Iraqi J Pharm Sci, Vol.23(1) 2014 Synthesis of prodrugs of amoxicillin and metronidazole 1 Synthesis and Antimicrobial Study of Possible Mutual Prodrugs of Amoxicillin and Metronidazole by Direct and Indirect Coupling through Spacer Ammar A. Ali Beg * and Ahlam J. Qasir **,1 * Department of Pharmaceutical Chemistry, College of Pharmacy, University of Kufa, Najaf, Iraq. ** Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract Amoxicillin have been conjugated with metronidazole as possible mutual prodrug to get a wider spectrum of activity by acting on aerobic and anaerobic bacteria, have antifungal activity, to provide protection for beta lactam ring of amoxicillin and also to improve patient compliance as it given as a single dose therapy. The structures of the synthesized compound were confirmed and characterized using elemental microanalysis (CHN), IR and some physiochemical properties. Biological study was done by using disc diffusion method against different bacterial strains which are , Staphylococcus aureus , Salmonella typhie , Pseudomonas aeruginosa , E. coli , Klebsiella pneumonia and fungi ( Candida albicans) . using nutrient agar medium and 5 mm diameter paper discs. The synthesized compounds showed maintenance or improvement of the antimicrobial activity especially compound (II) showed superior activity in comparison with compounds (I, III) . Key words: Amoxicillin , Metronidazole , Mutual prodrug . تبادليت لعقاري االموكسيسلين تخليق ودراست الفعاليت المضادة للميكروباث كمقذماث دوائيت زول بطريقت مباشرة وغير مباشرة بواسطت رراعذاوالمتروني عًاس عبذ انعضٌض عهً بٍج * و احالو جًٍم لظٍش **،1 * . انعشاقانكىفت،اننجف،جايعت ،كهٍت انظٍذنت،ًٍاء انظٍذالنٍتٍفشع انك ** . انعشاق،بغذاد،جايعت بغذاد ،كهٍت انظٍذنت،ًٍاء انظٍذالنٍتٍفشع انك الخالصة عماس االيىكسٍسٍهٍن يع عماس انًخشونٍذاصول كًمذياث دوائٍت نكً نحظم عهى فعانٍت راث طٍف أوسع فً هزا انبحذ نمىو بشبظ و ين خالل عًهه عهى انبكخشٌا انهىائٍت و انالهىائٍت , نذٌه فعانٍت ضذ انفطشٌاث , حىفٍش حًاٌت نحهمت انبٍخا الكخاو فً االيىكسٍسٍهٍن نخحسٍن اسخساغت انًشٌض نهعالج . حى حشخٍض انًشكباث انًظنعت باسخخذاو انخحهٍم انذلٍك نهعناطش انًكىنت نها و ين خالل األشعت ححج انحًشاء و بعض انخىاص انفٍضٌائٍت وانكًٍٍائٍت . دساست انفعانٍت انحٍىٌت حًج باسخخذاو طشٌمت االنبعاد انمشطً ضذ بعض Staphylococcus aureus , Salmonella typhie , Pseudomonasخهفت و انفطشٌاث و انخً هً سالالث انبكخشٌا انًخ aeruginosa , E. coli , Klebsiella pneumonia and Candida albicans . باسخخذاو وسظ يغزي و ألشاص وسلٍت لطش ( حٍذ اظهش فعانٍت IIنمذ أظهشث انًشكباث انًظنعت ححسنا أو حفاظا عهى انفعانٍت انًضادة نهًٍكشوباث وخظىطا انًشكب ) . يم 5 .( I,IIIعانٍت يماسنت بانًشكباث ) .تبادليت مقذماث دوائيت، المترونيذازول ، االموكسيسيلين الكلماث المفتاحيت : Introduction Anaerobic infections characteristically are polymicrobial (mixed) and include both anaerobic and facultative organisms (1) . The organisms tend to be acquired endogenously. The particular mix of pathogens reflects the combined influence of the complex commensal flora at a specific body site and the unique micro biota of the underlying conditions (2) . Because these organisms are generally of low pathogenicity, anaerobic or mixed infections generally develop as a consequence of either structural alterations in the normal mucosal barrier or tissue ischemia with lowered oxidation - reduction potential (3) . Knowledge of the anatomic location of the primary source of infection and the underlying condition of the host, therefore, is essential in predicting the probable organisms causing anaerobic and mixed infections associated with the indigenous micro flora (4) . Microorganism in mixed infections may respond to antimicrobial agents differently than do those in monomicrobial infections, and it may not be necessary to eradicate every bacterial species in mixed infection to achieve cure (5) . In the treatment of mixed aerobic anaerobic infection we use antibiotics that act on both type of bacteria so here we used metronidazole with amoxicillin as there is synergestic effect between them as shown In 1989, by van Winkelhoff et al. (6) who showed that mechanical treatment followed by a regimen of metronidazole and amoxicillin for 7 days is effective in eliminating A. actinomycetem- comitans from the infected sites. Recently, this was confirmed in a large patient group, in which more than 1 Corresponding author E-mail:ahlamqasir@yahoo.com Received:28 /1/2013 Accepted:15 /6/2013 Iraqi J Pharm Sci, Vol.23(1) 2014 Synthesis of prodrugs of amoxicillin and metronidazole 2 An explanation for the in vivo efficacy may be the in vitro synergism against 95% eradication of the microorganism was reported (7) . A.actinomycetem - comitans not only between metronidazole and amoxicillin but also between metronidazole and its hydroxymetabolite as well as between amoxicillin and the hydroxymetabolite of metronidazole (8) . However, there are potential advantages in giving such coadmi -nistered drugs having comple -mentary pharmacological activities in the form of a single chemical entity. Such agents are named as mutual prodrugs which are designed with improved physicochemical property . In the view of this background, the present study was conducted to the design, synthesis, and preliminary biological study of mutual prodrugs of amoxicillin with metronidazole to get antibiotic with broader spectrum of activity and given by single dose. Chemistry The synthetic pathways for the designed target compounds (1a- c and I, II and III) are illustrated in (schemes 1-6). Scheme (1) synthesis of MTZ-Cl Scheme (2) synthesis of compound I Scheme(3)synthesis of compound 1B Scheme (4) synthesis of compound II Iraqi J Pharm Sci, Vol.23(1) 2014 Synthesis of prodrugs of amoxicillin and metronidazole 3 Scheme (5) synthesis of compound 1c Scheme (6) synthesis of compound III Experimental A - Chemistry All reagents and anhydrous solvents were of analytical grade and were used as received from the commercial suppliers (Merck- Germany, Riedal-Dehaen-Germany, BDH- England and Fluka-Switzerland) . Amoxicillin and Metronidazole were purchased from SDI Company, Iraq. Thin layer chromatography (TLC) was run on Kieslgel GF254 (60), Merck (Germany), to check the purity of the products as well as monitoring the progress of reactions. FT-IR spectra were recorded at College of Pharmacy, Kufa University by using Shimadzu - Japan spectro - photometer and the determination of the spectra were performed by using KBr discs. CHNO microanalysis has been done at the central laboratory of kufa uniusing euroEA Shimadzu-Japan elemental analyzer . Synthesis of 1-(2-chloroethyl)-5-meth -yl-2- nitro-1H-imidazole, (1a ) (9) : To a solution of metronidazole ( 5 mmol , 0.858 gm) in THF ( 20 ml ) was added thionyl chloride (6.5 mmol , 0.75 ml) dropwise under stirring. then the reaction mixture was refluxed for 7 hrs. Excess thionyl chloride was removed azeotropically. The residue was taken into ethyl acetate (30 mL) and washed with water (10 mL x 3). Organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide a brown solid . The percent yield, physical appearance, melting point and Rf value are listed in table (1). Synthesis of compound (I) (10) : A suspension of amoxicillin trihydrate (1 mmol ,0.4195gm) in 5 ml DMF was prepared and (1 mmol ,0.01 gm) of potassium bicarbonate and [(0.203) mL (2 mmol)] benzaldehyde were added at 0 o C ( to provide protection for amine group0 . The mixture was stirred at 0 o C for 3.5 h. At the end of this time (1 mmol ,0.1 gm) of potassium bicarbonate and (1 mmol ) of compound (1a) were added and stirred at 0 o C for additional 6 hrs. The mixture was poured onto ice water and was extracted with (5 mL x 3) of ethyl -acetate. After washing the organic phase with 10% sodium chloride and drying over anhydrous sodium sulfate, it was evaporated to dryness in vacuo at 20 o C and a yellow oily residue was obtained. The residue was dissolved in 5 mL of acetonitrile. The pH of the solution was adjusted to 2 with 1N HCl and it was stirred for 30 min at 0-5 o C . In order to remove acetonitrile, 6 mL of water was added and the solution was evaporated in vacuo at 20 oC . After washing the aqueous solution with (6 mL x 3) of ethylacetate and saturating with Iraqi J Pharm Sci, Vol.23(1) 2014 Synthesis of prodrugs of amoxicillin and metronidazole 4 sodium chloride it was extracted with (6 mL x 3) of dichloro -methane. The organic phase was dried over anhydrous sodium sulfate and it was left overnight at 5 o C The residue was filtered and dried . The percent yield, physical appearance, melting point and Rf value are listed in table (1) . Synthesis of 4– (2– (5– methyl – 2 – nitro - 1H -imidazol – 1 –yl ) ethoxy)-4-oxo butanoic acid ( compound Ib ) (11) : Metronidazole (3 mmol ,0.5135gm) was dissolved in 30 mL of acetonitrile and (3 mmol , 0.3 gm) of the Succinic anhydride followed by 4-dimethyl aminopyridine (0.3 mmol , 0.037 gm) were added. The mixture was left at ambient temperature for 48 hrs. until the reaction was completed as evidenced by TLC , the products was precipitated after solvent evaporation and addition of cold water. The percent yield, physical appearance, melting point and Rf value are listed in table (1) . Synthesis of compound (II) (12) : Compound (Ib) (1.133 mmol , 0.303 g ) was dissolved in 10 ml of anhydrous DMSO and (1.699 mmol , 0.24 ml ) of TEA was added under stirring ; then ( 2.266 mmol , 0.260 gm) of NHS and then (2.266 mmol, 0.4675 gm) of DCC were added . The reaction was let under stirring overnight at room temperature in the dark . Dicyclohexylurea ( DCU ) was filtered out and the solution was dropped into 200 ml of diethyl ether. After 4 hrs. At 4 0 C the activated ( Ib- NHS ) was recovered by filtration and washed with Diethyl ether and then dried under vacuum . To ( 1 mmol , 0.4194 gm ) of Amoxicillin dissolved in 10 ml DMSO , ( 5 mmol ) of ( Ib- NHS ) dissolved in 6 ml of DMSO was added . To the mixture ( 0.02 ml ) of Et3N was added and the reaction was let to react over night under stirring at room temperature in the dark . Then 5 ml of water was added and the PH adjusted to 7.5 and 5ml of DMF was also added to help phase separation . the ( compound II ) was extracted from aqueous phase by ethyl acetate ( 50 ml x 4 ) . The organic layer was dried over anhydrous sodium sulphate and evaporate the ethyl acetate to get the compound (II). The percent yield, physical appearance, melting point and Rf value are listed in table (1) . To an ice-cooled solution of metro -nidazole (1.1 mmol , 0.1883 gm) and TEA (2.2 mmol , 0.3 ml) in dichloromethane (10 mL) , chloroacetyl chloride (2.2 mmol, 0.18 ml) was added under stirring. The reaction mixture was further stirred at 0-5 o C for 30 min. Synthesis of Chloro– acetic acid 2– ( 5– methyl–2-nitro – 1 H–imidazol – 1 - yl ) ethyl ester ( compound 1c ) (9) : Dichloromethane was distilled off and the residue was taken in ethyl acetate (20 mL). The ethyl acetate layer was washed with water (10 mL x 3) and dried over anhydrous sodium sulfate. Sodium sulfate was filtered off and washed with ethyl acetate (5 mL x 2). The filtrate was concentrated to give a brown semisolid . The percent yield, physical appearance, melting point and Rf value are listed in table (1) . Synthesis of compound (III) (10) : A suspension of amoxicillin trihydrate (1 mmol ,0.4195gm) in 5 ml DMF was prepared and (1 mmol ,0.01 gm) of potassium bicarbonate and [(0.203) mL (2 mmol)] benzaldehyde were added at 0 o C . The mixture was stirred at 0 o C for 3.5 h. At the end of this time (1 mmol ,0.1 gm) of potassium bicarbonate and (1 mmol ) of compound (1c) were added and stirred at 0 o C for additional 6 hrs. The mixture was poured onto ice water and was extracted with (5 mL x 3) of ethyl - acetate. After washing the organic phase with 10% sodium chloride and drying over anhydrous sodium sulfate, it was evaporated to dryness in vacuo at 20 o C and a yellow oily residue was obtained. The residue was dissolved in 5 mL of acetonitrile. The pH of the solution was adjusted to 2 with 1N HCl and it was stirred for 30 min at 0-5 o C . In order to remove acetonitrile, 6 mL of water was added and the solution was evaporated in vacuo at 20 o C . After washing the aqueous solution with (6 mL x 3) of ethylacetate and saturating with sodium chloride it was extracted with (6 mL x 3) of dichloro -methane. The organic phase was dried over anhydrous sodium sulfate and it was left overnight at 5 o C The residue was filtered and dried . The percent yield, physical appearance, melting point and Rf value are listed in table (1) . Iraqi J Pharm Sci, Vol.23(1) 2014 Synthesis of prodrugs of amoxicillin and metronidazole 5 Table (1) The percent yield, physical appearance, melting point and Rf of the intermediate and final products. A =(dichloromethane : Ethanol : ethylacetate) (6:1:3) Polarity index= 3.7 B=(dichloromethane : Ethanol : ethylacetate) (1:6:3) Polarity index= 4.75 1-(2-chloroethyl)-5-methyl-2-nitro-1H- imidazole (1a ) : IR (KBr): 2976 cm -1 [C-H] , 1532 cm -1 [N- O] , 1460 cm -1 [C=N] , 1365 cm -1 [N-O] , 756 cm -1 [C-Cl] . 4-(2-(5-methyl-2-nitro-1H-imidazol-1-yl) ethoxy)-4-oxo butanoic acid (Ib ) : IR (KBr): 3181 cm -1 [O-H] , 2962 cm -1 [C-H] , 1732 cm -1 [C=O] of ester , 1531 cm -1 [N-O] , 1476 cm -1 [C=N] , 1266 cm -1 [C-O] . Chloro-acetic acid 2-(2-methyl-5-nitro-1H- imidazol-1-yl)ethyl ester ( compound 1c ) : IR (KBr): 3019 cm -1 [C-H] , 1747 cm -1 [C- O] , 1529 cm -1 [N-O] , 1471 cm -1 [C=N] , 1262 cm -1 [C-N] , 825 cm -1 [C-Cl] . Compound (I) : IR (KBr): 3506 cm -1 asy. Str. [N-H] of amine , 3444cm -1 sy. Str. [N-H] of amine, 3151 cm -1 [O-H] of phenol , 1780 cm -1 [C=O] of lactam , 1738 cm -1 [C=O] of ester , 1697 cm -1 [C=O] of amide , 1550 cm -1 [N-O] of nitro , 1261 & 1174 cm -1 [C-O] . CHN Calculated: C, 50.96; H, 5.05; N,16.21; O, 21.6; Found: C, 51.21; H, 5.13; N, 16.18; O, 21.27. Compound (II) : IR (KBr): 3334 -1 [N-H] of amide , 3230 cm -1 [O-H] of phenol , 2966 cm -1 [C-H] , 1778 cm -1 [C=O] of lactam , 1742 cm -1 [C=O] of ester , 1674 cm -1 [C=O] of amide , 1638 cm - 1 [C=N] , 1519 cm -1 asy. Str. [N-O] of nitro , 1371 cm -1 sy. Str. [N-O] of nitro , 1273 & 1174 cm -1 [C-O] . CHN Calculated: C, 50.48; H, 4.89; N,13.59; O, 25.86; Found: C, 50.67; H, 5.02; N, 13.87; O, 25.46. Compound (III) : IR (KBr): 3511 cm -1 asy. Str. [N-H] of amine, 3441 cm -1 sy. Str. [N-H] of amine, 3207 cm -1 [O-H] of phenol, 1778 cm -1 [C=O] of lactam , 1751 & 1740 cm -1 [C=O] of ester , 1671 cm -1 [C=O] of amide , 1553 cm -1 [N-O] of nitro , 1275 & 1175 cm -1 [C-O] . CHN Calculated: C, 49.99; H, 4.89; N,14.58; O, 24.97; Found: C, 51.21; H, 5.21; N, 14.03; O, 24.42. B - Antimicrobial studies Antibacterial activity for the synthesized compounds was invest -tigated by disc diffusion method against different bacterial strains and fungi such as, Staphylococcus aureus , Salmonella typhie , Pseudomonas aeruginosa , E.coli , Klebsiella pneumonia and Candida albicans, using nutrient agar medium and 5 mm diameter paper discs (Whatman No. 1). The investigated compounds i.e. the final products, were dissolved in DMSO at a concentration of 5000 µg/ml. The filter paper disc were soaked in solutions of the test compounds, dried, and then placed in Petri plates previously seeded with the test organisms. The plates were incubated for 24 h at 37 °C and the inhibition zone in the region of each disc was measured . The results obtained are tabulated in table (2) . Compounds and intermediates Empirical formula Molecular weight Description % yield Melting point o C Rf value 1a C6H8ClN3O2 189.6 yellow crystals 60.1 77-79 *A=0.33 B=0.42 1b C10H13N3O6 271.23 White crystals 65.3 125-128 A=0.21 B=0.33 1c C8H10ClN3O4 247.64 Semisolid brown 76.3 A=0.3 B=0.41 I C22H26N6O7S 518.5 Faint yellow powder 59.4 189-191 A=0.29 B=0.41 II C26H30N6O10S 618.6 White powder 66.9 203-205 A=0.25 B=0.37 III C24H28N6O9S 576.58 Off white- yellow powder 55.3 197-199 d A=0.27 B=0.38 Iraqi J Pharm Sci, Vol.23(1) 2014 Synthesis of prodrugs of amoxicillin and metronidazole 6 Results and Discussion The synthesis of the designed compounds has been successfully achieved. Characterization and struct -ural formulas of the synthesized compounds were confirmed by melting point determination, Rf values, FTIR spectroscopy and elemental micro - analysis. The antimicrobial study for the synthesized compounds showed maintenance or improvement of the antibacterial activity against both gram negative and gram positive bacteria with activity against fungi . Especially compound II show superior activity in comparison with the other synthesized compounds. Table (2) Antimicrobial activities (a) of the compounds (I-III) . (a) Growth inhibition diameter (mm) References 1. Yoonseon Park, Jun Young Choi, Dongeun Yong , Clinical Features and Prognostic Factors of Anaerobic Infections: A 7-Year Retrospective Study , The Korean Journal of Internal Medicine , 2009;24:1- 13 . 2. Nagy E. Anaerobic infections: update on treatment considerations. Drugs. 2010;70 :841-58 . 3. zianabos AO et al : Anaerobic infections: General concepts, in Principles and Practice of Infectious Diseases, 6th ed, GL Mandell et al (eds). Elsevier , 2005, pp 2810–2816 4. Anthony W. Chow, m.d., f.a.c.p, f.r.c.p.c. 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Compound S.aureus Escherichia coli Pseudomonas aeruginos Klebsiella pneumoniae Candida albicans Control (DMSO) - - - - - Amoxicillin 25 22 23 23 - Metronidazole 6 - 14 - 17 I 31 23 32 21 19 II 29 25 31 29 20 III 28 24 30 28 19 http://www.ncbi.nlm.nih.gov/pubmed?term=Pasut%20G%5BAuthor%5D&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=Canal%20F%5BAuthor%5D&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=Dalla%20Via%20L%5BAuthor%5D&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=Arpicco%20S%5BAuthor%5D&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=Arpicco%20S%5BAuthor%5D&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=Veronese%20FM%5BAuthor%5D&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=Schiavon%20O%5BAuthor%5D&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed/18346806