Synthesis and Anti-Inflammatory Activity of N(2)-Arylindazol-3(2H)-One Derivatives: Copper-Promoted Direct N-Arylation via Chan-Evans-Lam Coupling.

Inflammatory-related diseases are becoming increasingly prevalent, leading to a growing focus on the development of anti-inflammatory agents, with a particular emphasis on creating novel structural compounds. In this study, we present a highly efficient synthetic method for direct N-arylation to produce a variety of N(2)-arylindazol-3(2H)-ones 3, which exhibit anti-inflammatory activity. The Chan-Evans-Lam (CEL) coupling of N(1)-benzyl-indazol-3-(2H)-ones 1 with arylboronic acids 2 in the presence of a copper complex provided the corresponding N(2)-arylindazol-3(2H)-ones 3 in good-to-excellent yields, as identified with NMR, MS, and X-ray crystallography techniques. The cell viability and anti-inflammatory effects of the synthesized compounds (3 and 5) were briefly assessed using the MTT method and Griess assay. Among them, compounds 5 exhibited significant anti-inflammatory effects with negligible cell toxicity.

Identification of a Novel ERK5 (MAPK7) Inhibitor, MHJ-627, and Verification of Its Potent Anticancer Efficacy in Cervical Cancer HeLa Cells.

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase (MAPK) family, is involved in key cellular processes. However, overexpression and upregulation of ERK5 have been reported in various cancers, and ERK5 is associated with almost every biological characteristic of cancer cells. Accordingly, ERK5 has become a novel target for the development of anticancer drugs as inhibition of ERK5 shows suppressive effects of the deleterious properties of cancer cells. Herein, we report the synthesis and identification of a novel ERK5 inhibitor, MHJ-627, and verify its potent anticancer efficacy in a yeast model and the cervical cancer HeLa cell line. MHJ-627 successfully inhibited the kinase activity of ERK5 (IC50: 0.91 µM) and promoted the mRNA expression of tumor suppressors and anti-metastatic genes. Moreover, we observed significant cancer cell death, accompanied by a reduction in mRNA levels of the cell proliferation marker, proliferating cell nuclear antigen (PCNA), following ERK5 inhibition due to MHJ-627 treatment. We expect this finding to serve as a lead compound for further identification of inhibitors for ERK5-directed novel approaches for oncotherapy with increased specificity.

Synthesis of 1,4-Dialkoxynaphthalene-Based Imidazolium Salts and Their Cytotoxicity in Cancer Cell Lines.

In this study, we designed and synthesized novel 1,4-dialkoxynaphthalene-2-alkyl imidazolium salt (IMS) derivatives containing both 1,4-dialkoxynaphthalene and imidazole, which are well known as pharmacophores. The cytotoxicities of these newly synthesized IMS derivatives were investigated in order to explore the possibility of using them to develop anticancer drugs. It was found that some of the new IMS derivatives showed good cytotoxic activities. In addition, an initial, qualitative structure-activity relationship is presented on the basis of observations of activity changes corresponding to structural changes.

Scalable Synthesis of TRPV1 Antagonist Bipyridinyl Benzimidazole Derivative via the Suzuki-Miyaura Reaction and Selective SeO2 Oxidation.

In this study, a kilogram-scale synthesis of a potent TRPV1 antagonist, 1, is described. To synthesize bipyridinyl benzimidazole derivative 1, we have developed a scalable Suzuki-Miyaura reaction capable of providing a key intermediate, 6'-methyl-3-(trifluoromethyl)-2,3'-bipyridine 4, on a kilogram scale. Then, unlike the existing oxidation reaction pathway, two synthetic routes that can be applied to mass production of bipyridinyl carboxylic acid intermediate 5 or aldehyde intermediate 6 were developed by appropriately controlling the oxidation reaction using a selenium dioxide oxidizing agent. Using our developed synthetic procedure, which includes Suzuki-Miyaura coupling, selective selenium dioxide oxidation, and benzimidazole formation, multi-kilogram-scale bi-pyridinyl benzimidazole derivative 1 can be synthesized.

Identification of a potent NAFLD drug candidate for controlling T2DM-mediated inflammation and secondary damage in vitro and in vivo.

Accumulation of glucose/sugar results in the formation of reactive di-carbonyl compounds such as MGO and GO that interact with several amino acids and proteins to form toxic advanced glycation end products (AGEs). Induction of AGEs breakdown can control symptoms and severity in T2DM and other related complications like NAFLD where AGEs are the key players. Therefore, an AGE cross-link breaker has been suggested for preventing the onset/progression of NAFLD. In this study, we reported novel synthetic naphthalene-2-acyl thiazolium derivatives (KHAGs). Among synthesized KHAG derivatives, we observed that a novel KHAG-04, a 1,4-dimethoxynaphthalen-2-acyl thiazolium salt which is an analog of alagebrium, dramatically cleaves MGO/GO-AGE cross-links, and it also inhibited inflammation by lowering the level of nitric oxide production and IL-1ß and TNF-α secretion in LPS and/or MGO-AGE-activated macrophage. Moreover, it also reduced FFA and MGO-AGE-induced lipogenesis in Hep-G2 cells. In mice, KHAG-04 significantly reduced the level of glyoxal in the liver, which was induced by DMC. Furthermore, KHAG-04 treatment significantly reduced blood glucose levels, lipid accumulation, and inflammation in the NAFLD/T2DM animal model. Novel KHAG-04-mediated induction of AGEs breakdown could be the possible reason for its anti-inflammatory, antihyperglycemic, and anti-lipidemic effects in cells and NAFLD in the T2DM animal model, respectively. Further research might explore the pharmacological efficacy and usefulness and consider the ability of this compound in the treatment strategy against various models of NAFLD in T2DM where MGO/GO-AGEs play a key role in the pathogenesis.

Synthesis and anti-inflammatory effects of Δ7-Cholestenol and Δ8(14)-Cholestenol derivatives.

In this study, O-glycosides of cholesterol derivatives Δ7-cholestenol and Δ8(14)-cholestenol were prepared by Schmidt glycosylation with various glycosyl imidates. In addition, 1,2,3-triazole-linked N-glycosides of Δ8(14)-cholestenol were prepared via the "click" reaction of proparzylic Δ8(14)-cholestenol with glycosyl azides. The anti-inflammatory activities of these molecules were investigated for inhibitory mRNA expression activity of CCL17 and CCL22, which are important biomarkers of atopic dermatitis. Both the O- and N-glycosides Δ7-cholestenol and Δ8(14)-cholestenol were observed to exhibit good anti-inflammatory activity in vitro. In particular, the 1,2,3-triazole-linked N-glycoside of Δ8(14)-cholestenol exhibited anti-inflammatory activity similar to that of O-glycoside in vitro and is expected to be more potent in vivo due to better stability.

Synthesis of Novel (S)-3-(1-Aminoethyl)-8-pyrimidinyl-2-phenylisoquinolin-1(2H)-ones by Suzuki-Miyaura Coupling and Their Cell Toxicity Activities.

A series of (S)-3-(1-aminoethyl)-8-pyrimidinyl-2-phenylisoquinoline-1(2H)-ones 3a-3k was synthesized in 40-98% yield through Suzuki-Miyaura coupling using Pd(PPh3)2Cl2, Sphos, and K2CO3 in THF/H2O mixed solvent. All newly synthesized compounds were evaluated for cell viability (IC50) against MDA-MB-231, HeLa, and HepG2 cells. The antitumor activities of 3a-3k were improved when various pyrimidine motifs were introduced at position C-8 of the isoquinolinone ring.

Δ8(14)-Ergostenol Glycoside Derivatives Inhibit the Expression of Inflammatory Mediators and Matrix Metalloproteinase.

Arthritis is a chronic inflammatory disease accompanied by pathological reactions such as swelling, redness, fever, and pain in various joint areas. The drugs currently available to treat arthritis are associated with diverse side-effects. Therefore, there is a need for safer and more effective treatments to alleviate the inflammation of arthritis with fewer side-effects. In this study, a new sterol, Δ8(14)-ergostenol, was discovered, and its glycosides were synthesized and found to be more efficient in terms of synthesis or anti-inflammatory activity than either spinasterol or 5,6-dihydroergosterol is. Among these synthetic glycosides, galactosyl ergostenol inhibited the expression of inflammatory mediators in TNF-α-stimulated FLS and TNF-α-induced MMPs and collagen type II A1 degradation in human chondrocytes. These results suggest the new galactosyl ergostenol as a treatment candidate for arthritis.

Anti-Inflammatory Activity of a Medicinal Herb Extract Mixture, HM-V, on an Animal Model of DNCB-Induced Chronic Skin Inflammation.

Chronic inflammatory skin diseases, such as atopic dermatitis, are caused by the accumulation of immune cells and the overproduction of chemokines, including CCL17 and CCL22, due to the activation of pro-inflammatory cytokines secreted from keratinocytes. In the present study, the inhibitory activity of HM-V on tumor necrosis factor alpha (TNF-α)/interferon gamma (IFN-γ)-induced pro-inflammatory cytokines was examined in human keratinocytes (HaCaTs) and 2,4-dinitrofluorobenzene (DNCB)-induced chronic skin contact dermatitis animal models. Traditional Asian medicinal herb extracts mixture (HM-V), which have been extensively used in Asian medicine, were utilized. In TNF-α/IFN-γ-induced HaCaTs, HM-V strongly inhibited mRNA and protein expression of CCL17 and CCL22 in a concentration-dependent manner. The expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 was also inhibited. Therefore, localized administration of HM-V in the DNCB-induced animal model alleviated immune cell deposition and skin inflammation. The results indicate that HM-V exerts inhibitory effects on keratinocyte production of CCL17 and CCL22. Furthermore, HM-V may be a useful anti-inflammatory agent for the prevention and treatment of inflammatory skin diseases.

Antifungal Activity of 1,4-Dialkoxynaphthalen-2-Acyl Imidazolium Salts by Inducing Apoptosis of Pathogenic Candida spp.

Even though Candida spp. are staying commonly on human skin, it is also an opportunistic pathogenic fungus that can cause candidiasis. The emergence of resistant Candida strains and the toxicity of antifungal agents have encouraged the development of new classes of potent antifungal agents. Novel naphthalen-2-acyl imidazolium salts (NAIMSs), especially 1,4-dialkoxy-NAIMS from 1,4-dihydroxynaphthalene, were prepared and evaluated for antifungal activity. Those derivatives showed prominent anti-Candida activity with a minimum inhibitory concentration (MIC) of 3.125 to 6.26 µg/mL in 24 h based on microdilution antifungal susceptibility test. Among the tested compounds, NAIMS 7c showed strongest antifungal activity with 3.125 µg/mL MIC value compared with miconazole which showed 12.5 µg/mL MIC value against Candida spp., and more importantly >100 µg/mL MIC value against C. auris. The production of reactive oxygen species (ROS) was increased and JC-1 staining showed the loss of mitochondrial membrane potential in C. albicans by treatment with NAIMS 7c. The increased release of ultraviolet (UV) absorbing materials suggested that NAIMS 7c could cause cell busting. The expression of apoptosis-related genes was induced in C. albicans by NAIMS 7c treatment. Taken together, the synthetic NAIMSs are of high interest as novel antifungal agents given further in vivo examination.

Ribose conversion with amino acids into pyrraline platform chemicals - expeditious synthesis of diverse pyrrole-fused alkaloid compounds.

One-pot conversion of sustainable d-ribose with l-amino acid, methyl esters produced pyrrole-2-carbaldehydes 5 in reasonable yields (32-63%) under pressurized conditions of 2.5 atm at 80 °C. The value-added pyrraline compounds 5 as platform chemicals were utilized for quick installation of poly-heterocyclic cores for the development of pyrrole-motif natural and artificial therapeutic agents. A pyrrole-fused piperazin-2-one scaffold 6 was prepared by reductive amination of pyrralines 5 with benzylamine. While further cyclization of pyrralines 5 with ethane-1,2-diamine produced pyrrolo-piperazin-2-ones 7 with an extra imidazolidine ring, the reaction with 2-amino alcohols derived from natural l-amino acids, alanine, valine, and phenylalanine, respectively provided pyrrolo-piperazin-2-ones 8, 9, and 10 with oxazolidine as the third structural core. Cell viability and an anti-inflammatory effect of the synthesized compounds were briefly tested by the MTT method and the Griess assay, among which 8h and 10g exhibited significant anti-inflammatory effects with negligible cell toxicity.

New Pyrimidinone-Fused 1,4-Naphthoquinone Derivatives Inhibit the Growth of Drug Resistant Oral Bacteria.

BACKGROUND: Dental caries is considered to be a preventable disease, and various antimicrobial agents have been developed for the prevention of dental disease. However, many bacteria show resistance to existing agents. METHODS/PRINCIPAL FINDINGS: In this study, four known 1,4-naphthoquinones and newly synthesized 10 pyrimidinone-fused 1,4-naphthoquinones, i.e. KHQ 701, 702, 711, 712, 713, 714, 715, 716, 717 and 718, were evaluated for antimicrobial activity against Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Actinomyces viscosus and Fusobacterium nucleatum. Pyrimidinone-fused 1,4-naphthoquinones were synthesized in good yields through a series of chemical reactions from a commercially available 1,4-dihydroxynaphthoic acid. MIC values of KHQ 711, 712, 713, 714, 715, 716, 717 and 718 were 6.25-50 µg/mL against E. faecalis (CCARM 5511), 6.25-25 µg/mL against E. faecium (KACC11954) and S. aureus (CCARM 3506), 1.56-25 µg/mL against S. epidermidis (KACC 13234), 3.125-100 µg/mL against S. mutans (KACC16833), 1.56-100 µg/mL against S. sobrinus (KCTC5809) and P. gingivalis (KCTC 5352), 3.125-50 µg/mL against A. viscosus (KCTC 9146) and 3.125-12.5 µg/mL against F. nucleatum (KCTC 2640) with a broth microdilution assay. A disk diffusion assay with KHQ derivatives also exhibited strong susceptibility with inhibition zones of 0.96 to 1.2 cm in size against P. gingivalis. Among the 10 compounds evaluated, KHQ 711, 712, 713, 715, 716 and 717 demonstrated strong antimicrobial activities against the 9 types of pathogenic oral bacteria. A pyrimidin-4-one moiety comprising a phenyl group at the C2 position and a benzyl group at the N3 position appears to be essential for physiological activity. CONCLUSION/SIGNIFICANCE: Pyrimidinone-fused 1,4-naphthoquinones synthesized from simple starting compounds and four known 1,4-naphthoquinones were synthesized and showed strong antibacterial activity to the 9 common oral bacteria. These results suggest that these derivatives should be prospective for the treatment of dental diseases caused by oral bacteria, including drug-resistant strains.

Comparative Study of the Effect of 5,6-Dihydroergosterol and 3-epi-5,6-dihydroergosterol on Chemokine Expression in Human Keratinocytes.

5,6-Dihydroergosterol-glucose is an organic synthetic derivative of spinasterol-glucose, which has potent anti-inflammatory activity. We previously synthesized alpha and beta anomers of DHE-glycosides and compared their inhibitory activity on CCL17 and CCL22 mRNA expression induced by TNF-α/IFN-γ in activated HaCaTs. Recently, we synthesized a new type of DHE-glycosides, 3-epi-5,6-dihydroergosterol(3-epi-DHE)-glycosides, and compared its inhibitory activity on mRNA expression levels of CCL17 and CCL22 in TNF-α/IFN-γ-induced HaCaT. DHE-Xly did not affect TNF-α/IFN-γ induced CCL17 and CCL22 mRNA expression in HaCaTs, however, 3-epi-DHE-Xly strongly inhibited TNF-α/IFN-γ induced CCL17 and CCL22 mRNA expression levels in human keratinocytes. These results provide important clues for development of chronic dermatitis treatment via inhibition of chemokine expression using DHE derivatives.

Development of a carbamate-conjugated catechol ligand and its application to Cs extraction from contaminated soil by using supercritical CO2.

Extraction of radioactive Cs from contaminated soil is a crucial aspect of remediation after nuclear accidents. For this purpose, we have developed a new type of ligand, carbamate-conjugated catechol, to assist in metal extraction by using supercritical CO2 (SCCO2). The synthesis process for this ligand is relatively simple, and the carbamate-conjugated catechol ligand dissolves well in SCCO2. The measured ligand distribution coefficient increased according to a power law with an exponent of 1.7 as the ligand concentration increased, indicating that approximately two ligand molecules are needed to extract one Cs ion. The roles of additives (ligand, co-ligand, and water) were limited when they were used separately, but the combination of these additives was important. We tested 27 combinations of these three additives for extracting Cs from artificially contaminated sea sand. A quantitative analysis indicated that the ligand had the strongest influence on Cs extraction, followed by water, and the co-ligand. The carbamate-conjugated catechol ligand was then used for Cs extraction from artificially contaminated real soil. Three types of soil were prepared: coarse soil (particle size = 0.5-1 mm), medium soil (particle size = 0.2-0.5 mm), and fine soil (particle size < 0.2 mm). The Cs fractions extracted from the coarse, medium, and fine soils were measured to be 95%, 91% and 70% of the Cs fraction extracted from sea sand, respectively, which indicates the existence of a surface area effect. Additionally, we suspect that Cs undergoes chemical interaction on the surface of real soil.

Comparison of the Inhibitory Activities of 5,6-Dihydroergosterol Glycoside α- and ß-Anomers on Skin Inflammation.

Chronic skin inflammatory diseases, such as atopic dermatitis, are associated with a dysfunctional skin barrier due to an increase in various inflammatory stimuli, for instance inflammatory cytokines and chemokines. In particular, CCL17 and CCL22 expression is increased in patients with chronic skin inflammation. In this study, we synthesized several α- and ß-anomers of dihydroergosterol (DHE)-glycosides and assessed their effects on CCL17 and CCL22 expression. We confirmed that the ß-anomers of DHE-glycosides were superior to α-anomers of DHE-glycosides in inhibiting CCL17 and CCL22 mRNA and protein expression. In addition, we determined that DHE-glycoside ß-anomers showed strong inhibitory activity towards pro-inflammatory cytokine mRNA and protein expression, including that of TNF-α, IL-6, and IL-1ß- in stimulated HaCaT cells. These results imply that DHE-glycoside α- and ß-anomers should be separated during synthesis of drugs for chronic skin inflammation. Our results also suggest that ß-anomers of DHE-glycosides may play an important role as new drugs for chronic skin inflammation because of their ability to inhibit the skin inflammatory biomarker proteins CCL17 and CCL22.

Trapping of Stable [4n+1] π-Electron Species from Peripherally Substituted, Conformationally Rigid, Antiaromatic Hexaphyrins.

Peripherally substituted antiaromatic naphthorosarins have been synthesized for the first time. The synthesis was accomplished by acid-catalyzed condensation of naphthobipyrrole building blocks with aromatic aldehydes. The naphthobipyrrole building blocks were synthesized by simple oxidative coupling of the corresponding pyrrole substituted aromatics. Solid-state structural analyses of the synthesized naphthorosarins revealed that the presence of meso-2,6-dichlorophenyl- and 5,6-difluoro-substitution substantially alter the geometry and properties of the naphthorosarins. The substituents affect the redox potentials as well and, in turn, the proton-coupled electron-transfer processes leading to the formation of one- and two-electron reduced forms of the corresponding naphthorosarins. One particular naphthorosarin that bears both peripheral fluorine and meso-2,6-dichlorophenyl substituents forms a stable 25 π-electron species upon treating with TFA that was characterized by single-crystal X-ray diffraction analysis. The current study underscores how structural modifications can be used to fine-tune the electronic features of naphthorosarins, including stabilization of odd electron species.

YH18968, a Novel 1,2,4-Triazolone G-Protein Coupled Receptor 119 Agonist for the Treatment of Type 2 Diabetes Mellitus.

G protein-coupled receptor 119 (GPR119) is expressed in the pancreas and gastrointestinal tract, and its activation promotes insulin secretion in the beta cells of the pancreatic islets as well as the secretion of glucagon-like peptide-1 (GLP-1) in intestinal L cells, consequently improving glucose-stimulated insulin secretion. Due to this dual mechanism of action, the development of small-molecule GPR119 agonists has received significant interest for the treatment of type 2 diabetes. We newly synthesized 1,2,4-triazolone derivatives of GPR119 agonists, which demonstrated excellent outcomes in a cyclic adenosine monophosphate (cAMP) assay. Among the synthesized derivatives, YH18968 showed cAMP=2.8 nM; in GLUTag cell, GLP-1secretion=2.3 fold; in the HIT-T15 cell, and insulin secretion=1.9 fold. Single oral administration of YH18968 improved glucose tolerance and combined treatment with a dipeptidyl peptidase 4 (DPP-4) inhibitor augmented the glucose lowering effect as well as the plasma level of active GLP-1 in normal mice. Single oral administration of YH18968 improved glucose tolerance in a diet induced obese mice model. This effect was maintained after repeated dosing for 4 weeks. The results indicate that YH18968 combined with a DPP-4 inhibitor may be an effective therapeutic candidate for the treatment of type 2 diabetes.

N,N-disubstituted azines attenuate LPS-mediated neuroinflammation in microglia and neuronal apoptosis via inhibiting MAPK signaling pathways.

BACKGROUND: Activated microglia interact with astrocytes and neuronal cells to induce neuroinflammation, which can contribute to the pathogenesis and progression of Alzheimer's and Parkinson's disease. To identify the most effective anti-neuroinflammatory agent, we designed and synthesized a family of 13 new azine derivatives and investigated their anti-neuroinflammatory activities in LPS-activated BV-2 microglial cells. RESULTS: Out of 13 derivatives, compound 3 [4,4'-(1E,1'E,3E,3'E)-3,3'-(hydrazine-1,2-diylidene) bis-(prop-1-ene-1-yl-3-ylidene) bis-(2-methoxyphenol)] exhibited excellent anti-neuroinflammatory activities (IC50 = 12.47 µM), which protected neurons from microglia-mediated neurotoxicity. Specifically, the anti-neuroinflammatory effects of compound 3 inhibited MAPK signaling pathways through the inhibition of p38 and JNK mediated signaling and the production of pro-inflammatory cytokines, and inflammatory mediators. Additionally, compound 3 strongly exhibited neuroprotective effect by inhibiting LPS-mediated necrosis and apoptosis. Preliminary SAR analysis suggests that the presence of methoxyphenol and the substitution pattern within hydrazine may influence the anti-neuroinflammatory activity. FACS analysis also strongly supports the neuroprotective effect of compound 3. CONCLUSIONS: Based on our results, the compound 3 exhibited excellent anti-neuroinflammatory activity against LPS-activated microglia, which resulted in the inhibition of neuronal apoptosis and neuronal degeneration.

Low-Affinity Zinc Sensor Showing Fluorescence Responses with Minimal Artifacts.

The study of the zinc biology requires molecular probes with proper zinc affinity. We developed a low-affinity zinc probe (HBO-ACR) based on an azacrown ether (ACR) and an 2-(2-hydroxyphenyl)benzoxazole (HBO) fluorophore. This probe design imposed positive charge in the vicinity of a zinc coordination center, which enabled fluorescence turn-on responses to high levels of zinc without being affected by the pH and the presence of other transition-metal ions. Steady-state and transient photophysical investigations suggested that such a high tolerance benefits from orchestrated actions of proton-induced nonradiative and zinc-induced radiative control. The zinc bioimaging utility of HBO-ACR has been fully demonstrated with the use of human pancreas epidermoid carcinoma, PANC-1 cells, and rodent hippocampal neurons from cultures and acute brain slices. The results obtained through our studies established the validity of incorporating positively charged ionophores for the creation of low-affinity probes for the visualization of biometals.

Gomisin N Inhibits Melanogenesis through Regulating the PI3K/Akt and MAPK/ERK Signaling Pathways in Melanocytes.

Gomisin N, one of the lignan compounds found in Schisandra chinensis has been shown to possess anti-oxidative, anti-tumorigenic, and anti-inflammatory activities in various studies. Here we report, for the first time, the anti-melenogenic efficacy of Gomisin N in mammalian cells as well as in zebrafish embryos. Gomisin N significantly reduced the melanin content without cellular toxicity. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, Gomisin N downregulated the expression levels of key proteins that function in melanogenesis. Gomisin N downregulated melanocortin 1 receptor (MC1R), adenylyl cyclase 2, microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). In addition, Gomisin N-treated Melan-A cells exhibited increased p-Akt and p-ERK levels, which implies that the activation of the PI3K/Akt and MAPK/ERK pathways may function to inhibit melanogenesis. We also validated that Gomisin N reduced melanin production by repressing the expression of MITF, tyrosinase, TRP-1, and TRP-2 in mouse and human cells as well as in developing zebrafish embryos. Collectively, we conclude that Gomisin N inhibits melanin synthesis by repressing the expression of MITF and melanogenic enzymes, probably through modulating the PI3K/Akt and MAPK/ERK pathways.