Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2 (PDE2) inhibitors: The neuroprotective and anxiolytic-like effects.

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders. Herein, based on the existing PDE2 inhibitors and their binding modes, a series of purin-6-one derivatives were designed, synthesized and evaluated for PDE2 inhibitory activities, which led to the discovery of the best compounds 6p and 6s with significant inhibitory potency (IC50: 72 and 81 nM, respectively). Docking simulation was performed to insert compound 6s into the crystal structure of PDE2 at the active site to determine the binding mode. Furthermore, compound 6s significantly protected HT-22 cells against corticosterone-induced cytotoxicity and rescued corticosterone-induced decreases in cAMP and cGMP levels. It also produced anxiolytic-like effect in the elevated plus-maze test and exhibited favorable pharmacokinetic properties in vivo. These results might bring significant instruction for further development of potent PDE2 inhibitors.

Novel butanehydrazide derivatives of purine-2,6-dione as dual PDE4/7 inhibitors with potential anti-inflammatory activity: Design, synthesis and biological evaluation.

A novel butanehydrazide derivatives of purine-2,6-dione designed using a ligand-based approach were synthesized and their in vitro activity against both PDE4B and PDE7A isoenzymes was assessed. The 7,8-disubstituted purine-2,6-dione derivatives 31, 34, 37, and 40 appeared to be the most potent PDE4/7 inhibitors with IC50 values in the range of that of the reference rolipram and BRL-50481, respectively. Moreover, docking studies explained the importance of N-(2,3,4-trihydroxybenzylidene)butanehydrazide substituent in position 7 of purine-2,6-dione core for dual PDE4/7 inhibitory properties. The inhibition of both the cAMP-specific PDE isoenzymes resulted in a strong anti-TNF-α effect. Compounds 31, 34, and 37 in the in vivo study in rats with LPS-induced endotoxemia decreased the maximum concentration of this proinflammatory cytokine by 53, 84 and 88%, respectively.

Synthesis and biological evaluation of 2-fluoro and 3-trifluoromethyl-phenyl-piperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione as potential antidepressant agents.

A series of 2-fluoro and 3-trifluoromethylphenylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (4-21) were synthesized and evaluated for their serotonin (5-HT1A/5-HT7) receptor affinity and phosphodiesterase (PDE4B and PDE10A) inhibitor activity. The study enabled the identification of potent 5-HT1A, 5-HT7 and mixed 5-HT1A/5-HT7 receptor ligands with weak inhibitory potencies for PDE4B and PDE10A. The tests have been completed with the determination of lipophilicity and metabolic stability using micellar electrokinetic chromatography (MEKC) system and human liver microsomes (HLM) model. In preliminary pharmacological in vivo studies, selected compound 8-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-1,3,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9) behaved as a potential antidepressant in forced swim test (FST) in mice. Moreover, potency of antianxiety effects evoked by 9 (2.5 mg/kg) is greater than that of the reference anxiolytic drug, diazepam. Molecular modeling revealed that fluorinated arylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione have major significance for the provision of lead compounds for antidepressant and/or anxiolytic application.

Discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as inhibitors of the human poly(A)-selective ribonuclease Caf1.

Eukaryotic mRNA contains a 3' poly(A) tail, which plays important roles in the regulation of mRNA stability and translation. Well-characterized enzymes involved in the shortening of the poly(A) tail include the multi-subunit Ccr4-Not deadenylase, which contains the Caf1 (Pop2) and Ccr4 catalytic components, and poly(A)-specific ribonuclease (PARN). Two Mg(2+) ions present in the active sites of these ribonucleases are required for RNA cleavage. Here, we report the discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as (sub)micromolar inhibitors of Caf1.

Synthesis and SAR studies of trisubstituted purinones as potent and selective adenosine A2A receptor antagonists.

A series of trisubstituted purinones was synthesized and evaluated as A(2A) receptor antagonists. The A(2A) structure-activity relationships at the three substituted positions were studied and selectivity against the A(1) receptor was investigated. One antagonist 12o exhibits a K(i) of 9nM in an A(2A) binding assay, a K(b) of 18nM in an A(2A) cAMP functional assay, and is 220-fold selective over the A(1) receptor.

Efficient synthesis and biological evaluation of 1,2,9-trisubstituted 1,9-dihydro-6H-purin-6-ones.

The carbodiimides 4, obtained from aza-Wittig reactions of iminophosphorane 3 with aromatic isocyanates, reacted with amines in the presence of a catalytic amount of RO(-)Na(+) to give the 1,2,9-trisubstituted 1,9-dihydro-6H-purin-6-ones 6 in good yields. Compound 6 exhibited cytotoxicity against various cancer cells. For example, compounds 6b showed the best inhibition activities against KB, HepG2 and OVCAR3 with IC(50) 9.5, 20.4 and 10.0 microM.

Structure-activity relationship studies of a new series of imidazo[2,1-f]purinones as potent and selective A(3) adenosine receptor antagonists.

We recently described the synthesis of 1-benzyl-3-propyl-1H,8H-imidazo[2,1-f]purine-2,4-diones, new potent and selective A(3) adenosine receptor antagonists containing a xanthine core. The present work can be considered an extension of our SAR studies on related structures in which the effect of different kind of substitutions at the 1-, 3- and 8-positions has been evaluated in order to improve both the potency and the hydrophilicity of the originally synthesised molecules. The A(3) binding disposition of these compounds was also investigated through docking and 3D-QSAR studies.

Synthesis, biological testing, and binding mode prediction of 6,9-diarylpurin-8-ones as p38 MAP kinase inhibitors.

Based on the purine scaffold of ATP, derivatives of 6,9-diarylpurine-8-one were prepared and tested for their ability to inhibit p38 MAP kinase, a key enzyme in the cellular regulation of proinflammatory cytokines. The inhibitor design combines the purine system of the authentic cosubstrate ATP with various phenyl moieties to explore the selectivity for the two hydrophobic regions of the kinase's ATP-binding cleft. The present study indicates a new binding mode of our scaffold to p38 MAP kinase, which comprises the desired structural features of ATP and the N-phenyl-N-purin-6-yl ureas previously published by Wan et al. Combinations of Autodock and FlexX docking with different scoring functions were used to assess the postulated binding mode. The predictive power of different docking-scoring combinations was determined. The presented results may form a solid basis for further optimization cycles since our theoretical findings are consistent with our experimental binding data and supported by the literature.

Inhibition of herpes simplex virus thymidine kinases by 2-phenylamino-6-oxopurines and related compounds: structure-activity relationships and antiherpetic activity in vivo.

Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.

Synthesis and incorporation into DNA fragments of the artificial nucleobase, 2-amino-8-oxopurine.

The nucleoside 2-amino-9-(2-deoxy-beta-d-ribofuranosyl)-7,8-dihydro-8-oxo-purine (dJ) was obtained in eight steps from 2'-deoxyguanosine. The appropriate protected phosphoramidite was synthesized and incorporated into DNA oligonucleotides. The thermal stability of heteroduplexes containing 2-amino-8-oxopurine (J) was investigated by UV-thermal denaturation experiments. The results obtained can be interpreted by the base pairing schemes involving the two edges of dJ depending on the anti and syn orientations.

Synthesis and cardiovascular activity of new 8-alkylamino-1,3-dimethyl-7-(2-hydroxy-3- piperazinopropyl)-3,7-dihydro-1H-purine-2,6-diones.

7-{2-Hydroxy-3-[4-(2-phenoxyethyl)-piperazinyl-1-yl]-propyl}-1,3-di-methyl-3,7-dihydro-1H-purine-2,6-dione dihydrochloride (2), and several of its 8-alkylamino substituted derivatives (11-17) were synthesized and tested for electrocardiographic, antiarrhythmic and hypotensive activity. Also their alpha(1)- and alpha(2)-adrenoreceptor affinities were determined. It was found that compound 2, and its analogue 15 with 8-(2-morpholin-4-yl-ethylamino) substituent displayed a strong prophylactic antiarrhythmic activity in experimentally induced arrhythmia (LD50/ED50 = 54.9 and 55.0, respectively). The hypotensive activity was observed for 8-benzylamino (11) or 8-(pyridin-2-yl-methylamino) (12) analogues. All the new derivatives (11-17) and 2 showed a weak affinity for alpha1-(Ki = 0.225-1.400 microM) and alpha2-(Ki = 0.152-4.299 microM) receptors.

Synthesis and benzodiazepine receptor binding activity of 2, 9-disubstituted quinolino[2', 3'-5, 4](3-pyrazolino)[3, 2-b]purin-4-ones.

2, 9-Disubstituted quinolino[2', 3'-5, 4](3-pyrazolino)pyrimidin-2-ones and purin-4-ones were synthesized and their benzodiazepine receptor activity was evaluated for their ability to displace [(3)H]R015-1788 from its specific binding in bovine brain membranes. Compound 5c caused 83 +/- 8 %inhibition in [(3)H]R015-1788 specific benzodiazepine receptor binding followed by compounds 5f, 5h, and 5i while other analogs were inactive at 10 microM concentration.

Pyrido[2,1-f]purine-2,4-dione derivatives as a novel class of highly potent human A(3) adenosine receptor antagonists.

1H,3H-Pyrido[2,1-f]purine-2,4-diones, which can be described as fused xanthine structures, have been synthesized by a novel synthetic procedure, and their affinities for the human adenosine A(1), A(2A), and A(3) receptors have been evaluated in radioligand binding studies. The synthetic procedure employed was developed in our laboratory and involved a two-step one-pot reaction that consists of the treatment of 6-aminouracil derivatives with N-bromosuccinimide to generate a 5,5-dibromo-6-imino intermediate that reacts "in situ" with pyridine, 4-methoxypyridine, 4-tert-butylpyridine, or 4-phenylpyridine to afford the corresponding 1H,3H-pyrido[2,1-f]purine-2,4-diones (2-5). Functionalization at the N(3) position in compounds 2-5 was performed by reaction with DBU and different alkyl, alkenyl, alkynyl, or benzyl halides. Binding studies at human adenosine A(1), A(2A), and A(3) receptors revealed significant antagonist effects in the low nanomolar range, in particular against the A(3) receptor. Thus, the 1-benzyl-3-propyl-1H,3H-pyrido[2,1-f]purine-2,4-dione derivative 6, which can be considered a lead compound in this series, exhibited a K(i) value of 4.0 +/- 0.3 nM against the hA(3) receptor. Because xanthine derivatives have traditionally been considered poor A(3) antagonists, the described pyrido[2,1-f]purine-2,4-dione derivatives represent a new family of adenosine receptor antagonists which deserves further exploration.

Imidazo[2,1-i]purin-5-ones and related tricyclic water-soluble purine derivatives: potent A(2A)- and A(3)-adenosine receptor antagonists.

A series of tricyclic imidazo[2,1-i]purinones and ring-enlarged analogues derived from xanthine derivatives have been prepared as adenosine receptor (AR) antagonists. In comparison with xanthines, the tricyclic compounds exhibit increased water solubility due to a basic nitrogen atom, which can be protonated under physiological conditions. Substituents were introduced that confer high affinity for A(2A) or A(3) ARs, respectively. A new capillary electrophoresis method was developed for the determination of the enantiomeric purity of selected chiral products using native and modified beta-cyclodextrins as chiral discriminators. The compounds were investigated in radioligand binding assays at rat brain A(1) and A(2A) ARs. Selected compounds were additionally investigated in radioligand binding assays at human recombinant A(3) ARs and in functional studies (adenylate cyclase assays) at A(1) ARs of rat fat cell membranes, A(2A) ARs of rat PC 12 cell membranes, and mouse A(2B) ARs of NIH 3T3 cell membranes. Structure-activity relationships were similar to those of corresponding xanthine derivatives. The 2-styrylimidazopurinones were less potent at A(2A) ARs as compared to 8-styrylxanthine derivatives. The most potent compound at A(2A) ARs was (S)-1,4-dimethyl-8-ethyl-2-styryl-imidazo[2,1-i]purinone (S-25) exhibiting a K(i) value of 424 nM at rat A(2A) ARs. The compound was highly selective for A(2A) receptors vs A(1) and A(3) ARs. Selectivity vs A(2B) ARs, however, was low. Among the 1-unsubstituted 2-phenyl-imidazo[2,1-i]purin-5-one derivatives, very potent and highly selective antagonists for human A(3) ARs were identified. The most potent A(3) antagonist of the present series was (R)-4-methyl-8-ethyl-2-phenyl-imidazo[2,1-i]purin-5-one (R-24) exhibiting a K(i) value of 2.3 nM and high selectivity for A(3) receptors vs all other AR subtypes.

A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and excretion in the urine of NPYR-treated rats.

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endogenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of alpha-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. alpha-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 degrees C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.00004% of the dose as adduct 8. The major differences in product formation in reactions of alpha-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.

Studies on the synthesis of the hypermodified base isolated from rat liver phenylalanine transfer ribonucleic acid.

Oxidation of methyl (S,E)-4-[4,9-dihydro-4,6-dimethyl-9-oxo-1- (phenylmethyl)-1H-imidazo[1,2-alpha]purine-7-yl]-2-[(methoxycarbonyl) amino]-3-butenoate (3) with osmium tetroxide/N-methylmorpholine N-oxide provided a mixture of diastereomers 4 and 7. Hydrogenolysis of the major dihydroxy compound 4 over Pd-C gave beta-hydroxywybutine [[R-(R*,S*)]-1]. The minor isomer 7 was transformed into [S-(R*,R*)]-1 through the cyclic carbonate 8.

Mesoionic purinone analogues as inhibitors of cyclic-AMP phosphodiesterase: a comparison of several ring systems.

Several simple alkyl and aralkyl derivatives of mesoionic thiazolopyrimidines (1) and mesoionic 1,3,4-thiadiazolopyrimidines (2) were found to possess theophylline-like activity as inhibitors of cyclic-AMP phosphodiesterase (PDE). Reduction of the C2-C3 double bond of 1 or replacement of the sulfur atom of 1 or 2 with an N-methyl group nearly abolishes activity. Optimal activity appears to be associated with a hydrophobic substituent at the N8 position. The five-membered ring of 1 can be replaced by a pyridine or isoquinoline nucleus without untoward effects. Preliminary kinetic data suggest that the type of enzyme inhibition produced by the mesoionic derivatives is similar to that observed for theophylline. Thus, several novel mesoionic ring systems display activity as inhibitors of cyclic-AMP PDE and can serve as lead compounds for further investigation.

Substituted 6,7-dihydroimidazo[1,2-a]purin-9(4H)-ones.

The synthesis of a series of substituted 6,7-dihydroimidazo[1,2-a]purin-9(4H)-ones is described. Several members of the series exhibit enhanced antiallergic and bronchodilator activity and reduced side effects as compared to theophylline. Structure-activity relationships and metabolic considerations are discussed for the series. Analogues substituted with a 4-(4-chlorobenzyl) moiety, such as 33 and 40, shown an optimal balance of antiallergic and bronchodilator activity and are of particular interest. Compound 33 is significantly more potent than theophylline against both metacholine- and antigen-induced bronchospasms, does not affect spontaneous motor activity, and shows minimal cardiovascular effects in the rat.

Antiallergic activity of 2-phenyl-8-azapurin-6-ones.

The synthesis and antiallergic activity in the rat passive cutaneous anaphylactic reaction of a series of 2-phenyl-8-azapurin-6-ones are described. Early in the investigation, a linear free-energy equation was established in which the activity was related to the size and hydrogen bonding capacity of the ortho substituent in the phenyl ring. This relationship was used to provide guidance and limits for subsequent work leading to 2-o-propoxyphenyl-8-azapurin-6-one which is 40 times more potent than disodium cromoglycate. It is suggested that good antiallergic activity in this series is associated with coplanarity of the phenyl group with the azapurin-6-one which would be favored by a high degree of hydrogen bonding.