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Glioma stem–like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1–ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo. Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1–ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219–30. ©2016 AACR.

Supplementary Table S1. Cohort demographics of 83, 84, 157, 1016, 28, 711 and 267 glioma spheres. Supplementary Table S2. STR analysis result of 83, 84, 157, 1016, 28, 711 and 267 glioma spheres. Supplementary Table S3. Physical chemical properties of GA11 and GA23, determined by BIOVIA Discovery Studio 4.5.

Supplement Fig. S1. Original film scan of western blots in indicated figures; Supplementary Fig. S2. FOXD1 is identified as a MES GSC-representative transcription factor, related to Figure 1.; Supplementary Fig. S3. Glycolysis assay by measuring relative lactic acid levels of MES83 glioma spheres transduced with shNT control or shFOXD1#1; Supplementary Fig. S4. FOXD1 transcriptionally regulates the expression of ALDH1A3 in MES83 GSCs, related to Figure 4.; Supplementary Fig. S5. RNA-interference mediated silencing of ALDH inhibits tumorigenicity of Drosophila eye neoplasia, related to Figure 5.; Supplementary Fig. S6. Analysis of Rembrandt database showing a higher expression of ALDH1A3 associated with poor prognosis in glioma patients, related to Figure 6.; Supplementary Fig. S7. ALDH inhibitor GA11 inhibits the tumorigenicity of MES83 glioma spheres, related to Figure 6.

Supplementary methods and materials

Melanoma is the most serious form of skin cancer but its medication is still far from being safe and thoroughly effective. The search of novel therapeutic approaches represents therefore a health emergency to push through eagerly. In this study, we describe a novel class of dual c-Kit/Aur inhibitors, characterized by a 1,2,4-triazole core and developed by a structure-based optimization of a previously developed hit, and report the evidence of their significance as drug candidates for the treatment of melanoma. Compound 6a, merging the best inhibitory profile against the target kinases, showed anti-proliferative efficacy against the human melanoma cell lines A2058, expressing the BRAF V600D mutation, and WM266-4, expressing BRAF V600E. Significantly, it displayed also a highly synergistic profile when tested in combination with vemurafenib, thus proving its efficacy not only per se but even in a combination therapy, which is nowadays acknowledged as the cornerstone approach of the forthcoming tumour management.

Aldose reductase is a key enzyme in the development of long term diabetic complications and its inhibition represents a viable therapeutic solution for people affected by these pathologies. Therefore, the search for effective aldose reductase inhibitors is a timely and pressing challenge. Herein we describe the access to a novel class of oxyimino derivatives, obtained by reaction of a 1,5-dicarbonyl substrate with O-(arylmethyl)hydroxylamines. The synthesised compounds proved to be active against the target enzyme. The best performing inhibitor, compound (Z)-8, proved also to reduce both cell death and the apoptotic process when tested in an in vitro model of diabetic retinopathy made of photoreceptor-like 661w cell line exposed to high-glucose medium, counteracting oxidative stress triggered by hyperglycaemic conditions.

The fungal pathways of melanin synthesis have so far been little considered as a source of bio-inspiration in the field of functional materials, despite the interesting properties exhibited by Ascomycetes melanins from 1,8-dihydroxynaphthalene (1,8-DHN), including the ability to shield organisms from ionizing radiation. Herein, the processing techniques and characterizations of mycomelanin thin films obtained from the solid state polymerization of 1,8-DHN is reported for the first time. Overall, the results highlighted the role of synthetic mycomelanin thin films as a prototype of next generation bioinspired interfaces featuring high structural regularity and ultrasmooth morphology, high robustness against peroxidative bleaching and adhesion under water conditions, good biocompatibility and unprecedented effects in inducing the spontaneous differentiation of embryonic stem cells prevalently towards the endodermal lineages in the absence of added factors. These data open up new avenues towards the applications of this biomaterial in the fields of tissue engineering and regenerative medicine.

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity against the target enzyme and selectivity toward highly homologous isoenzymes, representative examples of the series also showed a nanomolar to picomolar efficacy against patient-derived GBM stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a novel and promising way to combat GBM by striking its ability to divide immortally.

Nano-sized colloidal carriers represent innovative drug delivery systems, as they allow a targeted and prolonged release of poorly water-soluble drugs, improving their bioavailability and modifying their pharmacokinetic parameters. In this work we describe cyclodextrin-based nanosponges, obtained through polimerization of β-cyclodextrin with diphenyl carbonate as the cross-linking agent, loaded with a novel multi-effective heterocyclic compound, DB103, able to regulate key cellular events involved in the remodelling of vessels wall. Fabrication and drug-loading procedures, as well as physical-chemical characterization and drug-release profile of the novel colloidal system are reported. Results achieved demonstrate the ability of nanosponges to enclose efficiently the target drug and release it slowly and continuously, thus suggesting the exploitability of the novel system for the local therapy of vessels wall subjected to percutaneous intervention.

Aldose reductase (ALR2), a NADPH-dependent reductase, is the first and rate-limiting enzyme of the polyol pathway of glucose metabolism and is implicated in the pathogenesis of secondary diabetic complications. In the last decades, this enzyme has been targeted for inhibition but despite the numerous efforts made to identify potent and safe ALR2 inhibitors, many clinical candidates have been a failure. For this reason the research of new ALR2 inhibitors highly effective, selective and with suitable pharmacokinetic properties is still of great interest. In this paper some new N-(aroyl)-N-(arylmethyloxy)alanines have been synthesized and tested for their ability to inhibit ALR2. Some of the synthesized compounds exhibit IC50 in the low micromolar range and all have proved to be highly selective towards ALR2. The N-(aroyl)-N-(arylmethyloxy)-α-alanines are a promising starting point for the development of new ALR2 selective drugs with the aim of delaying the onset of diabetic complications.

The cycloisomerization of readily available 2-alkynylbenzamides has been conveniently carried out with CuCl2 as catalyst under basic conditions in 1-ethyl-3-methylimidazolium ethyl sulfate (EmimEtSO4) as an unconventional solvent. The process leads to (Z)-3-alkylideneisoindolin-1-ones in a chemo-, regio-, and stereoselective manner. The catalyst/solvent/base system could be recycled several times without appreciable loss of activity. The structures of representative products, that are, (Z)-3-benzylidene-2-phenylisoindolin-1-one and (Z)-3-pentylidene-2-phenylisoindolin-1-one, have been confirmed by X-ray diffraction analysis, and DFT (density functional theory) calculations have been carried out to clarify the roles exerted by the base and CuCl2 in favoring the reaction course leading to (Z)-3-alkylideneisoindolinones.

Carboxymethylcellulose (CMC) is a well-known pharmaceutical polymer, recently gaining attention in the field of nanomedicine, especially as a polyelectrolyte agent for the formation of complexes with oppositely charged macromolecules. Here, we report on the application of pH-sensitive pharmaceutical grade CMC-based nanoparticles (NP) for white blood cells (WBC) PET imaging. In this context and as an alternative to 99mTc-HMPAO SPECT labeling, the use of 68Ga3+ as PET radionuclide was investigated since, at early time points, it could provide the greater spatial resolution and patient convenience of PET tomography over SPECT clinical practices. Two operator-friendly kit-type formulations were compared, with the intention of radiolabeling within a short time (10 min), under mild conditions (physiological pH, room temperature) and in agreement with the actual clinically applied guidelines. NP were labeled by directly using 68Ga3+ eluted in HCL 0.05 N, from hospital suited 68Ge/68Ga generator and in absence of chelator. The first kit type approach involved the application of 68Ga3+ as an ionotropic gelation agent for in-situ forming NP. The second kit type approach concerned the re-hydration of a proper freeze-dried injectable NP powder. pH-sensitive NP with 250 nm average diameter and 80% labeling efficacy were obtained. The NP dispersant medium, including a cryoprotective agent, was modulated in order to optimize the Zeta potential value (&minus, 18 mV), minimize the NP interaction with serum proteins and guarantee a physiological environment for WBC during NP incubation. Time-dependent WBC radiolabeling was correlated to NP uptake by using both confocal and FT-IR microscopies. The ready to use lyophilized NP formulation approach appears promising as a straightforward 68Ga-WBC labeling tool for PET imaging applications.

Diabetes is a multi-factorial disorder that should be treated with multi-effective compounds. Here we describe the access to polyhydroxylated pyrrolidines, belonging to the d-gluco and d-galacto series, through aminocyclization reactions of two differentially protected d-xylo-hexos-4-ulose derivatives. The prepared compounds proved to inhibit both alpha-glucosidase, responsible for the emergence of hyperglycemic spikes, and aldose reductase, accountable for the development of abnormalities in diabetic tissues. Accordingly, they show the dual inhibitory profile deemed as ideal for diabetes treatment. Significantly, compound 17b reduced the process of cell death and restored the physiological levels of oxidative stress when tested in the photoreceptor-like 661w cell line, thus proving to be effective in an in vitro model of diabetic retinopathy.

This work aims to demonstrate the possibility to fabricate ultra-thin polymeric films loaded with an anti-restenotic drug and capable of tunable drug release kinetics for the local treatment of restenosis. Vascular nanopatches are composed of a poly(lactic acid) supporting membrane (thickness: ~250 nm) on which 20 polyelectrolyte bilayers (overall thickness: ~70 nm) are alternatively deposited. The anti-restenotic drug is embedded in the middle of the polyelectrolyte structure, and released by diffusion mechanisms. Nanofilm fabrication procedure and detailed morphological characterization are reported here. Barium titanate nanoparticles (showing piezoelectric properties) are included in the polymeric support and their role is investigated in terms of influence on nanofilm morphology, drug release kinetics, and cell response. Results show an efficient drug release from the polyelectrolyte structure in phosphate-buffered saline, and a clear antiproliferative effect on human smooth muscle cells, which are responsible for restenosis. In addition, preliminary evidences of ultrasound-mediated modulation of drug release kinetics are reported, thus evaluating the influence of barium titanate nanoparticles on the release mechanism. Such data were integrated with quantitative piezoelectric and thermal measurements. These results open new avenues for a fine control of local therapies based on smart responsive materials., Video abstract

Aldose reductase, the key enzyme of the polyol pathway, plays a crucial role in the development of long term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has failed, due to the emergence of side effects from compounds under development. Actually, aldose reductase is a Janus-faced enzyme which, besides ruling the polyol pathway, takes part to the antioxidant defence mechanism of the body. In this work we report the evidence that a novel class of compounds, characterized by a pyrazolo[1,5-a]pyrimidine core and a ionisable fragment, is able to modulate differently the catalytic activity of the enzyme, depending on the presence of specific substrates like sugar, toxic aldehydes and glutathione conjugates of toxic aldehydes. These compounds stand out as the first example of aldose reductase differential inhibitors (ARDIs), intended to target long term diabetic complications while leaving unaltered the detoxifying role of the enzyme.

Tyrosine kinase inhibitors (TKIs) are molecules that compete with ATP on tyrosine kinase receptors (TKRs), blocking tyrosine kinase (TK) activation and then oncogenic pathways; they have been studied, and some of them are right approved for the treatment of many types of cancer. Among TKIs, one of the most explored chemical template is the pyrazolo[3,4-d]pyrimidine (PP) heterocyclic core, which proved to be a useful scaffold for the obtainment of effective compounds. Actually, derivatives belonging to this structural class show a large spectrum of activity, thus standing out as multi-target agents. Different PP compounds have been shown to act as: a) ABL inhibitors and antiproliferative agents against human leukemia cell lines; b) Src kinase inhibitors in neuroblastoma, medulloblastoma and osteosarcoma; c) Phospholipase D inhibitors in different neoplasias; d) Urokinase plasminogen activator inhibitors, in breast cancer. In thyroid cancer (TC), PP1 and PP2 (inhibitors of RET, Hck, lck, and fynT kinases, and a good inhibitor of c-Src and platelet-derived growth factor receptor) showed antineoplastic actvity in human papillary TC cell lines that carry spontaneous RET/PTC1 rearrangements. More recently, new derivatives, (R)-1-phenethyl-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4- amine, namely, CLM3 and CLM29, have been demonstrated to exert a multiple signal transduction inhibition (including the RET-TK, BRAF, EGFR, and with antiangiogenic activity), showing antineoplastic activity, in vitro and in vivo, in papillary dedifferentiated, medullary and anaplastic TC. These data have shown the antineoplastic activity of PP in different neoplasias, opening the way to a future clinical evaluation in human cancers.

We investigated the ability of quercetin and apigenin to modulate platelet activation and aggregation, and compared the observed efficacy with that displayed by their synthetic analogues 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 1-4, and 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 5-7. Platelet aggregation was explored through a spectrophotometric assay on platelet-rich plasma (PRP) treated with the thromboxane A2 mimetic U46619, collagen and thrombin in presence/absence of various bioisosteres of flavonoids (12.5-25-50-100 μM). The platelet density, (mean platelet component, MPC), was measured by the Advia 120 Hematology System as a marker surrogate of platelet activation. The induced P-selectin expression, which reflects platelet degranulation/activation, was quantified by flow cytometry on PRP. Our synthetic compounds modulated significantly both platelet activation and aggregation, thus turning out to be more effective than the analogues quercetin and apigenin when tested at a concentration fully consistent with their use in vivo. Accordingly, they might be used as food supplements to increase the efficacy of natural flavonoids.

Background: Aldose reductase, the first enzyme of the polyol pathway, is the key determinant for the pathogenesis of long term diabetic complications. Accordingly, its inhibition represents the major therapeutic strategy to treat this kind of pathologies. Objectives: In this work we describe the synthesis and the functional evaluation of a number of spiro-oxazolidinone and spiro-morpholinone acetic acid derivatives, and their benzyloxy analogs, developed as aldose reductase inhibitors. Results: Most of them proved to inhibit the target enzyme, showing IC50 values in the micromolar/low micromolar range. SARs observed among the three different series allowed to highlight their key pharmacophoric elements, thus creating sound basis for the design of novel and more effective inhibitors. Conclusion: Although further substitution patterns are needed, the novel compounds here proposed represent a good starting point for the development of novel and effective ARIs.

Hyper-proliferation and migration of vascular smooth muscle cells and endothelial cell dysfunction are central events in the development of neo-intimal lesions. Pursuing our interest in the synthesis of bioisosters of flavonoids, we studied in depth a novel synthetic 2,3-diphenyl-4 H -pyrido[1,2- a ]pyrimidin-4-one derivative, examining its effects in vitro on induced-cell proliferation and activation in human aortic smooth muscle cells (HAoSMCs) and in human umbilical vein endothelial cells (HUVECs). Compared with two well known flavonoids, apigenin and quercetin, the novel compound, 2-(3,4-dimethoxyphenyl)-3-phenyl-4 H -pyrido[1,2- a ]pyrimidin-4-one, 3 , was not toxic for HUVECs, even at high concentrations and for long incubation times, while the two flavonoids were not tolerated, even at concentrations as low as 10 μmol/L. Compound 3 inhibited selectively, and in a concentration-dependent manner, the proliferation of HAoSMCs but not that of HUVECs. In HUVECs, it inhibited the cytokine-induced vascular cell adhesion molecule-1 expression, but not the cyclooxygenase-2 (COX-2) expression. Instead, in HAoSMC, it inhibited the induction of COX-2 expression and the relative release of prostaglandin E 2 . In addition, it inhibited the transcription of the matrix metalloproteinase-9 and its activity. Thanks to its multiple and tissue-specific function, 2-(3,4-dimethoxyphenyl)-3-phenyl-4 H -pyrido[1,2- a ]pyrimidin-4-one might replace or assist the action of current drugs eluted by coronary stents, in order to promote a functional repair of damaged wall.

A novel drug delivery vector, a free-standing polymeric ultrathin film (nanofilm) composed of PMMA and a polysaccharides multilayer, is presented. Chitosan and sodium alginate are alternatively deposited by spin-assisted LbL assembly onto a plasma-treated PMMA thin film. Hydrophobic anti-inflammatory drugs, an adenosine deaminase inhibitor (APP) and its fluorescent dansyl derivate (APP-Dns), are encapsulated inside the LbL multilayer using a simple casting deposition procedure. The resulting drug loaded nanofilm can be suspended in water upon dissolution of a PVA sacrificial layer. Morphological characterization of the nanofilm shows that PMMA/LbL nanofilms possess nanometric thickness (

Glioma stem–like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1–ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo. Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1–ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219–30. ©2016 AACR.

Three novel series of 1,2-benzisothiazole derivatives have been developed as inhibitors of carbonic anhydrase isoform IX. Compounds 5c and 5j, tested in vitro on the human colon cell line HT-29, blocked the growth of cells cultured under chemically induced hypoxic conditions, displaying a specific activity against cancer cells characterized by CAIX up-regulation. Moreover, a synergistic activity of 5c with SN-38 (the active metabolite of irinotecan) and 5-fluorouracil on cell proliferation under hypoxic conditions was demonstrated.

Recent advances in the knowledge of thyroid carcinomas development identified receptor tyrosine kinases, like VEGFR2 and RET, as viable and promising targets. Accordingly, their inhibition is emerging as the major therapeutic strategy to treat these pathologies. In this study we describe the synthesis and the functional evaluation of three different series of 4-substituted pyrazolo[3,4-d]pyrimidine derivatives, 8a-g, 9a-g, and 10a-g, designed exploiting a structure-based optimization of the previously developed inhibitor CLM3. Compared to the lead, the novel compounds markedly improved both their inhibitory profile against the target proteins, VEGFR2 and RET, and their antiproliferative efficacy against the medullary thyroid cancer cell line TT. Significantly, compounds 8b, 9c, and 10c proved to block the kinase activity of the mutant RET(V804L), which still lacks effective inhibitors.

Herein we report the synthesis, biological evaluation, and docking analysis of a class of cyclooxygenase-2 (COX-2) inhibitors with nitric oxide (NO)-releasing properties. In an earlier study, a number of selective COX-2 inhibitors/NO donors were developed by conjugating a diarylpyrrole scaffold endowed with selective COX-2 inhibitory properties with various nitrooxyalkyl side chains such as esters, α-amino esters, amides, α-amino amides, ethers, β-amino ethers, inverse esters, and amides. These candidates were found to have high in vitro potencies (COX-2 inhibition at 10 μm: ≥96 %), great efficacy in determining NO-vasorelaxing responses, and good antinociceptive activity in an abdominal writhing test. Among the compounds synthesized in the present work, derivative 2 b [2-(2-(1-(3-fluorophenyl)-2-methyl-5-(4-sulfamoylphenyl)-1H-pyrrol-3-yl)acetamido)ethyl nitrate] showed particularly outstanding activity, with efficacy similar to that of celecoxib even at very low concentrations.

This study investigated the effects of the new aldose reductase inhibitor benzofuroxane derivative 5(6)-(benzo[d]thiazol-2-ylmethoxy)benzofuroxane (BF-5m) on the prolongation of cardiac QT interval and increase of coronary perfusion pressure (CPP) in isolated, high glucose (33.3 mM D-glucose) perfused rat hearts. BF-5m was dissolved in the Krebs solution at a final concentration of 0.01 μM, 0.05 μM, and 0.1 μM. 33.3 mM D-glucose caused a prolongation of the QT interval and increase of CPP up to values of 190 ± 12 ms and 110 ± 8 mmHg with respect to the values of hearts perfused with standard Krebs solution (11.1 mM D-glucose). The QT prolongation was reduced by 10%, 32%, and 41%, respectively, for the concentration of BF-5m 0.01 μM, 0.05 μM, and 0.1 μM. Similarly, the CPP was reduced by 20% for BF-5m 0.05 μM and by 32% for BF-5m 0.1 μM. BF-5m also increased the expression levels of sirtuin 1, MnSOD, eNOS, and FOXO-1, into the heart. The beneficial actions of BF-5m were partly abolished by the pretreatment of the rats with the inhibitor of the sirtuin 1 activity EX527 (10 mg/kg/day/7 days i.p.) prior to perfusion of the hearts with high glucose + BF-5m (0.1 μM). Therefore, BF-5m supplies cardioprotection from the high glucose induced QT prolongation and increase of CPP.

Context and Objective: We have studied the antitumor activity of a novel cyclic amide, CLM94, with anti-vascular endothelial growth factor (VEGF) receptor-2 and antiangiogenic activity in primary anaplastic thyroid cancer (ATC) cells in vitro and in vivo. Design and Main Outcome Measures: CLM94 was tested: 1) in two human cell lines (HMVEC-d, dermal microvascular endothelial cells; and 8305C, undifferentiated thyroid cancer) at 0.001–100 μm; 2) in ATC cells at the concentrations of 10, 30, and 50 μm; and 3) in an ATC cell line (AF) in CD nu/nu mice. Results: CLM94 significantly inhibited VEGF receptor-2 and epidermal growth factor receptor phosphorylation in HMVEC-d and proliferation in HMVEC-d and 8305C cells. A significant reduction of proliferation with CLM94 in ATC cells (P < 0.01, ANOVA) and a slight but significant reduction of proliferation with CLM94 30 and 50 μm in normal thyroid follicular cells (P < 0.01, ANOVA) were shown. CLM94 increased the percentage of apoptotic ATC cells dose-dependently (P < 0.001, ANOVA) and inhibited migration (P < 0.01) and invasion (P < 0.001). AF cell line was injected sc in CD nu/nu mice, and tumor masses became detectable 25 d afterward. CLM94 (40 mg/kg · d) significantly inhibited tumor growth (starting 10 d after the beginning of treatment). CLM94 significantly decreased the VEGF-A gene expression in the AF cell line and the VEGF-A protein and microvessel density in AF tumor tissues. Conclusions: The antitumor and antiangiogenic activity of a new “cyclic amide” compound, CLM94, is very promising in ATC, opening the way to a future clinical evaluation.

Indolylglyoxylamides are a class of distinctive benzodiazepine receptor ligands, proposed in the mid-eighties as open analogues of -carbolines. Thorough and long-lasting studies of their structure-activity relationships led to the development of a great deal of derivatives, to satisfy increasingly structural and pharmacophoric requirements of the benzodiazepine binding site in the central nervous system. Efforts to pre-organize their flexible structure in the three-dimensional shape adopted when bound to the receptor led to the identification of two novel classes of rigid ligands, characterized by planar tricyclic heteroaromatic cores: the [1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one and the [1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-dione. The present review focuses on these selected classes of ligands, whose rational development, in terms of chemical structures and structure-activity relationships, will be fully discussed.

An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5- a ]pyrimidin-7-one.

Aims: To demonstrate the antiproliferative and pro-apoptotic activity of the novel pyrazolopyrimidine derivative multiple tyrosine kinase inhibitor CLM3, alone and in combination with SN-38 (the active metabolite of irinotecan), on endothelial and tumor cells and to show its mechanism of action. Methods: Proliferation and apoptotic assays were performed on microvascular endothelial (HMVEC-d) and lung (A549) and thyroid cancer (8305C, TT) cell lines exposed to CLM3 and to the simultaneous combination with SN38 for 72 h. Cell-based phospho-VEGFR-2, phospho-EGFR and phospho-RET inhibition assays were performed and ERK1/2 and Akt phosphorylation were quantified by ELISA kits. Cyclin D1 gene expression was performed with real-time PCR and cyclin D1 intracellular concentrations were measured by ELISA. Results: A strong effect on antiproliferative and pro-apoptotic activity was found with the CLM3 on endothelial and cancer cells, synergistically enhanced by SN38. Phospho-VEGFR-2, phospho-EGFR and phospho-RET levels significantly decreased after CLM3 treatments in activated endothelial and cancer cells; ERK1/2 and Akt phosphorylation were significantly inhibited by lower concentrations of the pyrazolopyrimidine drug in endothelial cells if compared to cancer cells. Moreover, CLM3 treatment greatly inhibited the expression of the cyclin D1 gene in endothelial and cancer cells, decreasing the cyclin D1 protein intracellular concentration. Conclusions: The pyrazolopyrimidine derivative CLM3 demonstrated a highly significant and promising antiproliferative and proapoptotic activity, alone and in combination with SN-38, for activated endothelial and cancer cells. These effects are mainly due to its inhibition of phosphorylation of VEGFR-2, EGFR and RET tyrosine kinases and their related signaling pathways. © 2011 Elsevier Inc. All rights reserved.

Adenosine kinase (AK) catalyzes the phosphorylation of adenosine (Ado) to AMP by means of a kinetic mechanism in which the two substrates Ado and ATP bind the enzyme in a binary and/or ternary complex, with distinct protein conformations. Most of the described inhibitors have Ado-like structural motifs and are nonselective, and some of them (e.g., the tubercidine-like ligands) are characterized by a toxic profile. We have cloned and expressed human AK (hAK) and searched for novel non-substrate-like inhibitors. Our efforts to widen the structural diversity of AK inhibitors led to the identification of novel non-nucleoside, noncompetitive allosteric modulators characterized by a unique molecular scaffold. Among the pyrrolobenzoxa(thia)zepinones (4a-qq) developed, 4a was identified as a non-nucleoside prototype hAK inhibitor. 4a has proapoptotic efficacy, slight inhibition of short-term RNA synthesis, and cytostatic activity on tumor cell lines while showing low cytotoxicity and no significant adverse effects on short-term DNA synthesis in cells.

Aldose reductase (AKR1B1), the key enzyme of the polyol pathway, plays a crucial role in the development of long-term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has failed, due to the emergence of side effects from compounds under development. Actually AKR1B1 is a Janus-faced enzyme which, besides ruling the polyol pathway, takes part in the antioxidant defense mechanism of the body. In this work we report the evidence that a class of compounds, characterized by a pyrazolo[1,5-a]pyrimidine core and an ionizable fragment, modulates differently the catalytic activity of the enzyme, depending on the presence of specific substrates such as sugar, toxic aldehydes, and glutathione conjugates of toxic aldehydes. The study stands out as a systematic attempt to generate aldose reductase differential inhibitors (ARDIs) intended to target long-term diabetic complications while leaving unaltered the detoxifying role of the enzyme.

Adenosine is a ubiquitous homeostatic substance which exerts its action by triggering four different cell membrane G protein-coupled receptors, classified as A1, A2A, A2B, and A3. As they are widely distributed and deeply involved in several physiological functions, as well as pathological disorders, these receptors represent an excellent drug target, and the development of specific ligands has been tested as a promising therapeutic concept. Among the obtainable ligands, allosteric modulators offer higher advantages with respect to classical orthosteric compounds, as they make it possible to achieve greater selectivity and better modulatory control at disease mediating receptors. Actually, when synergizing with adenosine bound to the primary binding site, these compounds may modify receptor functions through interaction with an additional binding site. As a consequence, their actions depend directly on the release of the endogenous agonist. A number of compounds have been developed as effective allosteric modulators. Most of them target adenosine A1 and A3 receptor subtypes whereas, to date, little or no research has been made to improve the field of A2A or A2B ligands. This review updates literature on the allosteric modulators that has appeared in the last few years, focusing its attention on medicinal chemistry, in terms of chemical structure and structure-activity relationships. This will provide new perspectives on existing data, and an exciting starting point for the development of novel and more effective modulators.

A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine spleen. The 2-arylalkyl derivatives exhibited excellent inhibitory activity, showing Ki values in the nanomolar/subnanomolar range. The most active compound, 1-(4-((4-oxo-4,5-dihydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea, 14d, was tested in rats with colitis induced by 2,4-dinitrobenzenesulfonic acid to assess its efficacy to attenuate bowel inflammation. The treatment with 14d induced a significant amelioration of both systemic and intestinal inflammatory alterations in animals with experimental colitis. Docking simulations of the synthesized compounds into the ADA catalytic site were also performed to rationalize the structure−activity relationships observed and to highlight the key pharmacophoric elements of these products, thus prospectively guiding the design of novel ADA inhibitors.

Vascular Endothelial Growth Factor (VEGF) pathway has emerged as one of the most important positive modulators of Angiogenesis, a central process implicated in tumour growth and metastatic dissemination. This led to the design and development of anti-VEGF monoclonal antibodies and small-molecule ATP-competitive VEGFR-inhibitors. In this study, we describe the synthesis and the biological evaluation of novel 2-aryl substituted benzothiopyrano-fused pyrimidines 1a-i, 2a-i and 3a-i. The ability of the compounds to target the VEGF pathway was determined in vitro exploiting the compounds' antiproliferative efficacy against HUVEC cells. The VEGFR-2 inhibition was confirmed by enzymatic assays on recombinant human kinase insert domain receptor (KDR), by cell-based phospho-VEGFR-2 inhibition assays, and by ex vivo rat aortic ring tests. The selectivity profile of the best performing derivatives belonging to series 2 was further explored combining modeling studies and additional assays in a panel of human cell lines and other kinases.

In continuing the search for more effective 5-arylidene-4-thiazolidinones as aldose reductase inhibitors, a new set of suitably substituted compounds (4, 5 and 8) was explored. Acetic acids 5, particularly 5a and 5h, proved to be interesting inhibitors of the enzyme as well as excellent antioxidant agents that are potentially able to counteract the oxidative stress associated with both diabetic complications as well as other pathologies. Molecular docking experiments supported SAR studies. © 2011 Elsevier Masson SAS. All rights reserved.

We report herein the synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl, sulfamoyl acetamides and ethyl acetates that selectively inhibit cyclooxygenase-2 (COX-2) isoform. Among the newly synthesized compounds, some of them were endowed with a good activity against COX-2 and a good selectivity COX-2/COX-1 in vitro as well as a desirable analgesic activity in vivo, proving that replacement of the ester moiety with an amide group gave access to more stable derivatives, characterized by a good COX-inhibition.

Context and Objective: We have studied the antitumor activity of a pyrazolo[3,4-d]pyrimidine compound (CLM3) proposed for a multiple signal transduction inhibition [including the RET tyrosine kinase, epidermal growth factor receptor, and vascular endothelial growth factor (VEGF) receptor and with antiangiogenic activity] in primary anaplastic thyroid cancer (ATC) cells, in the human cell line 8305C (undifferentiated thyroid cancer), and in an ATC-cell line (AF). Design and Main Outcome Measures: CLM3 was tested in primary ATC cells at the concentrations of 5, 10, 30, and 50 μM; in 8305C cells, in AF cells, at 1, 5, 10, 30, 50, or 100 μM; and in AF cells in CD nu/nu mice. Results: CLM3 significantly inhibited the proliferation of 8305C and AF cells, also inducing apoptosis. A significant reduction of proliferation with CLM3 in ATC cells (P < .01, ANOVA) was shown. CLM3 increased the percentage of apoptotic ATC cells dose dependently (P < .001, ANOVA) and inhibited migration (P < .01) and invasion (P < .001). The AF cell line was injected sc in CD nu/nu mice, and tumor masses became detectable 15 days later. CLM3 (50 mg/kg per die) significantly inhibited tumor growth (starting 16 d after the beginning of treatment). CLM3 significantly decreased the VEGF-A expression and microvessel density in AF tumor tissues. Furthermore, CLM3 inhibited epidermal growth factor receptor, AKT, and ERK1/2 phosphorylation and down-regulated cyclin D1 in 8305C and AF cells. Conclusions: The antitumor and antiangiogenic activity of a pyrazolo[3,4-d]pyrimidine compound (CLM3) is very promising in anaplastic thyroid cancer, opening the way to a future clinical evaluation.

The study investigated the effects of the aldose reductase (AR) inhibitor benzofuroxane derivative 5(6)-(benzo[d]thiazol-2-ylmethoxy) benzofuroxane (herein referred to as BF-5m) on the biochemical and tissue alterations induced by endotoxic uveitis in rats. BF-5m has been administered directly into the vitreous, in order to assess the expression and levels of (i) inflammatory markers such as the ocular ubiquitin-proteasome system, NF-κB, TNF-α, and MCP-1; (ii) prooxidant and antioxidant markers such as nitrotyrosine, manganese superoxide dismutase (MnSOD), and glutathione peroxidase (GPX); (iii) apoptotic/antiapoptotic factors caspases and Bcl-xl; (iv) markers of endothelial progenitor cells (EPCs) recruitment such as CD34 and CD117. 5 μL of BF-5m (0.01; 0.05; and 0.1 μM) into the right eye decreased in a dose-dependent manner the LPS-induced inflammation of the eye, reporting a clinical score 1. It reduced the ocular levels of ubiquitin, 20S and 26S proteasome subunits, NF-κB subunits, TNF-α, MCP-1, and nitrotyrosine. BF-5m ameliorated LPS-induced decrease in levels of MnSOD and GPX. Antiapoptotic effects were seen from BF-5m by monitoring the expression of Bcl-xl, an antiapoptotic protein. Similarly, BF-5m increased recruitment of the EPCs within the eye, as evidenced by CD34 and CD117 antibodies.

CLM29 (a pyrazolo[3,4-d]pyrimidine, that inhibits RET, epidermal growth factor receptor, vascular endothelial growth factor receptor, and has an anti-angiogenic activity) has anti-neoplastic activity in papillary dedifferentiated thyroid cancer. Here we tested CLM29 in medullary thyroid cancer (MTC), in primary MTC cells (P-MTC) obtained at surgery, and in TT cells harboring (C634W) RET mutation. CLM29 (10, 30, 50 μM) inhibited significantly (P

International audience; Driven by a multidisciplinary approach combination (Structure-Based (SB) Three-Dimensional Quantitative Structure-Activity Relationships (3-D QSAR), molecular modeling, organic chemistry and various biological evaluations) here is reported the disclosure of new thienopyrimidines 1-3 as inhibitors of KDR activity and human umbilical vein endothelial cell (HUVEC) proliferation. More specifically, compound 2f represents a new lead compound that inhibits VEGFR-2 and HUVEC at μM concentration. Moreover by the mean of an endothelial cell tube formation in vitro model 2f tartaric acid salt proved to block angiogenesis of HUVEC at μM level.

Diabetes mellitus is the major risk factor for cardiovascular disorders. Aldose reductase, the rate-limiting enzyme of the polyol pathway, plays a key role in the pathogenesis of diabetic complications. Accordingly, inhibition of this enzyme is emerging as a major therapeutic strategy for the treatment of hyperglycemia-induced cardiovascular pathologies. In this study, we describe a series of 5(6)-substituted benzofuroxane derivatives, 5a-k,m, synthesized as aldose reductase inhibitors. Besides inhibiting efficiently the target enzyme, 5a-k,m showed additional NO donor and antioxidant properties, thus emerging as novel multi-effective compounds. The benzyloxy derivative 5a, the most promising of the whole series, showed a well-balanced, multifunctional profile consisting of submicromolar ALR2 inhibitory efficacy (IC50 = 0.99 ± 0.02 μM), significant and spontaneous NO generation properties, and excellent hydroxyl radical scavenging activity. Computational studies of the novel compounds clarified the aldose reductase inhibitory profile observed, thus rationalizing structure-activity relationships of the whole series. © 2012 American Chemical Society.

Aldose reductase (ALR2) is a crucial enzyme in the development of the major complications of diabetes mellitus. Very recently it has been demonstrated that the ARL2 inhibitor, fidarestat, significantly prevents inflammatory signals (TNF-α, LPS) that cause cancer (colon, breast, prostate and lung), metastasis, asthma, and other inflammatory diseases. Currently, fidarestat is in phase III clinical trial for diabetic neuropathy and was found to be safe. Thus the finding of novel, potent ARL2 inhibitors is today more than in the past in great demand as they can pave the way for a novel therapeutic approach for a number of diseases besides the diabetes. Herein, starting from the virtual screening-derived ALR2 inhibitor S12728 (1), a rational receptor-based lead optimization has been undertaken. The design and synthetic efforts here reported led to the discovery of several new compounds endowed with low micromolar/submicromolar activities. © 2012 Elsevier Masson SAS. All rights reserved.