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Even though immunotherapy has radically changed the search for anticancer therapies, there are still many different pathways that are open to intervention with traditional small molecules. To expand our investigation in the anticancer field, we report here a new series of compounds in which our previous pyrazole and imidazopyrazole scaffolds are linked to a differently decorated phenyl ring through an acylhydrazone linker. Preliminary tests on the library were performed at the National Cancer Institute (USA) against the full NCI 60 cell panel. The best compounds among the imidazopyrazole series were then tested by immunofluorescence staining for their inhibition of cell proliferation, apoptosis induction, and their effect on the cell cycle and on microtubules. Two compounds, in particular 4-benzyloxy-3-methoxybenzyliden imidazopyrazole-7-carbohydrazide showed good growth inhibition, with IC 50 values in the low-micromolar range, and induced apoptosis. Both compounds altered the cell-cycle phases with the appearance of polyploid cells. Immunofluorescence analysis evidenced microtubules alterations; tubulin polymerization assays and docking studies suggested the tubulin system to be the possible, although not exclusive, target of the new acylhydrazone series reported here., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alzheimer's disease is the most common type of dementia, affecting millions of people worldwide. One of its main consequences is memory loss, which is related to downstream effectors of cyclic adenosine monophosphate (cAMP). A well-established strategy to avoid cAMP degradation is the inhibition of phosphodiesterase (PDE). In recent years, GEBR-32a has been shown to possess selective inhibitory properties against PDE type 4 family members, resulting in an improvement in spatial memory processes without the typical side effects that are usually correlated with this mechanism of action. In this work, we performed the HPLC chiral resolution and absolute configuration assignment of GEBR-32a. We developed an efficient analytical and semipreparative chromatographic method exploiting an amylose-based stationary phase, we studied the chiroptical properties of both enantiomers and we assigned their absolute configuration by 1 H-NMR (nuclear magnetic resonance). Lastly, we measured the IC 50 values of both enantiomers against both the PDE4D catalytic domain and the long PDE4D3 isoform. Results strongly support the notion that GEBR-32a inhibits the PDE4D enzyme by interacting with both the catalytic pocket and the regulatory domains., Competing Interests: O.B. and C.B. declare an Intellectual Property interest on GEBR-32a

Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure-activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets., Competing Interests: The authors have declared that there is no conflict of interests.

A new species, Alliumalbanicum , is described and illustrated from Albania (Balkan Peninsula). It grows on serpentines or limestone in open rocky stands with a scattered distribution, mainly in mountain locations. Previously, the populations of this geophyte were attributed to A.meteoricum Heldr. & Hausskn. ex Halácsy, described from a few localities of North and Central Greece. These two species indeed show close relationships, chiefly regarding some features of the spathe valves, inflorescence and floral parts. They also share the same diploid chromosome number 2 n =16 and similar karyotype, while seed testa micro-sculptures and leaf anatomy reveal remarkable differences. There are also several morphological features that allow them to be differentiated at specific level. The inclusion of both species into a newly described section Pseudoscorodon of the subgen. Allium is proposed. An analytic key to the species, included in the new section, is also provided.

Background: We screened a large library of differently decorated imidazo-pyrazole and pyrazole derivatives as possible new antitubercular agents and this preliminary screening showed that many compounds are able to totally inhibit Mycobacterium growth (>90 %). Among the most active compounds, we selected some new possible hits based on their similarities and, at the same time, on their novelty with respect to the pipeline drugs., Methods: In order to increase the potency and obtain more information about structure-activity relationship (SAR), we designed and synthesized three new series of compounds (2a-e, 3a-e, and 4a-l)., Conclusion: Performed tests confirmed that both new pyrazoles and imidazo-pyrazoles could represent a new starting point to obtain more potent compounds and further work is now underway to identify the protein targets of this new class of anti-TB agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Janus kinases (JAKs) are a family of non-receptor tyrosine kinases, composed by four members, JAK1, JAK2, JAK3 and TYK2. JAKs are involved in different inflammatory and autoimmune diseases, as well as in malignancies, through the activation of the JAK/STAT signalling pathway. Furthermore, the V617F mutation in JAK2 was identified in patients affected by myeloproliferative neoplasms. This knowledge prompted researchers from academia and pharmaceutical companies to investigate this field in order to discover small molecule JAK inhibitors. These efforts recently afforded to the market approval of four JAK inhibitors. Despite the fact that all these drugs are pyrrolo[2,3-d]pyrimidine derivatives, many compounds endowed with different heterocyclic scaffolds have been reported in the literature as selective or multi-JAK inhibitors, and a number of them is currently being evaluated in clinical trials. In this review we will report many representative compounds that have been published in articles or patents in the last five years (period 2013-2017). The inhibitors will be classified on the basis of their chemical structure, focusing, when possible, on their structure activity relationships, selectivity and biological activity. For every class of derivatives, compounds disclosed before 2013 that have entered clinical trials will also be briefly reported, to underline the importance of a particular chemical scaffold in the search for new inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Selected members of the large rolipram-related GEBR family of type 4 phosphodiesterase (PDE4) inhibitors have been shown to facilitate long-term potentiation and to improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, few if any structure-activity relationship studies have been performed to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degree of selectivity between the different PDE4 isoforms.

The limitation of targeting VEGF/VEGFR2 signalling to stop angiogenesis in cancer therapy has been blamed on re-activation of alternative receptor tyrosine kinases by compensatory angiogenic factors. Targeting MAPK and PI3K signaling pathways in endothelial cells may be an alternative or complementary approach. Herein we aimed to evaluate the antitumor and antiangiogenic potential of a novel pyrazolyl-urea kinase inhibitor, GeGe3, and to identify its kinase targets. We found GeGe3 to inhibit the proliferation of HUVEC and endothelial tube formation. GeGe3 impaired inter-segmental angiogenesis during development of zebrafish embryos. In mice, GeGe3 blocked angiogenesis and tumor growth in transplanted subcutaneous Lewis Lung Carcinomas. Screening for GeGe3-targeted kinases revealed Aurora B, Aurora C, NEK10, polo-like kinase (PLK)2, PLK3, DMPK1 and CAMK1 as candidate targets. Biochemical analysis of these kinases showed DMPK1 regulation upon VEGF challenge. Investigation of the role of DMPK1 in endothelial cells revealed DMPK1 as a novel mediator of angiogenesis that controls the activation of MAPK signaling, proliferation and migration. GeGe3 alters angiogenesis by targeting DMPK in tumor endothelial cells and pericytes. The pyrazolyl-urea GeGe3, a novel blocker of MAPK and PI3K pathways, strongly inhibits physiological and tumor angiogenesis. We also report GeGe3-targeted kinase DMPK as a novel mediator of angiogenesis., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

A new species of Bituminaria is described and illustrated: Bituminaria antiatlantica Brullo, C. Brullo, Cambria, Cristaudo & Giusso, sp. nov. , which is endemic to Anti-Atlas Mountains (Morocco). It is a true chasmophyte, characterized by a suffruticose habit, several woody branches, leaflets coriaceous, rounded to ovate, small, few-flowered inflorescences and corolla pale coloured.

Taking into account the structure activity relationship information given by our previous studies, we designed and synthesized a small library of pyrazolylureas and imidazopyrazolecarboxamides fluorinated on urea moiety and differently decorated on pyrazole nucleus. All compounds were preliminary screened by Western blotting technique to evaluate their activity on MAPK and PI3K pathways by monitoring ERK1/2, p38MAPK and Akt phosphorylation, and also screened with a wound healing assay to assess their capacity in inhibiting endothelial cell migration, using human umbilical vein endothelial cells stimulated with VEGF. Pyrazoles and imidazopyrazoles did not show the same activity profile. SAR consideration showed that specific substituents and their position in pyrazole nucleus, as well as the type of substituent on the phenylurea moiety play a pivotal role in determining increase or decrease of kinases phosphorylation. On the other hand the loss of flexibility in imidazopyrazole derivatives is responsible for activity potentiation. Screening of the compound library for inhibition of endothelial cell migration, a function required for angiogenesis, showed significant activity for compound 3. This compound might interfere with cell migration by modulating the activity of different upstream target kinases. Therefore, compound 3 represents a potential inhibitor of angiogenesis. Furthermore, it may be used as a tool to identify unknown mediators of endothelial migration and thereby unveiling new therapeutic targets for controlling pathological angiogenesis in diseases such as cancers., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Memory loss characterizes several neurodegenerative disorders, including Alzheimer's disease (AD). Inhibition of type 4 phosphodiesterase (PDE4) and elevation of cyclic adenosine monophosphate (cAMP) has emerged as a promising therapeutic approach to treat cognitive deficits. However, PDE4 exists in several isoforms and pan inhibitors cannot be used in humans due to severe emesis. Here, we present GEBR-32a, a new PDE4D full inhibitor that has been characterized both in vitro and in vivo using biochemical, electrophysiological and behavioural analyses. GEBR-32a efficiently enhances cAMP in neuronal cultures and hippocampal slices. In vivo pharmacokinetic analysis shows that GEBR-32a is rapidly distributed within the central nervous system with a very favourable brain/blood ratio. Specific behavioural tests (object location and Y-maze continuous alternation tasks) demonstrate that this PDE4D inhibitor is able to enhance memory in AD transgenic mice and concomitantly rescues their hippocampal long-term potentiation deficit. Of great relevance, our preliminary toxicological analysis indicates that GEBR-32a is not cytotoxic and genotoxic, and does not seem to possess emetic-like side effects. In conclusion, GEBR-32a could represent a very promising cognitive-enhancing drug with a great potential for the treatment of Alzheimer's disease.

In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US FDA and in other countries for the treatment of different diseases or are currently in phase I/II clinical trials. Herein we present the synthesis and the characterization of a novel series of pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme (GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity effect against U87 cell line with an IC 50 value of 7.1 μM., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The pyrrolo[2,3-d]pyrimidine nucleus is a deaza-isostere of adenine, the nitrogenous base of ATP, and is present in many ATP-competitive inhibitors of different kinases. In the last few years the number of articles and patents that have appeared involving this type of inhibitors has dramatically increased and some compounds have been approved for the treatment of inflammatory or myeloproliferative diseases. Other derivatives are currently being evaluated in clinical trials. This review deals with pyrrolo[2,3- d]pyrimidine derivatives active as kinase inhibitors that have been reported in the literature from 2011 to 2016, with a particular interest on the recently patented compounds. The molecules are classified depending on the inhibited kinase, focusing on their chemical structures., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Phosphodiesterase type 4D (PDE4D) has been indicated as a promising target for treating neurodegenerative pathologies such as Alzheimer's Disease (AD). By preventing cAMP hydrolysis, PDE4 inhibitors (PDE4Is) increase the cAMP response element-binding protein (CREB) phosphorylation, synaptic plasticity and long-term memory formation. Pharmacological and behavioral studies on our hit GEBR-7b demonstrated that selective PDE4DIs could improve memory without causing emesis and sedation. The hit development led to new molecule series, herein reported, characterized by a catechol structure bonded to five member heterocycles. Molecular modeling studies highlighted the pivotal role of a polar alkyl chain in conferring selective enzyme interaction. Compound 8a showed PDE4D3 selective inhibition and was able to increase intracellular cAMP levels in neuronal cells, as well as in the hippocampus of freely moving rats. Furthermore, 8a was able to readily cross the blood-brain barrier and enhanced memory performance in mice without causing any emetic-like behavior. These data support the view that PDE4D is an adequate molecular target to restore memory deficits in different neuropathologies, including AD, and also indicate compound 8a as a promising candidate for further preclinical development., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compounds showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compound 3b, chosen among the synthesized compounds as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analogue. Spontaneous locomotor activity, assessed in an open field apparatus, showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compounds 3b, 5b and 7b, did not affect locomotion, whereas compound 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fyn is a member of the Src-family of nonreceptor protein-tyrosine kinases. Its abnormal activity has been shown to be related to various human cancers as well as to severe pathologies, such as Alzheimer's and Parkinson's diseases. Herein, a structure-based drug design protocol was employed aimed at identifying novel Fyn inhibitors. Two hits from commercial sources (1, 2) were found active against Fyn with K(i) of about 2 μM, while derivative 4a, derived from our internal library, showed a K(i) of 0.9 μM. A hit-to-lead optimization effort was then initiated on derivative 4a to improve its potency. Slightly modifications rapidly determine an increase in the binding affinity, with the best inhibitors 4c and 4d having K(i)s of 70 and 95 nM, respectively. Both compounds were found able to inhibit the phosphorylation of the protein Tau in an Alzheimer's model cell line and showed antiproliferative activities against different cancer cell lines.

Introduction: Breakpoint cluster region Abelson (Bcr-Abl) tyrosine kinase (TK) is a constitutively activated cytoplasmic TK and is the underlying cause of chronic myeloid leukemia (CML). To date, imatinib represents the frontline treatment for CML therapy. The development of resistance has prompted the search for novel Bcr-Abl inhibitors., Areas Covered: This review presents a short overview of drugs already approved for CML therapy and of the compounds that are in clinical trials. The body of the article deals with Bcr-Abl inhibitors patented since 2008, focusing on their chemical features., Expert Opinion: The search for Bcr-Abl inhibitors is very active. We believe that a number of patented compounds could enter clinical trials and some could be approved for CML therapy in the next few years. Overall, Bcr-Abl inhibitors constitute a very appealing research field that can be expected to expand further.

c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells.

Introduction: The Bcr-Abl inhibitor imatinib was approved in 2001 for chronic myeloid leukemia therapy, and dramatically changed the lives of patients affected by this disease. Since it also inhibits platelet derived growth factor receptor (PDGFR) and c-Kit, imatinib is used for various other tumors caused by abnormalities of one or both these two enzymes., Areas Covered: This review presents an overview on imatinib formulations and derivatives, synthetic methodologies and therapeutic uses that have appeared in the patent literature since 2008., Expert Opinion: Innovative imatinib formulations, such as nanoparticles containing the drug, will improve its bioavailability. Moreover, oral solutions or high imatinib content tablets or capsules will improve patient compliance. Some solid formulations and innovative syntheses that have appeared in the last few years will reduce the cost of the drug, offering big advantages for poor countries. Some recently patented efficacious imatinib derivatives are in preclinical studies and could enter clinical trials in the next few years. Overall, Bcr-Abl inhibitors constitute a very appealing research field that can be expected to expand further.

A new series of 3-(cyclopentyloxy)-4-methoxyphenyl derivatives, structurally related to our hit GEBR-4a (1) and GEBR-7b (2), has been designed by changing length and functionality of the chain linking the catecholic moiety to the terminal cycloamine portion. Among the numerous molecules synthesized, compounds 8, 10a, and 10b showed increased potency as PDE4D enzyme inhibitors with respect to 2 and a good selectivity against PDE4A4, PDE4B2, and PDE4C2 enzymes, without both cytotoxic and genotoxic effects. The ability to enhance cAMP level in neuronal cells was assessed for compound 8. SAR considerations, also confirmed by in silico docking simulations, evidenced that both chain and amino terminal function characterized by higher hydrophilicity are required for a good and selective inhibitor-catalytic pocket interaction.

N-formyl-methionyl-leucyl-phenylalanine (fMLF), its methyl ester fMLF-OMe and interleukin 8 (IL8) play a pivotal role in neutrophil chemotaxis regulation in the latter and early stages, respectively, but the mechanisms through which the signal transduction pathways activate this function are not yet completely understood. Compounds 3l and 3r, a new class of arylcarbamoyl-imidazo-pyrazoles derivatives, were described as the first example of compounds able to inhibit human neutrophil chemotaxis induced by both fMLF-OMe and IL8. Here, we report their effects on superoxide production and lysozyme release. No inhibition was observed, thus they could be defined as "pure" chemotactic antagonists. Therefore, such molecules were used to highlight specific kinases involved in neutrophil chemotaxis. Our data provide support that compounds 3l and 3r strongly inhibit p38 MAPK with either fMLF-OMe or IL8 chemoattractants, while they show different signaling pathways regarding PKC isoforms suggesting that a fine tuning of the neutrophil activation occurs through differences in the activation of signaling pathways. Neither fMLF-OMe nor IL8 were able to obtain activation of the PI3K/Akt pathway. Since anomalous activation of neutrophil recruitment is one of the causes of many inflammatory diseases, the good versatility of our derivatives could represent the most important characteristic of these new molecules in the development of novel therapeutics., (© 2013 Elsevier B.V. All rights reserved.)

Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein tyrosine kinases. High levels of Hck are associated with drug resistance in chronic myeloid leukemia. Furthermore, Hck activity has been connected with HIV-1. Herein, structure-based drug design efforts were aimed at identifying novel Hck inhibitors. First, an in-house library of pyrazolo[3,4-d]pyrimidine derivatives, which were previously shown to be dual Abl and c-Src inhibitors, was analyzed by docking studies within the ATP binding site of Hck to select the best candidates to be tested in a cell-free assay. Next, the same computational protocol was applied to screen a database of commercially available compounds. As a result, most of the selected compounds were found active against Hck, with Ki values ranging from 0.14 to 18.4 μM, confirming the suitability of the computational approach adopted. Furthermore, selected compounds showed an interesting antiproliferative activity profile against the human leukemia cell line KU-812, and one compound was found to block HIV-1 replication at sub-toxic concentrations., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Starting from our in-house library of pyrazolo[3,4-d]pyrimidines, a cross-docking simulation was conducted on Bcr-Abl T315I mutant. Among the selected compounds (2a-e), the 4-bromo derivative 2b showed the best activity against the Bcr-Abl T315I mutant. Deeper computational studies highlighted the importance of the bromine atom in the para position of the N1 side chain phenyl ring for the interaction with the T315I mutant. A series of 4-bromo derivatives was thus synthesized and biologically evaluated. Compound 2j showed a good balance of different ADME properties, high activity in cell-free assays, and a submicromolar potency against T315I Bcr-Abl expressing cells. In addition, it was converted into a water-soluble formulation by liposome encapsulation, preserving a good activity on leukemic T315I cells and avoiding the use of DMSO as solubilizing agent. In vivo studies on mice inoculated with 32D-T315I cells and treated with 2j showed a more than 50% reduction in tumor volumes.

Pim-1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim-1 inhibitors, a previously identified ATP-competitive indolyl-pyrrolone scaffold was expanded to derive structure-activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP-competitive inhibitors as well as a series of 2-aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim-1. Notably, further evaluation of the 2-aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP-competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP-competitive and ATP-noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim-1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim-1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim-1 is associated with chemotherapeutic resistance., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The recent launch onto the market of five VEGFR inhibitors indicates the therapeutic value of these agents and the importance of the research in the field of angiogenesis inhibitors for future oncologic therapy. In this Perspective we briefly report the inhibitors that are in clinical use, while we dedicate two wider sections to the compounds that are in clinical trials and to the new derivatives appearing in the literature. We especially consider the medicinal chemistry aspect of the topic and report the structure-activity relationship studies and the binding mode of some inhibitors as well as the biological data of the compounds discovered in the past 5 years.

c-Src and Bcr-Abl are two cytoplasmatic tyrosine kinases (TKs) involved in the development of malignancies. In particular, Bcr-Abl is the etiologic agent of chronic myeloid leukemia, where Src is also involved; the latter is hyperactivated in several solid tumors. Because of the structural homology between Src and Abl, several compounds originally synthesized as Src inhibitors have also been shown to be Abl inhibitors, useful in overcoming the onset of some types of chronic myeloid leukemia resistances, which frequently appear in the advanced phases of pathology. In recent years, the development of such compounds has been promoted by both excellent preclinical and clinical results, and by the theory that dual or multi-targeted inhibitors might be more effective than selective inhibitors. This review is an update on the most important dual inhibitors already in clinical trials and includes information regarding compounds that have appeared in the literature in recent years.

In pursuing our research on some 2,4-diamino-benzopyranopyrimidines and 2-amino-5,6-dihydrobenzo[h]quinazolines, previously reported as antiplatelet and analgesic/anti-inflammatory agents respectively, we designed and synthesized a new series of 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted. The insertion of amino substituents at positions 2 and 4 of the benzoquinazoline scaffold resulted in compounds endowed with a potent ASA-like antiplatelet activity, combined with an anti-inflammatory activity comparable, in some cases, to that of indomethacin, used as a reference drug., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Anomalous activation of neutrophil recruitment is one of the causes of many inflammatory diseases. The chemoattractants N-formyl-methionyl-leucyl-phenylalanine (fMLP), and interleukine 8 (IL8) play a pivotal role in neutrophil chemotaxis regulation in the latter and early stages, respectively, probably by two independent mechanisms. We reported here synthesis and biological evaluation of new N-aryl-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyrazole-1-carboxamides 7-substituted which were designed as possible multi-target antiinflammatory agents. Many of the title compounds showed a good inhibition, in the nano molar range, of human neutrophil chemotaxis selectively acting toward fMLP-OMe (methylester of fMLP) or IL8 stimulus; whereas, two compounds showed an interesting dual activity inhibiting both fMLP-OMe and IL8-induced chemotaxis at nano molar concentration., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl inhibitors previously found active in NB cell lines (1-3), small modification of the original structures almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting antiproliferative activity in SH-SY5Y cells with an IC(50) of 80 nM and a favourable ADME profile. A 3D SAR analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents for NB treatment., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A(1) adenosine receptor antagonists have been proposed to possess an interesting range of potential therapeutic applications. We have already reported the synthesis and the biological characterization of a family of pyrazolo[3,4-b]pyridine derivatives as A(1) adenosine ligands endowed with an antagonistic profile. In the present work, we report the LC separation of enantiomers of our most active A(1) antagonists together with the determination of their absolute configuration by means of X-ray crystal structure analysis. Biological assays confirmed a different activity for the two enantiomers, with the R one showing the higher human A(1)AR affinity. We also developed a homology model of this receptor subtype in order to suggest a binding disposition of the ligands into the hA(1)AR. All of the obtained data suggest that the compound's chirality plays a key role in A(1) affinity.

A family of dual Src/Abl inhibitors characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold was previously reported by us and proved to be active against several tumor cell lines. Among these compounds, a promising antileukemia lead (1) has been recently identified, but, unfortunately, it suffers from substandard pharmaceutical properties. Accordingly, an approach for the optimization of the lead 1 is described in the present work. A series of more soluble pyrazolo[3,4-d]pyrimidine derivatives were rationally designed and proved to maintain the dual Src/Abl activity of the lead. Selected compounds showed an interesting activity profile against three different leukemic cells also in hypoxic conditions, which are usually characterized by imatinib-resistance. Finally, in vitro ADME properties (PAMPA permeation, water solubility, microsomal stability) for the most promising inhibitors were also evaluated, thus allowing the identification of a few optimized analogues of lead 1 as promising antileukemia agents.

Importance of the Field: c-Src and Bcr-Abl are two non-receptor or cytoplasmic tyrosine kinases (TKs) that play important roles in the development of solid and hematological malignancies. Indeed, Src is overexpressed or hyperactivated in a variety of solid tumors, while Bcr-Abl is the causative agent of chronic myeloid leukemia (CML), where Src is also involved. The two enzymes share significant sequence homology and remarkable structural resemblance., Areas Covered in This Review: ATP-competitive compounds originally developed as Src inhibitors, showed to be also potent Abl inhibitors. Dasatinib, the first dual Src/Abl inhibitor approved by the US FDA in 2006 for the treatment of imatinib-resistant CML, is currently being tested in several clinical trials for the treatment of different solid tumors. SKI-606 and AZD0530 are two other important dual Src/Abl inhibitors extensively tested in animal models and in clinical trials, but not entered into therapy yet., What the Reader Will Gain: In this review we will report the latest results regarding dasatinib, SKI-606 and AZD0530, but also the knowledge on new compounds that have appeared in the literature in the last few years, including AP24163, AP24534, XL228, DC2036. We will focus on the most recent clinical trials or on preclinical studies that are in progress on these small-molecule TK inhibitors that represent a targeted therapy with high potential against cancer., Take Home Message: Molecularly targeted therapies, including the inhibition of specific TKs hyperactivated or overexpressed in many human cancers, could be less toxic than the classical non-specific cytotoxic chemotherapeutic agents; they could offer important therapeutic effects, especially if used in association with other agents such as monoclonal antibodies.

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.

Quality QSAR: A combination of docking calculations and a statistical approach toward Abl inhibitors resulted in a 3D QSAR model, the analysis of which led to the identification of ligand portions important for affinity. New compounds designed on the basis of the model were found to have very good affinity for the target, providing further validation of the model itself.The X-ray crystallographic coordinates of the Abl tyrosine kinase domain in its active, inactive, and Src-like inactive conformations were used as targets to simulate the binding mode of a large series of pyrazolo[3,4-d]pyrimidines (known Abl inhibitors) by means of GOLD software. Receptor-based alignments provided by molecular docking calculations were submitted to a GRID-GOLPE protocol to generate 3D QSAR models. Analysis of the results showed that the models based on the inactive and Src-like inactive conformations had very poor statistical parameters, whereas the sole model based on the active conformation of Abl was characterized by significant internal and external predictive ability. Subsequent analysis of GOLPE PLS pseudo-coefficient contour plots of this model gave us a better understanding of the relationships between structure and affinity, providing suggestions for the next optimization process. On the basis of these results, new compounds were designed according to the hydrophobic and hydrogen bond donor and acceptor contours, and were found to have improved enzymatic and cellular activity with respect to parent compounds. Additional biological assays confirmed the important role of the selected compounds as inhibitors of cell proliferation in leukemia cells.

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.

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.

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.

In this study, we evaluated the activity of two novel pyrazolopyrimidine derivatives (Si 34 and Si 35) against ARO cells, a human anaplastic thyroid cancer cell line. ARO cells exposed to different concentrations of the drugs showed a reduced growth rate and an increase of mortality. After 72 h incubation, doses of 5 and 10 microM Si 34 determined a decrease of cell counts by approximately 25% and approximately 75% compared with those of control cells respectively. Similar findings were observed using Si 35. Treatment with both Si 34 and Si 35 at 10 microM increased cell mortality also ( approximately 29% and approximately 18% respectively). At these concentrations, a decrease in cyclin D1 levels was observed. To improve the biopharmaceutical properties, a liposome formulation was prepared. When entrapped in unilamellar liposomes, Si 34 exerted its cytotoxic effects even at lower doses (maximal inhibition at 5 microM) and after shorter incubation time (48 h) either in ARO or other thyroid cancer cell lines. The effects were associated with weak apoptotic death. Inhibition of epidermal growth factor-stimulated src and ERK phosphorylation, as well as reduction of migration properties of ARO cells was also observed. Moreover, the growth of tumor xenografts induced in severe combined immunodeficiency (SCID) mice was inhibited by i.v. administration of 25-50 mg/kg of the drug liposomal formulation. In conclusion, the liposomal preparation of this novel pyrazolopyrimidine derivative appears to be a promising tool for the treatment of anaplasic thyroid cancer.

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.

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.

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.

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.

A series of pyrazolo[3,4-d]pyrimidines, previously found to be Src inhibitors, was tested for their ability to inhibit proliferation of three Bcr-Abl-positive human leukemia cell lines (K-562, KU-812, and MEG-01), on the basis of the experimental evidence that various Src inhibitors are also active against Bcr-Abl kinase (the so called dual Src/Abl inhibitors). They reduce Bcr-Abl tyrosine phosphorylation and promote apoptosis of the Bcr-Abl-expressing cells. A cell-free enzymatic assay on isolated c-Abl confirmed that such compounds directly inhibit Abl activity. Finally, molecular modeling simulations were also performed to hypothesize the binding mode of the compounds into the Abl binding site.

New series of 5-alkoxy-benzopyranopyrimidine derivatives were developed from the chemical modulation of the substituent in position 2 of the scaffold, with the aim to produce analgesic/antiphlogistic agents more potent than analogues previously reported. The 2-hydrazino derivatives exhibited a good analgesic activity in writhing test; the analgesic doses of the compounds did not affect mice spontaneous locomotor activity thus any confounding sedative effect could be excluded. These derivatives revealed an aspirin-like profile with a strong inhibition of AA-induced platelet aggregation, probably due to a strong, non selective, inhibition of cyclooxygenases. In spite of the inhibition of COX activity displayed in vitro, the compounds did not cause gastric damage in rats after acute oral administration. A different pharmacological profile was observed for the 2-azido derivatives, particularly in vivo.

Two series of N-[5-oxo-4-(arylsulfonyl)-4,5-dihydro-1,3,4-thiadiazol-2-yl]-amides were synthesized and tested in vivo for their analgesic and anti-inflammatory activities. All the new compounds possess good antalgic action in the acetic acid writhing test and some terms of the series showed also fair anti-inflammatory activity in the carrageenan rat paw edema test. Ulcerogenic and irritative action on the gastrointestinal mucose, in comparison with indomethacin is low.

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.

Atherothrombotic coronary artery disease, associated with deep vein thrombosis, is one of the most common causes of death worldwide. Recently, antiplatelet combination therapy using agents with different mechanisms of action, such as aspirin, dipyridamole, and thienopyridines, seems to be an attractive preventive approach. Moreover, several large, randomized clinical trials support combination therapy with aspirin plus warfarin in high-risk patients with atherosclerotic heart disease. Our research on the benzopyrano[4,3-d]pyrimidine system gave rise to the synthesis of a large number of compounds endowed with in vitro anti-aggregating activity. Several SAR considerations suggest that the benzopyranopyrimidine system is an appropriate scaffold to obtain molecules that are able to act simultaneously in different pathways of aggregation. Now, we report the synthesis of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones and the results of the pharmacological study on haemostasis. Some tested compounds showed a large-spectrum antiplatelet activity in vitro, and are more potent than aspirin as antithrombotics in vivo but, at variance with aspirin, they do not increase bleeding. This paper describes novel antithrombotic compounds with an interesting pharmacological profile and a potentially attractive benefit/risk ratio, with their mechanism of action generally, but not exclusively, dependent on antiplatelet activity, deserving further investigations.