Cecilia Martelli

The functions and structure of ABC transporters: implications for the design of new inhibitors of Pgp and MRP1 to control multidrug resistance (MDR).

Multidrug resistance (MDR) is a kind of acquired resistance of microorganisms and cancer cells to chemotherapic drugs that are characterized by different chemical structure and different mechanism of action. Classic MDR is the consequence of the over-expression of a variety of proteins that extrude the chemotherapic from the cell, lowering its concentration below the effective one. The ABC (ATP Binding Cassette) is a ubiquitous and important family of such transporter proteins. Members of this super family are present in mammals as well as in prokaryotic organisms and use ATP as the energy source to activate the extrusion process. P-glycoprotein (Pgp) and Multidrug Resistance Proteins (MRP1 and sister proteins) are the most important and widely studied members of ABC super family. Our knowledge about the structures and functions of transporter proteins has definitely improved in recent years, following the resolution of the structure of bacterial pumps which opened the way to the building of homology models for the more complex Pgp and MRP. It can be anticipated that these results will have a strong impact on the design of more potent and safer MDR reverters. A huge number of small molecules, many of natural origin, are able to reverse multidrug resistance by inhibiting the functions of Pgp, MRP1 and sister proteins and their action has been considered a possible way to reverse MDR. However, while a few compounds have reached clinical trials, none of them has, so far, been cleared for therapeutic use. Two main reasons are at the base of this difficulty: i) MDR is a complex phenomenon that may arise from several different biochemical mechanisms, with the consequence that inhibition of transporter proteins may be insufficient to reverse it; ii) the physiological role of Pgp and sister proteins requires more potent modulators with proper selectivity and pharmacokinetic in order to avoid unwanted side effects. This paper first reviews the most recent discoveries on the structures and functions of the ABC super family, in particular Pgp and MRP. Then, the medicinal chemistry of MDR reverters, in light of these findings, is discussed and the molecules that are presently in development are reviewed.

N,N-bis(cyclohexanol)amine aryl esters: a new class of highly potent transporter-dependent multidrug resistance inhibitors.

A new series of Pgp-dependent MDR inhibitors having a N,N-bis(cyclohexanol)amine scaffold was designed on the basis of the frozen analogue approach. The scaffold chosen gives origin to different geometrical isomers. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells in the pirarubicin uptake assay. The most interesting compounds (isomers of 3) were studied further evaluating their action on the ATPase activity present in rat small intestine membrane vesicles and doxorubicin cytotoxicity potentiation on K562 cells. The latter assay was performed also on the isomers of 4. The four isomers of each set present different behavior in each of these tests. Compound 3d shows the most promising properties as it was able to completely reverse Pgp-dependent pirarubicin extrusion at low nanomolar concentration, inhibited ATPase activity at 5 x 10(-9) and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 36.4 at 3 microM concentration.

Structure-Activity Relationships Studies in a Series of N,N-Bis(alkanol)amine Aryl Esters as P-Glycoprotein (Pgp) Dependent Multidrug Resistance (MDR) Inhibitors

As a continuation of a previous research, a series of N,N-bis(alkanol)amine aryl esters, as Pgp-dependent MDR inhibitors, was designed and synthesized. The aromatic ester portions are suitably modulated, and new aryl rings (Ar(1) and Ar(2)) were combined with trans-3-(3,4,5-trimethoxyphenyl)vinyl, 3,4,5-trimethoxybenzyl and anthracene moieties that were present in the most potent previously studied compounds. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Selected compounds (5, 6, 8, 9, and 21) were further studied, evaluating their action on doxorubicin cytotoxicity potentiation on K562 cells; they significantly enhanced doxorubicin cytotoxicity on K562/DOX cells, confirming the results obtained with pirarubicin. Compound 9 shows the most promising properties as it was able to nearly completely reverse Pgp-dependent pirarubicin extrusion at nanomolar doses and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 19.1 at 3 microM dose.

Inhibition of P-glycoprotein-mediated Multidrug Resistance (MDR) by N,N-bis(cyclohexanol)amine aryl esters: Further restriction of molecular flexibility maintains high potency and efficacy

Conformational modulation of the aryl portion of a set of N,N-bis(cyclohexanol)amine aryl esters (1a-d) that are potent Pgp-dependent MDR inhibitors has been performed. Toward this end the trans-3-(3,4,5-trimethoxyphenyl)acrylic acid present in set 1 was substituted with 3-(3,4,5-trimethoxyphenyl)propanoic and 3-(3,4,5-trimethoxyphenyl)propiolic moieties to give sets 2 and 3, respectively. While the introduction of 3-(3,4,5-trimethoxyphenyl)propanoic moiety resulted in a definite drop in potency and efficacy, esterification with 3-(3,4,5-trimethoxyphenyl)propiolic acid gave four isomers (3a-d) that maintain high potency and possess optimal efficacy. These results are discussed in terms of conformational flexibility of the different sets of compounds.

New structure-activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR).

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure-activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis). The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I](0.5)) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a-d and 6d, showed excellent efficacy with a α(max) close to 1. Selected compounds (2d, 3a, 3b, 5a-d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells. The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site. In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,(29) are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.

Design, synthesis and nootropic activity of new analogues of sunifiram and sapunifiram, two potent cognition-enhancers

A series of amides and sulfonamides, structurally related to DM235 (sunifiram) and MN19 (sapunifiram), derived by ring expansion or contraction, or by inversion of the exocyclic amide function, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, and with a potency similar to the parent compounds.

2-Pyrrolidinone moiety is not critical for the cognition-enhancing activity of piracetam-like drugs

Following the indications of previous work, 2-pyrrolidinone moiety of piracetam and piracetam-like compounds has been opened to the corresponding amide derivatives. As found previously in the case of 1,4-diazabicyclo[4.3.0]nonan-9-one compounds, the cognition-enhancing activity of 2-pyrrolidinone compounds is maintained in most cases, suggesting that this moiety is not crucial for activity.

Synthesis, Affinity Profile and Functional Activity of Potent Chiral Muscarinic Antagonists with a Pyrrolidinylfuran Structure

Starting from the structure of previously studied muscarinic agonists, characterized by a pyrrolidinylfuran scaffold, a new series of muscarinic antagonists was synthesized by substituting the 5-position of the furane cycle with bulky hydrophobic groups. Both tertiary amines and the corresponding iodomethyl derivatives were obtained and studied. All the new compounds show high affinity toward cloned human muscarinic M(1)-M(5) receptors expressed in Chinese hamster ovary (CHO) cells and behave as competitive antagonists on classical models of muscarinic receptors. The diastereoisomeric mixture of the highest affinity compound of the series was resolved into the four optical isomers by chiral HPLC. The relative and absolute configuration of the obtained compounds was established by means of a combined strategy based on X-ray crystallography and chiroptical techniques. Although generally fairly potent, the compounds showed only modest subtype selectivity, with the exception of 2a and 6a, which in functional assays presented clear-cut selectivity for the muscarinic receptors present in rabbit vas deferens.

The Novel Potent Multidrug Resistance Inhibitors N,N-bis(cyclohexanol)amine Aryl Esters Are Devoid of Vascular Effects

The aim of this study was to investigate the effects of the four isomers (3a, 3b, 3c and 3d) of a novel multidrug resistance-reverting agent – 3,4,5-trimethoxybenzoic acid 4-(methyl-{4-[3-(3,4,5-trimethoxyphenyl)acryloyloxy]cyclohexyl}amino)cyclohexyl ester – on vascular functions in vitro. A comparison of their mechanical and electrophysiological actions in rat aorta rings and single rat tail artery myocytes, respectively, was performed. In rat aorta rings, 3a–d antagonized both 60 mmol/l K+- and phenylephrine-induced contraction in a concentration-dependent manner, with maximal relaxation values averaging 50% of controls, 3d being the most effective of the series. The vasorelaxing effect was similar either in presence or absence of intact endothelium. In rat tail artery myocytes, out of the four isomers, only 3a consistently inhibited Ba2+ current through Cav1.2 channels. Our results provide functional evidence that 3a–d are weak vasorelaxing agents, although at concentrations much higher than those effective for multidrug resistance reversion in cancer cells.