All starting materials used in the experiments were of reagent chemical grade and used as received without further purification. The solvents used in preparation and physical measurements were of analytical reagent grade. Water used in preparations is Milli-Q purified water (Milli-Q system, Millipore, Bedford, MA, USA). 2, 4-D (Cl₂C₆H₃OCH₂CO₂H; 221.04 g· mol^-1; purity 97%), zinc chloride (ZnCl₂; 136.30 g· mol^-1; purity ≥ 99.99%), copper(Ⅱ ) chloride dihydrate (CuCl₂· 2H₂O; 170.48 g· mol^-1; purity ≥ 99.95%), nickel(Ⅱ ) chloride hexahydrate (NiCl₂· 6H₂O; 237.69 g· mol^-1; purity 99.9%), cobalt(Ⅱ ) chloride hexahydrate (CoCl₂· 6H₂O; 237.93 g· mol^-1; purity 98.0%), and manganese(Ⅱ ) chloride tetrahydrate (MnCl₂· 4H₂O; 197.91 g· mol^-1; purity ≥ 98.0%) were bought from Sigma-Aldrich Co., (St Louis, MO, USA).

Elemental analyzer (model PE 2400 CHN) was performed to conduct the microanalyses (C% and H%) for the synthesized complexes. The IR spectra were obtained by using an infrared spectrometer (model Bruker FT-IR) in the region 400~4 000 cm^-1. The ¹H NMR spectra were obtained by using a NMR spectrometer (model Bruker DRX-250; 600 MHz; DMSO-d₆ solvent). Electronic spectra were obtained by using an UV-Vis spectrometer (model UV2-Unicam; DMSO solvent) in the region 200~800 nm.

The complexes were synthesized according to the following procedure: An aqueous solution containing 1 mmol (20 mL) of a metal chloride (ZnCl₂, CuCl₂· H₂O, NiCl₂· 6H₂O, CoCl₂· 6H₂O, or MnCl₂· 4H₂O) was added to a MeOH solution containing 2 mol (20 mL) of 2, 4-D under continuous stirring. A few drops of conc. ammonium (NH₃) were added till the pH of the mixture reached 7. At this point a colored precipitate begins to formed. The mixture was stirred for 20 minutes at 60 ℃, then cooled at room temperature to ensure the completeness of the precipitation and filtered. All the complexes with the Zn(Ⅱ ), Cu(Ⅱ ), Ni(Ⅱ ), Co(Ⅱ ), and Mn(Ⅱ ) ions were prepared by the same procedure, and the color of the resulting precipitate was yellowish white, greenish blue, oily green, crimson red, brown, respectively. These colored precipitates were thoroughly washed and dried in an oven at 70 ℃.

The modified Bauer-Kirby disc diffusion method^{[37, 38, 39]} was applied to assay the antifungal and antibacterial properties of the synthesized complexes towards to fungi organisms C. albicans and A. flavus and two Gram-positive strains (B. subtilis and S. aureus) and two Gram-negative strains (P. aeruginosa and E. coli.).

The HL ligand was dissolved in methanol solvent, where the Zn(Ⅱ ), Cu(Ⅱ ), Ni(Ⅱ ), Co(Ⅱ ), and Mn(Ⅱ ) chlorides were dissolved in Milli-Q purified water. The reaction between the ligand and each metal ion was carried out under four conditions; ① solvent was MeOH∶ H₂O (1∶ 1), ② media was neutral (pH 7), ③ reaction temperature was 60 ℃, and ④ molar reaction was 2∶ 1 (ligand to metal). Under these conditions, the obtained complexes with Zn(Ⅱ ), Cu(Ⅱ ), Ni(Ⅱ ), Co(Ⅱ ), and Mn(Ⅱ ) ions have the gross formula of C₁₆H₁₄Cl₄O₈Zn (541.46), C₁₆H₁₄Cl₄O₈Cu (539.63), C₁₆H₁₆Cl₄O₉Ni₍552.77), C₁₆H₁₄Cl₄O₈Co (535), and C₁₆H₁₄Cl₄O₈Mn (531), respectively, based on the elemental analysis date listed in Table 1. These data suggest that the obtained complexes are formulated as [ZnL₂](2H₂O (1), [CuL₂(H₂O)₂] (2), [NiL₂](3H₂O (3), [CoL₂(H₂O)₂] (4), and [MnL₂(H₂O)₂] (5), respectively (L^-: Cl₂C₆H₃OCH₂· COO).

Table 1

Table 1

Table 1 Elemental analysis data of 1, 2, 3, 4, and 5 complexes Complex Results Found/% Calculated/% C H Metal C H Metal 1 35.67 2.41 12.22 35.46 2.59 12.07 2 35.69 2.72 11.61 35.58 2.59 11.78 3 34.55 3.05 10.84 34.73 2.89 10.62 4 36.06 2.76 10.82 35.89 2.62 11.01 5 35.96 2.81 10.20 36.16 2.64 10.35

Table 1 Elemental analysis data of 1, 2, 3, 4, and 5 complexes

Figure 1 shows the measured IR spectra for complexes 1, 2, 3, 4, and 5, where Table 2 lists the main vibrational assignments obtained from these spectra. Here we discussed the main vibrational assignments that characterized the ligand^[40] and the obtained complexes: The IR spectrum of HL was characterized by a broad band centered at 3 445 cm^-1 assigned to the carboxylic acid O— H stretching mode. The O— H in-plane plane bending vibration is vibrated at 1 393 cm^-1, where the out-of-plane bending vibration is vibrated at 796 cm^-1. In the obtained complexes, the band of 3 445 cm^-1 is absent, instead, broad absorption bands ranged from 3 400 to 3 260 cm^-1 are observed in the IR spectra of the obtained complexes. These bands were characterized to the OH group ν (O— H) of water suggesting the existence of latticed or coordinated water molecules. The IR spectrum of HL was characterized by a very intense absorption band at 1 733 cm^-1 due to the stretching vibrations of carboxyl C& #61; O of — COOH group, which was no longer found in the IR spectra of the obtained complexes. After the deprotonation and complexation of — COOH group of HL, two new bands were appeared for the obtained complexes at around 1 479~1 445 cm^-1 due to the ν _sym(COO^-) vibrations, and at around 1 610~1 571 cm^-1 due to the ν _asym(COO^-) vibrations of the carboxylate group. The existing of the characteristic carboxylate bands suggests that ligand-metal binding occurs only through oxygen atoms in the carboxylate group while not through the oxygen atom of ether group or the Cl atoms. According to the Nakamoto criterion, the carboxylate group (COO^-) can adopt various coordination modes based oo the differences between the ν _as(COO^-) and ν _s(COO^-) values [Δ ν =ν _as-ν _s] of the complex compared to the free ligand anion^[41]. The band frequency differences (( for the complexes are in the range of (Δ ν =165~118), which are lower than that of the ligand sodium salt (NaL), suggests a bidentate corrdination mode for the carboxylate group (COO^-) in all the obtained complexes. In the IR spectra of the obtained complexes, the stretching vibrations of Zn— O, Cu— O, Ni— O, Co— O, and Mn— O bond were observed as a weak bands at 555, 552, 580, 550, and 567 cm^-1, respectively^[42].

Fig.1

	Figure Option ViewDownloadNew Window
	Fig.1 IR spectra of complexes 1, 2, 3, 4, and 5

Table 2

Table 2

Table 2 FT-IR data (cm-1) for HL ligand and complexes 1, 2, 3, 4, and 5 HL 1 2 3 4 5 Assignments* 3 445 3 361, 3 265 3 323, 3 263 3 375, 3 283 3 407, 3 283 3 405 ν (O— H) 3 076 3 172 3 179 3 180 3 186 3 173 ν asym(CH2) 3 070 3 074 3 040 3 088 - 3 075 ν asym(CH); ring 2 979 2 997 - 2 977 2 983 - ν sym(CH); ring 2 945 2 940 2 931 2 938 2 919 2 912 ν sym(CH2) - 2 364 2 365 2 359 2 359 2 365 Overtone and combination of δ def(CH) 1 733 - - - - - ν (C& #61; O) 1 580 - - - - - ν (C& #61; C); ring - 1 610 1 609 1 571 1 590 1 592 ν as(COO-) - 1 445 1 479 1 447 1 472 1 450 ν s(COO-) 1 449 1 425 1 421 1 422 1 424 1 426 δ sciss(CH2) 1 393 1 333 1 329 1 343 1 344 1 349 δ (O— H); in-plane 1 298 1 284 1 291 1 290 1 284 1 284 δ rock(CH2) 1 235 1 238 1 245 1 251 1 246 1 245 δ def(CH); ring 1 145 1 108 1 108 1 111 1 102 1 108 ν (C— C) 1 091 1 072 1 070 1 076 1 069 1 070 ν (C— O); ether 842 867 860 867 866 874 ν (C— Cl) 796 805 802 804 802 803 δ (O— H); out-of-plane 745 769 769 750 763 765 δ wag(CH2) 721 692 704 711 692 694 ν (C— Cl) 647 648 613 652 646 - O— H torsion 570 580 - 607 614 614 δ twist(CH2) - 555 552 580 550 567 ν (M— O) Note: * ν : Stretching; ν s: Symmetrical stretching; ν as: Asymmetrical stretching; δ : Bending

Table 2 FT-IR data (cm-1) for HL ligand and complexes 1, 2, 3, 4, and 5

The ¹H NMR spectrum of complex 1 was determined in DMSO-d₆ solvent at room temperature and compared with the free ligand. The ¹H NMR chemical shift for the HL molecule is: δ =4.36 (s, 2H, CH₂), 6.65 (d, J=9.00, 1H, C₆H phenyl group), 7.04 (d, J=2.4, 1H, C₅H phenyl group), 7.23 (s, 1H, C₃H phenyl group), 12.55 (s, 1H, COOH). The chemical shift for complex 1 is: δ =4.57 (s, 4H, 2CH₂), 6.85 (d, J=9.00, 2H, C₆H phenyl group), 7.14 (d, J=2.4, 2H, C₅H phenyl group), 7.33 (s, 2H, C₃H phenyl group). The HL molecule produced six signals in its ¹H NMR spectrum, and all of these proton resonances were found in the spectrum of the complex 1, except that of the COOH group which was no longer observed. In the spectrum of the free ligand, the aromatic protons were resonated in the 6.65~7.23 ppm range, the methylene protons were observed at 4.36 ppm, and the proton of the COOH group was observed at 12.55 ppm. In the spectrum of complex 1, all the aromatic protons from carbons numbered C3, C5, and C6 were represented down-field shifts. The methylene protons were undergone down-field shifted, and exhibited a definite singlet at 4.57 ppm. The protons of methylene group and aromatic protons from carbons numbered C6 which are close to the COO group showed the strongest down-field shift because of ligand to metal charge transfer which further confirmed the complex formation^[28].

The UV-Vis. spectra of complexes 1, 2, 3, 4, and 5 were recorded in DMSO solvent over the wavelength range from 200 nm to 800 nm at room temperature. The absorption spectrum of HL exhibits three bands at 228, 282 and 291 nm corresponded to the groups C— Cl, benzene C& #61; C, and C— O or C& #61; O in its molecular structure^[43]. After HL complexed with the metal ions, a very strong broad band was appeared with two maxiums at 274 nm and at 296~300 nm. The first maxium was corresponding to the π → π ^* transitions, where the second one was corresponding to the n→ π ^* transitions. The weak broad band at 660, 650, and 525 nm found in the spectrum of complex 2, 3, and 4, respectively, may be assigned to the ligand-to-metal charge transfer bands (LMCTs)^{[44, 45]}. The μ _eff values for complexes 2, 3, 4, and 5 were 1.9, 3.8, 4.8, and 4.5 B.M., respectively. These values suggest a paramagnetic property for complexes 2, 4, and 5 with an octahedral structure^{[46, 47, 48]}. Complex 4’ s higher μ _eff value suggests that it is likely a high spin complex^[49]. At room temperature, the μ _eff value for tetrahedral complexes is > 3.3 B.M., usually ranged from 3.4 to 3.9 B.M. The μ _eff value of complex containing Ni(Ⅱ ) ions falls within in this range (3.8 B.M.), indicating that complex 3 has a tetrahedral stereochemistry. Complex containing Zn(Ⅱ ) ions is diamagnetic, as expected for d¹⁰ configuration. Structures of complexes 1, 2, 3, 4, and 5 were proposed based on the spectral, magnetic, and elemental results and presented in Figure 2.

Fig.2

	Figure Option ViewDownloadNew Window
	Fig.2 Proposed structures of the synthesized complexes

All the obtained complexes as well as the free ligand were screened in vitro for antifungal properties towards to fungi organisms C. albicans and A. flavus., and for antibacterial properties towards to two Gram-positive organisms (B. subtilis and S. aureus) and two Gram-negative organisms (P. aeruginosa and E. coli.). The antibiotic drug Amphotericin B was used to compare the antifungal results of free ligand and the obtained complexes. Zones of inhibition (in mm· mg^-1) observed for this antibiotic were 19 and 18 mm· mg^-1 against C. albicans and A. flavus, respectively. The antibiotic drug Tetracycline was used to compare the antibacterial results of free ligand and the obtained complexes. Zones of inhibition (in mm· mg^-1) observed for this antibiotic were 34, 30, 34, and 32 mm· mg^-1 against B. subtilis, S. aureus, P. aeruginosa, and E. coli, respectively. The results showed that all of the complexes as well as the free ligand were found inactive against A. flavus microbe, except complex 3, which show strong activity against this microbe, with a zone of inhibition of 16 mm· mg^-1, which was equal to 89% of the activity of the antifungal drug (Amphotericin B). Interestingly, complex 3 is the only complex that exhibited excellent activity against C. albicans microbe, showing maximum zone of inhibition of 20 mm· mg^-1, which was higher activity than that of the antifungal drug (Amphotericin B). Complexes 2, 4, and 5 showed moderate activity against this microbe with zone of inhibition in the range 10~12 mm· mg^-1. All of the complexes were found active against all the tested bacterial strains, but with moderate level of lethality, with zone of inhibition in the range 12~19 mm· mg^-1. Only complex 1 and 4 and exhibited good activity against P. aeruginosa microbe, with a zone of inhibition of 21 and 22 mm· mg^-1, respectively. Complexes 1 and 4 were the most potent complexes showing activity against all the tested bacterial strains compared with the other complexes. It’ s important to note that the synthesized complexes are more potent than the free ligand against all the tested microbes.