The pure chemicals and reagents used in this study were purchased from “ Sigma Aldrich Chemical Corporation, St. Louis, Mo, USA” . These chemicals are summarized as: GaCl₃; GeCl₄; SiCl₄ and pipemidic acid. The microanalytical, physical and spectroscopic techniques as well as their models which were used in the interpretations of the solid synthesized complexes can be listed as follows:

Type of analysis Models Elemental analyses Perkin Elmer CHN 2400 Conductance Jenway 4010 conductivity meter FTIR spectra Bruker FTIR Spectrophotometer Electronic spectra UV2 Unicam UV/Vis Spectrophotometer Magnetic susceptibility Magnetic Susceptibility Balance, 1H-NMR spectra Varian Mercury VX-300 NMR spectrometer, 300 MHz XRD X 'Pert PRO PANanalytical, with copper target

The solid pipc complexes [Ga(pipc)₂(H₂O)(Cl)]· 4H₂O(1), Ge(pipc)₂(Cl)₂]· 4H₂O (2) and [Si(pipc)₂(Cl)₂]· 4H₂O (3) with yellow and white colors, respectively were synthesized by the same procedure: A mixtures of 2.0 mmol of GaCl₃, GeCl₄ or SiCl₄ and 4.0 mmol of pipcwere stirred in 25 mL of CH₃OH solvent. The mixtures were neutralized within pH=7~8 using ammonia solution, then refluxed for about three hours. The solutions of these mixtures were reduced by left them for one week. The solid yields of pipc are located within 60%~66% with higher melting point > 300 ℃. The micro analytical analyses of the calculated (Calc. ) and experimental data (Found) of the three synthesized complexes are collected in compacted as follows:

Complex Element Calc. Found 1 %C 42.04 41.87 %H 5.29 5.20 %N 17.51 16.75 %Cl 4.43 4.39 2 %C 41.00 39.83 %H 4.92 4.36 %N 17.08 17.03 %Cl 8.64 8.58 3 %C 43.36 43.31 %H 5.20 4.99 %N 18.06 17.37 %Cl 9.14 8.98

Kirby-Bauer disc diffusion modified method was employed to assessment of theantimicrobial activity of the metal complexity of pipc samples^[16]. In vitro, the cytotoxicity assay of the gallium(Ⅲ ) pipc complex was performed on the human Hepatocellular carcinomacell lines(HepG-2)^{[17, 18]}. The HepG-2 cell line was received from VACSERA Tissue Culture Unit.

The molar ratios and conductancebehaviors of gallium(Ⅲ ), germanium(Ⅳ ) and silicon(Ⅳ ) complexes of pipc drug have been studied. The elemental analyses are in well agreement with 1∶ 2 stoichiometry (metal-to-ligand) as refereed in experimental section. All the synthesizedpipc complexes are stable at room temperature, with higher melting points. These complexes are insoluble in polar solventsbut soluble in commonly organic solvents (DMF & DMSO) with gently heating. Gallium(Ⅲ ), germanium(Ⅳ ) and silicon(Ⅳ ) pipc solutions which were dissolved in DMSO showed slightly low conductance (Λ _m=20~29 ohm^-1· cm²· mol^-1) that confirmed the non-electrolyte nature of these respected complexes^[19].

Fig.2

	Figure Option ViewDownloadNew Window
	Fig.2 FT-IR spectra of pipcfree ligand and its complexes

An important information concerning the coordination mode of pipc ligand towards metal ions can be obtained in a first approach by comparative FTIR spectral analysis of the pipcchelate and itsgallium(Ⅲ ), germanium(Ⅳ ) and silicon(Ⅳ ) complexes (Fig.2) and the data assignments are listed in (Table 1).

Table 1

Table 1

Table 1 Infrared frequencies (cm-1) bands of pipcligand and its complexes FTIR assignments Compounds Pipc Ga(Ⅲ ) Ge(Ⅳ ) Si(Ⅳ ) ν (O— H)COOH or H2O 3 460 3 386 3 433 3 460 ν (C=O)COOH - - - ν (C=O) (carbonyl) 1 634 1 626 1 618 1 626 ν as(COO) 1 579 1 572 1 548 1 572 ν s(COO) 1 362 1 370 1 347 1 353 Δ ν =[ν as(COO)-ν s(COO)] 217 202 201 219

Table 1 Infrared frequencies (cm-1) bands of pipcligand and its complexes

The FTIR spectrum of the free pipc has two distinguish bands at 3 460 and 3 378 cm^-1 due to ν (OH) and ν (NH) ofthe carboxylic and imino piperazinyl groups^[20]. In case of pipc complexes, it was found the broad band in this region due to the existence of the water molecules as a coordinated and/or uncoordinated modes^[21]. The IR spectrum of pipc hasn’ t a ν (C=O) stretching frequency band, based on the placed of pipcmolecule in zwitterionic form^{[22, 23]}. it is worthy mention that the proton of carboxylic group is more acidic than proton of the piperazinyl aromatic moiety. Therefore, the character nature of the carbonyl carboxylic group is absent at 1 700 c^-1[24]and two new vibration bands at 1 579 and 1 362 cm^-1 are presents due to ν _as(COO) and ν _s(COO) stretching vibration motions (see Table 1). Regarding the spectra of pipc complexes, these bands were shifted toward lower wavenumbers because of the participation of the carboxylate oxygen in the chelation process. The difference of Δ ν =[ν _as(COO)-ν _s(COO)] is led to recognized the coordination mode of carboxylate group. The calculated of Δ ν for Ga^Ⅲ , Ge^Ⅳ and Si^Ⅳ pipc complexes (Table 1) located within the range of 201~219 cm^-1, this confirmed that carboxylate group is acts as a monodentate chelate^{[25, 26]}. The band with strong intensity at 1634 cm^-1 is attributed to ν (C=O) of the pyridinering in case of free pipc ligand. After coordination towards metal ions, this band is shifted to lower wavenumber with changed inteosity. These assignments were supported the place of coordination between pipc chelator and central metal ions through oxygen’ s of carboxylate and carbonyl groups^[27]. The new vibration bands flanked between 600~500 cm^-1 are assigned to the ν (M— O) stretching vibrations^[28].

UV-Vis spectrum of the pipc ligand was showed strong-to-shoulder peaks within the region of (36 496~30 864 cm^-1) and (25 000~24 331 cm^-1), thatassigned to π → π ^* and n→ π ^* respectively of the N— H, C=N, C=O and COOH groups. The new intense absorption band spresenceat 37 037~31 153 cm^-1 is attributed to π → π ^* transitions of the aromatic or carboxylate organic moieties. The complexes showed bands at 28 409~25 707 cm^-1 due to the L→ M_CT “ charge-transfer transitions” that assigned to octahedral geometry^[29].

The spectral data of ¹H-NMR analysis concerningpipc free chelator and Ga^Ⅲ and Si^Ⅳ complexes are assigned in (Table 2). By comparison between the pipcligand, [Ga(pipc)₂(H₂O)(Cl)]· 4H₂O and [Ge(pipc)₂(Cl)₂]· 4H₂O complexes, it was found a significant unrest on the chemical shift in the region of aromatic protons (H7 and H4) which are nearing the coordination sites^[30]. The signal characteristic for piperazine proton (H-2) is exhibited at δ =8.85~8.95 ppm (unchanged), this meaning that nitrogen of piperazine ring doesn’ t sharing in the coordination process. So, the pipc ligand coordinated towards respected metal ions through both oxygen atoms of pyridone and carboxylate rings.

Table 2

Table 2

Table 2 1H-NMR proton signals of the pipc ligand and its complexes Assignments Compounds pipc Ga(Ⅲ ) complex Ge(Ⅲ ) complex H7 and H4 8.90 8.16 8.16 8a 4.39 4.39 4.40 8b 1.36 1.35 1.35 2a, 2a’ 3.14~3.25 3.15~3.23 3.15~3.26 2b, 2b 3.86 3.81 3.70 H4’ 9.19 8.91 8.91

Table 2 1H-NMR proton signals of the pipc ligand and its complexes

Gallium(Ⅲ ), germanium(Ⅳ ) and silicon(Ⅳ ) pipc synthesized complexes have been assigned using micro analytical analysis, ¹H-NMR, FT-IR and UV-Vis spectral studies as well as thermo gravimetric analyses. Molar ratio of prepared complexes were 1:2 of (metal:ligand). The complexes which have the general formula [Ga(pipc)₂(H₂O)(Cl)]· 4H₂O, [Ge(pipc)₂(Cl)₂]· 4H₂O and [Si(pipc)₂(Cl)₂]· 4H₂O (Fig.3).

Fig.3

	Figure Option ViewDownloadNew Window
	Fig.3 Suggested structures of pipc complexes for the Ga(Ⅲ ), Ge(Ⅳ ) and Si(Ⅳ )

The average size of the synthesized pipc complexes for the gallium(Ⅲ ), germanium(Ⅳ ) and silicon(Ⅳ ) were determined from the Full width at half maximum of the respected XRD peaks (Fig.4) by Scherrer equation^[31].

The data obtained from X-ray diffraction patterns refer to crystalline structures of the synthesized solid pipc metal complexes. Scherrer’ s formula (DRX=0.9λ /Δ cosθ ) calculation is declared to the crystal size. Where 0.9 is a term constant of the equipment, λ is the wavelength of radiation dueto the Cu Kα , peak and θ is the Bragg’ s angle. The grain size of the synthesized pipc complexes for the Ga(Ⅲ ), Ge(Ⅳ ) and Si(Ⅳ ) is < 100 nanometer.

Fig.4

	Figure Option ViewDownloadNew Window
	Fig.4 XRD pattern of pipc complexes for the GaⅢ , GeⅣ and SiⅣ

In vitro, the antibacterial and antifungal evaluations of the pipc complexes for the gallium(Ⅲ ), germanium(Ⅳ ) and silicon(Ⅳ ) were assessed against Staphylococcus Aureus, Bacillus subtilis(G(+) bacteria) and Escherichia Coli, Pseudomonas aeruginosa(G(-) bacteria) and (Candida Albicans and Aspergillus flavus) fungi (Table 3). The results indicate that the gallium(Ⅲ ), germanium(Ⅳ ) and silicon(Ⅳ ) complexes have a significant active but less than the standard drugs Tetracycline and Amphotericin B. It is worthy mentioned that Si(Ⅳ ) complex has an significant efficient against (22 mm· mg^-1) Candida Albicans fungi species rather than standard Amphotericin B drug (19 mm· mg^-1). The higher activity of silicon(Ⅳ ) pipc complex against fungirevealed tothe nanosized form of this complex to make penetration during lipid membranes and blocking the sites of enzymes inthe microorganisms^[32].

Table 3

Table 3

Table 3 Antimicrobial assessments of GaⅢ , GeⅣ and SiⅣ pipc complexes Sample Inhibition zone diameter (mm· mg-1 sample) B. subtilis E. coli P. aeruginosa S. aureus A. flavus C. albicans Control solvent “ DMSO” 0.0 0.0 0.0 0.0 0.0 0.0 Standard Antibacterial drug “ Tetracycline” 34 32 34 30 - - Antifungal Drug “ Amphotericin” - - - - 18 19 Ga(Ⅲ ) 14 11 10 14 0.0 0.0 Ge(Ⅳ ) 15 14 15 17 0.0 11 Si(Ⅳ ) 15 17 16 21 0.0 22 Note: G: Gram reaction; Solvent: DMSO

Table 3 Antimicrobial assessments of GaⅢ , GeⅣ and SiⅣ pipc complexes

The cytotoxicity (IC50) of the gallium(Ⅲ ) pipc complex was assessed against human hepatocellular carcinoma and tabulated in (Table 4). The experimental of IC50 data of the gallium(Ⅲ ) complex in vitro is equal to 123 μ g· mL^-1, which can be modified in future to become a promising anticancer agent.

Table 4

Table 4

Table 4 The inhibitory activityof gallium(Ⅲ ) pipc complex against HepG-2 cell line Con in μ g· mL-1 HEPG Ab %C Ab %C Ab %C Abs Av Ave % C SE 0 0.453 97.419 35 0.476 102.365 6 0.466 100.215 1 0.465 100 1.431 899 0.1 0.455 97.849 46 0.444 95.483 87 0.450 96.566 52 0.449 667 96.702 51 0.683 703 1 0.421 90.537 63 0.423 90.967 74 0.419 90.107 53 0.421 90.537 63 0.248 323 10 0.418 89.892 47 0.414 89.032 26 0.412 88.602 15 0.414 667 89.175 63 0.379 319 100 0.253 54.408 6 0.260 55.913 98 0.266 57.204 3 0.259 667 55.842 29 0.807 844 1 000 0.081 17.419 35 0.078 16.774 19 0.09 19.354 84 0.083 17.849 46 0.775 387

Table 4 The inhibitory activityof gallium(Ⅲ ) pipc complex against HepG-2 cell line