ANTICANCER RESEARCH 25: 2857-2868 (2005)
Biological Evaluation of ˆ-(Dialkylamino)alkyl Derivatives of 6H-indolo[2,3-b]quinoline – Novel Cytotoxic DNA Topoisomerase II Inhibitors JOANNA GODLEWSKA1,2, WOJCIECH LUNIEWSKI1, BOGDAN ZAGRODZKI1, LUKASZ KACZMAREK1, ALEKSANDRA BIELAWSKA-POHL2, DANUTA DUS2, JOANNA WIETRZYK2, ADAM OPOLSKI2,6, MAGDALENA SIWKO3, ANNA JAROMIN3, ANNA JAKUBIAK5, ARKADIUSZ KOZUBEK3 and WANDA PECZYÑSKA-CZOCH4 1Pharmaceutical
Research Institute, 8 Rydygiera St., 01-783 Warszawa; of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigla St., 53-114 Wroclaw; 3Institute of Biochemistry and Molecular Biology, University of Wroclaw, 63/77 Przybyszewskiego St., 51-148 Wroclaw; 4Institute of Organic and Polymer Technology and 5Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology, 27 Wybrzeze Wyspiañskiego St., 50-370 Wroclaw; 6J. Dlugosz Academy, Al. Armii Krajowej 13/15, 42-201 Czestochowa, Poland 2Institute
Abstract. A series of novel 6H-indolo[2,3-b]quinoline derivatives, substituted at C-2, C-9 or N-6 position with dialkyl(alkylamino)alkyl chains differing in the number of methylene groups, was prepared. These compounds were evaluated in vitro for their antimicrobial and cytotoxic activity against several cell lines of different origin and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. Liphophilic and calf thymus DNA-binding properties of these compounds were also investigated. All the compounds tested inhibited the growth of Gram-positive bacteria and fungi at MIC values ranging between 0.25 and 1 mM. They also showed cytotoxic activity against KB (human cervix carcinoma) cells (ID50 varied from 2.1 to 9.0 ÌM) and were able to overcome multidrug resistance in colorectal adenocarcinoma LoVo/DX, uterine sarcoma MES-SA/DX5 and promyelocytic leukemia HL-60/MX2 cells (the values of the resistance index RI fell between 0.54 and 2.4). The compounds induced G2M-phase cell cycle arrest in Jurkat T-cell leukemia cells, revealed DNA-binding properties and inhibited topoisomerase II activity. Indolo[2,3-b]quinolines are a group of synthetically obtained analogues of the natural alkaloid, neocryptolepine. They share many biological properties with this compound,
Correspondence to: Adam Opolski, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigla St., 53-114 Wroclaw, Poland. e-mail:
[email protected] Key Words: Indolo[2,3-b]quinoline, cell cycle, topoisomerase II, MDR, DNA-binding, cytotoxicity, lipophilicity, antimicrobial.
0250-7005/2005 $2.00+.40
including the ability to interact with DNA as intercalators and to inhibit topoisomerase II activity. The indolo[2,3b]quinoline derivatives also revealed antimicrobial and cytotoxic potential (17-19). Our previous research on 6H-indolo[2,3-b]quinolines was directed towards the synthesis and characterization of derivatives bearing three kinds of substituents: alkyl-, (alkylamino) alkyl- and 4-(3-chlorophenyl) piperazi-1-ylpropyl at the N-6 position. We found that only the presence of an (alkylamino) alkyl chain introduced into the indolo[2,3-b]quinoline core would be a suitable structural requirement for cytotoxic activity, antimicrobial properties and topoisomerase II inhibition (1). As a part of our programme, which aimed at exploring the effect of the introduction of (alkylamino) alkyl chains of different length, we synthesized novel 6H-indolo[2,3b]quinoline derivatives bearing (alkylamino) alkyl substituents at the C-2, C-9 or N-6 positions. The obtained compounds were evaluated in vitro for their cytotoxicity against the KB cervical cancer cell line, as well as against three other cancer cell lines and their drug-resistant sublines: human colorectal adenocarcinoma LoVo and doxorubicin-resistant LoVo/Dx (P-gp-dependent1-, MRP2-, LRP3-dependent multidrug resistance), human uterine sarcoma MES-SA and
1P
- glycoprotein-dependent; - multidrug resistance-related protein; 3LRP - lung resistance-related protein; 2MRP
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ANTICANCER RESEARCH 25: 2857-2868 (2005) MES-SA/DX5 (P-gp- dependent resistance to doxorubicin) and human acute promyelocytic leukemia HL-60 and mitoxanthrone-resistant HL-60/ MX-2 (P-gp-independent and topoisomerase II-dependent MDR4). The antimicrobial activities of these compounds were determined against procaryotic organisms represented by Gram-negative and Gram-positive bacteria and against yeasts used as an example of eucaryotic organisms. All the indoloquinolines were also examined for their ability to influence the cell cycle using Jurkat acute T-cell leukemia cells. The interaction of the tested compounds with calf thymus DNA, the stability of the indoloquinoline complexes with DNA and the ability of indoloquinolines to inhibit topoisomerase II activity were assessed.
Materials and Methods Instrumentation. Melting points (m.p.’s), determined on a Koflertype equipment, were uncorrected. The IR spectra were recorded with a Perkin-Elmer 1640 FTIR spectrometer in KBr pellets. The 1H NMR spectra were measured with a Varian Gemini 200 MHz spectrophotometer in CDCl; the chemical shifts were expressed in ppm (‰) with TMS as an internal standard. The MS spectra were recorded with an Inectra AMD-604 equipment (–70eV). The elemental analyses were performed by the Analytical Laboratory, Institute of Organic Chemistry of the Polish Academy of Sciences. UV absorption spectra were taken with the Cary 3 UV-VIS Varian Spectrometer. The purity and identity of the products were checked by thin layer chromatography (TLC) with Merck DCAlufolien Kieselgel 60 F254. Column chromatography was performed on Merck silica gel (230-400 mesh). The chemicals and solvents were purchased from Sigma-Aldrich. Determination of antimicrobial activity. The antimicrobial activity of the indoloquinolines was determined by establishing their minimal inhibitory concentration (MIC) against pro- (Escherichia coli; Micrococcus luteus) and eucaryotic (Saccharomyces cerevisiae) microorganisms with the serial dilution method (2). Antiproliterative assay in vitro Cell lines: We applied human cervix carcinoma KB, human colorectal adenocarcinoma LoVo and LoVo/DX, human uterine sarcoma MES-SA and MES-SA/DX5, human acute promyelocytic leukemia HL-60 and HL-60/MX2 and human acute T-cell leukemia Jurkat cell lines, all established in vitro. The MES-SA, MESSA/DX5, HL-60/MX2 and LoVo cell lines were obtained from the American Type Culture Collection (Rockville, Maryland, USA).
4atypical
multidrug resistance (with the absence of P-glycoprotein overexpression and altered topoisomerase II catalytic activity as well as reduced levels of topoisomerase II alpha and beta proteins.
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The LoVo/DX cell line was kindly provided by Prof. E. Borowski (Technical University of Gdan’ sk, Poland) and the HL-60 cell line was obtained from the European Type Culture Collection by courtesy of Prof. Spik and Dr. Mazurier (Laboratory of Biological Chemistry USTL, Lille, France). All the lines were maintained in the Cell Culture Collection of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland. The cells were cultured in RPMI 1640 + Opti-MEM (1:1) (KB, LoVo LoVo/DX, MES-SA, MES-SA/DX5) or RPMI 1640 (Jurkat, HL-60, HL-60/MX2) medium supplemented with 2 mM glutamine (Sigma-Aldrich Chemie GmbH), streptomycin (100 mg/ml), penicillin (100 U/ml) (both antibiotics from Polfa, Tarchomin, Poland), 1 mM sodium pyruvate (Sigma-Aldrich Chemie GmbH) (HL-60, HL-60/MX2), 4.5 g/l glucose (POCh, Gliwic, Poland) (HL-60/MX2) and 5% (KB, LoVo LoVo/DX, MES-SA, MES-SA/DX5) or 10% (Jurkat, HL-60, HL-60/MX2) fetal calf serum (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The cell cultures were maintained at 37ÆC in a humid atmosphere saturated with 5% CO2. Cytotoxicity assays: Test solutions of the compounds (1 mg/ml) were prepared by dissolving the substances in 100 Ìl of DMSO (POCh) completed with 900 Ìl of tissue culture medium. Afterwards, the test compounds were diluted in culture medium to reach the final concentrations of 100, 10, 1 and 0.1 Ìg/ml. The cytotoxicity assay for adherent cells (solid cancers) was performed using the SRB method (3), while the MTT procedure was applied for non-adherent leukemia cells (4). Twenty-four hours before addition of the test agents, the cells were plated in 96-well plates (Sarstedt - Sarsted Inc., Newton, USA – plates for adherent cells and Costar - Corning Inc., Corning, USA – plates for non-adherent cells) at a density of 1x104 cells per well. The cytotoxicity assay was performed after 72-hour exposure of the cultured cells to varying concentrations (from 0.1 to 100 Ìg/ml) of the test agents. The SRB assay: The cells attached to the plastic were fixed by gently layering cold 50% TCA (trichloroacetic acid, SigmaAldrich Chemie GmbH) on the top of the culture medium in each well. The plates were incubated at 4ÆC for 1 hour and then washed 5 times with tap water. The background optical density was measured in the wells filled with culture medium, without the cells. The cellular material fixed with TCA was stained with 0.4% sulforhodamine B (SRB, Sigma-Aldrich Chemie GmbH) and dissolved in 1% acetic acid (POCh) for 30 minutes. Unbound dye was removed by rinsing (4x) with 1% acetic acid. The protein-bound dye was extracted with 10 mM unbuffered Tris base (POCh). The MTT assay: After 24 hours of incubation, 20 Ìl of the tetrazolium salt (MTT, Sigma-Aldrich Chemie GmbH) (5 mg/ml) were added to each well and the samples were incubated for a further 4 hours. Next, 80 Ìl of lysing mixture (225 ml of DMF, 67.5 g of SDS, 275 ml of distilled water) were added to each well for 24 hours. For the determination of optical density (at 540 and 570 nm for the SRB and MTT procedure, respectively), a computer-interfaced, 96-well microtiter plate reader Multiskan RC photometer (Labsystems, Helsinki, Finland) was applied. Each compound at the given concentration was tested in triplicate in each experiment. Each of the experiments was repeated 3 to 5 times. The results converted into ÌM concentrations were expressed as ID50 – the dose of the compound (in ÌM) that inhibits the proliferation rate of the tumor cells by 50% as compared to control untreated cells.
Godlewska et al: Novel Cytotoxic DNA Topoisomerase II Inhibitors
Table I. Structures of the 6H-indolo[2,3-b]quinolines tested.
Compound
Chemie GmbH) (1 Ìg/ml, 30 minutes, 37ÆC) and propidium iodide (Sigma Chemical Co., St. Louis, USA) (50 Ìg/ml, 30 minutes, 4ÆC). The Beckton Dickinson FACSCalibur cytofluorimeter and CELLQuest software were used for data collection (488 nm), while ModFit software was used for analysis.
R1
R2
R3
23
-NH(CH2)2 N(CH3)2
-H
-CH3
24
-H
-NH(CH2)2 N(CH3)2
-CH3
25
-NH(CH2)3 N(CH3)2
-H
-CH3
26
-H
-NH(CH2)3 N(CH3)2
-CH3
27
-NH(CH2)2 N(C2H5)2
-H
-CH3
28
-H
-NH(CH2)2 N(C2H5)2
-CH3
29
-H
-H
-NH(CH2)2 N(CH3)2
30
-H
-H
-NH(CH2)3 N(CH3)2
31
-H
-H
-NH(CH2)2 N(C2H5)2
RI (resistance index) values estimation: RI values were calculated according to the formula RI=(ID50 estimated against resistant cell line) / (ID50 estimated against non-resistant cell line); values range: 0
