In vitro and in silico analysis of the vascular effects of asymmetrical N,N-bis(alkanol)amine aryl esters, novel multidrug resistance-reverting agents.

Asymmetrical N,N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca(2+) antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Cav1.2 channel current (I Ca1.2) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α1C subunit of the Cav1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1 µM (FRA77, GDE6, GDE19) and >10 µM (verapamil) in EA.hy926 cells, or >10 µM (FRA77, GDE6, GDE19) and at 100 µM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60 mM K(+) (K60)-induced contraction at concentrations ≥1 and ≥3 µM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10 µM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α1C subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited I Ca1.2 with IC50 values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers.

Design, synthesis and preliminary evaluation of a series of histone deacetylase inhibitors carrying a benzodiazepine ring.

A series of new histone deacetylase inhibitors were designed and synthesized based on hybridization between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines. The compounds were tested for their enzyme inhibitory activity on HeLa nuclear extracts, and on human recombinant HDAC1 and HDAC6. Antiproliferative activity was tested on different cancer cells types, while proapoptotic activity was primarily tested on NB4 cells. The compounds showed IC50 values similar to those of SAHA. Compound (S)-8 displayed interesting activity against hematological and solid malignancies.

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

Multidrug resistance (MDR) is a kind of acquired resistance of microorganisms and cancer cells to chemotherapeutic drugs that are characterized by different chemical structure and different mechanism of action. Classic MDR is due to a lower intracellular concentration of cytotoxic drugs that is associated with accelerated efflux of the chemotherapeutic drugs and is the consequence of the over expression of transporter proteins that act as extrusion pumps. P-glycoprotein (P-gp/ABCB1) is the most important and studied member of such proteins belonging to the ATP Binding Cassette (ABC) superfamily of transporters that use ATP as energy source. Inhibition of the functions of P-gp and other ABC proteins could represent a way to circumvent appearance of MDR in cancer cells and the most classical pharmacological strategy is the administration of agents able to modulate the P-gp function. On the basis of the known characteristics of the recognition site of P-gp, we have designed a new class of P-gp-mediated MDR reverters. These compounds are flexible molecules carrying a basic nitrogen atom flanked, at properly modulated distance, by two aromatic moieties; most of them possess MDR inhibitory activity on anthracycline-resistant erytroleukemia K562 cells. By applying the frozen analog approach to that series of very flexible MDR reverters, we identified a new series of N,N-bis(cyclohexanol)amine aryl esters that show very interesting MDR-reversing properties. Among them, compound 15d, that consistently shows low nanomolar potency and high efficacy in all the tests used, appears as a new pharmacological tool for P-gp studies and a promising lead for the development of potent, efficient and safe MDR reverters.

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.

The medicinal chemistry of multidrug resistance (MDR) reversing drugs.

Multidrug resistance (MDR) is a kind of resistance of cancer cells to multiple classes of chemotherapic drugs that can be structurally and mechanistically unrelated. Classical MDR regards altered membrane transport that results in lower cell concentrations of cytotoxic drug and is related to the over expression of a variety of proteins that act as ATP-dependent extrusion pumps. P-glycoprotein (Pgp) and multidrug resistance protein (MRP1) are the most important and widely studied members of the family that belongs to the ABC superfamily of transporters. It is apparent that, besides their role in cancer cell resistance, these proteins have multiple physiological functions as well, since they are expressed also in many important non-tumoural tissues and are largely present in prokaryotic organisms. A number of drugs have been identified which are able to reverse the effects of Pgp, MRPI and sister proteins, on multidrug resistance. The first MDR modulators discovered and studied in clinical trials were endowed with definite pharmacological actions so that the doses required to overcome MDR were associated with unacceptably high side effects. As a consequence, much attention has been focused on developing more potent and selective modulators with proper potency, selectivity and pharmacokinetics that can be used at lower doses. Several novel MDR reversing agents (also known as chemosensitisers) are currently undergoing clinical evaluation for the treatment of resistant tumours. This review is concerned with the medicinal chemistry of MDR reversers, with particular attention to the drugs that are presently in development.

Structure-activity relationships and optimisation of the selective MDR modulator 2-(3,4-dimethoxyphenyl)-5-(9-fluorenylamino)-2-(methylethyl) pentanenitrile and its N-methyl derivative.

Several ring-substituted derivatives of previously studied MDR inhibitors 2-(3,4-dimethoxyphenyl)-5-(9-fluorenylamino)-2-(methylethyl)pentanenitrile and 2-(3,4-dimethoxyphenyl)-5-[(9-fluorenyl)-N-methylamino]-2-(methylethyl)pentanenitrile have been synthesised and studied with the aim of optimising activity and selectivity. The results show that MDR inhibition is scarcely sensitive to modulation of the electronic properties of the fluorene ring. Even if dramatic improvement was not obtained, one of the compounds (2) showed improved potency and selectivity with respect to the leads and appears to be a better candidate for drug development.

Fluorescent verapamil analogue for monitoring acidic intracellular organelles in multidrug resistant and sensitive cells.

Resistance to chemotherapeutic agent is a major cause of treatment failure in patients with cancer. In many cases, the primaly mechanism leading to a multidrug-resistant phenotype is the plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein. However, acidic intracellular organelles seem also to participate in resistance to chemotherapeutic drugs and the determination of the pH of these organelles is of importance. In the present study we have used a new fluorescent derivative of verapamil, 2-2-diphenyl-5-[(methylaminomethyl)anthracene] pentanenitrile (EDP 96), and show that it is an efficient inhibitor of the P-gp-mediated efflux of anthracycline in K562 resistant cells. The fluorescence of EDP 96 is environmental and pH sensitive. EDP 96 is a weak base (pKa=6.0) and its accumulation into K562 cells is accompanied by a significant fluorescence increase due to its entry of the drug into acidic regions in the cells. We have used this properties to develop a new method to accurately determine the pH of acidic organelle.

Design, synthesis, and preliminary pharmacological evaluation of 1, 4-diazabicyclo[4.3.0]nonan-9-ones as a new class of highly potent nootropic agents.

Several 4-substituted 1,4-diazabicyclo[4.3.0]nonan-9-ones have been synthesized and tested in vivo on mouse passive avoidance test, to evaluate their nootropic activity. The results show that they represent a new class of nootropic drugs with a pharmacological profile very similar to that of piracetam, showing much higher potency with respect to the reference. Among the compounds studied, 7 (DM 232) shows outstanding potency, being active at the dose of 0. 001 mg kg(-1) sc.

Design, synthesis, and in vitro activity of catamphiphilic reverters of multidrug resistance: discovery of a selective, highly efficacious chemosensitizer with potency in the nanomolar range.

On the basis of the results obtained in previous research, three series of compounds (A-C), derived from verapamil, were designed and synthesized to obtain drugs able to revert multidrug resistance (MDR), an acquired resistance that frequently impairs cancer chemotherapy. The ability of the obtained compounds to revert MDR was evaluated on anthracycline-resistant erythroleukemia K 562 cells, measuring the uptake of THP-adriamycin (pirarubicin) by continuous spectrofluorometric monitoring of the decrease of the fluorescence signal of the anthracycline at 590 nm (lambdaex = 480 nm), after incubation with cells. Cardiovascular activity, which is responsible for unwanted side effects, was also evaluated. The results obtained show that many of the compounds studied are potent reverters of MDR and are endowed with reduced cardiovascular activity. One of the compounds (7, MM36) presents a pharmacological profile (unprecedented nanomolar potency, high reversal of MDR, low cardiovascular activity) that makes it a promising drug candidate to treat MDR and a useful tool for studying P-glycoprotein.

Study of P-glycoprotein functionality in living resistant K562 cells after photolabeling with a verapamil analogue.

To our knowledge, this is the first study to investigate the modification of P-glycoprotein functionality in living resistant cells after photolabeling. For this purpose, four new photoactive verapamil analogues were synthesized. These compounds have the same efficacy as verapamil to increase pirarubicin (pira) incorporation into living multidrug resistant (MDR) K562 cells and to sensitize them to the cytotoxic effect of this anthracycline derivative, indicating that they act as typical MDR modifiers in MDR cells. These compounds were used to photolabel P-glycoprotein (P-gp) in living resistant cells. Irradiation did not result in photodamage to cells, and P-gp functionality was verified by the ability of living cells to incorporate pira. The irradiation of resistant cells, 10(6)/mL, in the presence of a verapamil analogue at concentrations equal to or higher than 3 microM yielded 70% inhibition of P-gp functionality. Our data provide the first evidence that the binding of a verapamil analogue to P-gp is not sufficient to completely inhibit the efflux of this anthracycline. The cells were, subsequently, cultured for several days. Resistance was progressively recovered with time, with the treated cells being just as resistant as before photolabeling after 6 days.

Reversal of multidrug resistance by verapamil analogues.

The basic distinguishing feature of multidrug resistant (MDR) cells is a decrease in steady-state drug levels as compared to drug-sensitive controls. It is well-known that verapamil increases the sensitivity of MDR cells to drugs, thus reverting drug resistance. A limiting factor for its clinical use is the pronounced cardiovascular effects of the calcium channel antagonist which occur at the high plasma concentrations required to block P-glycoprotein transport efficiently. From a clinical point of view, it is important to find verapamil derivatives with low calcium channel blocking activity and high reverting activity. This was the aim of the present study. In this context we have investigated the ability of 20 verapamil analogues with restricted molecular flexibility to increase cellular accumulation of anticancer drugs and overcome resistance, and their inotropic, chronotropic, and slow calcium channel antagonistic activity. In this study an anthracycline derivative 4'-O-tetrahydropyranyl adriamycin, and an erythroleukaemia K562 cell line were used. Three of the 20 derivatives checked were completely devoid of calcium channel blocking activity while exhibiting MDR reverting ability comparable to that of verapamil. These derivatives could be useful for the treatment of MDR in cancer patients and for the design and development of other verapamil derivatives.