Neuropeptide†S receptor: recent updates on nonpeptide antagonist discovery.

Neuropeptide S (NPS) is a 20-amino acid peptide of great interest due to its possible involvement in several biological processes, including food intake, locomotion, wakefulness, arousal, and anxiety. Structure-activity relationship studies of NPS have identified key points for structural modifications with the goal of modulating NPS receptor (NPSR) agonist activity or achieving antagonism at the same receptor. Only limited information is available for nonpeptide NPSR antagonists. In the last year, several studies have been reported in literature which present various series of small molecules as antagonists of this receptor. The results allow a comparison of the structures and activities of these molecules, leading to the design of new ligands with increased potency and improved pharmacological and pharmacokinetic profiles. This work presents a brief overview of the available information regarding structural features and pharmacological characterization of published nonpeptide NPSR antagonists.

Molecular modeling studies on the human neuropeptide S receptor and its antagonists.

Neuropeptide S (NPS) is a 20-residue peptide of great interest due to its potential involvement in several biological processes such as arousal, anxiety, and food intake. The NPS receptor belongs to the rhodopsin-like G-protein-coupled receptor superfamily, and several polymorphisms and isoforms of this receptor are associated with asthma, allergies, and bronchial hyper-responsiveness, in particular the Asn 107 Ile mutation. Limited structural information is available for this peptide-receptor system, particularly regarding the NPS receptor structure, its nonpeptide ligands, and the molecular aspects of agonist and antagonist binding processes. In this work, rhodopsin-based homology models of the NPS receptor and its Asn 107 Ile variant were built and refined in a membrane bilayer model, and binding modes for nonpeptide antagonists were simulated. This study provides the first structural study of the human NPS receptor, and the results provide a starting point for further characterization of the binding modes of its antagonists, and for the rational design of new NPS receptor ligands.

Adenine-based acyclic nucleotides as novel P2X3 receptor ligands.

A new series of acyclic nucleotides based on the adenine skeleton and bearing in 9-position a phosphorylated four carbon chain has been synthesized. Various substituents were introduced in 2-position of the adenine core. The new compounds were evaluated on rat P2X3 receptors, using patch clamp recording from HEK transfected cells and the full P2X3 agonist alpha,beta-meATP as reference compound. The results suggest that certain acyclic nucleotides, in particular compounds 28 and 29, are endowed with modest partial agonism on P2X3 receptors. This is an interesting property that can depress the function of P2X3 receptors, whose activation is believed to be involved in a number of chronic pain conditions including neuropathic pain and migraine. In fact, the new acyclic nucleotides are able to persistently block (by desensitization) P2X3 receptor activity after a brief, modest activation, yet leaving the ability of sensory neurons to mediate responses to standard painful stimuli via a lower level of signaling.

8-Bromo-9-alkyl adenine derivatives as tools for developing new adenosine A2A and A2B receptors ligands.

Importance of making available selective adenosine receptor antagonists is boosted by recent findings of adenosine involvement in many CNS dysfunctions. In the present work a series of 8-bromo-9-alkyl adenines are prepared and fully characterized in radioligand binding assays or functional cyclase experiments in respect to their interaction with all the four adenosine receptor subtypes. Results show that the presence of the bromine atom in 8-position of 9-substituted adenines promotes in general the interaction with the adenosine receptors, in particular at the A(2A) subtype. The present study also demonstrates that adenine derivatives could be a good starting point to obtain selective adenosine A(2B) receptor antagonists.

Design, synthesis, and biological evaluation of new mitonafide derivatives as potential antitumor drugs.

A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-6-{[omega-(alkylamino)alkyl]amino}-1H-benzo[de]isoquinolin-1,3(2H)-diones and 1,7-bis{6-[(omega-(dimethylamino)alkyl)amino]-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl}-4-methyl-4-azaheptanes, have been prepared as mitonafide derivatives. Their DNA-binding ability and cytotoxic activity have been evaluated. Some of the target compounds have shown high DNA affinity as well as relevant cytotoxic properties.

Synthesis and antitumor evaluation of bis aza-anthracene-9,10-diones and bis aza-anthrapyrazole-6-ones.

The good results obtained as potential antitumor drugs with aza-anthracenediones and aza-anthrapyrazoles, e.g. pixantrone, 1a, and 1b (Chart 1), prompted us to design and synthesize a series of symmetrical bis derivatives, compounds 7-10 (Chart 1). These compounds are dimers of different aza-anthracenedione and aza-anthrapyrazolone monomers connected by the linker found to be the most appropriate among potential bis intercalators synthesized by us. The DNA-binding properties of bis derivatives 7 and 8 have been examined using fluorometric techniques: these target compounds are excellent DNA ligands, with a clear binding site preference for AT-rich duplexes. In vitro cytotoxic activity of all target compounds 7-10 and of reference compound pixantrone toward human cancer adenocarcinoma cell line HT29 is also described. Two selected compounds have been investigated for their capacity of inducing early apoptosis.

Synthesis and biological evaluation of new asymmetrical bisintercalators as potential antitumor drugs.

The good results obtained in the past decade with various types of potential bisintercalating agents, e.g., LU 79553, DMP 840, BisBFI, MCI3335, WMC-26, BisAC, BisPA, and the asymmetrical derivative WMC-79 (Chart 1), prompted us to investigate a new series of asymmetrical bisintercalators, compounds 1a-t (Chart 2), which can combine the potentiality of bisintercalation with a possible different mechanism of action due to two diverse chromophores. The DNA-binding properties of these compounds have been examined using fluorometric techniques: target compounds are excellent DNA ligands, with a clear preference for binding to AT-rich duplexes. In vitro cytotoxicity of these derivatives toward human hormone-refractory prostate adenocarcinoma cell line (PC-3) is described. Apoptosis assays of four selected compounds are also reported. Very potent cytotoxic compounds, some of them capable of inducing early apoptosis, have been identified.

2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives, novel type of cytotoxic agents active on multidrug-resistant cell lines. Synthesis and biological evaluation.

We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the analog's ability to overcome multidrug resistance of tumor cells [J. Med. Chem.1999, 42, 3494]. High cytotoxic activity of obtained anthrapyridazones [Bioorg. Med. Chem.2003, 11, 561] toward the resistant cell lines, prompted us to synthesize the similarly modified acridine compounds. A series of pyridazinoacridin-3-one derivatives (2b-h) were prepared from the reaction of 9-oxo-9,10-dihydroacridine-1-carboxylate with POCl(3), followed by addition of the appropriate (alkylamino)alkylhydrazines. In vitro cytotoxic activity toward sensitive and resistant leukemia cell lines: L1210, K562, K562/DX, HL-60, HL-60/VINC, and HL-60/DX, with various type of multidrug resistance (MDR and MRP) was determined. The compounds studied exhibited in comparison to the reference cytostatics (DX, MIT) desirable very low resistance indexes (RI). Variations have been observed depending upon the substituent and the type of drug exporting pump. The cytotoxic activities of examined compounds, as well as of model anthrapyridazone derivative PDZ, were lower than those of reference drugs (DX, MIT) due to their diminished affinity to DNA.

Synthesis and biological evaluation of indazolo[4,3-bc][1,5]naphthyridines (10-aza-pyrazolo[3,4,5-kl]acridines): a new class of antitumor agents.

A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridines (2a-f), 10-aza derivatives of PZA, has been prepared by condensation of 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one (6) with the appropriate (omega-aminoalkyl)hydrazine in tetrahydrofuran/methanol. Compound 6 was obtained by heating at 100 degrees C in H(2)SO(4)5, yielded by the condensation of 2,6-dichloro-3-nitrobenzoic acid (4) and 6-methoxy-3-pyridinamine (3). The non-covalent DNA-binding properties of 2 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives against human hormone-refractory prostate adenocarcinoma cell line (PC-3) are described and compared to that of parent drug PZA. We selected the most cytotoxic target derivatives 2c,d, the in vitro inactive 2f, and reference compound PZA to investigate whether in vitro treatment with these drugs was able to induce necrotic and/or apoptotic cell death. To this purpose, we evaluated the percentage of apoptotic cells in PC-3 treated with the target compounds 2c,d,f and reference compound PZA, by Annexin V staining and Propidium iodide (PI)/Annexin V, biparametric flow cytometric analysis and agarose gel electrophoresis.

Rational design, synthesis, and biological evaluation of bis(pyrimido[5,6,1-de]acridines) and bis(pyrazolo[3,4,5-kl]acridine-5-carboxamides) as new anticancer agents.

The good results obtained with pyrimido[5,6,1-de]acridines 7 and with pyrazolo[3,4,5-kl]acridinecarboxamides 8 prompted us to the synthesis of two new series of bis acridine derivatives: the bis(pyrimidoacridines) 5 and the bis(pyrazoloacridinecarboxamides) 6. Compounds 5 can be regarded also as cyclized derivatives of bis(acridine-4-carboxamides) 3 and compounds 6 as cyclized derivatives of bis(acridine-4-carboxamides) 4. The noncovalent DNA-binding properties of these compounds have been examined using fluorometric techniques. The results indicate that (i) the target compounds are excellent DNA ligands; (ii) the bis derivatives 5 and 6 are more DNA-affinic than corresponding monomers 7 and 8; (iii) the new bis 5 and 6 result always less efficient in binding than related bis(acridine-4-carboxamides) 3 and 4; and (iv) in both series 5 and 6 a clear, remarkable in some cases, preference for binding to AT rich duplexes can be noted. In vitro cytotoxic potency of these derivatives toward the human colon adenocarcinoma cell line (HT29) is described and compared to that of reference drugs. Structure-activity relationships are discussed. We could identify six very potent cytotoxic compounds for further in vitro studies: a cytotoxic screening against six human cancer cell lines and the National Cancer Institute (NCI) screening on 60 human tumor cell lines. Finally, compound 6a was selected for evaluation in a NCI in vivo hollow fiber assay.

The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells.

The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer. Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient. Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs). The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line. The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells. It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC). The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake. In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein. Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.

Design, synthesis, and biological properties of new bis(acridine-4-carboxamides) as anticancer agents.

To enhance the outstanding biological response shown by the corresponding monomers 4 and 5, two classes of bis-acridine-4-carboxamides, 9, with a linker between the 4,4' positions, and 13, with a linker between the 1,1' positions, have been prepared as DNA-binding and potential antitumor agents. The noncovalent DNA-binding properties of these compounds have been examined using gel-electrophoresis and fluorometric techniques. The results indicate that (i). target compounds intercalate DNA; (ii). the bis derivatives with the optimal linker are considerably more DNA-affinic than corresponding monomers; (iii). overall affinity is sensitive to the nature of the linker, of the chromophores, and of the substituents at 7,7'; (iv). often, the bis derivatives show a marked AT-preferential binding. In vitro cytotoxic potency of these derivatives toward the human colon adenocarcinoma cell line (HT29) is described and compared to that of reference drugs. Structure-activity relationships are discussed. Some highly DNA-affinic and potent cytotoxic compounds, 9b,f and 13b,c, have been selected for the National Cancer Institute (NCI) screening on 60 human tumor cell lines and identified as new leads in the antitumor strategies.

Rational design, synthesis and biological evaluation of thiadiazinoacridines: a new class of antitumor agents.

A series of potential DNA-binding antitumor agents, 3-[omega-(alkylamino)alkyl]-6-nitro-thiadiazino[3,4,5-kl]acridines 12 and 1,3-di[omega-(alkylamino)alkyl]-6-nitro-thiadiazino[3,4,5-kl]acridines 13, has been prepared by cyclization with SOCl(2) of 1-[[omega-(alkylamino)alkyl]amino]-9-imino-4-nitro-9,10-dihydroacridines 16 or 1-[[omega-(alkylamino)alkyl]amino]-9-[omega-(alkylamino)alkyl]imino-4-nitro-9,10-dihydroacridines 17, respectively. The non-covalent DNA-binding properties of 12, 13 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives toward six tumor cell lines, including human colon adenocarcinoma (HT29) and human ovarian carcinoma (A2780 sensitive, A2780cisR cisplatin-resistant, CH1, CH1cisR cisplatin-resistant, and SKOV-3) cells, are described and compared to that of reference drugs. In vivo antitumor activity of some selected derivatives, endowed with relevant cytotoxic activity against murine leukemia P388 are reported. The 3-[2-(dimethylamino)ethyl]-6-nitro-2,7-dihydro-3H-2 lambda(4)-thiadiazino[3,4,5-kl]acridin-2-one (12d) has been identified as a new lead in the development of anticancer tetracyclic acridine derivatives.

DNA-binding antitumor agents: from pyrimido[5,6,1-de]acridines to other intriguing classes of acridine derivatives.

In the field of antitumor DNA-binding agents, the class of acridine derivatives play an important role either as number of compounds or as importance of their anticancer properties. We have synthesized a number of acridine derivatives as potential antitumor drugs, in which the chromophore is fully or partially constituted by acridine or by 9-acridone ring systems: from the pyrimido[5,6,1-de]acridines, to the pyrimido[4,5,6-kl]acridines, the bis(amine-functionalized) 9-acridone-4-carboxamides, the bis(amine-functionalized) acridine-4-carboxamides, and the pyrazolo[3,4,5-kl]acridine-5-carboxamides. In the present revue we will describe the rational design, the synthesis, and the salient biological characteristics of these classes of acridine derivatives.

Binding free energy of selected anticancer compounds to DNA--theoretical calculations.

Many studies have elucidated structures and thermodynamics of complexes formed by different ligands with DNA. However, in most cases structural and free energy binding studies were not correlated with each other because of the problem of identifying which experimental free energy of binding corresponds to which experimental DNA-ligand structure. In the present work, Poisson-Boltzmann and solvent-accessible surface area methods were used to predict unknown modes of interaction between DNA and three different ligands: mitoxantrone and two pyrimidoacridine derivatives. In parallel, experimental measurements of binding free energy for the studied complexes were performed to compare experimental and calculated values. Our studies showed that the calculated values of free energy are only close to experimental data for some models of interaction between ligands and DNA. Based on this correlation, the most likely models of DNA-ligand complexes were postulated: (i) mitoxantrone and one derivative of pyrimidoacridine, both with two charged side chains, intercalate from the minor groove of DNA and bind with both chains in this groove; (ii) pyrimidoacridine, with only one side chain, very likely does not intercalate into DNA at all. Additionally, the non-electrostatic and electrostatic parts of the calculated binding free energy for the DNA-ligands studied are discussed.

The ability to overcome multidrug resistance of tumor cell lines by novel acridine cytostatics with condensed heterocyclic rings.

Two recently synthesized groups of acridine cytostatics containing fused heterocyclic ring(s): pyrazoloacridines (PAC) and pyrazolopyrimidoacridines (PPAC) were tested in regard to their in vitro cytotoxic activity towards a panel of sensitive and resistant human tumor cell lines. The obtained results corroborate our earlier hypothesis on the essential role of heterocyclic ring fused to the acridine moiety in the ability of acridine cytostatics to overcome multidrug resistance of tumor cells. The presence, location and kind of substituents considerably influenced both the cytotoxic activity of the derivatives and their ability to overcome multidrug resistance. The same factors also affected the cytostatics ability to differentiate between tumor cell lines with various types of drug exporting pumps.

2,6-Di(omega-aminoalkyl)-2,5,6,7-tetrahydropyrazolo[3,4,5-mn]pyrimido[5,6,1-de]acridine-5,7-diones: novel, potent, cytotoxic, and DNA-binding agents.

DNA-binding agents with potential antitumor activities bearing two cationic side chains, the 2,6-di(omega-aminoalkyl)-2,5,6,7-tetrahydropyrazolo[3,4,5-mn]pyrimido[5,6,1-de]acridine-5,7-diones (4a-r), have been prepared either by reaction of the appropriate 2-(omega-aminoalkyl)-6-chloro-2,3-dihydro-1H,7H-pyrimido[5,6,1-de]acridine-1,3,7-trione with the appropriate (omega-aminoalkyl)hydrazine or by cyclization of the requisite N-6,2-di(omega-aminoalkyl)-2,6-dihydropyrazolo[3,4,5-kl]acridine-6-carboxamide with phosgene. In vitro cytotoxic properties of these derivatives against three human colon adenocarcinoma cell lines (HT29, LoVo, and LoVo/Dx) and against some cell lines of the NCI panel are described and compared to that of reference drugs. Some of the new compounds showed outstanding potency while lacking cross-resistance with anthracyclines. Structure-activity relationships are discussed, and a mechanistic analysis is performed using the COMPARE procedure. The mechanism and efficiency of noncovalent DNA binding of these compounds are examined using gel electrophoresis and fluorometric techniques. The 2,6-di(omega-aminoalkyl)-2,5,6,7-tetrahydropyrazolo[3,4,5-mn]pyrimido[5,6,1-de]acridine-5,7-diones (4) constitute a new class of potent, cytotoxic DNA-binding agents not cross-resistant with doxorubicin.