Synthesis and antiproliferative activity in vitro of new derivatives of 3-aminopyrazolo[3,4-b]pyridine. Part 1. Reaction of 3-aminopyrazolo[3,4-b]pyridine with 1,3-, 1,4-diketones and alpha,beta-unsaturated ketones

The synthesis of several new pyrazolo[3,4-b]pyridine, pyrido[2',3':3,4]-pyrazolo[1,5-a]pyrimidine and imidazo[1',2':1,5]pyrazolo[3,4-b]pyridine derivatives is described. The obtained compounds were tested for their antiproliferative activity in vitro. One of them, 4-phenyl-2-(3,4,5-trimethoxy-beta-styrylo)pyrido- [2',3':3,4]pyrazolo[1,5-a]pyrimidine (9), revealed cytotoxic properties against the cells of all three human cancer cell lines applied. Another one, 2,4-dimethyl-pyrido[2',3':3,4]pyrazolo[1,5-a]-pyrimidine (2), revealed weak cytotoxic activity only against the cells of human bladder cancer cell line HCV29T. All other compounds tested did not reveal any cytotoxic activity.     

Studies on condensed-heterocyclic azolium cephalosporins. II. Synthesis and antibacterial activity of 7 beta-[2-(2-aminothiazol-4-yl)-2(Z)-alkoxyiminoacetamido]-3-(condensed- heterocyclic azolium)methyl-3-cephem-4-carboxylates.

From our series of studies on cephalosporins bearing condensed-heterocyclic azolium methyl groups at the 3 position in the cephalosporin nucleus, we describe here the synthesis and antibacterial activity of 7 beta-[2-(2-aminothiazol-4-yl)-2(Z)-alkoxyiminoacetamido]ceph alosporins containing imidazo[1,5-a]pyridinium, imidazo[1,2-b]pyridazinium, imidazo[1,2-a]pyrimidinium, imidazo[1,2-c]pyrimidinium, and pyrazolo[1,5-a]pyridinium methyl groups at the 3 position. Among the cephalosporins tested, 7 beta-[2-(2-aminothiazol-4-yl)-2(Z)-methoxyiminoacetamido]-3- (imidazo[1,5-a]pyridinium-2-yl) (1), (imidazo[1,2-b]pyridazinium-1-yl) (2), and (pyrazolo[1,5-a]-pyridinium-1-yl) (3)methyl-3-cephem-4-carboxylates showed potent antibacterial activity and broad antibacterial spectrum. The antibacterial activity of these cephalosporins (1 approximately 3) was superior to that of ceftazidime (CAZ). These results imply that the delocalization of the positive charge of the imidazo[1,5-a]pyridinium, pyrazolo[1,5-a]pyridinium and imidazo[1,2-b]pyridazinium groups leads to an expanded antibacterial spectrum and increased activity and that these condensed-heterocyclic compounds as well as imidazo[1,2-a]pyridine are effective moieties for improving antibacterial activity and spectrum.     

Reactions with heterocyclic amidines (VI): Synthesis of some new sym. and assym. pyrazolotriazines and pyrazolo[4,5-e]pyrimidine derivatives

Several new pyrazolo[1,5-a]-S-triazine, pyrazolo[4,5-e]pyrimidine, pyrazolo [1,5-a]pyrimidine derivatives were synthesizedvia condensation of 3-antipyrinyl-5-aminopyrazole (2) with β-bifunctional reagents. The azo analogues of pyrazolo[1,5-a] pyrimidines;i.e pyrazolo[5,1-c]-as-triazine and pyrazolo[5,1-c]-as-benzotriazine were synthesized by coupling of diazotized 2 with agents containing active hydrogen.     

Synthesis and enzymic activity of various substituted pyrazolo[1,5-a]-1,3,5-triazines as adenosine cyclic 3',5'-phosphate phosphodiesterase inhibitors

A series of various pyrazolo[1,5-a]-1,3,5-triazines have been prepared and studied as inhibitors of cAMP phosphodiesterase isolated from bovine brain, bovine heart, and rabbit lung. A number of compounds were found to be superior to theophylline. 2-Ethyl-7-phenylpyrazolo[1,5-a]-1,3,5-triazine (35) was found to be 97 times more potent than theophylline as an inhibitor of bovine brain PDE. 8-Bromo-2,4-dimethyl-7-phenylpyrazolo[1,5-a]-1,3,5-triazine (52) showed alpha lung = 40 compared to alpha heart = 3.0. Thus, various substituents could increase or decrease the inhibition relative to the type and source of tissue from which the PDE was isolated. The most active compound was 8-bromo-4-(diethylamino)-7-phenylpyrazolo[1,3-a]-1,3,5-triazine (25), which was 185 times more potent than theophylline as an inhibitor of PDE isolated from rabbit lung. The stepwise synthesis via ring-closure procedures of requisite pyrazole intermediates, followed by electrophilic substitution in the pyrazole ring and/or nucleophilic substitution in the 1,3,5-triazine moiety, resulted in the various pyrazolo[1,5-a]1,3,5-triazines listed in Tables I and II. Structure-activity relationships are reviewed.     

Pharmacokinetics of the new antiplatelet agent 2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine in laboratory animals.

KC-764 (2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo [1,5-a]pyridine CAS 94457-09-7) and its metabolites in serum and urine were determined after intravenous and oral administration in mice, rats, rabbits and dogs at a dose of 5 mg/kg. KC-764 was rapidly eliminated from serum in all species. The biological half-lives of unchanged KC-764 after intravenous administration in mice, rats, rabbits and dogs were 1.31, 0.29, 1.94 and 1.20 h, respectively. 2-Methyl-3-(1,4,5,6-tetrahydro-6-oxonicotinoyl)pyrazolo-[1,5-a]pyr idine was a common major metabolite in serum of all species, although 6,7-dihydro-6,7-dihydroxy-2-methyl-3-(1,4,5,6- tetrahydro-6-oxonicotinoyl) pyrazolo-[1,5-a]pyridine (M-8) was more abundant in rabbits. Urinary recovery of unchanged KC-764 was as low as 0.4-2.2% in all species. The major urinary metabolite was 2-methyl-3-(1,4,5,6-tetrahydro-6-ureidonicotinoyl)pyrazolo-[1,5-a] pyridine in mice, rats and dogs, but M-8 was in rabbits. KC-764 was rapidly and well absorbed by oral administration, and extensively metabolized in all species tested.     

Adenosine cyclic 3',5',-monophosphate phosphodiesterasr inhibitors. 2.3-Substituted 5,7-dialkylpyrazolo [1,5-a]pyrimidines.

A number of 3-bromo-, 3-nitro-, and 3-ethoxycarbonyl-5,7-dialkylpyrazolo[1,5-a]pyrimidines were synthesized and screened as in vitro cAMP phosphodiesterase inhibitors. The condensation of 3-aminopyrazole with symmetrical beta-diketones (acetylacetone, heptane-3,5-dione, etc.) afforded symmetrical dialkylpyrazolo[1,5-a]pyrimidines (5). The reaction of 3-aminopyrazole with unsymmetrical beta-diketones (hexane-2,4-dione, heptane-3,5-dione, etc.) gave a mixture of 5-methyl-7-alkylpyrazolo[1,5-a]pyrimidine (3) and 5-alkyl-7-methylpyrazolo[1,5-a]pyrimidines (4). The technique for the separation of 3 from 4 is described. The inhibition constants, alpha (the ratio of the molar I50 of theophylline to the molar I50 of the test compounds), were subjected to a Hansch correlation analysis. The results indicated that PDE isolated from beef heart tissue was most sensitive to changes in the length of the alkyl group in the 5 position of the pyrazolo[1,5-a]pyrimidine ring, whereas the PDE isolated from rabbit lung tissue was more sensitive to changes in the length of the 7-alkyl group. Experimentally and theoretically, the n-propyl group was found to approximate the ideal size for the alkyl group in both the 5 and 7 positions;5,7-di-n-propyl-3-ethoxycarbonylpyrazolo[1,5-a]pyrimidine (5e) was the most potent inhibitor of both lung and heart PDE.     

Synthesis of new pyrazolo[1,5-a]pyrimidine derivative using 5-aminouracil and ketene dithioacetal

Novel 3-cyano-2-(3-uracilyl-5-amino) pyrazolo [1,5-a]pyrimidine has been synthesized using 5-aminouracil as a starting material.     

5,7-二取代吡唑并[1,5-a]嘧啶类化合物的合成及其抗肿瘤活性

目的设计并合成吡唑并[1,5-a]嘧啶类化合物,并评价其抗肿瘤活性。方法根据吡唑并[1,5-a]嘧啶类抗肿瘤药物的基本结构,设计了14个5-胺甲基-7-苯胺基吡唑并[1,5-a]嘧啶类化合物,并以丙二腈和原甲酸三乙酯为起始原料,经5步反应得到目标产物。采用MTT法,顺铂为阳性对照药,以Bel-7402和HT-1080为测试细胞株对目标化合物进行抗肿瘤活性评价。结果与结论合成了14个未见文献报道的新化合物,结构经MS、IR和1H-NMR确证。体外活性实验表明:化合物12显示出较好的抗癌活性。     

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.     

Pharmacological activity of some pyrazolo[1,5-a]pyrimidines.

The paper reports the pharmacological activity in vitro and in vivo of a series of pyrazolo[1,5-a]pyrimidine derivatives. Some of these compounds have proved to be active in decreasing the body temperature. They are also very active inhibitors of the guinea-pig lung prostaglandin-sintetase.     

Synthesis and structure-activity relationships of pyrazolo[1,5-a]pyridine derivatives: potent and orally active antagonists of corticotropin-releasing factor 1 receptor

Design, synthesis, and structure-activity relationships of a series of 3-dialkylamino-7-phenyl pyrazolo[1,5-a]pyridines (I) as selective antagonists of the corticotropin-releasing factor 1 (CRF(1)) receptor are described. The most prominent compound to emerge from this work, 46 (E2508), exhibits potent in vitro activity, excellent drug-like properties, and robust oral efficacy in animal models of stress-related disorders. It has advanced into clinical trials.     

Potent and selective inhibitors of platelet-derived growth factor receptor phosphorylation. 3. Replacement of quinazoline moiety and improvement of metabolic polymorphism of 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives

We have previously reported that a series of 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives were potent and selective inhibitors of platelet-derived growth factor receptor (PDGFR) phosphorylation and demonstrated several biological effects such as suppression of neointima formation following balloon injury in rat carotid artery by oral administration. Here, we investigated structure-activity relationships of the 6,7-dimethoxyquinazolinyl moiety. In regard to 6,7-dimethoxy groups, ethoxy analogues showed potent activity (IC(50) of 16b is 0.04 microM; IC(50) of 17a is 0.01 microM) and further extension of the alkyl group reduced activity. Interestingly, methoxyethoxy (IC(50) of 16j is 0.02 microM; IC(50) of 17h is 0.01 microM) and ethoxyethoxy (IC(50) of 17j is 0.02 micro M) analogues showed the most potent activity, suggesting that the inserted oxygen atom significantly interacts with beta-PDGFR. Among tricyclic quinazoline derivatives, the 2-oxoimidazo[4,5-e]quinazoline derivative 21a showed potent activity (IC(50) = 0.10 microM). Regarding replacements of quinazoline by other heterocyclic rings, pyrazolo[3,4-d]pyrimidine (39a, IC(50) = 0.17 microM) and quinoline (IC(50) of 40a is 0.18 microM; IC(50) of 40b is 0.09 microM) derivatives showed potent activity. Isoquinoline and some pyridopyrimidine derivatives were completely inactive; therefore, 1-aza has an important role. Also 7-aza and 8-aza substitution on the parent quinazoline ring has a detrimental effect on the interaction with beta-PDGFR. We also demonstrated that the substituents on the quinazoline ring possess major consequences for metabolic polymorphism. Although there existed extensive metabolizers and poor metabolizers in Sprague-Dawley rats administrated 6,7-dimethoxyquinazoline derivatives (1b and 1c), 6-(2-methoxy)ethoxy-7-methoxyquinazoline analogue 16k showed no metabolic polymorphism.     

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.     

Impurity profile study of zaleplon

Zaleplon is a pyrazolopyrimidine derivative and possesses sedative and hypnotic properties. Seven unknown impurities in zaleplon bulk drug at levels below 0.1% were detected by reverse-phase high performance liquid chromatography (HPLC). The starting material, 3-amino-4-cyanopyrazole and an intermediate, N-[3-[3-(dimethylamino)-1-oxo-2-propenyl]-phenyl]-N-ethylacetamide (DOPEA) were also present in the sample at a level below 0.1%. The molecular weights of impurities were determined by LC-MS analysis. These impurities were isolated from crude samples of zaleplon using reverse-phase preparative HPLC. Based on the spectral data the structures of these impurities were characterized as, N-(3-(3-(4-amino-2H-pyrazolo [3,4-d]pyrimidin-6-yl) pyrazolo[1,5-a] pyrimidin-7-yl)phenyl)-N-ethylacetamide (impurity I); N-[3-(3-carboxamidopyrazolo[1,5-a]pyrimidin-7-yl)phenyl]-N-ethylacetamide (impurity II); N-[3-(3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)phenyl]acetamide (impurity III); N-[3-(3-cyanopyrazolo [1,5-a]pyrimidin-7-yl)phenyl]-N-methylacetamide (impurity IV); N-[3-(3-cyanopyrazolo[1,5-a] pyrimidin-5-yl)phenyl]-N-ethylacetamide (impurity V); N-[3-(3-cyanopyrazolo[1,5-a] pyrimidin-7-yl)phenyl]-N-ethylamine (impurity VI); N-[3-(3-cyano-6-[(E)-3-((N-ethyl-N-acetyl)amino)phenyl-3-oxoprop-1-enyl] pyrazolo[1,5-a]pyrimidin-7-yl) phenyl]-N-ethylacetamide (impurity VII). Structural elucidation of all impurities by spectral data ((1)H NMR, (13)C NMR, MS and IR) and formation of these impurities are discussed in detail.     

A novel selective GABA(A) alpha1 receptor agonist displaying sedative and anxiolytic-like properties in rodents

In our pursuit to identify selective ligands for Bz/GABA(A) receptor subtypes, a novel pyrazolo[1,5-a]pyrimidine derivative (4), the azaisostere of zolpidem, was synthesized and evaluated in vitro on bovine brain homogenate and on recombinant benzodiazepine receptors (alphaxbeta2/3gamma2, x = 1-3, 5) expressed in HEK293 cells. Compound 4 displayed affinity only for alpha1beta2gamma2 subtype (K(i) = 31 nM), and in an in-depth, in vivo study it revealed sedative and anxiolytic-like properties without any amnesic and myorelaxant effects in rodents.     

7-Aminopyrazolo[1,5-a]pyrimidines as potent multitargeted receptor tyrosine kinase inhibitors

7-Aminopyrazolo[1,5- a]pyrimidine urea receptor tyrosine kinase inhibitors have been discovered. Investigation of structure-activity relationships of the pyrazolo[1,5- a]pyrimidine nucleus led to a series of 6-(4- N, N'-diphenyl)ureas that potently inhibited a panel of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases. Several of these compounds, such as 34a, are potent inhibitors of kinase insert domain-containing receptor tyrosine kinase (KDR) both enzymatically (<10 nM) and cellularly (<10 nM). In addition, compound 34a possesses a favorable pharmacokinetic profile and demonstrates efficacy in the estradiol-induced murine uterine edema (UE) model (ED 50 = 1.4 mg/kg).     

Synthesis and structure-activity relationship of a new series of COX-2 selective inhibitors: 1,5-diarylimidazoles

The synthesis and the pharmacological activity of a series of 1,5-diarylimidazoles developed as potent and selective cyclooxygenase-2 (COX-2) inhibitors are described. The new compounds were evaluated both in vitro (COX-1 and COX-2 inhibition in human whole blood) and in vivo (carrageenan-induced paw edema, air-pouch, and hyperalgesia tests). Modification of all the positions of two regioisomeric imidazole cores led to the identification of 4-[4-chloro-5-(3-fluoro-4-methoxyphenyl)imidazol-1-yl]benzenesulfonamide (UR-8880, 51f) as the best candidate, which is now undergoing Phase I clinical trials.     

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

Piperazine derivatives of 2-furanyl[1,2,4]triazolo[1,5-a][1,3,5]triazine have recently been demonstrated to be potent and selective adenosine A(2a) receptor antagonists with oral activity in rodent models of Parkinson's disease. We have replaced the piperazinyl group with a variety of linear, monocyclic, and bicyclic diamines. Of these diamines, (R)-2-(aminomethyl)pyrrolidine is a particularly potent and selective replacement for the piperazinyl group. With this diamine component, we have been able to prepare numerous analogues with low nanomolar affinity toward the A(2a) receptor and good selectivity with respect to the A(1) receptor (>200-fold in some cases). Selected analogues from this series of [1,2,4]triazolo[1,5-a][1,3,5]triazine have now been shown to be orally active in the mouse catalepsy model.     

2-Phenylpyrazolo[1,5-a]pyrimidin-7-ones. A new class of nonsteroidal antiinflammatory drugs devoid of ulcerogenic activity

Syntheses of some pyrazolo[1,5-a]pyrimidines were performed in order to study the relationship between structural modifications on the parent 4,7-dihydro-2-phenylpyrazolo[1,5-a]pyrimidin-7-one (1) and their antiinflammatory properties. The modifications carried out were introduction and functionalization of a longer side chain at the 4-position, substitution of the hydrogen atom at the 3-position, and replacement of the phenyl group with a 4-methylphenyl, methyl, or hydrogen substituent. 4-Ethyl-4,7-dihydro-2-phenylpyrazolo [1,5-a]pyrimidin-7-one (3) showed the highest activity and a better therapeutic index than phenylbutazone and indomethacin, used as reference drugs. All other changes at the 3-, 5-, and 6-positions, as well as the replacement of the phenyl group at position 2, caused a marked decrease of activity. Compound 3 was found devoid of ulcerogenic activity and was probably endowed with antiulcerogenic properties.