J. Med. Chem. 2006, 49, 3049-3051
New Potent P-Glycoprotein Inhibitors Carrying a Polycyclic Scaffold
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Chart 1
Alessandra Bisi,*,† Silvia Gobbi,† Angela Rampa,† Federica Belluti,† Lorna Piazzi,† Piero Valenti,† Nora Gyemant,‡ and Joseph Molna´r‡ Department of Pharmaceutical Sciences, UniVersity of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy, and Department of Medical Microbiology and Immunobiology, UniVersity of Szeged, Do´ m te´ r 10, 6720 Szeged, Hungary
Table 1. Structures of Target Compounds
ReceiVed January 17, 2006 Abstract: New pentacyclic inhibitors of the P-glycoprotein carrying nitrogen-containing alkyl chains were synthesized and evaluated for MDR reverting activity on mouse lymphoma cells infected with pHa MDR1/A retrovirus. The activity of the compounds proved to be up to 5-fold higher than verapamil, used as reference compound.
The development of drug resistance in neoplastic cells is a major obstacle to successful anticancer chemotherapy, and the search for pharmacological agents able to counteract the mechanisms of drug resistance in oncology has remained a major goal for the past 10 years. Multidrug resistance (MDR) appears after prolonged exposure of cells to a single drug, and it is characterized by resistance to a series of structurally unrelated compounds with different subcellular targets.1 The typical MDR in tumor cells is mainly associated with a reduced intracellular drug accumulation and an increased cellular drug efflux. This phenomenon can be related to the overexpression of the energy-dependent efflux pump, P-glycoprotein (P-gp),2 a 170 kDa protein that belongs to the ATP binding cassette superfamily of transporters. Molecular investigations in MDR resulted in the isolation and characterization of genes encoding for several transport proteins including P-gp, the multidrug resistance associated protein (MRP1), the lung resistance protein (LRP), and the breast cancer resistance protein (BCRP).3,4 The different expressions and tissue/tumor specificities of P-gp and MRP1 (the first described proteins of this series) have been recently reviewed.5-9 Overexpression of P-gp has been documented in several tumor types, in particular after the patient has received chemotherapy, and several studies proved that P-gp expression could be considered a prognostic indicator in certain malignancies. Therefore it seems that overexpression of P-gp is clinically more significant than elevation of MRP1 levels. A large variety of compounds (anticancer agents, calcium channel blockers, neuroleptics, antiarrhythmics, antimalarial drugs, and antifungal agents) with the properties of inhibiting P-gp were selected by different approaches, and some specific potent inhibitors of P-gp entered into clinical evaluation,10-13 but the promise of this field of investigations has not been fulfilled yet, since there are currently no clinically available reversal agents. However, these studies have led to the definition of limited common features for chemosensitizers: protonable nitrogen, aromatic rings, high lipophilicity, and H-bond interactions. Moreover, there are indications that, besides the overall lipophilicity of the molecule, weak interactions, such as those produced by the overlapping * Corresponding author. Phone: +39 051 2099710. Fax: +39 051 2099734. E-mail:
[email protected]. † University of Bologna. ‡ University of Szeged.
of π orbitals of aromatic rings, can play an important role in stabilizing the binding of MDR reverting agents to P-gp.14-18 In this paper, we report the synthesis and in vitro data for a series of new P-gp inhibitors having the 17-azapentacyclo[6,6,5,02,7,09,14,015,19]-nonadeca-2,4,6,9(14),10,12-esene-16,18dione scaffold, 1 (Chart 1). This structure was selected considering the presence of two aromatic residues and two carbonyl groups, providing good π orbital overlapping and having H-bond accepting properties, respectively. Other polycyclic structures are described in the literature as MDR reverting agents (i.e., the antidepressant maprotiline).19 Moreover, this scaffold could structurally resemble a series of anthracene derivatives recently reported as MDR modulators.20 The compounds were designed by introducing on this scaffold the side chain of verapamil and other residues, described as structural modifications of this well-known calcium antagonist and first generation reverting agent.21 This also led to the introduction of a protonable nitrogen, another key feature for MDR reverting activity. The structures of the compounds are collected in Table 1. All the synthesized compounds have been prepared starting from intermediate 1, which was obtained by standard procedures.22 According to Scheme 1, the key intermediate 1 was alkylated with propargyl bromide (2a) or R-bromo-ω-chloropropane/etane (2b and 2c, respectively), by means of potassium tert-butoxide in DMSO. Compound 3a was then obtained via the Mannich reaction, refluxing 2a with N-methylhomoveratrylamine and formaldehyde, while compounds 3b and 3c were synthesized by reacting 2b and 2c with the same amine. All the final compounds were characterized by 1H NMR, mass spectra, and elemental analyses. Because the increase in the efflux of drugs is the major feature of P-gp mediated MDR and considering that rhodamine efflux is more sensitive than that of anticancer drugs, the efflux inhibition by the new derivatives with rhodamine 123 as substrate was chosen to quantify the MDR reversal activity. This test was carried out on L5178 mouse T-cell lymphoma cells infected with pHa MDR1/A retrovirus.23 In this assay, the calcium entry blocker verapamil was used as reference drug.24
10.1021/jm060056p CCC: $33.50 © 2006 American Chemical Society Published on Web 05/02/2006
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Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Scheme 1a
a Reagents and conditions: (a) potassium tert-butoxide, propargyl bromide, DMSO, rt; (b) N-methylhomoveratrylamine, formaldehyde, CuSO4, EtOH/H2O, reflux; (c) potassium tert-butoxide, R-bromo-ω-chloropropane/ ethane, DMSO, rt; (d) N-methylhomoveratrylamine, toluene, reflux.
Table 2. Reversal of Multidrug Resistance by Studied Compounds compound 3a 3b 3c verapamil PARa control MDRb control DMSO (20 µL)
concn (µg/mL) 1 0.1 1 0.1 1 0.1 10
FSC
SSC
FL-1
516.92 519.70 528.45 512.86 523.22 511.30 511.27 482.07 520.21 511.59
214.93 222.93 215.41 216.60 222.98 223.39 219.71 159.65 213.20 203.31
592.43 17.58 189.74 52.49 317.26 18.30 282.21 960.82 7.65 5.65
fluorescence activity ratio 89.35 2.65 28.62 7.91 47.85 2.76 42.56 0.85
a
PAR: L5178Y mouse T cell lymphoma drug-sensitive cell line. b MDR: L5178Y transformed subline with human MDR1 gene.
A preliminary cytotoxicity assay in a long-term antiproliferative effect was also performed. In the studied compounds, the side chains of verapamil and some analogues were inserted on a polycyclic scaffold. Verapamil is a well-known chemosensitizer; however, plasma concentrations needed to reverse MDR may cause major cardiovascular side effects to arise, due to its calcium antagonist activity. Starting from this indication, extensive SAR studies were performed on this molecule, and an increase in chemosensitizing effect was seen with the introduction of a rigid side chain.21,25,26 This modification also led to a reduced heart effect and to a loss of its allosteric effect on dihydropyridine binding.27 The new compounds all show inhibitory activity on P-gp mediated transport that is considerably higher than that of verapamil, taken as reference (Table 2). It is interesting to note that the compounds were evaluated at concentrations 10 times and 100 times lower than that of verapamil, which was tested at the concentration regularly used for in vitro studies on mouse lymphoma cells. In particular, compound 3a, showing the highest value of fluorescence activity ratio, appeared to be the most active one, able to counteract the P-gp-mediated rhodamine efflux much better than Verapamil, when used at a 10 times lower concentration. At the same concentration, compounds 3b and 3c both maintain a fairly good activity. These results could be related to the π interactions that the two aromatic rings of the polycyclic scaffold would establish with P-gp, as postulated for other reverting agents as well. Moreover, the two hydrogenbond accepting (HBA) carbonyl groups could enhance the activity, since in a recent paper the presence of HBA groups at an appropriate distance was shown to play an important role in the reverting effect.28 These additional interactions could counterbalance the low lipophilicity of the new compounds; still their relative potency seems to increase with their log P values,
Letters
which range from 4 to 4.5 and proved to be lower than the log P value for the reference verapamil (5.5). The high activity of compound 3a could be predicted from the effect of the same modification performed on Verapamil (EDP42)26 and, even if clear SAR cannot be drawn with such a small number of derivatives, it should be noticed that the presence of a rigid 3- or 4-atom side chain linking the polycyclic nucleus to the basic nitrogen led to an increase in activity in different classes of MDR reverting agents, that is, phenothiazines.29 The antiproliferative effect of the compounds was also assessed on human mdr1 gene transfected mouse lymphoma cell line. These preliminary data (see Supporting Information) showed for all the compounds a significant cytotoxicity only at a concentration higher than that used for evaluation of MDR reverting activity. It should be noted that cytotoxicity and MDR reversal activity cannot be directly compared, because in the cytotoxicity study a few thousand cells were exposed to the compounds for 24 or 48 h, while for the MDR study a larger amount of cells were treated with the compounds for a short period of time (30 min). Therefore, in the time required for MDR assay, cytotoxicity did not seem to influence the results. Further preliminary data indicate that no calcium antagonist activity was observed for these compounds (data not shown). In summary, some very potent P-gp inhibitors were synthesized using a new scaffold. The promising results reported here prompt us to further investigate this new class of compounds by the introduction of different residues from other molecules showing important reverting activities. Further pharmacological investigations are in progress to better evaluate the cytotoxicity, assess the full biological profile, and exclude any calcium antagonist action or any other adverse effect of the compounds. Supporting Information Available: Experimental procedures and characterization of intermediates and final compounds. This material is available free of charge via the Internet at http:// pub.acs.org.
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