1-Methyl and 1-(2-hydroxyalkyl)-5-(3-alkyl/cycloalkyl/phenyl/naphthylureido)-1H-pyrazole-4-carboxylic acid ethyl esters as potent human neutrophil chemotaxis inhibitors.

In this paper we report the synthesis and the chemotaxis inhibitory activity of a number of 1H-pyrazole-4-carboxylic acid ethyl esters 2 functionalized in N1 with a methyl group or different hydroxyalkyl chains and in position 5 with a series of 3-substituted urea groups. These compounds were designed as development of previous pyrazole-urea derivatives that resulted potent IL8-induced neutrophil chemotaxis inhibitors in vitro. Most of the new compounds revealed a potent inhibition of both IL8- and fMLP-OMe-stimulated neutrophil chemotaxis. The most active compounds in the fMLP-OMe induced chemotaxis test showed IC(50) in the range 0.19 nM-2 microM; but we observed a very strong inhibition in the IL8-induced chemotaxis test, having the most active compounds IC(50) at pM concentrations. In vivo compounds 2e and 2f, although to a lesser extent, at 50mg/kg os decreased granulocyte infiltration in zymosan-induced peritonitis in mice.

Structure-based optimization of pyrazolo[3,4-d]pyrimidines as Abl inhibitors and antiproliferative agents toward human leukemia cell lines.

Results from molecular docking calculations and Grid mapping laid the foundations for a structure-based optimization approach to improve the biological properties of pyrazolo-pyrimidine derivatives in terms of inhibition of Abl enzymatic activity and antiproliferative properties toward human leukemia cells. Insertion of halogen substituents with various substitution patterns, suggested by simulations, led to a significant improvement of leukemia cell growth inhibition and to an increase up to 1 order of magnitude of the affinity toward Abl.

Synthesis and biological evaluation of N-pyrazolyl-N'-alkyl/benzyl/phenylureas: a new class of potent inhibitors of interleukin 8-induced neutrophil chemotaxis.

Neutrophils chemotaxis is a complex multistep process that, if upregulated, causes acute inflammation and a number of autoimmune diseases. We report here the synthesis of a new N-(4-substituted)pyrazolyl-N'-alkyl/benzyl/phenylureas that are potent inhibitors of interleukin-8 (IL8)-induced neutrophil chemotaxis. The first series of compounds, obtained by functionalization with a urea moiety of the 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl ester 3, blocked the IL8-induced neutrophil chemotaxis, while they did not block N-formylmethionylleucylphenylalanine-mediated chemotaxis. The most active compounds, 3-benzyl- (4d), 3-(4-benzylpiperazinyl)- (4i), 3-phenyl- (4k) and 3-isopropylureido (4a) derivatives, showed an IC50 of 10, 14, 45, and 55 nM, respectively. Several different molecules were then synthesized to obtain more information for SAR study. Compounds 4a, 4d, and 4k were inactive in the binding assays on CXCR1 and CXCR2 (IL8 receptors), whereas they inhibited the phosphorylation of PTKs (protein tyrosine kinases) in the 50-70 kDa region. Moreover, in the presence of the same derivatives, we observed a complete block of F-actin rise and pseudopod formation.

Identification of a novel pyrazolo[3,4-d]pyrimidine able to inhibit cell proliferation of a human osteogenic sarcoma in vitro and in a xenograft model in mice.

New pyrazolo[3,4-d]pyrimidines were synthesized and found to inhibit Src phosphorylation in a cell-free assay. Some of them significantly reduced the growth of human osteogenic sarcoma (SaOS-2) cells. The best compound, in terms of inhibitory properties toward both Src and SaOS-2 cells, was further investigated and found to reduce bone resorption when used to treat mouse osteoclasts, without interfering with normal osteoblast growth. Moreover, its metabolic stability prompted its study on a human SaOS-2 xenograft tumor model in nude mice, where the compound reduced significantly both the volume and weight of the tumor. These experimental findings make the new compound an interesting hit in the field of bone-related diseases.

Pyrazolo[3,4-d]pyrimidines as potent antiproliferative and proapoptotic agents toward A431 and 8701-BC cells in culture via inhibition of c-Src phosphorylation.

We report here the synthesis of new pyrazolo[3,4-d]pyrimidine derivatives along with their biological properties as inhibitors of isolated Src and cell line proliferation (A431 and 8701-BC cells). Such compounds block the growth of cancer cells by interfering with the phosphorylation of Src, and they act as proapoptotic agents through the inhibition of the anti apoptotic gene BCL2. Several of them were found to be more active than the reference compound (1-(tert-butyl)-3-(4-chlorophenyl)-4-aminopyrazolo[3,4-d]pyrimidine, PP2) in inhibiting cell proliferation and in inducing apoptosis, and as active as PP2 in the inhibition of the phosphorylation of isolated Src. Moreover, molecular modeling simulations have been performed to hypothesize the way, at the molecular level, by which the inhibitors were able to act as antiproliferative agents.

Synthesis and 3D QSAR of new pyrazolo[3,4-b]pyridines: potent and selective inhibitors of A1 adenosine receptors.

A number of 4-aminopyrazolo[3,4-b]pyridines 5-carboxylic acid esters (2-8) were synthesized and evaluated for their binding affinity at the A1, A2A, and A3 adenosine receptors (AR), in bovine cortical membranes, as well as for their affinity toward human A1AR (hA1AR). Some of the new compounds were characterized by a high affinity and selectivity toward the A1 receptor subtype, showing a significant improvement in comparison with other pyrazolo-pyridines previously reported in the literature. In particular the methyl ester 2h as well as the isopropyl ester 5h, both of them bearing a p-methoxyphenylethylamino side chain at the position 4, presented Ki values of 6 and 7 nM, respectively. To rationalize the relationships between structure and affinity of the novel compounds, a 3D QSAR model was also generated starting from compounds belonging to different classes of known A1AR antagonists.

Structure-based design, parallel synthesis, structure-activity relationship, and molecular modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase inhibitor isosteres of phenethylthiazolylthiourea derivatives.

In this paper we describe our structure-based ligand design, synthetic strategy, and structure-activity relationship (SAR) studies that led to the identification of thiocarbamates (TCs), a novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), isosteres of phenethylthiazolylthiourea (PETT) derivatives. Assuming as a lead compound O-[2-(phthalimido)ethyl]phenylthiocarbamate 12, one of the precursors of the previously described acylthiocarbamates (Ranise, A.; et al. J. Med. Chem. 2003, 46, 768-781), two targeted solution-phase TC libraries were prepared by parallel synthesis. The lead optimization strategy led to para-substituted TCs 31, 33, 34, 39, 40, 41, 44, 45, and 50, which were active against wild-type HIV-1 in MT-4-based assays at nanomolar concentrations (EC50 range: 0.04-0.01 microM). The most potent congener 50 (EC50 = 0.01 microM) bears a methyl group at position 4 of the phthalimide moiety and a nitro group at the para position of the N-phenyl ring. Most of the TCs showed good selectivity indices, since no cytotoxic effect was detected at concentrations as high as 100 microM. TCs 31, 37, 39, 40, and 44 significantly reduced the multiplication of the Y181C mutant, but they were inactive against K103R and K103N + Y181C mutants. Nevertheless, the fold increase in resistance of 41 was not greater than that of efavirenz against the K103R mutant in enzyme assays. The docking model predictions were consistent with in vitro biological assays of the anti-HIV-1 activity of the TCs and related compounds synthesized.

Design, synthesis, SAR, and molecular modeling studies of acylthiocarbamates: a novel series of potent non-nucleoside HIV-1 reverse transcriptase inhibitors structurally related to phenethylthiazolylthiourea derivatives.

A novel series of potent, selective HIV-1 N-acylthiocarbamate (ATC) nonnucleoside reverse transcriptase inhibitors (NNRTIs) is described. The title compounds were synthesized through a highly convergent, one-pot procedure. In cell-based assays, the lead compound (17c) prevented the HIV-1 multiplication with an EC(50) of 8 microM. The lead optimization strategy was developed by single or multiple modifications of the three molecular portions, in which 17c was notionally divided. Molecular modeling studies led to the synthesis of O-(2-phthalimidoethyl)-N-(p-substituted phenyl)-N-acylthiocarbamates, which showed in vitro activities against HIV-1 in the low nanomolar range. Nevertheless, the title compounds retained low potency against HIV-1 strains carrying mutations (K103R, Y181C, and K103N/Y181C) responsible for NNRTI resistance. The hypothetical docking model of RT/17c and RT/25c, derived from X-ray crystallographic structure of a PETT derivative in complex with HIV-1 RT, revealed that the model structures of ATCs do not approximate the NNRTI butterfly-like conformation. Analysis of these hypotetical complexes helps to rationalize some SARs and resistance data.

Pyrazolo[3,4-d]pyrimidines endowed with antiproliferative activity on ductal infiltrating carcinoma cells.

Novel 1,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (better than the reference compound) of Src phosphorylation of the breast cancer cells 8701-BC, known to overexpress Src. The ability of such compounds to significantly reduce 8701-BC cell proliferation suggests that this scaffold could be a promising lead for the development of antitumoral agents able to block Src phosphorylation of breast cancer cells.

Synthesis, molecular modeling studies, and pharmacological activity of selective A(1) receptor antagonists.

We present a combined computational study aimed at identifying the three-dimensional structural properties required for different classes of compounds to show antagonistic activity toward the A(1) adenosine receptor (AR). Particularly, an approach combining pharmacophore mapping, molecular alignment, and pseudoreceptor generation was applied to derive a hypothesis of the interaction pathway between a set of A(1) AR antagonists taken from the literature and a model of the putative A(1) receptor. The pharmacophore model consists of seven features and represents an improvement of the N(6)-C8 model, generally reported as the most probable pharmacophore model for A(1) AR agonists and antagonists. It was used to build up a pseudoreceptor model able to rationalize the relationships between structural properties and biological data of, and external to, the training set. In fact, to further assess its statistical significance and predictive power, the pseudoreceptor was employed to predict the free energy of binding associated with compounds constituting a test set. While part of these molecules was also taken from the literature, the remaining compounds were designed and synthesized by our research group. All of the new compounds were tested for their affinity toward A(1), A(2a), and A(3) AR, showing interesting antagonistic activity and A(1) selectivity.

Synthesis of 1-(2-chloro-2-phenylethyl)-6-methylthio-1H-pyrazolo[3,4-d]pyrimidines 4-amino substituted and their biological evaluation.

A new series of 4-amino-6-methylthio-1H-pyrazolo[3,4-d]pyrimidines (2a-m) bearing the 2-chloro-2-phenylethyl chain at the N1 position, has been synthesized. The affinity of these compounds for A1 adenosine receptor (A1AR) was measured. The compounds showed poor affinity. A more interesting result was obtained by 2a, 2d, 2g, which demonstrated inhibitory activity on cell proliferation of the A-431 cell line stimulated by epithelial growth factor (EGF) and on EGF receptor tyrosine kinase (EGFR-TK) phosphorylation.

Antiproliferative activity of new 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives toward the human epidermoid carcinoma A431 cell line.

Synthesis and biological evaluation of a new class of 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives are reported. A preliminary cellular assay system using the tumor cell line A431 responding to epidermal growth factor (EGF) for its growth, shows that the new compounds are potent inhibitors of cell growth. The inhibition of tumor cell proliferation is not associated with blockage of EGF receptor (EGFR), but substantially due to the interference with the signalling pathway at the level of Src tyrosine kinase and at the level of the downstream effector signal mitogen activated protein kinases (MAPKs), ERK1-2.

Progress in 5H[1]benzopyrano[4,3-d]pyrimidin-5-amine series: 2-methoxy derivatives effective as antiplatelet agents with analgesic activity.

A series of 2-methoxy-5H[1]benzopyrano[4,3-d]pyrimidin-5-amines were prepared and screened for their in vitro antiplatelet activity inducing the aggregation by ADP, arachidonic acid (AA) and collagen. In vivo experiments were performed in order to evaluate their antiphlogistic, analgesic and antipyretic activities. Title compounds showed antiplatelet activity in aggregation AA or collagen-induced, and a good analgesic activity without any gastric toxicity. Comparison with a number of analogue benzopyrano[4,3-d]pyrimidine derivatives and some SAR consideration were reported.

Synthesis and biological evaluation of neutrophilic inflammation inhibitors.

In several non-infectious human diseases, such as ulcerous colitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), the extravasal recruitment of neutrophils plays a crucial role in the development of tissue damage, which, when persistent, can lead to the irreversible organ dysfunction. The neutrophil activation is controlled by a number of intracellular pathways, particularly by a cAMP-dependent protein kinase A (PKA) which also acts on phosphodiesterase IV (PDE4) gene stimulating the synthesis of this enzyme, able to transform cAMP to inactive AMP. PDE4 inhibitors enhance intracellular cAMP and decrease inflammatory cell activation. Several 3-cyclopentyloxy-4-methoxybenzaldehyde and 3-cyclopentyloxy-4-methoxybenzoic acid derivatives were synthesized and studied by us to evaluate their ability to inhibit the superoxide anion production in human neutrophils. These compounds were found able to inhibit the neutrophil activation and some of them increased the cAMP level on tumor necrosis factor-alpha-stimulated neutrophils. Moreover, they also inhibited selectively the human PDE4 enzyme, although they are less potent than the reference compound Rolipram. We report here synthesis, biological studies and some SAR considerations concerning the above mentioned compounds.

Synthesis of N-substituted-N-acylthioureas of 4-substituted piperazines endowed with local anaesthetic, antihyperlipidemic, antiproliferative activities and antiarrythmic, analgesic, antiaggregating actions.

Three series of N-acyl and N-cyclohexyl- or N-methyl or N-phenyl-thioureas of 4-substituted (methyl, phenyl, 2-pyridyl)piperazines (4-12) were synthesised according to a highly convergent one-pot procedure and tested in vivo (local anaesthetic, anti-hyperlipoproteinemic, analgesic, anti-inflammatory, antiarrythmic activities) and in vitro (antiaggregating and, for some selected derivatives, antiproliferative activities) experiments. All the test compounds showed local anaesthesia in particular 4Ar(4), 5Ar(4), 12Ar(3) (after 5 min) and 5Ar(2), 5Ar(3), 9Ar(4) (after 30 min) were equipotent to lidocaine. In lowering triglyceride levels, compounds 6Ar(4) and 7Ar(3) were more active than nicotinic acid, whereas 7Ar(4) and 11Ar(4) were approximately equipotent. As concerns analgesic activity, 5Ar(2) and 5Ar(4) were as active as indomethacin. Appreciable anti-inflammatory activity was found in 8Ar(1), 5Ar(2) and 11Ar(2), but inferior to that of indomethacin. High levels of antiarrythmic activity, comparable with that of quinidine, were found in derivatives 4Ar(2) and 10Ar(1). Compounds 4Ar(2) and 8Ar(2), assayed in antitumor in vitro screening system at National Cancer Institute (NCI), showed significant antiproliferative activity against ACHN cell line (GI50: 0.13 microM) and NCI-H226 cell line (GI50: 1.03 microM), respectively.

2-aryl-3-phenylamino-4,5-dihydro-2h-benz[g]indazoles with analgesic activity.

A series of 2-aryl-3-phenylamino-4,5-dihydro-2H-benz[g]indazoles was synthesized and tested for antiarrhythmic, local anaesthetic and analgesic activity. The title compounds showed a good antinociceptive activity.

New selective phosphodiesterase 4D inhibitors differently acting on long, short, and supershort isoforms.

The lack of selective inhibitors toward the long, short, or supershort phosphodiesterases (PDE4s) prevented researchers from carefully defining the connection between different enzyme isoforms, their brain localization, and their role in neurodegenerative diseases such as Alzheimer's disease (AD). In the search for new therapeutic agents for treating memory and learning disorders, we synthesized new rolipram related PDE4 inhibitors, which had some selectivity toward the long form PDE4D3. The first series was synthesized as racemate and then resolved by semipreparative HPLC on chiral supports. Herein we report the synthetic pathways to obtain compounds 1a-c, 2a-c, 3a-c, 4a-f, 5a,b, 6a,b, 7a,b, the chiral analytical study to resolve compounds 1a-c, 2a-c, 3a-c, the molecular docking study for compound 1c, and the biological results and some SAR considerations that provide some insights and hints for the structural requirements for PDE4D subtype selectivity and enzyme inhibition.

Synthesis, biological evaluation and docking studies of 4-amino substituted 1H-pyrazolo[3,4-d]pyrimidines.

The synthesis of new 4-amino substituted pyrazolo[3,4-d]pyrimidines along with their activity in cell-free enzymatic assays on Src and Abl tyrosine kinases is reported. Some compounds emerged as good dual inhibitors of the two enzymes, showed antiproliferative effects on two Bcr-Abl positive leukemia cell lines K-562 and KU-812, and induced apoptosis, as demonstrated by the PARP assay. Docking studies have been also performed to analyze the binding mode of compounds under study and to identify the structural determinants of their interaction with both Src and Abl.

Substituted pyrazolo[3,4-b]pyridines as potent A1 adenosine antagonists: synthesis, biological evaluation, and development of an A1 bovine receptor model.

Sixty-eight new substituted pyrazolo[3,4-b]pyridine derivatives were synthesized and tested for enriching a library of active A(1) adenosine receptor (AR) antagonists belonging to the same class. These compounds were also used as an external test set to check the reliability of a 3D QSAR model recently reported by us. To investigate the binding mode of pyrazolopyridine derivatives, a model of the bovine A(1)AR (bA(1)AR) was developed by a novel homology modeling approach and used to evaluate the main interactions of the ligands with the receptor through docking studies. Results suggest important interactions of the ligands mainly with L3.33(88), T3.36(91), Q3.37(92) and H6.52(251), in agreement with mutagenesis data. The racemic mixture of the most active compound was separated into the corresponding enantiomers which showed a bA(1)AR affinity in the nanomolar range, with the R enantiomer sevenfold more active than the S enantiomer, according to results derived from calculations on the receptor model. Analysis of the bovine/human A(1)AR affinity profile of ligands supported the hypothesis that such receptors should be characterized by a different size of their binding site, responsible for the different affinity of the antagonists.

2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives: new potent inhibitors of fMLP-induced neutrophil chemotaxis.

It is well known that both acute and chronic autoimmune inflammatory disorders arise following a breakdown in control of neutrophil activation and recruitment. In the search for new anti-inflammatory agents, we synthesized some new 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives and tested them in vitro in order to evaluate their ability to interfere with human neutrophil functions. All tested compounds showed strong inhibition of fMLP-OMe-induced chemotaxis, although they appeared unable to block degranulation and the fMLP-OMe-induced respiratory burst, and were inactive in binding experiments.