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.     

The screening and characterization of 6-aminopurine-based xanthine oxidase inhibitors

Xanthine oxidase (XO) is a key enzyme which can catalyze xanthine to uric acid causing hyperuricemia in humans. By using the fractionation technique and inhibitory activity assay, an active compound that prevents XO from reacting with xanthine was isolated from wheat leaf. It was identified by the Mass and NMR as 6-aminopurine (adenine). A structure-activity study based on 6-aminopurine was conducted. The inhibition of XO activity by 6-aminopurine (IC(50)=10.89+/-0.13 microM) and its analogues was compared with that by allopurinol (IC(50)=7.82+/-0.12 microM). Among these analogues, 2-chloro-6(methylamino)purine (IC(50)=10.19+/-0.10 microM) and 4-aminopyrazolo[3,4-d] pyrimidine (IC(50)=30.26+/-0.23 microM) were found to be potent inhibitors of XO. Kinetics study showed that 2-chloro-6(methylamino)purine is non-competitive, while 4-aminopyrazolo[3,4-d]pyrimidine is competitive against XO.     

Lead optimization of isocytosine-derived xanthine oxidase inhibitors

We report our attempts at improving the oral efficacy of low-nanomolar inhibitors of xanthine oxidase from isocytosine series through chemical modifications. Our lead compound had earlier shown good in vivo efficacy when administered intraperitoneally but not orally. Several modifications are reported here which achieved more than twofold improvement in exposure. A compound with significant improvement in oral efficacy was also obtained.     

Synthesis and characterization of CAPE derivatives as xanthine oxidase inhibitors with radical scavenging properties

Inhibitors of the enzyme xanthine oxidase (XO) with radical scavenging properties hold promise as novel agents against reperfusion injuries after ischemic events. By suppressing the formation of damaging reactive oxygen species (ROS) by XO or scavenging ROS from other sources, these compounds may prevent a buildup of ROS in the aftermath of a heart attack or stroke. To combine these two properties in a single molecule, we synthesized and characterized the non-purine XO inhibitor caffeic acid phenethylester (CAPE) and 19 derivatives using a convenient microwave-assisted Knoevenagel condensation protocol. Varying systematically the number and positions of the hydroxyl groups at the two phenyl rings, we derived structure-activity relationships based on experimentally determined XO inhibition data. Molecular docking suggested that critical enzyme/inhibitor interactions involved π-π interactions between the phenolic inhibitor ring and Tyr914, hydrogen bonds between inhibitor hydroxyl groups and Glu802, and hydrophobic interactions between the CAPE phenyl ring and non-polar residues located at the entrance of the binding site. To effectively scavenge the stable radical DPPH, two hydroxyl groups in 1,2- or 1,4-position at the phenyl ring were required. Among all compounds tested, E-phenyl 3-(3,4-dihydroxyphenyl)acrylate, a CAPE analog without the ethyl tether, showed the most promising properties.     

Glycosides and xanthine oxidase inhibitors from Conyza bonariensis

Fractionation of the xanthine oxidase inhibitory methanol extract of Conyza bonariensis afforded three glycosides, in addition to nine known compounds including amyrin, beta-sitostero1 daucosterol, syringic acid 3-hydroxy-5-methoxybenzoic acid, eugenol 4-O-glucopyranoside, and luteolin, apigenin and takakin 8-O-glucuronide. The structures of the glycosides were established by a combination of spectroscopic methods (IR, MS, 1H and 13C NMR, DEPT, COSY, HMQC and HMBC) as 4-hydroxypyridin-3-carboxylic acid 4-O-glucopyranoside, 8-hydroxy-6,7-dihydrolinalool 8-O-glucopyranoside and bonaroside [viz. 1,3,4,12-tetrahydroxy-2-(9-hexadecenoylamino)octadecane 1-O-glucopyranoside]. The in vitro enzyme assay showed that syringic acid and takakin 8-O-glucuronide displayed weak inhibitory activity against xanthine oxidase with IC50 values of 500+/-41 microM and 170+/-12 microM, respectively.     

Pteridines as inhibitors of xanthine oxidase: structural requirements

Different pteridine derivatives were investigated for their inhibitory action on xanthine oxidase. From 27 investigated compounds, 13 showed concentration-dependent inhibition of the enzyme. Concentrations necessary for 50% inhibition ranged from <0.1 up to >100 microM. Different types of inhibition were found concerning xanthine and pterin as substrates: competitive, noncompetitive and mixed type. Out of 18 aromatic compounds tested, 12 were inhibitors. Only one out of nine reduced derivatives served as inhibitor. A simple regression model was used to specify the structural requirements for a pteridine to be an inhibitor. The most characteristic features of an inhibitor are aromaticity and no substitution at position 7 of the pteridine ring.     

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.     

Purification and characterization of mouse liver xanthine oxidase

Xanthine oxidase (EC 1.1.3.22) is purified to homogeneity from mouse liver after induction with bacterial lipopolysaccharide. The enzyme has an apparent molecular weight of 300,000 in its native state and it is suggested to be constituted of two identical subunits of Mr 150,000 each. The isoelectric point is 6.7 and the apparent Km value for xanthine is 3.4 microM. The amino acid composition of mouse xanthine oxidase is quite similar to that of Drosophila xanthine dehydrogenase.     

9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors

A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme–substrate complex than for the free enzyme, and inhibition constant of 55.1 ± 9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.     

Biochemical characterization of mouse d-aspartate oxidase

D-amino acids research field has recently gained an increased interest since these atypical molecules have been discovered to play a plethora of different roles. In the mammalian central nervous system, d-aspartate (D-Asp) is critically involved in the regulation of glutamatergic neurotransmission by acting as an agonist of NMDA receptor. Accordingly, alterations in its metabolism have been related to different pathologies. D-Asp shows a peculiar temporal pattern of emergence during ontogenesis and soon after birth its brain levels are strictly regulated by the catabolic enzyme d-aspartate oxidase (DASPO), a FAD-dependent oxidase. Rodents have been widely used as in vivo models for deciphering molecular mechanisms and for testing novel therapeutic targets and drugs, but human targets can significantly differ. Based on these considerations, here we investigated the structural and functional properties of the mouse DASPO, in particular kinetic properties, ligand and flavin binding, oligomerization state and protein stability. We compared the obtained findings with those of the human enzyme (80% sequence identity) highlighting a different oligomeric state and a lower activity for the mouse DASPO, which apoprotein species exists in solution in two forms differing in FAD affinity. The features that distinguish mouse and human DASPO suggest that this flavoenzyme might control in a distinct way the brain D-Asp levels in different organisms.     

New xanthine oxidase inhibitors from the fruiting bodies of Tyromyces fissilis

Excessive uric acid production, which causes gout and hyperuricemia, can be blocked by inhibiting xanthine oxidase (XO). However, some agents to block on XO often cause side effects, thereby necessitating the identification of new inhibitors. During the screening of XO inhibitors from various mushroom extracts, we found that a methanolic extract of the fruiting bodies of Tyromyces fissilis, an inedible and non-toxic fungus, showed inhibitory activity. Both n-hexane and ethyl acetate layers, obtained by partitioning this extract exhibited XO inhibitory activity. Subsequently, using an activity-guided separation method, eight active compounds (1–8) were isolated. The structures of five of the new compounds, 2–4, 6, and 7, were elucidated by spectral analysis and chemical derivatization. All compounds had a salicylic acid moiety with an aliphatic group at the C-6 position. Notably, 2-hydroxy-6-pentadecylbenzoic acid (1) showed the highest level of XO noncompetitive inhibition (58.9 ± 2.2% at 25 µM).     

Synthesis and structure-activity relationships of 1-phenylpyrazoles as xanthine oxidase inhibitors

A series of 1-phenylpyrazoles was evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds prepared, 1-(3-cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid (Y-700) had the most potent enzyme inhibition and displayed longer-lasting hypouricemic action than did allopurinol in a rat model of hyperuricemia induced by the uricase inhibitor potassium oxonate.     

Novel thiazolo-pyrazolyl derivatives as xanthine oxidase inhibitors and free radical scavengers

Xanthine oxidase (XO) is a complex metalloflavoprotein, overproduction of which usually leads to a pathological condition called Gout. XO inhibitors may prove to be promising antigout agents. Present investigation describes synthesis, characterization and evaluation of 26 thiazolo-pyrazolyl derivatives V(a-z) for XO inhibitory and free radical scavenging activities. Derivatives Vq, Vo and Vh showed most promising XO inhibitory and free radical scavenging activities on the basis of their IC(50) values ranging from (6.5-9 μM). Significant dock scores compared with Allopurinol have been figured out using molecular docking. Evaluation of Vq, Vo and Vh for both the activities for first time may provide a new approach for antigout research.