Coumarin Derivatives with Tumor-specific Cytotoxicity and ... - In Vivo

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Both 6-hydroxy-7-methoxy-4-methyl-3- isopropylcoumarin [C43] and 3-ethyl-6-hydroxy-7-methoxy-4- methylcoumarin [C44] showed the highest tumor-specific.
in vivo 19: 705-712 (2005)

Coumarin Derivatives with Tumor-specific Cytotoxicity and Multidrug Resistance Reversal Activity MASAMI KAWASE1, HIROSHI SAKAGAMI2, NOBORU MOTOHASHI3, HERMANN HAUER4, SHYAM S. CHATTERJEE4, GABRIELLA SPENGLER5, ANIKO VARADI VIGYIKANNE5, ANNAMARIA MOLNAR5 and JOSEPH MOLNAR5 1Faculty

of Pharmaceutical Sciences, Josai University, Saitama; of Dental Pharmacology, Meikai University School of Dentistry, Saitama; 3Meiji Pharmaceutical University, Kiyose, Tokyo, Japan; 4Department of Research and Development, Dr. Willmar Schwabe GmbH & Co. KG, Willmar-Schwabe Str. 4, 76227 Karlsruhe, Germany; 5Faculty of Medicine, Institute of Medical Microbiology, Albert Szent-Gyorgyi Medical Centrum, University of Szeged, Szeged, Dom ter 10, H-6720, Hungary 2Department

Abstract. A preliminary exploration of coumarin derivatives as novel multidrug resistance (MDR) modulators was carried out to determine the basic features of the structure responsible for the MDR reversal activity. Among 44 coumarins, 14 compounds moderately induced reversal of MDR (fluorescence activity ratio (FAR) values >1). The most active compound, 6-hydroxy-3-(2-hydroxyethyl)-4-methyl-7-methoxycoumarin [C34], was equally potent as a MDR modulator verapamil. These data show a relationship between the chemical structure and MDR-reversal effect on tumor cells. All coumarins tested were more cytotoxic against tumor cells than normal cells. Several compounds displayed potent cytotoxic activities (CC50 15-29 Ìg/mL in HSC cells), comparable with that of gallic acid (CC50=24 Ìg/mL). Both 6-hydroxy-7-methoxy-4-methyl-3isopropylcoumarin [C43] and 3-ethyl-6-hydroxy-7-methoxy-4methylcoumarin [C44] showed the highest tumor-specific cytotoxicity (SI value=4.1 and 3.6, respectively). We conclude that coumarins are potentially potent new MDR modulators with low toxicity against normal cells. A deeper understanding of the relationship between their structures and their potency will contribute to the design of optimal agents. Although recent developments in molecular cancer chemotherapeutics has been successful and encouraging,

Correspondence to: Dr. Masami Kawase, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 3500295, Japan. Tel: (+)81-492-86-2233 (Ext. 455), Fax: (+)81-49271-7984, e-mail: [email protected] Key Words: Coumarin, cytotoxic activity, multidrug resistance, oral tumor cells, structure-activity relationship.

0258-851X/2005 $2.00+.40

effectiveness has often been limited by cytotoxic effects on normal tissues and by drug resistance of tumors. Our studies have focused on molecular cancer chemotherapeutics, mainly in the areas of drug resistance and tumor-specific cytotoxic drugs. Multidrug resistance (MDR) of human tumors is one of the major reasons for the failure of chemotherapy in refractory cancer patients (1). Reversal of MDR has been accomplished by a number of agents such as verapamil and cyclosporin A (2, 3). Unfortunately, the concentration of many of these agents necessary to reverse drug resistance is difficult to achieve in vivo (4). Thus, there is considerable interest in the search for new P-glycoprotein (P-gp) inhibitors that do not show significant toxicity at doses required for P-gp inhibition. Coumarins constitute a major class of widely distributed O-heterocyclic natural products isolated from citrus fruits and vegetables (5). Naturally occurring coumarins possess a variety of biological activities, including antitumor activity (6). In contrast, little information is available on the MDRmodulating potency of simple coumarins, which are considered to exert low mammalian toxicity. Due to the presence of coumarins in the human diet and medicinal plants, a study on the MDR modulatory effects of coumarins is of significance (7). Three coumarin derivatives, 6-methylcoumarin, 7-methylcoumarin and ethyl 3-coumarincarboxylate, were not effective on the MDR efflux pump P-gp of mouse lymphoma cells in vitro (8). 7-Hydroxycoumarin and 8-nitro-7-hydroxycoumarin were shown to be potent cytotoxic agents against the human renal cell carcinoma cell line. However, these compounds were not a substrate for P-gp-mediated MDR (9, 10). Pyranocoumarin derivatives, racemic cis-3’-angeloyl-4’-acetoxy-khellactone,

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in vivo 19: 705-712 (2005) were suggested to possess the ability to circumvent the MDR phenotype conferred by the overexpression of P-gp (11). In drug combination studies, the pyranocoumarins increased intracellular doxorubicin accumulation in the P-gpoverexpressing human oral epidermoid carcinoma cell line KB-V1, but not in the drug-sensitive human oral epidermoid carcinoma cell line KB-3-1. In our previous paper, coumarin derivatives [C1-23] were screened for their cytotoxic activity against human tumor cells and some compounds were found to exhibit potent cytotoxic activity (12). These studies led to the identification of a 6,7-dihydroxycoumarin derivative as a lead molecule with tumor cell-specific cytotoxicity. It is also suggested that proper substitution at the 3 and/or 4 positions of the molecule makes it possible to design more cytotoxic agents. In a continuing search for potent and selective cytotoxic antitumor agents, we prepared another 22 coumarin derivatives [C24-45] and evaluated their cytotoxic effects against human oral tumor cells. In the present work, we also investigated the MDR reversal activities of 45 coumarins [C1-45] against mouse lymphoma cells transfected with the human MDR 1 gene.

Materials and Methods General. Melting points were determined by an Electrothermal or a Büchi B-545 instrument. 1H- and 13C-NMR spectra were determined using a Bruker AC 200 or Avance 200 spectrometer (200 MHz for 1H and 50.3 MHz for 13C both) in DMSO-d6. The chemical shifts refer to TMS (1H-NMR) or to DMSO-d6 (13C-NMR, ‰=39.5 ppm), respectively. Combustion analyses were carried out on "Mikroanalytisches Labor Hein", Moembris, Germany. TLC (Thin layer chromatography) was performed using Merck Kieselgel 60 F254 (Merck 5549, USA). Materials. The following chemicals were obtained from each indicated company: 6-nitro-3,4-benzocoumarin [C25], 7methoxycoumarin-4-acetic acid [C26], 4-hydroxy-3-nitrocoumarin [C27], ethyl coumarin-3-carboxylate [C28] and 4-trifluoromethyl-7dimethylaminocoumarin [C32] (Tokyo Kasei Co., Tokyo, Japan); Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY, USA); fetal bovine serum (FBS) (JRH Biosci., Lenexa, KS, USA). Coumarins [C1-23] were previously synthesized (13). Preparation of coumarins [C24, 29-31, 33-44]. The following coumarins were prepared by the Pechmann reaction (14), unless otherwise noted. One equivalent of the (2-substituted) ‚-ketoester was added dropwise to a solution of the appropriate phenol in sulfuric acid (75% in water) at 0 to 25ÆC. The mixture was kept at room temperature for at least 30 min and was then placed on ice or diluted with ice/water. The product was filtered off and recrystallized. 7,8,9,10-Tetrahydro-2-hydroxy-3-methoxy-6H-dibenzo[b,d]pyran6-one [C24]: yield 86%; mp. 184-186ÆC (2-propanol) (15). 6-Hydroxy-7-methoxy-4-phenyl-2H-1-benzopyran-2-one [C29]: yield 77%; mp. 209-210ÆC (2-propanol/methanol)(16).

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6-Hydroxy-7-methoxy-4-methyl-3-phenyl-2H-1-benzopyran-2-one [C30]: yield 99%; mp. 258-260ÆC (ethanol); 1H NMR (DMSO-d6, 200 MHz) ‰: 2.17 (s, 3H), 3.90 (s, 3H), 7.05 (s, 1H), 7.11 (s, 1H), 7.27-7.48 (m, 5H), 9.40 (s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 16.4 (CH3), 56.1 (CH3), 99.7 (CH), 109.7 (CH), 112.7 (C), 123.2 (C), 127.6 (CH), 128.0 (CH x 2), 130.3 (CH x 2), 135.0 (C), 143.5 (C), 146.7 (C), 147.8 (C), 151.3 (C), 160.4 (C). Anal. Calculated for C17H14O4: C, 72.33; H, 5.00. Found: C, 72.09; H, 4.93. 6-Hydroxy-7-methoxy-4-trifluoromethyl-2H-1-benzopyran-2-one [C31]: purification by chromatography over silica gel before recrystallization; yield 13%; mp. 190-192ÆC (TBME/petrol ether); 1H NMR (DMSO-d , 200 MHz) ‰: 3.90 (s, 3H), 6.82 (s, 1H), 7.05 6 (q, 1H, J=2.0 Hz), 7.19 (1H, s), 9.81 (1H, s). 13C NMR (DMSOd6, 50.3 MHz) ‰: 56.3 (CH3), 100.9 (CH), 105.4 (C), 108.0 (CH), 112.8 (CH), 121.8 (CF3), 139.4 (C), 144.2 (C), 149.1 (C), 152.8 (C), 159.0 (C). Anal. Calculated for C11H7F3O4: C, 50.78; H, 2.71. Found: C, 50.33; H, 2.57. 7-Hydroxy-6-{3-[4-(2-methoxyphenyl)-1-piperazinyl]propoxy}3,4-dimethyl-2H-1-benzopyran-2-one [C33]: step1: (6-Hydroxy-3,4dimethyl-7-(phenylmethoxy)-2H-1-benzopyran-2-one): 48.5 mmol 6,7-dihydroxy-3,4-dimethyl-2H-1-benzopyran-2-one [C16], 53.4 mmol potassium carbonate and 48.5 mmol benzyl bromide were stirred for 18 h at room temperature in 80 mL DMF. The mixture was filtered. The product crystallized from the filtrate by concentration; yield 43%; mp 216.5-217ÆC (ethanol). Anal. Calculated for C18H16O4: C, 72.96; H, 5.44. Found: C, 73.07; H, 5.58. step2: (6-{3-[4-(2-Methoxyphenyl)-1-piperazinyl]propoxy}-3,4dimethyl-7-(phenylmethoxy)-2H-1-benzopyran-2-one: A solution of 23.8 mmol potassium hydroxide in 100 mL ethanol was added to 11.9 mmol 1-(3-chloropropyl)-4-(2-methoxyphenyl)piperazine dihydrochloride in 200 mL ethanol. 11.9 mmol potassium carbonate, 2 mmol potassium iodide, 10.8 mmol 6-hydroxy-3,4dimethyl-7-(phenylmethoxy)-2H-1-benzopyran-2-one and 200 mL ethanol were added. The mixture was refluxed for 30 h. The precipitate formed on cooling to room temperature was filtered off, purified by chromatography over silica gel (acetone) and recrystallized; yield 69%; mp. 135-135.5ÆC (ethanol/TBME); Anal. Calculated for C32H36N2O5: C, 72.70; H, 6.86; N, 5.30. Found: C, 72.68; H, 6.95; N, 5.13. step 3: (7-Hydroxy-6-{3-[4-(2methoxyphenyl)-1-piperazinyl]propoxy}-3,4-dimethyl-2H-1benzopyran-2-one [C33]: 1.9 mmol 6-{3-[4-(2-Methoxyphenyl)-1piperazinyl]propoxy}-3,4-dimethyl-7-(phenylmethoxy)-2H-1benzopyran-2-one were dissolved in 150 mL acetone and hydrogenated in the presence of 500 mg Pd/C (5 %) at room temperature (16 h; 8 bar). The mixture was filtered, evaporated, purified by chromatography over silica gel (ethyl acetate/ethanol 95/5) and recrystallized; yield 82%; mp. 168-168.5ÆC (ethanol/petrol ether); 1H NMR (DMSO-d6, 200 MHz) ‰: 1.94 (quint., 2H, J=6.7), 2.05 (s, 3H), 2.34 (s, 3H), 2.57 (m, 6H), 3.00 (m, 4H), 3.77 (s, 3H), 4.07 (t, 2 H, J=6.1 Hz), 6.76 (s, 1H), 6.856.95 (m, 4H), 7.21 (s, 1H), 10.6 (br s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 12.9 (CH3), 15.1 (CH3), 25.8 (CH2), 49.1 (CH2 x 2), 53.0 (CH2 x 2), 55.0 (CH2), 55.3 (CH3), 69.0 (CH2), 102.8 (CH), 110.4 (CH), 111.7 (C), 116.9 (C), 111.9 (CH), 117.8 (CH), 120.8 (CH), 122.3 (CH), 141.1 (C), 144.3 (C), 147.0 (C), 147.6 (C), 151.3 (C), 151.9 (C), 161.5 (C). Anal. Calculated for C25H30N2O5: C, 68.47; H, 6.90; N, 6.39. Found: C, 68.26; H, 6.92; N, 6.22. 6-Hydroxy-3-(2-hydroxyethyl)-7-methoxy-4-methyl-2H-1benzopyran-2-one [C34]: starting material 2-acetyl-Á-butyrolactone; yield 54%; mp. 229-235ÆC (methanol)(17).

Kawase et al: Structure-activity Relationships of Coumarins

7,8-Dihydroxy-3-(2-hydroxyethyl)-4-methyl-2H-1-benzopyran-2one [C35]: starting material 2-acetyl-Á-butyrolactone; yield 66%; mp. 221-222ÆC (ethanol/water)(18). 7-Hydroxy-3-(2-hydroxyethyl)-6-methoxy-4-methyl-2H-1benzopyran-2-one [C36]: starting material 2-acetyl-Á-butyrolactone; yield 70%; mp. 225-227ÆC (dec.)(2-propanol); 1H NMR (DMSOd6, 200 MHz) ‰: 2.40 (s, 3H), 2.72 (t, 2H, J=6.9 Hz), 3.50 (td, 2H, J=6.7, 5.9 Hz), 3.86 (s, 3H), 4.68 (t, 1H, J=5.5 Hz), 6.74 (s, 1H), 7.13 (s, 1H), 10.12 (s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 15.2 (CH3), 30.9 (CH2), 56.2 (CH3), 59.4 (CH2), 102.5 (CH), 107.1 (CH), 111.9 (C), 118.6 (C), 145.1 (C), 147.2 (C), 148.4 (C), 150.0 (C), 161.3 (C). Anal. Calculated for C13H14O5: C, 62.39; H, 5.64. Found: C, 62.32; H, 5.71. 5,7-Dihydroxy-3-(2-hydroxyethyl)-4-methyl-2H-1-benzopyran-2one [C37]: starting material 2-acetyl-Á-butyrolactone; yield 89%; mp. 243-245ÆC (ethanol/water)(17). 2-(6-Hydroxy-7-methoxy-2-oxo-2H-1-benzopyran-4-yl)acetic acid methylester [C38]: yield 11%; mp. 186.5-188.5ÆC (methanol); 1H NMR (DMSO-d6, 200 MHz) ‰: 3.66 (s, 3H), 3.88 (s, 3H), 3.92 (s, 2H), 6.30 (s, 1H), 6.96 (s, 1H), 7.05 (s, 1H), 9.44 (s, 1 H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 36.9 (CH2), 52.2 (CH3), 56.1 (CH3), 100.2 (CH), 109.1 (CH), 111.5 (C), 113.2 (CH), 143.5 (C), 147.9 (C), 149.1 (C), 151.8 (C), 160.3 (C), 169.6 (C). Anal. Calculated for C13H12O6: C, 59.09; H, 4.58. Found: C, 58.52; H, 4.72. 7-Ethoxy-6-hydroxy-3,4-dimethyl-2H-1-benzopyran-2-one [C39]: yield 72%; mp. 198-200ÆC (ethanol/water 9/1); 1H NMR (DMSOd6, 200 MHz) ‰: 1.38 (t, 3H, J=7.0 Hz), 2.05 (s, 3H), 2.28 (s, 3H), 4.10 (q, 2H, J=6.9 Hz), 6.92 (s, 1H), 7.04 (s, 1H), 9.19 (s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 12.9 (CH3), 14.4 (CH3), 14.9 (CH3), 64.2 (CH2), 100.3 (CH), 109.1 (CH), 112.7 (C), 117.8 (C), 143.4 (C), 145.8 (C), 146.4 (C), 149.6 (C), 161.5 (C). Anal. Calculated for C13H14O4: C, 66.66; H, 6.02. Found: C, 66.47; H, 5.93. 6-Hydroxy-7-methoxy-4-propyl-2H-1-benzopyran-2-one [C40]: yield 68%; mp. 163-164ÆC (2-propanol/ethanol); 1H NMR (DMSO-d6, 200 MHz) ‰: 0.98 (t, 3H, J=7.3 Hz), 1.64 (sext, 2H, J=7.5 Hz), 2.66 (t, 2H, J=7.5 Hz), 3.88 (s, 3H), 6.12 (s, 1H), 7.01 (s, 1H), 7.09 (s, 1H), 9.36 (s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 13.7 (CH3), 21.1 (CH2), 33.1 (CH2), 56.1 (CH3), 100.2 (CH), 108.8 (CH), 110.3 (CH), 111.5 (C), 143.4 (C), 147.9 (C), 151.5 (C), 156.4 (C), 160.6 (C). Anal. Calculated for C13H14O4: C, 66.66; H, 6.02. Found: C, 66.26; H, 5.78. 6-Hydroxy-7-methoxy-4-(1-methylethyl)-2H-1-benzopyran-2-one [C41]: purification by chromatography over silica gel before recrystallisation; yield 38%; mp. 126-127ÆC (TBME/2-propanol); 1H NMR (DMSO-d , 200 MHz) ‰: 1.24 (d, 6H, J=6.8 Hz), 3.20 6 (sept, 1H, J=6.8 Hz), 3.88 (s, 3H), 6.12 (s, 1H), 7.03 (s, 1H), 7.15 (s, 1H), 9.37 (s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 21.4 (CH3 x 2), 28.2 (CH), 56.0 (CH3), 100.3 (CH), 107.4 (CH), 108.5 (CH), 110.9 (C), 143.4 (C), 147.9 (C), 151.4 (C), 160.9 (C), 162.0 (C). Anal. Calculated for C13H14O4: C, 66.66; H, 6.02. Found: C, 66.26; H, 6.04. 3-Butyl-6-hydroxy-7-methoxy-4-methyl-2H-1-benzopyran-2-one [C42]: yield 59%; mp. 174-175ÆC (ethanol); 1H NMR (DMSO-d6, 200 MHz) ‰: 0.91 (t, 3H, J=7.0 Hz), 1.28-1.43 (m, 4H), 2.31 (s, 3H), 2.52 (t, 2H, J=7.8 Hz), 3.86 (s, 3H), 6.97 (s, 1H), 7.05 (s, 1H), 9.28 (s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 13.8 (CH3), 14.7 (CH3), 22.1 (CH2), 26.6 (CH2), 30.5 (CH2), 56.0 (CH3), 99.7 (CH), 109.3 (CH), 112.9 (C), 122.5 (C), 143.3 (C), 145.4 (C), 145.9 (C), 150.5 (C), 161.2 (C). Anal. Calculated for C15H18O4: C, 68.68; H 6.92. Found: C, 68.81; H, 6.81.

6-Hydroxy-7-methoxy-4-methyl-3-(1-methylethyl)-2H-1benzopyran-2-one [C43]: purification by chromatography over silica gel; yield 7%; mp. 180-181.5ÆC (washed with TBME); 1H NMR (DMSO-d6, 200 MHz) ‰: 1.26 (d, 6H, J=6.9 Hz), 2.34 (s, 3H), 3.26 (sept, 1H, J=6.9 Hz), 3.86 (s, 3H), 6.96 (s, 1H), 7.09 (s, 1H), 9.27 (s, 1H). 13C NMR (DMSO-d6, 50.3 MHz) ‰: 14.5 (CH3), 19.8 (CH3 x 2), 27.8 (CH), 56.0 (CH3), 99.5 (CH), 109.6 (CH), 112.9 (C), 126.7 (C), 143.2 (C), 145.8 (C), 146.1 (C), 150.5 (C), 159.4 (C). Anal. Calculated for C14H16O4: C, 67.73; H, 6.50. Found: C, 67.57; H, 6.55. 3-Ethyl-6-hydroxy-7-methoxy-4-methyl-2H-1-benzopyran-2-one [C44]: yield 74%; mp. 179-180ÆC (ethanol/TBME); 1H NMR (DMSO-d6, 200 MHz) ‰: 1.04 (t, 3H, J=7.5 Hz), 2.31 (s, 3H), 2.55 (q, 2H, J=7.4), 3.87 (s, 3H), 6.96 (s, 1H), 7.05 (s, 1H), 9.29 (s, 1H). 13C NMR (DMSO-d , 50.3 MHz) ‰: 13.0 (CH ), 14.4 (CH ), 20.2 6 3 3 (CH2), 56.0 (CH3), 99.7 (CH), 109.2 (CH), 112.8 (C), 123.7 (C), 143.3 (C), 145.9 (C), 146.1 (C), 150.5 (C), 161.0 (C). Anal. Calculated for C13H11O4: C, 66.66; H, 6.02. Found: C, 66.69; H, 5.95. Cell culture. Normal cells, human gingival fibroblast (HGF), human pulp cell (HPC) and human periodontal ligament fibroblast (HPLF), were obtained from human periodontal tissue after informed consent, according to the guidelines of Meikai University Ethics Committee, Japan (No. 0206). Since normal cells have a limited lifespan (19), cells at 3-7 population doubling level (PDL) were used for the present study. The human oral squamous cell carcinoma cell lines (HSC-2, HSC-3) were supplied by Prof. Nagumo, Showa University and Dr. Fukuda, Meikai University, Japan, respectively. All cells used were cultured as a monolayer culture at 37ÆC in DMEM supplemented with 10% heatinactivated FBS in a humidified 5% CO2 atmosphere, and subcultured by trypsinization. Cytotoxic activity. The relative viable cell number of adherent cells was determined by MTT methods. In brief, the cells were treated for 24 h without (control) or with various concentrations of the test samples. The cells were washed once with phosphate-buffered saline without Mg2+ or Ca2+ (PBS), and further incubated for 4 h with 0.2 mg/mL MTT in DMEM + 10% FBS. After removal of the medium, the cells were lysed with 0.1 mL of dimethyl sulfoxide (DMSO). The absorbance at 540 nm of the solubilized formazan pellet (which reflects the relative viable cell number) was then determined by microplate reader (Biochromatic Labsystem, Helsinki, Finland). From the dose-response curve, the 50% cytotoxic concentration (CC50) was determined (20). Tumor-specific cytotoxicity (SI value) was determined by the following equation: SI=CC50 (HGF + HPC + HPLF)/CC50 (HSC-2 + HSC-3) x 2/3. Cell and fluorescence uptake. The MDR1/A-expressing cell lines were selected by culturing the infected cells with 60 ng/mL colchicine to maintain the expression of the MDR phenotype (21). The L5178 MDR cell line and the L5178 Y parent cell line were grown in McCoy’s 5A medium supplemented with 10% heatinactivated horse serum, L-glutamine and antibiotics. The cells were adjusted to a density of 2 x 106/mL and resuspended in serumfree McCoy’s 5A medium, and 0.5 mL aliquots of the cell suspension were distributed into each Eppendorf centrifuge tube. Then, 10 ÌL of 2 mg/mL test compounds were added and incubated for 10 min at room temperature. Then, 10 ÌL rhodamine 123 (R123), as an indicator of drug accumulation, was added to the extracts (5.2 ÌM final concentration) and the cells were incubated

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in vivo 19: 705-712 (2005)

Figure 1. Chemical structures and calculated log p-values of coumarin derivatives [C1-44].

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Kawase et al: Structure-activity Relationships of Coumarins

Table I. Cytotoxic activity of coumarin derivatives [C24-44] against cultured human tumor and normal cells. Compound

50% Cytotoxic concentration (CC50, Ìg/mL)

Human tumor cell lines

C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35 C36 C37 C38 C39 C40 C41 C42 C43 C44 Gallic acid A540a

HSC-2

HSC-3

75 >200 >200 195 120 15 29 61 >200 48 100 53 154 67 150 20 19 34 33 19 24 24 0.966

>200 >200 >200 150 181 85 >200 >200 197 >200 >200 78 >200 >200 >200 >200 108 157 169 68 65 38 0.752

SI = CC50 (normal) / CC50 (tumor)

Normal cells HGF 133 >200 >200 >200 192 100 >200 >200 >200 153 >200 152 >200 >200 >200 >200 157 147 >200 161 171 74 0.331

HPC

HPLF

>200 180 >200 >200 >200 133 >200 >200 >200 154 >200 >200 >200 >200 >200 >200 157 162 >200 190 163 76 0.307

>200 >200 >200 >200 179 127 >200 >200 >200 133 >200 158 >200 >200 >200 >200 173 148 >200 188 151 70 0.355

>1.2 >1.3 2.4 >2.0 4.1 3.6 2.4

Near confluent cells were incubated for 24 hours without or with various concentrations of each compound, and the relative viable cell number (absorbance at 540 nm of the lysate of MTT-stained cells) was determined by the MTT method. The CC50 was determined from the dose-response curve. Each value represents the mean from duplicate determinations. aAbsorbance at 540 nm of the lysate of MTT-stained control cells.

for a further 20 min at 37ÆC, washed twice and resuspended in 0.5 mL PBS (pH 7.4) for analysis. The fluorescence of the cell population was measured by flow cytometry using the Beckton Dickinson FACScan instrument. (±)-Verapamil was used as the positive control in R123 accumulation experiments (22). The R123 accumulation was calculated from the fluorescence of one height values. Then, the percentage of mean fluorescence intensity was calculated in treated MDR1 and parental cell lines, compared to untreated cells. The fluorescence activity ratio (FAR) was calculated by the following equation (21, 22): MDR1 reversal activity = (MDR1-treated/MDR1 control)/ (parental-treated/parental control)

Results Cytotoxic activity. The in vitro cytotoxicities of 21 coumarin derivatives [C24-44] (see structural formulae in Figure 1) were evaluated in two human tumor cells (HSC-2, HSC-3) and three normal cells (HGF, HPC, HPLF) by the MTT assay and the results are summarized in Table I. In general, the coumarin derivatives were more cytotoxic against HSC-2

than HSC-3 cells. Five compounds, C29, C39, C40, C43 and C44, displayed potent cytotoxic activities, ranging from CC50 15 to 24 Ìg/mL in HSC-2 cells. Their potencies were comparable with that of gallic acid (CC50=24 Ìg/mL). Both C43 and C44 showed the highest tumor-specific cytotoxicity (SI value=CC50(HGF + HPC + HPLF)/CC50(HSC-2 + HSC-3) x 2/3=4.1 and 3.6, respectively). MDR reversal on tumor cells. The MDR-reversing effect of 44 coumarins [C1-44] was compared to that of (±)verapamil, using a mouse lymphoma cell line (L-5178 cells) (Table II). The effects were measured by determination of the fluorescence activity ratio (FAR) between treated and untreated group of cells. Among 44 coumarins, fourteen compounds [C5, 19, 21, 22, 24, 28, 33, 34, 36, 39, 40, 42, 43 and 44] were able to moderately increase the amount of rhodamine 123 accumulated by resistant lymphoma cells at 40 Ìg/mL concentration (FAR values >1) (data not shown). However, most compounds, except C34 and C43, had a 10

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in vivo 19: 705-712 (2005) Table II. Effect of coumarins [C34 and C43] on the multidrug resistance of L-5178 cells. Compounds Par (control)b MDR+R123 (mean)c (dl)-verapamil

DMSO C34

C43

Concentration (Ìg/mL) 4 10 40 80 20 4 40 80 4 40

FSCa

SSCa

461.75 493.34 469.55 517.66 482.29 438.87 455.39 481.45 484.59 481.89 486.24 489.01

193.80 196.92 200.59 199.05 203.07 182.62 192.75 193.87 202.70 204.23 197.50 176.91

FL-1a

FARa

6.12 6.29 19.40 115.42 234.01 395.48 5.87 17.19 113.12 134.67 6.21 35.66

3.08 18.34 37.20 62.87 0.93 2.73 17.98 21.41 0.98 5.66

aFSC:

Forward scatter count; SSC: Side scatter count; FL-1: Fluorescence intensity; FAR: Fluorescence activity ratio. a parental cell without MDR gene. cMDR: a parental cell transfected with MDR gene. bPar:

times lower FAR value than that of the MDR modulator verapamil (used as a positive control at concentration of 10 Ìg/mL). Another thirty compounds were not active at 40 Ìg/mL concentration (FAR values 1) (data not shown). However, the majority of the compounds, except C34 and C43, had a 10 times lower FAR value than that of the MDR modulator verapamil (used as a positive control at a concentration of 10 Ìg/mL). The most active compound C34, was equally potent to verapamil. It is suggested that the presence of the 2-hydroxyethyl group is favorable for the activity. Then, C34 might be an anti-MDR inducing agent of great interest (3, 24). It has often been suggested that hydrophobicity is an important feature of MDR modulators (25, 26), which is why the log P values of the coumarins studied was calculated. The most active C34 showed low hydrophobicity concerning the log P value. Therefore, hydrophobicity alone was not an essential parameter for the direct MDRmodulating activity of the present series of compounds.

Kawase et al: Structure-activity Relationships of Coumarins

Although a number of agents have been developed to modify MDR, none of them are currently used in the clinic due to their lack of potency or of side-effects at that concentrations necessary for efficacy. At the same time, there is an urgent need to improve cancer chemotherapy for resistant tumors. Therefore, we have to find and select effective compounds, at least in vitro. To fulfil this need, the coumarins were tested in this study and, after further modifications, the coumarins might be MDR reversal compounds.

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Received February 23, 2005 Accepted April 21, 2005

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