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

A new series of P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors having a N,N-bis(cyclohexanol)amine scaffold have been designed, following the frozen analog approach. With respect to the parent flexible molecules, the new compounds show improved potency and efficacy. Among them, compound 1d, on anthracycline-resistant erythroleukemia K562 cells, is able to completely reverse Pgp-dependent MDR at low nanomolar concentration.     

The cytotoxic effect and the multidrug resistance reversing action of lignans from Phyllanthus amarus

Multidrug resistance (MDR) constitutes the major obstacle to the successful treatment of cancer. In several cancer cells, MDR is thought to be mediated by the super-expression of P-glycoprotein (Pgp). Pgp extrudes drugs from the cells, therefore reducing their cytotoxicity, and its activity inhibition may reverse the MDR phenotype. The present study evaluated the possible cytotoxic effect and MDR reversing properties of the extract and compounds isolated from Phyllanthus amarus. To this purpose, two human leukaemia cell lines were employed: K-562 and its vincristine-resistant counterpart Lucena-1, a Pgp-overexpressing subline. We report here that Lucena-1 was significantly more resistant to the cytotoxicity of P. amarus derivatives: the hexane extract (HE, 100 microg/mL), the lignans-rich fraction (LRF, 100 microg/mL) and the lignans nirtetralin (NIRT, 43.2 microg/mL), niranthin (NIRA, 43 microg/mL) or phyllanthin (PHYLLA, 43 microg/mL) exerted cytotoxic effects on K-562 cells with 40.3, 66.0, 62.0, 61.0 or 24.1% of cell death, respectively. The cellular toxicity observed on Lucena-1 was 16.3, 40.4, 29.4, 30.2, or 24.8%, respectively. However, cell treatment with the lignan phyltetralin (PHYLT) up to 41.6 microg/mL had no cytotoxic action on either of the cell lines. P. amarus derivatives were also found to be effective in inhibiting Pgp activity as assessed by rhodamine accumulation in Lucena-1 cells, as were the classical Pgp inhibitors, cyclosporine A (160 nM), PSC-833 (2 microM) and verapamil (5 microM). The lignan NIRT produced the most potent inhibition (EC (50) = 29.4 microg/mL) followed by NIRA (44.3 microg/mL), LRF (49.1 microg/mL), PHYLT (99.4 microg/mL), PHYLLA and HE (> 100 microg/mL). Lucena-1 cells were more resistant to daunorubicin-induced cell death (LC (50) = 50 microM) than K562 cells (LC (50) = 4.95 microM). Of note, the P. amarus derivatives significantly potentiated 5 microM daunorubicin-induced cell death in Lucena-1 cells (P < 0.01) but not in K562 cells. After treatment only with P. amarus derivatives (100 microg/mL HE, 30 microg/mL LRF, 12.9 microg/mL NIRA, 43.2 microg/mL NIRT, 43 microg/mL PHYLLA or 41.6 microg/mL PHYLT), the Lucena-1 cellular viability was 83.7, 85.3, 101, 69.7, 75.6 or 88.7%, respectively, whereas the in the presence of daunorubincin, which was not cytotoxic PER SE, the cell viability decreased to 42.9, 42.2, 64.2, 35.4, 30.4 or 52.6%, respectively. Together, these results suggest a potential action of P. amarus derivatives as MDR reversing agents, mainly due to their ability to synergize with the action of conventional chemotherapeutics.     

咯萘啶逆转肿瘤多药耐药及其作用机制

目的:利用mdr1~ 的人白血病和乳腺癌多药耐药(MDR)细胞系K562/A02和MCF-7/ADR研究咯萘啶(pyronaridine,PND)对MDR的逆转作用及其机制.方法:采用MTT法、荧光分光光度法、荧光显微镜法、流式细胞仪法和RT-PCR法分别测定PND单独或与阿霉素(DOX)合用,对肿瘤细胞的生长抑制、诱导凋亡、细胞内药物浓度、mdr1基因表达的影响.结果:PND对敏感及耐药细胞均具有生长抑制作用,半数抑制剂量(IC_(50))根据不同细胞在5.10-18.66μmol/L之间;低毒剂量PND显著增强DOX对耐药细胞的细胞毒和诱导凋亡作用,且增加DOX在耐药细胞内的蓄积及减少罗丹明(Rh123)的外排.RT-PCR结果显示,PND对mdr1基因无下调作用.结论:PND可作为第三代P-糖蛋白(P-gp)抑制剂,通过下调P-gp药物外排泵功能而产生强大的逆转MDR效应.     

Modulation by LY335979 of P-glycoprotein function in multidrug-resistant cell lines and human natural killer cells

Resistance to chemotherapy by some human tumors may be due to overexpression of membrane-associated transport proteins. The best characterized of these is the multidrug resistance (MDR) transporter, P-glycoprotein (Pgp). The aim of this study was to measure the inhibitory effects of a potent new MDR modulator, (2R)-anti-5-(3-[4-(10,11-difluoromethanodibenzo-suber-5-yl) piperazin-1-yl]-2-hydroxypropoxy)quinoline trihydrochloride (LY335979), in the drug-resistant cell line HL60/VCR and in normal, human CD56(+) lymphocytes. We used flow cytometric methods to detect the accumulation of rhodamine 123 and daunorubicin, fluorescent MDR substrates, in these cells. Our results indicate that LY335979 was 500-1500 times more potent than cyclosporin A or verapamil in restoring Pgp substrate accumulation in the MDR cell line HL60/VCR. Moreover, LY335979 could effectively block Pgp function on isolated CD56(+) lymphocytes (IC(50) = 1.2 nM) or CD56(+) lymphocytes in whole blood (IC(50) = 174 nM). We conclude that LY335979 is among the most potent Pgp inhibitors described and that it maintains significant potency in whole-human blood. These latter findings are important for establishing the dosing regimens of LY335979 for future clinical studies.     

Modulation of P-glycoprotein activity by the substituted quinoxalinone compound QA3 in adriamycin-resistant K562/A02 cells

QA3 is a derivative of the substituted 1,3-dimethyl-1H-quinoxalin-2-ones, which are compounds that may selectively antagonize P-glycoprotein (P-gp) in multidrug resistance (MDR) cancer cells. Our previous work identified QA3 as a candidate compound for reversing MDR in cancer cells. In the present study, we found that QA3 significantly decreases the intracellular level of ATP, stimulates ATPase activity in membrane microsomes and decreases protein kinase C (PKC) activity. These results indicated that QA3 inhibits P-gp activity by blocking ATP hydrolysis and ATP regeneration. Furthermore, QA3 triggered and increased adriamycin-induced K562/A02 cell apoptosis as evidenced by Annexin V-FITC plus PI staining. Western blot analysis showed that the levels of cleaved caspase-9 and cleaved caspase-3 proteins increased, and similarly, the levels of procaspase-9 and procaspase-3 decreased after QA3 treatment. Consequently, poly ADP-ribose polymerase (PARP) activity increased as evidenced by the presence of the PARP cleavage product in K562/A02 cells. QA3 also enhanced the potency of adriamycin against K562/A02 cells as demonstrated by increased apoptosis and activation of caspase-9,-3 and PARP. These data support the observation that P-gp activity is inhibited after QA3 treatment. Moreover, these results indicate that QA3 is a novel MDR reversal agent with potent inhibitory action against P-gp MDR cancer cells.     

Synthesis and evaluation of (2-(4-methoxyphenyl)-4-quinolinyl)(2-piperidinyl)methanol (NSC23925) isomers to reverse multidrug resistance in cancer

Development of multidrug resistance (MDR) during chemotherapy is a fundamental obstacle associated with cancer care. Prior studies have identified (2-(4-methoxyphenyl)-4-quinolinyl)(2-piperidinyl)methanol (5) (NSC23925) to be a small molecule agent that reverses MDR in cancer cells. We synthesized all four isomers of 5 and analyzed them by liquid chromatography-mass spectrometry (LCMS). Structure-activity relationships for reversing MDR were evaluated. Isomer 11 demonstrated the most potent activity. 11 reversed MDR in several drug-resistant cell lines expressing Pgp, including ovarian, breast, colon, uterine, and sarcoma cancer. 11 resensitized these cell lines to paclitaxel, doxorubicin, mitoxantrone, vincristine, and trabectedin with no effect on cell sensitivity to cisplatin, topotecan, and methotrexate. 11 significantly enhanced in vivo antitumor activity of paclitaxel in MDR xenograft models, without increasing the level of paclitaxel toxicity. In conclusion, 11 and derivatives of this compound may hold therapeutic value in the treatment of MDR-dependent cancers.     

Reversal of P—glycoprotein—mediated multidrug resistance by pyronaridine

An association between P-glycoprotein(Pgp) level and poor clinical outcome has been found.Efforts have been made to search for the modulators of tumor multidrug resistance (MDR) from the components of Chinese herbs and the molecules developed in China.Pyronaridine (PND)was found to be able to reverse MDR to doxorubicine(DOX) in K562/A02 and MCF7/ADR,expressing Pgp with more efficacy than verapamil.PDN increased the accumulation of DOX and reduced efflux of Rh123 in the two cell lines.The reversibility prersisted for at least 24h after removel of the drug from the culture medium.When administered orally or parenterally,PND significantly enhanced the in vivo antitumor activity of DOX in K562/A02 xenografts,but did not significantly increase the toxicity or alter the plasma pharmacokinetics of DOX.In view of PND has been safely used in clinic for the treatment of malaria for more than 20 years at high dose,the modulator might be the promision in the reversal of MDR in the clinic.     

C-7 analogues of progesterone as potent inhibitors of the P-glycoprotein efflux pump.

The P-glycoprotein product (Pgp) of the MDR1 gene has been implicated in the multiple drug resistance phenotype expressed by many cancers. Functioning as an efflux pump, P-glycoprotein prevents the accumulation of high intracellular concentrations of substrates. We have taken a rational approach to designing inhibitors of P-glycoprotein function, selecting a natural substrate (progesterone) as our lead compound. We hypothesized that progesterone, substituted at C-7 with an aromatic moiety(s), would exhibit reduced Pgp affinity, significantly increased antiPgp activity, and reduced affinity for progesterone receptors (PGR). We synthesized 7 alpha-[4'-(aminophenyl)thio]pregna-4-ene-3,20-dione (2), which comprises a C-7 alpha thiol bridge linking an aminophenyl moiety to progesterone, from pregna-4,6-diene-3,20-dione (1). The subsequent addition reaction of 2 with the appropriate isocyanate produced an initial series of compounds (3-6). Compounds 3-5 (respectively, -CH(2)CH(2)Cl; -CH(2)CH(3); and -CH(CH(3))C(6)H(5)) exhibit a significantly increased ability to inhibit P-glycoprotein. Potency for restoring doxorubicin accumulation in MDR1-transduced human breast cancer cells is increased up to 60-fold as compared with progesterone. Compound 5 has greater potency than verapamil and is equipotent with cyclosporin A, for inhibiting P-glycoprotein function. Furthermore, 5 does not bind to PGR, implying a potential reduction in in vivo toxicity. These data identify C-7-substituted progesterone analogues and 5, in particular, as rationally designed antiPgp compounds worthy of further evaluation/development.     

以K562细胞为靶点的抗肿瘤药物筛选模型的建立

目的：建立以白血病细胞系K562为靶点的高通量抗肿瘤药物筛选模型。方法：在96孔细胞培养板上,运用四唑氯化合物（MTS）和电子耦联剂（PMS）连用的方法,对K562细胞增殖情况进行检测。对在化合物影响下K562细胞增殖变化的检测条件进行了优化。结果：采用这一细胞水平的高通量抗肿瘤药物筛选模型,完成了800个小分子有机化合物的筛选,每个化合物的用量是500ng.11个化合物在浓度为5mg/L时可抑制细胞增殖达80％以上,其中9个通过多浓度复筛得到了确认。抑制活性最强的化合物的IC50为170nmol/L,共有7个化合物显示IC50低于10μmol/L。结论：采用K562细胞系进行高通量筛选是快速、经济、有效、实用的发现新型抗肿瘤药物的方法。     

Reversal of P-glycoprotein and multidrug resistance-associated protein 1 mediated multidrug resistance in cancer cells by HZ08 Isomers, tetrataisohydroquinolin derivatives

Overexpression of P-glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1) by tumors results in multidrug resistance (MDR) to structurally unrelated anti-tumor agents. HZ08, a chiral compound, was a newly synthesized tetraisohydroquinoline derivative to reverse Pgp and MRP1 mediated MDR. In present studies, R, S-HZ08 and their racemate reversed the resistance to adriamycin and vincristine of adriamycin-selected human leukemia (K562/ADM) cells that overexpress Pgp. R, S-HZ08 and their racemate modulated adriamycin cytotoxicity when R, S-HZ08 and their racemate were removed 12 h prior to the cytotoxicity assay. In addition, R, S-HZ08 and their racemate increased intracellular accumulation of Rhodamine123 in Caco-2 cells that overexpress Pgp. Furthermore, using a DNA content analysis and an annexin V binding assay, R, S-HZ08 and their racemate effectively reversed the resistance to adriamycin-induced apoptosis in K562/ADM cells. R, S-HZ08 and their racemate also moderately reversed the resistance to adriamycin and vincristine of MCF-7/ADM cells that overexpress MRP1. However, R, S-HZ08 and their racemate hardly affected intracellular glutathione (GSH) levels and glutathione S-transferase (GST) activities in MCF-7/ADM cells. The result showed that R, S-HZ08 and their racemate possibly reverse MDR1 mediated multidrug resistance by a direct interaction with MRP1, not interaction with MRP1 via GSH. Thus, R, S-HZ08 and their racemate should be useful for treating patients with tumors that overexpress both Pgp and MRP1.     

Design and synthesis of new symmetrical derivatives of dihydropyridine containing a pyridyl group on the 3, 5-positions and evaluation of their cytotoxic and multidrug resistance reversal activity

Today, chemotherapy is an important part in the treatment of several kinds of cancer; however, the development of drug resistance remains one of the major obstacles in successful chemotherapy. Several types of agents have been recognized as multidrug resistance (MDR) inhibitors, among which the 1,4-dihydropyridines (DHPs) have been investigated the most. P-glycoprotein inhibition has been reported as the main MDR reversal mechanism of DHPs, whilst other mechanisms such as inhibition of topoisomerase II have received less attention. Therefore, in this study new derivatives of DHP have been synthesized. Their cytotoxic activity and their effects in reversing atypical MDR have been evaluated. The results confirmed the appropriate effect of these compounds on atypical MDR. Although it was observed that these compounds had a moderate cytotoxic effect, the cytotoxicity of one compound on the K562 cell line (IC50 = 6.61 microM) was comparable with that of doxorubicin (IC50 = 4.17 microM). Finally, the Ca(2+)-channel antagonistic activity, an undesired effect for these compounds, was evaluated.     

Resistance of CHO cells expressing P-glycoprotein to cyclopropylpyrroloindole (CPI) alkylating agents.

Several new antitumor agents belonging to the class of minor groove binders that are able to form covalent bonds with DNA via a cyclopropylpyrroloindole (CPI) group are susceptible to a multidrug resistance (MDR) phenotype in Chinese hamster ovary (CHO) cells. The multidrug resistant CCHR-C5 cell line was 16-, 23- and 13-fold more resistant to the analogs U-73,975, U-77,779 and U-80,244, respectively, although its cytotoxic response to the parent compound CC-1065 was similar to the response of the drug-sensitive wild-type cells (AuxB1). For a sequence of MDR cell lines showing increasing expression of P-glycoprotein (Pgp) there were corresponding increments in the level of resistance to U-73,975, arguing that Pgp is the key determinant in resistance of the MDR cells to CPI agents. MDR cells treated with U-73,975 showed diminished generation of covalent adducts on DNA as well as increased resistance to cytotoxicity.     

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.     

Reversal of vinblastine resistance in human leukemic cells by haloperidol and dihydrohaloperidol.

Haloperidol, an antipsychotic, was investigated in cells overexpressing P-glycoprotein to detemine whether it was a clinically effective drug to reverse for reversing multidrug resistance (MDR) mediated by P-glycoprotein. A nontoxic concentration of haloperidol (1-30 microM) enhanced the cytotoxic effects of vinblastine (VBL) concentration-dependently in VBL-resistant human leukemia (K562/VBL) cells, but had no effect in the parent cells. Haloperidol also enhanced the cytotoxicities of epirubicin, doxorubicin and actinomycin D in the K562/VBL cells, but not those of idarubicin or cisplatin; this enhancement was less than that of the VBL toxicity in the VBL-resistant tumor line. Haloperidol increased the intracellular accumulation of VBL in the K562/VBL cells, and the binding of [3H]-azidopine to the cell-surface protein, P-glycoprotein, was inhibited by haloperidol in a concentration-dependent manner. Haloperidol was less potent than verapamil. Thus, haloperidol appeared to potentiate anticancer agents through the reversal of MDR by competitively inhibiting drug-binding to P-glycoprotein. In contrast, the main metabolite of haloperidol, dihydrohaloperidol, without antipsychotic activity, had less of an effect. Therefore, haloperidol might be useful in reversing drug-resistance.     

Aureobasidins: structure-activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC-transporter

Cyclic depsipeptide cyclo-[D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([D-Hiv(1)]-AbA), AbC ([Val(6)]-AbA), and AbD [gammaHOMeVal(9)]-AbA). The replacement of the [Phe(3)-MePhe(4)-Pro(5)] tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the [D-Hmp(1)-L-MeVal(2)-L-Phe(3)] and [L-aIle(6)-L-MeVal(7)-L-Leu(8)-] tripeptides, does not critically depend on the occurrence of the [L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)] type II' beta-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [betaHO-MeVal(9)] residue alterations, with some data suggesting a negative impact of the [L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)] gamma-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal(9)]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.     

Effects of isoprenoid analogues of SDB-ethylenediamine on multidrug resistant tumor cells alone and in combination with chemotherapeutic drugs

Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) remains the major obstacle for successful treatment of cancer. Inhibition of Pgp transport is important for higher efficacy of anticancer drugs. Lipophilic cationogenic amines with at least one tertiary N atom, such as verapamil, are classical PgP-blocking agents. In a search for novel accessible compounds potent against MDR tumor cells, we synthesized a series of arylalkylamines that contain isoprenoid side chains of different length. Two out of seven new analogues of the known N,N'-bis(3,4-dimethoxybenzyl)-N-solanesylethylenediamine (SDB-ethylenediamine), namely, compounds with C10 and C15 side chains, at low micromolar concentrations were preferentially toxic for several mammalian tumor cell lines that acquired MDR during prolonged drug selection. Moreover, at noncytotoxic concentrations, these compounds potently sensitized MDR cells to Pgp substrates vinblastine and adriamycin. We conclude that these analogues of SDB-ethylenediamine may have dual therapeutic advantage because (i) they are preferentially toxic for MDR cells when administered alone and (ii) they potentiate the cytotoxicity of Pgp-transported anticancer drugs.     

Structural Optimization of Indole Derivatives Acting at Colchicine Binding Site as Potential Anticancer Agents

相似文献