Design,synthesis, and biological evaluation of C9- and C2-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as new A2A and A3 adenosine receptors antagonists

In the past few years, our group has been involved in the development of A(2A) and A(3) adenosine receptor antagonists which led to the synthesis of SCH58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, 61), potent and very selective at the A(2A) receptor subtype, and N(8)-substituted-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N(5)-urea or amide (MRE series, b), very selective at the human A(3) adenosine receptor subtype. We now describe a large series of C(9)- and C(2)-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines to represent an extension of structure-activity relationship work on this class of tricyclic compounds. The introduction of a substituent at 9 position of the tricyclic antagonistic structure led to retention of receptor affinity but a loss of selectivity in respect to the lead compounds b, N(8)-substituted-pirazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N(5)-urea or -amide. The substitution of the furanyl moiety of compound 61, necessary for receptor binding, with a phenyl or a substituted aromatic ring (compounds 5a-d, 6-8), caused a complete loss of the affinity at all the adenosine receptor subtypes, demonstrating that the furanyl ring is a necessary structural element to guarantee interaction with the adenosine receptor surface. The introduction of an ethoxy group at the ortho position of the aromatic ring to mimic the oxygen of the furan (compound 5c, 5-amino-7-(2-phenylethyl)-2-(2-ethoxyphenyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) did not enhance affinity. The introduction of the cycloaminomethyl function by Mannich reaction at the 5' position of the furanyl ring of 61 and the C(9)-substituted compound 41 (5-amino-8-methyl-9-methylsulfanyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) resulted in complete water solubility but a loss of receptor affinity. We can conclude that modifications or substitutions at the furanyl ring are not allowed and the introduction of a substituent at the 9-position of the core pyrazolo-triazolo-pyrimidine structure caused a severe loss of selectivity, probably due to an increased steric hindrance of the radical introduced.     

Synthesis, biological activity, and molecular modeling investigation of new pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as human A(3) adenosine receptor antagonists

A new series of pyrazolotriazolopyrimidines bearing different substitutions on the phenylcarbamoyl moieties at the N5 position, being highly potent and selective human A(3) adenosine receptor antagonists, is described. The compounds represent an extension and an improvement of our previous work on this class of compounds (J. Med. Chem. 1999, 42, 4473-4478; J. Med. Chem. 2000, 43, 4768-4780). All the synthesized compounds showed A(3) adenosine receptor affinity in the subnanomolar range and high levels of selectivity in radioligand binding assays at the human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the substitution and its position on the phenyl ring have been studied. From binding data, it is evident that the unsubstituted derivatives on the phenyl ring (e.g., compound 59, hA(3) = 0.16 nM, hA(1)/hA(3) = 3713, hA(2A)/hA(3) = 2381, hA(2B)/hA(3) = 1388) showed the best profile in terms of affinity and selectivity at the human A(3) adenosine receptors. The introduction of a sulfonic acid moiety at the para position on the phenyl ring was attempted in order to design water soluble derivatives. However, this substitution led to a dramatic decrease of affinity at all four adenosine receptor subtypes. A computer-generated model of the human A(3) receptor was built and analyzed to better interpret these results, demonstrating that steric control, in particular at the para position on the phenyl ring, plays a fundamental role in the receptor interaction. Some of the synthesized compounds proved to be full antagonists in a specific functional model, where the inhibition of cAMP-generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor with IC(50) values in the nanomolar range, with a statistically significative linear relationship with the binding data.     

Fluorosulfonyl- and bis-(beta-chloroethyl)amino-phenylamino functionalized pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives: irreversible antagonists at the human A3 adenosine receptor and molecular modeling studies

A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective human A(3) adenosine receptor antagonists (Baraldi et al. J. Med. Chem. 2000, 43, 4768-4780). A derivative having a methyl group at the N(8) pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N(5) position, displayed a K(i) value at the hA(3) receptor of 0.2 nM. We now describe chemically reactive derivatives which act as irreversible inhibitors of this receptor. Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(beta-chloroethyl)amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes containing the recombinant hA(3) receptor were preincubated with the compounds and washed exhaustively. The loss of ability to bind radioligand following this treatment indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N(8) pyrazole nitrogen. The bis-(beta-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the human A(3) receptor was built and analyzed to help interpret these results. The model of the A(3) transmembrane region was derived using primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using the recently published crystal structure of rhodopsin as a template. According to our model, sulfonyl fluoride derivatives could dock within the hypothetical TM binding domain, adopting two different energetically favorable conformations. We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic partners of the irreversible binding to the receptor.     

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine ligands, new tools to characterize A3 adenosine receptors in human tumor cell lines

Increased concentrations of extracellular adenosine are reached in ischemic or inflamed tissues but have also been detected inside tumoral masses. If this finding may account for an important role of adenosine in the pathogenesis of tumors remains to be determined in view of its contradictory effects on cell survival and proliferation. In particular, adenosine was found to exert its effects on proliferation and on cell death mainly through the A(3) adenosine receptor. Therefore, a complete pharmacological characterization of the subtype and number of the expressed A(3) adenosine receptors is necessary for the elucidation of the role of adenosine via A(3) receptors in a specific cell subtype. The lack of potent and selective radiolabelled A(3) receptor antagonists has been, in the past, the major obstacle in the characterization of structure, function and regulation of this adenosine receptor subtype. Recently, our group has identified a series of substituted pyrazolotriazo-lopyrimidine derivatives as potent and selective antagonists to human A(3) adenosine receptors. The most recent results obtained in this field will be summarized in the present review. Furthermore, the review will report the results of the biochemical and pharmacological characterization of A(3) receptors in different human tumor cell lines and the multiple A(3) receptor-sustained ways that could prime tumor development.     

Synthesis and properties of 7-methyl-5-oxo-1,5-dihydro-8-[1,2,4]-triazolo [4,3-c]pyrimidinecarboxylic acid derivatives

The synthesis of the new triazolo[4,3-c]pyrimidines is described, starting from derivatives of 5-carboxy-2-hydroxy-4-hydrazino-6-methylpyrimidine. The 4-methyl-2,3-dihydropyrazolo[3,4-d]pyrimidine-3,6-dione was also obtained. Some of triazolo[4,3-c]pyrimidines tested for biological activity were found inactive.     

Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as adenosine receptor antagonists. Influence of the N5 substituent on the affinity at the human A 3 and A 2B adenosine receptor subtypes: a molecular modeling investigation

A new series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines bearing various substituents at both the N5-pyrimidinyl and N8-pyrazolyl positions have been synthesized, and their binding affinities at the four human adenosine receptor subtypes (hA(1), hA(2A), hA(2B), and hA(3)) have been evaluated. All the described compounds contain arylacetyl moieties at the N5 position and arylalkyl substituents at the N8 position. Surprisingly, all the compounds present their most potent affinities at the hA(2B) adenosine receptor with a range of selectivities against the other subtypes. When bulky groups are present simultaneously at the N5 and N8 positions (e.g., compound 9), the best selectivity for the hA(2B) receptor was observed (K(i)(hA(1)) = 1100 nM; K(i)(hA(2A)) = 800 nM; K(i)(hA(2B)) = 20 nM; K(i)(hA(3)) = 300 nM, K(i)(hA(1)/A(2B)) = 55, K(i)(hA(2A)/A(2B)) = 40, K(i)(hA(3)/hA(2B)) = 15). To understand the molecular significance of these results, we compared the putative TM (transmembrane) binding motif of compound 9 on both hA(2B) and hA(3) receptors. From our docking studies, compound 9 fits neatly inside the TM region of the hA(2B) receptor but not in the corresponding hA(3) region, illustrating significant differences between the two subtypes. The study herein presented permits an understanding of why the bioisosteric replacement of an -NH, present in previously reported hA(3) receptor antagonists, with a -CH(2) group at the N5 position induces such large differences in hA(2B)/hA(3) affinity. In the molecular structure of the hA(3) receptor, two residues, Ser243 (TM6) and Ser271 (TM7), create a hydrophilic region, which seems to permit a better accommodation of the phenylurea series into this putative hA(3) binding site than the phenylacetyl series.     

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.     

Combined target-based and ligand-based drug design approach as a tool to define a novel 3D-pharmacophore model of human A3 adenosine receptor antagonists: pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as a key study

A combined target-based and ligand-based drug design approach has been carried out to define a novel pharmacophore model of the human A(3) receptor antagonists. High throughput molecular docking and comparative molecular field analysis (CoMFA) have been used in tandem to assemble a new target based pharmacophore model. In parallel, to provide more accurate information about the putative binding site of these A(3) inhibitors, a rhodopsin-based model of the human A(3) receptor was built and a novel Y-shape binding motif has been proposed. Docking-based structure superimposition has been used to perform a quantitative study of the structure-activity relationships for binding of these pyrazolo-triazolo-pyrimidines to adenosine A(3) receptor using CoMFA. Both steric and the electrostatic contour plots obtained from the CoMFA analysis nicely fit on the hypothetical binding site obtained by molecular docking. On the basis of the combined hypothesis, we have designed, synthesized, and tested 17 new derivatives. Consistently, the predicted K(i) values were very close to the experimental values.     

Synthesis and biological studies of a new series of 5-heteroarylcarbamoylaminopyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines as human A3 adenosine receptor antagonists. Influence of the heteroaryl substituent on binding affinity and molecular modeling investigations

Some pyrazolotriazolopyrimidines bearing different heteroarylcarbamoylamino moieties at the N5-position are described. We previously reported the synthesis of a water soluble compound with high potency and selectivity versus the human A3 adenosine receptor as antagonist, and herein we present an enlarged series of compounds related to the previously mentioned one. These compounds showed A3 adenosine receptor affinity in the nanomolar range and different levels of selectivity evaluated in radioligand binding assays at human A1, A2A, A2B, and A3 adenosine receptors. In particular, the effect of the heteroaryl substituents at the N5 position has been analyzed. This study allows us to recognize that the presence of a pyridinium moiety in this position not only increases water solubility but also improves or retains potency and selectivity at the human A3 adenosine receptors. In contrast, replacement of pyridine with different heterocycles produces loss of affinity and selectivity at the human A3 adenosine receptors. A molecular modeling study has been carried out with the aim to explain these various binding profiles.     

Synthesis and biological evaluation of a new series of 1,2,4-triazolo[1,5-a]-1,3,5-triazines as human A(2A) adenosine receptor antagonists with improved water solubility

The structure-activity relationship (SAR) of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives related to ZM241385 as antagonists of the A(2A) adenosine receptor (AR) was explored through the synthesis of analogues substituted at the 5 position. The A(2A) AR X-ray structure was used to propose a structural basis for the activity and selectivity of the analogues and to direct the synthetic design strategy to provide access to solvent-exposed regions. Thus, we have identified a point of substitution for the attachment of solubilizing groups to enhance both aqueous solubility and physicochemical properties, maintaining potent interactions with the A(2A) AR and, in some cases, receptor subtype selectivity. Among the most potent and selective novel compounds were a long-chain ether-containing amine congener 20 (K(i) 11.5 nM) and its urethane-protected derivative 14 (K(i) 17.8 nM). Compounds 20 and 31 (K(i) 11.5 and 16.9 nM, respectively) were readily water-soluble up to 10 mM. The analogues were docked in the crystallographic structure of the hA(2A) AR and in a homology model of the hA(3) AR, and the per residue electrostatic and hydrophobic contributions to the binding were assessed and stabilizing factors were proposed.     

Synthesis and structure-activity relationships of pyrazolo[4,3-d]pyrimidin-7-ones as adenosine receptor antagonists

A series of 21 1,3-dialkylpyrazolo[4,3-d]pyrimidin-7-ones substituted in the 5-position with various phenyl substituents has been synthesized and found to have affinity for the adenosine A1 receptor. The potency pattern due to substituents of the phenyl ring was found to parallel that found in a previously reported 1,3-dialkyl-8-phenylxanthine series. A quantitative structure-activity relationship was developed between these two series that correctly predicted the potencies of six additional 5-substituted pyrazolo[4,3-d]pyrimidines that were synthesized during the course of the analysis. With use of the correlation as a guide, one additional 5-phenylpyrazolo[4,3-d]pyrimidine containing a 4-[[(dimethylamino)ethyl]amino]sulfonyl substituent to improve aqueous solubility was prepared. On the basis of the high correlation between adenosine binding affinities of analogously substituted xanthines and pyrazolo-[4,3-d]pyrimidines and the close superposition of the heterocyclic rings and substituents that is apparent from molecular models of these two series, it is hypothesized they fit the receptor in an analogous fashion.     

Benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidines: synthesis, characterization, antimicrobial activity, and incorporation into solid lipid nanoparticles

Fused triazolothienopyrimidines were prepared from the corresponding 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile. These precursors were intern prepared by employing the Gewald's reaction. All the newly synthesized compounds were characterized by spectral and analytical data. Title compounds displayed promising antibacterial and antifungal activities. Compound 3h which exhibited good antimicrobial activity was incorporated into SLN and characterized for particle size, entrapment efficiency (EE%), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and in-vitro release studies. It showed narrow particle size distribution with high entrapment efficiency. In-vitro release study of compound loaded SLNs in phosphate buffer of pH 7.4, exhibited a biphasic pattern with an initial burst and prolonged release over 24 h.     

Synthesis, characterization, and in vitro antimicrobial evaluation of new 5-chloro-8-bromo-3-aryl-1,2,4-triazolo[4,3-c]pyrimidines

A series of new 5-chloro-8-bromo-3-aryl-1,2,4-triazolo[4,3-c]pyrimidines (4a–j) have been accomplished in excellent yields by the oxidative cyclization of pyrimidinylhydrazines (3a–j) of various aryl aldehydes with one equivalents of iodobenzene diacetate in methanol. The chemical structures of the synthesized compounds were confirmed by elemental analyses, FT-IR, ¹H NMR, ¹³C NMR, and mass spectral studies. Ten new compounds (4a–j) were tested in vitro for their antimicrobial activity against clinically isolated strains. Variable and modest activities were observed against the investigated strains of bacteria and fungi. Compounds 4f, 4i, and 4j demonstrated good antimicrobial activity against all the tested microbial strains.     

2-(Alkylthio)-1,2,4-triazolo[1,5-a]pyrimidines as adenosine cyclic 3',5'-monophosphate phosphodiesterase inhibitors with potential as new cardiovascular agents

A series of new 2-(alkylthio)-5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines have been prepared as inhibitors of cAMP phosphodiesterase from various tissues. These derivatives were prepared via ring closure of various requisite 3-amino-1,2,4-triazole intermediates. 2-(Benzylthio)-5-methyl-7-(dimethylamino)-1,2,4-triazolo[1,5-a]pyrimidine (15a) is 6.3 times as potent as theophylline in inhibiting cAMP PDE isolated from rabbit heart. Treatment of dogs intravenously with 5 (mg/kg)/h of 15a gave a cardiac output increase of 69%, which was largely sustained for a 2-h period after administration of drug had ceased. There was no significant increase in heart rate upon administration of 15a. Related studies with 5,7-di-n-propyl-2-(benzylthio)-1,2,4-triazolo[1,5-a]pyrimidine (22a) in five dogs showed a 31.5% increase in cardiac output with an increase in stroke volume of 34.4% with no increase in heart rate. The specificity of action of these PDE inhibitors could be due to selective binding at a certain cAMP PDE site in the cardiovascular system. Several of these compounds are candidates for further studies with a view to clinical evaluation.     

New 2-arylpyrazolo[4,3-c]quinoline derivatives as potent and selective human A3 adenosine receptor antagonists

In this paper we report the synthesis and biological evaluation of a new class of 2-phenyl-2,5-dihydro-pyrazolo[4,3-c]quinolin-4-ones as A(3) adenosine receptor antagonists. We designed a new route based on the Kira-Vilsmeier reaction for the synthesis of this class of compounds. Some of the synthesized compounds showed A(3) adenosine receptor affinity in the nanomolar range and good selectivity as evaluated in radioligand binding assays at human (h) A(1), A(2A), A(2B), and A(3) adenosine receptor subtypes. We introduced several substituents on the 2-phenyl ring. In particular substitution at the 4-position by methyl, methoxy, and chlorine gave optimal activity and selectivity 6c (K(i)hA(1), A(2A)>1000 nM, EC(50)hA(2B)>1000 nM, K(i)hA(3) = 9 nM), 6d (K(i)hA(1), A(2A)>1000 nM, EC(50)hA(2B)>1000 nM, K(i)hA(3) = 16 nM), 6b (K(i)hA(1), A(2A) >1000 nM, EC(50)hA(2B)>1000 nM, K(i)hA(3) = 19 nM). In conclusion, the 2-phenyl-2,5-dihydro-pyrazolo[4,3-c]quinolin-4-one derivatives described herein represent a new family of in vitro selective antagonists for the adenosine A(3) receptor.     

Synthesis and antimicrobial activity of acyclo C-nucleosides: 3-(alditol-1-yl)-7-oxo-5-phenyl-1,2,4-triazolo[4,3-a]pyrimidines

Condensation of 2-hydrazino-4-oxo-6-phenylpyrimidine (1) with aldopentoses 2a-d or aldohexoses 2e-g gave the corresponding aldehydo-sugar (4-oxo-6-phenylpyrimidin-2-yl)hydrazones 3a-g which were acetylated to the corresponding poly-O-acetyl-aldehydo-sugar (3-acetyl-4-oxo-6-phenylpyrimidin-2-yl)hydrazones 4a-g. The latter compounds underwent oxidative cyclization with bromine in acetic acid and in the presence of sodium acetate to the corresponding 8-acetyl-3- (poly-O-acetyl-alditol-1-yl)-7-oxo-5-phenyl-1,2,4-triazolo[4,3-a]pyrimid ines 6a-g. Compounds 6a-g were also obtained by consecutive one-pot oxidative cyclization/acetylation in which the parent hydrazones 3a-g were treated with bromine/acetic acid/sodium acetate followed by acetic anhydride. Deacetylation of 6a-g with ammonium hydroxide in methanol gave the title compounds 7a-g. The antibacterial and antifungal activities of compounds 3c, 3f, 7c and 7f are reported.     

Piperazine derivatives of [1,2,4]triazolo[1,5-a][1,3,5]triazine as potent and selective adenosine A2a receptor antagonists

The [1,2,4]triazolo[1,5-a]triazine derivative 3, more commonly known in the field of adenosine research as ZM-241385, has previously been demonstrated to be a potent and selective adenosine A2a receptor antagonist, although with limited oral bioavailability. This [1,2,4]triazolo[1,5-a]triazine core structure has now been improved by incorporating various piperazine derivatives. With some preliminary optimization, the A2a binding affinity of some of the best piperazine derivatives is almost as good as that of compound 3. The selectivity level over the adenosine A1 receptor subtype for some of the more active analogues is also fairly high, > 400-fold in some cases. Many compounds within this piperazine series of [1,2,4]triazolo[1,5-a]triazine have now been shown to have good oral bioavailability in the rat, with some as high as 89% (compound 35). More significantly, some piperazines derivatives of [1,2,4]triazolo[1,5-a]triazine also possessed good oral efficacy in rodent models of Parkinson's disease. For instance, compound 34 was orally active in the rat catalepsy model at 3 mg/kg. In the 6-hydroxydopamine-lesioned rat model, this compound was also quite effective, with a minimum effective dose of 3 mg/kg po.     

Synthesis of 2-substituted-7-heptyloxy-4,5-dihydro-[1,2,4]-triazolo[4,3-a]quinolin-1(2H)-ones with anticonvulsant activity

A series of 2-substituted-7-heptyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolin-1(2H)-ones was synthesized. The anticonvulsant effect and neurotoxicity of the compounds were calculated with maximal electroshock (MES) test, subcutaneous pentylenetetrazole (sc-PTZ), and rotarod tests with intraperitoneally injected mice. Among the synthesized compounds, 2-propionyl-7-heptyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline-1(2H)-one 4b was the most active one and also had the lowest toxicity. In the anti-MES potency test, it showed median effective dose (ED(50)) of 8.2 mg/kg, median toxicity dose (TD(50)) of 318.3 mg/kg, and the protective index (PI) of 39.0 which is much greater than the PI of the reference drugs phenytoin and carbamazepine.