Synthesis, structural characterization and biological studies of some nalidixic acid–metal complexes: Metalloantibiotic complexes of some divalent and trivalent metal ions

Accepted Manuscript Synthesis, structural characterization and biological studies of some nalidixic acid-metal complexes: Metalloantibiotic complexes of some divalent and trivalent metal ions Fatima A.I. Al-Khodir, Moamen S. Refat PII: DOI: Reference:

S0022-2860(15)00298-7 http://dx.doi.org/10.1016/j.molstruc.2015.03.063 MOLSTR 21447

To appear in:

Journal of Molecular Structure

Received Date: Revised Date: Accepted Date:

25 January 2015 28 March 2015 30 March 2015

Please cite this article as: F.A.I. Al-Khodir, M.S. Refat, Synthesis, structural characterization and biological studies of some nalidixic acid-metal complexes: Metalloantibiotic complexes of some divalent and trivalent metal ions, Journal of Molecular Structure (2015), doi: http://dx.doi.org/10.1016/j.molstruc.2015.03.063

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Synthesis, structural characterization and biological studies of some nalidixic acid-metal complexes: Metalloantibiotic complexes of some divalent and trivalent metal ions Fatima A.I. Al-Khodir1,2 and Moamen S. Refat 3,4 * 1

Department of Chemistry, College of Science, Princess Nora Bint Abdul Rahman University 2 Deanship of Scientific Research, Princess Nora Bint Adul Rahman University 3 Department of Chemistry, Faculty of Science, Port Said University, Port Said, Egypt 4 Department of Chemistry, Faculty of Science, Taif University, Al-Hawiah, Taif, P.O. Box 888 Zip Code 21974, Saudi Arabia Email: [email protected]

Abstract This article describes the synthesis, characterization, computational and biological assessments of some divalent and trivalent metal (Ca(II), Fe(III), Pd(II) and Au(III)) complexes of nalidixic acid (nixH). The structures of these complexes were assigned using elemental analyses and spectral measurements e.g., IR, Raman, 1HNMR, 13C-NMR and electronic techniques. These results indicated that, nalidixic acid reacts as a bidentate ligand bound to the metal ion through the oxygen atoms of carbonyl and carboxylate groups. The molar conductance measurements of the complexes in DMSO correspond to be non-electrolyte nature. Thus, these complexes may be formulated as [Ca(nix)(Cl)(H2O)3]. H2O, [Fe(nix)(Cl)2(H2O)2].3H2O, [Pd(nix)(Cl)(H2O)] and [Au(nix)(Cl)2]. Base on the Coats–Redfern and Horowitz– Metzeger methods, the kinetic thermodynamic parameters (E*, ΔS*, ΔH* and ΔG*) of the thermal decomposition reactions have been calculated from thermogravimetric curves of TG and DTG. The nano-scale range of the nalidixic acid complexes have been discussed using X-ray powder diffraction (XRD), scanning electron microscope (SEM) and transmission electron microscopy (TEM) analyzer. The computational studies for the synthesized complexes have been estimated. Key words: Metalloantibiotics, nalidixic acid, metal ions, complexation, anticancer, nano-scale.

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1- Introduction Nalidixic acid (nixH, Fig. 1) was commonly named as 1-Ethyl-1,4-dihydro-7methyl-4-oxo-1,1,8- naphthyridine-3-carboxylic acid and its belongs to a 4-quinolone antibiotic drug families. The 4-quinolones is a class of antibacterial agents which has been known for over 40 years [1]. Quinolones were comprised of a large family of antibacterial agents such as nalidixic acid, perfoxacin, norfloxacin, ofloxacin, and ciprofloxacin. Some of fluoroquinolones and their derivatives have been used in the improvement of antimicrobial, anticancer, anti-inflammatory, antiviral, and anti-HIV activities [2-4]. Nalidixic acid has been used in the treatment of several diseases as a powerful antibiotic drug for decontamination of the gut infections [5-12].

N

N

HO

O

O

Fig. 1: Nalidixic acid (1-Ethyl-1,4-dihydro-7-methyl-4-oxo-1,1,8- naphthyridine-3carboxylic acid) free drug The different metal ions constituted the backbone for the metalloantibiotic skeletons, which were helpful to enhancement of biological and medical functions [13-17]. Metalloantibiotics interact with DNA, RNA, proteins, receptors and lipids, making them very unique and specific. Metal contamination potentially contributes to the maintenance and spread of antibiotic resistance factors. Antibiotics metal complexes as well as mixed antibiotics metal complexes were found more effective as chemotherapy agents than their parent antibiotics [13-18]. Quinolones drugs are good complexing agents for different transition and nontransition metal ions. The synthesis and characterization of metal complexes with quinolone antibacterial agents were of great importance for understanding the drugmetal ion interaction and for their potential pharmacological usage [18, 2, 3]. Precise dissociation constants as well as stability constants for the binding of the nalidixate anion using several divalent metal ions were reported [19]. Nakano et al., [20] have been reported the ability of nalidixic acid drug to form complexes with aluminum(III), magnesium(II) and calcium(II) ions. Complex formation between nalidixic acid, metal ion and DNA has been discussed [21]. Behrens et al., have been synthesized and characterized the transition metal complexes of nalidixic acid. The nalidixic acid was used in the clinical treatment of urinary tract infections caused by gram-negative bacteria. The mode of coordination of the drug was investigated by spectroscopic studies. From the spectral data, nalidixic acid anion binds through the carboxylate group either as a chelate or as bridge to give polymeric structure [22]. The most metal complexes of fluoroquinolone prefer to coordinate as bi-dentate statement ligand- tometal through the carbonyl and one oxygen atoms of the carboxylic group. The antimicrobial activities of these complexes were greater than free ligand against the 2

tested organisms [16-20]. The studied metal complexes of fluoroquinolones have the potential of being used as drugs. The chemistry of metal-drug coordination compounds is more popular now than before in importance particularly in the design of more biologically active drugs [23]. Metal ions are known to affect the action of many drugs. The efficacies of the drugs on coordination with a metal are enhanced in many cases [24]. Metal ions play an essential role in a vast number of widely differing biological processes and depending on their concentration, they may either contribute towards the health of the organism or cause toxicity [25, 26]. In literature survey, the great attention has been drawn to studies of the antitumor activities of inorganic especially metal complexes [27, 28]. The transfer of metal ion from the ligand to the viruses associated with cancer is a mechanism for releasing the anticancer drug in the locality of the tumor [28]. For the continuation of the methodology for our research group, this deals with metal-drug interactions [29-34]. Metal ions play an essential role in the design of more biologically active drugs, so, this article aimed to synthesis, theoretical calculations, spectroscopic characterizations, thermal stabilities and their biological evaluations of new nalidixic complexes with some of divalent and trivalent metal ions (Ca(II), Fe(III), Pd(II) and Au(III)). The experimental studies have been accompanied by theoretical calculations (PM3) due to their important role in understanding of the probably behavior of the compound during reactions and identification of the important information about the compounds under investigations, like total energy, binding energy, electronic energy, dipole moment, bond lengths, LUMO and HOMO. 2- Experimental 2-1- Chemicals Nalidixic acid mentioned in this article was received from the Aldrich chemical company. All of chemicals used for this study were of analytically reagent grade, commercially available from BDH and used without previous purification like CaCl2, FeCl3.6H2O, PdCl2 and sodium tetrachloroaurate(III) dehydrate (NaAuCl4 .2H2O). 2-2- Synthesis The Ca(II), Fe(III), Pd(II) and Au(II) nalidixate complexes were prepared by refluxing 1:1 molar ratio for the mixture of the nixH ligand (1 mmol) with each metal chloride salt in methanolic medium on a hotplate for 2–3 hrs at ~ 70 oC. The precipitate was filtered off washed with methanol and diethyl ether and finally dried in a vacuum desiccator's over anhydrous calcium chloride. The yields of the final products were 70-75%. 2-3-Instruments Carbon, hydrogen and nitrogen analyses have been carried out in Vario EL Fab. CHNS. The amount of water and the metal content percentage were determined by gravimetric analysis method. Infrared spectra of the nix complexes were recorded on Bruker infrared spectrophotometer in the range of 400-4000 cm-1, while Raman laser spectra of samples were measured on the Bruker FT-Raman with laser 50 mW. The molar conductances of 10-3 M solutions of the complexes in DMSO solvent were measured on a HACH conductivity meter model. All the measurements were taken at room temperature for freshly prepared solutions. The electronic spectrum of the complexes were measured in DMSO solvent with concentration of 1×10-3 M, in rang 200-800 nm by using Unicam UV/Vis spectrometer. The mass susceptibility (Xg) of

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complexes was measured at room temperature using Gouy's method by a magnetic susceptibility balance from Johnson Metthey and Sherwood model. The effective magnetic moment (μeff) value was obtained using the following equations (1, 2 and 3) [35]. Xg =

CBal L (R  R0 ) 109 M

(1)

Where: Ro = Reading of empty tube L = Sample length (cm) M = Sample mass (gm) R = Reading for tube with sample CBal = balance calibration constant = 2.086 XM = X g x M . W t . (2) The values of XM as calculated from equation (2) are corrected for the diamagnetism of the ligand using Pascal's constants, and then applied in Curie's equation (3). μeff = 2.84

XM x T

(3)

Where T= t (°C) + 273 1

H-NMR and 13C-NMR was recorded as DMSO solutions on a Bruker 600 MHz spectrometer using TMS as the internal standard. Thermogravimetric analysis (TGA) experiments were conducted using Shimadzu TGA-50H thermal analyzers. All experiments were performed using a single loose top loading platinum sample pan under nitrogen atmosphere at a flow rate of 30 ml/min and a 10 °C/min heating rate for the temperature range 25-800 °C. SEM images were obtained using a Jeol Jem1200 EX II Electron microscope at an acceleration voltage of 25 kV. X-ray diffraction (XRD) patterns of the samples were recorded on X Pert Philips X-ray diffractometer. All the diffraction patterns were obtained by using CuK1 radiation, with a graphite monochromator at 0.02 /min scanning rate. The transmission electron microscopy images were performed using JEOL 100s microscopy. 2-4- Antimicrobial assessments Antimicrobial activity of the tested samples was determined using a modified Kirby-Bauer disc diffusion method [36]. Briefly, 100 μL of the test bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately108 cells/mL for bacteria or (105 cells/mL) for fungi [37]. 100 μL of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Isolated colonies of each organism that might be playing a pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method [38, 39]. Of the many media available, National Committee for Clinical Laboratory Standards (NCCLS) recommends Mueller-Hinton agar due to it results in good batch-to-batch reproducibility. Disc diffusion method for filamentous fungi tested by using approved standard method (M38-A) developed by the NCCLS [40] for evaluating the susceptibility of filamentous fungi to antifungal agents. Disc diffusion method for yeast developed standard method (M44-P) by the NCCLS [41]. Plates inoculated with filamentous fungi as Aspergillus Flavus at 25 oC for 48 hours;

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Gram (+) bacteria as Staphylococcus Aureus, Bacillus subtilis; Gram (-) bacteria as Escherichia Coli, Pseudomonas aeruginosa they were incubated at 35-37 oC for 2448 hours and yeast as Candida Albicans incubated at 30 oC for 24-48 hours and, then the diameters of the inhabitation zones were measured in millimeters [36]. Standard discs of Tetracycline (Antibacterial agent), Amphotericin B (Antifungal agent) served as positive controls for antimicrobial activity but filter disc impregnated with 10 μL of solvent (distilled water and DMSO) were used as a negative control. The agar used is Meuller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhabitation have been determined for susceptible values. Blank paper disks (Schleicher & Schuell, Spain) with a diameter of 8.0 mm were impregnated 10 μL of tested concentration of the stock solutions. When a filter paper disc impregnated with a tested chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a "Zone of inhibition" or "Clear zone". For the disc diffusion, the zone diameters were measured with slipping calipers of the National for Clinical Laboratory Standers [38]. Agar-based methods such as Etest disk diffusion can be good alternatives because they are simpler and faster than broth methods [42, 43]. 2-5- Anti-cancer activities Human colon carcinoma (HCT-116) cells and human hepatocellular carcinoma (HepG-2) cells were obtained from the American type culture collection ATCC, Rockvill, MD). The cells were grown on RPMI-1640 medium supplemented with 10 % inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37 oC in a humidified atmosphere with 5 % CO2 and were subcultured two to three times a weak. The cells were grown as monolayers in growth RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The monolayers of 10000 cells adhered at the bottom of the wells in a 96-well micro titer plate incubated for 24 h at 37 oC in a humidified incubator with 5 % CO2. The monolayers were then washed with sterile phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells were treated with 100 µL from different dilutions of the test sample in fresh maintenance medium and incubated at 37 oC. A control of untreated cells was made in the absence of the test sample. Six wells were used for each concentration of the test sample. Every 24 h the observation under the inverted microscope was made. The number of the surviving cells was determined by staining the cells with crystal violet [44, 45] followed by cell lysing using 33% glacial acetic acid and read the absorbance at 490 nm using ELISA reader (Sun Rise, TECAN, Inc, USA) after well mixing. The absorbance values from untreated cells were considered as 100% proliferation. The number of viable cells was determined using ELISA reader as previously mentioned before and the percentage of viability was calculated as [1 – (ODt/ODc)] 100% where; ODt is the mean optical density of wells treated with the test sample and ODc is the mean optical density of untreated cells. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effect in 50% of inactivated cells, was estimated from graphic plots.

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3- Results and Discussion 3-1- Analytical and physical data The analytical and physical data of the nalidixic acid free ligand and their Ca(II), Fe(III), Pd(II) and Au(III) complexes are listed in Table 1. The melting point of the nixH ligand has 227-230 oC. The isolated complexes are stable in air, high melting points, insoluble in H2O and most organic solvents except DMSO and DMF with gently heating. The coordination behavior of nixH towards some of divalent and trivalent metal ions was investigated via the (IR, Raman, 1H-NMR) spectra, thermal analyses and molar conductance. The elemental analysis of the complexes show 1:1 (metal: ligand) stoichiometry for Ca(II), Fe(III), Pd(II) and Au(III) complexes. The magnetic moment of all nix complexes were measured at room temperature and have diamagnetic character except for [Fe(nix)(Cl)2(H2O)2].3H2O complex has a paramagnetic situation. The low molar conductance values of the complexes reveal the non-electrolytic behavior [46], which elucidates the proposal of the absence or presence of coordinated anion but covalently attached with central metal ion. This is in a good agreement with the elemental analysis data for the isolated complexes. The results are in closed matched data with the suggested formulas; [Ca(nix)(Cl)(H2O)3].H2O, [Fe(nix)(Cl)2(H2O)2].3H2O, [Pd(nix)(Cl)(H2O)] and [Au(nix)(Cl)2] (Fig. 2). Table1: Analytical and physical data of nixH free drug ligand and their metal complexes Empirical formula

Color

nixH [Ca(nix)(Cl)(H2O)3]. H2O [Fe(nix)(Cl)2(H2O)2]. 3H2O

White

[Pd(nix)(Cl)(H2O)] [Au(nix)(Cl)2]

White Reddish brown Yellowish white Light brown

m.p/ o C 278 >250 >250 >250 >250

Λm (S) 6 12 16 8 14

6

Elemental analysis, % Found % (Calcd.) C

H

N

Cl

M

62.06 38.02 (38.05) 32.10 (32.17) 36.64 (36.85) 28.43 (28.88)

5.21 5.01 (5.06) 4.56 (4.72) 3.21 (3.35) 2.05 (2.22)

12.06 7.22 (7.39) 6.11 (6.25) 7.09 (7.16) 5.41 (5.61)

9.30 (9.36) 15.77 (15.83) 9.02 (9.06) 14.08 (14.21)

10.43 (10.58) 12.33 (12.46) 27.10 (27.21) 39.33 (39.46)

N

N

N

N

O

.3H2O

O

O

O

H2O

O

O

.H2O

Fe

Ca H2O

H2O

Cl

Cl

Cl OH2

OH2

N

N N

N

O O

O

O O

Pd Cl

O Au

OH2 Cl

Cl

Fig. 2: Suggested formulas of [Ca(nix)(Cl)(H2O)3].H2O, [Fe(nix)(Cl)2(H2O)2].3H2O, [Pd(nix)(Cl)(H2O)] and [Au(nix)(Cl)2] complexes 3-2-Infrared and Raman spectra The infrared spectra of nixH free drug ligand and their Ca(II), Fe(III), Pd(II) and Au(III) complexes are exhibited in Fig. 3 and the characteristic bands are listed in Table 2. The nixH molecule has two essential moieties as 4-oxo[1,8]naphthyridine (carbonyl group) and 3-carboxylic acid group which have a definite absorption peaks. So, we can discuss firstly vibration motions of free ligand to facilitate the coordination modes of the respected metal ions. 3-2-1-Nalidixic acid (nixH) free ligand The nalidixic acid free ligand has one absorption band with strong broad intensity at the 3431 cm-1 due to the –OH stretching vibration of carboxylic group. This band is absorbed at higher frequency arise by intermolecular hydrogen bonding [46-48]. The carboxylic group of nixH molecule also has a carbonyl stretching vibration motion at the higher frequency than other carbonyl group of 4-oxo[1,8]naphthyridine moiety; this band is exhibited at 1714 cm-1 [47]. The stretching vibration band of the carbonyl group in case of 4-oxo-[1,8]naphthyridine moiety is exhibit at 1617 cm-1, this band under hydrogen bonding is shifted to lower value [47, 48]. There is another type of bands which are presence within the region of 13001200 cm-1 in nixH ligand, these bands arise due to coupling between C-N and C-O

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stretching vibrations. The asymmetric and symmetric stretching vibrations of the CH3 and CH2 groups attached to the 4-oxo-[1,8]naphthyridine moiety nucleus have some weak-to-medium bands in the region 3050-2945 cm-1. Infrared absorption bands characteristic of deformation of both CH3 and CH2 groups has medium-to-strong bands at 1443 and 1328 cm-1 [48]. The stretching vibration of C=N group is observed at 1539 cm-1. The stretching vibration bands exhibited at 1519 and 1474 cm-1 are assigned to aromatic ring stretching of benzene ring. 3-2-2-Nalidixate complexes Herein, this article discussed four nalidixate complexes formed between nalidixic acid drug and some divalent and trivalent metal ions (CaCl2, FeCl3.6H2O, PdCl2 and NaAuCl4.2H2O). The infrared spectra of naldixate complexes under investigation were illustrated in Fig. 3 and briefly assigned in Table 2. The spectra of nalidixate complexes are similar but there are some differences which could give indication on the mode of chelation as suggested in Fig. 2. The infrared spectra of Ca(II), Fe(III) and Pd(II) nalidixate complexes have a broad bands observed at 3419, 3406 and 3400 cm-1, respectively, these bands are assigned to the ν(OH) stretching vibration of the coordinated and uncoordinated water molecules. As listed before, the infrared spectrum of free nixH has two main very strong frequencies bands at 1714 and 1617 cm-1 due to the stretching vibration motions ν(C=O) of the carboxylic group and the carbonyl of 4-oxo-[1,8]naphthyridine moiety [47]. The Ca(II), Fe(III), Pd(II) and Au(III) complexes haven't absorption band at 1718 cm-1, that is discussed upon the deprotonation of COOH group. The stretching absorption band of ν(C=O) carbonyl group is shifted by ~ 10 cm-1 upon ligation [47]. The characteristic bands of nalidixate complexes have two characteristic bands at (1578-1355), (1558-1355), (1617-1375) and (1618-1407) cm-1 for the Ca(II), Fe(III), Pd(II) and Au(III) nalidixate complexes, respectively. These spectral bands are assigned to ν(COO-) asymmetric and symmetric stretching vibrations of the chelate carboxylate group, respectively. The carboxylic group can be coordinated with metal ions by three fashions, so, we can be identified, the binding geometry of the carboxylate ligand. Deacon and Phillips [49] have studied the criteria that can be used to distinguish between the three binding states of the carboxylate complexes. These criteria as; aΔν>200 cm-1 (where Δν = [νas(COO-)-νs(COO-)]) this relation was found in case of unidentate geometry, b- bidentate carboxylate complexes exhibit Δν significantly smaller than ionic values (Δν<100 cm-1), and finally, c- bridging complexes show Δν comparable to ionic values (Δν ~150 cm-1). Therefore, the difference values of Δν for Ca(II), Fe(III), Pd(II) and Au(III) complexes (Table 2) existed in the range of 203-242 cm-1 are assigned to unidentate complexation mode for the carboxylate group [47, 49]. It is worthy mentioned that the infrared frequencies within the 1100 and 600 cm-1 region are discussed as angular deformation motions of the coordinated water molecules. The nalidixate complexes have new bands at 543-446 cm-1, these bands are assigned to the ν(M-O) stretching vibration motions [48, 49]. Results of infrared spectra can be summarized, that nalidixic acid is coordinated toward metal ions as bidentate ligand via one carboxylate oxygen atom and oxygen atom of the carbonyl group.

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Table 2: Selected Infrared/Raman spectral bands and their assignments of nixH free ligand and their complexes. nixH 3431 3047 2984 2945 1714 1617 1519 1474 1443 1328 1296 1260 1230 -

Ca(II) 3419 3063 2981 2936 1626 1578 1355 223 1503

Fe(III) 3406 3157 3043

Pd(II) 3148 3046 2989

1624 1558 1355 203 1489

-

Au(III) 3139 3046 2810 -

1617

1618

1375 242 1473

1407 211 1473

ν(CO); COOH ν(CO); keto group νas(COO) νs(COO) Δν = [νas(COO)- νs(COO)] ν(C=C); aromatic

1446 1321 1292 1261 1230 543 496 370, 320

1445 1316 1290 1256

1441 1326 1295 1256 1228 539 507 370, 320

1329

CH-deformation

1296 1229

ν(C-O)+ν(C-N)

486 456 360, 320

ν(M-O)

545 511 364, 320

Assignments ν(OH); COOH+H2O νas(CH)+ νs(CH); CH2+CH3

ν(M-Cl); Raman

1.2

1.0

%

0.8

0.6

0.4

0.2

0.0 4000

nixH Ca(II) Fe(III) Pd(II) Au(III) 3500

3000

2500

2000

cm

1500

1000

500

-1

Fig. 3: Infrared spectra of nixH free ligand and their complexes The Raman spectra (Fig. 4) of Ca(II), Fe(III), Pd(II) and Au(III) complexes are helpful to identified the stretching vibration motions of ν(M-Cl) bands which are observed at (400-300 cm-1).

9

Raman intenisty

0.020

0.015

320

0.010

370

0.005

364 355 Ca(II) Fe(III) Pd(II)

0.000 400

380

360

340

cm

320

300

-1

Fig. 4: Raman spectra of [Ca(nix)(Cl)(H2O)3].H2O, [Fe(nix)(Cl)2(H2O)2].3H2O and [Pd(nix)(Cl)(H2O)] complexes 3-3-Electronic spectra and magnetic measurements The UV–visible spectra (200-1000 nm) of the free nixH ligand, Fe(III) and Au(III) nalidixate complexes are shown in Fig. 5. The electronic spectrum of free nixH has two characteristic absorption bands existed at 245 and 315 nm due to π→π* and n→π* transitions, respectively. These transitions presence in case of unsaturated hydrocarbons, that contain carbon atom attached with oxygen atoms like carboxylic and ketone groups [50, 51]. The electronic spectrum of Ca(II) complex has two absorption bands exhibited at 240 and 318 nm due to π→π* and n→π* transitions, respectively. These bands are presence at the same wavelengths of free nixH with small shift in both wavelengths and intensities of absorbance; this can be assigned to chelation process. The spectrum of Pd(II) complex exhibits d-d spin due to the transition from the lower d orbital to empty dx2–y2 orbital. The d-d transition bands are 280, 370 and 402 nm attributed to charge-transfer, 1E1g→1g and 1A1g→1g, respectively. The electronic spectrum of gold(III) nalidixate complex (Fig. 5) gave three essential absorption bands at 410, 375 and 320 nm due to 1A1g→2g, 1A1g→1g and 1A1g→g, respectively. The fourth band refereed at 280 nm is assigned to charge transfer transition (Table 3). These bands are in matched with low-spin square planar configuration [52]. The Fe(III) nalidixate complex has high spin with two absorption bands at 405 and 315 nm (Fig. 5) due to 6A1g→4T2g and 6A1g→4T1g transitions, respectively [53, 54]. The third band at 280 nm was assigned to charge transfer (M→L and L→M charge transfer band). The observed magnetic moment value is 5.30 B.M. within the accepted range and hence an octahedral geometry [55, 56]. The shift in π–π* and charge transfer regions of the complexes may be assigned to π– interaction between metal ions and nalidixic acid orbital. The magnetic moments of Ca(II), Pd(II) and Au(III) nalidixate complexes calculated from the corrected magnetic susceptibilities determined at room temperature indicate the diamagnetic nature (Table 3). The palladium(II) complex have eff = 0.10 BM, respectively, indicate a diamagnetic geometry that the central

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metal ion possess. The gold(III) nalidixate complex also has a diamagnetic nature (eff 0.22) as expected for low spin d8 complexes, which assigned to square planar geometry [57].

Fig. 5: Electronic spectra of nixH ligand, [Fe(nix)(Cl)2(H2O)2].3H2O and [Au(nix)(Cl)2] complexes Table 3: Electronic spectral bands and magnetic moments of nalidixate complexes Sample nix Fe(III) complex Pd(II) complex Au(III) complex

eff -5.30 (paramagnetic) 0.10 (diamagnetic) 0.22 (diamagnetic)

Electronic bands/ nm 245, 315 280, 315, 336, 405 280, 370, 402 280, 320, 354, 375, 410

Geometry -Octahedral Square planar Square planar

3-4- 1H-NMR and 13C-NMR spectra 1 H-NMR spectra of nixH free ligand (Scheme 1) and their [Ca(nix)(Cl)(H2O)3].H2O and [Au(nix)(Cl)2] complexes (Fig. 6) were recorded in DMSO-d6 taking TMS as internal standards (Table 4). The 1H-NMR spectrum of nixH ligand show singlet peak due to -CH3 proton of 2-methyl pyridine moiety at 2.55 ppm. The –CH3 and –CH2 protons of 1-ethyl-7-methyl-4-oxo-[1,8]naphthyridine moiety were observed at 1.00 and 3.10 ppm as a multiples, respectively. The aromatic protons of the 4-oxo-[1,8]naphthyridine moiety were observed in their expected regions in the spectrum at 6.61, 7.85 and 8.14 ppm. The –OH proton of carboxylic group shows singlet peak at 11.00 ppm. In the 13C-NMR (DMSO-d6) spectrum of nixH ligand, the signals assigned as: δ ppm 20.90(CH3), 13.40(CH3CH2), 48.80(CH3CH2), 187.00(C=O), 113.10, 163.10, 138.20, 113.80, 110.90, and 161.10 (aromatic rings), and 170.00(carboxylic acid).

11

1.00 3.10 8.14 H

N

2.55

N

11.0 HO

6.61 7.85 O

O

13.4 48.8

161.1

N

N

163.1 113.8

HO 170.0 O

110.9

20.9

163.1 113.0

187.0 138.2 O

Scheme 1: 1H-NMR and 13C-NMR positions and δ(ppm) of free nixH ligand. The [Ca(nix)(Cl)(H2O)3].H2O complex: 1H-NMR (DMSO-d6): δppm singlet peak due to -CH3 proton of 2-methyl pyridine moiety is presence at 2.66 ppm. The –CH3 and –CH2 protons of 1-ethyl-7-methyl-4-oxo-[1,8]naphthyridine moiety were observed at 1.37 and 3.34 ppm as a multiples, respectively. The aromatic protons of the 4-oxo-[1,8]naphthyridine moiety were observed in their expected regions in the spectrum at 7.22, 7.23 and 7.24 ppm. The –OH proton of carboxylic group was disappeared due to complexation through deprotonation of carboxylic acid. The new singlet peak exhibited at 2.80 ppm is assigned to protons of water molecules. The [Au(nix)(Cl)2] complex: 1H-NMR (DMSO-d6): δppm singlet peak due to -CH3 proton of 2-methyl pyridine moiety was refereed at 2.71 ppm. The –CH3 and –CH2 protons of 1-ethyl-7-methyl-4-oxo-[1,8]naphthyridine moiety were observed at 1.41 and 3.35 ppm as a multiples, respectively. The aromatic protons of the 4-oxo[1,8]naphthyridine moiety were observed in their expected regions in the spectrum at 7.62, 8.62 and 9.18 ppm. The –OH proton of carboxylic group was absence due to complexation via deprotonation of carboxylic acid. The 13C-NMR (DMSO-d6) spectral data (Scheme 2) can be assigned as: δppm δ ppm 20.90(CH3), 13.40(CH3CH2), 49.10(CH3CH2), 53.00(C=O), 155.40, 111.60, 138.40, 162.00, 114.90, and 145.90 (aromatic rings), and 170.00 (carboxylic acid).

12

Fig. 6A: 1H-NMR spectrum of [Ca(nix)(Cl)(H2O)3].H2O complex

Fig. 6B: 1H-NMR spectrum of [Au(nix)(Cl)2] complex 13.4 49.1

145.9

N

N

155.4

162.0 114.9

O 170

114.1

O

53

20.9

111.6 138.4

O Au

Cl

Cl

Scheme 2: 13C-NMR positions and δ(ppm) of [Au(nix)(Cl)2] complex.

13

Table 4: 1H-NMR spectral data of free nixH ligand and their Ca(II) and Au(IIII) complexes. Assignments –CH3 (2-methyl pyridine) –CH3 1-ethyl-7-methyl-4-oxo –CH2 1-ethyl-7-methyl-4-oxo Aromatic protons –OH (COOH)

nixH 2.55 1.00 3.10 6.61, 7.85, 8.14 11.00

δ(ppm) Ca(II) 2.66 1.37 3.34 7.22, 7.23, 7.24 -

Au(II) 2.71 1.41 3.35 7.62, 8.62, 9.18 -

3-5- Thermal analyses The thermal degradation steps, maximum differential thermal analyses, decomposition assignments and weight loss (found/calculated) of the nalidixate complexes (Fig. 7a-d) are listed in Table 5. 70 0.0000

-0.0005

50 40

-0.0010

DTA (uV)

DTG (mg/sec)

60

30 20 -0.0015 10

DTG DTA -0.0020 0

100

200

300

400

500

600

700

0 800

o

Temp. ( C)

Fig. 7a: DTG and DTA curves of [Ca(nix)(Cl)(H2O)3].H2O complex

The thermal decomposition of Ca(II) nalidixate complex with the general formula [Ca(nix)(Cl)(H2O)3].H2O occurs at two steps. The first degradation step take place at a temperature range of 150-310 oC at DTGmax= 250 oC and DTAmax= 200 oC (endo) and it correspond to the loss of 4H2O (3H2O coordinated + one H2O uncoordinated water molecules), ½Cl2 and C2H4 gas with an observed weight loss 36.00% (calcd.= 35.77%). The second step occur within a temperature range 310-550 o C at two DTGmax= 400 and 505 oC and one DTAmax= 450 oC (exo) due to the loss of naldixate moiety with a weight loss (obs.= 49.50%, calcd.= 49.20%) The calcium(II) oxide CaO is the final product remains stable till 800 oC as final residue.

14

60 0.0000

DTG DTA

40

30

-0.0010

20

DTA (uV)

DTG (mg/sec)

-0.0005

50

-0.0015 10 -0.0020 0

-0.0025 0

100

200

300

400

500

600

700

800

-10 900

o

Temp. ( C)

Fig. 7b: DTG and DTA curves of [Fe(nix)(Cl)2(H2O)2].3H2O complex The thermal degradation of [Fe(nix)(Cl)2(H2O)2].3H2O complex exhibited in four continuously steps. The first and second decomposition steps occurs at a temperature range of 30-115 and 115-285 oC with DTGmax= 70 and 215 oC as well as DTAmax= 150 oC (exo) and it assigned to the loss of 5H2O, Cl2, C2H4 molecules with an observed weight loss 41.70% (calcd.= 42.18%). The second step occur within a temperature range 285-400 and 400-500 oC at DTGmax= 340 and 435 oC as well as DTAmax= 350 and 435 oC, which assigned to the decomposition of nalidixate molecule with a weight loss (obs.= 39.70%, calcd.= 40.00%). The FeO1½ is the final product remains stable till 800oC as final residue. The thermal decomposition of [Pd(nix)(Cl)(H2O)] complex existed in three steps. The first degradation exhibited at a temperature range of 30-150 oC with DTGmax= 60 oC and DTAmax= 60 oC that is correspond to the loss of H2O, ½Cl2 and C2H4 molecules with an observed weight loss 21.00% (calcd.= 20.84%). The second and third step occur within a temperature range 150-600 oC at DTGmax= (220, 360 and 515) oC and DTAmax= (410 and 515) oC which assigned to the loss of nalidixate molecule with weight loss (obs.= 47.98%, calcd.= 47.86%). The palladium(II) oxide is the final product remains stable till 800 oC as final residue.

15

70

DTG DTA

60 50

-0.0005

40 -0.0010

30

DTA (uV)

DTG (mg/sec)

0.0000

20

-0.0015

10 -0.0020

0 0

200

400

600

800

o

Temp. ( C)

Fig. 7c: DTG and DTA curves of [Pd(nix)(Cl)(H2O)] complex The thermal decomposition of [Au(nix)(Cl)2] complex occurs at three DTGmax = (219, 365 and 532) oC steps and three DTAmax= 196, 478 and 533 oC steps. The first degradation step take place at a temperature range of 30-300 oC at DTGmax= 219 oC and DTA= 196 oC (endo) and it correspond to the loss of C2H4 molecule with an observed weight loss 5.70 % (calcd.= 5.61%). The second and third steps occur within a temperature range 300-600 oC at DTGmax= (365 and 532) oC and DTAmax= (478 and 533) oC which assigned to the loss of Cl2 and nix molecules with a weight loss (obs.= 54.84%, calcd.= 54.93%). The gold metal is the final product remains stable till 800 o C as final residue.

0.0000 30

24

-0.0010

18

-0.0015

12

6

-0.0020

DTG DTA

-0.0025 0

100

200

300

400

500

600

700

o

Temp. ( C)

Fig. 7d: DTG and DTA curves of [Au(nix)(Cl)2] complex

16

800

0

900

DTA (uV)

DTG (mg/sec)

-0.0005

Table 5: Thermogravimetric data of the nalidixate complexes Complex

Steps

DTG peak/ (°C)

DTA peak/ (°C)

Assignments

Ca(II)

1st 2nd residue 1st 2nd residue 1st 2nd residue 1st 2nd residue

200 450

250 400, 505

70, 215 340, 435

150 350, 435

60 220, 360, 515

60 410, 515

219 365, 532

196 478, 533

4H2O+½Cl2+C2H4 nix CaO 5H2O+Cl2+C2H4 nix FeO1½ H2O+½Cl2+C2H4 nix PdO C2H4 Cl2+nix Au metal

Fe(III)

Pd(II)

Au(III)

Weight loss Found (Calcd. %) 36.00(35.77) 49.50 (49.20) 14.50(14.80) 41.70(42.18) 39.70(40.00) 18.60(17.82) 21.00(20.84) 47.98(47.86) 31.02(31.30) 5.70(5.61) 54.84(54.93) 39.46(39.46)

3-6- Kinetic thermodynamic parameters The kinetic parameters were calculated dependent on the integral method of Coats & Redfern [58] with the helpful of the following equations:

1  1   1n   AR ln   ln  2   E  (1  n )T    ln 1      AR ln    ln  2 T  E  

 E   RT

 E   RT

for n ≠ 1

(4)

for n = 1

(5)

Where, A, is the pre- exponential factor. The correlation coefficient, r, was computed using the least square method for different values of n, by plotting the left – hand side of equ. (4) or (5) versus 1000/T (Fig. 8). The n value which gave the best fit (r ≈ 1) was chosen as the order parameter for the decomposition stage of interest. From the intercept and linear slop of such stage, A and E values were determined. The other kinetic parameters ∆H, ∆S and ∆G were computed using the relationships; ∆H = E – RT, ∆S = R [ln(Ah/kT)] and ∆G = ∆H - T∆S, where, k is the Boltzmann’s constant and h is the Planck’s constant. The kinetic parameters are listed in Table 6. The following remarks can be pointed out: 1- The value of ∆G increases significantly for the subsequently decomposition stages of a given complex. This is due to increasing the values of T∆S significantly from one stage to another which overrides the values of ∆H. Increasing the values of ∆G of a given complex as going from one decomposition step subsequently to another reflects that the rate of removal of the subsequent ligand will be lower than that of the precedent ligand [59, 60]. This may be attributed to the structural rigidity of the remaining complex after the expulsion of one and more ligands, as compared with the precedent complex, which require more energy, T∆S, for its rearrangement before undergoing any compositional change.

17

2- The negative values of activation entropies ∆S indicate a more ordered activated complex than the reactants and/ or the reactions are slow [61]. The positive values of ∆H mean that the decomposition process is endothermic. -12.0

2

ln(-ln(1-) )

-12.5 -13.0 -13.5 -14.0 -14.5 -15.0 -15.5 -16.0

Ca(II) Fe(III) Pd(II) Au(III) 0.00120 0.00125 0.00130 0.00135 0.00140 0.00145 0.00150 1000/T (K)

Fig. 8: Coats–Redfern curves of nalidixate complexes

18

Table 6: Kinetic parameters using the Coats–Redfern equation for the nalidixate complexes Complexes

DTGmax

Thermodynamic parameters Parameters

Ca(II) complex

250 oC

Fe(III) complex

170 oC

Pd(II) complex

220 oC

Au(III) complex

270 oC

E (kJmol-1) A (s-1) ΔS (Jmol-1 K-1) ΔH (kJmol-1) ΔG (kJmol-1) r E (kJmol-1) A (s-1) ΔS (Jmol-1 K-1) ΔH (kJmol-1) ΔG (kJmol-1) r E (kJmol-1) A (s-1) ΔS (Jmol-1 K-1) ΔH (kJmol-1) ΔG (kJmol-1) r E (kJmol-1) A (s-1) ΔS (Jmol-1 K-1) ΔH (kJmol-1) ΔG (kJmol-1) r

Values 75 6.00*104 -160 69 155 0.9872 71 3.00*108 -85 68 100 0.9919 22 2.80 -240 175 100 0.9840 142 145*108 -95 140 210 0.9991

3-7- X-ray powder diffraction, SEM and TEM studies The X-ray solid powder diffractions of [Pd(nix)(Cl)(H2O)] and [Au(nix)(Cl)2] complexes are shown in Fig. 9. These diffraction patterns are included of the starting materials and have crystalline feature. The crystallization for resulted Pd(II) and Au(III) nalidixate complexes was calculated from the major diffraction patterns of the respective complex using the Deby-Scherrer formula (equation 6) [62]. D=

K  cos 

(6)

Where λ is the wavelength of x-ray (1.5418 Å) for Cu Kα radiation, K is constant taken as 0.94, β full width at half maximum (FWHM) of prominent intensity peak (100% relative intensity peak), θ is a peak position. The calculated grain sizes using Deby-Scherrer formula were found to be 6.00 and 5.00 nm for Pd(II) and Au(III) complexes, respectively. The lower grain size of both Pd(II) and Au(III) complexes ~ 5.5 nm can be discussed according to the increasing of nalidixate chelates around metal ions (ratio 1:1) [63]. The collected data of XRD like 2, intensities and dspacing are listed in Table 7. The crystallite sizes (D) were calculated using the

19

Scherrer formula [62] from the full-width half-maximum (FWHM) (). The strain () was calculated from the slope of  cos versus sin  plot using the relation (equation 7).

6000 1400

Counts

5000

1200

4000

1000 800

Counts

3000

2000

600 400

1000 200 0

0

Pd(II) complex 20

40

60

80

100

2

Au(III) complex 20

40

60

80

100

2

Fig. 9: X-ray solid powder diffraction patterns of [Pd(nix)(Cl)(H2O)] and [Au(nix)(Cl)2] complexes

β=

 D cos 

  tan

(7)

Since the dislocation density and strain are the manifestation of dislocation network in the complexes, the decrease in the strain and dislocation density indicates the formation of high quality complexes. The dislocation density () was evaluated as equation (8) [64] and listed in Table 8. =

1 D2

(8)

20

Table 7: The XRD collected data of 2, intensities and d-spacing. [Pd(nix)(Cl)(H2O)] complex 2Theta 4.27 6.28 10.09 10.66 11.2 11.77 12.22 12.55 13.54 14.41 14.92 15.91 16.9 17.83 18.55 19.3 19.66 20.32 21.1 21.55 22.51 24.07 24.91 25.39 25.69 26.59 27.13 27.97

Intensity 186.7316 185.7662 293.4199 1011.139 179.8355 1432.13 390.2338 471.619 336.0563 775.9177 199.5281 399.9913 306.3939 346.4675 1825.217 182.9481 179.0779 180.2554 194.8139 265.7619 545.1082 5103.58 275.1255 226.2078 254.8139 293.8658 3030.974 201.1515

2-theta 4.18 10 10.54 11.62 14.32 16.93 18.43 19.21 20.23 21.25 22.36 22.93 23.86 24.88 26.53 27.01 27.97 28.6 29.14 29.95 31.09 31.72 32.62

intensity 257.9827 187.697 300.5628 352.7792 153.8918 114.9524 143.0216 117.7706 105.3463 98.6797 202.3593 341.2424 367.2078 126.8961 129.6926 187.1948 103.5758 212.8312 120.6926 252.0866 120.1472 343.9091 1309.775

d- Spacing 2Theta Intensity 20.6769 28.81 1184.342 14.0627 30.01 1312.745 8.7596 32.74 257.671 8.2924 33.34 144.5281 7.8938 34.09 401.7186 7.5128 36.04 163.2078 7.2371 38.17 123.7489 7.0475 40.3 126.1385 6.5344 41.02 116.0433 6.1418 43.96 244.1991 5.933 44.56 147.5887 5.5659 45.22 127.8745 5.242 56.74 108.3766 4.9707 60.01 86.5498 4.7793 60.97 81.2208 4.5953 61.54 69.0216 4.5119 64.81 59.9351 4.3668 65.05 62.2165 4.2071 72.82 63.1039 4.1203 76.81 61.9567 3.9467 78.13 61.8312 3.6943 79.21 58.0952 3.5716 84.19 54.5844 3.5052 84.73 54.2597 3.4649 85.36 52.3896 3.3496 85.72 54.9394 3.2842 86.32 55.1169 3.1874 88.36 53.987 [Au(nix)(Cl)2] complex d-spacing 2-theta intensity 21.1219 44.35 139.2424 8.8382 45.43 202.8701 8.3866 45.82 76.013 7.6094 46.78 229.8398 6.1802 48.67 61.0823 5.2328 52.72 137.9394 4.8102 55.12 60.0649 4.6166 57.28 73.4632 4.3861 58.18 185.0043 4.1778 61.33 58.8961 3.9728 61.63 58.1082 3.8753 64.48 95.6017 3.7264 64.69 100.6623 3.5758 65.29 58.0476 3.3571 67.09 50.8139 3.2985 67.66 53.5844 3.1874 68.2 77.5455 3.1186 68.47 64.0649 3.0621 71.35 41.0303 2.9811 71.71 38.2294 2.8743 72.1 43.8095 2.8186 73.18 64.8528 2.7429 74.11 47.6753

21

d- Spacing 3.0964 2.9752 2.7331 2.6853 2.6279 2.4901 2.3559 2.2361 2.1985 2.0581 2.0317 2.0036 1.6211 1.5404 1.5184 1.5057 1.4374 1.4327 1.2978 1.24 1.2223 1.2083 1.1491 1.1431 1.1363 1.1324 1.1261 1.1053 d-spacing 2.0409 1.9948 1.9788 1.9404 1.8693 1.7349 1.6649 1.6071 1.5844 1.5103 1.5037 1.4439 1.4398 1.428 1.394 1.3836 1.374 1.3692 1.3208 1.3151 1.3089 1.2923 1.2783

33.1 33.4 33.67 33.88 34.24 34.63 35.29 35.59 35.98 38.2 38.68 40.21

136.3593 126.1212 118.619 131.7965 131.4329 123.8615 125.9394 118.0303 114.0779 303.7576 93.1255 107.8182

2.7042 2.6806 2.6597 2.6437 2.6167 2.5882 2.5412 2.5205 2.4941 2.3541 2.326 2.2409

74.47 75.22 76.84 77.77 79.96 81.79 82.33 83.59 83.89 85.96 86.83 87.16

41.1732 51.2035 43.2251 105.6537 43.7403 50.1342 48.039 42.8312 46.1688 38.671 42 44.4416

1.273 1.2622 1.2396 1.2271 1.1989 1.1766 1.1703 1.1558 1.1524 1.1299 1.1208 1.1174

Table 8: Data of crystallite sizes (D), dislocation density (), and strain () Complexes [Pd(nix)(Cl)(H2O)] [Au(nix)(Cl)2]

(1012.lin.m-2) 0.032 0.038

D(nm) 5.57 5.13

(10-4) 0.800 0.066

The SEM images of the solid [Pd(nix)(Cl)(H2O)] and [Au(nix)(Cl)2] complexes are shown in Fig. 10A&B. These figures give an impression about the images of NPs grown by complexation process. It is obviously from the SEM images that the synthesized products are NPs, which grown as uniform shape. These figures exhibit the high resolution images with regular segments and solid touch shapes for Pd(II) and Au(III) complexes, respectively. The diameter size ranged 20-100 micrometer.

Fig. 10A: SEM image of [Pd(nix)(Cl)(H2O)] complex

22

Fig. 10B: SEM image of [Au(nix)(Cl)2] complex The TEM morphology of solid [Au(nix)(Cl)2] complex after grinding well was presented in the Fig. 11. The TEM analysis technique confirmed the presence of the nanometric gold(III) ions inclusion in nalidixate molecule. TEM photograph showed spherical NPs of iron oxide which appeared as dark spots. The diameter of Au(III) complex is in the range of ~ 6.0 nm, these data matched with the XRD data.

Fig. 11: TEM image of [Au(nix)(Cl)2] complex 3-8- Molecular modeling studies The quantum chemical calculations and molecular modeling studies for the nixH and ([Ca(nix)(Cl)(H2O)3].H2O, [Fe(nix)(Cl)2(H2O)2].3H2O and

23

[Pd(nix)(Cl)(H2O)]) complexes are useful to support the suggestion structures [65, 66], which depending upon the elemental analyses, spectroscopic and thermal characterizations. The geometry optimization and conformational analysis has been performed (Fig. 12) using of semi-empirical PM3 level [65, 66] as implemented in software of hyperchem 7.5 program [67]. HOMO

LUMO

Fig. 12a: HOMO and LUMO structure of nixH free ligand.

HOMO

LUMO

Fig. 12b: HOMO and LUMO structure of [Ca(nix)(Cl)(H2O)3].H2O complex.

HOMO

LUMO

Fig. 12c: HOMO and LUMO structure of [Fe(nix)(Cl)2(H2O)2].3H2O complex.

24

HOMO

LUMO

Fig. 12d: HOMO and LUMO structure of [Pd(nix)(Cl)(H2O)] complex. The quantum chemical parameters; the highest occupied molecular orbital energy (EHOMO), the lowest unoccupied molecular orbital energy (ELUMO), the difference between HOMO and LUMO energy levels (ΔE), Mulliken electronegativity (χ), chemical potential (Pi), global hardness (η), global softness (S), global electrophilicity (ω), absolute softness (σ) and electronic charge (ΔNmax) have been estimated [68-72] and listed in Table 9 using semi-empirical PM3 method as implemented in HyperChem [67]. The mentioned quantum chemical parameters were calculated with the help of the following equations; ΔE = (ELUMO) – (EHOMO)

(9)

χ=

 ( EHOMO  ELUMO ) 2

(10)

η=

( ELUMO  EHOMO ) 2

(11)

1 σ=



(12)

Pi = – χ

S=

(13)

1 2

(14)

2

Pi ω= 2

(15)

25

ΔNmax =

 Pi

(16)



Table 9: The quantum chemical parameters of [Ca(nix)(Cl)(H2O)3].H2O, [Fe(nix)(Cl)2(H2O)2].3H2O complexes Parameters Total energy (a.u) Binding energy (a.u) Heat formation (a.u) Electronic energy (a.u) Dipole moment/ debye EHOMO (eV) ELUMO (eV) ΔE (eV) χ (eV) η (eV) σ (eV) Pi (eV) S (eV) ω (eV) ΔNmax (eV)

nixH -95.81 -3.27 0.639 -483 3.71 -9.280 -6.953 2.327 8.116 1.163 0.8598 -8.116 0.4299 28.317 6.978

Ca(II) complex -151 -6.77 -1.21 -1077 5.55 -8.871 -8.039 0.832 8.455 0.416 2.404 -8.455 1.202 85.927 20.324

nixH and

ligand and their [Pd(nix)(Cl)(H2O)]

Fe(III) complex -115 -3.43 0.64 -614 6.17 -9.349 -6.791 2.558 8.07 1.279 0.782 -8.07 0.391 25.46 6.3096

Pd(II) complex -134 -3.62 0.43 -665 5.81 -9.141 -6.182 2.959 7.6615 1.4795 0.6759 -7.6615 0.3379 19.84 5.18

 The high value of energy gap (ΔE) led to hard molecules as well as low

      

reactivity, but, the low energy gap data led to soft molecules, which is more reactivity and high flexibility transfer of electrons from donor (ligand) to acceptors (metal ions). The lower values of ELUMO give the highly acceptability for electrons [66], as well as the highly values of EHOMO which easily to electrons to liberated. The high values of EHOMO were confirmed that the nixH ligand has a powerful donation behavior. The difference between energy gap for ligand rather that Ca(II), Fe(III), and Pd(II) complexes reflect to the presence of complexation status. The global electrophilicity (ω) of ligand and complexes arranged as follows; Ca(II) complex (85.927 eV)> Fe(III) complex (25.46 eV)> Pd(II) complex (19.84 eV). The increasing in the global electrophilicity value attributed to highest capacity of accepted electrons. The calculation of both global hardness (η) and absolute softness (σ) parameters are useful to recognize the molecular stability and reactivity. The negative data of both ELUMO and EHOMO were assigned to the stability of synthetic complexes.

3-9- Antimicrobial assessments In vitro antibacterial and antifungal assessments of the free nixH drug ligand and their Ca(II), Fe(III), Pd(II) and Au(III) complexes were tested against Gram (+)

26

bacteria as Staphylococcus Aureus, Bacillus subtilis; Gram (-) bacteria as Escherichia Coli, Pseudomonas aeruginosa and Fungi as Candida Albicans and Aspergillus flavus. Standard discs of tetracycline (antibacterial agent) and amphotericin B as antifungal agents were utilized in this study. The results of antibacterial and antifungal activities of the compounds are listed (Table 10). The complexes able to attach sufficient portion of the bacterial ribosome, and in doing so, cause a misreading of messenger RNA. Thus faulty proteins are synthesized and are incapable of sustaining vital cell functions [73], then, in a short time the cell is killed [74]. The gold(III) complex show higher activity than standards (tetracycline and amphotericin B). In comparison between nixH free ligand and their complexes indicates that the gold(III) complex exhibit higher antibacterial and antifungal activities than the free ligand and other complexes. The higher activity of gold(III) complex can be discussed upon the increasing of lipophilic character of gold(III) ion than the free ligand and other complexes. The breakthrough of complexes via lipid layer of the cell membranes deactivates diverse cellular enzymes, which play essential role in various metabolic systems of these organisms [73]. Such increased activity of complexes can also be explained on the basis of overtone’s concept of cell permeability [75], according to which the lipid membrane that surrounds the cell favors the passage of only the lipid soluble materials due to which liposolubility is an important factor, which controls the antibacterial activity. The higher activity of gold(III) complex can also be explained on the basis of Tweedy’s chelation theory [75], according to which chelation increases the activity. On chelation, the polarity of gold(III) ion will be reduced to a greater extend due to the overlap of the ligand orbital and partial sharing of the positive charge of the gold(III) ion with donor groups. Further, it increases the delocalization of π-electrons over the whole chelate ring and enhances the lipophilicity of the complex. The increased lipophilicity enhances the penetration of the complexes into lipid membranes and blocking of the metal biding sites in the enzymes of microorganisms. The complex also disturbs the respiration process of the cell and thus blocks the synthesis of the proteins that restricts further growth of the organism. In vitro cytotoxicity assessment of the gold(III) complex was performed on human colon carcinoma (HCT-116) cell line and human hepatocellular carcinoma (HepG-2) cell line in the presence of doxorubicin standard drug. The results evaluated upon the determination of inhibitory concentration of 50 % (IC50), the data was listed in Table 11 and introduced in Fig. 13. In comparison between data of gold(III) complex and doxorubicin standard, the gold(III) complex has IC50 equal 0.45 and 0.77 μg for HepG-2 and HCT-116 cell line, respectively. From these data we can deduced that gold(III) complex is more effective for HepG-2 cell line rather than HCT-116 cell line, so, gold(III) complex can be used as anti-tumor drug.

27

Table 10: The inhibition zone diameter (mm/mg sample) of nixH and their complexes against some kind of bacteria and fungi

Inhibition zone diameter (mm / mg sample)

Sample

Standard

Control: DMSO Tetracycline Antibacterial agent Amphotericin B Antifungal agent nixH ligand Ca(II) complex Fe(III) complex Pd(II) complex Au(III) complex

Bacillus subtilis (G+)

Escherichia coli (G-)

Pseudomonas Staphylococcus aeruginosa aureus (G-) (G+)

Aspergillus flavus (Fungus)

0.0

0.0

0.0

0.0

0.0

0.0

34

32

34

30

--

--

--

--

--

--

18

19

28 31 30 39 52

22 24 21 18 34

31 32 26 21 44

12 11 14 14 32

10 9 8 11 19

9 14 12 10 22

Table 11: The inhibitory activities against colon carcinoma and hepatocellular carcinoma cells for the gold(III) complex and doxorubicin drug Viability Sample conc.( μg) 50 25 12.5 6.25 3.125 1.56 0.78 0.39 0 IC50

HepG-2 cell line doxorubicin Au(III)complex 4.91 3.40 8.87 7.24 14.83 10.43 16.16 13.89 25.28 21.83 34.64 30.51 45.79 39.42 51.08 51.94 100 100.00 0.467 µg 0.45 µg

28

HCT-116 cell line doxorubicin Au(III) complex 6.82 4.62 8.89 9.31 14.83 14.52 16.16 18.95 22.28 24.17 34.64 38.29 45.78 49.76 51.28 62.34 100 100.00 0.471 µg 0.77 µg

Candida albicans (Fungus)

100

Viability (%)

80

Doxrubcin HepG-2 cell line Au(III) complex HepG-2 cell line Doxrubcin HCT-116 cell line Au(III) HCT-116 cell line

60

40

20

0 0

10

20

30

40

50

Sample conc.( g)

Fig. 13: Curves of the inhibitory activities against colon carcinoma and hepatocellular carcinoma cells for the gold(III) complex and doxorubicin drug. Acknowledgment This work was funded by Deanship of Scientific Research at university of Princess Nora Bint Abdul Rahman.

29

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Graphical abstract

TEM image of [Au(nix)(Cl)2] complex

33

Highlights     

Some divalent and trivalent complexes of nalidixic acid were discussed. Structures of complexes were fully spectroscopic characterizations. Nalidixic acid reacts as a bidentate ligand. The gold(III) complex has nano-scale range. Anticancer activity was checked.

34