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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.

Small-molecules acting as positive allosteric modulators (PAMs) of the A 2B adenosine receptor (A 2B AR) could potentially represent a novel therapeutic strategy for pathological conditions characterised by altered bone homeostasis, including osteoporosis. We investigated a library of compounds ( 4 - 13 ) exhibiting different degrees of chemical similarity with three indole derivatives ( 1 - 3 ), which have been recently identified by us as PAMs of the A 2B AR able to promote mesenchymal stem cell differentiation and bone formation. Evaluation of mineralisation activity of 4 - 13 in the presence and in the absence of the agonist BAY60-6583 allowed the identification of lead compounds with therapeutic potential as anti-osteoporosis agents. Further biological characterisation of one of the most performing compounds, the benzofurane derivative 9 , confirmed that such a molecule behaves as PAM of the A 2B AR.

Glioblastoma Multiforme (GBM) is a highly invasive primary brain tumour characterized by chemo- and radio-resistance and poor overall survival. GBM can present an aberrant functionality of p53, caused by the overexpression of the murine double minute 2 protein (MDM2) and its analogue MDM4, which may influence the response to conventional therapies. Moreover, tumour resistance/invasiveness has been recently attributed to an overexpression of the chemokine receptor CXCR4, identified as a pivotal mediator of glioma neovascularization. Notably, CXCR4 and MDM2-4 cooperate in promoting tumour invasion and progression. Although CXCR4 actively promotes MDM2 activation leading to p53 inactivation, MDM2-4 knockdown induces the downregulation of CXCR4 gene transcription. Our study aimed to assess if the CXCR4 signal blockade could enhance glioma cells' sensitivity to the inhibition of the p53-MDMs axis. Rationally designed inhibitors of MDM2/4 were combined with the CXCR4 antagonist, AMD3100, in human GBM cells and GBM stem-like cells (neurospheres), which are crucial for tumour recurrence and chemotherapy resistance. The dual MDM2/4 inhibitor RS3594 and the CXCR4 antagonist AMD3100 reduced GBM cell invasiveness and migration in single-agent treatment and mainly in combination. AMD3100 sensitized GBM cells to the antiproliferative activity of RS3594. It is noteworthy that these two compounds present synergic effects on cancer stem components: RS3594 inhibited the growth and formation of neurospheres, AMD3100 induced differentiation of neurospheres while enhancing RS3594 effectiveness preventing their proliferation/clonogenicity. These results confirm that blocking CXCR4/MDM2/4 represents a valuable strategy to reduce GBM proliferation and invasiveness, acting on the stem cell component too., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Several indole derivatives have been disclosed by our research groups that have been collaborating for nearly 25 years. The results of our investigations led to a variety of molecules binding selectively to different pharmacological targets, specifically the type A γ-aminobutyric acid (GABA A ) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A 2B adenosine receptor (A 2B AR) and the Kelch-like ECH-associated protein 1 (Keap1). Herein, we describe how these works were conceived and carried out thanks to the versatility of indole nucleus to be exploited in the design and synthesis of drug-like molecules.

The osteoblast generation from Mesenchymal stem cells (MSCs) is tightly coordinated by transcriptional networks and signalling pathways that control gene expression and protein stability of osteogenic "master transcription factors". Among these pathways, a great attention has been focused on p53 and its physiological negative regulator, the E3 ligase Murine double minute 2 (Mdm2). Nevertheless, the signalling that regulates Mdm2-p53 axis in osteoblasts remain to be elucidated, also considering that Mdm2 possesses numerous p53-independent activities and interacts with additional proteins. Herein, the effects of Mdm2 modulation on MSC differentiation were examined by the use of short- and long-lasting inhibitors of the Mdm2-p53 complex. The long-lasting Mdm2-p53 dissociation was demonstrated to enhance the MSC differentiation into osteoblasts. The increase of Mdm2 levels promoted its association to G protein-coupled receptors kinase (GRK) 2, one of the most relevant kinases involved in the desensitization of G protein-coupled receptors (GPCRs). In turn, the long-lasting Mdm2-p53 dissociation decreased GRK2 levels and favoured the functionality of A 2B Adenosine Receptors (A 2B ARs), a GPCR dictating MSC fate. EB148 facilitated cAMP accumulation, and mediated a sustained activation of extracellular signal-regulated kinases (ERKs) and cAMP response element-binding protein (CREB). Such pro-osteogenic effects were not detectable by using the reversible Mdm2-p53 complex inhibitor, suggesting the time course of Mdm2-p53 dissociation may impact on intracellular proteins involved in cell differentiation fate. These results suggest that the long-lasting Mdm2 binding plays a key role in the mobilization of intracellular proteins that regulate the final biological outcome of MSCs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We designed new 3-arylthio- and 3-aroyl-1H-indole derivatives 3-22 bearing a heterocyclic ring at position 5, 6 or 7 of the indole nucleus. The 6- and 7-heterocyclyl-1H-indoles showed potent inhibition of tubulin polymerization, binding of colchicine to tubulin and growth of MCF-7 cancer cells. Compounds 13 and 19 inhibited a panel of cancer cells and the NCI/ADR-RES multidrug resistant cell line at low nanomolar concentrations. Compound 13 at 50 nM induced 77% G2/M in HeLa cells, and at 20 nM caused 50% stable arrest of mitosis. As an inhibitor of HepG2 cells (IC 50  = 20 nM), 13 was 4-fold superior to 19. Compound 13 was a potent inhibitor of the human U87MG glioblastoma cells at nanomolar concentrations, being nearly one order of magnitude superior to previously reported arylthioindoles. The present results highlight 13 as a robust scaffold for the design of new anticancer agents., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The phosphoinositide-dependent kinase-1 (PDK1) is one of the main components of the PI3K/Akt pathway. Also named the "master kinase" of the AGC family, PDK1 plays a critical role in tumorigenesis, by enhancing cell proliferation and inhibiting apoptosis, as well as in cell invasion and metastasis formation. Although there have been done huge efforts in discovering specific compounds targeting PDK1, nowadays no PDK1 inhibitor has yet entered the clinic. With the aim to pick out novel and potent PDK1 inhibitors, herein we report the design and synthesis of a new class of molecules obtained by merging the 2-oxo-indole nucleus with the 2-oxo-pyridonyl fragment, two moieties with high affinity for the PDK1 hinge region and its DFG-out binding site, respectively. To this purpose, a small series of compounds were synthesised and a tandem application of docking and Molecular Dynamic (MD) was employed to get insight into their mode of binding. The OXID-pyridonyl hybrid 8, possessing the lower IC50 (IC50 = 112 nM), was also tested against recombinant kinases involved in the PI3K/PDK1/Akt pathway and was subjected to vitro studies to evaluate the cytotoxicity and the inhibition of tumour cell migration. All together the results let us to consider 8, as a lead compound of a new generation of PDK1 inhibitors and encourage us to further studies in this direction., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The steroidogenic 18 kDa translocator protein (TSPO) is an emerging, attractive therapeutic tool for several pathological conditions of the nervous system. Here, 13 high affinity TSPO ligands belonging to our previously described N,N-dialkyl-2-phenylindol-3-ylglyoxylamide (PIGA) class were evaluated for their potential ability to affect the cellular Oxidative Metabolism Activity/Proliferation index, which is used as a measure of astrocyte well-being. The most active PIGA ligands were also assessed for steroidogenic activity in terms of pregnenolone production, and the values were related to the metabolic index in rat and human models. The results showed a positive correlation between the increase in the Oxidative Metabolism Activity/Proliferation index and the pharmacologically induced stimulation of steroidogenesis. The specific involvement of steroid molecules in mediating the metabolic effects of the PIGA ligands was demonstrated using aminoglutethimide, a specific inhibitor of the first step of steroid biosynthesis. The most promising steroidogenic PIGA ligands were the 2-naphthyl derivatives that showed a long residence time to the target, in agreement with our previous data. In conclusion, TSPO ligand-induced neurosteroidogenesis was involved in astrocyte well-being.

The development of multi-target drugs and irreversible modulators of deregulated signalling proteins is the major challenge for improving glioblastoma multiforme (GBM) treatment. Reversible single-target drugs are not sufficient to sustain a therapeutic effect over time and may favour the activation of alternative signalling pathways and the onset of resistance phenomena. Thus, a multi-target compound that has a long-lasting mechanism of action might have a greater and longer life span of anti-proliferative activity. Recently, a dual-target indol-3ylglyoxyldipeptide derivative, designed to bind to the Translocator Protein (TSPO) and reactivate p53 function via dissociation from its physiological inhibitor, murine double minute 2 (MDM2), has been developed as a potent GBM pro-apoptotic agent. In this study, this derivative was chemically modified to irreversibly bind MDM2 and TSPO. The new compound elicited a TSPO-mediated mitochondrial membrane dissipation and restored p53 activity, triggering a long-lasting apoptosis of GBM cells. These effects were sustained over time, involved a stable activation of extracellular signal regulated kinases and were specifically observed in cancer cells, in which these protein kinases are deregulated. Dual-targeting and irreversible binding properties combined in the same molecule may represent a useful strategy to overcome the time-limited effects elicited by classical chemotherapies.

The pharmacological activation of the cholesterol-binding Translocator Protein (TSPO) leads to an increase of endogenous steroids and neurosteroids determining benefic pleiotropic effects in several pathological conditions, including anxiety disorders. The relatively poor relationship between TSPO ligand binding affinities and steroidogenic efficacies prompted us to investigate the time (Residence Time, RT) that a number of compounds with phenylindolylglyoxylamide structure (PIGAs) spends in contact with the target. Here, given the poor availability of TSPO ligand kinetic parameters, a kinetic radioligand binding assay was set up and validated for RT determination using a theoretical mathematical model successfully applied to other ligand-target systems. TSPO ligand RT was quantified and the obtained results showed a positive correlation between the period for which a drug interacts with TSPO and the compound ability to stimulate steroidogenesis. Specifically, the TSPO ligand RT significantly fitted both with steroidogenic efficacy (Emax) and with area under the dose-response curve, a parameter combining drug potency and efficacy. A positive relation between RT and anxiolytic activity of three compounds was evidenced. In conclusion, RT could be a relevant parameter to predict the steroidogenic efficacy and the in vivo anxiolytic action of new TSPO ligands.

Aggressive behavior and diffuse infiltrative growth are the main features of Glioblastoma multiforme (GBM), together with the high degree of resistance and recurrence. Evidence indicate that GBM-derived stem cells (GSCs), endowed with unlimited proliferative potential, play a critical role in tumor development and maintenance. Among the many signaling pathways involved in maintaining GSC stemness, tumorigenic potential, and anti-apoptotic properties, the PDK1/Akt pathway is a challenging target to develop new potential agents able to affect GBM resistance to chemotherapy. In an effort to find new PDK1/Akt inhibitors, we rationally designed and synthesized a small family of 2-oxindole derivatives. Among them, compound 3 inhibited PDK1 kinase and downstream effectors such as CHK1, GS3Kα and GS3Kβ, which contribute to GCS survival. Compound 3 appeared to be a good tool for studying the role of the PDK1/Akt pathway in GCS self-renewal and tumorigenicity, and might represent the starting point for the development of more potent and focused multi-target therapies for GBM., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer.

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 Ki 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 L1 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.

Ligands addressed to the mitochondrial Translocator Protein (TSPO) have been suggested as cell death/life and steroidogenesis modulators. Thus, TSPO ligands have been proposed as drug candidates in several diseases; nevertheless, a correlation between their binding affinity and in vitro efficacy has not been demonstrated yet, questioning the specificity of the observed effects. Since drug-target residence time is an emerging parameter able to influence drug pharmacological features, herein, the interaction between TSPO and irDE-MPIGA, a covalent TSPO ligand, was investigated in order to explore TSPO control on death/life processes in a standardized glioblastoma cell setting. After 90 min irDE-MPIGA cell treatment, 25 nM ligand concentration saturated irreversibly all TSPO binding sites; after 24 h, TSPO de-novo synthesis occurred and about 40 % TSPO binding sites resulted covalently bound to irDE-MPIGA. During cell culture treatments, several dynamic events were observed: (a) early apoptotic markers appeared, such as mitochondrial membrane potential collapse (at 3 h) and externalization of phosphatidylserine (at 6 h); (b) cell viability was reduced (at 6 h), without cell cycle arrest. After digitonin-permeabilized cell suspension treatment, a modulation of mitochondrial permeability transition pore was evidenced. Similar effects were elicited by the reversible TSPO ligand PIGA only when applied at micromolar dose. Interestingly, after 6 h, irDE-MPIGA cell exposure restored cell survival parameters. These results highlighted the ligand-target residence time and the cellular setting are crucial parameters that should be taken into account to understand the drug binding affinity and efficacy correlation and, above all, to translate efficiently cellular drug responses from bench to bedside.

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., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC₅₀ = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes.

A number of neurosteroids have been demonstrated to exert anxiolytic properties by means of a positive modulation of inhibitory GABAergic neurotransmission. The observation that neurosteroid synthesis can be pharmacologically regulated by ligands to the mitochondrial translocator protein (TSPO) has prompted the search for new, more selective TSPO ligands able to stimulate steroidogenesis with great efficacy. In the present study, the potential anxiolytic activity of a selective TSPO ligand, N,N-di-n-propyl-2-(4-methylphenyl)indol-3-ylglyoxylamide (MPIGA), was tested by means of the elevated plus maze paradigm. Moreover, the in vitro effects on synaptoneurosomal GABA(A) receptor (GABA(A)R) activity exerted by the conditioned salt medium from MPIGA-treated ADF human glial cells were investigated. MPIGA (30mg/kg) was found to affect rats' performance in the elevated plus maze test significantly, leading to an increase in both entries and time spent in the open arms. This same dose of MPIGA had no effect on rats' spontaneous exploratory activity. The conditioned salt medium from MPIGA-treated ADF cells potentiated the (36)Cl(-) uptake into cerebral cortical synaptoneurosomes. The exposure of ADF cells to MPIGA stimulated the production of pregnelonone derivatives including allopregnanolone, one of the major positive GABA(A)R allosteric modulator. In conclusion, the TSPO ligand MPIGA is a promising anxiolytic drug. The mechanism of action by which MPIGA exerts its anxiolytic activity was identified in the stimulation of endogenous neurosteroid production, which in turn determined a positive modulation of GABA(A)R activity, thus opening the way to the potential use of this TSPO ligand in anxiety and depressive disorders., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A novel series of N(1)-methyl-(2-phenylindol-3-yl)glyoxylamides, 19-31, designed in accordance with our previously reported pharmacophore/topological model, showed high affinity for the 18 kDa translocator protein (TSPO) and paved the way for developing a new radiolabeled probe. Thus ligand 31, N,N-di-n-propyl-(N(1)-methyl-2-(4'-nitrophenyl)indol-3-yl)glyoxylamide, featuring the best combination of affinity and lipophilicity, was labeled with carbon-11 for evaluation with positron emission tomography (PET) in monkey. After intravenous injection, [(11)C]31 entered brain to give a high proportion of TSPO-specific binding. These findings augur well for the future application of [(11)C]31 in humans. Consequently, the binding of 31 to human TSPO was tested on samples of brain membranes from deceased subjects who through ethically approved in vitro study had previously been established to be high-affinity binders (HABs), mixed-affinity binders (MABs), or low-affinity binders (LABs) for the known TSPO ligand, PBR28 (2). 31 showed high affinity for HABs, MABs, and LABs. In conclusion, [(11)C]31 represents a promising new chemotype for developing novel TSPO radioligands as biomarkers of neuroinflammation.

Novel N-substituted indol-3-ylglyoxylamides (10-37) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR). In an effort to achieve affinity-based selectivity among BzR subtypes, these compounds were designed to probe the LDi and L2 lipophilic regions. Taking the alpha1-selective benzylindolylglyoxylamides Ia and Ib as leads, we varied the substituent on the benzylamide phenyl ring (compounds 10-23) or replaced the benzyl moiety with alkyl groups (compounds 24-37). The above structural changes gave no shift of selectivity from the alpha1 toward the alpha2 or alpha5 subtypes, thus confirming that a ligand which occupies the LDi region probably exhibits alpha1 selectivity, despite its interactions with other lipophilic areas in the receptor binding cleft. Compound 11 (N-(p-methylbenzyl)-5-nitroindol-3-ylglyoxylamide), which selectively binds with a full agonist efficacy at the alpha1 receptor subtype and displays sedative action, can be regarded as an interesting potential zolpidem-like sedative-hypnotic agent.

Fluorescent ligands for the peripheral-type benzodiazepine receptor (PBR) featuring the 7-nitrobenz-2-oxa-1,3-diazol-4-yl moiety were synthesized, based on N,N-dialkyl-2-phenylindol-3-ylglyoxylamides, a potent, selective class of PBR ligands previously described by us. All the new ligands are moderately to highly potent at the PBR, with a complete selectivity over the central benzodiazepine receptor. Results from fluorescence microscopy showed that these probes specifically labeled the PBR at the mitochondrial level in C6 glioma cells.

N-(heteroarylmethyl)indol-3-ylglyoxylamides (1-26) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR) to probe the hydrogen bonding properties of the so-called S(1) site of the BzR by means of suitable heterocyclic side chains. SARs were developed in light of our hypothesis of binding modes A and B. Pyrrole and furan derivatives adopting mode A (2, 8, 10, 20, 22) turned out to be more potent (K(i) values < 35 nM) than their analogues lacking hydrogen bonding heterocyclic side chains. These data suggest that the most potent indoles interact with a hydrogen bond acceptor/donor (HBA/D) group located within the S(1) site of the BzR. Compounds 1, 2, 8, 19, 20, and 22, tested at recombinant rat alpha(1)beta(2)gamma(2), alpha(2)beta(2)gamma(2), and alpha(5)beta(3)gamma(2) BzRs, elicited selectivity for the alpha(1)beta(2)gamma(2) isoform. On the basis of published mutagenesis studies and the present SARs, we speculate that the S(1) HBA/D group might be identified as the hydroxyl of alpha(1)-Tyr209 or of other neighboring amino acids.

Mitochondrial benzodiazepine-receptor (mBzR) ligands constitute a heterogeneous class of compounds that show a pleiotropic spectrum of effects within the cells, including the modulation of apoptosis. In this paper, a novel synthetic 2-phenylindol-3-ylglyoxylamide derivative, N,N-di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide (PIGA), which shows high affinity and selectivity for the mBzR, is demonstrated to induce apoptosis in rat C6 glioma cells. PIGA was able to dissipate mitochondrial transmembrane potential (DeltaPsim) and to cause a significant cytosolic accumulation of cytochrome c. Moreover, typical features of apoptotic cell death, such as caspase-3 activation and DNA fragmentation, were also detected in PIGA-treated cells. Our data expand the knowledge on mBzR ligand-mediated apoptosis and suggest PIGA as a novel proapoptotic compound with therapeutic potential against glial tumours, in which apoptosis resistance has been reported to be involved in carcinogenesis.

We report the synthesis and the affinity data at both the peripheral (PBR) and the central benzodiazepine receptors of a series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamide derivatives III, designed as conformationally constrained analogues of 2-phenylindole-3-acetamides II such as FGIN-1-27. Most of the new compounds showed a high specificity and affinity for PBR, with K(i) in the nanomolar to subnanomolar range. The most potent ligands (4-7, 9, 13-27) stimulated steroid biosynthesis in rat C6 glioma cells with a potency similar to or higher than that of classical ligands. The SARs of this new class of compounds are discussed.

A series of 3-benzylamino-and 3-arylalkylaminocarbonyl [1, 2, 4]triazino [4, 3-a]benzimidazoles 1-12 were synthesized and biologically assayed as geometrically constrained analogues of N-benzylindolylglyoxylylamides II, which are high affinity ligands at the benzodiazepine receptor (BzR). The intermediate 3-ethoxycarbonyl [1, 2, 4]triazino [4, 3-a]benzimidazol-4(10H)-one 14 and its N(10)-methyl analogue 15 closely related to 3-alkoxycarbonyl-beta-carbolines I were also investigated. The title compounds exhibited a lower affinity compared with the corresponding indolylglyoxylylamide derivatives II. Attempts were made to rationalize these results taking into account the possible tautomeric equilibria involving these ligands.

A series of N-(arylalkyl)indol-3-ylglyoxylylamides (4-8) was synthesized as ligands of the benzodiazepine receptor (BzR) and tested for their ability to displace [(3)H]flumazenil from bovine brain membranes. The new compounds, bearing a branched (4) or a geometrically constrained benzyl/phenylethyl amide side chain (5-8), represent the continuation of our research on N-benzylindol-3-ylglyoxylylamides 1 (Da Settimo et al., 1996), N'-phenylindol-3-ylglyoxylohydrazides 2 (Da Settimo et al., 1998), and N-(indol-3-ylglyoxylyl)alanine derivatives 3 (Primofiore et al., 1989). A few indoles belonging to the previously investigated benzylamides 1 and phenylhydrazides 2 were synthesized and tested to enrich the SARs in these two series. The affinities and the GABA ratios of selected compounds for clonal mammalian alpha(1)beta(2)gamma(2), alpha(3)beta(2)gamma(2), and alpha(5)beta(3)gamma(2) BzR subtypes were also determined. It was hypothesized that the reduced flexibility of indoles 4-8 would both facilitate the mapping of the BzR binding cleft and increase the chances of conferring selectivity for the considered receptor subtypes. In the series of indoles 4, the introduction of a methyl group on the benzylic carbon with the R configuration improved affinity of the 5-substituted (5-Cl and 5-NO(2)) derivatives, whereas it was detrimental for their 5-unsubtituted (5-H) counterparts. All S enantiomers were less potent than the R ones. Replacement of the methyl with hydrophilic substituents on the benzylic carbon lowered affinity. The isoindolinylamide side chain was tolerated if the 5-position was unsubstituted (K(i) of 5a = 123 nM), otherwise affinity was abolished (5b, c). All the 2-indanylamides 6 and (S)-1-indanylamides 8 were devoid of any appreciable affinity. The 5-Cl and 5-NO(2) (R)-1-indanylamides 7b (K(i) 80 nM) and 7c (K(i) 28 nM) were the most potent among the indoles 5-8 geometrically constrained about the side chain. The 5-H (R)-1-indanylamide 7a displayed a lower affinity (K(i) 675 nM). The SARs developed from the new compounds, together with those collected from our previous studies, confirmed the hypothesis of different binding modes for 5-substituted and 5-unsubstituted indoles, suggesting that the shape of the lipophilic pocket L(1) (notation in accordance with Cook's BzR topological model) is asymmetric and highlighted the stereoelectronic and conformational properties of the amide side chain required for high potency. Several of the new indoles showed selectivity for the alpha(1)beta(2)gamma(2) subtype compared with the alpha(3)beta(2)gamma(2) and alpha(5)beta(3)gamma(2) subtypes (e.g.: 4t and 7c bind to these three BzR isoforms with K(i) values of 14 nM, 283 nM, 239 nM, and 9 nM, 1960 nM, 95 nM, respectively). The GABA ratios close to unity exhibited by all the tested compounds on each BzR subtype were predictive of an efficacy profile typical of antagonists.

A series of N'-phenylindol-3-ylglyoxylohydrazides, isosters of the N-benzylindol-3-ylglyoxylamide derivatives previously described by us, were synthesized and tested for their ability to displace [3H]Ro 15-1788 from bovine brain membranes. These compounds were designed with the aim of obtaining products which could exert an in vivo activity, thanks to a higher hydrosolubility and consequently a better bioavailability. Affinity was restricted to the derivatives unsubstituted in the 5 position of the indole nucleus (1, 6, 9, 12, 15, 18, 23, and 26), with Ki values ranging from 510 to 11 nM. The most active compounds (6, 9, 23, and 29) proved to be effective in antagonizing pentylenetetrazole-induced seizures. Molecular modeling studies were performed to rationalize the lack of affinity of hydrazides with a chloro or a nitro group in the 5 position of the indole nucleus. It was hypothesized that the conformational preference of the hydrazide side chain, characterized by a gauche disposition of lone pairs and substituents about the N-N bond, prevents all hydrazides from binding to the receptor similarly to other classes of indole analogues previously investigated. The potency of 5-H hydrazides was attributed to a binding mode which is not feasible for 5-Cl and 5-NO2 counterparts. This theoretical model of ligand-receptor interaction permitted a more stringent interpretation of structure-affinity relationships of hydrazides and of recently described benzylamide derivatives (Da Settimo et al. J. Med. Chem. 1996, 39, 5083-5091).

A number of benzyl and phenylethyl esters of indol-3-ylglyoxylic acid were synthesized and tested for their ability to displace [3H]Ro 15-1788 binding from bovine brain membranes. In these new compounds the oxygen atom of the ester function replaced the amide NH group of a class of previously described indolylglyoxylylamides, since it is reported in literature that in the beta-carboline series an ester function is more favourable to the activity than an amide group. However, none of the compounds showed an affinity at the Benzodiazepine receptor higher than that of the corresponding amides, demonstrating that the presence of the amide NH group is favourable to the interaction of ligands with the receptor site.

A number of N-(indol-3-ylglyoxylyl)benzylamine derivatives were synthesized and tested for [3H]flunitrazepam displacing activity in bovine brain membranes. Some of these derivatives (9, 12, 14, 15, 17, 27, 34, 35, 38, 41, and 45) exhibited high affinity for the benzodiazepine receptor (BzR) with Ki values ranging from 67 to 11 nM. The GABA ratio and [35S]-tert-butylbicyclophosphorothionate binding data, determined for the most active compounds, showed that they elicit an efficacy profile at the BzR which depends on the kind of substituent present on the phenyl ring of the benzylamine moiety. Moreover, lengthening (propylamine derivatives 1-3) and shortening (aniline derivatives 46-54) of the distance between the phenyl ring and the amide group of the side chain gave compounds with a drastically lower binding potency. The biological results are discussed in the light of a recently proposed pharmacophore model and compared, by molecular modeling studies, with those obtained from effective BzR ligands.

A number of furoylindoles were synthesized with the aim of obtaining structurally more restrained analogues of the previously described indoleglyoxylylamides, which are high affinity ligands at the benzodiazepine receptor. In these new compounds, the oxygen atom of the oxalyl CO(2) is inserted into the rigid furan ring. However, unlike the glyoxylylamides, they proved to be incapable of interacting with the benzodiazepine receptor. To rationalize these results, molecular electrostatic potentials were calculated; these indicated a positive electrostatic potential region for the furan oxygen, which thus prevents the formation of a hydrogen bond necessary for interaction with the receptor. Nevertheless, these findings confirmed that the CO(2) of the indoleglyoxylylamide derivatives represents one of the principal points of interaction with the receptor site for these kinds of ligands, as previously hypothesized by us.

Several N-(indol-3-ylglyoxylyl)alanine and glycine ester derivatives were prepared to evaluate the influence of the ester group size both on affinity and on activity at the benzodiazepine receptor. Moreover, indol-3-ylglyoxylic acid esters, closely related to 3-alkoxy-beta-carbolines, were synthesized and tested in vitro on the benzodiazepine receptor.

Recently, several derivatives, in which tryptamine, tyramine, and dopamine moieties are linked to the indole nucleus by an oxalyl bridge, were tested for their affinity and efficacy at the benzodiazepine receptor (BzR). To better define the structure-activity relationships (SAR) several phenylethylamine derivatives were also synthesized and tested for their affinity at the BzR. Compounds bearing a protic group on the aromatic system of the side chain show a pharmacological profile of inverse agonist, while the products lacking this group behave as partial agonist. We now report the affinity data at the BzR of new compounds in which the distance between the phenyl ring and the amide group of the side chain has been changed. The benzylamine derivatives showed a good affinity at the BzR, generally higher than that of the phenylethylamine derivatives. In this series the pharmacological profile showed to be opposite to that of the corresponding phenylethylamine derivatives, since the compounds substituted with protic groups on the phenyl ring behaved as partial agonists. Moreover, a probable interaction with the receptor site is hypothesized.

Several N-(indol-3-ylglyoxylyl)dipeptide derivatives 1-12 were synthesized and tested for their affinity at the benzodiazepine receptor in bovine cortical membranes. They proved to bind with low or no affinity at the receptor site. It was hypothesized that this result was not due to the steric hindrance of the dipeptide side chain, but to the establishment of intramolecular hydrogen bonds involving the indole N-H and/or the glyoxylyl C = O(2). Conformational analysis indicated that coiled conformations, with intramolecular hydrogen bonds, were energetically more favoured than the staggered, completely unfolded ones. Therefore, the low or no affinity of these compounds should be attributed to the unavailability of the N-H and/or C = O(2) groups for the binding, again confirming that both these groups are necessary for interaction with the receptor.

Several derivatives, in which tryptamine, tyramine, and dopamine moieties are linked to the indole nucleus by an oxalyl bridge, were tested for their ability to displace the specific binding of [3H]flunitrazepam from bovine brain membranes. GABA ratio and in vivo tests for the most potent compounds showed they behave as inverse agonists at the benzodiazepine receptor (BzR). To better define the structure-activity relationship (SAR) of this kind of ligand, several phenylethylamine derivatives were synthesized to evaluate their affinity to BzR. Some of these derivatives (17, 21, 24, 26, and 30) were found to exhibit high affinity (Ki = 0.51-0.085 microM) for BzR and possessed a partial agonist activity, although their chemical structure is closely related to tryptamine 2-6, tyramine 7-11, and dopamine 12-16 derivatives. A different interaction of these ligands to the receptor site is hypothesized. Moreover, all the prepared 1-methyl derivatives exhibited very low binding affinity to BzR.

Several N-(2-methyl or 1,2-dimethylindol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for their affinity for the benzodiazepine receptor in bovine cortical membranes. The 2-methyl derivatives showed a lower affinity than the unmethylated analogues, and the 1,2-dimethyl derivatives practically lacked any affinity for the benzodiazepine receptor. The importance of the indole N-H group is therefore evidenced for an optimal interaction of these ligands with receptor site.

Some 3-amino- and 3-hydroxy-8-halosubstituted 6H-indolo[2,3-b] [1,8]naphthyridines were synthesized and tested for their affinity for the benzodiazepine receptor in bovine cortical membranes. All prepared compounds were more active than the corresponding 8-unsubstituted derivatives. Moreover, among these compounds the 8-chloroindolonaphthyridines were clearly the most potent.

Several optically active N-(indol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for [3H]flunitrazepam displacing activity in bovine brain membranes. IC50 values were measured and revealed that the D form of the amino acid moiety of the compounds was more potent than both the L form and racemic form, suggesting a key role of the amino acid stereochemistry on the affinity to the benzodiazepine receptors. GABA ratio and proconvulsant/convulsant data reported for the most active compounds reveal they behave as inverse agonists at the benzodiazepine receptor.

Several N-(indol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for their affinity for the benzodiazepine receptor in bovine cortical membranes. From these compounds, the N-[(5-chloro-, 5-bromo-, or 5-nitroindol-3-yl)glyoxylyl]glycine or -alanine esters were clearly the most potent, while the 5-methoxy analogues were considerably less active. Moreover, esters were more active than the corresponding acids. It is concluded that the affinity of these derivatives for the benzodiazepine receptor is profoundly dependent on amino acid molecular size, as well as the hydrophobic and electronic properties of the compounds.