Xanthine oxidase and adenosine deaminase in commercial bovine spleen phosphodiesterase preparations.

Commercial bovine spleen phosphodiesterase preparations contain xanthine oxidase activity; the xanthine oxidase in such preparations mediates the oxidation of a pteridine derivative as well as a standard purine substrate (hypoxanthine). The xanthine oxidase activity in the phosphodiesterase preparations is inhibited strongly by allopurinol (4-hydroxypyrazolo(3,4-d) pyrimidine). The reported ability of phosphodiesterase preparations to catalyze the deamination of adenosine derivatives appears to be due to contamination with a conventional adenosine deaminase in view of the observations that this activity is inhibited by an established inhibitor of adenosine deaminase and that the relative rates of deamination of N¹-methyladenosine and adenosine are similar with both the phosphodiesterase preparation and calf intestine adenosine deaminase.     

Development of the pteridine pathway in the zebrafish, Danio rerio

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.     

Discovery and biological evaluation of some (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety as potent xanthine oxidase inhibitors

A series of (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety were synthesized and identified as novel xanthine oxidase inhibitors. Among them, the most promising compounds 1h and 1k were obtained with IC₅₀ values of 0.6 μM and 0.8 μM, respectively, which were more than 10-fold potent compared with allopurinol. The Lineweaver-Burk plot revealed that compound 1h acted as a mixed-type xanthine oxidase inhibitor. SAR analysis showed that the benzaldehyde moiety played a more important role than the anthraquinone moiety for inhibition potency. The basis of significant inhibition of xanthine oxidase by 1h was rationalized by molecular modeling studies.     

3,5,2',4'-Tetrahydroxychalcone, a new non-purine xanthine oxidase inhibitor

Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid and the overproduction of uric acid will lead to hyperuricemia which is an important cause of gout. In the present study, three chalcone derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, only Compound 1, 3,5,2′,4′-tetrahydroxychalcone, exhibited a significant inhibitory activity on xanthine oxidase with an IC₅₀ value of 22.5 μM. Lineweaver–Burk transformation of the inhibition kinetics data demonstrated that it was a competitive inhibitor of xanthine oxidase and Ki value was 17.4 μM. In vivo, intragastric administration of Compound 1 was able to significantly reduce serum uric acid levels and inhibited hepatic xanthine oxidase activities of hyperuricemic mice in a dose-dependent manner. Acute toxicity study in mice showed that Compound 1 was very safe at a dose of up to 5 g/kg. These results suggest that Compound 1 is a novel competitive xanthine oxidase inhibitor and is worthy of further development.     

Inhibitor studies of methionyl-tRNA transformylase of Euglena gracilis

Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH3-H4PteGlu) (KI = 3 . 10(-6) M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.     

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.     

Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC50 = 0.600 +/- 0.009 microM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC50 = 1.326 +/- 0.013 microM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC50 = 1.564 +/- 0.065 microM) also showed comparable inhibitory activity to that of allopurinol (IC50 = 0.776 +/- 0.012 microM). All three compounds showed competitive type of inhibition with comparable Ki values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophenolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound was inhibiting enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.     

Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC₅₀=0.600±0.009 µM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC₅₀=1.326±0.013 µM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC₅₀=1.564±0.065 µM) also showed inhibitory activity comparable to that of allopurinol (IC₅₀ = 0.776 ± 0.012 µM). All three compounds showed competitive type of inhibition with comparable K_i values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophe nolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound inhibited enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.     

Xanthine oxidase inhibitory activity of constituents of Cinnamomum cassia twigs

A methanol extract of the twigs of Cinnamomum cassia was found to inhibit xanthine oxidase. Purification of the methanol extract afforded three new phenolic glycosides, cinnacasolide A-C (11-13), together with 10 known compounds (1-10). The structures of the three new compounds were determined by interpretation of spectroscopic data. Cinnamaldehyde derivatives 1-5 and 7 were significant inhibitors of xanthine oxidase, with IC(50) values ranging from 7.8 to 36.3 μg/mL. The results indicate that the acyl group of these cinnamaldehyde derivatives plays an important role in the inhibition of xanthine oxidase.     

Discovery of novel xanthone derivatives as xanthine oxidase inhibitors

Xanthine oxidase is the key enzyme that catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. In this study, a series of xanthone derivatives were synthesized as effective and a new class of xanthine oxidase inhibitor. Compounds 8a, 8c, 8i, 8g and 8r showed good inhibition against xanthine oxidase. The presence of a cyano group at the para position of benzyl moiety turned out to be the preferred substitution pattern. Molecular modeling studies were performed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for further structure-guided design of new non-purine xanthine oxidase inhibitors associated with the xanthone framework.     

Design,synthesis and bioevaluation of 3-oxo-6-aryl-2,3-dihydropyridazine-4-carbohydrazide derivatives as novel xanthine oxidase inhibitors

In view of expanding the structure activity relationship of xanthine oxidase inhibitors, a series of 3-oxo-6-aryl-2,3-dihydropyridazine-4-carbohydrazide/carboxylic acid derivatives were designed by molecular docking and synthesized. All the target compounds were evaluated for their in vitro XO inhibition by using febuxostat and allopurinol as the standard controls. Most of the hydrazide derivatives exhibited potency levels in the micromolar range. From the view of docking study, hydrazide derivatives bind to the active site of XO through a novel interaction mode, which is different from that of febuxostat bearing a carboxyl group. The most promising compound 8b was further subjected to kinetic analysis to deduce their modes of inhibition.     

New 3-substituted xanthines : Potent inhibitors of cell plate formation

Caffeine and some related compounds are known to inhibit cell plate formation (CPF), but little is known about their mechanism of action. In this connection, the effects of xanthine derivatives on CPF in Tradescantia stamen hair cells were examined. Caffeine (1,3,7-trimethylxanthine) and 1,3-dimethyl-xanthine were more effective inhibitors of CPF than 1-methyl- or 1,7-dimethyl-xanthine. Four compounds with a substituent at the three position of 1,7-dimethyl-xanthine were found to be more effective than caffeine as inhibitors of CPF. Among them, 1,7-dimethyl-3-(3-ethylbutyl) xanthine was the most effective and the concentration required for inhibition of CBF was one tenth of that of caffeine. Breakdown of cell plates could be partially reversed using guanosine or adenosine, but reversibility was not detected using cytokinins.     

Synthesis and antioxidant,antixanthine oxidase,and antielastase activities of novel N,S‐substituted polyhalogenated nitrobutadiene derivatives

In this study, three substituted polyhalogenated nitrobutadiene derivatives were synthesized. Compound 1‐[(2,3‐dibromopropyl)sulfanyl]‐1,3,4,4‐tetrachloro‐2‐nitrobuta‐1,3‐diene ( 4 ) was synthesized before by our group. Compounds 8‐{[1‐[(2,3‐dibromopropyl)sulfany]‐3,4,4‐trichloro‐2‐nitrobuta‐1,3‐butadien‐1‐yl}‐1,4‐dioxa‐8‐azaspiro[4.5]decane ( 5 ) and 1‐[(2,3‐dibromopropyl)sulfanyl]‐3,4,4‐trichloro‐N‐(4‐methylpiperazin‐1‐yl)‐2‐nitrobuta‐1,3‐diene‐1‐amine ( 6 ) were synthesized in this work as original compounds. Xanthine oxidase, elastase inhibition abilities, and antioxidant activities were investigated in this work for compounds 4 , 5 , and 6 . In this study, compounds 4 , 5 , and 6 exhibited antixanthine oxidase, antielastase, and antioxidant activities. Among the compounds screened, compound 4 exhibited xanthine oxidase and elastase inhibitor effect similar to the standard compound. Among the three tested compounds, compound 6 showed potent DPPH radical scavenging and reducing power activities. Therefore, these three compounds ( 4 , 5 , and 6 ) may be useful as an antixanthine oxidase, antielastase, and antioxidant agent in pharmaceutical and cosmetic industry.     

Synthesis and evaluation of hydroxychalcones as multifunctional non-purine xanthine oxidase inhibitors for the treatment of hyperuricemia

A series of hydroxychalcone derivatives have been designed, synthesized and evaluated for human xanthine oxidase (XO) inhibitory activity. Most of the tested compounds acted moderate XO inhibition with IC₅₀ values in the micromolar rang. Molecular docking and kinetic studies have been performed to explain the binding modes of XO with the selected compounds. In addition, in vitro antioxidant screening results indicated that some of the hydroxychalcones possessed good anti-free radical activities. Furthermore, the preferred compounds 16 and 18 were able to significantly inhibit hepatic xanthine oxidase activity and reduced serum uric acid level of hyperuricemic mice in vivo. In summary, compounds 16 and 18 with balanced activities of antioxidant, XO inhibition and serum uric acid reduction, which are promising candidates for the treatment of hyperuricemia and gout.     

1-Isoamyl-3-isobutylxanthine: a remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices.

1-Isoamyl-3-isobutylxanthine (EC₅₀ t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.     

Characterization of the antioxidant activity of aglycone and glycosylated derivatives of hesperetin: an in vitro and in vivo study

The flavonoids are mainly present in Citrus fruits as their glycosyl derivatives. This study was conducted comparing in vitro xanthine oxidase inhibitory activity of the aglycone hesperetin and its glycosylated forms (hesperidin and G‐hesperidin) and their effects on the plasma lipid profile and the oxidative–antioxidative system (TBARS and antioxidant enzymes) in rats. The concentrations of the major conjugated metabolites in rat plasma after oral administration of these compounds were also determined. Wistar male rats were randomly assigned to three groups (n = 6) supplemented for 30 days with 1 mmol/kg body mass of hesperetin, hesperidin or G‐hesperidin. Hesperetin was a stronger xanthine oxidase inhibitor (IC₅₀ = 53 μM and K_i = 17.3 μM) than the glycosylate derivatives. Supplementation with the three compounds led to a lower (more favorable) atherogenic index, and an antioxidant preventive effect from the increase of hepatic superoxide dismutase was observed associated to HT supplementation, possibly because of the higher level of hesperetin‐glucuronide in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular modifications on carboxylic acid derivatives as potent histone deacetylase inhibitors: Activity and docking studies

In the light of known HDAC inhibitors, 33 carboxylic acid derivatives were tested to understand the structural requirements for HDAC inhibition activity. Several modifications were applied to develop the structure–activity relationships of carboxylic acid HDAC inhibitors. HDAC inhibition activities were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Molecular docking was also carried out for the human HDAC8 enzyme in order to predict inhibition activity and the 3D poses of inhibitor–enzyme complexes. Of these compounds, caffeic acid derivatives such as chlorogenic acid and curcumin were found to be highly potent compared to sodium butyrate, which is a well-known HDAC inhibitor.     

Rat intestinal peroxidase: inhibition by endogenous xanthine and xanthine oxidase

The high-speed supernatant from homogenates of rat small intestine contains a heat-stable, dialyzable factor which showed a time-dependent inhibition of peroxidase activity in salt extracts of the tissue. The inhibitor was purified by chromatography on Dowex 50W-X8 and identified as xanthine. The inhibition of peroxidase by xanthine was prevented by allopurinol, an inhibitor of xanthine oxidase, and hypoxanthine was also found to be inhibitory. H2O2, produced in the reaction catalyzed by xanthine oxidase, was shown to be directly responsible for the observed inhibition. The time-dependent loss of peroxidase activity in the presence of xanthine or hypoxanthine occurred more rapidly in NH4Cl than in CaCl2 extracts of small intestine and was due to the difference in the initial concentration of H2O2 in these two extracts. The possible relationship between peroxidase and xanthine oxidase in the rat small intestine is discussed.     

Microwave assisted synthesis of naphthopyrans catalysed by silica supported fluoroboric acid as a new class of non purine xanthine oxidase inhibitors

A series of naphthopyrans was synthesized employing silica supported fluoroboric acid under solvent free conditions in a microwave reactor. The catalytic influence of HBF₄–SiO₂ was investigated in detail to optimize the reaction conditions. The synthesised compounds were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure–activity relationship analyses have also been presented. Among the synthesised compounds, NP-17, NP-19, NP-20, NP-23, NP-24, NP-25 and NP-26 were the active inhibitors with an IC₅₀ ranging from 4 to 17 μM. Compound NP-19 with a thiophenyl ring at position 1 emerged as the most potent xanthine oxidase inhibitor (IC₅₀ = 4 μM) in comparison to allopurinol (IC₅₀ = 11.10 μM) and febuxostat (IC₅₀ = 0.025 μM). The basis of significant inhibition of xanthine oxidase by NP-19 was rationalized by its molecular docking at MTE binding site of xanthine oxidase.     

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.