Engineering Streptomyces sp. CPCC 204095 for the targeted high-level production of isatropolone A by elucidating its pathway-specific regulatory mechanism.

BACKGROUND: Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. RESULTS: Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield-980.8 mg/L of isatropolone A-achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. CONCLUSION: In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.

Semisynthetic derivatives of the fungal metabolite eupenifeldin via targeting the tropolone hydroxy groups.

Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2-6), carbonate (7-8), sulfonate (9-16), carbamate (17-20), and ether (21-30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1-30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2-8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue (6), which was acylated on the secondary hydroxy at the 11 position.

Discovery of colchicine aryne cycloadduct as a potent molecule for the abrogation of epithelial to mesenchymal transition via modulating cell cycle regulatory CDK-2 and CDK-4 kinases in breast cancer cells.

In this study, we synthesized a new-generation library of colchicine derivatives via cycloaddition of colchicine utilizing position C-8 and C-12 diene system regioselectivity with aryne precursor to generate a small, focused library of derivatives. We assessed their anticancer activity against various cancer cell lines like MCF-7, MDA-MB-231, MDA-MB-453, and PC-3. Normal human embryonic kidney cell line HEK-293 was used to determine the toxicity. Among these derivatives, silicon-tethered compound B-4a demonstrated the highest potency against breast cancer cells. Subsequent mechanistic studies revealed that B-4a effectively modulates cell cycle regulatory kinases (CDK-2 and CDK-4) and their associated cyclins (cyclin-B1, cyclin-D1), inducing apoptosis. Additionally, B-4a displayed a noteworthy impact on tubulin polymerization, compared to positive control flavopiridol hydrochloride in a dose-dependent manner, and significantly disrupted the vimentin cytoskeleton, contributing to G1 arrest in breast cancer cells. Moreover, B-4a exhibited substantial anti-metastatic properties by inhibiting breast cancer cell migration and invasion. These effects are attributed to the down-regulation of major epithelial to mesenchymal transition (EMT) factors, including vimentin and Twist-1, and the upregulation of the epithelial marker E-cadherin in an apoptosis-dependent manner.

Investigation of the activity of a novel tropolone in osteosarcoma.

Osteosarcoma (OS) is a primary malignant bone tumor characterized by frequent metastasis, rapid disease progression, and a high rate of mortality. Treatment options for OS have remained largely unchanged for decades, consisting primarily of cytotoxic chemotherapy and surgery, thus necessitating the urgent need for novel therapies. Tropolones are naturally occurring seven-membered non-benzenoid aromatic compounds that possess antiproliferative effects in a wide array of cancer cell types. MO-OH-Nap is an α-substituted tropolone that has activity as an iron chelator. Here, we demonstrate that MO-OH-Nap activates all three arms of the unfolded protein response (UPR) pathway and induces apoptosis in a panel of human OS cell lines. Co-incubation with ferric chloride or ammonium ferrous sulfate completely prevents the induction of apoptotic and UPR markers in MO-OH-Nap-treated OS cells. MO-OH-Nap upregulates transferrin receptor 1 (TFR1) protein levels, as well as TFR1, divalent metal transporter 1 (DMT1), iron-regulatory proteins (IRP1, IRP2), ferroportin (FPN), and zinc transporter 14 (ZIP14) transcript levels, demonstrating the impact of MO-OH-Nap on iron-homeostasis pathways in OS cells. Furthermore, MO-OH-Nap treatment restricts the migration and invasion of OS cells in vitro. Lastly, metabolomic profiling of MO-OH-Nap-treated OS cells revealed distinct changes in purine and pyrimidine metabolism. Collectively, we demonstrate that MO-OH-Nap-induced cytotoxic effects in OS cells are dependent on the tropolone's ability to alter cellular iron availability and that this agent exploits key metabolic pathways. These studies support further evaluation of MO-OH-Nap as a novel treatment for OS.

Tropolone-Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism, Enzymatic Incorporation into DNA and HeLa Cell Internalization.

Tropolone is a non-benzenoid aromatic scaffold with unique photophysical and metal-chelating properties. Recently, it has been conjugated with DNA, and the photophysical properties of this conjugate have been explored. Tropolonyl-deoxyuridine (tr-dU) is a synthetic fluorescent DNA nucleoside analogue that exhibits pH-dependent emissions. However, its solvent-dependent fluorescence properties are unexplored owing to its poor solubility in most organic solvents. It would be interesting to incorporate it into DNA primer enzymatically. This report describes the solvent-dependent fluorescence properties of the silyl-derivative, and enzymatic incorporation of its triphosphate analogue. For practical use, its cell-internalization and cytotoxicity are also explored. tr-dU nucleoside was found to be a potential analogue to design DNA probes and can be explored for various therapeutic applications in the future.

Carbocycloaddition Strategies for Troponoid Synthesis.

Tropone is the prototypical aromatic 7-membered ring, and can be found in virtually any undergraduate textbook as a key example of non-benzenoid aromaticity. Aside from this important historical role, tropone is also of high interest as a uniquely reactive synthon in complex chemical synthesis as well as a valuable chemotype in drug design. More recently, there has been growing interest in the utility of tropones for catalysis and material science. Thus, synthetic strategies capable of synthesizing functional tropones are key to fully exploiting the potential of this aromatic ring system. Cycloaddition reactions are particularly powerful methods for constructing carbocycles, and these strategies in turn have proven to be powerful for generating troponoids. The following review article provides an overview of strategies for troponoids wherein the 7-membered carbocycle is generated through a cycloaddition reaction. Representative examples of each strategy are also provided.

Pseudomonas bohemica strain ins3 eliminates antibacterial hinokitiol from its culture broth.

A bacterial strain, Pseudomonas bohemica strain ins3 was newly isolated as a resistant strain against high concentrations of hinokitiol. This strain was revealed not only to show resistance but also completely remove this compound from its culture broth. In addition, its mechanism was revealed to be independent of conventional aromatic dioxygenases, ie catechol-1,2- or 2,3-dioxygenases.

Effects of Troponoids on Mitochondrial Function and Cytotoxicity.

The α-hydroxytropolones (αHTs) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target HBV RNase H with submicromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays that assess mitochondrial function. Previous studies suggest that tropolones induce cytotoxicity through inhibition of mitochondrial respiration. Therefore, we screened 35 diverse troponoids for effects on mitochondrial function, mitochondrial/nuclear genome ratios, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass interfered with these processes. The troponoids had no impact on the mitochondrial DNA/nuclear DNA ratio after 3 days of compound exposure. The patterns of troponoid-induced cytotoxicity among three hepatic cell lines were similar for all compounds, but three potent HBV RNase H inhibitors were not cytotoxic in primary human hepatocytes. Tropolones and αHTs increased ROS production in cells at cytotoxic concentrations but had no effect at lower concentrations that efficiently inhibit HBV replication. Troponoid-mediated cytotoxicity was significantly decreased upon the addition of the ROS scavenger N-acetylcysteine. These studies show that troponoids can increase ROS production at high concentrations within cell lines, leading to cytotoxicity, but are not cytotoxic in primary hepatocytes. Future development of αHTs as potential therapeutics against HBV may need to mitigate ROS production by altering compound design and/or by coadministering ROS antagonists to ameliorate increased ROS levels.

Direct deuteration of hinokitiol and its mechanistic study.

Hinokitiol has a broad antibacterial activity against bacteria and fungi. While its biosynthetic pathway has been intensively studied, its dynamics in natural environments, such as biodegradation pathway, remain unclear. In this study, the authors report a direct deuterium labeling of hinokitiol as a traceable molecular probe to serve those studies. Hinokitiol was subjected to the H2-Pd/C-D2O conditions and deuterated hinokitiol was obtained with excellent deuteration efficiencies and in moderate yield. The 1H and 2H NMR spectra indicated that all ring- and aliphatic hydrogens except that on C-6 were substituted by deuterium. According to the substrate scope and computational chemistry, deuteration on tropolone ring was suggested to proceed via D+-mediated process, and which was supported by the results of the experiment with trifluoroacetic acid and Pd(TPP)4. On the other hand, the deuteration on aliphatic group was predicted to be catalyzed by Pd(II) species.

Discoveries and Syntheses of Highly Potent Antimalarial Troponoids.

Novel derivatives of puberulic acid were synthesized and their antimalarial properties were evaluated in vitro against the Plasmodium falciparum K1 parasite strain, cytotoxicity against a human diploid embryonic cell line MRC-5, and in vivo efficacy using a Plasmodium berghei-infected mouse model. From previous information that three hydroxy groups on the tropone framework were essential for antimalarial activity, we converted the carboxylic acid moiety into the corresponding esters, amides, and ketones. These derivatives showed antimalarial activity against chloroquine-resistant Plasmodium in vitro equivalent to puberulic acid. We identified that the pentane-3-yl ester, cyclohexyl ester, iso-butyl ketone, cyclohexyl methyl ketone all show an especially potent antiparasitic effect in vivo at an oral dose of 15 mg/kg without any apparent toxicity. These esters were more effective than the existing commonly used antimalarial drug, artesunate.

Thallium(I) Tropolonates: Synthesis, Structure, Spectral Characteristics, and Antimicrobial Activity Compared to Lead(II) and Bismuth(III) Analogues.

Synthesis, single-crystal X-ray determination diffraction and FT-IR, NMR (1H, 13C, 19F and 205Tl), UV-vis, and luminescence spectra characteristics were described for series of thallium(I) compounds: thallium(I) triflate (Tl(OTf)), 1:1 co-crystals of thallium(I) triflate and tropolone (Htrop), Tl(OTf)·Htrop, as well as simple thallium(I) chelates: Tl(trop) (1), Tl(5-metrop) (2), Tl(hino) (3), with Htrop, 5-methyltropolone (5-meHtrop), 4-isopropyltropolone (hinokitiol, Hhino), respectively, and additionally more complex {Tl@[Tl(hino)]6}(OTf) (4) compound. Comparison of their antimicrobial activity with selected lead(II) and bismuth(III) analogs and free ligands showed that only bismuth(III) complexes demonstrated significant antimicrobial activity, from two- to fivefold larger than the free ligands.

Understanding and Engineering the Stereoselectivity of Humulene Synthase.

The non-canonical terpene cyclase AsR6 is responsible for the formation of 2E,6E,9E-humulene during the biosynthesis of the tropolone sesquiterpenoid (TS) xenovulene A. The structures of unliganded AsR6 and of AsR6 in complex with an in crystallo cyclized reaction product and thiolodiphosphate reveal a new farnesyl diphosphate binding motif that comprises a unique binuclear Mg2+ -cluster and an essential K289 residue that is conserved in all humulene synthases involved in TS formation. Structure-based site-directed mutagenesis of AsR6 and its homologue EupR3 identify a single residue, L285/M261, that controls the production of either 2E,6E,9E- or 2Z,6E,9E-humulene. A possible mechanism for the observed stereoselectivity was investigated using different isoprenoid precursors and results demonstrate that M261 has gatekeeping control over product formation.

Tropolone alkaloids from Colchicum kurdicum (Bornm.) Stef. (Colchicaceae) as the potent novel antileishmanial compounds; purification, structure elucidation, antileishmanial activities and molecular docking studies.

Natural compounds played an important role for prevention and treatment of the disease as well as are the important compounds for the design of the new bioactive compounds. In this study, eight tropolone alkaloids were isolated from Colchicum kurdicum including colchicoside, 2-demethyl colchicine, 3-demethyl colchicine, demecolcine, colchifoline, N-deacetyl-N-formyl colchicine, colchicine and cornigerine by column and preparative thin layer chromatography. The chemical structures were identified by 1H NMR and 13C NMR spectroscopy. Moreover, the antileishmanial activity on Leishmania major, anti-inflammatory activity, iron chelating activity and toxicity studies including hemolytic activity, brine shrimp toxicity, cytotoxicity and acute toxicity and docking study of all isolated bioactive compounds were evaluated. As result, colchicoside and colchicine had potent leishmanicidal effects and N-deacetyl-N-formyl colchicine and cornigerine had the highest anti-inflammatory effects. All compounds had the significant iron chelating activity. According to toxicity studies, isolated compounds showed the low hemolytic activity and cytotoxicity, high LC50, LC90 and LD50. In the molecular docking study, colchicoside had the high dockscore. According to the study, with future studies all isolated compounds could be used for design the novel antileishmanial drugs.

Synthetic Biology Driven Biosynthesis of Unnatural Tropolone Sesquiterpenoids.

Tropolone sesquiterpenoids (TS) are an intriguing family of biologically active fungal meroterpenoids that arise through a unique intermolecular hetero Diels-Alder (hDA) reaction between humulene and tropolones. Here, we report on the combinatorial biosynthesis of a series of unprecedented analogs of the TS pycnidione 1 and xenovulene A 2. In a systematic synthetic biology driven approach, we recombined genes from three TS biosynthetic gene clusters (pycnidione 1, xenovulene A 2 and eupenifeldin 3) in the fungal host Aspergillus oryzae NSAR1. Rational design of the reconstituted pathways granted control over the number of hDA reactions taking place, the chemical nature of the fused polyketide moiety (tropolono- vs. monobenzo-pyranyl) and the degree of hydroxylation. Formation of unexpected monobenzopyranyl sesquiterpenoids was investigated using isotope-feeding studies to reveal a new and highly unusual oxidative ring contraction rearrangement.

Investigations of the hydrogen bond in the crystals of tropolone and thiotropolone via car-parrinello and path integral molecular dynamics.

Car-Parrinello and path integrals molecular dynamics (CPMD and PIMD) simulations were carried out for the 10π-electron aromatic systems: 2-hydroxy-2,4,6-cycloheptatrien-1-one, commonly known as Tropolone (I) and 2-hydroxy-2,4,6-cycloheptatriene-1-thione, called Thiotropolone (II) in vacuo and in the solid state. The extremely fast proton transfer (FPT) and "prototropy" tautomerism in the keto-enol (thione-enethiol) systems have been analyzed on the basis of CPMD and PIMD methods level. Comparisons of two-dimensional (2D) free-energy landscapes of reaction coordinate δ-parameter and RO…O or RO…S distances shows that the OH… tautomer to be more favorable in the Thiotropolone. The hydrogen between the oxygen and the sulfur atoms adopts a starkly asymmetrical position in the double potential well. The values of the energy barriers for the FPT were calculated and suggested a strong hydrogen bond with low barrier for FPT mechanism. These studies and the 2D average index of π-delocalization 〈λ〉 landscape of time evolutions of RO1…O2 and RC7O2 or RC7S1 distances for the both crystals indicate that hydrogen bonds in the crystals of Tropolone (I) and Thiotropolone (II) have characteristic properties for the type of bonding model resonance-assisted hydrogen bonds and also low-barrier hydrogen bonds. In the crystal of the Thiotropolone (II), we found the hydrogen bond OH…S existing without the equilibrium of the two tautomers whereas in the crystal of the Tropolone (I) has been confirmed the hydrogen bond OH…O existing with the equilibrium of the two tautomers. It was also found the significant differences in frequency, speed, and the image of the FPT in the studied crystals. © 2018 Wiley Periodicals, Inc.

Tropolone-Conjugated DNA: Fluorescence Enhancement in the Duplex.

Tropolone (2-hydroxycyclohepta-2,4,6-triene-1-one and tautomer) is a non-benzenoid bioactive natural chromophore with pH-dependent fluorescence character and extraordinary metal binding affinities, especially with transition-metal ions Cu2+ /Zn2+ /Ni2+ . This report describes the syntheses and biophysical studies of a new tropolonyl thymidine [(4(5)-hydroxy-5(4)-oxo-5(4)H-cyclohepta-1,3,6-trienyl)thymidine] (tr-T) nucleoside and of corresponding tropolone-conjugated DNA oligonucleotides that form B-form DNA duplex structures with a complementary DNA strand, although their duplex structures are less stable than that of the control. Furthermore, the stabilities of those DNA duplex structures are lowered by the presence of increasing numbers of tr-T residue or by decreasing pH of their environments. Most importantly, these duplex structures are made fluorescent because of the presence of the tropolone moieties conjugated to the thymidine residues. The fluorescence behavior of those duplex structures exhibits pH dependence, with stronger fluorescence at lower pH and weaker fluorescence at high pH. Importantly, the fluorescence characters of tr-DNA oligonucleotides are significantly enhanced by nearly threefold after duplex structure formation with their complementary control DNA oligonucleotide. Further, the fluorescence behavior of these tr-DNA duplex structures is also dependent on the pH conditions. Hence, tropolonyl-conjugated DNA represents a class of new fluorescent analogues that might be be employed for sensing DNA duplex formation and provide opportunities to improve fluorescence properties further.

A fast, miniaturised in-vitro assay developed for quantification of lipase enzyme activity.

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL's properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.

Biosynthesis of Tropolones in Streptomyces spp.: Interweaving Biosynthesis and Degradation of Phenylacetic Acid and Hydroxylations on the Tropone Ring.

Tropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated from Streptomyces species is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. In this study, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and biotransformation. Nine trl genes and some of the aerobic phenylacetic acid degradation pathway genes (paa) located outside the trl biosynthetic gene cluster are required for the heterologous production of DHT. The trlA gene encodes a single-domain protein homologous to the C-terminal enoyl coenzyme A (enoyl-CoA) hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it toward the formation of heptacyclic intermediates. TrlB is a 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhanced de novo biosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT in Streptomyces spp. Six seven-membered carbocyclic compounds were identified from the trlC, trlD, trlE, and trlF deletion mutants. Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone, and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. The monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to produce DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones in Streptomyces spp.IMPORTANCE Tropolonoids are promising drug lead compounds because of the versatile bioactivities attributed to their highly oxidized seven-membered aromatic ring scaffolds. Our present study provides clear insight into the biosynthesis of 3,7-dihydroxytropolone (DHT) through the identification of key genes responsible for the formation and modification of the seven-membered aromatic core. We also reveal the intrinsic mechanism of elevated production of DHT and related tropolonoids in Streptomyces spp. The study on DHT biosynthesis in Streptomyces exhibits a good example of antibiotic production in which both anabolic and catabolic pathways of primary metabolism are interwoven into the biosynthesis of secondary metabolites. Furthermore, our study sets the stage for metabolic engineering of the biosynthetic pathway for natural tropolonoid products and provides alternative synthetic biology tools for engineering novel tropolonoids.

Expanding the Rubterolone Family: Intrinsic Reactivity and Directed Diversification of PKS-derived Pyrans.

We characterized two key biosynthetic intermediates of the intriguing rubterolone family (tropolone alkaloids) that contain a highly reactive pyran moiety (in equilibrium with the hydrolyzed 1,5-dione form) and undergo spontaneous pyridine formation in the presence of primary amines. We exploited the intrinsic reactivity of the pyran moiety and isolated several new rubterolone derivatives, two of which contain a unique thiazolidine moiety. Three rubterolone derivatives were chemically modified with fluorescence and biotin tags using peptide coupling and click reaction. Overall, eight derivatives were fully characterized by HRMS/MS and 1D and 2D NMR spectroscopy and their antimicrobial, cytotoxic, anti-inflammatory and antiparasitic activities evaluated.

Synthesis, structure-activity relationships, and mechanistic studies of 5-arylazo-tropolone derivatives as novel xanthine oxidase (XO) inhibitors.

Xanthine oxidase (XO) is an enzyme that contains molybdenum at the active site and catalyzes the oxidation of purine bases to uric acid. Even though XO inhibitors are widely used for the treatment of hyperuricemia and gout, only very few such compounds are clinically used as drugs for the treatment of these diseases. Given the unique physicochemical properties of tropolone, i.e., its chelating effect and the pKa value that is similar to that of carboxylic acid, we have synthesized 22 5-arylazotropolone derivatives as potential XO inhibitors. In vitro enzyme-inhibitory assays for XO revealed that 3-nitro derivative 1j showed the most potent XO inhibitory activity, which is by one order of magnitude more potent than allopurinol. An enzyme-kinetic study revealed that 1j inhibited the production of uric acid by XO both competitively and non-competitively. A docking-simulation study of 1j with XO suggested that the carbonyl and hydroxyl groups of the tropolone ring interact with the hydroxy group that acts as a ligand for molybdenum and the amino acid residues around the active site of XO.