Iraqi J Pharm Sci, Vol.24(1) 2015 Synthesis of new derivatives of cephalexin 85 Design, Synthesis and Preliminary Antimicrobial Evaluation of New Derivatives of Cephalexin Shakir M. Alwan *,1 and Sarrah S. Jabbar * * Department of Pharmaceutical Chemistry, Collage of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract There is a continuous and massive need for newer cephalosporins that should have resistance against β-lactamases and can be used orally. An approach of using cephalexin, as a well-studied and potent antibacterial compound is considered to prepare new designed derivatives. These derivatives include the incorporation of amino acid moiety linked through an amide bond with the α-amino group of cephalexin. Certain aliphatic amino acids were used, such as glycine, alanine, valine and proline. The chemical structures of these derivatives were confirmed by IR spectroscopy and elemental analyses. All the synthesized compounds were subjected for preliminary evaluation of antimicrobial activity using well diffusion method, against certain microbes. Most of the synthesized compounds were found to possess significant antibacterial activities. Compound 1 (125 μg and 250μg) showed significant activity against P. aeruginosa. Compound 2 (125 and 250μg) exhibited significant activity against P. aeruginosa and Bacillus cereus. Compound 3 (125 and 250μg) demonstrated very significant activity against E. coli, P. aeruginosa and Bacillus cereus and slight activity towards S. aureus. Compound 4 (250μg) showed significant activity against P. aeruginosa and no antibacterial activities against E. coli, S. aureus and Bacillus cereus, as compared with cephalexin as the standard compound. Keywords: Cephalosporins, Cephalexin, Glycine, Alanine, Valine, Proline. اليت المضادة للبكتيريا لمشتقاث جديدةلفعاالولي لتقييم التحضير وتصميم و يهلعقار السيفالكس شاكر محمود علوان ،*1 و سارة ستار جبار * * فزع انكيًيبء انصيذالَيت ، .انعزاق ،بغذاد ،جبيعت بغذاد ،كهيت انصيذنت الخالصة احعح يٍ انذراسبث ٔانبحٕد انعهًيّ ببٌ ُْبنك حبجّ كبيزة نًعبداث حيٕيّ يٍ َٕع انسيفبنٕسبٕريُبث انًمبٔيّ نهبكخزيب انًُخجت الَزيًبث انبيخبالكخًيز ٔانخي لذ حسخعًم عٍ غزيك انفى. اسخخذو انسيفبالكسيٍ نخحعيز يشخمبث جذيذة بٕاسطت ربػ االحًبض االييُيّ عٍ غزيك يجًٕعّ انكبربٕكسيم بًجًٕعت االييٍ االٔنيّ )حزِ( انًٕجٕدِ في جزيئت انسيفبنكسيٍ ٔحكٕيٍ اصزة ايبيذ اسطت ببسخعًبل االحًبض االييُيت االنيفبحيت يثم انكاليسيٍ ,األنُيٍ , فبنيٍ ٔانبزٔنيٍ. حى حشخيص انخزاكيب انكيًيبٔيت نٓذِ انًشخمبث بٕ في )االشعت ححج انحًزاء ( ٔانخحهيم انذليك نهعُبصز )انكبربٌٕ ٔانٓيذدرٔجيٍ ٔانُخزٔجيٍ( ٔكبَج انُخبئج يطببمّ انخحهيم انطي نهخزكيببث انكيًيبٔيّ انًفخزظّ. حًج دراست انفعبنيّ انًعبدة نهبكخزيب ببسخعًبل بعط انًيكزٔببث انًزظيت ٔاظٓزث انُخبئج ببٌ يبيكزٔغزاو( فمذ اظٓز 252-125) 2يّ فعبنيّ يخًيزة ظذ انزائفت انزَجبريت , بيًُب انًزكب يبيكزٔغزاو( نذ 252-125) 1انًزكب يبيكزٔغزاو( اظٓز فعبنيّ جذا يخًيزة 252-125) 3فعبنيّ ظذ انزائفت انزَجبريت ٔانبكخزيب انعصٕيت انشًعيت. في حيٍ اٌ انًزكب يبيكزغزاو( فمذ اظٓز فعبنيّ يخًيزة 252) 4شزيكيت انمٕنَٕيت. ايب انًزكب ظذ انزائفت انزَجبريت ٔانبكخزيب انعصٕيت انشًعيت ٔ اال ظذ انزائفت انزَجبريت ٔنى يظٓز أي فعبنيّ نبميّ انًبيكزٔببث ٔلذ اسخعًم عمبر انسيفبنكسيٍ كًبدة ليبسيت نهًمبرَّ. روليه.فاليه والب ،االلنيه ،كاليسيه ،السيفالكسيه ،الكلماث المفتاحيت:السيفالوسبوريه Introduction The wide use of antibiotics in man and animals and their extensive use in areas other than the treatment and prophylaxis of diseases have resulted in a serious problem of drug resistance. More and more bacterial strains have become resistant to available drugs. A relation between the structure of the complexes and their anti-bacterial activity can be observed (1) . In the last two decades, antimicrobial resistance has become one of the greatest health problems, mainly in hospitals (2) . Despite the continuous development and introduction of new antibiotics, resistance continues to increase progressively in several microbes (3) . Preparation of different semisynthetic derivatives of cephalosporins based on structure-activity relationship has been one of the best approaches. Intensive search for new cephalosporins that may have broader antibacterial spectrum and resistance toward β-lactamase–producing bacteria and could be used orally are of great interest (4) . Cephalexin is a β- lactam antibacterial and has bactericidal action and acts similarly to 1 Corresponding Author E-mail: shakmawales@yahoo.co.uk Received: 17/12/ 2014 Accepted: 1/6/2015 mailto:shakmawales@yahoo.co.uk Iraqi J Pharm Sci, Vol.24(1) 2015 Synthesis of new derivatives of cephalexin 86 benzyl penicillin by inhibiting synthesis of the bacterial cell wall. It is most active against G (+) cocci and has moderate activity against some G (-) bacilli. Sensitive G (+) cocci includes both penicillinase- and non- penicillinase-producing staphylococci, although methicillin-resistant staphylococci are resistant; most streptococci are also sensitive, but not penicillin-resistant Streptococci Pneumonia; Enterococci, which are usually resistant. Some G (+) anaerobes are also susceptible. Cephalexin is usually inactive against Listeria monocytogenes. Among G (-) bacteria, cephalexin has activity against some Enterobacteriaceae including strains of E. coli, K. pneumoniae, Proteus mirabilis, Salmonella and Shigella spp., but not active against Enterobacter, indole-positive Proteus and Serratia spp. It is also active against Moraxella catarrhalis (Branhamella catarrhalis) and Neisseria spp., though Haemophilus influenzae is moderately resistant. Bacteroides fragilis and P. aeruginosa are not sensitive and neither, mycobacteria, mycoplasma nor fungi (5) . The incorporation of privileged chemical moieties, such as, amino acids has been found to have great potential in the field of antimicrobial agents. Amino acid linked to cephalexin through amide bond can be of great benefits and may add appreciable activity, resistance to β-lactamases and/ or improved pharmacokinetic properties . The proposed compounds may serve for injectable purposes, when prepared as sodium salt or could be used orally due to expected stability in aqueous acidic condition due to the presence of primary amine group at the α-carbon of the acyl side chain. Experimental Work Materials and Methods General Melting points (uncorrected) were determined using electrical melting point apparatus, Electro-thermal 9300, USA. The infrared spectra were performed in KBr disc by FT-IR spectrophotometer/ Shimadzu. Elemental micro- analyses (CHN) were performed by Euro-vector EA 3000A, Italy. Checking the purity of the products as well as monitoring the progress of the reaction was achieved by Thin layer chromatography using Silica gel F254 aluminum sheets, Merck, Germany. Chemicals and Reagents Ethyl chloroformate (ECF) was purchased from Sigma Aldrich/ Germany, Boc- glycine; Boc-L-alanine, Boc-L-valine and Boc-L- proline were purchased from Shanghai World Yang Chemical/China. Trifluoroacetic acid (TFA) was obtained from Sigma Aldrich/Germany. Cephalexin monohydrate was from SDI Samarra/Iraq General procedure for synthesis of the intermediates of cephalexin (1a-d). The new intermediates of cephalexin were synthesized by the mixed anhydride method (7-9) , as shown in (schemes 1and 2). Boc-amino acid (11.41 mmol) was dissolved in Tetrahydrofuran, THF (20mL) containing TEA (11.41 mmol) and this mixture was cooled in an ice bath at (-10°C). Ethyl chloroformate, ECF (11.41 mmol) was added drop wise over a period of 10 min and the mixture was continuously stirred for further 30 min. Cephalexin (11.41 mmol) in distilled water (10ml), containing TEA (11.41 mmol) was cooled to 0 °C and added at once to the above solution and the mixture was stirred for 4 hrs at -10 °C and for 2 hrs at room temperature. The solvent was evaporated and the resultant precipitate was washed with diluted HCl (0.1N) and filtered. The precipitate was collected and was washed with water several times with stirring, then washed with ether, recrystallized from ethanol/ toluene (1:9). Synthesis of 1a, 7-(2-(2-((t-butoxy carbonyl)- amino)acetamido)-2-phenylacetamido)-3- methyl–8-oxo-5-thia-1-azabicyclo {4.2.0}oct-2- ene-carboxylate This compound 1a was synthesized, as previously described and as shown in (scheme 1). Chemical structure of compound 1a Boc-glycine (11.416mmol, 5.75g) in THF (20mL) containing TEA (11.416 mmol, 1.153 g) was reacted with ECF (11.416 mmol, 1.24 g). Cephalexin (11.416 mmol, 4.16 g) in distilled water (10ml) containing TEA (11.416 mmol, 1.153 g) was added. The reaction mixture was treated as described earlier. The physical appearance, percent yield and Rf value are listed on table (1). http://www.sdisamarra.com/ Iraqi J Pharm Sci, Vol.24(1) 2015 Synthesis of new derivatives of cephalexin 87 Synthesis of 1b, 7-(2-2-((t-butoxy carbonyl) amino) propanamido)-2-phenylacetamido)-3- methyl-8-oxo-5-thia-1-azabicyclo {4.2.0} oct-2- ene-carboxylate This compound 1b was synthesized, as follows and as shown in (scheme 1): Chemical structure of compound 1b BOC-alanine (11.416 mmol, 5.91 g) in 20 ml of THF containing TEA (11.416 mmol, 1.153g) was reacted with ECF (11.416 mmol, 1.24 g). Cephalexin (11.416mmol, 4.16 g) in distilled water (10ml) containing TEA (11.416 mmol, 1.153g) was added. The reaction mixture was treated as previously described. The physical appearance, percent yield and Rf value are listed on table (1). Synthesis of 1c, 7-(2-2- ((t-butoxy carbonyl) amino)-3-methylbutanamido)-2-phenyl acetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo {4.2.0} oct-2-ene carboxylate Compound 1c was synthesized, as follows and as shown in (scheme 1): Chemical structure of compound 1c Boc-valine (11.416 mmol, 6.23 g) in 20 ml of THF containing TEA (11.416 mmol, 1.153g) reacted with ECF (11.416 mmol, 1.24g). Cephalexin (11.416 mmol, 4.16 g) in distilled water (10ml) containing TEA (11.416 mmol, 1.153g) was added. The mixture was treated as previously described. The physical appearance, percent yield and Rf value are listed on table (1). Synthesis of 1d, 7-(2-(1-(t-butoxy carbonyl) pyrrolidin-2-carboxamido)-2-phenylacet amido)-3-methyl-8-oxo-5-thia-1-azabicyclo {4.2.0} oct-2-ene-carboxylate. Compound 1d was synthesized, as follows and as shown in (scheme 2): Chemical structure of compound 1d Boc-proline (11.416mmol, 6.22g) in THF (20ml) containing TEA (11.416 mmol, 1.153g) was reacted with ECF (11.416mmol, 1.24g). Cephalexin (11.416 mmol, 4.16 g) in distilled water (10ml) containing TEA (11.416 mmol, 1.153g) was added. The mixture was treated as previously described. The physical appearance, percent yield and Rf value are listed on table (1). Scheme (1): Synthesis of intermediates 1a-c Scheme (2): Synthesis of intermediate 1d General procedure for synthesis of the new derivatives of cephalexin (1-4). These compounds were obtained by deprotection (10) of the amino group of compounds 1a-d, to afford the new derivatives of cephalexin 1-4, as follows and as shown in (schemes 3 and 4). Compounds 1a-d, (1.984 mmol) was suspended in dichloromethane (DCM) (10ml) and cooled Iraqi J Pharm Sci, Vol.24(1) 2015 Synthesis of new derivatives of cephalexin 88 to 0 °C in an ice bath and TFA (15ml) was added with continuous stirring for 1hr at 0 o C in presence of anisole (3ml). The completion of the reaction was monitored by TLC using the mobile phase methanol: chloroform (1:1). Diethyl ether (100ml) was added to the reaction mixture and the resulting precipitate was collected, suspended in methanol (30) ml and the pH was adjusted to 7 with 5% methanolic solution of NaOH. A precipitate was formed after the addition of ether, which was filtered and washed with acetone and recrystallized from ethyl acetate: petroleum ether (9:1). The precipitate was collected and dried in an oven at 50 o C. Synthesis of 1, 7-(2-(2-aminoacetamido)-2 phenylacetamido)-3-methyl-8-oxo-5-thia-1- azabicyclo{4.2.0}oct-2-ene-carboxylate Sodium. Compound 1 was obtained by deprotection of the amino group of compound 1a, as follows and as shown in (scheme 3): Chemical structure of compound 1 Compound 1a (1.984 mmol, 1 g) in DCM (10ml) was reacted with TFA (15ml) in presence of anisole (3ml). The mixture was treated as previously described. A yellowish crystalline powder was collected. The physical appearance, percent yield and Rf value are listed on table (1). Synthesis of 2, 7-(2-2-amino propan amido)2- phenylacetamido)-3-methyl-8-oxo-5-thia-1- azabicyclo{4.2.0}oct-2-ene-carboxylate Sodium. Compound 2 was obtained by deprotection of the amino group of compound 1b, as follows and as shown in (scheme 3): Chemical structure of compound 2 Compound 1b (1.984mmol, 1.027 g) in DCM (10ml) was reacted with TFA (15ml) in presence of anisole (3ml). The mixture was treated as previously described. The physical appearance, percent yield and Rf value are listed on table (1). Synthesis of 3, 7- (2- (2-amino-3-methylbutan amido) 2-phenylacetamido)-3-methyl-8-oxo -5- thia-1-azabicyclo {4.2.0}oct-2-ene-carboxylate Sodium. Compound 3 was obtained by deprotection of the amino group of compound 1c, as follows and as shown in (scheme 3): Chemical structure of compound 3 Compound 1c (1.984 mmol, 1.083 g) in DCM (10ml) was reacted with TFA (15ml) in the presence of anisole (3ml) and the mixture was treated as previously described. The physical appearance, percent yield and Rf value are listed on table (1). Synthesis of 4, 7-(2-phenyl-2- (pyrrolidin -2 - carboxamido) acetamido)-5-thia-1-azabicyclo {4.2.0} oct - 2 - ene-carboxylate Sodium. Compound 4 was obtained by deprotection of the amino group of compound 1d, as follows and as shown in (scheme 4): Chemical structure of compound 4 Compound 1d (1.984 mmol, 1.081 g) in DCM (10ml) was reacted with TFA (15) in presence of anisole (3ml) and the mixture was treated as previously described. The physical appearance, percent yield and Rf value are listed in table (1). Iraqi J Pharm Sci, Vol.24(1) 2015 Synthesis of new derivatives of cephalexin 89 Table (1): Physical parameters and percent yields of the synthesized compounds. Scheme (3): Synthesis of the target compounds 1-3 Scheme (4): Synthesis of the target compound 4 Results and Discussion The use of two solvent systems A and B was to differentiate between reactants and products and to follow progress of reactions. A-Chloroform: Methanol (3:1) B-Chloroform: Methanol (1:3), as illustrated on Table (1). The IR characteristic bands of compound 1 are 1543, 1400 (C=O stretching of carboxylate anion), 3060 (N-H stretching 2-amide, 3473, 3397 (N-H stretching of primary amine group), 1656 (C=O stretching 2-amide), 1761(C=O stretching β-lactam), 1543(N-H bending 2- amide). The elemental analysis (CHN) was calculated for C18H19N4SO5Na (426); calculated: C; 50.70, H; 4.46, N; 13.145. Found C; 48.35, H; 4.684, N; 13.36. The IR characteristic bands of compound 2 are 1549, 1400 (C=O stretching of carboxylate anion), 3061 (N-H stretching 2-amide), 3494, 3399 (N-H stretching primary amine group), 1664 (C=O stretching 2-amide), 1759 (C=O stretching β-lactam), 1527 (N-H bending 2- amide). CHN analysis was calculated for C19H21N4SO5Na (440); calculated C: 51.8, H: 4.77, N: 12.72. Found C: 49.91, H: 4.56, N: 12.28. The IR characteristic bands of compound 3 are as follows; 1543, 1400 C=O stretching of carboxylate anion, 3063 (N-H stretching of 2- amide), 3473, 3397 (N-H stretching of primary amine), 1653 (C=O stretching of 2-amide), 1759 (C=O stretching of β-lactam), 1570 (N-H bending of 2-amide). CHN analysis was calculated for C21H25N4SO4Na (429) calculated; C: 53.846, H: 5.341, N: 11.965. Found C: 52.04, H: 5.16, N: 11.71. Compound Physical appearance % Yield m.p. ( o C) Rf value 1a White 63.8 172-177 0.60 (A) 1b White 72 182-186 0.44 (A) 1c White 74 160-166 0.55 (A) 1d Off white 82 180-185 0.50 (A) 1 Pale yellow 95 185 (decomposed) 0.41 (B) 2 Pale yellow 44 200 (decomposed) 0.46 (B) 3 Off white 70 214 (decomposed) 0.57(B) 4 Off white 40 212 (decomposed) 0.55(B) Cephalexin Off white -- 182-186 0.11(A) Iraqi J Pharm Sci, Vol.24(1) 2015 Synthesis of new derivatives of cephalexin 90 The IR characteristic bands of compound 4 are 1550, 1400 C=O stretching of carboxylate anion, 3034.13N-H stretching of 2-amide, 3373, 3317 N-H stretching of primary amine group, 1662.69 C=O stretching of 2-amide, 1755.28 C=O stretching of β-lactam, 1527 N- H bending of 2-amide. CHN analysis was calculated for C21H25N4SO5Na (445), calculated; C: 54.663, H: 3.904, N: 12.147. Found C: 52.20, H: 3.78, N: 12.68. Preliminary Antimicrobial Evaluation The synthesized compounds were subjected to antimicrobial evaluation by well-diffusion method (11) . The zone of inhibition (mm) was measured in comparison with cephalexin. The antimicrobial activity was performed in nutrient agar medium containing E. coli, P. aeruginosa and Bacillus cereus, S. aureus and the compounds used at concentrations (125 and 250 μg/well).The activity was determined after incubation for 24h at 37 o C by the comparison of inhibition of growth of bacteria by cephalexin using dimethyl sulfoxide (DMSO) as the solvent. No inhibition zone was observed for the control (dimethylsulfoxide). These compounds were subjected to preliminary antimicrobial evaluation against four types of microbes (E. coli, P. aeruginosa, and Bacillus cereus, S. aureus). Table ( 2) :The preliminary antibacterial activity of the new derivatives of cephalexin. Key to symbols: (-) = no inhibition. The antimicrobial evaluation revealed that the newly synthesized compounds, 1-4 showed reasonable antibacterial activities against G (-) bacteria, such as P. aeruginosa and G (+) bacteria, such as Bacillus cereus in comparison with cephalexin, which has no activity against these type of microbes. Compound 1 (125 and 250μg) showed significant activity against P. aeruginosa and reduction in antibacterial activities against S. aureus and E. coli as compared with cephalexin. Compound 2 (125 μg, 250μg) showed significant activity against P. aeruginosa and Bacillus cereus, as compared with cephalexin. Furthermore, at concentration (125 μg) compound 2 exhibited no activity against S. aureus and E. coli. However, at concentration of (250 μg) the activity was less against S. aureus as compared with cephalexin. Compound 3 (125 and 250μg) showed very significant activity against E. coli, P. aeruginosa and Bacillus cereus and slight activity towards S. aureus. This result indicates that compound 3 has a broader spectrum of antibacterial activities, i.e. against both G (+) and G (-) bacteria. Compound 4 (125μg) has no antibacterial activities against all strains of bacteria used. However, Compound 4 at concentration (250 μg) showed significant activity against P. aeruginosa and no antibacterial activities against E. coli, S. aureus and Bacillus cereus as compared with cephalexin. This chemical modification at the acyl side chain positioned at alpha to β- lactam ring supposed to provide some protection against β-lactamses. This model contains a primary amino group of the amino acid, which may provide certain stability in the acidic condition of stomach (4). Conclusion A series of new derivatives of cephalexin have been synthesized successfully in appreciable yields and screened for their antimicrobial activity using well diffusion method against bacterial strains (E. coli, P. aeruginosa and Bacillus cereus, S. aureus). It is concluded that the new derivatives of cephalexin linked with certain amino acids were found to possess moderate antibacterial activities. Furthermore, the new derivative of cephalexin linked with valine has a significant activity against p. aeruginosa and Bacillus cereus. Compound Concentration µg/ml Escherichia coli. Pseudomonas aeruginosa Staphylococcus aureus Bacillus cereus. DMSO -- 9 - 10 - cephalexin 125 10 - 12 - 250 12 14 1 125 - 11 - - 250 - 12 - - 2 125 - 11 - 6 250 - 14 8 10 3 125 8 15 9 15 250 9 15 9 17 4 125 - - - - Iraqi J Pharm Sci, Vol.24(1) 2015 Synthesis of new derivatives of cephalexin 91 References 1. Brown, A.G. and S.M. Roberts, (Ed.). Recent Advances in the Chemistry of β- lactam Antibiotics. The Royal Society of Chemistry, London – 1984. 2. Gary A. Thiobodeas, Kelvin T. Patton, Elsevier. Synthesis and Biological Evaluation of Some Novel Schiff Bases of Cephalexin. Inter. J. Pharmaceut. 2004, 7; 703. 3. Furtado G. H. C., Perdiz L. B. and Medeiros E. A. 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R., and Osato, R.L., Preparation of peptides using mixed carbonic–carboxylic acid anhydride. J. Am. Chem. Soc., 1952, 74 (3), 676-678. 10. Cephalosporin antibiotic. Synthesis and antimicrobial activity of cephalosporin derivatives I. Csendes, B.W. Muller and W. Toch. The J. Antibiotic, 1983, 36 (8), 1032. 11. Fingegold ,S.M.Martin , W J.Diagnostic Microbiology ,6 th ed.:Mosby London :p.450;1982