Fabrizio Bordi

Dual mechanisms of action of the 5-benzylidene-hydantoin UPR1024 on lung cancer cell lines

In this study, we examined the mechanism of action of the novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor 5-benzylidene-hydantoin UPR1024, whose structure was designed to interact at the ATP-binding site of EGFR. The compound had antiproliferative and proapoptotic effects when tested on the non–small cell lung cancer cell line A549. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 induced significant level of DNA strand breaks associated with increased expression of p53 and p21WAF1 proteins, suggesting an additive mechanism of action. The presence of wild-type p53 improved the drug efficacy, although the effect was also detectable in p53 null cells. We also noted apoptotic cell death after treatment with UPR1024 at concentrations above 10 μmol/L for >24 h, with involvement of both the extrinsic and intrinsic pathways. The present data show that UPR1024 may be considered a combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage. UPR1024 or its derivatives might serve as a basis for development of drugs for the treatment of lung cancer in patients resistant to classic tyrosine kinase inhibitors. [Mol Cancer Ther 2008;7(2):361–70]

Novel irreversible epidermal growth factor receptor inhibitors by chemical modulation of the cysteine-trap portion.

Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATP-competitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group, which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic exploration of the role for the warhead group, introducing different cysteine-trapping fragments at position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups, including epoxyamides (compounds 3-6) and phenoxyacetamides (compounds 7-9), were able to irreversibly inhibit EGFR. In particular, at significant lower concentrations than gefitinib (1), (2R,3R)-N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(piperidin-1-ylmethyl)oxirane-2-carboxamide (6) inhibited EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.

Irreversible inhibition of epidermal growth factor receptor activity by 3-aminopropanamides.

Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead, effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-anilinoquinazoline or 4-anilinoquinoline-3-carbonitrile driving portions. Some of these compounds proved to be as efficient as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative which can react with thiol groups.

Interaction of selective compounds with muscarinic receptors at dispersed intestinal smooth muscle cells

1 The characterization of muscarinic receptors on single cells of the guinea‐pig ileum longitudinal smooth muscle, devoid of neuronal elements, was functionally studied by estimating the affinities of muscarinic antagonists on acetylcholine‐induced contractions. 2 Atropine (5 × 10−11 to 5 × 10−6 m), 4‐diphenylacetoxy‐N‐methyl‐piperidine methiodide (4‐DAMP, 5 × 10−8 to 5 × 10−6 m), cyclohexyl(4‐fluoro‐phenyl) (3‐piperidinopropyl) silanol (pFHHSiD, 5 × 10−7 to 5 × 10−5 m) as well as pirenzepine (5 × 10−7 to 5 × 10−5 m) competitively antagonized the acetylcholine‐dependent contractions with different affinities (atropine > 4‐DAMP > pFHHSiD > pirenzepine). 3 Methoctramine (5 × 10−7 to 5 × 10−5 m), and AF‐DX 116 (5 × 10−6 and 5 × 10−5 m) also showed antagonist properties but these deviated from simple competition. These compounds, which discriminate between M2 and M3 receptors, showed a potency lower than that of pirenzepine, the rank order of potencies being pirenzepine > methoctramine > AF‐DX 116. When concentrations of AF‐DX 116, methoctramine and pirenzepine were increased an unspecific contractile effect occurred. 4 McN‐A‐343, a partial agonist on intact guinea‐pig longitudinal smooth muscle strips, on this preparation induced a weak contraction (about 7% in comparison to control) that was not reversed by antimuscarinic agents. 5 These data indicate that M3 rather than M2 receptor sites are present on this tissue.

5-Benzylidene-hydantoins: synthesis and antiproliferative activity on A549 lung cancer cell line.

Benzylidene hydantoins have been recently reported as a new class of EGFR inhibitors. We describe here a simple and efficient methodology for the parallel solution-phase synthesis of a library of 5-benzylidene hydantoins, which were evaluated for antiproliferative activity on the human lung adenocarcinoma A549 cell line. Various substituents at positions 1, 3 and 5 on the hydantoin nucleus were examined. In the presence of a 5-benzylidene group and of a lipophilic substituent at position 1, most of the tested compounds inhibited cell proliferation at a concentration of 20 microM. Compound 7 (UPR1024), bearing 1-phenethyl and (E)-5-p-OH-benzylidene substituents, was found to be the most active derivative of the series. It inhibited EGFR autophosphorylation and induced DNA damage in A549 cells. Compound 7 and other synthesized 5-benzylidene hydantoin derivatives increased p53 levels, suggesting that the dual mechanism of action was a common feature shared by compound 7 and other member of the series.

Heteroarylaminoethyl and heteroarylthioethyl imidazoles. Synthesis and H3-receptor affinity

Summary -- The synthesis of new H3-receptor antagonists, 4-(2-heteroarylaminoethyl) and 4-(2-heteroarylthioethyl) imidazoles and their H3-receptor affinity obtained from competitive binding curves vs [3H]-NC~-methylhistamine ([3H]NAMHA) on rat brain cortex membranes are described. These compounds are derived from structural modulations of thioperamide and were synthesized in order to study binding interactions with H3-receptors and find alternative lead compounds with H3-receptor antagonist activity. The new compounds differ from thioperamide by the following features: 1) the N-cyclohexylcarbothioamide moiety of thioperamide has been replaced by a benzothiazole (1); 2) the piperidine ring has been replaced by more flexible aminoethyl and thioethyl chains, in order to lower the excessive rigidity of 1 and to test the importance of the tertiary piperidine nitrogen; and 3) the benzothiazole moiety of 1 has been replaced by other heterocyclic nuclei, endowed with different lipophilic, steric and hydrogen-bonding features. Some of the compounds tested showed good affinity for central H3-receptors (pKi range: 5.89-7.96) and can be considered as lead compounds for further optimization studies. The most lipophilic compounds showed higher affinities among benzo-condensed compounds, while imidazolylthioethyl imidazoles were more potent in displacing [3H]NAMHA than thiazolylthioethyl and thiazolylaminoethyl imidazoles which suggests an interaction between the annular NH of the imidazolylthioethyl moiety and the binding site. heteroarylglaminoethylimidazole / heteroarylthioethylimidazole / histamine H3-receptor affinity / [3H]-Na-methylhistamine / rat brain cortex membrane

Pharmacological profile of new thioperamide derivatives at histamine peripheral H1-, H2-, H3-receptors in guinea-pig

The recent availability of potent and selective ligands, namely R-(α)-methylhistamine and thioperamide, led to conclusive progresses as regards histamine H3-receptor knowledge. The aim of this work is to investigate byin vitro tests the pharmacological properties of new amino and methyl derivatives of the H3-antagonist thioperamide. Such original compounds, developed by the modulation of the thioperamide imidazolyl moiety, were assayed at guinea-pig ileal contractile H1-, atrial chronotropic H2- and enteric neuronal H3-receptors.None of the drugs exhibited interaction with H1 or H2 sites. On electrically stimulated ileum, two of the thioperamide methyl derivatives competitively antagonized the inhibitory effect of the H3-agonist R-(α)-methylhistamine. On the basis of the Schild analysis, the more active isomer (compound IV) displayed an affinity at H3-receptors only five times lower than thioperamide. These results could contribute to elucidate further the structural features required to develop potent and selective H3-antagonists. On the other hand, to prove the hypothesized apparent heterogeneity between peripheral and central H3-sites, as emerged by pharmacological and binding studies, autoradiographic investigations are in progress.

Synthesis and biological assays of new H3-antagonists with imidazole and imidazoline polar groups

New histamine H3-receptor antagonists were synthesised and tested on rat brain membranes and on electrically stimulated guinea-pig ileum. The new compounds have a central polar group represented by a 2-alkylimidazole or a 2-thioimidazoline nucleus. The effect of the polar group basicity on the optimal length of the alkyl chain, connecting this group to a 4(5)-imidazolyl ring, was investigated. The best affinity values, obtained by displacement of [3H]-RAMHA from rat brain, were obtained for the 2-alkylimidazole derivatives (2a-f) with tetramethylene chain (pKi 8.03-8.97), having an intermediate basicity between that of the previously reported 2-thioimidazoles (1a-i) and that of 2-alkylthioimidazolines (3a-h). In contrast, a general lowering of affinity (pKi 5.90-7.63) was observed for compounds of the last series (3a-h), with a complex dependence on the terminal lipophilic group and chain length.

Synthesis and stability in biological media of 1H-imidazole-1-carboxylates of ROS203, an antagonist of the histamine H3 receptor.

A series of carbamate derivatives of the H3 antagonist ROS203 (1) were prepared, and their lipophilicity and steric hindrance were modulated by introducing linear or branched alkyl chains of various lengths. In vitro stability studies were conducted to evaluate how structural modulations affect the intrinsic reactivity of the carbamoyl moiety and its recognition by metabolic enzymes. Linear alkyl carbamates were the most susceptible to enzymatic hydrolysis, with bioconversion rates being higher in rat liver and plasma. Chain ramification significantly enhanced the enzymatic stability of the set, with two derivatives (1g and 1h) being more stable by a factor of 8–40 than the ethyl carbamate 1a. Incubation with bovine serum albumin (BSA) showed a protective role of proteins on chemical and porcine‐liver esterase (PLE)‐catalyzed hydrolysis. Ex vivo binding data after i.v. administration of 1h revealed prolonged displacement of the labeled ligand [3H]‐(R)‐α‐methylhistamine ([3H]RAMHA) from rat‐brain cortical membranes, when compared to 1. However, the high rates of bioconversion in liver, as well as the chemical instability of 1h, suggest that further work is needed to optimize the enzymatic and chemical stability of these compounds.

Are histamine receptors involved in the stimulant activities of thiazolylethylamines supposed as cyclic models of dimaprit

A representative group of 2-aminothiazolylethylamine derivatives, in which the gastric acid secretion stimulatingS-aminoalkylisothiourea moiety can be recognized, was tested. The quite different responses observed suggest thatin vivo but notin vitro some events mimicking an H2-receptor agonist-like activity rather than a direct interaction with H2-receptors could take place. On the basis of structure-activity relationships, it can be speculated that the active conformation of dimaprit is not that resembled by these compounds which have been considered as cyclic models of one of its possible conformations.