Studies on depigmenting activities of dihydroxyl benzamide derivatives containing adamantane moiety

Six diphenolic compounds containing adamantane moiety were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. The inhibitory activity of 4-adamantyl resorcinol 1 was similar to that of 4-n-butyl resorcinol in both assays. However, dihydroxyl benzamide derivatives 6a–e showed different inhibitory patterns. All derivatives significantly suppressed the cellular melanin formation without tyrosinase inhibitory activities. These behaviors indicated that the introduction of amide bond changes the binding mode of dihydroxyl groups to tyrosinase. Among derivatives, 6d (3,4-dihydroxyl compound) and 6e (2,3-dihydroxyl compound) showed stronger inhibitory activities (IC₅₀ = 1.25 μM and 0.73 μM, respectively) as compared to 4-n-butyl resorcinol (IC₅₀ = 21.64 μM) and hydroquinone (IC₅₀ = 3.97 μM). This study showed that the position of dihydroxyl substituent at aromatic ring is important for the intercellular inhibition of melanin formation, and also amide linkage and adamantane moiety enhance the inhibition.     

Highly potent tyrosinase inhibitor,neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson’s disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC₅₀ of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC₅₀ = 500 nM), significantly. Another potent inhibitor 1 (IC₅₀ = 2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: K_i^app = 1.48 nM, k₃ = 0.0033 nM⁻¹ min⁻¹ and k₄ = 0.0049 min⁻¹. Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC₅₀. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.     

Design,synthesis and biological evaluation of hydroxy substituted amino chalcone compounds for antityrosinase activity in B16 cells

A series of hydroxy substituted amino chalcone compounds have been synthesized. These compounds were then evaluated for their inhibitory activities on tyrosinase and melanogenesis in murine B16F10 melanoma cell lines. The structures of the compounds synthesized were confirmed by ¹H NMR, ¹³C NMR, FTIR and HRMS. Two novel amino chalcone compounds exhibited higher tyrosinase inhibitory activities (IC₅₀ values of 9.75 μM and 7.82 μM respectively) than the control kojic acid (IC₅₀: 22.83 μM). Kinetic studies revealed them to act as competitive tyrosinase inhibitors with their K_i values of 4.82 μM and 1.89 μM respectively. Both the compounds inhibited melanin production and tyrosinase activity in B16 cells. Docking results confirm that the active inhibitors strongly interact with mushroom tyrosinase residues. This study suggests that the depigmenting effect of novel amino chalcone compounds might be attributable to inhibition of tyrosinase activity, suggesting amino chalcones to be a promising candidate for use as depigmentation agents or as anti-browning food additives.     

Development of hydroxylated naphthylchalcones as polyphenol oxidase inhibitors: Synthesis,biochemistry and molecular docking studies

Polyphenol oxidase (Tyrosinase) has received great attention, since it is the key enzyme in melanin biosynthesis. In this study, novel hydroxy naphthylchalcone compounds were synthesized, and their inhibitory effects on mushroom tyrosinase activity were evaluated. The structures of the compounds synthesized were confirmed by ¹H NMR, ¹³C NMR, FTIR and HRMS. Two of the compounds synthesized inhibited the diphenolase activity of tyrosinase in a dose dependent manner and exhibited much higher tyrosinase inhibitory activities (IC₅₀ values of 10.4 μM and 14.4 μM, respectively) than the positive control, kojic acid (IC₅₀: 27.5 μM). Kinetic analysis showed that their inhibition was reversible. Both the novel compounds displayed competitive inhibition with their K_i values of 3.8 μM and 4.5 μM, respectively. Docking results confirmed that the active inhibitors strongly interacted with the mushroom tyrosinase residues. This study suggests hydroxy naphthylchalcone compounds to serve as promising candidates for use as depigmentation agents.     

Design,synthesis and biological evaluation of hydroxy- or methoxy-substituted 5-benzylidene(thio) barbiturates as novel tyrosinase inhibitors

Here a new class of hydroxy- or methoxy-substituted 5-benzylidene(thio)barbiturates were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that several compounds had more potent tyrosinase inhibitory activities than the widely used tyrosinase inhibitor kojic acid (IC₅₀ = 18.25 μM). In particular, 3′,4′-dihydroxylated 1e was found to be the most potent inhibitor with IC₅₀ value of 1.52 μM. The inhibition mechanism analysis revealed that the potential compounds 1e and 2e exhibited such inhibitory effects on tyrosinase by acting as the irreversible inhibitors. Structure–activity relationships’ (SARs) analysis also suggested that further development of such compounds might be of interest.     

Design and synthesis of thiourea derivatives with sulfur-containing heterocyclic scaffolds as potential tyrosinase inhibitors

Tyrosinase is a key enzyme during the production of melanins in plants and animals. A class of novel N-aryl-N′-substituted phenylthiourea derivatives (3a–i, 6a–k) were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed some 4,5,6,7-tetrahydro-2-[[(phenylamino)thioxomethyl]amino]-benzo[b]thiophene-3-carboxylic acid derivatives (3a–i) exhibited moderate inhibitory potency on diphenolase activity of tyrosinase. When the scaffold of 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid was replaced with 2-(1,3,4-thiadiazol-2-yl)thio acetic acid, the inhibitory activity of compounds (6a–k) against tyrosinase was improved obviously; especially, the inhibitory activity of compound 6h (IC₅₀ = 6.13 μM) is significantly higher than kojic acid (IC₅₀ = 33.3 μM). Moreover, the analysis on inhibition mechanism revealed that compound 6h might plays the role as a noncompetitive inhibitor.     

Discovery of a new type of scaffold for the creation of novel tyrosinase inhibitors

Tyrosinase is known as the key enzyme for melanin biosynthesis, which is effective in preventing skin injury by ultra violet (UV). In past decades, tyrosinase has been well studied in the field of cosmetics, medicine, agriculture and environmental sciences, and a lot of tyrosinase inhibitors have been developed for their needs. Here, we searched for new types of tyrosinase inhibitors and found phenylbenzoic acid (PBA) as a unique scaffold. Among three isomers of PBA, 3-phenylbenzoic acid (3-PBA) was revealed to be the most potent inhibitor against mushroom tyrosinase (IC₅₀ = 6.97 μM, monophenolase activity; IC₅₀ = 36.3 μM, diphenolase activity). The kinetic studies suggested that the apparent inhibition modes for the monophenolase and diphenolase activities were noncompetitive and mixed type inhibition, respectively. Analyses by in silico docking studies using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid group of the 3-PBA could adequately bind to two cupric ions in the tyrosinase. To prove this hypothesis, we examined the effect of modification of the carboxylic acid group of the 3-PBA on its inhibitory activity. As expected, the esterification abrogated the inhibitory activity. These observations suggest that 3-PBA is a useful lead compound for the generation of novel tyrosinase inhibitors and provides a new insight into the molecular basis of tyrosinase catalytic mechanisms.     

Cytotoxic,antimicrobial activities,AChE and BChE inhibitory effects of compounds from Tanacetum chiliophyllum (Fisch. & Mey.) Schultz Bip. var. oligocephalum (D.C.) Sosn. and T. chiliophyllum (Fisch. & Mey.) Schultz Bip. var. monocephalum Grierson

Tanacetum L. species traditionally used for insecticidal purposes as well as in folk medicine for their antitumor, antimicrobial, antifungal activities. In our previous study a novel sesquiterpene lactone and triterpene lactone together with 12 known flavonoids, coumarin and a triterpene were isolated from T. chiliophyllum var. oligocephalum and T. chiliophyllum var. monocephalum extracts which have insecticidal and antimicrobial activity. In this study, cytotoxic, antimicrobial activities and acetylcholinesterase (AChE), butyrylcholinesterase (BChE) inhibitory effects of pure compounds isolated from these plants were investigated. The tested compounds showed AChE and BChE inhibition which ranged between 7.20–80.37% and 9.19%–76.99% respectively. The highest AChE and BChE inhibition was observed for ulubelenolide which afforded 80.37% and 76.99% inhibition respectively. The cytotoxic effect of the compounds ranged between 22.34–49.77 μg/mL IC₅₀ values. Highest cytotoxic activity was observed against MCF-7 and HEK 293 cell line by 5–hydroxy-3′,4′,7-trimethoxy flavone and 5-hydroxy-3′,4′,6,7-tetramethoxyflavone that produced 25.80 ± 0.17 and 22.34 ± 0.70 IC₅₀ values respectively. Compounds eupatilin, cirsilineol, 5–hydroxy-3′,4′,7-trimethoxy flavone and ulubelenolide showed significant antimicrobial effect on C. albicans with 7.8 μg/mL MIC. The new compound ulubelenolide afforded high AChE and BChE inhibition as well as high antifungal activity. In our opinion activity of this substance should be evaluated further against other fungal species.     

Inhibitory effect of novel tetrahydropyrimidine-2(1H)-thiones on melanogenesis

The series of imidazoldine-2-thiones 2 and tetrahydropyrimidine-2-thiones 3 were discovered as inhibitor of α-MSH-induced melanin production in melanoma B16 cells. The primary bioassay showed that 1-(4-ethylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3e (>100% inhibition at 10 μM, IC₅₀ = 1.2 μM) and 1-(4-tert-butylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3f (>100% inhibition at 10 μM, IC₅₀ = 0.76 μM) exhibited potent inhibitory effect against α-MSH-induced melanin production. Compounds 3 inhibit the biosynthesis of tyrosinase without affecting its catalytic activity in melanogenesis.     

Synthesis and biological activity of oxadiazole and triazolothiadiazole derivatives as tyrosinase inhibitors

A series of 16 oxadiazole and triazolothiadiazole derivatives were designed, synthesized and evaluated as mushroom tyrosinase inhibitors. Five derivatives were found to display high inhibition on the tyrosinase activity ranging from 0.87 to 1.49 μM. Compound 5 exhibited highest tyrosinase inhibitory activity with an IC₅₀ value of 0.87 ± 0.16 μM. The in silico protein–ligand docking using autodock 4.1 was successfully performed on compound 5 with significant binding energy value of ?5.58 kcal/mol. The docking results also showed that the tyrosinase inhibition might be due to the metal chelating effect by the presence of thione functionality in compounds 1–5. Further studies revealed that the presence of hydrophobic group such as cycloamine derivatives played a major role in the inhibition. Piperazine moiety in compound 5 appeared to be involved in an extensive hydrophobic contact and a 2.9 Å hydrogen bonding with residue Glu 182 in the active site.     

Synthesis of novel azo-resveratrol,azo-oxyresveratrol and their derivatives as potent tyrosinase inhibitors

Ten azo compounds including azo-resveratrol (5) and azo-oxyresveratrol (9) were synthesized using a modified Curtius rearrangement and diazotization followed by coupling reactions with various phenolic analogs. All synthesized compounds were evaluated for their mushroom tyrosinase inhibitory activity. Compounds 4 and 5 exhibited high tyrosinase inhibitory activity (56.25% and 72.75% at 50 μM, respectively). The results of mushroom tyrosinase inhibition assays indicate that the 4-hydroxyphenyl moiety is essential for high inhibition and that 3,5-dihydroxyphenyl and 3,5-dimethoxyphenyl derivatives are better for tyrosinase inhibition than 2,5-dimethoxyphenyl derivatives. Particularly, introduction of hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mushroom tryosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (5) showed the most potent mushroom tyrosinase inhibition with an IC₅₀ value of IC₅₀ = 36.28 ± 0.72 μM, comparable to that of resveratrol, a well-known tyrosinase inhibitor.     

Synthesis,antioxidant capacity,and structure–activity relationships of tri-O-methylnorbergenin analogues on tyrosinase inhibition

A series of tri-O-methylnorbergenin analogues 1–9 were synthesized and their antioxidant activities and inhibitory effects on tyrosinase were evaluated. Among tested analogues, compound 4 bearing cathechol moiety exhibited greater antioxidant activity and excellent inhibition on tyrosinase with IC₅₀ value of 9.1 μM, comparable to that of corresponding positive controls. The inhibition mechanism analysis of compound 4 demonstrated that it was a mixed-type inhibitor on tyrosinase. These results suggest that these compounds may serve as a useful clue for further designing and development of novel potential tyrosinase inhibitors.     

Molecular docking studies and biological evaluation of 1,3,4-thiadiazole derivatives bearing Schiff base moieties as tyrosinase inhibitors

1,3,4-Thiadiazole derivatives bearing Schiff base moieties were designed, synthesized, and their tyrosinase inhibitory activities were evaluated. Some compounds displayed potent tyrosinase inhibitory activities, especially, 4-(((5-mercapto-1,3,4-thiadiazol-2-yl)-imino)methyl)-2-methoxy-phenol (14) exhibited superior inhibitory effect to the other compounds with an IC₅₀ value of 0.036 μM. The structure–activity relationships (SARs) were preliminarily discussed and docking studies showed compound 14 had strong binding affinity to mushroom tyrosinase. Hydroxy might be the active groups. The inhibition kinetics study revealed that compounds (13 and 14) inhibited tyrosinase by acting as uncompetitive inhibitors. The LD₅₀ value of the compound 14 was 5000 mg/kg.     

De novo tyrosinase inhibitor: 4-(6,7-Dihydro-5H-indeno[5,6-d]thiazol-2-yl)benzene-1,3-diol (MHY1556)

In this study, we have synthesized and studied de novo tyrosinase inhibitor, MHY1556, which showed significantly better efficacy than other pre-existing tyrosinase inhibitors in vitro experiments. The IC₅₀ value of MHY1556 was 0.50 μM which was significantly lower than that of kojic acid (IC₅₀ = 53.95 μM), which is a well-known tyrosinase inhibitor and was used as a positive control in this study. We predicted the tertiary structure of tyrosinase, simulated the docking with compound MHY1556 and confirmed that the compound strongly interacts with mushroom tyrosinase residues. Substitutions with a hydroxy group at both R1 and R3 of the phenyl ring indicated that these groups play a major role in the high binding affinity to tyrosinase, especially through the hydrogen bonding interaction of the hydroxyl group at R1 with GLY281. In addition, MHY1556 showed concentration-dependent inhibitory effects in melanin content assay where B16F10 melanoma cells were treated with α-melanocyte stimulating hormone (α-MSH), and also there is no significant cytotoxicity of this compound in cell viability assay conducted in B16F10 melanoma cells. The tyrosinase activity assay results with MHY1556 also support its potent inhibitory effects. Therefore, our data strongly suggest MHY1556 suppresses the melanogenesis via a tyrosinase inhibitory effect.     

Selective monoamine oxidase B inhibition by an Aphanizomenon flos-aquae extract and by its constitutive active principles phycocyanin and mycosporine-like amino acids

Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga that has been traditionally used for over 25 years for its health-enhancing properties. Recent studies have shown the ability of a proprietary AFA extract (Klamin^®) to improve mood, counteract anxiety, and enhance attention and learning. Aim of this study was to test the monoamine oxidase (MAO) inhibition activity of the same AFA extract and of its constituents phycocyanin (AFA-PC) and mycosporine-like aminoacids (AFA-MAAs). All compounds showed a dose-dependent selective inhibition of MAO-B activity as compared to MAO-A. The IC₅₀ values of the AFA extract (concentration 10 mg/ml), AFA-PC and AFA-MAAs were 6.4 μl/ml, 1.33 μM and 1.98 μM, respectively, evidencing a mixed-type of inhibition for the AFA extract (K_i 0.99 μl/ml), a non-competitive inhibition for AFA-PC (K_i 1.06 μM) and a competitive inhibition for AFA-MAAs (K_i 0.585 μM). These results are important to explain the neuromodulating properties of the AFA extract Klamin^®, which is rich in phenylethylamine, a general neuromodulator, that would nevertheless rapidly destroyed by MAO-B enzymes without the inhibitory activity of the synergic active principles AFA-PC and AFA-MAAs. The present investigation thus proposes the extract as potentially relevant in clinical areas such as mood disorders and neurodegenerative diseases.     

Natural ortho-dihydroxyisoflavone derivatives from aged Korean fermented soybean paste as potent tyrosinase and melanin formation inhibitors

Natural o-dihydroxyisoflavone (ODI) derivatives with variable hydroxyl substituent at the aromatic ring of isoflavone and three known isoflavones were isolated from five-year-old Korean fermented soybean paste (Doenjang) and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells comparing with other known isoflavones, 7,8,4′-trihydroxyisoflavone (1) and 7,3′,4′-trihydroxyisoflavone (2) inhibited tyrosinase by 50% at a concentration of 11.21 ± 0.8 μM and 5.23 ± 0.6 μM (IC₅₀), respectively, whereas, 6,7,4′-trihydroxyisoflavone (3), daidzein (4), glycitein (5) and genistein (6) showed very low inhibition activity. Furthermore, those compounds significantly suppressed the cellular melanin formation by 50% at a concentration of 12.23 ± 0.7 μM (1), 7.83 ± 0.7 μM (2), and 57.83 ± 0.5(6) and show more activity than arbutin. But, compounds 3, 4, and 5 showed lower inhibition activity. This study shows that the position of hydroxyl substituent at the aromatic ring of isoflavone plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Three new geranyl aurones from the leaves of Artocarpus altilis

From the MeOH extract of the leaves of Artocarpus altilis (Moraceae), three new aurones, altilisin H (1), I (2), and J (3), have been isolated together with two known flavonoids. Their structures were elucidated on the basis of spectroscopic data. All compounds possessed tyrosinase inhibitory activity with IC₅₀ values less than 100 μM, while compounds 1–3 displayed potent α-glucosidase inhibitory activity with IC₅₀ values ranging from 4.9 to 5.4 μM.     

Three new benzophenone glycosides with MAO-A inhibitory activity from Hypericum thasium Griseb.

Purification of n-BuOH fraction from 80% ethanol extract of Hypericum thasium Griseb. resulted in the isolation of three new compounds 3′,4,5′-trihydroxy-6-methoxy-2-O-α-l-arabinosylbenzophenone (1), 3′,4,5′,6-tetrahydroxy-2-O-α-l-arabinosylbenzophenone (2), and 3′,4-dihydroxy-5′-methoxy-2-O-α-l-arabinosyl-6-O-β-d-xylosylbenzophenone (3) along with a known flavonoid glycoside quercetin-3-O-α-l-arabinofuranoside (4). The structures of the new compounds were elucidated by 1D and 2D NMR analysis as well as HRESIMS. The isolated compounds (1–4), as well as quercetin, and kaempferol previously isolated from EtOAc fraction were screened against MAO-A inhibitory activity. When tested against the MAO-A quercetin and kaempferol displayed IC₅₀ values of 19.6, and 17.5 μM, respectively. The IC₅₀ values for MAO-A inhibition by compounds (1–4) were 310.3, 111.2, 726.0, and 534.1 μM, respectively. Standard inhibitor (clorgyline) exhibited MAO-A inhibition with an IC₅₀ value of 0.5 μM.     

A newly synthesized,potent tyrosinase inhibitor: 5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol

In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with a potent tyrosinase inhibitory activity. 5HNB inhibited mushroom tyrosinase with an IC₅₀ value of 2.95 μM, which is more potent than the well-known anti-tyrosinase activity of kojic acid (IC₅₀ = 38.24). The results of the enzymatic inhibition kinetics by Lineweaver–Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when l-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that 5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential for treating skin pigmentation disorders.     

Antioxidant, α-amylase inhibitory and oxidative DNA damage protective property of Boerhaavia diffusa (Linn.) root

Boerhaavia diffusa Linn. of family Nyctaginaceae is a known traditional medicinal plant and has been used in the treatment of many free radical mediated diseases. Excessive formation of free radicals, either reactive oxygen species (ROS) or reactive nitrogen species (RNS) is responsible for damaging various biomolecules like DNA, lipids and proteins. The present investigation was initially carried out to explore the possible link between antioxidant, oxidative DNA damage protective and α-amylase inhibitory property of B. diffusa root extract and their bioactive fraction. Our results illustrated an enhanced DPPH radical scavenging activity/antioxidant power of methanol root extract (IC₅₀ < 250 μg/ml) than ethanol (IC₅₀ = 250 μg/ml) and aqueous extract (IC₅₀ > 250 μg/ml). In addition, the methanol root extract also showed better oxidative DNA damage protective activity and α-amylase inhibitory property than ethanol and aqueous root extract. Phytochemical screening of B. diffusa ethanol and methanol root extract showed the presence of saponins, alkaloids, flavonoids, glycosides and terpenoids in large amount. By means of repetitive preparatory TLC and HPLC the potent antioxidant and α-amylase inhibitory fraction was isolated from methanol root extract. Our results illustrated that DPPH radical scavenging activity (IC₅₀ < 250 μg/ml) and oxidative DNA damage protective and α-amylase inhibitory activity of the isolated/purified bioactive compound from methanol extract were significantly closer to that of crude extract, which in turn confirm that antioxidant and antidiabetic property of methanol root extract resides in this fraction and established a significant correlation between antioxidant and inhibitory α-amylase property of this bioactive fraction compound. These profound effects of B. diffusa methanol root extract and its purified fraction against oxidative plasmid DNA damage, antioxidant and α-amylase inhibitory activity may explain its extensive use in daily life and possible health benefits.