Identification of small-molecule urea derivatives as PTPC modulators targeting the c subunit of F1/Fo-ATP synthase.

Maintaining a high percentage of living and functional cells in those pathologies in which excessive cell death occurs, such as neurodegenerative disorders and cardiovascular diseases, is one of the most intriguing challenges in the field of biochemical research for drug discovery. Here, mitochondrial permeability transition-driven regulated cell death is the main mechanism of mitochondrial impairment and cell fate; this pathway is still lacking of satisfying pharmacological treatments to counteract its becoming; for this reason, it needs continuous and intense research to find new compounds as modulator of the permeability transition pore complex (PTPC) activity. In this study, we report the identification of small-molecule urea derivatives able to inhibit PTPC opening following calcium overload and selected for future use in cytoprotection.

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

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

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening.

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.

Tetrabranched Hetero-Conjugated Peptides as Bifunctional Agonists of the NOP and Mu Opioid Receptors.

The general aim of the work was the validation of a new synthetic methodology designed for obtaining bifunctional heterotetrabranched peptide ligands. Applying an easily accessible synthetic route, we provided a small series of heteromultimeric peptide conjugates targeting the nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) and mu opioid receptors. Among these, H-PWT1-N/OFQ-[Dmt1]dermorphin demonstrated a similar and high agonist potency at the NOP and mu receptors. The achieved results confirmed the robustness of the approach that is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity can be combined for generating dual acting multimeric compounds. These innovative pharmacological tools will be extremely helpful for investigating the consequences of the simultaneous activation and/or blockage of different peptidergic receptors.

The acyl-glucuronide metabolite of ibuprofen has analgesic and anti-inflammatory effects via the TRPA1 channel.

Ibuprofen is a widely used non-steroidal anti-inflammatory drug (NSAID) that exerts analgesic and anti-inflammatory actions. The transient receptor potential ankyrin 1 (TRPA1) channel, expressed primarily in nociceptors, mediates the action of proalgesic and inflammatory agents. Ibuprofen metabolism yields the reactive compound, ibuprofen-acyl glucuronide, which, like other TRPA1 ligands, covalently interacts with macromolecules. To explore whether ibuprofen-acyl glucuronide contributes to the ibuprofen analgesic and anti-inflammatory actions by targeting TRPA1, we used in vitro tools (TRPA1-expressing human and rodent cells) and in vivo mouse models of inflammatory pain. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited calcium responses evoked by reactive TRPA1 agonists, including allyl isothiocyanate (AITC), in cells expressing the recombinant and native human channel and in cultured rat primary sensory neurons. Responses by the non-reactive agonist, menthol, in a mutant human TRPA1 lacking key cysteine-lysine residues, were not affected. In addition, molecular modeling studies evaluating the covalent interaction of ibuprofen-acyl glucuronide with TRPA1 suggested the key cysteine residue C621 as a probable alkylation site for the ligand. Local administration of ibuprofen-acyl glucuronide, but not ibuprofen, in the mouse hind paw attenuated nociception by AITC and other TRPA1 agonists and the early nociceptive response (phase I) to formalin. Systemic ibuprofen-acyl glucuronide and ibuprofen, but not indomethacin, reduced phase I of the formalin response. Carrageenan-evoked allodynia in mice was reduced by local ibuprofen-acyl glucuronide, but not by ibuprofen, whereas both drugs attenuated PGE2 levels. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited the release of IL-8 evoked by AITC from cultured bronchial epithelial cells. The reactive ibuprofen metabolite selectively antagonizes TRPA1, suggesting that this novel action of ibuprofen-acyl glucuronide might contribute to the analgesic and anti-inflammatory activities of the parent drug.

NOP-Targeted Peptide Ligands.

The nociceptin/orphanin FQ (N/OFQ)-N/OFQ peptide (NOP) receptor system is widely distributed at both the peripheral and central level where it modulates important biological functions with increasing therapeutic implications. This chapter wants to provide a comprehensive and updated overview focused on the available structure-activity relationship studies on NOP receptor peptide ligands developed through different rational approaches. Punctual modifications and cyclizations of the N/OFQ sequence have been properly combined furnishing potent NOP selective ligands with different pharmacological activities (full and partial agonists, pure antagonists) and enhanced metabolic stability in vivo. The screening of peptide libraries provided a second family of NOP ligands that have been successfully optimized. Moreover, recent findings suggest the possibility to apply different multimerization strategies for the realization of multi-target NOP/opioid receptor ligands or tetrabranched N/OFQ derivatives with extraordinarily prolonged duration of action in vivo. The diverse approaches led to the identification of important pharmacological tools along with drug candidates currently in clinical development such as Rec 0438 (aka UFP-112) for the treatment of overactive bladder and SER 100 (aka ZP120) for the clinical management of systolic hypertension.

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

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

Naphthoquinone amino acid derivatives, synthesis and biological activity as proteasome inhibitors.

The ubiquitin-proteasome system has been largely investigated for its key role in protein degradation mechanisms that regulate both apoptosis and cell division. Because of their antitumour activity, different classes of proteasome inhibitors have been identified to date. Some of these compounds are currently employed in the clinical treatment of several types of cancer among which multiple myeloma. Here, we describe the design, chemistry, biological activity and modelling studies of a large series of amino acid derivatives linked to a naphthoquinone pharmacophoric group through variable spacers. Some analogues showed interesting inhibitory potency for the ß1 and ß5 subunits of the proteasome with IC50 values in the sub-µm range.

Ozone-Induced Hypertussive Responses in Rabbits and Guinea Pigs.

Cough remains a major unmet clinical need, and preclinical animal models are not predictive for new antitussive agents. We have investigated the mechanisms and pharmacological sensitivity of ozone-induced hypertussive responses in rabbits and guinea pigs. Ozone induced a significant increase in cough frequency and a decrease in time to first cough to inhaled citric acid in both conscious guinea pigs and rabbits. This response was inhibited by the established antitussive drugs codeine and levodropropizine. In contrast to the guinea pig, hypertussive responses in the rabbit were not inhibited by bronchodilator drugs (ß2 agonists or muscarinic receptor antagonists), suggesting that the observed hypertussive state was not secondary to bronchoconstriction in this species. The ozone-induced hypertussive response in the rabbit was inhibited by chronic pretreatment with capsaicin, suggestive of a sensitization of airway sensory nerve fibers. However, we could find no evidence for a role of TRPA1 in this response, suggesting that ozone was not sensitizing airway sensory nerves via activation of this receptor. Whereas the ozone-induced hypertussive response was accompanied by a significant influx of neutrophils into the airway, the hypertussive response was not inhibited by the anti-inflammatory phosphodiesterase 4 inhibitor roflumilast at a dose that clearly exhibited anti-inflammatory activity. In summary, our results suggest that ozone-induced hypertussive responses to citric acid may provide a useful model for the investigation of novel drugs for the treatment of cough, but some important differences were noted between the two species with respect to sensitivity to bronchodilator drugs.

History and perspectives of A2A adenosine receptor antagonists as potential therapeutic agents.

Growing evidence emphasizes that the purine nucleoside adenosine plays an active role as a local regulator in different pathologies. Adenosine is a ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1 , A2A , A2B , and A3 adenosine receptors (ARs). At the present time, the role of A2A ARs is well known in physiological conditions and in a variety of pathologies, including inflammatory tissue damage and neurodegenerative disorders. In particular, the use of selective A2A antagonists has been reported to be potentially useful in the treatment of Parkinson's disease (PD). In this review, A2A AR signal transduction pathways, together with an analysis of the structure-activity relationships of A2A antagonists, and their corresponding pharmacological roles and therapeutic potential have been presented. The initial results from an emerging polypharmacological approach are also analyzed. This approach is based on the optimization of the affinity and/or functional activity of the examined compounds toward multiple targets, such as A1 /A2A ARs and monoamine oxidase-B (MAO-B), both closely implicated in the pathogenesis of PD.

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

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

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

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

Bifunctional octadentate pseudopeptides as Zirconium-89 chelators for immuno-PET applications.

BACKGROUND: Positron emission tomography (PET) is a highly sensitive method that provides fine resolution images, useful in the field of clinical diagnostics. In this context, Zirconium-89 (89Zr)-based imaging agents have represented a great challenge in molecular imaging with immuno-PET, which employs antibodies (mAbs) as biological vectors. Indeed, immuno-PET requires radionuclides that can be attached to the mAb to provide stable in vivo conjugates, and for this purpose, the radioactive element should have a decay half-life compatible with the time needed for the biodistribution of the immunoglobulin. In this regard, 89Zr is an ideal radioisotope for immuno-PET because its half-life perfectly matches the in vivo pharmacokinetics of mAbs. RESULTS: The main objective of this work was the design and synthesis of a series of bifunctional octadentate pseudopeptides able to generate stable 89Zr complexes. To achieve this, here we investigated hydroxamate, N-methylhydroxamate and catecholate chelating moieties in complexing radioactive zirconium. N-methylhydroxamate proved to be the most effective 89Zr-chelating group. Furthermore, the increased flexibility and hydrophilicity obtained by using polyoxyethylene groups spacing the hydroxamate units led to chelators capable of rapidly forming (15 min) stable and water-soluble complexes with 89Zr under mild reaction conditions (aqueous environment, room temperature, and physiological pH) that are mandatory for complexation reactions involving biomolecules. Additionally, we report challenge experiments with the competitor ligand EDTA and metal ions such as Fe3+, Zn2+ and Cu2+. In all examined conditions, the chelators demonstrated stability against transmetallation. Finally, a maleimide moiety was introduced to apply one of the most promising ligands in bioconjugation reactions through Thiol-Michael chemistry. CONCLUSION: Combining solid phase and solution synthesis techniques, we identified novel 89Zr-chelating molecules with a peptide scaffold. The adopted chemical design allowed modulation of molecular flexibility, hydrophilicity, as well as the decoration with different zirconium chelating groups. Best results in terms of 89Zr-chelating properties were achieved with the N-methyl hydroxamate moiety. The Zirconium complexes obtained with the most effective compounds were water-soluble, stable to transmetallation, and resistant to peptidases for at least 6 days. Further studies are needed to assess the potential of this novel class of molecules as Zirconium-chelating agents for in vivo applications.

Spiropiperidine-Based Oligomycin-Analog Ligands To Counteract the Ischemia-Reperfusion Injury in a Renal Cell Model.

Finding a therapy for ischemia-reperfusion injury, which consists of cell death following restoration of blood flowing into the artery affected by ischemia, is a strong medical need. Nowadays, only the use of broad-spectrum molecular therapies has demonstrated a partial efficacy in protecting the organs following reperfusion, while randomized clinical trials focused on more specific drug targets have failed. In order to overcome this problem, we applied a combination of molecular modeling and chemical synthesis to identify novel spiropiperidine-based structures active in mitochondrial permeability transition pore opening inhibition as a key process to enhance cell survival after blood flow restoration. Our results were confirmed by biological assay on an in vitro cell model on HeLa and human renal proximal tubular epithelial cells and pave the way to further investigation on an in vivo model system.

The 'headache tree' via umbellulone and TRPA1 activates the trigeminovascular system.

The California bay laurel or Umbellularia californica (Hook. & Arn.) Nutt., is known as the 'headache tree' because the inhalation of its vapours can cause severe headache crises. However, the underlying mechanism of the headache precipitating properties of Umbellularia californica is unknown. The monoterpene ketone umbellulone, the major volatile constituent of the leaves of Umbellularia californica, has irritating properties, and is a reactive molecule that rapidly binds thiols. Thus, we hypothesized that umbellulone stimulates the transient receptor potential ankyrin 1 channel in a subset of peptidergic, nocioceptive neurons, activating the trigeminovascular system via this mechanism. Umbellulone, from µM to sub-mM concentrations, selectively stimulated transient receptor potential ankyrin 1-expressing HEK293 cells and rat trigeminal ganglion neurons, but not untransfected cells or neurons in the presence of the selective transient receptor potential ankyrin 1 antagonist, HC-030031. Umbellulone evoked a calcium-dependent release of calcitonin gene-related peptide from rodent trigeminal nerve terminals in the dura mater. In wild-type mice, umbellulone elicited excitation of trigeminal neurons and released calcitonin gene-related peptide from sensory nerve terminals. These two responses were absent in transient receptor potential ankyrin 1 deficient mice. Umbellulone caused nocioceptive behaviour after stimulation of trigeminal nerve terminals in wild-type, but not transient receptor potential ankyrin 1 deficient mice. Intranasal application or intravenous injection of umbellulone increased rat meningeal blood flow in a dose-dependent manner; a response selectively inhibited by systemic administration of transient receptor potential ankyrin 1 or calcitonin gene-related peptide receptor antagonists. These data indicate that umbellulone activates, through a transient receptor potential ankyrin 1-dependent mechanism, the trigeminovascular system, thereby causing nocioceptive responses and calcitonin gene-related peptide release. Pharmacokinetics of umbellulone, given by either intravenous or intranasal administration, suggest that transient receptor potential ankyrin 1 stimulation, which eventually results in meningeal vasodilatation, may be produced via two different pathways, depending on the dose. Transient receptor potential ankyrin 1 activation may either be caused directly by umbellulone, which diffuses from the nasal mucosa to perivascular nerve terminals in meningeal vessels, or by stimulation of trigeminal endings within the nasal mucosa and activation of reflex pathways. Transient receptor potential ankyrin 1 activation represents a plausible mechanism for Umbellularia californica-induced headache. Present data also strengthen the hypothesis that a series of agents, including chlorine, cigarette smoke, formaldehyde and others that are known to be headache triggers and recently identified as transient receptor potential ankyrin 1 agonists, utilize the activation of this channel on trigeminal nerves to produce head pain.

Non-neuronal TRPA1 encodes mechanical allodynia associated with neurogenic inflammation and partial nerve injury in rats.

BACKGROUND AND PURPOSE: The pro-algesic transient receptor potential ankyrin 1 (TRPA1) channel, expressed by a subpopulation of primary sensory neurons, has been implicated in various pain models in mice. However, evidence in rats indicates that TRPA1 conveys nociceptive signals elicited by channel activators, but not those associated with tissue inflammation or nerve injury. Here, in rats, we explored the TRPA1 role in mechanical allodynia associated with stimulation of peptidergic primary sensory neurons (neurogenic inflammation) and moderate (partial sciatic nerve ligation, pSNL) or severe (chronic constriction injury, CCI) sciatic nerve injury. EXPERIMENTAL APPROACH: Acute nociception and mechanical hypersensitivity associated with neurogenic inflammation and sciatic nerve injury (pSNL and CCI) were investigated in rats with TRPA1 pharmacological antagonism or genetic silencing. TRPA1 presence and function were analysed in cultured rat Schwann cells. KEY RESULTS: Hind paw mechanical allodynia (HPMA), but not acute nociception, evoked by local injection of capsaicin or allyl isothiocyanate, the TRP vanilloid 1 (TRPV1) or the TRPA1 activators was mediated by CGRP released from peripheral sensory nerve terminals. CGRP-evoked HPMA was sustained by a ROS-dependent TRPA1 activation, probably in Schwann cells. HPMA evoked by pSNL, but not that evoked by CCI, was mediated by ROS and TRPA1 without the involvement of CGRP. CONCLUSIONS AND IMPLICATIONS: As found in mice, TRPA1 mediates mechanical allodynia associated with neurogenic inflammation and moderate nerve injury in rats. The channel contribution to mechanical hypersensitivity is a common feature in rodents and might be explored in humans.

Novel Aryl Sulfonamide Derivatives as NLRP3 Inflammasome Inhibitors for the Potential Treatment of Cancer.

The NLRP3 inflammasome is a critical component of innate immunity that senses diverse pathogen- and host-derived molecules. However, its aberrant activation has been associated with the pathogenesis of multiple diseases, including cancer. In this study, we designed and synthesized a series of aryl sulfonamide derivatives (ASDs) to inhibit the NLRP3 inflammasome. Among these, compounds 6c, 7n, and 10 specifically inhibited NLRP3 activation at nanomolar concentrations without affecting the activation of the NLRC4 and AIM2 inflammasomes. Furthermore, we demonstrated that these compounds reduce interleukin-1ß (IL-1ß) production in vivo and attenuate melanoma tumor growth. Moreover, metabolic stability in liver microsomes of 6c, 7n, and 10 was studied along with plasma exposure in mice of the most interesting compound 6c. Therefore, we generated potent NLRP3 inflammasome inhibitors, which can be considered in future medicinal chemistry and pharmacological studies aimed at developing a new therapeutic approach for NLRP3 inflammasome-driven cancer.

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

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

Synthesis and biological evaluation of 2-substituted-4-(3',4',5'-trimethoxyphenyl)-5-aryl thiazoles as anticancer agents.

Antitumor agents that bind to tubulin and disrupt microtubule dynamics have attracted considerable attention in the last few years. To extend our knowledge of the thiazole ring as a suitable mimic for the cis-olefin present in combretastatin A-4, we fixed the 3,4,5-trimethoxyphenyl at the C4-position of the thiazole core. We found that the substituents at the C2- and C5-positions had a profound effect on antiproliferative activity. Comparing compounds with the same substituents at the C5-position of the thiazole ring, the moiety at the C2-position influenced antiproliferative activities, with the order of potency being NHCH(3) > Me >> N(CH(3))(2). The N-methylamino substituent significantly improved antiproliferative activity on MCF-7 cells with respect to C2-amino counterparts. Increasing steric bulk at the C2-position from N-methylamino to N,N-dimethylamino caused a 1-2 log decrease in activity. The 2-N-methylamino thiazole derivatives 3b, 3d and 3e were the most active compounds as antiproliferative agents, with IC(50) values from low micromolar to single digit nanomolar, and, in addition, they are also active on multidrug-resistant cell lines over-expressing P-glycoprotein. Antiproliferative activity was probably caused by the compounds binding to the colchicines site of tubulin polymerization and disrupting microtubule dynamics. Moreover, the most active compound 3e induced apoptosis through the activation of caspase-2, -3 and -8, but 3e did not cause mitochondrial depolarization.