Introduction Iraqi J Pharm Sci , Vol.18 (Suppl . ), 2009 Quinolone’s cytotoxic study 13 Preliminary Cytotoxic Study of Some Novel Furo-2-quinolone Compounds Nohad A.AlOmari *.1 , Adnan O. Omar ** and Iklas M. Taher ** *College of pharmacy , university of Mosul , Mosul , Iraq **College of science , university of Mosul , Mosul , Iraq Abstract In this research, new series of Furo-2-quinolone [FQ] compounds have been synthesized. These novel [FQ] compounds were prepared from coumarin derivatives (Furocoumarins: psoralen and isopsoralen).Identifications of these FQ compounds were performed by using infrared spectrum (I.R), Ultraviolet spectrum (U.V) and Nuclear Magnetic Resonance spectrum (H 1 -NMR) besides some physical data. The cytotoxic screening involves ;using HEP-2 cell line which gave differential responses against tested compounds : 4,6 Dimethyl furo[2, 3-g] coumarin (C1), 1-(2`, 4`, Dimethoxy benzylideneimino)-2,6-dimethyl Furo [2, 3-g] quinoline-2-one (C3) and the angular psoralen of the same derivative with the chemical name; 1-(2`,4`-Dimethoxybenzylidenimine)-4,8,9-trimethyl furo[3,2-h] quinoline-2-one (C3A).These preliminary studies using different cell lines in addition to full cytotoxic screening may facilitate generation of better structural activity relationship and then shed the light for new lead promising anticancer compounds. Keyword: Coumarin, Angelicin, HEP-2 Cell line, cytotoxicity, الخالصة اممحايثتكي حمم يثتضاتضممرينةممي يومم يو مم يي[FQ] دن تمم -2-تممفي مماياممحثيثتحضمم سيتضةممديية ممم ريباتمماويومم يوي حمم ي دمم ي(IR)ت ممصدهياممحايثتكي حمم يتممفي ةمم صاثةيتدممايثمرمم ريتضمم يثتضكمميث ييي).د ي ثتد ي ثتز ممم ثتمممثتك ومم ت يي دمم ي ومم ت ي ند يثتنمم ايثتكان تدممماي ة مم ريضتممصي ممليثتيمم يثت دزت تممرايتةممكن ي ثةممريثتمممكدري ثتمميي(UV) تدممايثمرمم ري مم فيثتحن ممم دري ي-[2,3 ثايوض تم ي دم يي,6ي4 ثت مايعطتم يثةم يوص مري مايثتكي حم يخدمايثمخ حم ي امايHEP-2ت صالت ي ة صاثةيخثيخالت ي ي(C3يع -2ي– ي دنمم تد يgي[3,2- دمم يي– ثايودض تمم يي-6,2-ثتكدنمم يي- ثايودض ممماي نزثت ممات ي1-(2' ,4' يي(C1) ومم ت يي]بمما ايامحاي(C3A)ث يي-2- دن تم يي]يH -[2,3ثالثمايودض تم ي دم يي-9,8,4ي-ثتكندم ي- ثايودض مماي نزث مات ي-2',4'ي-1 ة م ريضتمصي ي ظد دمرايتممي ي م ويت الخمري ة م ريضتمصيوممدي مايةمكايرم و يت م يخميك ن طمرويةميت ندريثتا ثةريثأل تدمري ةم صاثةيختم تيتصالتم ايةيت ندريو خ ري ثطاوايذث ي تدريتر و يثفيضزثنريثتة يتكي ح يخد ت ي ت ي دبيثتكدك اي Introduction Furoquinoline-2-one(I) compounds are considered as a main derivatives of furocoumarines(II)(1). Coumarin(III) is being the main nucleus of these compounds which classified into linear furocoumarin ; psoralen(II) and angular furocoumarin ; angelicin(IV) [Fig.1]. The investigation of coumarin compounds revealed that a wide spectrum of medicinal plant extracts that were in use as early as 1000 A. D. contain a high content of coumarins. Subsequent analysis of scientific literature revealed numerous reports on the antiproliferalive and antitumor activities of a variety of coumarin compounds(2-5), and have demonstrated activity against several types of animal tumors(6-10). These compounds have also been reported in clinical trials to demonstrate activity against prostate cancer, malignant melanoma and metastic renal cell carcinoma(11, 12). Recently, series of Furo-2-quinolone compounds [Fig. 2] were synthesized(13) and identified by using infrared (FT/IR), Ultraviolet (U.V.) and Nuclear Magnetic Resonance Spectroscopy (1HNMR) besides some physical data . 1 Corresponding author E-mail : Nohad_alomari@yahoo.com Received : 28/12/2008 Accepted : 26/5/2009 Iraqi J Pharm Sci , Vol.18 (Suppl . ), 2009 Quinolone’s cytotoxic study 13 This previous literature survey urged us to direct this paper towards preliminary cytotoxic study of the "building block" (4,6 Dimethyl furo[2, 3-g] coumarin (C1) , one of linear psoralen ; 1-(2`, 4`, Dimethoxy benzylideneimino)-2-6-dimethyl Furo [2, 3-g] quinoline-2-one (C3) and the angular psoralen of the same derivative with the chemical name; 1-(2`,4`-Dimethoxybenzylidenimine)-4,8,9- trimethyl furo[3,2-h] quinoline-2-one (C3A)These three selected lead compounds were screened against human larynx epidermoid carcinoma (HEP-2) cell line. The preliminary results indicate that HEP-2 cell differs in its sensitivity towards these compounds. Figure (1) Figure (2) O O O O O O OO II III IV O OO CH3 CH3 O O CH3 O CH3 CH3 N H 2 N H 2 .H 2 O N O CH3 O CH3 CH3 NH2N O NH2 CH3 O CH3 R C H O , A b s. E th a n o l N O CH3 O CH3 CH3 N CH RN O N CH3 O CH3 CH R OCH3 O CH3 R = I O N O H C1 C1A C2 C2A C3 C3A Iraqi J Pharm Sci , Vol.18 (Suppl . ), 2009 Quinolone’s cytotoxic study 11 Experimental Chemistry General Method Melting points were uncorrected & determined by using electrothermal 9300. The identification of the prepared compounds were done by using FT/IR spectrophotometer tensor 27 as KBr disc. Ultraviolet by schimadzu-UV visible UV. 1650 PC. Nuclear Magnetic Resonance spectrum were carried out by using Bruker-Avance 400 MHz / France. Synthesis 1-Amino-4,6-dimethyl furo[2, 3-g] quinoline-2-one (14) (C2). 1-Amino-4,8,9-trimethylfuro[3,2-h] quinoline-2-one (13) ( C2A). Dissolve (0.02 M) of Furocoumarin in (30 ml) of dry pyridine with continuous stirring and gentile heating, then cool the mixture and add to it (0.025M, 1-3 ml) of aqueous hydrazine drop wise, reflux for 6 hours. Cool the mixture until formation of precipitate, filter and wash many times with cold water and recrystallized from ethanol. Yield 42%, the melting point is (C2= 240 - 241 o C) & (C2A 166-169 o C). 1-(2`,4`-Dimethoxy benzylidene imino)-4,6- dimethyl furo[2, 3-g] quinoline-2-one (C3). 1-(2`, 4`-Dimethoxy benzylidene imino)-4, 8, 9-trimethyl furo[2,3-h] quinoline-2- one(C3A). Dissolve an equimolar (0.003 M) of C2 or C2A with 2, 4-dimethoxy benzaldehyde in 50ml of absolute ethanol, reflux the reaction mixture for 1 hr. cool and filter. The precipitate is washed with cold ethanol and recrystallized by methanol. M.Pt. (202-204 o C, pale brown) and (90-93 o C, yellow) for C3and C3A respectively C=O C-N N-N N-H U.V. EtOH nm. IR cm -1 KBr C2 1654 1252 1184 3107 300 C2A 1672 1244 1173 3188 304 CH3(s) CH= NH2 Phenyl 1 HNMR ppm/CDCl3 C2 2.2, 2.4 6.3 , 6.5 5.3 7.2 , 7.4 C2A 2.2, 2.4, 2.5 6.5 5.8 7.0 - 7.4 complex C=O C=N N-N U.V/ EtOH nm. I.R. /cm -1 KBr C3 1675 1575 1180 296 nm C3A 1673 1562 1182 304 nm CH3 (S) CH= (S) OCH3 (S) CH=N (S) Phenyl 1 H NMR ppm/CDCl3 C3 2.22, 2.42 6.3,6.5 3.74, 3.81 8.1 7.2 ,7.4 (S) (S) C3A 2.22, 2.42 ,2.5 6.3, 6.5 3.78, 3.82 8.21 7.0 -7.4 complex Iraqi J Pharm Sci , Vol.18 (Suppl . ), 2009 Quinolone’s cytotoxic study 13 II.Cytotoxic screening II.Material Bovine serum (BS), Phosphate buffered saline (PBS) PH 7.2, HEP-2 cell line and all other solutions and media for cytotoxicity study were kindly provided by the Iraqi Center for Cancer and Medical Genetics Research / Baghdad. Cell line synthesis for cytotoxicity study (17) The growth medium was decanted off and the cell sheet was washed twice with PBS. Two to three ml of trypsin-versene was added to the cell sheet and the flask was rocked gently. After approximately 30 seconds most of the trypsin was poured off and the cells incubated at 30 o C until they had detached from the flask. Afterwards, 200 l of cells in growth medium were added to each well of a sterile 96-well microtitration plate. The plates were incubated at 37 o C in 5% CO2 humidified atmosphere incubator, the medium was removed and serial dilutions of the compound (500, 250, 125 g/m) under assay in SFM were added to the well. The cell line was exposed to cisplatin (EBEWE, Austria Europe) as a reference (positive control); where the negative control were the cells treated with SFM only. The cells in each well were washed with PBS exposed to diluted formalin exposed to crystal violet dye then washed and left to dry (18) . Reading the result was accomplished using a multi-well plate reader at 492 nm. Results and Discussion Preparation of C2 & C2A from reaction of Furocoumarin with hydrazine hydrate is a nucleophilic substitution lead to cyclic cleavage followed by recyclization with removal of one molecule of water. This reaction product shows three singlet bands at (2.2, 2.4, 2.5 ppm), due to trimethyl substituent at C4, 8, 9 respectively of C2A ,and the singlet proton of C3 appears at 6.5 ppm.The two methyl groups in C2 appears at (2.2 and 2.4 ppm) and the two singlet protons of this compound at C3 and C7 are interfere with the aromatic region at (6.3 and 6.5 ppm) respectively, due to the aromatic properties of this compound. The FT/IR spectra of the compounds C3 and C3A (Schiff bases derivatives) shows a significant C = N band between (1575 and 1562 cm-1) respectively. The 1HNMR of these compounds appears approximately not far from the values of compounds C2 and C2A..The U.V. spectra showed max between 296nm and 304 nm respectively The other spectral data of these compounds U.V., I.R. and 1HNMR are appear in the previous Tables. Cytotoxicity screening Compounds C1, C3 & C3A were screened for their cytotoxicity testing. Morphological changes of HEP-2 cell upon exposure to selected compounds [Fig. 3] accompanied with reduction in size i.e. loss of contact with neighboring cells as the apoptotic cells shrank and become detached from the adjacent cells.The dose-response curve of C1, shows a dramatic positive downward curve [Fig. 4 , Table1] and highly HEP-2 sensitivity comparing with resistance cisplatin (positive control), indicate delivering of good prominent lead compound, this significant difference from cisplatin shown at lower concentrations 62.5& 125 g/10ml.A different response [Fig. 5, Table 2] could be exploited from C3 although a cross-resistance at lower doses, but a decrease in % survival appeared with higher dose which may be clearer with time-course interval higher then 48 hours. HEP-2 cell shows a gradual sensitivity with cisplatin C3A [Fig.6, Table3] and significant difference from cisplatin at P < 0.001 at 5 different dilution doses.From the collective dose response curves [Fig.7] C1, C3 & C3A we can conclude that insertion of Iraqi J Pharm Sci , Vol.18 (Suppl . ), 2009 Quinolone’s cytotoxic study 13 ( R = ) to C3 may considered as non essential modification to linear psoralen while angular psoralen "Angelicin" may considered as a good lead compound, and collective informations obtained from SAR yield from cytotoxic screening of all synthesized compounds may accumulate informations & shed light to new promising anti-proliferative agents. Fig 3 : The morphologicl effects exerted by C1,C3, and C3A on HEP-2 , photographs were taken using a Nikon inverted light microscope (20x objective) - ve Control C3 OCH3 OCH3 Drug - ve Control C1 C3 C3A Iraqi J Pharm Sci , Vol.18 (Suppl . ), 2009 Quinolone’s cytotoxic study 13 ** Significant difference from cisplatin at p<0.01, *** at p<0.001 Fig 4: Graphical representation of concentration dependent effect of C1 on HEP-2 cells. Table 1: The cell survival as percentage of the control for the HEP-2 cell when the cells were treated with C1. Concentratio n (µg/10 ml) % of Survival (Mean  SD) Cisplatin C1 62.5 6.77  1.27 106.01  9.33 125 10.15  1.88 73.79  6.74 250 32.42  4.29 36.83  6.59 500 54.88  4.60 36.96  3.64 * Significant difference from cisplatin at p<0.05, *** at p<0.001 Fig 5: Graphical representation of concentration dependent effect of C3 on HEP-2 cells. Table 2: the cell survival as percentage of the control for the HEP-2 cell when the cell were treated with C3. Concentration (g/10 ml) % of Survival (Mean  SD) Cisplatin C3 62.5 6.77  1.27 71.34  46.35 125 10.15  1.88 97.09  12.01ي 250 32.42  4.29 100.77  8.97 ي500 54.88  4.60 70.68  5.82 *** Significant difference from cisplatin at p<0.001 Fig 6 : Graphical representation of concentration dependent effect of C3A on HEP-2 cells. Table 3: The cell survival as percentage of the control for the HEP-2 cell when the cell were treated with C3A. Concentrati on (g/10 ml) % of Survival (Mean  SD) Cisplatin C3A 62.5 6.77  1.27 107.66  10.88 125 10.15  1.88 109.46  5.21 250 32.42  4.29 103.76  4.69 ي500 54.88  4.60 89.92  6.10 0 20 40 60 80 100 120 10 100 1000 Concentration % S u rv iv a l Cisplatin C1 *** *** ** 0 20 40 60 80 100 120 10 100 1000Concentration % S u rv iv a l Cisplatin C3 * *** * *** 0 20 40 60 80 100 120 10 100 1000 Concentration % S u rv iv a l Cisplatin C3A *** *** *** *** Iraqi J Pharm Sci , Vol.18 (Suppl . ), 2009 Quinolone’s cytotoxic study 13 Fig 7: Three dimensional histograph of over all tested compounds (C1,C3,C3A) on HEP- 2 cells. References 1. S. M. Sentha, Chem. Rev., 1945. 36, 1-62. 2. M. E. Marshall, J. L. Mohler, K. Edmonds, B. Williams, K. Butler, M. Ryles, L. Weiss, D. Urban, A. Beuschen, and M. Markiewicz., J. cancer Res. Clin. Oncol., 1994, 120, 39. 3. I. Kostova , synthetic & natural coumarins as cytotoxic agent , Curr. Med. Chem. Anticancer Agent , 2005, 5, 29-46. 4. C. P. 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