Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 35 Synthesis of new derivatives of Ceftazidime as possible Prodrugs Shakir M. Alwan *,1 and Abdul-Hafeedh H. Abdul-Wahab * * Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad,Iraq. Abstract Five new ceftazidime derivatives were designed and synthesized in an attempt to improve the acid stability and may increase the spectrum of ceftazidime. The synthesized compounds included; Schiff base of ceftazidime (compound 1), ceftazidime lysine amide Schiff base (compound 2), ceftazidime lysine amide (compound 3), ceftazidime-di-lysine amide Schiff base (compound 4) and ceftazidime-di- lysine amide (compound 5). New ceftazidime derivatives were successfully prepared characterized and identified using spectral and elemental microanalysis (CHNS) analyses and the results comply with the calculated measurements. Compounds 1 and 2 were subjected to a stability study in phosphate buffer (0.2M, pH 7.4) and in KCl/HCl buffer (0.2M, pH 1.2) at different time intervals (0 – 240 min) incubated at 37 °C. This revealed that both compounds in phosphate buffer (0.2M, pH 7.4) are significantly stable with t 1/2 of 18hrs and 24hrs respectively. However, compounds 1 and 2 are less stable in KCl/HCl buffer (0.2M, pH 1.2) with t1/2 of 3.48hrs and 3.13hrs respectively. The antibacterial evaluation of the new ceftazidime derivatives showed various degrees of antibacterial activities when compared with ceftazidime. The chemical modifications of ceftazidime showed slight effect on activities and most of compounds retained the antibacterial activities. Compounds 2 and 4 afforded comparable antibacterial action. However, compounds 3 and 5 were equipotent with ceftazidime with respect to E.coli and Staph. aureose. Compound 4 has better activity than ceftazidime with respect to Pseudomonas aeruginosa. Schiff's base derivative of lysine (2, 6-bis- (benzylideneamino) hexanoic acid) gave a reasonable antibacterial action towards Escherichia coli and Streptococcus Spp; as compared with lysine which has no antibacterial activity. Key words: Ceftazidime, Schiff bases, Lysine. تخليق ودراست الفعاليت البكتيريت لمشتقاث جذيذة لعقار السفتازديم شاكر محمود علوان *،1 و عبذ الحفيظ حميذ عبذ الوهاب * * فزع انكًٍٍاء انظٍذالٍَت ،كهٍت انظٍذنت ،جايؼت بغذاد، بغذاد ، انؼزاق . الخالصة حى حظًٍى ٔححضٍز خًست يشخماث جذٌذة نؼمار انسفخاسٌذٌى ححٕي ػهى لٕاػذ شف ٔلٕاػذ شف نالسٍٍ ٔثُائً انالٌسٍٍ فً ج انًزكباث انًحضزة لٕاػذ شف نهؼمار يحأنت نشٌادة انفؼانٍت انبإٌنٕجٍت ٔححضٍز يشخماث حؼطى ػٍ طزٌك انفى. حضًُ -ثُائً االٌسٍٍ-( ٔسفخاسٌذٌى3( ٔسفخاسٌذٌى الٌسٍٍ اياٌذ )يزكب 2( ٔسفخاسٌذٌى الٌسٍٍ اياٌذ لاػذة شف )يزكب 1انسفخخاسٌذٌى)يزكب ٍ انخابؼت (. حضزث انًشخماث انجذٌذة بٕاسطت حفاػم يجًٕػت االي5ٍثُائً انالٌسٍٍ )يزكب –( ٔسفخاسٌذٌى 4لٕاػذ شف )يزكب ( .حضًٍ انبحث كذنك حفاػم انالٌسٍٍ يجًٕػخً 1نًجًٕػت االيٍُٕثٍاسٔل ٔانؼمار انًذكٕر يغ انبُشنذْاٌذ نخكٌٍٕ لٕاػذ شف )يزكب االيٍٍ االحادي يغ انبُشنذْاٌذ نخكٌٍٕ لٕاػذ شف نالٌسٍٍ ٔانذي حى ربطّ بًجًٕػت االيٍٍ انخابؼت نأليٍُٕثٍاسٔل بٕاسطت آطزة أياٌذ ( بٕاسطت ححهم 3(. كًا حى ححضٍز سفخاسدٌى انًزحبظ بانالٌسٍٍ )يزكب 2ٌٍ لٕاػذ شف نالٌسٍٍ انًزحبطت بانسفخاسدٌى )يزكب نخكٕ يجًٕػت لٕاػذ شف بٕاسطت حايض انٍٓذرٔكهٕرٌك. كًا حى ححضٍز سفخاسدٌى يزحبظ بثُائً انالٌسٍٍ انحأي ػهى لٕاػذ انشف بٕاسطت حايض 4. ٔبطزٌمت يشابٓت اجزٌج ػًهٍت ححهم نًجًٕػت لٕاػذ شف نًزكب (4بٕاسطت اطزة االياٌذ نخكٌٍٕ )يزكب ( .5انٍٓذرٔكهٕرٌك نخكٌٍٕ )يزكب حى ححضٍز انًزكباث بشكم َاجح ٔحى حشخٍض انخزكٍبّ انكًٍٍأٌت نهًزكباث انجذٌذة بٕاسطت انطزق انطٍفٍت ٔححهٍم انؼُاطز ٔدرجت فً يحانٍم انبفز 2ٔ 1فٍا انزلائك ٔجاءث انُخائج يطابمت نهخزكٍبت انًمخزحت. انًزكبٍٍ االَظٓار ٔانخحهٍم بٕاسطت كزٔياحٕكزا 254ٌ.و. بانخخابغ ٔانخً حخخهف ػٍ درجت ايخظاص انسفخاسٌذٌى) 222ٔ 222انفٕسفاث ٔانبٕحاسٍى اظٓزٔا درجت ايخظاص فً ٔكذنك يحهٕل كهٕرٌذ انذارئت (M, pH 7.4 0.27ٕو )( فً يحانٍم بفز فٕسفاث انظٕد2ٌٔ 1ٌ.و.(, درجت ثباث انًزكباٌ ) (. اظٓزث انذراست درجت ثباحٍت ٔاضحت ٔيؼمٕنت بانُسبت نهًزكبٍٍ اػالِ فً 0.2M pH 7.4انبٕحاسٍٕو ٔحايض انٍٓذرٔكهٕرٌك ) درجت 32ٔدرجت حزارة دلٍمت( 240-0يحهٕل انفٕسفاث ٔكاَج درجت ثباحٍت انًزكبٍٍ الم بانُسبت نهًحهٕل االخزنفخزاث سيٍُت ) يؤٌت.كًا حى حمٍٍى انفؼانٍت انًضادة نهبكخزٌا يغ إَاع يخخهفت يٍ انبكخزٌا كًا يٕضح فً انجذأل ٔكاَج انُخائج يُاسبت، حٍث اظٓزث يؼظى انًزكباث فؼانٍت يمبٕنت ٔجٍذة يمارَت يغ ػمار انسٍفخاسٌذو. ( فكاَج يخماربت يغ 5ٔ 3ت يغ ػمار انسفخاسٌذٌى ,ايا َخائج حمٍٍى فؼانٍت انًزكبٍٍ )( يخمارب4ٔ 2كاَج َخائج حمٍٍى انفؼانٍت نهًكزبٍٍ ) ( نذٌّ فؼانٍت يثهى ضذ بكخٍزٌا 4يزكب. ).auroseStaphylococcusٔبكخٍزٌا Escherichia coliانسفخاسدٌى فًٍا ٌخض بكخزٌا Pseudomonasaeruginosaٌسٍٍ ٔكاَج َخائجّ يمبٕنت ٔجٍذة ضذ بكخزٌا ( انذي ًٌثم لٕاػذ شف نال 2. ايا) انًزكب Escherichia coli ٔStreptococcus pyogene ٍْٔذا افضم بكثٍز يٍ انالٌسٍٍ نٕحذِ انذي نٍس نّ فؼانٍّ ضذ ْذِ االَٕاع ي انبكخزٌا. . الاليسيه ، قواعذ شف، السفتازيذيم الكلماث المفتاحيت: 1 Corresponding author E-mail:Shakmawales@yahoo.co.uk Received:28/1/2013 Accepted:22/9/2013 Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 36 Introduction Chemical modifications of the C-3 carboxyl group and the C-7 amino group in the cephem nucleus produce various semi- synthetic cephalosporins that have different antibacterial spectra, β-lactamase sensitivity/resistance and pharmacokinetic properties (1) . Most of the known cephalosporins are prepared for parental administration, and still there is a great need for parental cephalosporin that has β- lactamase-resistant and anti-pseudomonal activity (2) . Oral activity conferred by the phenylglycyl substituent is attributed to increased acid stability of the β- lactam ring, resulting from the presence of a protonated amino group on the C-7 acylamino portion of the molecules. Carrier mediated transport of the dipeptide-like and zwitterion cephalosporin are important factors in achieving oral absorption (3) . A series of monofunctional and bifunctionalprodrugs of Cefizoxime was synthesized and shown to have improved physicochemical properties and oral absorption (4) . Ceftazidime is a semi- synthetic, β-lactam antibiotic containing a pyridinium ring and used for parenteral administration. Ceftazidime is stable to most β-lactamases produced by G (+) and G (-) organisms, and consequently is active against many ampicillin and cephalothin resistant strains, except methicillin resistant strains (5) . Schiff bases have been shown to exhibit a wide range of biological activities including antimicrobial 6,7) , anti-inflammatory (8,9) , analgesic (9) , anti-tubercular (10-12) , antimycobecterial (13,14) , antioxidant (15) , antiviral (16) and enzyme inhibitors (17) . Lysine based prodrugs are widely used for different purposes, such as targeting certain drugs to a specific site and improve pharmacokinetic properties. L-lysine linked with amphetamine was found useful for treatment of Attention Deficit Hyperactivity Disorder (18) . L-lysine was linked with aminoflavon through an amide bond in order to improve pharmacokinetic properties affording marked antitumor effect (19) . A new series of 8-aminoquinolons derivatives prepared by conjugation with amino acids, dipeptides and pseudo-dipeptide have been synthesized and showed promising cytotoxic, anti-leshmenial and broad spectrum of antifungal activities (20) . L-lysine linked to Cefizoxime resulted in significant improvement in oral absorption (4) . A prodrug of cephalosporin (RWJ-333441) was prepared by linking lysine, through an amide bond, resulted in an improvement in aqueous solubility that is useful for parenteral administration (21) . Di/tripeptides and peptide- like drugs are absorbed across the brush- border membrane (BBM) by H + - coupled peptide transporter (PEPT1), intracellular peptides and peptide-like drugs exit from cells across the basolateral membrane (BLM) by an unidentified peptide transporter (22). In an attempt to produce new derivatives of ceftazidime that may have broader spectrum of activities, acid stable and could be used orally, a new series was designed and synthesized, as new Schiff bases of ceftazidime and ceftazidime linked L-lysine Schiff bases and ceftazidime-linked di-lysine Schiff bases. Ceftazidime is also designed to be linked with lysine and di-lysine through amide bonds and these derivatives may target the amino acid and dipeptide transport systems and may provide an approach of producing an oral ceftazidime preparation. Experimental General Melting points were determined (uncorrected) by using electrical melting point apparatus, Electro-thermal 9300, USA. Recording of the infrared spectra were performed in KBr disk using FT- IR spectrophotometer/Shimadzu. Elemental micro-analyses were performed by Euro- vector EA 3000A. UV Spectra were recorded by UV Spectrophotometer type AnalytikjenaSpecord 40/ Germany. The ascending TLC was run on silica gel F-254 (type 60) pre-coated aluminum sheets, for checking the purity of the products as well as monitoring the progress of the reaction. Chemical synthesis Five derivatives of ceftazidime (compounds 1-5) were designed and synthesized as Schiff bases of ceftazidime, Schiff bases of L-lysine linked to ceftazidime, L-lysine and di-lysine linked to ceftazidime that may improve pharmacokinetic properties and few could be applied for oral administration of ceftazidime and may broaden the antibacterial spectrum. Benzaldehyde was used to prepare the Schiff bases. Synthesis of a new Schiff base of ceftazidime sodium (compound 1) Disodium mono(7-((Z)-2-(2-((E)- benzylideneamino)thiazol – 4 – yl) – 2-(2- carboxylato propan – 2 – yl – oxyimino) -8- oxo-3-(pyridinium – yl – methyl) – 5-thia-1- azabicyclo(4.2.0) oct –ene – carboxylate).bicarbonate. This derivative was prepared by reaction of ceftazidime sodium and benzaldehyde in methanol at 60 °C (23) . Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 37 Benzaldehyde (1.56 mmol, 0.18g) in methanol (10 mL) was added to ceftazidime (1.56 mmol, 1g) dissolved in methanol (100 mL) and to the above solution TEA (1.56nmol, 0.48mL) was added. The mixture was refluxed for 6 hrs and was then acidified with (0.1N) HCl and filtered to remove the unreacted ceftazidime. The precipitate was washed with (2x30 mL) of distilled water and was dissolved in a solution of sodium bicarbonate solution (5%, 20mL) and dry acetone (60mL) was added and the solution was placed in a refrigerator to precipitate the product as yellowish-brown sodium salt of the Schiff base of ceftazidime. The physical appearance, percent yield and Rf values are listed on table 1. The IR characteristic bands (ѵ, cm -1 ) are recorded as; 3191 (N-H stretching of secondary amid), 1745 (carbonyl of β-lactam), 1647 (carbonyl stretching of carboxylate) and 1585 (C=N stretching). Elemental microanalysis of compound 1 was listed on Table 2. Synthesis of Schiff base of lysine, 2, 6-bis (benzylidene amino) hexanoic acid (2a) The two aliphatic amino groups of lysine were reacted with benzaldehyde to form Schiff' bases (24) . Schiff' bases were collected as solids and characterized by chemical and spectral analysis. Lysine mono hydrochloride (26.3 mmol, 4.8g) was dissolved in distilled water (35ml) and NaOH (26.3 mmol, 1g) was added and the mixture was cooled in an ice bath. Benzaldehyde (52.6 mmol, 5.6 g) was added drop wise and the mixture was stirred for 2 hrs at room temperature. The obtained solid was excessively washed with distilled water and was recrystallized from methanol: ether (1:4) to afford a white needle-like crystals of the lysine Schiff' bases (2a). The physical appearance, percent yield and Rf values are listed on table (1). The IR characteristic bands (ѵ, cm -1 ); 3251 (O-H stretching of carboxylic acid), 3082, 3028 (C- H aromatic stretching), 1703 (carbonyl group of carboxylic acid) and 1649 (C=N stretching). Synthesis of new Schiff bases of ceftazidime- lysineamide disodium (compound 2) Disodium mono (7-((Z)-2-(2-(2,6-bis((E)- benzylidene amino) hexaneamido)thiazol-4- yl)-2-(2-carboxylato propan-2-yl- oximino)acetamido)-8-oxo-3-(pyridinum-1- ylmethyl) -5-thia-1-azabicyclo(4.2.0)oct-2- ene carboxylate) bicarbonate. The synthesis of compound (2) was achieved by reaction of compound 2a (lysine Schiff base) and ceftazidime by the mixed anhydride method (25) , and as stated below. Schiff base of Lysine 2a (3.41mmol, 1.098gm) was suspended in (75 ml) of a mixture of dry acetone and DMF (1:2). To the above suspension, TEA (3.41mmol, 0.48mL) was added and the mixture was placed in an ice bath at (-5 to -10C°). A solution of ethylchloroformate, ECF (3.41mmol 0.33ml) was added drop wise over a period of 10 min with continuous stirring, which was continued for further 30 min at room temerature. Ceftazidime (3.41mmol, 2.171gm) was dissolved in distilled water (20ml) containing TEA (3.41mmol, 048mL) and the solution was cooled to (0°C) and was added at once to the above mixture containing ECF with vigorous stirring for 4 hrs. The solvent was then evaporated and the resultant precipitate was washed with (0.1N) HCl. The suspension was filtered and the precipitate was collected and washed with ethanol (99.5%) to afford the hydrochloride salt. This was re-dissolved in a solution of sodium bicarbonate (25mL, 5%) and the pH of the solution was adjusted to 8. A cold dry acetone (60 mL) was added and the solution was placed in a refrigerator to precipitate compound 2 as sodium salt. The physical appearance, percent yield and Rf values are listed on table (1). The IR characteristic bands (ѵ, cm -1 ); 3292 (N-H amide stretching), 1750 (carbonyl group of β- lactam), 1660 (carbonyl group of carboxylate), 1620 (C=N stretching of imine) and 1573 (stretching of carbonyl of carboxylate anion). Elemental microanalysis of compound 1 is listed on Table 2. Synthesis of ceftazidime-lysineamide disodium (compound 3) Disodium mono (7-((Z)-2-(2- carboxylatopropan-2-yloxyimino) 2(2)-2,6- diaminohexane amido) thiazol-4-yl) acetamido)-8-oxo-3-(pyridinuim-1-ylmethyl)- 5-thia-1-aza bicyclo (4,2,0)oct-2-ene-2- carboxylate)bicarbonate. This compound was obtained by cleavage of the Schiff' bases of compound 2 by reaction with hydrochloric acid solution at pH 2 incubated at 5-10˚C (26) , as shown in scheme (2). Compound 2 was dissolved in distilled water (20mL), placed in an ice bath at (10°C) and a cold solution of HCl (1N, 10mL) was added drop wise to adjust the pH to 2 and the mixture was stirred for 4hrs. Cold acetone (40 mL) was added to precipitate compound 3 and the precipitate was collected and washed with ethanol to afford a fine white powder of the hydrochloride salt. The IR characteristic bands (ѵ, cm -1 ); 3325, 3273 (N-H stretching Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 38 of primary aliphatic amine), 3197 N-H (stretching of secondary amide), 1758 (carbonyl group of β-lactam), 1665 (carbonyl group of carboxylate anion) and 1614 (N=C stretching of imine). Elemental microanalysis of compound 3 is listed on Table 2. Synthesis of ceftazidime-di- lysineamideSchiff bases di sodium (compound 4) Disodium mono (7-((Z)-2-((2-(2-((E)- benzylideneamino)-(hexanamido) hexanamido) thiazol-4-yl)-2-(carboxylate propan-2-yl oxyimino) acetamido)-8-oxo-3- (pyridinum-1-ylmethyl)-5-azabicyclo (4,2,0)oct-2-ene-2-carboxylate) bicarbonate. The synthesis of this compound was achieved by reaction of compound 2a with compound 3 using the following procedure, as shown in scheme (2).Schiff base of Lysine 2a (3.41mmol, 1.098gm) was suspended in a mixture (75 ml) of dry acetone and DMF (1:2), and to the above mixture TEA (3.41mmol, 0.48mL) was added and placed in an ice bath at (-5 to -10°C). A solution of ECF (3.41mmol 0.33ml) was added over a period of 10 min and the mixture was continuously stirred for further 30 min at room temperature. Compound 3 (1.705mmol, 1.226gm) was dissolved in distilled water (20ml) containing TEA (1.705mmol, 0.238mL) and was cooled to 0 °C and added at once to the solution of ECF-Schiff base of lysine and the mixture was vigorously stirred for 4 hrs. The solvent was evaporated and the resultant precipitate was suspended with diluted HCl and was filtered and the precipitate was collected as the hydrochloride salt, which was washed with ethanol. The precipitate was dissolved in a solution of sodium bicarbonate (5%, 15mL) and the pH of the solution was adjusted to 8. A cold dry acetone (60 mL) was added and the mixture was placed in a refrigerator to precipitate compound 4, as sodium salt. The physical appearance, percent yield and Rf value are listed on table (1). The IR characteristic bands (ѵ, cm -1 ); 3327 (N-H stretching vibration), 1758 (carbonyl group β-lactam), 1678 (carbonyl group stretching of carboxylate anion) and 1626 (C=N stretching of imine). Elemental microanalysis of compound 3 is listed on Table 2. Synthesis of ceftazidime-di-lysineamide di sodium (compound 5) Disodium mono(7-((Z)-2-(2-(R-2,6-bis(2,6- diaminohexanamido)-thiazol-4-yl)-2- (carboxylate-propan-2-yl- oxyimino)acetamido)-8-oxo-3-(pyridinium-1- yl-methyl)-5-thia-1-azabicyclo(4,2,0)oct-2- carboxylate) bicarbonate. This derivative was prepared by reaction of compound 4 with hydrochloric acid solution at pH 2 and the mixture was incubated at 5-10 o C. Compound 4 (1.08 mmol, 1.5gm) was dissolved in distilled water (20mL) and placed in an ice bath at 5- 10°C. A cold solution of hydrochloric acid (1N, 10mL) was added drop wise and the pH was adjusted to 2. The mixture was stirred for 4 hrs and was filtered and the product was separated as the dihydrochloride salt in the filtrate. Compound 5 as the dihydrochloride salt was neutralized by sodium bicarbonate (5%) and the pH of the solution was adjusted to 8. A cold dry acetone (60mL) was added to precipitate compound 5 as the sodium salt. The physical appearance, percent yield and Rf value are listed on table (1). The IR characteristic bands (ѵ, cm -1 ); 3329, 3226 (N- H stretching of primary aliphatic amine), 3194 (N-H stretching of amide), 1759 (carbonyl group of β-lactam) and 1622 (C=N stretching of imine). Elemental microanalysis of compound 3 is listed on table 2. C H O METHANOL TEA 1- 2- NaHCO3 Reflux HCO3. N+S N O H N O N OO S N NH2 O OH O- N+S N O H N O N O O S N N O O- O- Na+ Na+ 5% Scheme 1. Synthesis of Schiff base of ceftazidime sodium (compound 1). Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 39 O HO N N ECF,1- DMF, Acetone TEA 2- NaHCO3 HCO3 . N+S N O H N O N OO OH S N H2N O O - Na+ Na+ N+ S N O HN ON O O O- S N H N O O- O N N 5% HCl 5-10 c NaHCO31- 2- HCO3. N+ S N O N H O N O O S N H N O O H2N NH2 +Na-O +Na-O 5% Compound 1 Compound 2 Scheme 2. Synthesis of compounds 2 and 3. Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 40 NOH N N+ S N N H O N O O N S N H O H2N H2N O O -O O- Na+ Na+ 1- DMF, Acetone 2- NaHCO3 ECF, TEA .HCO3 - HCO3. HN O N O O N SH N O HN N H O N N O N N +Na-O N S O O-Na+ O N+ 5% HCl 5-10 c NaHCO31- 2- 5% Compound 4 Compound 5 HCO 3 . N+ S N O HN ON O O O- S N H N O O- O NHN H O H2N NH2 O H2N H2N Na+ Na+ Scheme 3. Synthesis of compounds 4 and 5. Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 41 NOH N N + S N N H O N O O N S N H O H2N H2N O O -O O- Na + Na+ 1- DMF, Acetone 2- NaHCO3 ECF, TEA .HCO 3 - HCO 3. HN O N O O N SH N O HN N H O N N O N N + Na - O N S O O - Na + O N+ 5% Scheme 4. Synthesis of compound 4. Results and Discussion The IR characteristic bands shown in the spectra of compounds were used to identify and confirm their structures depending on the appearance and disappearance of certain chemical groups and consequently their bands (27, 28) . The IR spectra of the synthesized compounds showed characteristic bands of absorption, which were consistence with the proposed structures of the compounds. The IR spectrum of compound 1 (Schiff base of ceftazidime) showed characteristic bands and are previously listed. The disappearance of the amine band of the aminothiazole moiety in the IR spectra gives a good indication for the formation of the newly synthesized amide bond (29) . The newly synthesized compounds (1,2 and 4) contain an extra imine bond with regard to the already existing imines, as shown clearly in their chemical structures. However, the presence of such number of imines resulted in very close bands in the IR spectra of these compounds. The IR characteristic bands for compound 2a (Schiff base of lysine) showed imine group at 1649 cm -1 , which don’t exist in lysine and at the same time, disappearance of the primary amine group in the region 3400- 3300 cm -1 . Ceftazidime contain a primary aromatic amine of the aminothiazole moiety which appears at 3500-3400 cm -1 , while, compounds 3 and 5 contain primary aliphatic amine groups and their IR absorption bands appear at (3300-3200cm -1 ). The IR spectra of (C=O) of carboxylic acid of ceftazidime, which appeared at 1705 cm -1 , while the asymmetric vibration of (COO-) of these compounds appeared at (1647-1678 cm -1 ). Elemental microanalyses were performed for the target compounds (1-5) to confirm their basic chemical formulas. The results were presented on table (2). The percent deviations of the observed to the calculated values were found to be acceptable and within the range of 0.4%. Stability of compounds 1 and 2 in aqueous buffer solutions UV spectra of the aqueous solutions of the sodium salts of the parent and the new derivatives of ceftazidime were recorded on a double-beam UV-spectrophotometer and their λmax were determined. The λmax of ceftazidimepentahydrate is at 254nm, while λmax of ceftazidime Schiff base (compound 1) is at 299 nm. λmax of ceftazidime- lysine Schiff base (compound 2) is at 277nm. Under the experimental conditions (30), the behavior of the hydrolysis of compounds 1 and 2 follow pseudo-first order kinetic, since plot of log concentration vs. time resulted in a straight line and from the slope of this plot, the observed rate constant of hydrolysis was calculated. The degree of hydrolysis of compound 1(28 g ml) in Cl Cl buffer (0.2 ,p 1.2) and in phosphate buffer (0.2 , p 7.4) incubated at 37 C was studied at different time intervals (zero, 15, 30, 60,120 and 240 min).The half-life values were calculated from the pseudo-first order kinetic Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 42 law and found to be t½ 3.48hrs and 18.78hrs respectively. A similar procedure was conducted to study the stability of ceftazidime-lysine Schiff base (compound 2) in KCl/HCl buffer (0.2M,pH 1.2) and in phosphate buffer (0.2M, pH 7.4) and the t½ values were calculated using the pseudo-first order kinetic law and the values equal to 3.13 hrs and 24hrs respectively. Accordingly, the above studies have indicated significant increase in the values of t1/2 (18.78hrs) of compound 1 at slightly basic media (pH 7.4), which is much longer than the t1/2 of ceftazidime (2hr) (31) . However, the stability of compound 1 in KCl/HCl buffer (0.2M, pH 1.2) showed that thet1/2 was 3.48hrs compared with ceftazidime (2hr) (31) . Compound 2 has a t1/2 value of 24hrs when incubated in phosphate buffer (0.2M, pH 7.4), which is a clear significant result that indicate a great stability of compound 2 at the slightly basic conditions (resembles the physiological pH). However, the stability of compound 2 in KCl/HCl buffer (0.2M, pH 1.2) was slightly better and reaching 3hrs, when compared with ceftazidime (31). Table (1) Physical parameters and percent yield of the synthesizedcompounds Compound Physical appearance Yield % m.p. o C Rf value 1 Brown to yellow powder 70 165 Decomposed 0.75 ( A ) 2a White powder 50 195-197 0. 69 (B ) 2 Pale brown powder 68 210 Decomposed 0.62 (B ) 3 White to pale yellow powder 52 266 Decomposed 0.68 ( B ) 4 Brown powder 58 225 Decomposed 0.65 (A ) 5 Pale yellow powder 50 284 Decomposed 0.62 ( B ) Table (2) Elemental microanalysis of the synthesized compounds. Elemental microanalysis % Chemical formula Compounds found Calculated Element Molecular weight 48.57 48.65 C C30H26N6Na2O10S2 740.09 1 3.46 3.54 H 11.24 11.35 N 8.88 8.66 S 53.36 53.97 C C43H42N8Na2O11S2 956.95 2 4.57 4.42 H 11.84 11.71 N 6.92 6.70 S 44.79 44.61 C C29H34N8Na2O11S2 780.74 3 4.50 4.39 H 14.10 14.35 N 8.48 8.21 S 59.48 59.64 C C69H74N12Na2O13S2 1389.51 4 5.57 5.37 H 12.45 12.10 N 4.38 4.62 S 47.59 47.48 C C41H58N12Na2O13S2 1037.08 5 5.49 5.64 H 16.55 16.21 N 6.37 6.18 S Iraqi J Pharm Sci, Vol.22(2) 2013 Synthesis of new derivative of ceftazidime 43 Antimicrobial Activity Assessment The newly synthesized derivatives were tested for their antimicrobial activity by disc- diffusion method (32) against the following microorganisms: (a) Gram-negative: Escherichia coli and Pseudomonas aeruginosa (b) Gram-positive: Streptococcus spp and Staphylococcusaureus. Type of media used: (Nutrient Medium), which contain 1g/L distilled water, peptone (5gm) and meat extract (3gm), and the pH was adjusted to 7.0. Each of the synthesized compounds (30 g) was dissolved in dimethylsulphoxide to prepare the test solutions. Ceftazidime (30 g) was used as the standard antibacterial drug. Dimethylsulphoxide: water mixture (1:30) was used as the solvent. The results are shown on Table (3). Table (3) Antimicrobial activity evaluation of the synthesized compounds. compounds S. pyogen. S. Aureus. E. coli P. aeruginosa DMSO - - - - Ceftazidime - + ++ ++ 1 + + ++ ++ 2 ++ ++ ++ ++ 3 - + +++ ++ 4 ++ + +++ +++ 5 - + ++ + 2a + - + - Key to symbols: (-) = no inhibition, (14 mm) = +, (15-17 mm) = + + (more than 18 mm) = +++. The antimicrobial screening revealed that the newly synthesized compounds (2 and 4) showed reasonable antibacterial activities against G (+) Strep. Spp. in comparison with ceftazidime, which has no activity against this type of microorganism. Compound 2 showed good activity against all 4 strains of bacteria used, as compared with ceftazidime. Compound 4 showed good and reasonable activities against E. coli and P. aeruginosa comparable with ceftazidime. While, it showed good activity against Strep. Spp. and moderate activity against Staph. aureus. Compound 1 showed moderate activity against Strep. Spp. and staph Spp. and good activity against E. coli and P. aeruginosa. Compound 3 showed good and reasonable activity against E. coli and P. aeruginosa and moderately active towards staph. Spp. However, compound 3 showed no activity against Strep. Spp. Compound 5 showed good activity against E. coli and moderate activity against P. aeruginosa. Generally, all the Schiff bases of ceftazidime (compounds 1, 2 and 4) showed good and reasonable antimicrobial activity against the tested microorganisms especially G (+) bacteria. This increase in activity may be due to the incorporation of extra imine groups. 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