Preparation, characterization and cytotoxicity studies of some transition metal complexes with ofloxacin and 1,10-phenanthroline mixed ligand

Accepted Manuscript Preparation, characterization and cytotoxicity studies of some transition metal complexes with ofloxacin and 1,10-phenanthroline mixed ligand S.A. Sadeek, S.M. Abd El-Hamid PII:

S0022-2860(16)30570-1

DOI:

10.1016/j.molstruc.2016.05.101

Reference:

MOLSTR 22615

To appear in:

Journal of Molecular Structure

Received Date: 24 February 2016 Revised Date:

27 May 2016

Accepted Date: 30 May 2016

Please cite this article as: S.A. Sadeek, S.M.A. El-Hamid, Preparation, characterization and cytotoxicity studies of some transition metal complexes with ofloxacin and 1,10-phenanthroline mixed ligand, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.05.101. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

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Preparation, characterization and cytotoxicity studies of some transition metal complexes with ofloxacin and 1,10-phenanthroline mixed ligand

Abstract [Zn(Ofl)(Phen)(H2O)2](CH3COO).2H2O

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S.A. Sadeeka,*, S.M. Abd El-Hamida a Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt

(1),

[ZrO(Ofl)(Phen)(H2O)]NO3.2H2O (2) and [UO2(Ofl)(Phen)(H2O)](CH3COO).H2O (3) complexes of fluoroquinolone antibacterial agent ofloxacin (HOfl), containing

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a nitrogen donor heterocyclic ligand, 1,10-phenathroline monohydrate (Phen), were prepared and their structures were established with the help of elemental

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analysis, molar conductance, magnetic properties, thermal studies and different spectroscopic studies like IR, UV, 1H NMR and Mass. The IR data of the HOfl and Phen ligands suggested the existing of a bidentate binding involving carboxylate O and pyridone O for HOfl ligand and two pyridine N atoms for Phen ligand. The coordination geometries and electronic structures are determined from electronic

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absorption spectra and magnetic moment measurements. From molar conductance studies reveals that metal complexes are electrolytes and of 1:1 type. The calculated bond length and force constant, F(U=O), in the uranyl complex are

investigated

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1.751 Å and 641.04 Nm-1. The thermal properties of the complexes were by

thermogravimetry

(TGA)

technique.

The

activation

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thermodynamic parameters are calculated using Coats– Redfern and HorowitzMetzger methods. Antimicrobial activity of the compounds was evaluated against some bacteria and fungi species. The activity data show that most metal complexes have antibacterial activity than that of the parent HOfl drug. The in vitro cytotoxicities of ligands and their complexes were also evaluated against human breast and colon carcinoma cells. Keywords: Fuoroquinolone; 1,10-phenanthroline; IR; 1H NMR; Thermal; Cytotoxicity ∗ Corresponding author. Tel.: +20 01220057510; fax: +20 0553208213. E-mail addresses: [email protected]. 1

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1. Introduction Fluoroquinolones are a large and constantly expanding group of synthetic antimicrobial agents [1,2]. This class of compounds, when compared to existing

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bactericidal drugs, shows improved pharmacokinetic properties and a broad spectrum of activity against parasites, bacteria and mycobacteria, including resistant strains; in addition to that they displayed significant in vitro antibacterial activity against many bacteria species through inhibition of their DNA gyrase [3].

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Ofloxacin (HOfl) (Scheme 1 A) is one of the most popular members of secondgeneration quinolone, which characterized by good to excellent activity against

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Gram-positive and Gram-negative bacteria. It is indicative for many infections such as those of the sinuses, lungs, ears, skin, bones and many others caused by susceptible bacteria, urinary infections and prostatitis [4,5]. O

O

F

N

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OH

N

N

O

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(A)

H

.H2O N

N

(B)

Scheme 1. (A) (RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-3,7-

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dihydro-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (HOfl), (B) 1,10phenanthroline monohydrate (Phen).

Due to their wide use, there has been an increasing menace of bacterial

resistance to quinolones [6], which led to the need to improve existing antimicrobial drugs and/or develop new ones, pushing forwards the concept that metal complexes could be an alternative to conventional drugs, as novel derivatives of fluoroquinolones [7,8]. Therefore, numerous studies regarding the 2

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interaction between quinolones with several metallic cations have reported in the literature [9-11]. Recently, a relatively new approach to the rational design of antitumor agents has been introduced based on some new fluoroquinolones

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molecules that display a novel mode of action [12,13]. Metal complexes containing phen and related ligands have been intensively investigated because of their numerous biological activities such as antitumor, antibacterial and antimicrobial [14-16]. Little articles have been reported on the coordination properties of mixed

and

the

known

mixed

ligand

metal

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ligand metal complexes of ofloxacin with the hetero ligand 1,10-phenanthroline complex

has

the

formula

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[Cu(oflo)(phen)(H2O)](NO3).2H2O [17]. The complex shows stronger suppression effects against liver cancer BEL-7402 cell line and lung cancer A549 cell line, with suppression ratios reaching approximately 100% for complex concentrations of 10-4-10-5 mol L-1 also, the complex shows stronger antibacterial activities against S. enteriditis, E. coli, S. aureus, P. aeruginosa, and B. subtilis [17]. in

this

article,

synthesis,

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Therefore,

spectroscopic

and

thermal

characterization of (1), (2) and (3) complexes of ofloxacin in presence of 1,10phenanthroline

monohydrate

(Phen,

Scheme

1

B)

are

reported

using

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physicochemical techniques such as elemental analysis, molar conductance, magnetic susceptibility, thermal analysis, IR, UV–Vis., 1H NMR and mass spectral

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studies. The antimicrobial activity of HOfl, Phen and their metal complexes has been screened against some Gram-positive and Gram negative bacteria. Antifungal activity against three different fungi has been evaluated. Furthermore, the antitumor activities were investigated in vitro against human breast carcinoma cell line (MCF-7) and human colon carcinoma cell line (HCT-116).

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2. Materials and methods 2.1. Materials All chemicals used for the preparation of the complexes were of analytical

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reagent grade, commercially available from different sources and used without further purification. Ofloxacin used in this study was purchased from Egyptian Company for Chemicals & Pharmaceuticals (ADWIA). 1,10-phenanthroline, NaOH, acetone, ethanol, FeCl3.6H2O, FeSO4, Zn(CH3COO)2.2H2O, ZrO(NO3)2,

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and UO2(CH3COO)2.2H2O were commercial products(from Fluka and Aldrich Chemical Co.) and were used without further purification.

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2.2. Synthesis of mixed ligand metal complexes

The white solid complex (1) was prepared by mixing 1 mmol (0.361 g) of hot saturated ethanolic solution of HOfl with 1mmol (0.04 g) NaOH and1mmol (0.198 g) of Phen with the same ratio 1 mmol (0.219 g) of zinc(II) acetate dihydrate. The mixture was refluxed for 3 h. The white precipitate was filtered off and dried under

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vacuum over anhydrous CaCl2. The orange and dark yellow solid complexes (2) and (3) were prepared in a similar manner described above by using acetone as a solvent and ZrO(NO3)2 and UO2(CH3COO)2.2H2O, respectively, in 1:1:1:1 molar

ratio.

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(Mn+:HOfl:NaOH:Phen)

Single

crystal

suitable

for

X-ray

crystallographic measurements was not obtained.

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Mn++HOfl+NaOH+Phen

[M(Ofl)(Phen)]n++H2O+Na salt

Mn+=Zn(II), Zr(IV) and U(VI)

The chemical Structures of synthesized metal complexes were confirmed as follows:

Complex (1): White; Yield 81.22%; m.p.: 302οC; Elemental analysis: found, C 52.11, H 5.18, N 9.47, M 8.85. Calc. for ZnC32H38FN5O10 (737.1) C 52.14%, H 5.20%, N 9.50%, M 8.87%; Λm=76.6 S cm2 mol-1; µ eff: diam.; IR (KBr): ν=3420mbr (O-H, H2O, COOH), 1622s (asymmetric COO-), 1581s (C=O, 4

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pyridone group), 1525ms (C=N), 1370w (symmetric COO-), 641m, 544vw, 504w (M-O) and (M-N). UV-Vis. (DMSO- d6): λ=(274 nm) (36,496 cm-1) (π-π* transition), (310 nm) (32,258 cm-1) (n-π* transition), λ=(530nm) (18,867 cm-1)

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(ε=150 M-1*cm-1) Ligand-metal charge transfer. 1H NMR (DMSO- d6):d = 1.15 (2) (d, 3H, -CH3), 2.24 (10) (s, 3H, -CH3), 2.44-2.51 (7,9) (t, 2H, -CH2), 3.33 (3) (m, 1H, -CH), 3.77 (6,8) (t, 2H, -CH2), 4.37 (4) (d, 2H, -CH2), 4.51-4.84 (s, 2H, H2O), 7.56-8.00 (1,5) (s, 2H, HAr), 8.16-8.91 (1//-8//) (m, 8H, Hpy). (Scheme 2 (A)).

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Complex (2): Orange; Yield 92.6%; m.p.: 310 οC; Elemental analysis: found, C 47.13, H 4.32, N 11.02, M 11.92. Calc. for ZrC30H33FN6O11 (763.8) C 47.17%, H

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4.35%, N 11.00%, M 11.94%; Λm=73.2 S cm2 mol-1; µ eff: diam.; IR (KBr): ν=3430mbr (O-H, H2O, COOH), 1623s (asymmetric COO-), 1576m (C=O, pyridone group), 1527ms (C=N), 1381vs (symmetric COO-), 810m (Zr=O), 688vw, 631w, 544vw, 503w (M-O) and (M-N).UV-Vis. (DMSO- d6): λ=(275 nm) (36,363 cm-1) (π-π* transition), (352 nm) (28,409 cm-1) (n-π* transition), λ=(541

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nm) (18,484 cm-1) (ε=76 M-1*cm-1) Ligand-metal charge transfer. 1H NMR (DMSO- d6):d = 1.16 (2) (d, 3H, -CH3), 2.30 (10) (s, 3H, -CH3), 2.41-2.52 (7,9) (t, 2H, -CH2), 3.40 (3) (m, 1H, -CH), 3.64 (6,8) (t, 2H, -CH2), 4.38 (4) (d, 2H, -CH2), (Scheme 2 (B)).

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4.57-5.02 (s, 2H, H2O), 7.02-8.00 (1,5) (s, 2H, HAr), 8.49-8.96 (1//-8//) (m, 8H, Hpy).

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Complex (3): Dark yellow; Yield 84.88%; m.p.: 274οC; Elemental analysis: found, C 42.41, H 3.75, N 7.70, M 26.25. Calc. for UC32H34FN5O10 (905.7) C 42.44%, H 3.78%, N 7.73%, M 26.28%; Λm=82.4 S cm2 mol-1; µ eff: diam.; IR (KBr): ν=3424mbr (O-H, H2O, COOH), 1620m (asymmetric COO-),1562m (C=O, pyridone group), 1524m (C=N), 1369w (symmetric COO-), 916vs (asymmetric U=O), 814m (symmetric U=O), 674s, 632vw,562vw, 504m (M-O) and (M-N). UV-Vis. (DMSO- d6): λ=(276 nm) (36,231 cm-1) (π-π* transition), (304 nm) (32,894 cm-1) (n-π* transition), λ=(535 nm) (18,691 cm-1) (ε=200 M-1*cm-1) 5

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Ligand-metal charge transfer. 1H NMR (DMSO- d6):d = 1.15 (2) (d, 3H, -CH3), 2.28 (10) (s, 3H, -CH3), 2.50-2.52 (7,9) (t, 2H, -CH2), 3.39 (3) (m, 1H, -CH),3.70 (6,8) (t, 2H, -CH2), 4.35 (4) (d, 2H, -CH2), 4.50-5.09 (s, 2H, H2O), 7.77-8.01 (1,5)

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(s, 2H, HAr), 8.19-8.52 (1//-8//) (m, 8H, Hpy). (Scheme 2 (C)). 2.3. Instruments

The elemental analyses were performed using a Perkin Elmer 2400CHN elemental analyzer. The percentage of the metal ions were determined

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gravimetrically by transforming the solid products into metal oxide and also determined by using atomic absorption method. Spectrometer model PYE-

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UNICAM SP 1900 fitted with the corresponding lamp was used for this purposed. FT-IR spectra in KBr discs were recorded in the range from 4000-400 cm-1 with FTIR 460 PLUS Spectrophotometer. 1H NMR spectra were recorded on Varian Mercury VX-300 NMR Spectrometer using DMSO-d6 as solvent. TGA-DTG measurements were run under N2 atmosphere within the temperature range from

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room temperature to 1000 οC using TGA-50H Shimadzu, the mass of sample was accurately weighted out in an aluminum crucible. Electronic spectra were obtained using UV-3101PC Shimadzu. The absorption spectra were recorded as solutions in

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DMSO-d6. Mass spectra were recorded on GCMS-QP-1000EX Shimadzu (ESI70ev) in the range from 0-1090. Room temperature Magnetic susceptibilities of the

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powdered samples were done on a Sherwood scientific magnetic balance using Gouy balance at room temperature using Hg[Co(CSN)4] as calibrant. The molar conductance of 1×10-3 M solutions of the ligands and metal complexes in DMF was measured at room temperature using CONSORT K410. All measurements were carried out at ambient temperature with freshly prepared solutions. Melting points were recorded on a Buchi apparatus.

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2.4. Antimicrobial Investigation Antibacterial activity of the ligands and their metal complexes was investigated by a previously reported modified method of Beecher and Wong [18]

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against different bacterial species, such as Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and antifungal screening was studied against three species, Candida albicans (C. Albicans), Aspergillus awamori (A. awamori) and Alternaria

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species. The tested microorganisms isolates were isolated from Egyptian soil and water then identified according to the standard mycological and bacteriological

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keys for identification of fungi and bacteria as stock cultures in the microbiology laboratory, water treatment station. The nutrient agar medium for antibacterial was (0.5% Peptone, 0.1% Beef extract, 0.2% Yeast extract, 0.5% NaCl and 1.5% AgarAgar) and Czapeks Dox medium for antifungal (3% Sucrose, 0.3% NaNO3, 0.1% K2HPO4, 0.05% KCl, 0.001% FeSO4, 2% Agar-Agar) was prepared [19] and then

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cooled to 47 οC and seeded with tested microorganisms. Sterile water agar layer was poured then solidified, the prepared growth medium for fungi and bacteria (plate of 12 cm diameter, 15 ml medium plate). After solidification 5 mm diameter

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holes were punched by a sterile cork-borer. The investigated compounds, i.e., ligands and their complexes, were introduced in petri-dishes (only 0.1 ml) after

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dissolving in DMSO at 1.0×10-3 M. These culture plates were then incubated at 37 οC for 20h for bacteria and for seven days at 30 οC for fungi. The activity was determined by measuring the diameter of the inhibition zone (in mm). Microbial growth inhibition was calculated with reference to the positive control, i.e., HOfl. The activity index for the complex was calculated by the formula below [20]: Zone of inhibition by test compound (diameter) Activity Index= ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Zone of inhibition by standard (diameter) 7

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2.5. Cytotoxic activity Mammalian cell lines: The cell lines that used in this study were human breast carcinoma cell line (MCF-7 cells) and human colon carcinoma cell line

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(HCT-116 cells) were obtained from American Type Tissue Culture Unit.

The mammalian cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI-1640 depending on the type of cell line supplemented

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with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50µg/ml gentamycin. All cells were maintained at 37 ºC in a humidified atmosphere with 5% CO2 and were subcultured two times a week along

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experimentation.

2.5.1. Anticancer activity evaluation using viability assay The extracts or pure compounds were tested against two cancer cell lines i.e., breast carcinoma cell line (MCF-7) and colon carcinoma cell line (HCT-116). All the experiments concerning the cytotoxicity evaluation were performed and

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analyzed by tissue culture unit at the Regional Center for Mycology and Biotechnology RCMB, Al-Azhar University, Cairo, Egypt. Anticancer viability

[21]. Procedure

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assay was carried according to the method described by Saintigny and MonnatJr

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The cancer cell lines were seeded in 96-well plate in 100 µl of growth

medium at a cell concentration of 1×104 cells per well. After 24h of seeding, 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. Different two-fold dilutions of the tested compound (100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 µg/ml) were added to confluent cell monolayers dispensed into 96-well, flat8

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bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. The microtiter plates were incubated at 37 ºC in a humidified incubator with 5% CO2 for a period of 24h. Untreated cells were served as controls. Three independent

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experiments were performed each containing six replicates for each concentration of the tested samples. The cytotoxic effects of the tested compounds were then measured using crystal violet staining viability assay. Briefly, after 24h of treatment, the medium was removed, 100 µl of 0.5% of crystal violet in 50%

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methanol was added to each well and incubated for 20 minutes at room temperature and subsequently excess dye was washed out gently by distilled water.

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The plate was allowed to dry then the viable crystal violet-stained cells were lysed using 33% glacial acetic acid solution [22,23]. Absorbance at 570 nm was then measured in each well using microplate reader (SunRise, TECAN, Inc, USA). Doxorubicin was used as positive control. The absorbance is proportional to the number of surviving cells in the culture plate. Thus, using this colorimetric

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procedure, the tested compounds-mediated cell lysis and the cytotoxic effect of doxorubicin (used as a positive control) were measured and compared to the viability of untreated cells [24]. Because the stock solutions to prepare the different

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concentrations from the tested compounds were solubilized in DMSO, controls with DMSO alone were performed in parallel for each concentration.

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Calculation

The percentage cell viability was calculated using the Microsoft Excel.

Percentage cell viability was calculated as follows: according to the following calculation: the percentage of cell viability = [1 − (ODt/ODc)] × 100%, where ODt is the mean optical density of wells treated with the tested compound and ODc is the mean optical density of untreated cells. The test compounds were compared using the IC50 value, i.e., the concentration of an individual compound leading to

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50% cell death that was estimated from graphical plots of surviving cells versus compound concentrations. 3. Results and discussion

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The three new complexes (1), (2) and (3) formed from the reaction of HOfl with Zn(II), Zr(IV) and U(VI) in presence of NaOH and Phen in ethanol and acetone as a solvents. The results of elemental analyses with molecular formulas of the chelates are in good agreement with that calculated for the suggested formulas.

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This study was used many techniques of analysis to prove the structure of the synthesized complexes. According to IR, UV–Vis.,

1

H NMR, mass and

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thermogravimetric data, the complexes of the bidentate ligands had one or two coordinated water molecules. The molar conductance values of the complexes were found to be in the range from 73.2-82.4 S cm2 mol-1 at room temperature, which indicates its ionic nature and it’s 1:1 electrolyte [25,26]. Qualitative reactions revealed the presence of nitrate and acetate ions as counter ions. The

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magnetic moments (as B.M.) of the complexes were measured at room temperature. The complexes are found in diamagnetic character with molecular geometries octahedral. The thermodynamic parameters of decomposition processes

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of complexes, namely, activation energy (∆Ea*), enthalpy (∆H*), entropy (∆S*) and Gibbs free energy, (∆G*), were evaluated by employing Coats-Redfern and

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Horowitz-Metzeger equations. Microbiological studies were carried out to compare the antibacterial and antifungal efficacy of the synthesized mixed ligand metal complexes with that of HOfl free ligand against Gram-negative and Gram-positive bacteria in addition to three species of fungi. The complexes as well as the free ligands were tested for their in vitro cytotoxicity on human breast carcinoma cell line (MCF-7) and human colon carcinoma cell line (HCT-116).

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3.1. IR absorption spectra The coordination mode of HOfl and Phen ligand may be discussed on the basis of the comparison of the IR spectra of the free ligands and are presented in

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Fig. S1. The IR spectrum of HOfl ligand shows the ν(C=O) stretching vibration at 1716 cm-1 as sharp intense band. This band is disappeared in the complexes indicating the participation of carboxylic oxygen atom in coordination (M–O) [27]. Two characteristic bands are present in the 1620–1623 cm-1 and 1369-1381

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cm-1 range assigned to ν(O–C–O) asymmetric and symmetric stretching vibrations, respectively, while the pyridone stretch ν(C=O)is shifted from 1620 cm-1 to 1562cm-1

range

upon

coordination.

The

values

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1581

of

band

shift

∆ν=νas(COO-)-νs(COO-) are all higher than 200 cm-1, indicating that the carboxylate group in HOfl chelated in a monodentate manner to the metal ions with proton displacement [28]. The peak of ring vibration from free Phen was at 1586 cm-1 and the peak of phen in the complexes was in the range of

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1524-1527 cm-1 indicating that the phen was coordinated with metal ions [29]. The medium band in HOfl at 3426 cm-1 due to ν(O-H); completely vanished in the spectra of the metal complexes indicating deprotonation of the carboxylic

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proton [30] and coordination to all metal ions, assisting the formation of a bond between the metal ions and the carboxylate oxygen [31]. Finally, the bands in the

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range 3420-3430 cm-1 in the IR spectra of the complexes can be attributed to the ν(O–H) vibration of the water ligand [32]. New multiple bands in region of 641– 503 cm-1 seemed to be in the spectra of complexes which are assigned to ν(M–O) and ν(M–N) [33].

It is concluded that HOfl behaves as a bidentate ligand with OO donor coordination sites and coordinated to the metal ions via pyridone oxygen and deprotonated carboxylic oxygen. Phen behaves as a bidentate ligand with NN donor sites and coordinated to the metal ion via pyridyl nitrogen. 11

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The proposed structures for complexes are represented in Scheme 2. The data show that ν(Zr=O) is a medium band at 810 cm-1. The data show that the νas(U=O) occurs at 916 cm-1 as very strong singlet and νs(U=O) is observed as

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medium at 814 cm-1. These assignments for uranyl agree with those for many dioxouranium(VІ) complexes [34-36]. The νs(U=O) value was used as according to the known method [37,38], to calculate both the U=O bond stretching force

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values are 1.751 Å and 641.04 Nm-1, respectively.

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constant, F(U=O), and bond length. The calculated bond length and force constant

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+

+

10

10

N 6

N

8

F

H2O

4 3

1"

O

H

O

1

3" 4"

3

Zn 5"

N

H

O

2

O

2"

3"

N

4"

Zr

H2O

5"

N 6"

8" 7"

(B) +

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(A)

N

O

1

7"

10

N

6"

8"

1"

O

4

H2O

O

5

O

N

N

2

2"

F

N

8

5

O

6

9

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9

7

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7

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N

7

6

9

N

EP

8

O

F

5

H 2O

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4

3

N

O

2

O

H 1

1"

O

2" 3"

O N

4"

U O

5"

N 6"

8" 7"

(C) Scheme 2. The coordination mode of Zn(II), Zr(IV) and U(VI) with mixed ligand. 13

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3.2. Electronic absorption spectra The electronic absorption spectra are often very helpful in the evaluation of results furnished by other methods of structural investigation. The electronic

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spectral measurements were used for assigning the stereo-chemistries of metal ions in the complexes based on the position and number of transition peaks [39,40]. The electronic absorption spectra of HOfl, Phen and their metal complexes, are shown in Fig. 1.

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The electronic absorption spectra for HOfl and Phen (Fig. 1) showed two bands at λ1= 297 and 326 nm and λ2= 273 and 350 nm, respectively. The first one

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band at 297 and 273 nm may be attributed to π-π* transitions, respectively and the band observed at 326 and 350 nm is assigned to n-π* transitions, respectively. These transitions are known for unsaturated hydrocarbons that contain pyridone groups [41-43]. The UV–Vis. spectra of the complexes are slightly shifted than those of the HOfl and Phen ligands indicative of coordination of the ligand to

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metal ions.

The electronic spectra of complexes recorded the absorption bands in the range 530-541 nm which refer to ligand-metal charge transfer [44-46]. The molar

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absorptivity (ε) values of the prepared complexes with the metal ions under investigation were determined using 1.0×10-3 M DMSO solution of the synthesized

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complexes, by using the relation: A= εcl, where, A= absorbance, c=1.0×10-3 M, l= length of cell (1 cm) 3.3. 1H NMR spectra

The 300 MHZ 1H NMR spectra of ligands and the synthesized complexes

were measured in DMSO-d6 at room temperature and are shown in Fig. 2. The band present in the range between 4.50-5.09 ppm assigned to water molecules, which participate in coordination mode [47]. The results clearly indicate that the ligand coordinates to metal ions via carboxylic (Fig. 2) [48]. The signals at 6.6814

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7.96 ppm and 7.26-8.81 ppm (m, Ar-H) assigned to aromatic protons observed in HOfl and Phen ligands show shifts in the complexes (7.02-8.96 ppm). This shift can be attributed to variation in electron density upon chelation [49].

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The singlet at 11.0 ppm due to the proton of carboxylic acid group observed in the spectrum of the free HOfl ligand is not observed in the complexes spectra. The disappearance of this proton comes in agreement with the coordination through the carboxylic group of HOfl. This conclusion support the suggestion that

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HOfl deprotonated during the reaction with Zn(II), Zr(IV) and U(VI) comes in consistence with the data previously obtained from the infrared spectrum [50-52].

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Peaks of HOfl free ligand are present in spectra of the complexes, but shifted to lower or higher upon coordination with the metal. 3.4. Mass spectra

The electron impact mass spectra of (1), (2) and (3) complexes showed the molecular ion peaks at m/z 737 (26.67%), 763 (43.04%) and 905 (31.53%) amu,

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respectively, which confirm the stoichiometry as being of [ML1L2] type of these complexes. The observed peaks were in good agreement with their proposed formulae as indicated by the micro-analytical data. The fragmentation patterns of

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our studied complexes were obtained from the mass spectra (Fig. S2). The fragmentation pattern and mass spectrum of complex (2) as

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representative example (Scheme 3). The molecular ion peak [a] appeared at m/z=763 (43.04%) loses NO3 to give [b] at m/z=701 (74.68%) and it loses C5H11N2 to give [c] at m/z=664 (56.33%). The molecular ion peak [a] loses CH5FNO4 to give [d] at m/z=649 (36.08%) and it loses C6H14FN2 to give [e] at m/z=630 (43.04%). The molecular ion peak [a] loses C5H15FN3O5 to give fragment [f] at m/z=547 (41.77%) and it loses C6H18FN3O5 to give fragment [g] at m/z=532 (51.27%).

15

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N N O O O

H

O

O

N

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N

Zr H2O

O

N

O O

H

O

N

Zr

.H2O [d] 649 (36.08%)

H2O

O

N

-C

H

O

14

FN

5

FN

2

H

SC

NO3.H2O [e] 630 (43.04%)

N F N

.

O

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-C

6

4

N N

N

F

F

.

O N

O O

N

-NO3

Zr O

O

H

O

H2O

N

O O

H

O

H 5

-C

EP

-C

N

O

O

H2O

N

3O 5

N

O O

H

O

N

Zr

N

O

H 2O

N

[g] 532 (51.27%)

[f] 547 (41.77%)

Scheme 3. Fragmentation pattern of complex (2).

16

N

NO3.2H2O [c] 664 (56.33%)

Zr

H

H 2O

O 6H 18 F

N

Zr

O

AC C O O

N

-C5H11N2

N

NO3.2H2O [a] m/z 763 (43.04%)

15

FN

3

O

5

.2H2O [b] 701 (74.68%)

O

N

Zr

N

H2O

O

O O

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H

N

O

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3.5. Thermal studies Several reports in the literature demonstrate the importance of thermal analysis by thermogravimetry (TGA) and differential thermogravimetry (DTG) in

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the characterization, polymorphism identification, purity evaluation of drugs, compatibility studies for the pharmaceutical formulation, stability and drugs thermal decomposition [41]. Table 1 and Fig. S3 show TGA and DTG results of thermal decomposition of HOfl, Phen and their metal chelates.

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The thermal decomposition process of HOfl drug involves one decomposition step. Decomposition of HOfl drug started at 30 °C and finished at

8C2H2+HF+NH3+2NO+2CO.

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783 °C and is accompanied by a weight loss of 99.37% involves loss of

The TGA curve of Phen shows two successive steps of decomposition. The first estimated mass loss of 8.02% (calc. 8.07%) at 25–121 οC. In the final stage within the temperature range of 121-298 οC, the estimated mass loss of 91.98%

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(calc. 91.93%), may be attributed to the decomposition of 5C2H2+C2N2 fragment loss with a complete decomposition [53,54]. Thermal decomposition of complex (1) proceeds in three ranged main

EP

stages. The hydrated water molecules are associated with complex formation and found outside the coordination sphere. The dehydration of this type of water takes

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place in the temperature range 33–95οC. The weight loss corresponds to two water molecules (found 4.31%, calc. 4.89%). The water of coordination and Phen molecules were lost in the temperature range 95–320 οC, corresponding to loss of two coordination water and Phen molecules (found 29.64%, calc. 29.34%). The final stage is showing the decomposition of Ofl. The final decomposition products are ZnO with residual two carbon atoms (found 14.56%, calc.14.30%).

17

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The TGA curve of chelate (2) shows three decomposition steps within the temperature range 30–1000 οC. The first step corresponds to the loss of two water molecules at maximum temperature 48 οC with mass loss of 4.52% (calc. 4.72%).

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The second step occurs within temperature range 55-420 °C and shows loss of 5C2H2+C2N2+0.5O2 with mass loss 25.24% (calc. 25.95%). The final stage occurs at maximum temperature 535 °C corresponds to the removal of the organic part of the Ofl leaving metal oxide as a residue with mass loss of 16.46% (calc. 16.13%).

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The diagram of complex (3) reveals mass loss in temperature range 30–564 °C; three stages are shown in TGA curve, the first stage corresponding to

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the dehydration of one water molecule (found 1.66%, calc. 1.99%). The second stage was discussed concerning the loss of one coordinated water and Phen molecules, weight loss (found 21.77%, calc. 21.89%). The following stage is in between 276–564 °C, showing the decomposition of Ofl drug. The final decomposition product is UO2+2C (found 32.69%, calc. 32.46%).

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3.6. The kinetics studies

The kinetic parameters were calculated for the complexes in order to know the effect of the structural properties of the HOfl and Phen ligands and the type of

EP

the metal on the thermal behaviour of the complexes and the heat of activation Ea of the various decomposition stages were determined from TGA thermograms

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using Coats-Redfern [55] and Horowitz-Metzger [56] equations. Coats–Redfern equations

−lnሺ1-αሻଵି୬ -E* AR ln X=ln ቈ 2 +ln ൤ ቉= ൨ for n≠1ሺ1ሻ RT φE* T ሺ1 − ݊ሻ where (n=0, 0.33, 0.5 and 0.66) ln X=ln ቈ

−lnሺ1-αሻ -E* AR = +ln ቉ ൤ ൨ for n=1ሺ2ሻ RT φE* T2

18

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Horowitz-Metzger equations

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−lnሺ1-αሻଵି୬ -E* AR ln X=ln ቈ 2 +ln ൤ ቉= ൨ for n≠1ሺ3ሻ RT φE* T ሺ1 − ݊ሻ where (n=0, 0.33, 0.5 and 0.66) −lnሺ1-αሻ -E* AR ln X=ln ቈ = +ln ቉ ൤ ൨ for n=1ሺ4ሻ RT φE* T2 From the intercept and linear slope of each stage, A values were determined.

relationships. ∆S*=R ln(Ah/kBTs) ∆H*=E*–RT

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The other kinetic parameters, ∆H*, ∆S* and ∆G* were calculated using the

(5)

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(6)

∆G*=∆H*–T∆S*

(7)

Where k is the Boltzmann's constant and h is the Planck's constant. The linearization curves of Coats–Redfern and Horowitz–Metzger methods are shown in Fig. S4. The kinetic parameters for the complexes are listed in Table 2. The

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following remarks can be pointed out. The value of ∆G* increases for the subsequently decomposition stages of a given complex. This is due to increasing the values of T∆S* from one stage to another. Increasing the values of ∆G* of a

EP

given complex as going from one decomposition step to another indicates that the rate of removal of the subsequent ligand will be lower than that of the precedent

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ligand. 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 change. The high values obtained for the activation energies reflect the thermal stability of the complexes. The negative values of activation entropies ∆S* indicate a more ordered activated complex than the reactants and/or the reactions are slow. The positive values of ∆H* mean that the decomposition processes are endothermic [57,58]. The correlation coefficient of the Arrhenius 19

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plot of the thermal decomposition steps lie in the range 0.996-0.999, showing a good fit with linear function. 3.7. Antimicrobial activity

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In testing the antibacterial and antifungal activity of our compounds, more than one test organism was used to increase the chance of detecting antibiotic principles in tested materials. The free HOfl ligand and its metal chelates with Phen were screened against different fungi species and different types of Gram-

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negative (G-) and Gram-positive (G+) bacteria and the data are listed in Table 3. DMSO having no effect on the microorganisms in the concentration studied. The

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activity index of the ligands and the complexes was calculated (Table 3). Comparisons of the biological activity of the synthesized complexes with reference standard (HOfl) shows that complex (1) exhibits very highly significant (inhibition zone) than HOfl, Phen, (2) and (3) compounds against S. aureus. Complex (1) has higher activity index than other compounds against all bacterial species. Complex

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(2) showed a significant result against C. albicans with high activity index. Complex (3) is non significant against E. Coli and B. Subtilis and significant against S. aureus compared with other compounds.

EP

It is clear from Table 3 and Fig. S5 that HOfl, Phen and all the complexes have no fungal activity towards A. awamori and Alternaria species organisms. The

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biological activity against C. albicans shows that the complexes together with HOfl and Phen ligands have antifungal activity. The high sensitivity of the complexes have been attributed to hyper-

conjugation of the coordinated aromatic Lewis bases, which increases the net electron density on the coordinated metal ion and consequently higher antimicrobial activity [59]. In general, metal complexes are more active than the ligand because metal complexes may serve as a vehicle for activation of ligands as the principle cytotoxic species [60]. 20

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3.8. Cytotoxic activity The effect of the free ligands and their metal complexes on the viability of human breast cancer cell line (MCF7) and human colon cancer cell line (HCT-116)

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were assayed by using crystal violet staining viability assay. The values of concentration at which half of the maximal effect is observed (IC50) with the numerical data summarized in Table 4.

From these results, it is notable that the free ligands HOfl and Phen were

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active against breast carcinoma cell line (MCF7) with IC50 values of 19.3 and 4.73 µg/ml, respectively and colon carcinoma cell line (HCT-116) with IC50 values of

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31.8 and 4.96 µg/ml, respectively. Complexes (2) and (3) showed higher antitumor activity than HOfl free ligand against the breast cell line (with IC50 value 7.54 and 5.58 µg/ml, respectively) and the colon cell line (with IC50 value 8.58 and 9.2 µg/ml, respectively) (Fig. 3). Complex (1) was less active than free ligands against the two carcinoma cell lines tested. It has shown that HOfl, Phen, (2) and (3)

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compounds are significantly more active, but complex (1) is less active anti-cancer agents than cisplatin against breast cancer cell line (MCF7). HOfl and complex (1) are less active but, other compounds are more active than cisplatin against colon

EP

cancer cell line (HCT-116). The phen ligand performs better antitumoral activity than all complexes.

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Conclusion

The synthesis and the characterization of three novel metal complexes with

fluoroquinolone antibacterial drug HOfl in presence of a nitrogen-donor ligand Phen, has been synthesized with physicochemical, spectroscopic and thermal methods. The infrared spectra confirmed that HOfl binds to the metal ions via the pyridone oxygen and carboxylate oxygen and Phen binds via the pyridine nitrogen atoms. The results of this investigation support the suggested octahedral structure of the metal complexes and form a favourable molecular arrangement. The thermal 21

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behaviour of the complexes revealed that they decomposed in three steps, they loss water molecules in the first steps followed by the decomposition of Phen and HOfl molecules in the subsequent steps. The anti-microbial activity results show that

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most of the synthesized complexes possess good antibacterial activity against Gram negative and Gram-positive bacteria tested. The complexes were also screened for its in vitro anticancer activity against (MFC7) and (HCT-116) cell lines and the results obtained throw more light on the activity of (2) and (3)

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complexes. Acknowledgments

under project Grants No. 442. References

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This work was supported by grants from Zagazig University, Zagazig, Egypt

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N.N.

Greenwood,

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Earnshaw,

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of

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(2nd Ed.). Butterworth-Heinemann. 1997, 320–321. [55] A.W. Coats, J.P. Redfern, Nature 201 (1964) 68-69. [56] H.H. Horowitz, G. Metzger, Anal. Chem. 35 (1963) 1464-1468. 25

Elements

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[57] W.H. Mahmoud, G.G. Mohamed, M.M.I. El-Dessouky, Spectrochimica Acta Part A 122 (2014) 598-608. [58] S.M. El-Megharbel, R.Z. Hamza, M.S. Refat, Chem. Biol. Interact. 220

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(2014) 169-180. [59] D.H. Petering, H. Sigel, Marcel Dekker, New York, 2 (1973) 167-172.

[60] A.A. Osowole, J.A.O. Woods, O.E. Fagade, O.A. Odunola, Bio. Sci. Res.

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Commun. 14 (2002) 531-535.

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Table 1: The maximum temperature Tmax(οC) and weight loss values of the decomposition stages for (HOfl), (Phen); (1), (2) and (3). Decomposition

Tmax(οC)

(HOfl) (C18H20FN3O4)

First step Total loss Residue First step Second step Total loss Residue First step Second step Third step Total loss Residue First step Second step Third step Total loss Residue First step Second step Third step Total loss Residue

334, 572

(3)

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48 252, 335 535

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(2)

55 274 516

51 258 419, 479

EP

(1)

95 278

AC C

(Phen) (C12H10N2O)

Weight loss (%) Calc. Found 100 99.37 100 99.37 8.07 8.02 91.93 91.98 100 100 4.89 4.31 29.34 29.64 51.47 51.49 85.70 85.44 14.30 14.56 4.72 4.52 25.95 25.24 53.20 53.78 83.87 83.54 16.13 16.46 1.99 1.66 21.89 21.77 43.66 43.88 67.54 67.31 32.46 32.69

Lost species

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Compounds

8C2H2+HF+NH3+2NO+2CO 0.5O2 5C2H2+C2N2 2H2O 6C2H2+2NO 8C2H2+HF+2NO+2CO+NH3+H2O ZnO+2C 2H2O 5C2H2+C2N2+0.5O2 9C2H2+HF+2N2O3

ZrO2 H2O 5C2H2+C2N2+0.5O2 8C2H2+HF+2NO+NH3+CO+CO2+H2O UO2+2C

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Ts (K) 607

method

E* (KJ/ mol) 100.73 107.98 117.83 146.78 50.99 54.88 67.71 72.61 16.29 31.62 135.58 155.67 87.89 97.78 248.26 202.83

A (s−1) 1.57×106 1.15×107 2.03×109 7.97×1011 4.19×105 5.63×106 5.98×103 4.18×104 0.062 3.22 1.93×106 5.54×107 2.60×106 2.89×107 3.63×1012 8.83×1011

EP

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CR HM 394-572 551 CR HM 306-368 328 CR HM 368-655 547 CR HM (2) 328-534 525 CR HM 695-909 808 CR HM (3) 334-549 531 CR HM 714-837 752 CR HM a=correlation coefficients of the Arrhenius plots and b=standard deviation

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HOfl (C18H20FN3O4) Phen (C12H10N2O) (1)

Decomposition Range (K) 477-670

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compounds

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Table 2: Thermal behavior and Kinetic parameters determined using Coats–Redfern (CR) and Horowitz–Metzger (HM) operated for HOfl, Phen and their complexes (1), (2) and (3). parameter ∆S* (KJ/mol.K) -0.1322 -0.1156 -0.0718 -0.0222 -0.1381 -0.1165 -0.1777 -0.1615 -0.2727 -0.2399 -0.1328 -0.10496 -0.1269 -0.1069 -0.0122 -0.0239

∆H* (KJ/mol) 95.68 102.93 113.25 142.20 48.26 52.16 63.16 68.07 11.92 27.26 128.86 148.95 83.47 93.36 242.00 196.57

∆G* (KJ/mol) 175.93 173.12 152.84 154.42 93.55 90.36 160.34 156.40 155.12 153.20 236.20 233.76 150.85 150.11 251.15 214.56

Ra

SDb

0.997 0.997 0.996 0.998 0.998 0.998 0.997 0.996 0.996 0.997 0.998 0.997 0.999 0.999 0.998 0.998

0.064 0.072 0.120 0.076 0.050 0.057 0.073 0.093 0.056 0.066 0.059 0.083 0.026 0.026 0.056 0.065

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Table 3: The inhibition diameter zone values (mm) for (HOfl), (Phen) and their metal complexes (1), (2) and (3). Microbial species Bacteria AI B. subtilis AI S. aureus 30 ±0.31 46 ±0.38

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Tested compounds

Fungi C. albicans 16 ±0.14

28 ±0.10

0.61

35 ±0.27

2.19

1.13

55+3 ±0.68

1.2

20+1 ±0.77

1.25

0.97

50+2 ±0.86

1.09

21+1 ±0.87

1.31

48+1 ±1.07

1.04

17NS ±0.64

1.06

AI -

P. aeruginosa 28 ±0.17

(Phen)

29 ±0.33

1.16

31 ±0.18

1.11

36 ±0.32

1.2

(1)

31+2 ±0.37

1.24

29NS ±0.32

1.04

34NS ±1.25

(2)

30+1 ±0.72

1.2

23 ±0.44

0.82

29 ±0.31

(3)

28NS ±0.77

1.12

25 ±0.41

0.89

33NS ±0.83

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(HOfl)

E. coli 25 ±0.05

1.1

AI -

AI -

EP

AI= Activity index.

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(Paired).

TE D

Zn(CH3COO)2.2H2O 0 0 0 0 0 0 0 0 0 0 ZrO(NO3)2 0 0 0 0 0 0 0 0 0 0 UO2(CH3COO)2.2H2O 0 0 0 0 0 0 0 0 0 0 Control (DMSO) 0 0 0 0 0 0 0 0 0 0 Statistical significance PNS P not significant, P >0.05; P+1 P significant, P<0.05; P+2 P highly significant, P<0.01; P+3 P very highly significant, P <0.001; student’st-test

ACCEPTED MANUSCRIPT Table 4: The in vitro inhibitory activity of tested compounds against tumor cell lines expressed as IC50 values (µg/ml) ± standard deviation from six replicates. Tested compounds

Tumor cell lines S.D. (±)

HCT-116

S.D. (±)

(HOfl)

19.3

0.02

31.8

0.11

(Phen)

4.73

0.26

4.96

0.62

(1)

78.7

0.24

94.5

0.16

(2)

7.54

0.09

8.58

0.12

(3)

5.58

0.14

9.2

0.27

Doxorubicin Standard

0.46

0.1

0.46

0.1

5-flurouracil standard

3.9

0.1

4.3

0.2

Cisplatin

25.2

1.5

25.4

7.4

AC C

EP

TE D

M AN U

SC

RI PT

MCF-7

Fig.

1

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Electronic

absorption

AC C

EP

(C) (1), (D) (2) and (E) (3).

spectra

for

(A)

HOfl;

(B)

Phen,

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig.

2

1

H

NMR

(C) (1), (D) (2) and (E) (3).

spectra

for

(A)

HOfl;

(B)

Phen,

0

0.78

1.56

3.125

0

0.78

1.56

3.125

6.25

12.5

50

100

MCF-7

SC M AN U 12.5

25

50

100

HCT-116

EP

TE D

6.25

AC C

100 90 80 70 60 50 40 30 20 10 0

25

RI PT

100 90 80 70 60 50 40 30 20 10 0

Surviving Fraction %

Surviving Fraction %

ACCEPTED MANUSCRIPT

Fig. 3 The dose response curve showing the in vitro inhibitory activity of the tested compounds against (A) human breast carcinoma (MCF-7) and (B) human colon carcinoma (HCT-116) cell lines.

ACCEPTED MANUSCRIPT

Highlights Preparation, characterization and cytotoxicity studies of some transition

RI PT

metal complexes with ofloxacin and 1,10-phenanthroline mixed ligand • Mixed ligand metal complexes of ofloxacin and 1,10-phenanthroline were prepared.

• The isolated solid complexes were characterized by using elemental

SC

analysis, IR, UV-Vis., 1H NMR, mass spectra, and thermal analysis. • The activation kinetic parameters were calculated. antimicrobial

and

cytotoxicity

activity

M AN U

• The

of

AC C

EP

TE D

1,10-phenanthroline and their metal complexes were evaluated.

ofloxacin,