Synthesis and Characterization of Schiff Base Metal Complexes Derived from Cefotaxime with 1 H-indole-2 , 3-dione ( Isatin ) and 4N , N-dimethyl-aminobenzaldehyde

This work involved the synthesis of two Schiff base derivatives of cefotaxime antibiotic (CFX) namely: [sodium3-(acetoxymethyl)-7-((Z)-2-(methoxyimino)-2-(2-((E)-2–oxoindolin-3-ylide-neamino) thiazol-4-yl)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate]. (0.5) Methanol (LI) and [sodium3-(acetoxymethyl)-7-((2Z)-2-(2-(4-dimethylamino) benzylideneamino) thiazol-4yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylate]. (0.5) Methanol (LII) from the condensation reaction of the antibiotic with 1H-Indole-2,3-dione(isatin) and -N, N-dimethyl amino benzaldehyde respectively. Metal complexes of the two Schiff base ligands with Co(II), Ni(II), Cu(II), Cd(II), Pd(II) and Pt(IV) ions were prepared by reacting each ligand with the metal salts in refluxing ethanol. The chemical structures of the two ligands as well as the stereo-chemical structures and geometries of the studied metal complexes were suggested depending the results obtained from CHN and TG analysis, NMR, FTIR, and atomic absorption spectrophotometry, electronic spectra, magnetic moments and conductivity measurements. The mole ratio of the ligands to the metal ion was 1:1 with tridentate bonding behaviors of the coordinating ligands with the metal ions.


Materials and Methods
All chemicals were of analytical grades and were used as received from suppliers without further purification.Because of limited solubility of palladium (II) chloride, the salt was converted first to the complex dichlorobis (benzonitrile) palladium (II) [PdCl 2 (PhCN) 2 ] prior to use following the method reported by Rochow [13].Purity of products was detected by TLC techniques using a mixture of acetone: chloroform (1:1 and 1:2 v/v), ether: chloroform (1:1 v/v), ethanol: chloroform (1:2) v/v) and chloroform only as eluents, and iodine chamber for spot location.

Instruments
Melting points (uncorrected) were determined on a Gallenkamp M.F.B 600 -010f melting point apparatus.Fourier Transform Infrared (FTIR) spectra were obtained using a SHIMADZUE FT-IR 8400S Fourier transforms, within the wavenumber region between 4000 and 400 cm −1 using KBr disc and 4000 and 200 cm −1 using CsI disc.Electronic spectra for compounds in the (UV-Visible) region (200 -1100) nm were recorded using a SHIMADZUE 1800 Double Beam UV-Visible spectrophotometer.Elemental microanalysis was performed on Eurovector EA 3000A. 1 H-NMR and 13 C-NMR spectra were performed by using a Bruker Ultra Sheild 300 MHz NMR, Al al-Bayt University (Jordan) and Delta A2 NMR JMN-ECS 400 MHz Manchester Metropolitan University.Thermal analyses (TG and DTG) of samples were performed under helium and nitrogen atmospheres at heating range of (25˚C -900˚C) and (25˚C -800˚C) respectively and heating rate of 20˚C/min.by using Perkin Elmer TGA 4000.Molar conductivity measurements (S•mol −1 •cm 2 ) for metal complexes (10 -3 M) in DMF, DMSO at room temperature were carried out by using LASSCO Digital Conductivity Meter.Magnetic moments (eff.B.M) for the prepared complexes in the solid state at room temperature were measured according to Faraday's method by using Bruker Magnet B.M-6, Atomic Absorption measurements were performed using BUCK Scientific model 210 VGP USA Atomic Absorption Spectrophotometer.

Synthesis of Ligands
To a stirred solution of isatin (0.308 g, 2 m•mole) in hot methanol (10 ml) was added (1 g, 2 m•mole) of cefotaxime sodium salt in hot methanol (15 ml) followed by 2 ml of glacial acetic acid.The reaction mixture was heated under reflux for 6 h with continuous stirring.The color of solution was changed from orange to brown followed by the formation of a brown precipitate.The product was filtered, washed with hot mixture methanol: water (v: v) (80:20) % followed by cold methanol and ether and vacuum dried.
Synthesis of This ligand was previously prepared by A. H. kshash in ethanol at pH 4 [4].In this work we prepared the ligand adopting a different procedure.A hot solution of 4-dimethylamino benzaldehyde (DMAB) (0.312 g, 2 mmol) in methanol (3 ml) was added to a hot stirred solution of cefotaxime sodium salt (1 g, 2 m•mol) in the same solvent (15 ml).Then glacial acetic acid was added (1.5 ml) and the mixture was heated under reflux for 3 h to achieve complete precipitation.An olive green precipitate was formed.The product was filtered, washed several times with a hot mixture of methanol-water followed by cold methanol and ether and vacuum dried.

Synthesis of Metal Complexes
To a stirred solution of each ligand (0.2 g, 0.33 m•mol) in a mixture of ethanol-water (80:20)% (3 ml) heated to boiling point was added an alcoholic solution of the metal salts: and K 2 PtCl 6 (0.039 g, 0.039 g, 0.028 g, 0.036 g, 0.029 g and 0.0804 g respectively, 0.16 m•mol) with continuous stirring.Immediate precipitation of metal complexes (C 1 -C 6 respectively for L I complexes and C 7 -C 12 for L II complexes) was observed.Reflux was continued for 1 h for complete precipitation.The products were filtered off, washed with hot EtOH: H 2 O followed by ether and vacuum dried.

Physical Properties and Elemental Analyses
The physical properties and results obtained from C.H.N. analyses and metal contents of the prepared compounds are described in Table 1.The analytical data were almost agreeable with calculated values with some deviations attributed to incomplete combustion or technical errors.The molecular formula of the ligands and their metal complexes were suggested according to these data together with those obtained from spectral and thermal analyses as well as conductivity and magnetic susceptibility of metal complexes.

Infrared Spectra
Table 2 and Table 3 describe respectively the important vibrational modes of L I , L II and their metal complexes.Both L I and L II have several potential donor atoms but, due to steric constraints, the ligands can provide a maximum of three donor atoms to any one metal center [18].The infrared spectra of the two ligands exhibited the absence of absorption bands at (3348, 3252 cm −1 ) corresponding to stretching modes of NH 2 group of cefotaxime and at 1728, and 1664 cm −1 assigned to (C-3) carbonyl of isatin [25] [26] and C=O group of N, N-DMAB [5] which refers to the formation of the Schiff base ligands by condensation reaction between both isatin and N, N-DMAB with the antibiotic to form the azomethine CH=N linkage.This was confirmed by the appearance of new bands at υ 1636 and 1643 cm −1 respectively assignable to υ (C=N-) of azomethine group [1]- [5].A broad band was observed around 3565 cm −1 and 3418 cm −1 of the two ligands respectively attributed to vibrational modes of hydrogen bonded O-H group of methanol solvent embedded in the crystal lattice structure of the  ligands [26]- [28].The two tables describe also the positions of the bands assigned to vibrational modes of amide NH groups [15] [28]- [30], υ C=O of lactam ring of the antibiotic moiety [3] [31] [32], overlapped amide and ester carbonyls [18], the asymmetric and symmetric stretching vibrations respectively of carboxylate anion [3] for both ligands.The -NH-vibrations of the indole ring system [25] and υ C=O at C-2 [26] [33] of isatin moiety of L I (Table 2) are in agreement with those in the litrature.The band assigned to υ (C-S) stretching vibration was located at (580) cm −1 [1] [5].The C-N-C and the N-H stretching vibrations of the lactam amide residue in the two ligands were observed at (1180, 1177) cm −1 [34] and 3255 cm −1 respectively [3] [17] [29].The spectrum of the Schiff base ligand L II (Table 3) exhibited an additional band observed at (1335) cm −1 assigned to υ (C-N) stretching vibration of benzylic moiety [5] [15].All complexes exhibited shifts in the position of the bands related to the β-lactam carbonyl (ν C=O), azomethine groups, υ (-C=N) and carboxylate anion which refers to the coordination of these groups with the metal ions [33] [34].This was confirmed by the appearance of new low intensity bands at lower wavenumbers corresponding to stretching vibrations of M-N and M-O bonds respectively [33] [35].The values of frequency difference (∆υ) between the two bands of (COO − ) asymmetric and (COO − ) symmetric stretching vibration (˃200 cm −1 ) refers to monodentate coordination behavior of carboxylate group with the metal atoms [29] [34] [35].These observations suggest that the two Schiff base ligands behaved as tridentate ligands in the coordination with the metal ions [16] [36].The bands observed at frequency ranges 363 -370 cm −1 were assigned to stretching vibrations of (M-Cl) bonds [32] [35].Bands assigned to vibrational modes of lattice and coordinated waterbonds [35] were also detected and are described in Table 2 and Table 3.

Electronic Spectra, Magnetic Moments, and Conductivity Measurements
The electronic spectrum of Schiff base L I in DMSO (Figure 5  cm −1 respectively) with a shoulder at υ max 28,986 cm −1 attributed to the π → π * transition and a low intensity band at υ max 24,570 cm −1 attributed to the n → π * transition [5] [37].The spectral data together with molar conductivity and magnetic moments of all metal complexes of L I and L II in DMSO are described in Table 4 and Table 5 respectively.
The spectra of the metal complexes exhibited bathochromic or hypsochromic shifts of ligand bands.The bands assigned to intraligand π → π * for the Co(II), Ni(II), Cu(II), Cd(II), Pd(II) and Pt(IV) complexes (C 1 -C 6 ) were observed at υ max (38,320,34,483), (38,168,34,722), (38,462,34,483), (38,314,34,247), (38,314,34,483) and 21,231 cm −1 respectively, while those of L II complexes for the same metal ions (C 7 -C 12 ) were observed at (38,023,33,784), (38,314,33,784), (38,320,34,247), (38,314,33,557), (36,765,30,395) and 21,053 cm −1 respectively.The spectra of transition metal complexes showed additional low intensity bands in the visible and near IR regions related to ligand field d-d transitions [38]- [40].The spectra of the two Co(II) complexes (C 1 and C 7 ) exhibited two additional bands in the visible region attributed respectively to 4 T 1 g → 4 A 2 g (F) (υ 2 ) and 4  4 T 1 g (P) (υ 3 ) transitions of octahedral Co(II) complexes [20] [41].By applying the observed band energies and band ratio υ 3 /υ 2 (1.4 and 1.17 respectively) for the two complexes on Tanabe-Saugano diagram of d 7 configuration, the values of , Dq B B and 10 Dq were obtained (Table 4 and Table 5) and the energy of υ 1 assigned to the transition 4 T 1 g → 4 T 2 g for each complex was calculated.The spectra of the two Ni(II) complexes (C 2 and C 8 ) showed two additional bands appeared in the near IR attributed to the spin allowed transitions 3 A 2 g → 3 T 2 g(υ 1 ) and 3 A 2 g → 3 T 1 g(F)(υ 2 ) of octahedral Ni(II) complexes [20] [37].By applying the two observed band energies and band ratio υ 2 /υ 1 (1.45 and 1.39 respectively) for the two complexes on Tanabe-Saugano diagram of d 8 configuration, the values , Dq B B and 10 Dq were obtained and the energies of υ 3 assigned to the transition 3 A 2 g → 3 T 1 g (P) were also calculated.The values of β for the cobalt and nickel complexes indicated some covalent character [41].The values of magnetic moments for the two complexes support the paramagnetic high spin octahedral structures [20] [27] [41].The spectra of the two Cu(II) complexes (C 3 , C 9 ) showed one broad band in the visible region assigned to the 2 Eg → 2 T 2 g transition of tetragonally distorted octahedral Cu(II) complexes [38]- [40].The magnetic moment of the two Cu (II) complexes is within the expected values for one unpaired electron [38]- [40].No d-d transition bands were observed the spectra of the Cd (II) complexes (C 4 and C 10 ) which were quite common with the d 10 complexes [38]- [40].
The spectra of the two diamagnetic Pd(II) complexes (C 5 and C 11 ) exhibited two additional bands attributed to the transitions 1 A 1 g → 1 A 2 gand 1 A 1 g → 1 B 1 g respectively of square planar geometry [20] [42].The Pt(IV) complexes (C 6 , C 12 ) were diamagnetic and their spectra exhibited the appearance of one additional band attributed to  1 A 1 g → 3 T 2 g transitions of octahedral Pt(IV) complexes [42].The molar conductivity measurements of all metal complexes in DMSO (10 −3 M) suggested that the complexes were nonelectrolyte [43].According to these data, together with elemental analyses and I.R. spectra, the structures of metal complexes were suggested as illustrated in Scheme 2.

Thermo Gravimetric Analysis
The TGA analyses of the two ligands and some selected metal complexes described in (Table 6).The TG curves of the two ligands refer to three decomposition steps.In the first step the weight loss corresponds to the elimination of solvent molecules (alcohol and water molecules) embedded in the crystal lattice at peak temperature 65 −100˚C as is demonstrated by DTG curves data, which agrees with the elemental and IR analysis of the studied samples.The second and third steps involved the thermal decomposition of the ligands.The thermal decomposition  of L I showed similar behavior to other isatin Schiff bases mentioned in the literature [21] [22] [44].In the second step, the weight losses were mainly attributed to the removal of the isatinazomethinic moiety [22] [44] [45], as well as the methyl ester substituent at the fused six membered thiazin ring.The third step was attributed to the removal of the substituted thiazole ring and its side chain moiety by amide bond breaking, leaving the fused ring β-lactam sodium salt as the residual part of the ligand.The weight losses demonstrated by TG curves of the Co(II) and Cd(II) complexes (C 1 , C 4 ) were attributed to the loss of coordinated water together with coordinated isatinazomethinic moiety and methyl ester substituent.The third stage is similar to that of the original ligand.
The DTA curve of the Cd(C 4 ) complex (Figure 6(a)) shows a broad endothermic peak in range (301˚C -500˚C) which corresponds to the partial removal of the coordinated part of the complexes and an exothermic broad peak at temperature range (501 -808).The final residue of the complex corresponds to the metal ion bonded to the chloride and carboxylate anion at the fused β-lactam rings fragments.The total weight losses exhibited by the Pd(II)L I and Pt(II)L I complexes (C 5 and C 6 ) were (71% -73%) up to 839˚C are summarized in three steps for the former and four steps for the latter.The mass losses displayed by the thermal decomposition of L II and its selected Cd(II), Pd(II) and Pt(IV) complexes (C 10 , C 11 and C 12 ) in the first and second steps correspond to the departure of lattice solvent and to methyl ester + N, N-dimethyl benzyl imino moieties respectively in addition to coordinated water molecules in the case of Cd (II) L II complex.The maximum weight loss in the third step was due to the loss of thiazole ring and its side chain up to the remaining part of the β-lactam moiety.The DTA curve of the Cd(II) complex (C 10 ) (Figure 6(b)) showed two endothermic peaks at the temperature ranges (25˚C -310˚C) and (311˚C -420˚C) corresponding to the mass loss of the first and second steps.The exothermic peak at the temperature range (421˚C -800˚C) is attributable to the third step.The maximum weight loss of the Pd(II) complex observed in the third step refers to almost complete degradation of the ligand.The DTA curve of the Pt(IV) complex (C 12 ) (Figure 6(c)) indicated three steps.The first is an endothermic step in the range (25˚C -270˚C) corresponding to the loss of solvents together with the methyl ester and the coordinated N, N-dimethylaminobenzylimino moieties.The second step shows endothermic and exothermic decomposition reactions in the range (271˚C -600˚C) which corresponds to further decomposition of the uncoordinated parts of the ligand.In the third step an exothermic peak was observed at temperature range (600˚C -800˚C) corresponding to the formation of the final product.

Antibacterial Activity
Evaluation of antimicrobial activity of all compounds in DMSO (1 mg/ml) in vitro was carried out against E. coli, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumonia utilizing the agar diffusion technique.The antibiotic showed a selective antibacterial activity as it exhibited no inhibition zone against Pseudomonas aeroginosa, a weak growth inhibition against Streptococcus pneumonia, a moderate growth inhi-bition against Staphylococcus aureus and a high growth inhibition against E. coli.The two Schiff base ligands were completely inactive against all the studied cultures at the selected concentration, which indicates that the amino group of the original antibiotic plays an important role in growth inhibition activity.All metal complexes showed selective activity against one or two bacteria except the complex [Pt(L II )Cl 3 ]H 2 O•0.5EtOH which was active against all cultures and exhibited higher activities against Streptococcus pneumonia and Pseudomonas aeroginosacompared with the free antibiotic (Cefotaxim).

Conclusion
Two Schiff base derivatives of cefotaxime were successfully synthesized from the condensation reaction of the antibiotic with isatin (L I ) and 4-N, N-dimethylaminobenzaldehyde (L II ) and their structures were characterized by elemental and thermal analysis, NMR and FTIR spectra.The coordination of the two ligands with Co(II), Cd(II), Ni(II), Cu(II), Pd(II), and Pt(IV) ions showed tridentate behavior with M:L mole ratio of 1:1.All complexes were of octahedral geometries except the Pd(II) complexes which had square planar structures.The biological activity of the prepared compounds was controlled by type of bacteria, functional groups of ligands and type of metal ion.

Scheme 1 .
Scheme 1. Synthetic route of the two Schiff base derivatives of cefotaxime.

Figure 1 .
Figure 1. 1 HNMR spectrum of L I (a) before drying and (b) after vacuum drying.
per million: Proton

Figure 3 .
Figure 3. 1 HNMR spectrum of L II (a) before and (b) after vacuum drying.

Figure 5 .
Figure 5. Absorption spectra of (a) L I and (b) L II in DMSO.

2 .
Scheme 2. Suggested stereochemical structures of the cefotaxime Schiff base metal complexes.
The complexes [Co(L I )Cl•(H 2 O) 2 ]•0.5(EtOH), [Cu(L I )Cl•(H 2 O) 2 ]•0.5(EtOH), [Ni(L II )Cl•(H 2 O) 2 ]•0.5(EtOH) and; [Pd(L II )Cl]•H 2 O•EtOH were found moderately active against Staphylococcusaureus especially the copper complex which showed comparable bactericidal activity with the original drug.The other metal complexes showed selective activity against one type of bacteria at a range starting from highest activity of [Co(L I )Cl•(H 2 O) 2 ]•0.5EtOH against E. coli, [Pt(L I )Cl 3 ]•EtOH against Staphylococcusaureus, [Ni(L II )Cl•(H 2 O) 2 ]•0.5(EtOH), against E. coli to the lowest activity of [Cd(L II )(H 2 O) 2 Cl].EtO H against Pseudomonas aeroginosa.These results imply that the type of metal ions and type microorganism are the main controlling factors on antibacterial action.

Table 1 .
Physical properties and analytical data for L I , L II ligands and their metal complexes.

Table 2 .
Characteristic IR stretching vibrations (cm −1 ) of L I and its metal complexes.

Table 3 .
Characteristic IR stretching vibrations (cm −1 ) of L II and its metal complexes.

Table 4 .
Electronic spectra, spectral parameters, and molar conductivity of L I metal complexes.

Table 5 .
Electronic spectra, spectral parameters, and molar conductivity of L II metal complexes.

Table 6 .
Thermal decomposition of L I , L II and some selected metal complexes.