Hydrogen sulphide: biopharmacological roles in the cardiovascular system and pharmaceutical perspectives.

Hydrogen sulphide (H(2)S) is now viewed as an important endogenous gasotransmitter, which exhibits many beneficial effects on the cardiovascular system. H(2)S is biosynthesized in mammalian tissues by both non-enzymatic processes and several enzymatic pathways ensured by cystathionine-ß-synthase and cystathionine-γ-lyase. H(2)S is endowed with the antioxidant properties of inorganic and organic sulphites, being a scavenger of reactive oxygen species. Furthermore, H(2)S triggers other important effects and the activation of ATP-sensitive potassium channels (KATP) accounts for its vasorelaxing and cardioprotective effects. H(2)S also inhibits smooth muscle proliferation and platelet aggregation. Conversely, the impairment of H(2)S contributes to the pathogenesis of hypertension and is involved in cardiovascular complications associated with diabetes mellitus. There is also evidence of a link between H(2)S and endothelial nitric oxide (NO). Recent observations indicate a possible pathogenic link between deficiencies of H(2)S activity and the progress of endothelial dysfunction. These biological aspects of endogenous H(2)S led to consider this mediator as "the new NO" and to evaluate new attractive opportunities to develop innovative classes of drugs. In this review, the main roles played by H(2)S in the cardiovascular system and the first examples of H(2)S-donor drugs are discussed. Some hybrid drugs are also addressed in this review. In such compounds opportune H(2)S-releasing moieties are conjugated to well-known drugs to improve their pharmacodynamic profile or to reduce the potential for adverse effects.

Geometrically constrained derivatives of indolylglyoxylamides as ligands binding the GABAA/BzR complex.

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

Receptor tyrosine kinase kit and gastrointestinal stromal tumours: an overview.

Kit is a growth factor receptor of the type III tyrosine kinase family, whose gain-of-function mutations have been identified as driving causes of different kinds of tumours. It thus represents a viable drug target, and the development of Kit inhibitors has been shown to be a promising therapeutic concept. This review will focus on structural and signalling properties of both wild-type and mutant Kit, as well as its role in the development of human cancers. Special attention will be dedicated to gastrointestinal stromal tumours, GISTs. Progress in research on the aetiopathogenesis of GISTs and their therapeutic approaches will be fully discussed, focusing on the latest tendencies for the treatment of these kinds of tumours.

Recent advances in the development of dual topoisomerase I and II inhibitors as anticancer drugs.

DNA topoisomerases (topos) are essential enzymes that regulate the topological state of DNA during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Topoisomerase I (Topo I) is a ubiquitous nuclear enzyme which catalyzes the relaxation of superhelical DNA generating a transient single strand nick in the duplex, through cycles of cleavage and religation. Topoisomerase II (Topo II) mediates the ATP-dependent induction of coordinated nicks in both strands of the DNA duplex, followed by crossing of another double strand DNA through the transiently broken duplex. Although the biological functions of Topoisomerases are important for ensuing genomic integrity, the ability to interfere with enzymes or generate enzyme-mediated damage is an effective strategy for cancer therapy and, in this connection, DNA topos (I and II) proved to be the excellent targets of clinically significant classes of anticancer drugs. Actually, specific Topo I and Topo II inhibitors reversibly trap the enzyme-DNA complexes, thus converting Topos into physiological poisons, able to produce permanent DNA damage, which triggers cell death. Given that both enzymes are good targets, it would be desirable to jointly inhibit them, but use-limiting toxicity of sequential or simultaneous combinations of topo I and II poisons include severe to life-threatening neutropenia and anemia. Furthermore, the emergence of resistance phenomena to topo I inhibitors is often accompanied by a concomitant rise in the level of topo II expression and viceversa, leading to the failure of clinical therapies. In this regard, a single compound able to inhibit both Topo I and II may present the advantage of improving antitopoisomerase activity, with reduced toxic side effects, with respect to the combination of two inhibitors. Due to the high interest in such compounds, this review represents an update of previous works dealing with the development of dual Topo I and II inhibitors as novel anti-cancer agents. The newly collected derivatives have been described focusing attention on their chemical structures and their biological profiles.

Allosteric modulators for adenosine receptors: an alternative to the orthosteric ligands.

Adenosine is a ubiquitous homeostatic substance which exerts its action by triggering four different cell membrane G protein-coupled receptors, classified as A(1), A(2A), A(2B), and A(3). Being widely distributed and deeply involved in several physiological functions, as well as pathological disorders, these receptors represent an excellent drug target and the development of specific ligands has been tested as a promising therapeutic concept. Among the obtainable ligands, allosteric modulators offer higher advantages with respect to classical orthosteric compounds, as they possible to achieve greater selectivity and better modulatory control at disease mediating receptors. Actually, synergizing with adenosine bound to the primary binding site, these compounds may modify receptor functions through interaction with an additional binding site. As a consequence, their actions depend directly on the release of the endogenous agonist. A number of compound have been developed as effective allosteric modulators. Most of them target adenosine A(1) and A(3) receptor subtypes as, to date, little or no research attempt have been made to improve the field of A(2A) and A(2B) ligands. This review updates literature on the allosteric modulators that has appeared in the last few years, focusing its attention on medicinal chemistry, in terms of chemical structure and structure-activity relationships. This will provide new perspectives on existing data and an exciting starting point for the development of novel and more effective modulators.

Inhibition of metalloproteinases derived from tumours: new insights in the treatment of human glioblastoma.

Glioblastoma multiforme is the most commonly diagnosed malignant primary brain tumour in adults. Invasive behaviour is the pathological hallmark of malignant gliomas; consequently, its inhibition has been suggested as a therapeutic strategy. Tumour cell-derived gelatinases (matrix metalloproteinase-2, matrix metalloproteinase-9) can be considered prime factors in glioma invasiveness: their expression correlates with the progression and the degree of malignancy. Thus, broad spectrum matrix metalloproteinase inhibitors (MMP inhibitors) have been included in clinical trials. In the present study, the invasiveness, viability and progression of the human glioma cell line U87MG were investigated following treatment with N-O-isopropyl sulfonamido-based hydroxamates (compounds 1 and 2) as MMP-2 inhibitors used at nanomolar concentration. A standard broad spectrum MMP-inhibitor belonging to the classical tertiary sulfonamido-based hydroxamates family (CGS_27023A) was used too. The compounds 1 and 2 resulted in potent inhibition of cell invasiveness (P<0.0001) without affecting viability. In some clinical trials, the combined therapy of temozolomide (an alkylating agent used in glioma treatment) plus marimastat (a broad spectrum MMP inhibitor) has provided evidence of the importance of MMPs to tumor progression and invasiveness. On this basis, the effect on U87MG cells of a combined treatment with temozolomide, plus each of the two MMP inhibitors at nanomolar concentration, was investigated. The obtained data demonstrated the inhibition of cell invasiveness and viability after treatment. These results can help in developing clinical combined therapy using MMP inhibitors that, at low doses, increase the anticancer efficacy of chemotherapeutic drugs, probably without causing the side effects typical of broad-spectrum MMP inhibitors.

Translocator protein ligands as promising therapeutic tools for anxiety disorders.

The Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is an 18 kDa mitochondrial protein primarily involved in steroid biosynthesis in both peripheral and glial cells. It has been extensively reported that TSPO regulates the rate-limiting translocation of cholesterol from the outer to the inner mitochondrial membrane before its transformation by cytochrome P450(scc) into pregnenolone, which is further converted into an array of different steroids. In the brain, neurosteroids such as allopregnanolone and pregnenolone, acting as positive modulators of gamma-aminobutyric type A (GABA(A)) receptors, exert anxiolytic activity. Specific ligands targeting TSPO increase neurosteroid production and for this reason they have been suggested to play an important role in anxiety modulation. Unlike benzodiazepines (Bzs), which represent the most common anti-anxiety drugs administered around the world, selective TSPO ligands have shown anxiolytic effects in animal models without any of the side effects associated with Bzs. Therefore, specific TSPO ligands that are able to promote neurosteroidogenesis may represent the future of therapeutic treatment of anxiety disorders. Furthermore, TSPO expression levels are altered in several different psychiatric disorders in which anxiety is the main symptom. This article reviews the primary and patent literature over the last decade concerning the development of novel TSPO ligands that have resulted effective in various models of anxiety, taking into special consideration their structure-activity relationships.

GABA A/Bz receptor subtypes as targets for selective drugs.

The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory neuronal receptors in the mammalian brain. Their activation by GABA opens the intrinsic ion channel, enabling chloride flux into the cell with subsequent hyperpolarization. Several GABA(A) receptor subunit isoforms have been cloned, the major isoform containing alpha, beta, and gamma subunits, and a regional heterogeneity associated with distinct physiological effects has been suggested. As a variety of allosteric ligands can modulate GABA-gated conductance changes through binding to distinct sites, the development of subtype-selective ligands may lead to the selective treatment of GABA system-associated pathology. In particular, the best characterized binding site is the benzodiazepine site (BzR), localized at the alpha/gamma subunit interface, in which the alpha subunit is the main determinant of BzR ligand action selectivity. The alpha1-containing BzR have been proposed to be responsible for the sedative action; the alpha2 and/or the alpha3 subtypes have been suggested to mediate the anxiolytic activity and the myorelaxation effects, and the alpha5 subtype has been associated with cognition processes. The discovery of alpha-selective subtype ligands may help in the specific treatment of anxiety, sleep disorders, convulsions and memory deficits with fewer side effects. Selectivity may be achieved by two approaches: selective affinity or selective efficacy. Selective affinity needs a compound to bind with a higher affinity to one receptor subtype compared with another, whereas subtype-selective efficacy relies on a compound binding to all subtypes, but having different efficacies at various subtypes. The status of BzR ligands, subdivided on the basis of their main chemical structural features, is reviewed in relation to structure-activity relationships which determine their affinity or efficacy selectivity for a certain BzR subtype.

[1,2,4]Triazino[4,3-a]benzimidazole acetic acid derivatives: a new class of selective aldose reductase inhibitors.

Acetic acid derivatives of [1,2,4]triazino[4,3-a]benzimidazole (TBI) were synthesized and tested in vitro and in vivo as a novel class of aldose reductase (ALR2) inhibitors. Compound 3, (10-benzyl[1,2,4]triazino[4,3-a]benzimidazol-3,4(10H)-dion-2-yl)acetic acid, displayed the highest inhibitory activity (IC(50) = 0.36 microM) and was found to be effective in preventing cataract development in severely galactosemic rats when administered as an eyedrop solution. All the compounds investigated were selective for ALR2, since none of them inhibited appreciably aldehyde reductase, sorbitol dehydrogenase, or glutathione reductase. The activity of 3 was lowered by inserting various substituents on the pendant phenyl ring, by shifting the acetic acid moiety from the 2 to the 3 position of the TBI nucleus, or by cleaving the TBI system to yield benzimidazolylidenehydrazines as open-chain analogues. A three-dimensional model of human ALR2 was built, taking into account the conformational changes induced by the binding of inhibitors such as zopolrestat, to simulate the docking of 3 into the enzyme active site. The theoretical binding mode of 3 was fully consistent with the structure-activity relationships in the TBI series and will guide the design of novel ALR2 inhibitors.

3-Aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones: a new class of selective A1 adenosine receptor antagonists.

Radioligand binding assays using bovine cortical membrane preparations and biochemical in vitro studies revealed that various 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (ATBI) derivatives, previously reported by us as ligands of the central benzodiazepine receptor (BzR) (Primofiore, G.; et al. J. Med. Chem. 2000, 43, 96-102), behaved as antagonists at the A1 adenosine receptor (A1AR). Alkylation of the nitrogen at position 10 of the triazinobenzimidazole nucleus conferred selectivity for the A1AR vs the BzR. The most potent ligand of the ATBI series (10-methyl-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 12) displayed a Ki value of 63 nM at the A1AR without binding appreciably to the adenosine A2A and A3 nor to the benzodiazepine receptor. Pharmacophore-based modeling studies in which 12 was compared against a set of well-established A1AR antagonists suggested that three hydrogen bonding sites (HB1 acceptor, HB2 and HB3 donors) and three lipophilic pockets (L1, L2, and L3) might be available to antagonists within the A1AR binding cleft. According to the proposed pharmacophore scheme, the lead compound 12 engages interactions with the HB2 site (via the N2 nitrogen) as well as with the L2 and L3 sites (through the pendant and the fused benzene rings). The results of these studies prompted the replacement of the methyl with more lipophilic groups at the 10-position (to fill the putative L1 lipophilic pocket) as a strategy to improve A1AR affinity. Among the new compounds synthesized and tested, the 3,10-diphenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (23) was characterized by a Ki value of 18 nM which represents a 3.5-fold gain of A1AR affinity compared with the lead 12. A rhodopsin-based model of the bovine adenosine A1AR was built to highlight the binding mode of 23 and two well-known A1AR antagonists (III and VII) and to guide future lead optimization projects. In our docking simulations, 23 receives a hydrogen bond (via the N1 nitrogen) from the side chain of Asn247 (corresponding to the HB1 and HB2 sites) and fills the L1, L2, and L3 lipophilic pockets with the 10-phenyl, 3-phenyl, and fused benzene rings, respectively.

Synthesis, in vitro antiproliferative activity and DNA-interaction of benzimidazoquinazoline derivatives as potential anti-tumor agents.

The synthesis of benzimidazoquinazoline derivatives bearing different alkylamino side chains is reported. All new compounds tested by means of an in vitro assay exhibit antiproliferative activity toward human tumor cell lines. The cytotoxic effect depends on the type of side chain inserted in the planar nucleus and in some cases it is comparable to that of the well-known drug ellipticine. In order to understand the mechanism of action of these compounds, the interaction with DNA has been investigated. Linear flow dichroism measurements allowed us to verify the formation of a molecular complex with DNA and the corresponding geometry of interaction. Intrinsic binding constants have also been evaluated by performing fluorimetric titrations.

Pharmacological characterization of a new Ca(2+) sensitizer.

The benzimidazole molecule was modified to synthesize a Ca(2+) sensitizer devoid of additional effects associated with Ca(2+) overload. Newly synthesized compounds, termed 1, 2, 3, 4, and 5, were evaluated in spontaneously beating and electrically driven atria from reserpine-treated guinea pigs. Compound 3 resulted as the most effective positive inotropic agent, and experiments were performed to study its mechanism of action. In spontaneously beating atria, the inotropic effect of 3 was concentration-dependent (3.0 microM-0.3 mM). Compound 3 was more potent and more active than the structurally related Ca(2+) sensitizers sulmazole and caffeine, but unlike them it did not increase the heart rate. In electrically driven atria, the inotropic activity of 3 was well preserved and it was not inhibited by propranolol, prazosin, ranitidine, pyrilamine, carbachol, adenosine deaminase, or ruthenium red. At high concentrations (0.1-1.0 mM) 3 inhibited phosphodiesterase-III, whereas it did not affect Na(+)/K(+)-ATPase, sarcolemmal Ca(2+)-ATPase, Na(+)/Ca(2+) exchange carrier, or sarcoplasmic reticulum Ca(2+) pump activities of guinea pig heart. In skinned fibers obtained from guinea pig papillary muscle and skeletal soleus muscle, compound 3 (0.1 mM, 1 mM) shifted the pCa/tension relation curve to the left, with no effect on maximal tension and no signs of toxicity. Compound 3 did not influence the basal or raised tone of guinea pig isolated aorta rings, whose cells do not contain the contractile protein troponin. The present results indicate that the inotropic effect of compound 3 seems to be primarily sustained by sensitization of the contractile proteins to Ca(2+).

Effects of newly-developed benzodiazepine ligands on noise-induced mitochondrial damage in the rat.

In this study we measured the ability of three newly-synthesized N-arylalkylindol-3-ylglyoxylylamide derivatives, which have recently been characterized as partial agonists at central benzodiazepine binding sites, to prevent the rat cardiac mitochondrial alterations resulting from acute loud noise exposure. In particular, we evaluated the effects of these new compounds on the ultrastructural damage induced by noise stress on the right atrium and ventricle after 6 and 12 hr of loud noise exposure. In parallel experiments, we measured the affinity of these compounds for peripheral benzodiazepine binding sites. Following a single injection of the test products, we observed a cardioprotective effect which was more marked after 6 hr compared with 12 hr of noise exposure. Confirming our recent data showing that full agonists at benzodiazepine receptors produce cardioprotection, we demonstrate in this study that partial agonists, like indolylglyoxylylamides, can also produce a cardioprotective effect. Based on their greater affinity in binding studies, the protective activity seems to be related more to their action at central than at peripheral benzodiazepine receptors.

3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones: tricyclic heteroaromatic derivatives as a new class of benzodiazepine receptor ligands.

A series of 3-substituted [1,2,4]triazino[4,3-c]benzimidazoles V were prepared and tested at the central benzodiazepine receptor (BzR). These compounds were designed as rigid analogues of the previously described N-benzylindolylglyoxylylamide derivatives IV. The title compounds V showed an affinity which depended directly on the presence of the N(10)-H group and an aromatic ring at position 3. Some of them elicited a 2- or 3-fold higher affinity with respect to that of the indolylglyoxylylamide derivatives IV (R = H). The GABA ratio and [(35)S]-tert-butylcyclophosphorothionate binding data revealed an efficacy profile of partial inverse agonists/antagonists for compounds 1c,e,f,j,k, and of a partial agonist for 2c. This last compound proved to be effective in antagonizing pentylenetetrazole-induced seizures in mice. Attempts were made to interpret the structure-affinity relationships of compounds V in the light of possible tautomeric equilibria involving the ligands.

N'-Phenylindol-3-ylglyoxylohydrazide derivatives: synthesis, structure-activity relationships, molecular modeling studies, and pharmacological action on brain benzodiazepine receptors.

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).

Isosteric replacement of amide by ester function. Synthesis and benzodiazepine receptor affinity of indol-3-ylglyoxylyl ester derivatives.

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.

Benzisothiazole-1,1-dioxide alkanoic acid derivatives as inhibitors of rat lens aldose reductase.

Derivatives of 4-substituted 1,2-benzisothiazole-1,1-dioxide alkanoic acids were prepared and their in vitro aldose reductase inhibitory activity was tested in rat lens enzyme. The acetic derivatives 10, 12, and 16a-d proved to be much more potent inhibitors than the propionic derivatives 11, 13, and 17a-d. The presence of an acyl moiety on the amino group in position 4 of the acetic derivatives 16a-d led to a significant increase in activity with respect to the parent compound 14. One of the most active compounds in vitro, 10, was also evaluated in vivo as an inhibitor of glutathione lens depletion in galactosemic rats, but it did not show any activity in maintaining the rat lens glutathione level, probably due to problems of ocular bioavailability or metabolism.

Synthesis, structure-activity relationships, and molecular modeling studies of N-(indol-3-ylglyoxylyl)benzylamine derivatives acting at the benzodiazepine receptor.

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.

Synthesis and local anaesthetic activity of some 7-amino-2-dialkylaminoalkylpyrrolo[3,4-c]pyridine derivatives.

A number of 7-amino-2-dialkylaminoalkylpyrrolo[3,4-c] pyridin-1,3(2H)-dione derivatives were synthesized and their local anaesthetic activity was evaluated in vivo by corneal anaesthesia in rabbits. Only compounds 3,9 and 14 showed any activity, albeit lower than that of the reference drug lidocaine.

Acid derivatives of benzisothiazole-1,1-dioxide as inhibitors of rat lens aldose reductase.

A number of 6-substituted 1, 2-benzisothiazole-1, 1-dioxide alkanoic acids were synthesized and evaluated for crude rat lens aldose reductase inhibitory activity. The inhibitory potency of the acetic (6a, 10a), propionic (6b, 10b, 11b), and isopropionic (6c, 10c, 11c) derivatives was very similar and generally lower than that of the reference compound, Sorbinil. The presence of an acyl moiety on the amino group in position 6, as in the acetic and propionic derivatives 14a-f and 15a, b, respectively, resulted in a significant increase in activity. A good potency was shown by compounds 14g and 15g, in which a second carboxylic function is present on the 6-acylamino group. Also the open products 16, which contain the phenylsulfonyl fragment found in several known inhibitors of aldose reductase, were obtained and tested in the rat lens assay.