulin inhibitor 1 as an anti-neuroinflammatory agent via phenotype-based screening. A competitive photoaffinity labeling study revealed that compound 1 binds to tubulin at the colchicine-binding site. Structure-activity relationship analysis of 1's analogs identified SB26019 as a lead compound with enhanced anti-neuroinflammatory efficacy. Mechanistic studies revealed that upregulation of the tubulin monomer was critical for the anti-neuroinflammatory activity of SB26019. We serendipitously found that the tubulin monomer recruits p65, inhibiting its translocation from the cytosol to the nucleus and blocking NF-κB-mediated inflammatory pathways. Further in vivo validation using a neuroinflammation mouse model demonstrated that SB26019 suppressed microglial activation by downregulating lba-1 and proinflammatory cytokines. Intraperitoneal administration of SB26019 showed its therapeutic potential as an NCE for successful anti-neuroinflammatory regulation. Along with the recent growing demands on tubulin modulators for treating various inflammatory diseases, our results suggest that colchicine-binding site-specific modulation of tubulins can be a potential strategy for preventing neuroinflammation and treating CNS diseases.

Seizurogenic effect of perfluorooctane sulfonate in zebrafish larvae.

While the developmental neurotoxicity of perfluorooctane sulfonate (PFOS) has been reported, its seizurogenic potential has not been investigated. Behavior assessment was conducted in zebrafish larvae exposed to PFOS at concentrations of 0, 0.1, 1, 5, 10, and 20 µM. Changes in electrophysiological signals and in the concentration of 20 neurochemicals were measured. Behavior assessment revealed that PFOS altered larval behaviors and significantly increased the counts and duration of bursting (an irregular high-speed movement). Electrophysiological analysis showed that the number of seizure-like events and duration of seizure-like signals were significantly increased, corresponding to results observed using pentylenetetrazol as a positive seizurogenic agent. The outbreak of seizures detected via abnormal electrophysiological signals was confirmed by the increased expression of c-fos and bdnf, which are typical seizure-related genes. Analysis of neurochemicals indicated that PFOS dysregulated overall neurotransmission systems, and aberrant endogenous concentrations of various neurochemicals in the amino acid, cholinergic, dopaminergic, serotonergic and kynurenergic, and GABAergic systems were associated with seizure-like behavior and signals. This study, the first to demonstrate that exposure to PFOS provokes a seizurogenic effect in developing zebrafish larvae, should stimulate further research on the association between PFOS exposure and neurodevelopmental toxicity or neurological disorders.

Size-Dependent Effects of Polystyrene Nanoparticles (PS-NPs) on Behaviors and Endogenous Neurochemicals in Zebrafish Larvae.

Microplastics, small pieces of plastic derived from polystyrene, have recently become an ecological hazard due to their toxicity and widespread occurrence in aquatic ecosystems. In this study, we exposed zebrafish larvae to two types of fluorescent polystyrene nanoparticles (PS-NPs) to identify their size-dependent effects. PS-NPs of 50 nm, unlike 100 nm PS-NPs, were found to circulate in the blood vessels and accumulate in the brains of zebrafish larvae. Behavioral and electroencephalogram (EEG) analysis showed that 50 nm PS-NPs induce abnormal behavioral patterns and changes in EEG power spectral densities in zebrafish larvae. In addition, the quantification of endogenous neurochemicals in zebrafish larvae showed that 50 nm PS-NPs disturb dopaminergic metabolites, whereas 100 nm PS-NPs do not. Finally, we assessed the effect of PS-NPs on the permeability of the blood-brain barrier (BBB) using a microfluidic system. The results revealed that 50 nm PS-NPs have high BBB penetration compared with 100 nm PS-NPs. Taken together, we concluded that small nanoparticles disturb the nervous system, especially dopaminergic metabolites.

Size-dependent seizurogenic effect of polystyrene microplastics in zebrafish embryos.

The effects of polystyrene microplastic (PS-MP) size on neurotoxicity remain to be evaluated at various microsizes, and the seizurogenic effects of PS-MPs are unknown. This study aimed to evaluate the swimming behavior of zebrafish larvae under light-dark transitions after exposure to four PS-MP sizes (i.e., 1, 6, 10, and 25 µm) at concentrations of 500, 5,000, and 50,000 particles/mL. Changes in electroencephalographic signals, seizure-related gene expression, and neurochemical concentrations were measured. Locomotor activity was inhibited only by 10-µm PS-MPs. According to electroencephalographic signals, the number and total duration of seizure-like events significantly increased by 10-µm PS-MPs, which was confirmed by the altered expression of seizure-related genes c-fos and pvalb5. Additionally, an increase in the levels of neurochemicals choline, betaine, dopamine, 3-methoxytyramine, and gamma-aminobutyric acid indicated that the observed hypoactivity and seizure-like behavior were associated with the dysregulation of the cholinergic, dopaminergic, and GABAergic systems. Overall, these findings demonstrate that exposure to PS-MPs can potentially cause seizurogenic effects in developing zebrafish embryos, and we highlight that PS-MPs 10 µm in size dominantly affect neurotoxicity.

Chronic exposure to butyl-paraben causes photosensitivity disruption and memory impairment in adult zebrafish.

Limited studies on neurotoxicity following chronic exposure to butyl­paraben (BuP) have been conducted. In this study, neurobehavior in zebrafish adults was assessed using the novel tank test, photomotor response test, and T-maze test after exposure to BuP for 28 days at concentrations of 0, 0.01, 0.1, and 1.0 mg/L. To comprehensively understand the underlying molecular perturbations in the brain, alterations in transcripts, neurotransmitters, and neurosteroids were measured. We found that BuP penetrated the blood-brain barrier and impaired neurobehavior in photosensitivity at 1.0 mg/L and in memory at 0.1 and 1.0 mg/L. RNA-seq analysis showed that phototransduction, tight junctions, and neuroactive ligand receptor activity were significantly affected, which explains the observed abnormal neurobehaviors. Neurosteroid analysis revealed that BuP increased cortisol levels in a concentration-dependent manner and specifically reduced allopregnanolone levels at all tested concentrations, suggesting that cortisol and allopregnanolone are significant neurosteroid markers associated with photosensitivity and memory deficits. Collectively, we demonstrated that BuP can cross the blood-brain and modulate the levels of transcripts, associated with phototransduction and circadian rhythm, and neurosteroidal cortisol and allopregnanolone, resulting in abnormal neurobehavioral responses to light stimulation and learning and memory.

Mechanism of action and neurotoxic effects of chronic exposure to bisphenol F in adult zebrafish.

Although bisphenol F (BPF), the main replacement for bisphenol A, has been commonly used in polycarbonate production, its neurotoxicity and the underlying mechanisms remain poorly understood. To address this knowledge gap, this study aimed to assess the neurotoxicity caused by chronic exposure to BPF and to identify its underlying mechanisms. We exposed adult zebrafish chronically to BPF at environmentally relevant concentrations (0.001, 0.01, and 0.1 mg/L) for 4 weeks. The results revealed that with BPF crossing the blood-brain barrier and bioaccumulating in brain tissues, chronic exposure to BPF resulted in anxiety-like behaviors and disruptions in learning and memory function in adult zebrafish. Furthermore, BPF toxicity in the zebrafish brain involved the dysregulation of metabolic pathways for choline and kynurenine in neurotransmitter systems and for 17ß-estradiol, cortisol, pregnenolone-sulfate, and Dehydroepiandrosterone (DHEA)-sulfate in neurosteroid systems. RNA-seq analysis revealed that BPF exposure affected metabolic pathways, calcium signaling pathways, neuroactive ligand-receptor interactions, tight junctions, gap junctions, and the gonadotropin-releasing hormone signaling pathway. Our results indicate that chronic exposure to BPF alters the neurochemical profile of the brain and causes neurobehavioral effects, such as anxiety and cognitive decline. Overall, the multimodal approach, including behavioral and neurochemical profiling technologies, has great potential for the comprehensive assessment of potential risks posed by environmental pollutants to human and ecosystem health.

Novel Thioxothiazolo[3,4-a]quinazolin-5(4H)-one Derivatives as BKCa Channel Activators for Urinary Incontinence.

Overactive bladder (OAB) is a syndrome causing a sudden and unstoppable need to urinate with significant global prevalence. Several drugs are used to treat OAB; however, they have various side effects. Therefore, new treatment options for OAB are required. A series of novel 5-oxo-N-phenyl-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide derivatives were synthesized and evaluated for their large-conductance voltage- and Ca2+-activated K+ channel activation through a cell-based fluorescence assay and electrophysiological recordings. Several compounds, including a 7-bromo substituent on the heterocyclic system, showed increased channel currents. Among the derivatives, compound 12h exhibited potent in vitro activity with a half-maximal effective concentration (EC50) of 2.89 µM, good oral pharmacokinetic properties (area under the curve and half-life), and in vivo efficacy in a spontaneously hypertensive rat model.

Identification of New Non-BBB Permeable Tryptophan Hydroxylase Inhibitors for Treating Obesity and Fatty Liver Disease.

Serotonin (5-hydroxytryptophan) is a hormone that regulates emotions in the central nervous system. However, serotonin in the peripheral system is associated with obesity and fatty liver disease. Because serotonin cannot cross the blood-brain barrier (BBB), we focused on identifying new tryptophan hydroxylase type I (TPH1) inhibitors that act only in peripheral tissues for treating obesity and fatty liver disease without affecting the central nervous system. Structural optimization inspired by para-chlorophenylalanine (pCPA) resulted in the identification of a series of oxyphenylalanine and heterocyclic phenylalanine derivatives as TPH1 inhibitors. Among these compounds, compound 18i with an IC50 value of 37 nM was the most active in vitro. Additionally, compound 18i showed good liver microsomal stability and did not significantly inhibit CYP and Herg. Furthermore, this TPH1 inhibitor was able to actively interact with the peripheral system without penetrating the BBB. Compound 18i and its prodrug reduced body weight gain in mammals and decreased in vivo fat accumulation.

Synthesis and biological evaluation of tyrosine derivatives as peripheral 5HT2A receptor antagonists for nonalcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), attributed to excessive fat accumulation in the liver, is reportedly prevalent worldwide. NAFLD is one of the leading causes of chronic liver disease, including non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatic cellular carcinoma (HCC). The peripheral roles of serotonin (5-hydroxytryptamine, 5HT) were found to regulate hepatic lipid metabolism. Among serotonin receptor subtypes, 5HT2A receptor is known to regulate hepatic lipid metabolism. Hepatic lipid accumulation and hepatic triglyceride (TG) were reduced in liver-specific 5HT2A receptor knockout (5HT2A receptor LKO) mice upon high-fat diet (HFD) feeding. In the present study, we explored a series of new peripherally acting 5HT2A receptor antagonists. Compound 14a displayed good in vitro activity, with an IC50 value of 0.17 nM. Compound 14a exhibited good microsomal stability, no significant CYP and hERG inhibition, and 5HT receptor subtype selectivity. The brain-to-plasma ratio of 14a was below the lower limit of quantification, indicating limited blood-brain barrier (BBB) penetration. HFD-fed 14a treated mice showed decreased liver steatosis and lobular inflammation. These results demonstrate the potential of newly synthesized peripheral 5HT2A receptor antagonists for treating NAFLD.