Cephalosporins antibiotics are categorized into four generations according to the spectrum of their activity^[1]. These are belong to β -lactam antibiotics, the mechanism of action, resistance and other properties are similar to those of penicillin^{[1, 2, 3]}. The first generation of cephalosporins are active toward gram positive bacteria and limited activity against Gram-negative bacteria^[4]. Cephalosporins of the second generation show increased activity against Gram-negative microorganisms but are much less active than third and fourth generation agents^{[2, 3, 4, 5, 6]}. Mn(Ⅱ ), Co(Ⅱ ), Ni(Ⅱ ), Cu(Ⅱ ) and Cd(Ⅱ ) complexes of cefotaxime were synthesized with molar ratio M:L (1:2). The place of coordination take place through the oxygen of carboxylate, oxygen of beta-lactam ring and nitrogen of aminothiazole ring^[7]. Cefepime has been described as the fourth generation of broad-spectrum antibiotics^{[8, 9]}. It is effective against some bacteria that fight other antibiotics and is used to treat Gram-negative and Gram-positive bacteria, especially those that cause infections in the lungs, kidneys, bladder, skin, and abdomen^{[10, 11]}. Cefepime was reacted with some of transition metal(Ⅱ ) ions and gave a 1:1 (M:L) complexes, which were discussed using a physicochemical and spectroscopic analyses^[12]. Many metal-based compounds have been manufactured with promising anti-cancer properties, some of which are already using in clinical practice for diagnosis and treatment while some are undergoing clinical trials^{[13, 14, 15, 16]}.

In view of this point and as part of metal-drug interactions^{[17, 18, 19, 20, 21]}, we reported here the synthesis, spectroscopic characterizations and biological investigations of gold(Ⅲ ) complexes of a cefotaxime (ceph-3) and cefepime (ceph-4) drugs (Fig.1).

Fig.1(a)

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	Fig.1(a) Cefotaxime sodium (ceph-3) drug

Fig.1(b)

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	Fig.1(b) Cefepime hydrochloride (ceph-4) drug

The chemicals AuCl₃, cefotaxime sodium and cefepime hydrochloride were received from Aldrich chemical company (United States) and used without further purification. The solvents were used have an analytical grade.

A dissolved solutions of cefotaxime sodium and cefepime hydrochloride (1 mmol in 50 mL CH₃OH) were mixed with AuCl₃ (1 mmol in 20 mL CH₃OH). The brown solutions were neutralized at pH 7~8 by 0.1 mol· L^-1 of ammonia solution, then refluxed for 3 hrs with continuous stirring. The solid brown precipitates were isolated, washed with few amount of methanol solvent and dried under vacuum. Single crystals for X-ray structural studies were unsuccessful.

Antimicrobial and anticancer tests: The antimicrobial test of the Au(Ⅲ ) cephs complexes was scanned against (G(+)(Staphylococcus Epidermidis and Staphylococcus Aureus) and G(-)(Klebsiella and Escherichia coli) bacteria dependent on modified Kirby-Bauer method^{[22, 23]}. The anticancer assessments of gold complexes against Colorectal adenocarcinoma (Caco-2) and Breast cancer (MCF-7) cell lines were performed upon the standard neutral red uptake assay^[24].

Two synthesized gold(Ⅲ ) complexes are insoluble in more organic solvents but dissolved in two mentioned solvents e.g. (DMF and DMSO) and melting with decomposition at the temperature range -250 ℃. The stoichiometry and analytical data of the complexes reveal that the molecular formula of the compositions as [Au(ceph-3)(Cl)₂] (1) and [Au(ceph-4)(Cl)₂] (2). At room temperature the conductivity data was measured in DMSO and the results showed that the prepared complexes have a non electrolytic property (25~29 ohm^-1· cm²· mol^-1)^[17] due to the presence of chloride ion inside the coordination sphere. From the TGA curves, the synthesized complexes don’ t recorded any mass loss up to 120 ℃, this assigned to the absent of a crystalline water molecules. The microanalytical and stoichiometric results of the complexes are listed in Table 1.

Table 1

Table 1

Table 1 Elemental and physical data of gold(Ⅲ ) ceph-3 and ceph-4 complexes Complexes Color Content ((calculated) found) Λ M/ (Ω -1· cm2· mol-1) Yield/% %C %H %N %Au [Au(ceph-3)(Cl)2] (1) Brown (26.60) 26.11 (2.23) 2.17 (9.70) 9.46 (27.27) 26.98 25 72 Brown[Au(ceph-4)(Cl)2] (2) (30.49) 30.24 (3.23) 3.18 (11.23) 11.11 (26.32) 26.29 29 68

Table 1 Elemental and physical data of gold(Ⅲ ) ceph-3 and ceph-4 complexes

Both cefotaxime and cefepime ligands contains the — NH, — COOH, — NHCO and C=O functional groups and its molecular model reveals that its structure is able for complexation. The characteristic infrared spectral bands of ceph-3, ceph-4 free ligands and their gold(Ⅲ ) complexes are assingend in Table 2. The stretching vibration band of ν (C=O) β -lactam ring is exist at 1 757 and 1 771 cm^-1 in the spectra of ceph-3 and ceph-4, respectively^[25], this band is absence in the IR spectra of Au(Ⅲ ) complexes. In case of the free ceph ligand, the ν (CO) of amido group is presence at ~1 640 cm^-1, while after chetation with gold(Ⅲ ) ion, this band is detectable as the same frequency with not significant shift. Therefore the coordinationbetween gold(Ⅲ ) ion and ceph-3 or ceph-4occurs through the oxygen atom of β -lactam rather than oxygen of amido group. The asymmetric stretching frequency of carboxylate group ν _asCOO which is presence in free ceph-3 and ceph-4 at 1 531 and 1 560 c m-1[25⁃26], respectively is shifted to higher wavenumbers at 1 604 and 1 642 cm^-1 after complexation towards gold(Ⅲ ) ion, this meaning that carboxylate group is sharing in the coordination. Whereas, the vibration band of ν _sCOO carboxylate group is exhibit at 1 352 and 1 320 cm^-1 for the ceph-3 and ceph-4, respectively, also shifted after chelation to become at 1 348 cm^-1. The value of difference between Δ ν =ν _asCOO-ν _sCOO give an idea about the coordination behaviour of carboxylate group (mono or bidentate). Herein, in case of our study, the value of Δ ν is > 200 cm^-1, this confirm that the carboxylate group acts as a monodentate ligand. The presence of new vibration bands regarding ceph-3 and ceph-4 gold(Ⅲ ) complexes at 476 and 523 cm^-1, respectively, can be assigned to the ν M— O stretching vibration through the coordination bond between gold ion and oxygen atoms of β -lactam and carboxylate groups. It is noteworthy that this band doesn’ t exist in the spectra of free ligands^[26].

Table 2

Table 2

Table 2 FT-IR frequencies and assignments of the ceph-3, ceph-4 free ligands and their gold(Ⅲ ) complexes Assignments* Compounds ceph-3 ceph-4 Complex 1 Complex 2 ν (C=O); β -lactam 1 757 1 771 - - ν as(COO) 1 531 1 560 1 604 1 642 ν s(COO) 1 352 1 320 1 348 1 348 Δ ν 179 240 256 294 ν (Au— O) - - 476 523 Note: * ν s=stretching symmetry; ν as=stretching asymmetric; δ =bending

Table 2 FT-IR frequencies and assignments of the ceph-3, ceph-4 free ligands and their gold(Ⅲ ) complexes

¹HNMR spectrum of ceph-3 drug ligand is assigned (Table 3) as: δ (ppm) 1.99 (3H; — COOCH₃), 3.28~3.58 (2H; S— CH₂), 3.91 (3H; N— O— CH₃), 4.63~4.79 (2H; CH₂— O— CO), 5.11 (H; N— CH_{β -lactam}), 5.71 (H; CO— CH_{β -lactam}), 6.89 (H; Ph_{5-member ring}), and 9.03 (H; CONH_{amido group})^[27]. Regarding ¹HNMR spectrum of [Au(ceph-3)(Cl)₂] complex, the characteristic bands of β -lactam and the chemical shifts of six-remembered ring attached with carboxylate group!are moved to downfield δ (ppm). The ¹HNMR spectrum of the Au(Ⅲ ) complex changed in compared with the represented ceph-3 ligand and the signals are present with downfield, as expected, due to increased conjugation on coordination^[28].

Table 3

Table 3

Table 3 1H-NMR spectral data of ceph-3 drug and its gold(Ⅲ ) complex Assignments/ppm Compounds ceph-3 [Au(ceph-3)(Cl)2] 3H; — COOCH3 1.99 0.91 2H; S— CH2 3.28~3.58 3.34 3H; N— O— CH3 3.91 - 2H; CH2— O— CO 4.63~4.79 5.13 H; N— CHβ -lactam 5.11 6.11 H; CO— CHβ -lactam 5.71 6.12 H; Ph5-member ring 6.89 7.37 H; CONHamido group 9.03 8.81

Table 3 1H-NMR spectral data of ceph-3 drug and its gold(Ⅲ ) complex

The electronic spectra of ceph-3 and ceph-4 in DMSO solvent within the UV-Vis region were scanned. These spectra of free ligands have three absorption bands at wavelengths 237, 315 and 386 nm due to π -π ^* , n-π and intra-ligand charge transfer bands^{[25, 28]}. The absorption spectra of gold(Ⅲ ) complexes (Fig.2) corresponding to an electronic transition band at 240 nm, which is assigned to the absorption band of ¹A_1g (D)→ ¹E_u(D), while the band at 430 nm is due to the electronic transition of ¹A_1g(D)→ ¹A_2g (D). These absorption bands are revealed that the gold(Ⅲ ) complexes of ceph-3 and ceph-4 have a square planar geometry^[29]. The diamagnetic character of the synthesized gold(Ⅲ ) complexes revealed that the presence of complexes in a square-planar configuration.

Fig.2

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	Fig.2 UV-Vis spectra of ceph-3 and [Au(ceph-3)(Cl)2] complex

The XRD values of gold(Ⅲ ) complexes as referred in Figure 3(a, b) within range of 5° ~50° (2θ ). According to Figure 3, the 2θ values with maximum intensity of the peaks for Au(Ⅲ ) complex are observed to be 2θ =10.30° , 13.18° , 24.73° , 25.84° , 26.98° , 36.10° , and 37.39° which corresponds to d=8.581 4, 6.712 0, 3.597 1, 3.445 1, 3.302 1, 2.486 1, and 2.403 2, respectively. All these peaks calculated on the obtained values of interplanar distance are compared with the recorded ones^[30]. This implies that gold(Ⅲ ) complex has square-planar geometry in structure. The X-ray diffraction patterns of gold(Ⅲ ) complexes deduced the crystalline structure with nano size (20~25 nm) based on Debye-Scherrer equation^[31].

Fig.3

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	Fig.3 XRD patterns spectra of (a): Au3+ ceph-3 complex and (b): Au3+ ceph-4 complex

The SEM micrograph [Fig.4(a, b)] reveals the overall appearance of Au(Ⅲ ) ceph-3 and ceph-4 complexes. The particles are nearly rod or spherical in shape have uniform size and distribution with asymmetrical sizes, might be due to distribution of temperature during synthesized. It can be observed that product aggregation is constituted by many irregular particles with a variety of particle size due to the difference chemical structure of ligands. TEM images of Au(Ⅲ ) complexes are shown in Figure 4(c, d), with a nanoscale range between 20~24 nm in agreement with XRD data.

Fig.4

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	Fig.4 SEM and TEM images of (a & c): Au3+ ceph-3 complex and (b & d): Au3+ ceph-4 complex

The proposed structures of the synthesis gold(Ⅲ ) complexes concluded that the coordination of ceph-3 and ceph-4 occurs through the carboxylate and lactam carbonyl oxygen atoms as mentioned in Figure 5.

Fig.5

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	Fig.5 Suggested structures of [Au(ceph-3)(Cl)2] and [Au(ceph-4)(Cl)2] complexes

The susceptibility of strains of bacteria “ G(+)(Staphylococcus Epidermidis and Staphylococcus Aureus) and G(-)(Klebsiella and Escherichia coli)” towards cefotaxime (ceph-3) and cefepime (ceph-4) ligands and their gold(Ⅲ ) complexes were screened by measuring the size of inhibition diameter (Table 4). The cefotaxime and cefepime antibiotic drugs and the gold(Ⅲ ) complexes have a bactericide diameters > 19 mm with highly sensitive^{[32, 33]}. Generally, gold(Ⅲ ) complexes [Au(ceph-3)(Cl)₂] and [Au(ceph-4)(Cl)₂] were found to have higher antibacteria activities than that of corresponding ligands. The highest antibacterial activity was showed by the [Au(ceph-3)(Cl)₂] complex against Escherichia coli being inhibition zone diameter about 29 mm higher than ceph-3 activity.

Table 4

Table 4

Table 4 Inhibition zone (mm) of ceph-3 drug, ceph-4 drug, Au3+ ceph-3 complex and Au3+ ceph-4 complex Sample Inhibition zone diameter (mm· mg-1 sample) Bacteria S. Epidermidis, (G+)a S. Aureus, (G+) Klebsiella, (G-) Escherichia coli, (G-) Control: DMSO 0.0 0.0 0.0 0.0 Standard Tetracycline (Antibacterial agent) 27 26 29 25 Control (DMSO) 0.0 0.0 0.0 0.0 ceph-3 22 20 19 23 Au3+ ceph-3 23 21 22 29 ceph-4 21 18 20 22 Au3+ ceph-4 26 20 22 26 Note: a G: Gram reaction.

Table 4 Inhibition zone (mm) of ceph-3 drug, ceph-4 drug, Au3+ ceph-3 complex and Au3+ ceph-4 complex

The anticancer activities of gold(Ⅲ ) complexes of ceph-3 and ceph-4 drugs against the growth colorectal adenocarcinoma (Caco-2) and breast cancer (MCF-7) cell lines are listed in Table 5. The % cell inhibition and IC50 values (26.1, 110, 1.99, and 11.4 μ g· mL^-1) for the Au³⁺ ceph-3 complex against (Caco-2), Au³⁺ ceph-3 complex against (MCF-7), Au³⁺ ceph-4 complex against (Caco-2) and Au³⁺ ceph-4 complex against (MCF-7), respectively. They indicates that gold(Ⅲ ) complexes have a sensitivity towards the Caco-2 and MCF-7 cancer lines in comparable with cisplatin standard drug (5.71 and 3.67 μ g· mL^-1).

Table 5

Table 5

Table 5 Sample concentration and cell viability of Au3+ complexes and cisplatin against (Caco-2) and (MCF-7) cancer cell lines Sample conc. (μ g· mL-1) Viability/% Caco-2 cell lines MCF-7 cell lines cisplatin Au3+-ceph-3 Au3+-ceph-4 cisplatin Au3+-ceph-3 Au3+-ceph-4 500 3.72 5.87 2.76 3.08 11.83 4.89 250 4.98 11.24 4.08 4.31 28.75 9.73 125 7.83 19.40 7.31 6.75 43.59 16.49 62.5 14.68 28.83 12.68 12.39 70.82 23.85 31.25 23.79 40.67 20.93 22.98 86.13 30.47 15.6 34.62 69.21 28.32 31.87 95.08 41.72 7.8 46.71 88.13 36.48 40.62 99.46 56.94 3.9 52.85 97.02 42.91 47.89 100 70.31 2 61.74 100 49.87 60.75 100 84.19 1 70.88 100 62.45 68.17 100 92.3701001 0 0 100 100 100 100

Table 5 Sample concentration and cell viability of Au3+ complexes and cisplatin against (Caco-2) and (MCF-7) cancer cell lines