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Abstract: In 2018, James Allison and Tasuku Honjo received the Nobel Prize in physiology or medicine to discover tumor therapy by inhibition of negative immune regulation. Immunotherapy stimulates T-cells to fight cancer cells by blocking different immune checkpoint pathways. The interaction between programmed cell death 1 (PD-1) and its ligand PD-L1 (Programmed cell death ligand 1) is one of the main pathways. Of note, interfering with this pathway is already exploited in clinical cancer therapy, demonstrating that it is one of the key factors involved in the immune escape mechanism of cancer. The development of monoclonal antibodies (mAbs) that possess the ability to inhibit the interactions between PD-1/PD-L1 has radically made the difference in cancer immunotherapy. Yet, due to the many drawbacks of this therapy, the research shifted its efforts towards the development of novel small molecules. This may constitute hope and an arduous challenge in fighting cancer. This paper reviews the recent primary literature concerning the development of novel small molecules able to block the interaction between PD-1 and its ligand PD-L1.

BRAF represents one of the most frequently mutated protein kinase genes and BRAFV600E mutation may be found in many types of cancer, including hairy cell leukemia (HCL), anaplastic thyroid cancer (ATC), colorectal cancer and melanoma. Herein, a fluorescent probe, based on the structure of the highly specific BRAFV600E inhibitor Vemurafenib (Vem, 1) and featuring the NIR fluorophore cyanine-5 (Cy5), was straightforwardly synthesized and characterized (Vem-L-Cy5, 3), showing promising spectroscopic properties. Biological validation in BRAFV600E-mutated cancer cells evidenced the ability of 3 to penetrate inside the cells, specifically binding to its elective target BRAFV600E with high affinity, and inhibiting MEK phosphorylation and cell growth with a potency comparable to that of native Vem 1. Taken together, these data highlight Vem-L-Cy5 3 as a useful tool to probe BRAFV600E mutation in cancer cells, and suitable to acquire precious insights for future developments of more informed BRAF inhibitors-centered therapeutic strategies.

Glioblastoma (GBM) is the most aggressive and frequent primary brain tumor, with a poor prognosis and the highest mortality rate. Currently, GBM therapy consists of surgical resection of the tumor, radiotherapy, and adjuvant chemotherapy with temozolomide. Consistently, there are poor treatment options and only modest anticancer efficacy is achieved; therefore, there is still a need for the development of new effective therapies for GBM. Indole is considered one of the most privileged scaffolds in heterocyclic chemistry, so it may serve as an effective probe for the development of new drug candidates against challenging diseases, including GBM. This review analyzes the therapeutic benefit and clinical development of novel indole-based derivatives investigated as promising anti-GBM agents. The existing indole-based compounds which are in the pre-clinical and clinical stages of development against GBM are reported, with particular reference to the most recent advances between 2013 and 2022. The main mechanisms of action underlying their anti-GBM efficacy, such as protein kinase, tubulin and p53 pathway inhibition, are also discussed. The final goal is to pave the way for medicinal chemists in the future design and development of novel effective indole-based anti-GBM agents.

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer’s disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.

A library of variously decorated N-phenyl secondary sulphonamides featuring the bicyclic tetrahydroquinazole scaffold was synthesised and biologically evaluated for their inhibitory activity against human carbonic anhydrase (hCA) I, II, IV, and IX. Of note, several compounds were identified showing submicromolar potency and excellent selectivity for the tumour-related hCA IX isoform. Structure–activity relationship data attained for various substitutions were rationalised by molecular modelling studies in terms of both inhibitory activity and selectivity.

Carbonic anhydrases (CAs) are a family of ubiquitous metal enzymes catalyzing the reversible conversion of CO2 and H2O to HCO3− with the release of a proton. They play an important role in pH regulation and in the balance of body fluids and are involved in several functions such as homeostasis regulation and cellular respiration. For these reasons, they have been studied as targets for the development of agents for treating several pathologies. CA inhibitors have been used in therapy for a long time, especially as diuretics and for the treatment of glaucoma, and are being investigated for application in other pathologies including obesity, cancer, and epilepsy. On the contrary, CAs activators are still poorly studied. They are proposed to act as additional (other than histidine) proton shuttles in the rate-limiting step of the CA catalytic cycle, which is the generation of the active hydroxylated enzyme. Recent studies highlight the involvement of CAs activation in brain processes essential for the transmission of neuronal signals, suggesting CAs activation might represent a potential therapeutic approach for the treatment of Alzheimer’s disease and other conditions characterized by memory impairment and cognitive problems. Actually, some compounds able to activate CAs have been identified and proposed to potentially resolve problems related to neurodegeneration. This review reports on the primary literature regarding the potential of CA activators for treating neurodegeneration-related diseases.

Abstract: In the nervous system, inflammatory responses physiologically occur as defense mechanisms triggered by damaging events. If improperly regulated, neuroinflammation can contribute to the development of chronically activated states of glial cells, with the perpetuation of inflammation and neuronal damage, thus leading to neurological and neurodegenerative disorders. Interestingly, neuroinflammation is associated with the overexpression of the mitochondrial translocator protein (TSPO) in activated glia. Despite the precise role of TSPO in the immunomodulatory mechanisms during active disease states is still unclear, it has emerged as a promising target to promote neuroprotection. Indeed, TSPO ligands have been shown to exert beneficial effects in counteracting neuroinflammation and neuronal damage in several in vitro and in vivo models of neurodegenerative diseases. In particular, the regulation of neurosteroids’ production, cytokine release, metabolism of radical oxidative species, and cellular bioenergetics appear to be the main cellular events that underlie the observed effects. The present review aims to illustrate and summarize recent findings on the potential effect of TSPO ligands against neuroinflammation and related neurodegenerative mechanisms, taking into consideration some pathologies of the nervous system in which inflammatory events are crucial for the onset and progression of the disease and attempting to shed light onto the immunomodulatory effects of TSPO.

A series of novel 3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazine (BIT) derivatives were designed and synthesized. In vitro antiproliferative activity was detected toward two human colorectal adenocarcinoma cell lines (CaCo-2 and HT-29) and one human dermal microvascular endothelial cell line (HMVEC-d). The most active compounds, namely 2-4 and 8, were further investigated to clarify the mechanism behind their biological activity. Through immunofluorescence assay, we identified the target of these molecules to be the microtubule cytoskeleton with subsequent formation of dense microtubule accumulation, particularly at the periphery of the cancer cells, as observed in paclitaxel-treated cells. Overall, these results highlight BIT derivatives as robust and feasible candidates deserving to be further developed in the search for novel potent antiproliferative microtubule-targeting agents.

Abstract: The translocator protein 18kDa (TSPO) is expressed in the outer mitochondrial membrane and is implicated in several functions, including cholesterol transport and stereoidogenesis. Under normal physiological conditions, TSPO is present in very low concentrations in the human brain but is markedly upregulated in response to brain injury and inflammation. This upregulation is strongly associated with activated microglia. Therefore, TSPO is particulary suited for assessing active gliosis associated with brain lesions following injury or disease. For over three decades, TSPO has been studied as a biomarker. Numerous radioligands for positron emission tomography (PET) that target TSPO have been developed for imaging inflammatory progression in the brain. Although [¹¹C]PK11195, the prototypical first-generation PET radioligand, is still widely used for in vivo studies, mainly now as its single more potent R-enantiomer, it has severe limitations, including low sensitivity and poor amenability to quantification. Second-generation radioligands are characterized by higher TSPO specific signals but suffer from other drawbacks, such as sensitivity to the TSPO single nucleotide polymorphism (SNP) rs6971. Therefore, their applications in human studies have a burden of needing to genotype subjects. Consequently, recent efforts are focused on developing improved radioligands that combine the optimal features of the second-generation with the ability to overcome the differences in binding affinities across the population. This review presents essential principles in the design and development of TSPO PET ligands and discusses prominent examples among the main chemotypes.

Inhibition of Carbonic Anhydrases (CAs) has been clinically exploited for many decades for a variety of therapeutic applications. Within a research project aimed at developing novel classes of CA inhibitors (CAIs) with a proper selectivity for certain isoforms, a series of derivatives featuring the 2-substituted-benzimidazole-6-sulfonamide scaffold, conceived as frozen analogs of Schiff bases and secondary amines previously reported in the literature as CAIs, were investigated. Enzyme inhibition assays on physiologically relevant human CA I, II, IX and XII isoforms revealed a number of potent CAIs, showing promising selectivity profiles towards the transmembrane tumor-associated CA IX and XII enzymes. Computational studies were attained to clarify the structural determinants behind the activities and selectivity profiles of the novel inhibitors., Graphical Abstract

Over the years, researchers in drug discovery have taken advantage of the use of privileged structures to design innovative hit/lead molecules. The α-ketoamide motif is found in many natural products, and it has been widely exploited by medicinal chemists to develop compounds tailored to a vast range of biological targets, thus presenting clinical potential for a plethora of pathological conditions. The purpose of this perspective is to provide insights into the versatility of this chemical moiety as a privileged structure in drug discovery. After a brief analysis of its physical-chemical features and synthetic procedures to obtain it, α-ketoamide-based classes of compounds are reported according to the application of this motif as either a nonreactive or reactive moiety. The goal is to highlight those aspects that may be useful to understanding the perspectives of employing the α-ketoamide moiety in the rational design of compounds able to interact with a specific target.

The Translocator Protein 18 kDa (TSPO) has been discovered in 1977 as an alternative binding site for the benzodiazepine diazepam. It is an evolutionary well-conserved and tryptophan-rich 169-amino acids protein with five alpha helical transmembrane domains stretching the outer mitochondrial membrane, with the carboxyl-terminus in the cytosol and a short amino-terminus in the intermembrane space of mitochondrion. At this level, together with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT), it forms the mitochondrial permeability transition pore (MPTP). TSPO expression is ubiquitary, with higher levels in steroid producing tissues; in the central nervous system, it is mainly expressed in glial cells and in neurons. TSPO is implicated in a variety of fundamental cellular processes including steroidogenesis, heme biosynthesis, mitochondrial respiration, mitochondrial membrane potential, cell proliferation and differentiation, cell life/death balance, oxidative stress. Altered TSPO expression has been found in some pathological conditions. In particular, high TSPO expression levels have been documented in cancer, neuroinflammation, and brain injury. Conversely, low TSPO expression levels have been evidenced in anxiety disorders. Therefore, TSPO is not only an interesting drug target for therapeutic purpose (anticonvulsant, anxiolytic, etc.), but also a valid diagnostic marker of related-diseases detectable by fluorescent or radiolabeled ligands. The aim of this report is to present an update of previous reviews dealing with the medicinal chemistry of TSPO and to highlight the most outstanding advances in the development of TSPO ligands as potential therapeutic or diagnostic tools, especially referring to the last five years.

The oncogene KRAS is involved in the pathogenesis of many tumors such as pancreatic, lung and colorectal cancers, thereby representing a relevant target for the treatment of these diseases. The KRAS P1 promoter contains a nuclease hypersensitive, guanine-rich sequence able to fold into a G-quadruplex motif (G4). The stabilization of this G4 structure by small molecules is emerging as a feasible approach to downregulate KRAS expression. Here, a set of novel stabilizing molecules was identified through a virtual screening campaign on the NMR structure of the 22-mer KRAS G4. The most promising hits were then submitted to structure-activity relationships studies which allowed improving their binding affinity and selectivity over double helix DNA and different G4 topologies. The best derivative (19) underwent fluorescence titration experiments and further computational studies to disclose its binding mechanism to KRAS G4. Finally, biological assays showed that this compound is capable to reduce the viability of colorectal cancer cells in which mutated KRAS acts as a driver oncogene. Thus, 19 might represent the prototype of a new class of drugs for the treatment of tumors that, expressing mutated forms of KRAS, are refractory to current therapeutic regimens.

As a part of our efforts to expand chemical diversity in the carbonic anhydrases inhibitors (CAIs), three small series of polyheterocyclic compounds (4-6) featuring the primary benzenesulfonamide moiety linked to bi/tricyclic scaffolds were investigated. Highly effective inhibitors against the target tumor-associated hCA IX (low nanomolar/ subnanomolar potency levels) showing significant functional selectivity profile toward hCA I, II, and IV isozymes were identified. Molecular docking studies clarified the reasons behind the activity and selectivity of the new compounds.

Inhibition of angiogenesis via blocking vascular endothelial growth factor receptor (VEGFR) signaling pathway emerged as an established approach in anticancer therapy. So far, many monoclonal antibodies and ATP-competitive small molecule inhibitors have been clinically validated and approved. In this study, structure-activity relationships (SAR) within the 2-phenylamino-substituted benzothiopyrano[4,3-d]pyrimidine class of kinase inhibitors were further refined by the synthesis and biological evaluation of new compounds 1–21 featuring different substitution patterns on the pendant phenyl moiety, combined with H, OCH3, or Cl at 8-position. Most compounds showed a promising human kinase insert domain receptor (KDR) inhibition profile, with IC50 values in the submicromolar/low micromolar range, and promising antiproliferative activity on human umbilical vein endothelial cells (HUVECs) as well as on a panel of three human tumor cell lines. The angio-kinase selectivity profile was assessed for the most promising compound 16 against a set of six human kinases. Finally, computational studies allowed clarifying at molecular level the interaction pattern established by the compounds with KDR, highlighting key stable cation-π interactions, and thus providing the basis for further designing novel inhibitors.

Molecule interacting with CasL 2 (MICAL2), a cytoskeleton dynamics regulator, are strongly expressed in several human cancer types, especially at the invasive front, in metastasizing cancer cells and in the neo-angiogenic vasculature. Although a plethora of data exist and stress a growing relevance of MICAL2 to human cancer, it is worth noting that only one small-molecule inhibitor, named CCG-1423 (1), is known to date. Herein, with the aim to develop novel MICAL2 inhibitors, starting from CCG-1423 (1), a small library of new compounds was synthetized and biologically evaluated on human dermal microvascular endothelial cells (HMEC-1) and on renal cell adenocarcinoma (786-O) cells. Among the novel compounds, 10 and 7 gave interesting results in terms of reduction in cell proliferation and/or motility, whereas no effects were observed in MICAL2-knocked down cells. Aside from the interesting biological activities, this work provides the first structure–activity relationships (SARs) of CCG-1423 (1), thus providing precious information for the discovery of new MICAL2 inhibitors.

Carbonic Anhydrases (CAs) are pharmaceutically relevant targets for the treatment of several disease conditions. The ubiquitous localization of these enzymes and the high homology shared by the different isoforms represent substantial impediments for the discovery of potential drugs devoid of off-target side effects. As a consequence, substantial efforts are still needed to allow for the full realization of the pharmacological potential of CA modulators. In this contribution, starting from our previous studies, we describe the synthesis of a set of new bicyclic tetrahydroindazoles featuring a secondary sulfonamide. Biological evaluation of the inhibitory activity against the hCA I, II, IV, and IX isoforms allowed drawing a structure-activity relationship profile that was rationalized through theoretical studies. This allowed dissecting the new molecules into the single portions influencing the zinc chelation properties and the selectivity profile thereby offering a new platform for the discovery of new isotype selective CA inhibitors. Carbonic Anhydrases (CAs) are pharmaceutically relevant targets for the treatment of several disease conditions. The ubiquitous localization of these enzymes and the high homology shared by the different isoforms represent substantial impediments for the discovery of potential drugs devoid of off-target side effects. As a consequence, substantial efforts are still needed to allow for the full realization of the pharmacological potential of CA modulators. In this contribution, starting from our previous studies, we describe the synthesis of a set of new bicyclic tetrahydroindazoles featuring a secondary sulfonamide. Biological evaluation of the inhibitory activity against the hCA I, II, IV, and IX isoforms allowed drawing a structure-activity relationship profile that was rationalized through theoretical studies. This allowed dissecting the new molecules into the single portions influencing the zinc chelation properties and the selectivity profile thereby offering a new platform for the discovery of new isotype selective CA inhibitors.

The gasotransmitter hydrogen sulfide (H2S) is an important tuner of the cardiovascular homeostasis, and its deficiency is etiologically associated with a number of cardiovascular diseases. Therefore, the research of original moieties able to release H2S represents a timely issue for drug discovery. In this work, we developed a collection of iminothioethers (ITEs), exhibiting H2S-releasing properties and producing vasorelaxing effects on rat aortic rings. Derivatives 4 and 11, selected as representative of slow and fast rate H2S donors, respectively, produced a complete recovery of the basal coronary flow, reverting the AngII-induced effects in isolated rat hearts. In addition, studies on human aortic smooth muscle cells (HASMCs) demonstrated membrane hyperpolarizing effects, well related to the intracellular generation of H2S. Taken together, the results obtained support ITEs 4 and 11 as new pharmacological tools, as well as effective and innovative H2S donors for cardiovascular drug discovery.

[Image: see text] Protein kinases dysregulation is extremely common in cancer cells, and the development of new agents able to simultaneously target multiple kinase pathways involved in angiogenesis and tumor growth may offer several advantages in the treatment of cancer. Herein we report the discovery of new pyridothiopyranopyrimidine derivatives (2–4) showing high potencies in VEGFR-2 KDR inhibition as well as antiproliferative effect on a panel of human tumor cell lines. Investigation on the selectivity profile of the representative 2-anilino-substituted compounds 3b, 3i, and 3j revealed a multiplicity of kinase targets that should account for the potent antiproliferative effect produced by these pyridothiopyranopyrimidine derivatives.

New benzothiopyranoindoles (5a-l) and pyridothiopyranoindoles (5m-t), featuring different combinations of substituents (H, Cl, OCH3) at R2-R4 positions and protonatable R1-dialkylaminoalkyl chains, were synthesized and biologically assayed on three human tumor cell lines, showing significant antiproliferative activity (GI50 values spanning from 0.31 to 6.93 μM) and pro-apoptotic effect. Linear flow dichroism experiments indicate the ability of both chromophores to form a molecular complex with DNA, following an intercalative mode of binding. All compounds displayed a moderate ability to inhibit the relaxation activity of both topoisomerases I and II, reasonably correlated to their intercalative capacities. Cleavable assay performed with topoisomerase I revealed a significant poisoning effect for compounds 5g, 5h, 5s, and 5t. A theoretical model provided by hydrated docking calculations clarified the role of the R1-R4 substituents on the topoisomerase I poison activity, revealing a crucial role of the R2-OCH3 group.

Three A3 adenosine receptor (AR) antagonists (1–3) selected from 4-acylamino-6-alkyloxy-2- alkylthiopyrimidines previously investigated by us were modified by inserting a methyl group on their ether or thioether side chains. These compounds gave us the chance to evaluate whether their higher lipophilicity, reduced conformational freedom and chirality might improve the potency towards the A3 AR. Racemic mixtures of 1–3 were resolved using chiral HPLC methods and the absolute configurations of the enantiomers were assigned by chiroptical spectroscopy and density functional theory calculations. We measured the affinity for human A1, A2A, A2B and A3 ARs of the racemic mixtures and the pure enantiomers of 1–3 by radioligand competition binding experiments. Cell-based assays of the most potent enantiomers confirmed their A3 AR antagonist profiles. Our research led to the identification of (S)-1 with high potency (0.5 nM) and selectivity as an A3 AR antagonist. Moreover we built a docking-model useful to design new pyrimidine derivatives.

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N1 (1,2) or N10 (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A1 and A2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A1 and A2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs. Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A1 AR and A2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A1 and A2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.

The quinazoline class was exploited to search for a new translocator protein (TSPO) fluorescent probe endowed with improved affinity and residence time (RT). Computational studies on an "in-house" collection of quinazoline derivatives, featuring highly steric demanding groups at the amide nitrogen, suggested that, despite their molecular extension, these ligands are still easily lodged in the TSPO binding site. Binding assays supported this hypothesis, highlighting a low nanomolar/subnanomolar affinity of these ligands, together with a higher RT of the representative compound 11 with respect to our previously reported indole-based fluorescent probe. Thanks to the amenability of the amide nitrogen atom to be substituted with bulky groups, we developed quinazoline-based imaging tools by fluorescently labeling the scaffold at this position. Probes with relevant TSPO affinity, favorable spectroscopic properties, and improved RT were identified. The results from fluorescence microscopy showed that these probes specifically labeled the TSPO at the mitochondrial level in the U343 cell line.

Targeting neuroactive steroid biosynthetic pathway by specific 18 kDa Translocator Protein (TSPO) ligands may represent a therapeutic approach in a variety of neurodegenerative and neuropsychiatric diseases. However, the lack of correlation between the binding affinity and the in vitro steroidogenic efficacy has limited the identification of lead compounds by a traditional affinity-based drug discovery strategy. Our recent researches indicate that the key factor for robust steroidogenic TSPO ligand efficacy is not the binding affinity per se, but rather the time the compound spends into the target, namely its Residence Time (RT). The assessment of this kinetic parameter during the in vitro characterization of compounds appears mandatory in order to obtain structure-efficacy relationships suitable for the future development of novel molecules with promising pharmacological properties.

Structure-activity relationships (SARs) within the 4-phenylquinazoline-2- carboxamide series of translocator protein (TSPO) ligands have been explored further by the synthesis and TSPO binding affinity evaluation of N-benzyl-N-ethyl/methyl derivatives variously decorated at the 6-, 2′-, 4′-, and 4″-positions. Most of the compounds showed high affinity with Ki values in the nanomolar/subnanomolar range. A pharmacophore model was developed and employed to better address SAR data presented by the new TSPO ligands. A subset of the new compounds (5, 8, 12, and 19) were tested for their ability to inhibit the viability of human glioblastoma cell line U343. The observed antiproliferative effect was demonstrated to be specific for compound 19, endowed with the best combination of binding affinity and efficacy. Furthermore, the ability of 19 to induce mitochondrial membrane dissipation (Δψm) substantiated the intracellular pro-apoptotic mechanism activated by the binding of this class of ligands to TSPO. © 2014 American Chemical Society.

In glioblastoma multiforme (GBM), translocator protein (TSPO) and murine double minute (MDM)2/p53 complex represent two druggable targets. We recently reported the first dual binder 3 possessing a higher anticancer effect in GBM cells than the standards PK11195 1 or Nutlin-3 2 singularly applied. Herein, through a structure-activity relationship study, we developed derivatives 4-10 with improved potencies toward both TSPO and MDM2. As a result, compound 9: (i) reactivated the p53 functionality; (ii) inhibited the viability of two human GBM cells; (iii) impaired the proliferation of glioma cancer stem cells (CSCs), more resistant to chemotherapeutics and responsible of GBM recurrence; (iv) sensitized GBM cells and CSCs to the activity of temozolomide; (v) directed its effects preferentially toward tumor cells with respect to healthy ones. Thus, 9 may represent a promising cytotoxic agent, which is worthy of being further developed for a therapeutic approach against GBM, where the downstream p53 signaling is intact and TSPO is overexpressed.

Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the other hand, the carboxylates selectively inhibited hCA IX (KIs ranging between 40.8 and 92.7nM) without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring the design of isoform-selective CAIs with various pharmacologic applications.

As a continuation of our studies on 2-phenylindol-3-ylglyoxylamides as potent and selective translocator protein (TSPO) ligands, two subsets of novel derivatives, featuring hydrophilic group (OH, NH2, COOH) at the para-position of the pendent 2-phenyl ring (8-16) or different 2-aryl moieties, namely, 3-thienyl, p-biphenyl, 2-naphthyl (23-35), were synthesized and biologically evaluated, some of them showing K-i values in the subnanomolar range and the 2-naphthyl group performance being the best. The resulting SARs confirmed the key role played by interactions taking place between ligands and the lipophilic Li pocket of the TSPO binding site. Docking simulations were performed on the most potent compound of the present series (29) exploiting the recently available 3D structures of TSPO bound to its standard ligand (PK11195). Our theoretical model was fully consistent with SARs of the newly investigated as well of the previously reported 2-phenylindol-3-ylglyoxylamide derivatives.

We have disclosed a series of 1-benzyl-3-ketoindole derivatives acting as either positive or negative modulators of the human A(2B) adenosine receptor (A(2B) AR) depending on small differences in their side chain. The new compounds were designed taking into account structural similarities between AR antagonists and ligands of the GABA(A)/benzodiazepine receptor. All compounds resulted totally inactive at A(2A) and A₃ ARs and showed small (8a,b) or none (7a,b, 8c and 9a,b) affinity for A₁ AR. When tested on A(2B) AR-transfected CHO cells, 7a,b and 8a acted as positive modulators, whereas 8b,c and 9a,b acted as negative modulators, enhancing or weakening the NECA-induced increase of cAMP levels, respectively. Compounds 7-9 might be regarded as useful biological and pharmacological tools to explore the therapeutic potential of A(2B) AR modulators, while their 3-ketoindole scaffold might be taken as a reference to design new analogs.

In search for a novel chemotype to develop topoisomerase I (Top1) inhibitors, the pyrazolo[1,5-a]quinazoline nucleus, structurally related to the indenoisoquinoline system precursor of well-known Top1 poisons, was variously decorated (i.e., a substituted phenyl ring at 2- or 3-position, a protonable side chain at 4- or 5-position), affording a number of Top1 inhibitors with cleavage patterns common to CPT and MJ-III-65. SARs data were rationalized by means of an advanced docking protocol. © 2013 American Chemical Society.

A small library of arylthioamides 1-12 was easily synthesized, and their H2S-releasing properties were evaluated both in the absence or in the presence of an organic thiol such as l-cysteine. A number of arylthioamides (1-3 and 7) showed a slow and l-cysteine-dependent H2S-releasing mechanism, similar to that exhibited by the reference slow H2S-releasing agents, such as diallyl disulfide (DADS) and the phosphinodithioate derivative GYY 4137. Compound 1 strongly abolished the noradrenaline-induced vasoconstriction in isolated rat aortic rings and hyperpolarized the membranes of human vascular smooth muscle cells in a concentration-dependent fashion. Finally, a significant reduction of the systolic blood pressure of anesthetized normotensive rats was observed after its oral administration. Altogether these results highlighted the potential of arylthioamides 1-3 and 7 as H2S-donors for basic studies, and for the rational design/development of promising pharmacotherapeutic agents to treat cardiovascular diseases.

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.

In an effort to identify novel ligands possessing high affinity and selectivity for the A2B AR subtype, we further investigated the class of 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones V, previously disclosed by us as selective A1 AR antagonists. Preliminary assays on a number of triazinobenzimidazoles derived from our "in-house" collection revealed that all the derivatives selected showed significant affinity at A 2B AR, no affinity at A3 AR, and various degrees of selectivity toward A1 and A2A ARs. Investigation of a new series featuring modified substituents at the 10-position (4′-chlorophenyl or phenylethyl groups), and a chlorine atom at the 7-position (X) of the triazinobenzimidazole nucleus, yielded highly potent and selective A 2B AR antagonists. The presence of a pendant 3-phenyl ring appears to hamper the interaction with A2A AR, conferring high A 2B/A2A AR selectivity. Derivative 13 (X = Cl, R = C 6H5) is the most potent and selective compound, with an IC50 of 3.10 nM at A2B AR and no affinity at A 1, A2A, and A3 ARs. © 2012 American Chemical Society.