Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 49 Synthesis of some Novel Nitrogenous Heterocyclic Compounds with Expected Biological Activity as Antimicrobial and Cytotoxic Agents. Mohammed S. Farhan *,1 and Kawkab Y. Saour * * Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad,Baghdad,Iraq. Abstract This study includes synthesis of some nitrogenous heterocyclic compounds linked to amino acid esters or heterocyclic amines that may have a potential activity as antimicrobial and/or cytotoxic. Quinolines are an important group of organic compounds that possess useful biological activity as antibacterial, antifungal and antitumor .8-Hydroxyquinoline (8-HQ) and numerous of its derivatives exhibit potent activities against fungi and bacteria which make them good candidates for the treatment of many parasitic and microbial infection diseases. These pharmacological properties of quinolones aroused our interest in synthesizing several new compounds featuring heterocyclic rings of the quinoline derivatives linked to amino acid ester or heterocyclic amine with the aim of obtaining a pharmacologically active compounds.O-alkylation has been done on( 8-hydroxyquinoline ) to get (O-alkylated ester) derivatives which are deesterfied to get acetic acid derivatives, then coupled with amino acid that have protected carboxyl group (amino acid esters) or heterocyclic amine by using conventional solution method for peptide synthesis as a coupling method. The proposed analogues were successfully synthesized and the processing of the reactions confirmed by TLC ,the synthesized analogues with the proposed structures as they were characterized and proved by melting point, infrared spectroscopy (IR) and elemental microanalysis. The tested analogues showed cytotoxic activity on the HEp-2 cell line (tumor of larynx) with inhibitory concentration percent of (IC %) range (32.43 % - 49.55%) and showed that the tested compounds had variable antimicrobial activities against selected bacteria and yeast when compared with selected standard drugs. Keywords: Quinolones, 8-hydroxyquinoline , N-heterocycles biological activity. مع تىقع الحاويح على الىتروجيهومتجاوسح ر غيال الجذيذج تخليق تعض المركثاخ الحلقيح يحوسرطاو ميكروتيحكمضاداخ الفعاليح الحيىيح لها محمذ سىجار فرهان *،1 كىكة يعقىب ساعىر و * * . انعشاق ،بغذاد،خبيعت بغذاد،كهيت انصيذنت ،فشع انكيًيبء انصيذالَيت الخالصة غيش يشكببث حهميتأ حخعًٍ ْزِ انذساست حخهيك بعط انًشكببث انحهميت غيش انًخدبَست انًمخشَت بأسخشاث األحًبض االييُيت أٔ يعبداث نهسشغبٌ /انخي لذ حًخهك فعبنيت َشيطت كًعبداث نهدشاثيى ٔٔ يخدبَست حبٔيت عهى يدًٕعت اييُيت .ٔسشغبَيت ًعبداث بكخيشيت ٔفطشيتكحًخهك انكٕيُٕنيُبث اًْيت يًيزة كأحذ انًشكببث انععٕيت ٔانًسخحعشاث انطبيت ٔانخي حسخخذو يششحت خيذة نعالج انعذيذ يٍ ب ٔانعذيذ يٍ يشخمبحّ أَشطت فعبنت ظذ انفطشيبث ٔ انبكخيشيب انخي خعهٓ) ْيذسٔكسيكٕيُٕنيٍ-8)اظٓش ال ْٕ األكثش إثبسة نالْخًبو ، ٔرنك (ْيذسٔكسيكٕيُٕنيٍ-8)يٍ خًيع يشخمبث انٓيذسٔكسيكٕيُٕنيٍ .األيشاض انطفيهيت ٔ اندشثٕييت نذٔائيت أثبسث اْخًبيُب بخحعيش عذة يشكببث خذيذة حخًيز بحهمبث انغيش يخدبَست بسبب خصبئصّ انًخعذدة انٕظبئف ، ْزِ انخصبئص ا بٓذف انحصٕل غيش يخدبَست حبٔيت عهى يدًٕعت اييُيت يشكببث حهميتأ نًشخمبث انكٕيُٕنيٍ يشحبطت بإسخشاث األحًبض األييُيت .عهى انًشكببث انصيذالَيت انفعبنت ٔ يٍ ثى ححهم االسخش (O-Alkylated ester)نهحصٕل عهى يشخمبث )ْيذسٔكسيكٕيُٕنيٍ -8(عهى O-Alkylationحى إخشاء غيش يخدبَست حبٔيت يشكببث حهميتأ نهحصٕل عهى يشخمبث حبيط أنخهيك ٔيٍ ثى سبطٓب يع يخخهف اسخشاث األحًبض االييُيت ث كطشيمت ناللخشاٌ ٔحكٕيٍ أصشة اييذيت نهحصٕل عهى ببسخخذاو غشيمت انًحهٕل انخمهيذيت نصُبعت انببخيذا عهى يدًٕعت اييُيت ٔلذ حًج يشالبت سيش انخفبعالث .اٌ عًهيت حخهيك انًشكببث انًطهٕبت لذ حًج ببحببع غشيمت انخفبعم يخعذد انخطٕاث.انًشكببث انُٓبئيت نُٓبئيت ٔانخبكذ يُٓب يٍ خالل ليبس حى حشخيص انخشكيب انكيًيبئي نهًشكببث انٕسطيت ٔا .ببسخخذاو كشٔيبحٕغشافيب انطبمت انشليمت .دسخبث االَصٓبس ٔانخحهيم انطيفي نألشعت ححج انحًشاء ٔانخحهيم انذليك نهعُبصش بُسبت حشكيز يئٕيت (سو انحُدشةت )ٔأظٓشث انُخبئح انخي اخشيج عهى انًشكببث انًصُعت َشبغ سًي عهى َٕع يٍ انخاليب انسشغبَي بث ييكشٔبيت أحًخهك فعبني الحًخهكانًصُعت ٔكزنك اظٓشث انُخبئح اٌ انًشكببث % ( 5443 %- 6654) يثبطت حخشأذ يب بيٍ ال ب ٔانفطشيبث انًخخبسة ببنًمبسَت يع االدٔيت انميبسيت انًخخبسة ايعب في ْزِ انذساستبكخشيانظذ يخخهفت .الحلقيح الغير متجاوسح والحاويح على الىتروجيهالفعاليح الحيىيح للمركثاخ ، هيذروكسيكىيىىليه -8 ،كىيىىلىنالكلماخ المفتاحيح : 1 Corresponding author E-mail: mohmadsinjar@Gmail.com Received:10 /1/2015 Accepted: 28/4/2015 mailto:mohmadsinjar@Gmail.com Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 50 Introduction Heterocyclic compounds are acquiring more importance in recent years which display biological activities. (1) Heterocyclic compounds particularly five and six member heterocyclic have attracted the attention of pharmaceutical community over the years due to their therapeutic values. (2) Poly functionalized heterocyclic compounds containing nitrogen, sulphur, oxygen as heteroatom play important roles in the drug discovery process. (3) Analysis of drugs in late development stages or in the market shows that 68% of them are heterocycles. (4) N-heterocycles have found wide-spread applications as key components of a large number of biologically-active natural products. Examples include antibiotics such as penicillin and cephalosporin, alkaloids such as vinblastine and morphine and fungal natural product like cyclosporine A , all exhibiting interesting biological properties. However, synthetic N-heterocyclics have also found widespread use in pharmaceuticals as anticancer agents, analeptics, analgesics, hypnotics and antioxidants (5) .Quinoline is one of the most popular N-heteroaromatic compounds incorporated into the structures of many pharmaceuticals. Many quinoline- containing compounds exhibit a wide spectrum of pharmacological activities, such as antiplasmodial (6), , cytotoxic (7) , antibacterial (8) , antiproliferative (9), ,antimalarial (10) , and anticancer activity (11) . These pharmacological properties of quinolines and their derivatives had attracted worldwide attention in the last few decades because of their wide occurrence in natural products and drugs (12,13) . Quinoline derivatives also have been shown to exhibit awide variety of pharmacological activities including effects on cancer and nowadays it is reported that the incorporation of quinolone nucleus could alter the course of reaction as well as the biological properties of the synthesized compounds (14, 15). . Materials and Methods Materials 8-hydroxyquinoline was purchased from AVONCHEM (U.K). Absolute ethanol, Absolute methanol, Acetone, and Chloroform were purchased from GCC (Germany). Absolute isopropyl alcohol, ethylBromoacetate , Diethyl ether, Ethyl acetate, Hydrazine hydrate, Hydrochlorica cid, N-methyl morpholine (NMM), Petroleum spirit and Thionyl chloride were purchased from BDH (U.K).Coumarin was purchased from Himedia (India). D-Alanine, 1-Hydroxy benzotriazole (HOBt), and N,NDimethyl formamide (DMF), were purchased from Fluka AG(Switzerland). All other reagents were of analytical grade. Methods of Identification General methods were used for purification and identification of the synthesized analogues including: • Thin Layer Chromatography: • Melting Points: • Infrared Spectra: • Elemental Microanalysis Synthesis Esterification of amino acids synthesis of D- alanine methyl ester HCl compound (A.1) (16) A suspension of D-Alanine (5 mmol, 0.445g) in (100ml) of absolute methanol, was cooled down to –15 ºC then thionyl chloride (5 mmol, 0.37 ml) was added drop by drop(the temperature should be kept below –10 0 C) , the reaction mixture was left at 40 0 C for 3hr, then reflux started for other 3hr and left at room temperature overnight, the solvent was evaporated to dryness under vacuum, redissolved in methanol and evaporated, this process was repeated several times and recrystallize from methanol–ethyl acetate (3:1) Synthesis of 1-aminoquinolin-2(1H)-one (B.1) (17) A solution of (2.96g, 20 mmol) coumarin and excess hydrazine hydrate (99%), in 25 ml of absolute ethanol was refluxed for 24 h, left at room temperature(R.T.) for one week , then the solvent was evaporated to dryness and the solid product was washed with cold ethanole, and recrystallized from chloroform to give yellow crystals. TLC was done indicate the disappearance or lightening of coumarin spot and new spot was resulted. Synthesis of ethyl-quinoline-8-yl oxyacetate. compound 1 (18) A mixture of 8- hydroxyquinoline(12.5 mmol), ethyl bromoacetate (12.5 mmol) and K2CO3 (17.9 mmol) in 50ml dry acetone was refluxed for about 20 hr at 70 0 c. After cooling, the mixture was evaporated to dryness and the residue was partitioned between chloroform (50 ml) and water (50 ml). The organic phase was dried with anhydrous sodium sulphate (Na2SO4), filtered and evaporated to dryness. The residue was recrystallized from ethanol. Synthesis of 2(quinoline-8-yloxy) acetic acid (QAA) compound 2 (19) A solution of compound 1 (1.5g, 7.38mmol) in methanol (25 ml) and 5% NaOH (6 ml) was heated under reflux for 4 h. After cooling, the solution was evaporated to dryness and the residue was dissolved in 10 ml water and acidified with 1N HCl( 6 ml). The Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 51 white precipitate was filtered, dried and crystallized from methanol. Coupling method and reagents Conventional solution method for peptide synthesis was used as a coupling method between the carboxy protected amino acids or heterocyclic amine and carboxy derivatives of quinolone. Dicyclohexylcarbodiimide (DCC) was used in the peptide bond formation as the coupling reagent, while 1-Hydroxybenzotriazole(HOBt) was used to decrease racemization and to increase the yields (20) . Synthesis of methyl 2-(2-(quinolin-8- yloxy)acetamido)propanoate ( Comp.3 ) To a stirred solution of D-Ala. methyl ester HCl (comp. A.1) (2mmol, 0.28g) in (25ml) of Dimethylformamide (DMF), (2mmol, 0.22ml) of N-Methylmorpholine (NMM) was added with stirring for 10 min., then (2mmol,0.406 g) of (comp. 2) was also added, and the mixture was cooled down to (-10 0 C) then (4.0mmol, 0.540g) of HOBt and (2.0mmol,0.412 g ) of DCC were added with stirring, which was continued for 2 days at 0 0 C and then at room temperature for 5days. The reaction mixture evaporated to exclude DMF and redissolved in chloroform from which the N,N–Dicyclohexyl urea (DCU) was filtered off. The clear filterate washed three times with 5% sodium bicarbonate solution, 0.1N HCl, once with distilled water, and with saturated sodium chloride solution. The chloroform layer was dried with anhydrous magnesium sulphate and evaporated under vacuum; the resulted product was collected, recrystallized from (methanol: chloroform) (5:1). Synthesis of N-(2-oxoquinolin-1(2H)-yl)-2- (quinolin-8-yloxy)acetamide ( comp. 4) To a stirred solution of compound 2 (0.406g, 2.0 mmol) in (20ml) of DMF, (0.32 g, 2.0 mmol ) of compound B1 was added, the mixture was cooled down to (-10 0 C) then (0.56g, 0.4 mmol) of HOBt and (0.412g ,2 mmol) of DCC were added with stirring, which was continued for 2days at 0 0 C and then at room temperature for 5 days. The reaction mixture evaporated to exclude DMF and re dissolved in chloroform from which the N,N–Dicyclohexyl urea (DCU) was filtered off. The clear filterate washed three times with 5% sodium bicarbonate solution, 0.1N HCl, once with distilled water, and with saturated sodium chloride solution. The chloroform layer was dried with anhydrous magnesium sulphate and evaporated under vacuum; the resulted product was collected, recrystallized from (methanol: acetone) (2.5:1) . Biological activity (21,22) Antimicrobial activity The antimicrobial of the synthesized final products was done in IBN SENA Research Center / Research and Development Authority/ Ministry of Industry and Minerals_IRAQ. A preliminary antibacterial and antifungal activity has been carried out according to Agar well Diffusion Method The prepared compounds had been studied for their antimicrobial activity in vitro against three tested bacteria (Staphylococcus aureus) as gram positive bacteria and( E. Coli. ,Pseudomonas aeruginosa) as gram negative bacteria and the yeast (Candida albicans) were clinical isolated and maintained on nutrient agar medium for testing antibacterial activity and sabaroud agar medium for antifungal activity. Compounds were dissolved in DMSO.Ciprofloxacin, Gentamycin and Cephalexin were used as a standard antibiotic for antibacterial activity and Clotrimazole was used as a standard drug for antifungal activity. Sensitivity assay Agar well diffusion assay was carried out by using bacterial suspension of about (1.5×10 8 CFU/ml) obtained from McFarland turbidity standard (number 0.5).This was used to inoculate by swabbing the surface of Mueller Hinton agar (MHA) plates. Excess was air-dried under a sterile hood, and in each agar plate of tested bacteria five wells were made and (30µl) of each concentration was added in it. The plates were incubated at 28 °C for 48 hours (Fungi spp.) or 37 °C for 24 hours (bacteria) and the antimicrobial activity was evaluated by measuring the diameter of the inhibition zone (IZ) around the disc in mm. The assessment of antibacterial activity was based on measurement of the diameter of inhibition zone formed around the well, and show that the zone of inhibition varied with the increasing of concentration of the tested compounds, as show in Table (4) . Cytotoxic activity study ( 23) A preliminary in vitro cytotoxicity assay (Cell Viability Assay on Cancer Cell Line) for some of the final compounds (3 and 4) has been carried out in the Biotechnology Research Center / University of AL-Nahrain. Actually, the in vitro cytotoxicity assays with cultured cells are widely used to evaluate chemicals including cancer chemotherapeutics, pharmaceuticals, biomaterials , natural toxins, antimicrobial agents and industrial chemicals because they are rapid and economical. Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 52 Cytotoxicity assay has been done by using Neutral Red uptake assay method. This method has been used for cytotoxicity assay of compounds (3 and 4). A set of two fold in four concentrations (3, 1.5, 0.75, 0.375 μg/ml) was made for each product and the exposure time of the assay was 24hrs. Result and Discussion Synthetic part A. The reaction pathways The aim of our research is to synthesize quinoline derivatives coupled to different amino acid ester or heterocyclic amine (figure 1). The overall synthesis strategy based one four major lines: 1. Amino acid derivatives The amino acids were activated by thionyl chloride to get acyl chloride that attacks either ethanol or methanol to get ethyl or methyl esters of the selected amino acids. 2. alkylation of 8-hydroxyquinoline by ethyl bromoacetate in presence of anhydrous potassium carbonate base to get (O-alkylated ester) derivatives which are deesterfied by using NaOH solution to get acetic acid derivatives. 3. Peptide bond formation Conventional solution method for peptide synthesis used as a coupling method between the carboxy-protected amino acids or 1-amino quinolone derivatives with acetic acid side chain of quinolone. The DCC/ HOBt coupling reagents used for peptide bond formation. The overall reaction pathway is shown in the following figure 1 . Figure (1): Scheme of overall pathway of synthesis of N-heterocyclic compounds. Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 53 B. Strategy of synthesis The strategy of synthesis started from the esterification of amino acids then proceeded to the synthesis of quinolone-8-yloxy acetic acid from 8-hydroxyquinoline and finally coupling between the acid and the amino group of the amino acid esters, and as follows: 1) Esterification of amino acids The esterification of carboxyl group of amino acids is normally used as an amino acid protecting group. Esterification of carboxyl group enhances the nucleophilic character of amine group and allows its subsequent acylation (24) . 2) 8-hydroxyquinoline can be alkylated at hydroxyl group by alpha-haloester of ethylbromoacetate which is widely used as alkylating agent to O-, N- and S- groups. Triethylamine or potassium carbonate act as a base which make a nucleophilic attack to the hydroxl group (-OH) of the above heterocyclic and deprotonate it and convert it to negative charge group which will then make a nucleophilic attack to the ethylbromoacetate which have bromide group that is a good leaving group to get ethyl acetate ester derivative of the above heterocyclic (compound 1). (25) The mechanism of O-alkylation by alkyl halide is a nucleophilic substitution as shown in figure (2) (26) . Figure (2): Scheme of the O-alkylation of 8-hydroxyquinoline 3) Peptide Coupling Method: There is many ways to form a peptide bond (30) , however in this work; the direct coupling with DCC/HOBt method was used. This method is characterized as being simple, efficient, no racemization, rapid, and leading to a good yield at R. T. (31) . The overall reaction resembles the dehydration process to form the amide bond (figure 1) .The properties of synthesized new compounds are shown in table (1) while the data obtained from elemental microanalysis are shown in table (2).On the other hand the characteristic IR bands are shown in table (3). 4) Removal of ethoxy group from compounds 1 were done by alkaline catalyzed de- esterification (saponification) to get the corresponding conjugated bases, that acidified by mineral acid (HCl) to get the corresponding carboxylic acid (27,28) (compound 2). The mechanism of saponification was proposed by Ingold as shown in figure (3) 29 ). Figure (3): Scheme of saponification of compound 1. Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 54 Table (1): The identification parameters of the synthesized compounds Symp. Compound Name Yield % Physical appearance m.p. o C Rf value A1 D-Alanine methyl ester. HCl 78 Off-white crystals 98-102 0.73A 0.69B B1 1-amino-quinoline-2-(1H)-one 73 yellow crystals 128- 130 0.90A 0.63C 1 ethyl 2-(quinolin-8-yloxy)acetate 77 yellow crystals 52-54 52-53 0.74C 0.79D 2 2-(quinolin-8-yloxy)acetic acid 68 Palle-yellow crystals 210- 214 0.30C 0.25D 3 methyl 2-(2-(quinolin-8- yloxy)acetamido)propanoate 60 White crystals 208- 210 0.88B 0.36C 4 N-(2-oxoquinolin-1(2H)-yl)-2-(quinolin- 8-yloxy)acetamide 50 Faint-yellow powder 217- 220 0.90D Table( 2): The Elemental microanalysis of the synthesized compounds Table (3): The characteristic IR bands of the synthesized compounds; (measured in cm-1) Cpd No. M. Wt. Chemical Formula Calculated ∕ Found C% H% N% O% 3 288.30 C15H16N2O4 62.49 61.94 5.59 6.10 9.72 10.58 22.20 21.38 4 345.35 C20H15N3O3 69.56 70.44 4.38 4.25 12.17 12.26 13.90 13.05 A1 3325, 3277 asym. and sym. str. of NH2 ; 2928, 2850 asym. and sym. CH3, CH2 str.;1743 C=O Str. Ester; 1570NH bend.; 1215 C-O str. Ester; 1435 CH2 bend.; 1375 CH3 bend. . B1 3306, 3186 asym. and sym. str. of NH2; 3057 C-H ar. str of benzene; 1662 C=O str. of amide; 1514NHbend.; 1118 C-N str.;1597,1548,1458 C=Car. Str. . 1 (3053 C-Har Str.), (2989assym , 2875 sym. C-Halif. Str.), (1743.7 C=O Str. ester), (1504 , 1446 C=Car Str.), (1219 C-O Str. ester), (1118 Ar-O-C str.),1653 C=N str. . Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 55 Table (3): Continued the characteristic IR bands of the synthesized compounds; (measured in cm -1 ) Asym=asymmetric , sym.=symmetric , str.=stretching , bend.=bending , ar.=aromatic, alif=alifatic Table (4): The antibacterial activity of the tested compounds.(3 and 4 ) 2 (3462 OH str. Of COOH), (3053 C-Har. Str.), (2937assym,2833sym C-Halif. Str.), (1716 C=O Str. of COOH), (1600 , 1562,1454 C=Car Str.), (1118 Ar-O-C str.). 3 3323 NH str.), (3036 CH ar. Str.), (2928assym,2850symCHalif.Str.), (1743.7 C=O Str. Ester), (1678C=O Str. amid) ,(1624 C=N Str.), (1535-N-H bend.) ,(1572,1564,1502 C=Car Str.), (1242 Ar-O- C str.). 4 (3323 NH str.), (3032 CH ar str. ), (2928 assym. , 2850 sym CHalif. Str.), (1683 C=O str. amide), (1537 NH bend. amide II), (1570- 1435 C=C str.), (1242 Ar-O-C str.). Compound No. Zone of Inhibition in mm E. Coli ATCC10536 Staphy. Aureus ATCC6538 Pseud. aurogenosa ATCC15442 3 100µg/ml No activity No activity 13 200µg/ml 13 No activity 14.5 4 100µg/ml No activity No activity 15 200µg/ml 13.5 No activity 15 ciprofloxacin 100µg/ml 20.5 22 30 200µg/ml 26 32 35 Gentamycin 200µg/ml 20 20 14 Cephalexin 600µg/ml 24 36 No activity Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 56 Table (5): The antifungal activity of the tested compounds.( 3 and 4) According to the following equation, the cytotoxic effect for tested compounds expressed as IR % at transmittance wave length 492 nm as showed in table 6 Inhibition Rate% = x100 Table (6): The initial cytotoxic effect (cell viability assay ) on Hep - 2 cell line of compounds ( 3 and 4) by Neutral red assay method. Cpd. No. Concentration time 24 hrs. 3μg/ml 1.5μg/ml 0.75μg/ml 0.375μg/ml Control/ 0μg/ml 3 0.1396 88.49 11.51 0.1353 85.768 14.23 0.1066 67.575 32.43 0.1093 69.286 30.71 0.15775 100 0 Abs. at 492nm cell viability% I.R.% 4 0.1026 65.05 34.97 0.09 57.05 42.95 0.0796 50.45 49.55 0.0836 52.99 47.01 0.15775 100 0 Abs. at 492nm cell viability% I.R.% Abs.= Absorbance I.R.%= Inhibition Rate Percent Summary of the Results 1)From the result in table (4), all tested compounds had low or no activity against E.Coli and no activity against Staphylococcus aureus when compared with Ciprofloxacin, Gentamycin and Cephalexin. While all tested compounds showed high activity against Pseudomonas aeruginosa when compared to Cephalexin, and comparable activity against same bacteria when compared to Gentamycin and finally ,the tested compounds showed low activity against Pseudomonas aeruginosa when compared to Ciprofloxacin. From the results in Table (5), the tested compounds showed a moderate to good activity against Candida albicans. when compared to Clotrimazole. 2)The cytotoxic study was done on Hep-2 cell line passage (75), Exposure time =24hrs. Staining is Neutral red stain. When the cancer cell line (Hep-2) was treated with these products the result showed significant cytotoxic effect in tested samples in comparison with the control. The toxic effect varied from one sample to another, all samples showed a significant toxicity (P < 0.05) started from 3μg/ml to the 0.375μg/ml. The inhibitory concentration percent (IC %) was estimated, and the result was varied among samples as shown in table (6). Conclusions From the antimicrobial and cytotoxic activity studies, compound 4 showed the best activity. according to pharmacokinetics properties and cationic nature of comp.4, comp.4 may cause DNA intercalation through DNA topoisomerase/gerase chelation or other binding sites. References 1. Padmavathi V., Subbaiah DRCV, Mahesh K, Lakshmi TR.; Synthesis of and bioassay of amino-pyrozole, amino- isoxazolon and amino- pyrimidinone derivatives, Chem Pharm Bull, 2007; 55: 1704-1709. 2. Shinde DB, Aaglawe MJ, Dhule SS, Bahekar SS, Wakte PS.; Synthesis and antibacterial activity of some oxazolone Compound No. Zone of Inhibition in mm Candidia albicans 10231 3 100µg/ml 14 200µg/ml 11 4 100µg/ml 10 200µg/ml 11 Clotrimazole 100µg/ml 12.75 200µg/ml 14.5 Absorbance of control – Absorbance of test Absorbance of control Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 57 derivaties, J Kor Chem Sty, 2003; 47: 133-136 3. Bazgir A., Khanaposhtani MM, Sooski AA.; One pot synthesis and antibacterial of pyrazolo[4’,3’:5,6]pyrido[2,3- d]pyrmidine-dione derivatives, Bioorg Med Chem Lett, 2008;18: 5800-5803. 4. Dahiya R, Kumar A, Yadav R.; Synthesis and Biological Activity of Peptide Derivatives of Iodoquinazolinones /Nitroimidazoles, Molecules, 2008; 13: 956-976. 5. Shazia Zaman; Synthesis of Nitrogenous Heterocycles Using Transition Metal Catalysed Cyclization Reactions; Ph.D. thesis, University of Canterbury,2005; 2- 17. 6. Beagley P, Blackie MA, Chibale K, Clarkson C, Moss JR, Smith P, and Su H. Synthesis and antiplasmodial activity in vitro of new ferrocene-chloroquine analogues. Journal of the ChemicalSociety – Dalton Transactions. 2003; 3046-3051. 7. Ma Z, Hano Y, Nomura T, Chen Y. Novel quinazoline-quinolinealkaloids with cytotoxic and DNA topoisomerase II inhibitory activities. Bioorg Med Chem Lett. 2004 Mar 8;14(5):1193-6. 8. Fokialakis N, Magiatis P, Chinou L, Mitaku S, Tillequin F. “Megistoquinones I and II, two quinoline alkaloids with antibacterial activity from the bark of Sarcomelicopemegistophylla”, Chem. Pharm. Bull. 2002; 50: 413–414. 9. Fossa P, Mosti L, Menozzi G, Marzano C, Baccichetti F, Bordin F. “Novel angular furo and thieno-quinolinones: Synthesis and preliminary photobiological studies”, Bioorg. Med. Chem. 2002; 10: 743–751. 10. Ryckebusch A, Derprez-Poulain R, Maes L, Debreu-FontaineMA, Mouray E, Grellier P, Sergheraert C. “Synthesis and in vitro and in vivo antimalarial activity of N1-(7-chloro-4-quinolyl)- 1,4-bis(3- aminopropyl) piperazine derivatives”, J. Med. Chem. 2003; 46: 542–557. 11. Morgan LR, Jursic BS, Hooper CL, Neumann DM, Thangaraj K, Leblanc B. “Anticancer activity for 4,40- dihydroxybenzophenone-2,4- dinitrophenylhydrazone (A-007) analogues and their abilities to interact with lymphoendothelial cell surface markers”, Bioorg. Med. Chem. Lett. 2002; 12: 3407–3411. 12. Nithyanantham M, Vedachalam G U, Ramaiah S, Navaneetharaman A, and Joseph TL, Synthesis, Anticonvulsant and Antihypertensive Activities of 8- Substituted Quinoline Derivatives. Biol. Pharm. Bull. 2004; 27(10): 1683-1687 13. Tajana A, Leonardi A, Cappelletti R, Nardi D, Massarani E, Degen L. 7-Nitro- 8-benzoyloxyquinolines with antimicrobial and antifungal activity Bollettino chimico farmaceutico. 1973; 112(11):767-75. 14. Chikhalia KH, Patel MJ, Vashi DB. Design, synthesis and evaluation of novel quinolyl chalcones as antibacterial agents. Arkivoc, 2008; (xiii): 189-197. 15. Azad M, Munawar MA, and Siddiqui HL. Antimicrobial activity and synthesis of quinoline-based chalcones. J. Applied Sci., 2007; 7: 2485-2489. 16. AL-Ameri M. F., Synthesis of new analgesic peptides analogues with expected biological activity; M. sc. Thesis, College of Pharmacy, Baghdad University, Baghdad., 2000. 17. Redha, I.A.; Ahmed, A. H.; Yasmien, K. A.: Design, synthesis and bioassay of novel coumarins. African Journal of Pure and Applied Chemistry, 2010; 4(6): 74-86. 18. Supriya M.; Suroor A. Khan; Shamim Ahmad, synthesis, characterization ,antimicrobial and antioxidant activity of some novel Schiff bases derived from 8- hydroxy quinolone. IJPBS, 2012; 2(3): 90- 98 19. Najim, A. ; Iman, A,; Ibrahim, A.: Amino acid derivatives. Part I. Synthesis, antiviral and antitumor evaluation of new amino acid esters bearing coumarin side chain, Acta Pharm. , 2006; 59: 175–188. 20. Henklein P, Rapp W. Comparison of Microwave Mediated Peptide Synthesis in Comparison to Conventional Peptide Synthesis. J. Peptide Chemistry, 2008; 14(8):10401-10421. 21. Kuete, V.; Eyong, G.; Folefoc, V.: Antimicrobial activity of the methanolic extract and of the chemical constituents isolated from Newbouldia laevis. Pharmazie, 2007; 62: 552-556. 22. Neli, L.; Yogendra, K.; Berington, M.: in vitro antibacterial activity of alkaloid extract from stem bark of Mahonia manipurensis Takeda. Journal of Medicinal Plants Research. 2011; 5(5): 859-861. 23. Irena Kostova; Synthetic and Natural Coumarins as Cytotoxic Agents, Curr. Med. Chem - Anti-Cancer Agents, Bentham Science Publishers Ltd., 2005; 5: 29-46. 24. Cary F.; Chapter 25: Amino acids, peptides and proteins, Organic Chemistry, Iraqi J Pharm Sci, Vol.24(1) 2015 Novel nitrogenous heterocyclic compounds with biological activity 58 8th Ed., 2011, McGraw–Hill, pp. 1116 – 1173. 25. Carey. F.A., Sunberg. R.J.; Advance Organic Chemistry; part A: Structure and mechanisms, 4th ed., Plenum Press, New York, 2000, pp. 263-268 and 295-298. 26. Mc Murry J.; Chapter 11, Organic chemistry, 8th Ed., Brooks/Cole, Cengage Learning, USA, 2012, pp. 372-389. 27. McLaren, J. A.; Amino acids and peptides V: The alkaline saponification of N- benzyloxycarbonyl peptide esters, Aust. J. Chem., 1958; 11: 360-365. 28. Morrison R. T., Boyd R. N.; Chapter 18: Alkaline hydrolysis of esters, Organic Chemistry, 6th Ed., Prentice-Hall Inc., New Jersey, USA, 2002, pp. 677-680. 29. Ingold C. K.; Aliphatic substitution, Structure and Mechanism in Organic Chemistry, 2nd Ed., Cornell University Press, USA, 1969, pp. 457-463. 30. Boger, D. et al; Total synthesis and characterization of DNA binding properties, J. Am. Chem. Soc., 1996; 118: 1629-1644. 31. Bodansky M, Klauser Y S and Ondetti M A; Formation of peptide bond, peptide synthesis, 2nd Ed, John Wiley and Sons Inc., New York, 1976, pp.116-124.