Design, synthesis, and biological investigation of selective human carbonic anhydrase II, IX, and XII inhibitors using 7-aryl/heteroaryl triazolopyrimidines bearing a sulfanilamide scaffold.

A novel library of human carbonic anhydrase (hCA) inhibitors based on the 2-sulfanilamido[1,2,4]triazolo[1,5-a]pyrimidine skeleton modified at its 7-position was prepared by an efficient convergent procedure. These derivatives were evaluated in vitro for their inhibition properties against a representative panel of hCA isoforms (hCA I, II, IV, IX, and XII). The target tumour-associated isoforms hCA IX and XII were potently inhibited with KIs in the low nanomolar range of 5-96 nM and 4-72 nM, respectively. Compounds 1d, 1j, 1v, and 1x were the most potent hCA IX inhibitors with KIs of 5.1, 8.6, 4.7, and 5.1 nM, respectively. Along with derivatives 1d and 1j, compounds 1r and 1ab potently inhibited hCA XII isoform with KIs in a single-digit nanomolar range of 8.8, 5.4, 4.3, and 9.0 nM, respectively. Compounds 1e, 1m, and 1p exhibited the best selectivity against hCA IX and hCA XII isoforms over off-target hCA II, with selectivity indexes ranging from 5 to 14.

Synergistic Effects of A Combined Treatment of Glioblastoma U251 Cells with An Anti-miR-10b-5p Molecule and An AntiCancer Agent Based on 1-(3',4',5'-Trimethoxyphenyl)-2-Aryl-1H-Imidazole Scaffold.

(1) Background: In the development of new and more effective anticancer approaches, combined treatments appear of great interest. Combination therapy could be of importance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer of the central nervous system, with a median survival of 15 months. This study aimed to verify the activity on a glioblastoma cancer cell line of one of the most active compounds of a novel series of tubulin polymerization inhibitors based on the 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold, used in combination with a miRNA inhibitor molecule targeting the oncomiRNA miR-10b-5p. This microRNA was selected in consideration of the role of miR-10b-5p on the onset and progression of glioblastoma. (2) Methods: Apoptosis was analyzed by Annexin-V and Caspase 3/7 assays, efficacy of the anti-miR-10b-5p was assessed by determining the miR-10b-5p content by RT-qPCR. (3) Results: The results obtained show that a "combination therapy" performed by combining the use of an anti-miR-10b-5p and a 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole derivative is an encouraging strategy to boost the efficacy of anticancer therapies and at the same time to reduce side effects.

Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]thiophene derivatives as potent apoptosis-inducing agents.

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure-activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

Apoptosis Pathways Triggered by a Potent Antiproliferative Hybrid Chalcone on Human Melanoma Cells.

The World Health Organization reported that approximately 324,000 new cases of melanoma skin cancer were diagnosed worldwide in 2020. The incidence of melanoma has been increasing over the past decades. Targeting apoptotic pathways is a potential therapeutic strategy in the transition to preclinical models and clinical trials. Some naturally occurring products and synthetic derivatives are apoptosis inducers and may represent a realistic option in the fight against the disease. Thus, chalcones have received considerable attention due to their potential cytotoxicity against cancer cells. We have previously reported a chalcone containing an indole and a pyridine heterocyclic rings and an α-bromoacryloylamido radical which displays potent antiproliferative activity against several tumor cell lines. In this study, we report that this chalcone is a potent apoptotic inducer for human melanoma cell lines SK-MEL-1 and MEL-HO. Cell death was associated with mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage and was prevented by a non-specific caspase inhibitor. Using SK-MEL-1 as a model, we found that the mechanism of cell death involves (i) the generation of reactive oxygen species, (ii) activation of the extrinsic and intrinsic apoptotic and mitogen-activated protein kinase pathways, (iii) upregulation of TRAIL, DR4 and DR5, (iv) downregulation of p21Cip1/WAF1 and, inhibition of the NF-κB pathway.

Design, synthesis and biological evaluation of 2-alkoxycarbonyl-3-anilinoindoles as a new class of potent inhibitors of tubulin polymerization.

A new class of inhibitors of tubulin polymerization based on the 2-alkoxycarbonyl-3-(3',4',5'-trimethoxyanilino)indole molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. The results presented show that the methoxy substitution and location on the indole nucleus plays an important role in inhibition of cell growth, and the most favorable position for the substituent was at C-6. In addition, a small-size ester function (methoxy/ethoxycarbonyl) at the 2-position of the indole core was desirable. Also, analogues that were alkylated with methyl, ethyl or n-propyl groups or had a benzyl moiety on the N-1 indolic nitrogen retained activity equivalent to those observed in the parent N-1H analogues. The most promising compounds of the series were 2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-5-methoxyindole 3f and 1-methyl-2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-6-methoxy-indole 3w, both of which target tubulin at the colchicine site with antitubulin activities comparable to that of the reference compound combretastatin A-4.

Synthesis and Biological Evaluation of 2-Substituted Benzyl-/Phenylethylamino-4-amino-5-aroylthiazoles as Apoptosis-Inducing Anticancer Agents.

Induction of apoptosis is a common chemotherapeutic mechanism to kill cancer cells The thiazole system has been reported over the past decades as a building block for the preparation of anticancer agents. A novel series of 2-arylalkylamino-4-amino-5-(3',4',5'-trimethoxybenzoyl)-thiazole derivatives designed as dual inhibitors of tubulin and cyclin-dependent kinases (CDKs) were synthesized and evaluated for their antiproliferative activity in vitro against two cancer cell lines and, for selected highly active compounds, for interactions with tubulin and cyclin-dependent kinases and for cell cycle and apoptosis effects. Structure-activity relationships were elucidated for various substituents at the 2-position of the thiazole skeleton. Among the synthesized compounds, the most active analogues were found to be the p-chlorobenzylamino derivative 8e as well as the p-chloro and p-methoxyphenethylamino analogues 8f and 8k, respectively, which inhibited the growth of U-937 and SK-MEL-1 cancer cell lines with IC50 values ranging from 5.7 to 12.2 µM. On U-937 cells, the tested compounds 8f and 8k induced apoptosis in a time and concentration dependent manner. These two latter molecules did not affect tubulin polymerization (IC50 > 20 µM) nor CDK activity at a single concentration of 10 µM, suggesting alternative targets than tubulin and CDK for the compounds.

Synthesis and Biological Evaluation of New Antitubulin Agents Containing 2-(3',4',5'-trimethoxyanilino)-3,6-disubstituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine Scaffold.

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3',4',5'-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3',4',5'-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 µM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.

A3 Adenosine Receptors as Modulators of Inflammation: From Medicinal Chemistry to Therapy.

The A3 adenosine receptor (A3 AR) subtype is a novel, promising therapeutic target for inflammatory diseases, such as rheumatoid arthritis (RA) and psoriasis, as well as liver cancer. A3 AR is coupled to inhibition of adenylyl cyclase and regulation of mitogen-activated protein kinase (MAPK) pathways, leading to modulation of transcription. Furthermore, A3 AR affects functions of almost all immune cells and the proliferation of cancer cells. Numerous A3 AR agonists, partial agonists, antagonists, and allosteric modulators have been reported, and their structure-activity relationships (SARs) have been studied culminating in the development of potent and selective molecules with drug-like characteristics. The efficacy of nucleoside agonists may be suppressed to produce antagonists, by structural modification of the ribose moiety. Diverse classes of heterocycles have been discovered as selective A3 AR blockers, although with large species differences. Thus, as a result of intense basic research efforts, the outlook for development of A3 AR modulators for human therapeutics is encouraging. Two prototypical selective agonists, N6-(3-Iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; CF101) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA; CF102), have progressed to advanced clinical trials. They were found safe and well tolerated in all preclinical and human clinical studies and showed promising results, particularly in psoriasis and RA, where the A3 AR is both a promising therapeutic target and a biologically predictive marker, suggesting a personalized medicine approach. Targeting the A3 AR may pave the way for safe and efficacious treatments for patient populations affected by inflammatory diseases, cancer, and other conditions.

3-Aryl/Heteroaryl-5-amino-1-(3',4',5'-trimethoxybenzoyl)-1,2,4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization.

Many natural and synthetic substances are known to interfere with the dynamic assembly of tubulin, preventing the formation of microtubules. In our search for potent and selective antitumor agents, a novel series of 1-(3',4',5'-trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had different heterocycles, including thiophene, furan or the three isomeric pyridines, and they possessed a phenyl ring bearing electron-releasing or electron-withdrawing substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-two tested compounds showed moderate to potent antiproliferative activities against a panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing strong antiproliferative activity (IC50 < 1 µM) against selected cancer cells. Among them, several molecules preferentially inhibited the proliferation of leukemic cell lines, showing IC50 values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly inhibited tubulin assembly, with an IC50 value of 0.66 µM, half that obtained in simultaneous experiments with CA-4 (IC50 = 1.3 µM).

Design, synthesis, in vitro antiproliferative activity and apoptosis-inducing studies of 1-(3',4',5'-trimethoxyphenyl)-3-(2'-alkoxycarbonylindolyl)-2-propen-1-one derivatives obtained by a molecular hybridisation approach.

Inhibition of microtubule function using tubulin targeting agents has received growing attention in the last several decades. The indole scaffold has been recognized as an important scaffold in the design of novel compounds acting as antimitotic agents. Indole-based chalcones, in which one of the aryl rings was replaced by an indole, have been explored in the last few years for their anticancer potential in different cancer cell lines. Eighteen novel (3',4',5'-trimethoxyphenyl)-indolyl-propenone derivatives with general structure 9 were synthesized and evaluated for their antiproliferative activity against a panel of four different human cancer cell lines. The highest IC50 values were obtained against the human promyelocytic leukemia HL-60 cell line. This series of chalcone derivatives was characterized by the presence of a 2-alkoxycarbonyl indole ring as the second aryl system attached at the carbonyl of the 3-position of the 1-(3',4',5'-trimethoxyphenyl)-2-propen-1-one framework. The structure-activity relationship (SAR) of the indole-based chalcone derivatives was investigated by varying the position of the methoxy group, by the introduction of different substituents (hydrogen, methyl, ethyl or benzyl) at the N-1 position and by the activity differences between methoxycarbonyl and ethoxycarbonyl moieties at the 2-position of the indole nucleus. The antiproliferative activity data of the novel synthesized compounds revealed that generally N-substituted indole analogues exhibited considerably reduced potency as compared with their parent N-unsubstituted counterparts, demonstrating that the presence of a hydrogen on the indole nitrogen plays a decisive role in increasing antiproliferative activity. The results also revealed that the position of the methoxy group on the indole ring is a critical determinant of biological activity. Among the synthesized derivatives, compound 9e, containing the 2-methoxycarbonyl-6-methoxy-N-1H-indole moiety exhibited the highest antiproliferative activity, with IC50 values of 0.37, 0.16 and 0.17 µM against HeLa, HT29 and MCF-7 cancer cell lines, respectively, and with considerably lower activity against HL-60 cells (IC50: 18 µM). This derivative also displayed cytotoxic properties (IC50 values ∼1 µM) in the human myeloid leukemia U-937 cell line overexpressing human Bcl-2 (U-937/Bcl-2) via cell cycle progression arrest at the G2-M phase and induction of apoptosis. The results obtained also demonstrated that the antiproliferative activity of this molecule is related to inhibition of tubulin polymerisation. The presence of a methoxy group at the C5- or C6-position of the indole nucleus, as well as the absence of substituents at the N-1-indole position, contributed to the optimal activity of the indole-propenone-3',4',5'-trimethoxyphenyl scaffold.

Synthesis and biological evaluation of alpha-bromoacryloylamido indolyl pyridinyl propenones as potent apoptotic inducers in human leukaemia cells.

The combination of two pharmacophores into a single molecule represents one of the methods that can be adopted for the synthesis of new anticancer molecules. To investigate the influence of the position of the pyridine nitrogen on biological activity, two different series of α-bromoacryloylamido indolyl pyridinyl propenones 3a-h and 4a-d were designed and synthesized by a pharmacophore hybridization approach and evaluated for their antiproliferative activity against a panel of six human cancer cell lines. These hybrid molecules were prepared to combine the α-bromoacryloyl moiety with two series of indole-inspired chalcone analogues, possessing an indole derivative and a 3- or 4-pyridine ring, respectively, linked on either side of 2-propen-1-one system. The structure-activity relationship was also investigated by the insertion of alkyl or benzyl moieties at the N-1 position of the indole nucleus. We found that most of the newly synthesized displayed high antiproliferative activity against U-937, MOLT-3, K-562, and NALM-6 leukaemia cell lines, with one-digit to double-digit nanomolar IC50 values. The antiproliferative activities of 3-pyridinyl derivatives 3f-h revealed that N-benzyl indole analogues generally exhibited lower activity compared to N-H or N-alkyl derivatives 3a-b and 3c-e, respectively. Moreover, cellular mechanism studies elucidated that compound 4a induced apoptosis along with a decrease of mitochondrial membrane potential and activated caspase-3 in a concentration-dependent manner.

Inhibition of activated STAT5 in Bcr/Abl expressing leukemia cells with new pimozide derivatives.

STATs are transcription factors acting as intracellular signaling after stimulation with cytokines, growth factors and hormones. STAT5 is also constitutively active in many forms of cancers, including chronic myelogenous leukemia, acute lymphoblastic leukemia and Hodgkin's lymphoma. Recently, literature reported that the neuroleptic drug pimozide inhibits STAT5 phosphorylation inducing apoptosis in CML cells. We undertook an investigation from pimozide structure, obtaining simple derivatives with cytotoxic and STAT5-inhibitory activity, two of them markedly more potent than pimozide.

Synthesis and biological evaluation of novel 2-amino-3-aroyl-4-neopentyl-5-substituted thiophene derivatives as allosteric enhancers of the A1 adenosine receptor.

2-Amino-3-benzoyl thiophenes have been widely reported to act as allosteric enhancers at the A1 adenosine receptor. Their activity can be increased considerably by appropriate substitutions at the 4- and 5-positions of the thiophene ring. Substituent size at the thiophene C-4 position seemed to be a factor closely related to activity, with the 4-neopentyl (2,2-dimethylpropyl) substitution showing the greatest enhanced activity. A wide series of 2-amino-3-aroyl-4-neopentylthiophene derivatives with general structure 3, characterized by the presence of different substituents (bromine, aryl and heteroaryl) at the 5-position of the thiophene ring, have been identified as potent AEs at the A1AR. With only one exception, all of the synthesized compounds proved to be superior to the reference compound PD 81,723 in a functional assay. Derivatives 3p, 3u, 3am, 3ap and 3ar were the most active compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [(3)H]CCPA binding to the A1 receptor.

Design, synthesis and biological evaluation of 3,5-disubstituted 2-amino thiophene derivatives as a novel class of antitumor agents.

In search of new compounds with strong antiproliferative activity and simple molecular structure, we designed a novel series of agents based on the 2-amino-3-alkoxycarbonyl/cyano-5-arylethylthiophene scaffold. The presence of the ethyl spacer between the 2',5'-dimethoxyphenyl and the 5-position of the thiophene ring, as well as the number and location of methoxy substitutents on the phenyl ring, played a profound role in affecting the antiproliferative activity. Among the synthesized compounds, we identified the 2-amino-3-cyano-[2-(2,5-dimethoxyphenyl)ethyl] thiophene 2c as the most promising derivative against a wide panel of cancer cell lines (IC50=17-130 nM). The antiproliferative activity of this compound appears to correlate well with its ability to inhibit tubulin assembly and the binding of colchicine to tubulin. Moreover 2c, as determined by flow cytometry, strongly induced arrest in the G2/M phase of the cell cycle, and annexin-V and propidium iodide staining indicate that cell death proceeds through an apoptotic mechanism that follows the intrinsic mitochondrial pathway.

TR-644 a novel potent tubulin binding agent induces impairment of endothelial cells function and inhibits angiogenesis.

TR-644 is a novel combretastatin A-4 (CA-4) analogue endowed with potent microtubule depolymerizing activity superior to that of the lead compound and it also has high affinity to colchicines binding site of tubulin. We tested TR-644 anti-angiogenic effects in human umbilical endothelial cells (HUVEC). It showed no significant effects on the growth of HUVEC cells at concentrations below 1,000 nM, but at much lower concentrations (10-100 nM) it induced inhibition of capillary tube formation, inhibition of endothelial cell migration and affected endothelial cell morphology as demonstrated by the disruption of the microtubule network. TR-644 also increased permeability of HUVEC cells in a time dependent manner. The molecular mechanism for the anti-vascular activity of TR-644 was investigated in detail. TR-644 caused G2/M arrest in endothelial cells and this effect correlated with downregulation of the expression of Cdc25C and Cdc2(Tyr15). Moreover TR-644 inhibited VEGF-induced phosphorylation of VE-cadherin but did not prevent the VEGF-induced phosphorylation of FAK. In chick chorioallantoic membrane in vivo assay, TR-644 (0.1-1.0 pmol/egg) efficiently counteracted the strong angiogenic response induced by FGF. Also CA-4, used as reference compound, caused an antagonistic effect, but in contrast, it induced per se, a remarkable angiogenic response probably due to an inflammatory reaction in the site of treatment. In a mice allogenic tumor model, immunohistochemical staining of tumors with anti-CD31 antibody showed that TR-644 significantly reduced the number of vessel, after 24 h from the administration of a single dose (30 mg/Kg).

The new iodoacetamidobenzofuran derivative TR120 decreases STAT5 expression and induces antitumor effects in imatinib-sensitive and imatinib-resistant BCR-ABL-expressing leukemia cells.

The identification of novel compounds modulating the expression/activity of molecular targets downstream to BCR-ABL could be a new approach in the treatment of chronic myeloid leukemias (CMLs) resistant to imatinib or other BCR-ABL-targeted molecules. Recently, we synthesized a new class of substituted 2-(3,4,5-trimethoxybenzoyl)-2-N,N-dimethylamino-benzo[b]furans, and among these 3-iodoacetylamino-6-methoxybenzofuran-2-yl(3,5-trimethoxyphenyl)methanone (TR120) showed marked cytotoxic activity in BCR-ABL-expressing cells. Interestingly, TR120 was more potent than imatinib in cell growth inhibition and apoptosis induction in both BCR-ABL-expressing K562 and KCL22 cells. Moreover, it showed antitumor activity in imatinib-resistant K562-R and KCL22-R cells at concentrations similar to those active in the respective sensitive cells. Further, TR120 induced a marked decrease in signal transducer and activator of transcription 5 (STAT5) expression in K562 cells. Consistent with this effect, it determined a block of cells in the G0-G1 phase of the cell cycle, a decrease in the level of cyclin D1, and a reduction in Bcl-xL expression; however, it did not cause modifications in the Bcl-2 level. Of interest, TR120 had synergistic effects when used in combination with imatinib in both sensitive and resistant cells. Considering that STAT5 is a BCR-ABL molecular target that plays a key role in the pathogenesis of CML as well as in BCR-ABL-mediated resistance to apoptosis, TR120 could potentially be a useful novel agent in the treatment of imatinib-resistant CML.

Allosteric modulation of A1-adenosine receptor: a review.

Allosteric modulators of adenosine receptors represent an alternative to direct-acting adenosine agonists and nucleoside uptake blockers, preferably those can selectively modulate the response to adenosine in only those organs or localized areas of a given organ in which production of adenosine is increased. Allosteric enhancers at the adenosine A1 receptor have received attention as anti-arrhythmic cardiac agents, and, more recently, as anti-lipolytic agents. In addition, this class of compounds has therapeutic potential as analgesics and neuroprotective agents.

Design, synthesis and biological evaluation of novel 2,4-thiazolidinedione derivatives able to target the human BAG3 protein.

The Bcl-2-associated athanogene 3 (BAG3) protein plays multiple roles in controlling cellular homeostasis, and it has been reported to be deregulated in many cancers, leading tumor cell apoptosis escape. BAG3 protein is then an emerging target for its oncogenic activities in both leukemia and solid cancers, such as medulloblastoma. In this work a series of forty-four compounds were designed and successfully synthesized by the modification and optimization of a previously reported 2,4-thiazolidinedione derivative 28. Using an efficient cloning and transfection in human embryonic kidney HEK-293T cells, BAG3 was collected and purified by chromatographic techniques such as IMAC and SEC, respectively. Subsequently, through Surface Plasmon Resonance (SPR) all the compounds were evaluated for their binding ability to BAG3, highlighting the compound FB49 as the one having the greatest affinity for the protein (Kd = 45 ± 6 µM) also against the reference compound 28. Further analysis carried out by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) spectroscopy further confirmed the highest affinity of FB49 for the protein. In vitro biological investigation showed that compound FB49 is endowed with an antiproliferative activity in the micromolar range in three human tumoral cell lines and more importantly is devoid of toxicity in human peripheral mononuclear cell deriving from healthy donors. Moreover, FB49 was able to block cell cycle in G1 phase and to induce apoptosis as well as autophagy in medulloblastoma HD-MB03 treated cells. In addition, FB49 demonstrated a synergistic effect when combined with a chemotherapy cocktail of Vincristine, Etoposide, Cisplatin, Cyclophosphamide (VECC). In conclusion we have demonstrated that FB49 is a new derivative able to bind human BAG3 with high affinity and could be used as BAG3 modulator in cancers correlated with overexpression of this protein.

Development of a clot-adhesive coating to improve the performance of thrombectomy devices.

BACKGROUND: The first-pass complete recanalization by mechanical thrombectomy (MT) for the treatment of stroke remains limited due to the poor integration of the clot within current devices. Aspiration can help retrieval of the main clot but fails to prevent secondary embolism in the distal arterial territory. The dense meshes of extracellular DNA, recently described in stroke-related clots, might serve as an anchoring platform for MT devices. We aimed to evaluate the potential of a DNA-reacting surface to aid the retention of both the main clot and small fragments within the thrombectomy device to improve the potential of MT procedures. METHODS: Device-suitable alloy samples were coated with 15 different compounds and put in contact with extracellular DNA or with human peripheral whole blood, to compare their binding to DNA versus blood elements in vitro. Clinical-grade MT devices were coated with two selected compounds and evaluated in functional bench tests to study clot retrieval efficacy and quantify distal emboli using an M1 occlusion model. RESULTS: Binding properties of samples coated with all compounds were increased for DNA (≈3-fold) and decreased (≈5-fold) for blood elements, as compared with the bare alloy samples in vitro. Functional testing showed that surface modification with DNA-binding compounds improved clot retrieval and significantly reduced distal emboli during experimental MT of large vessel occlusion in a three-dimensional model. CONCLUSION: Our results suggest that clot retrieval devices coated with DNA-binding compounds can considerably improve the outcome of the MT procedures in stroke patients.

Pyrrolo- and pyrazolo-[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines as adenosine receptor antagonists.

The discovery and development of adenosine receptor antagonists have represented for years an attractive field of research from the perspective of identifying new drugs for the treatment of widespread disorders such as inflammation, asthma and Parkinson's disease. The present work can be considered as an extension of our structure-activity relationship studies on the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) nucleus, extensively investigated by us as a useful template, in particular, for the identification of A(2A) and A(3) adenosine receptor antagonists. In order to explore the role of the nitrogen at the 7-position, we performed a new synthetic strategy for the preparation of pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives which can be considered as 7-deaza analogues of the parent PTPs. We also synthesised a novel series of pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines as junction isomers of the reference compounds. In both cases we obtained some examples of potent antagonists (K(i) in the low nanomolar range) with variable selectivity profiles in relation to the nature of substituents introduced at the C(5)-, N(8)- and/or N(9)-positions. The pyrrolo-triazolo-pyrimidine derivative 9b appeared to be a potent A(3) adenosine receptor antagonist (K(i)=10 nM) with good selectivity over hA(1) (74-fold) and hA(2A) (20-fold) adenosine receptors combined with low activity at the hA(2B) subtype (IC(50)=906 nM). Moreover, some examples of high-affinity A(1)/A(2A) dual antagonists have been identified in both series. This work constitutes a new and important contribution for the comprehension of the interaction between PTPs and adenosine receptors.