N, N'-(1,2-Pheneylene) bis (3,4,5-Trihudroxybenzamide) as Bioactive Ligand  Synthesis, Analytical and Anti-Microbial Studies

Esraa O. Ahmed1; Abdou S. El-Tabl2; Eman A. Mohamed2; Islam M. I. Moustafa1

Original Articles: 2024 Vol: 16 Issue: 7

N, N'-(1,2-Pheneylene) bis (3,4,5-Trihudroxybenzamide) as Bioactive Ligand Synthesis, Analytical and Anti-Microbial Studies

Esraa O. Ahmed¹, Abdou S. El-Tabl², Eman A. Mohamed², Islam M. I. Moustafa^1*

¹Department of Chemistry, University of Banha, Banha, Egypt

²Department of Chemistry, University of Menofia, Shibin El Kom, Egypt

Corresponding Author:: Islam M. I. Moustafa
Department of Chemistry, University of Banha, Banha, Egypt

Received: 03-Jul-2024, Manuscript No. JOCPR-24-140579; Editor assigned: 05-Jul-2024, PreQC No. JOCPR-24-140579 (PQ); Reviewed: 19-Jul-2024, QC No. JOCPR-24-140579; Revised: 26-Jul-2024, Manuscript No. JOCPR-24-140579 (R); Published: 02-Aug-2024, DOI:10.37532/0975-7384.2024.16(7).170

Citation: Moustafa IMI, et al. 2024. N, N'-(1,2-Pheneylene) bis (3,4,5-Trihudroxybenzamide) as Bioactive Ligand; Synthesis, Analytical and Anti-Microbial Studies. J. Chem Pharm. Res. 16:170.

Copyright: © 2024 Moustafa IMI, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, providedthe original author and source are credited

Abstract

The synthesis and characterization of some metal complexes with new polyhydroxyl amide ligand derived from Gallic acid viz: N, N' -(1,2-phenylene) bis (3,4,5-trihydroxy benzamide), expected to have biological activity, had been synthesized and characterized by different spectroscopic and physical techniques. In solution, the study focused on the formation of Mn2+, Fe3+, Ni2+, Cu2+ and Zn2+ complexes with the newly prepared ligand. Firstly, the stoichiometry of the complexes was determined by conductometric titration then the proton-ligand ionization constants (log Kn H ) and metal-ligand stability constants (log βn M ) were determined by potentiometric titration. The former proved the formation of 1:1 and 2:1 (L:M) complexes while the stability constants were found to be dependent on the effective charge (e/r) ratio of the metal ion. In solid state, metal complexes with different metal ions (Mn2+, Fe3+, Ni2+, Cu2+ Zn2+, Ca2+ and Pb2+) and different anions (SO4 2- , AcO- ) had been successfully prepared and characterized by elemental and thermal analysis, mass spectra, IR, 1HNMR, ESR and TEM techniques. The biological activity of the prepared complexes towards two Gram positive bacterial species (Staphylococcus aureus and Bacillus subtilis), two Gram negative bacterial species (Escherichia coli, Proteus vulgaris), one filamentous fungus (Aspergillus fumigatus) and one yeast species (Candida albicans) was studied using the modified well diffusion method. The optained results showed higher activities of the solid complexes compared to that of the free ligand.

Keywords

Hydroxyl amide ligand; Gallic acid; Transition metal complexes; Conductometry; Potentiometry; Biological activity

Introduction

Gallic acid (3,4,5-trihydroxybenzoic acid) is one of the most abundant phenolic acids in the plant kingdom. It and many of its derivatives have extensive application in the food and pharmaceutical industries and can inhibit the oxidation and rancidity of oils and fats due to their free radical scavenging and antioxidant nature [1]. Many scientific researches on the biological and pharmacological activities of these phytochemicals were reported with emphasis on antioxidant, antimicrobial, anti-inflammatory, anticancer, cardio protective, gastro protective, and neuroprotective effects [2-6]. These compounds can disrupt the integrity of the cell membrane in both Gram positive and Gram negative bacteria and change the charge, hydrophobicity, and membrane permeability of Campylobacter jejune then elevate the antibiotic accumulation in the microorganism [7-10]. Gallic acid can interfere with the Chemo Dynamic Therapy (CDT) provides a new treatment strategy for tumor cells. A variety of CDT agents have been designed by taking advantage of the catalytic property of transition metals, e.g., Fe, Cu, and Mn. Among those nano substrates, Cu-based CDT agents are obviously superior to the other transition metal-based counterparts [11]. Metal complexes derived from bioactive compounds (Gallic acid and others) are being explored for antimicrobial actions and potentially are applicable to provide effective and nontoxic drug candidates having preferred and improved pharmacological activity [12-16]. It should be noted that the presence of metal ions in biological fluids could have a significant effect on the therapeutic action of such organic compounds [17]. Although there are many methods available to study the stability of metal-ligand complexes, the potentiometric method had been used extensively in many branches of solution chemistry and is most frequently used for the study of binary and ternary complexes of transition metals with molecules of biological and pharmaceutical interest [18,19]. In the present work, and in continuation of our study, we provide the synthesis and characterization of Mn²⁺, Fe²⁺, Ni²⁺, Cu²⁺, Zn²⁺, pb²⁺ and Ca (II) complexes with new polyhydroxyl amide ligand derived from Gallic acid. The stoichiometry and stability constants of the complexes are determined in solution by conductometric and potentiometric titrations. The biological activity towards some bacteria, fungi and yeast were also studied.

Materials and Methods

All chemicals used in the present study were of higher-grade Merck or Alderich and were used as received. Second distilled water was used whenever water is necessary. The organic polyhydroxyl amide ligand was prepared by esterification of Gallic acid with ethyl alcohol and condensation with o-phenelynediamine (Figure 1). Solid complexes were prepared by refluxing of metal ion salt (in least amount of ethanol) with ethanolic ligand solution in molar ratio 1:1 or 2:1 (L:M) for about 6 hrs. All techniques and instruments used for characterization of metal complexes are as described in our previous works [20,21].

chemical-pharmaceutical-research-alcohol

Figure 1: Gallic acid with ethyl alcohol and condensation with o-phenelynediamine.

Conductometric titration

It was carried out, using YSI Model 32 conductance meter, by titrating 50 mL of ligand solution (10^-2 M) with the metal ion solution (10^-3 M) added from micro burette with mechanical stirring after the addition of each increment of the titrant. Correction for the dilution effect on the measured conductance was performed by multiplying its value by the factor (V_o+V₁/V₁) where V_o is the original volume of the solution (50.0 mL) and V₁ is the volume of the titrant. The corrected conductance (in μ Mohs) is then plotted as a function of the molar ratio (ligand/metal).

Potentiometric titration

The following solutions were prepared:

(A): 5.0 mL of 0.1 M HCl and 5.0 mL of 1.0 M NaCl.

(B): Mixture (A) + 25 mL of 0.001 M ligand (in ethanol).

(C): Mixture (B) + 2.0 mL of 0.0025 M metal ion solution.

Each solution mixture was completed to 50.0 mL using ethanol and bi-distilled water so as to obtain 40% by volume ethanol. Each mixture was then titrated against 0.02 M NaOH solution containing the same percent of ethanol using an automatic micro burette with constant stirring.

Antimicrobial activity assay

The antimicrobial effect was tested against Staphylo-coccus aureus and Bacillus subtilis as Gram positive, Escherichia coli and Proteus vulgaris as Gram negative bacterial species in addition to one filamentous fungus; Aspergillus fumigatus and one yeast species; Candida albicans using a modified well diffusion method [22]. Briefly, samples are incubated at 37°C for 18-24 hours and are placed in the center of nutrient agar plates which have been inoculated with the test bacteria. The evaluation of this test is based on the level of growth both under and around the sample. The area of no growth is known as a "Zone of inhibition" or "Clear zone" which is proportional to the inhibitory action of the investigated compound [23]. The resulting inhibition zone diameters were measured in millimeters and used as criterion for the antimicrobial activity. Solvent controls (DMSO) were included in every experiment as negative control. Positive controls were also performed using gentamycin as standard antibacterial drugs and the ketoconazole as standard antifungal drug. The lowest concentration showing complete inhibition of growth was recorded as the MIC of the respective sample [24].

Results and Discussion

Part A: Studies on the metal complexes in solutions

In this part, the possibility of complex formation between ligand and Mn²⁺, Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺ metal ions in solution are discussed. The stoichiometry of the complexes formed was studied by conductometric titration while the proton-ligand stability constant, (log K_n^H), of the free ligands and the metal-ligand formation constants, (log β_n ^M), of the complexes were determined by potentiometric titration.

Conductometric titration: The stoichiometry of the complexes formed between Mn²⁺, Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺ and the ligand under study is determined by conductometric titration. In the present work, the common behavior of the conductance-molar ratio plots is the increase in conductance with increasing volume of titrant (Figure 2). Although the ligand solution has negligible conductance, its addition to the metal ion solution caused a continuous increase in the conductance. The slopes of equivalent-conductivity plots for all ligand-metal ions mixtures showed significant changes when the mole ratio (C_L:C_M) was about 1:1 and 2:1; thus indicating the formation of a relatively stable 1:1 and 2:1 complexes (L:M) between Mn⁺ and the ligand (Figure 2).

chemical-pharmaceutical-research-titration

Figure 2: Conductometric titration of ligand (HL) with, Mn²⁺, Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺.

Potentiometric titration: The potentiometric titrations were performed in presence of acid mixture (A) as medium consisting of 0.1 M HCl and 1.0 M NaCl as recommended by Bjerrum [25]. Mixture (A) was mixed with the chelating ligand (HL) of concentration of 1.0 × 10^-3 M called mixture (B). Mixture (B) was mixed with different metal ions of concentration 2.5 × 10^-3 M called C₁, C₂, C₃, C₄ and C₅ for Mn²⁺, Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺ ions respectively. In all mixtures A, B or Cn, absolute ethanol of 40% (v/v) is enough to keep the components of the mixture in soluble state. These mixtures were then titrated with 0.02 M NaOH freed from carbonate containing also 40% (v/v) ethanol to keep the ratio of ethanol in the titration vessel constant. The potentiometric titration curves of the mixtures are shown in Figure 3. These curves are S-shaped with a sharp jump in mixtures (A) and (B) but relatively smooth curves are obtained with mixtures C_n which contain the metal ions. The end points for the titration of the mixtures increase in the order A< B< C_n (Figure 3).

chemical-pharmaceutical-research-presence

Figure 3: Potentiometric titration of Ligand (HL) in presence of Mn²⁺, Fe³⁺, Ni²⁺, Cu²⁺ and Zn^2+.

The average number of protons associated with the reagent molecule (n'A) is determined at different pH values using the relation:

(1)

Where V₁ and V₂ the volumes of NaOH required to reach the same pH in the titration curve of acid mixture and the chelating drug, respectively, V^o the initial volume (50 mL), TC_Lo the total concentration of the drug, Y the total number of dissociable protons attached to the drug, N^o the normality of NaOH solution and E^o the initial concentration of the free acid. The values of n'_A are plotted against pH and the value of proton-ligand stability constants K_n^H is calculated by interpolation at half n'_A integrals (Figure 4).

Figure 4: Formation curve of proton Ligand (HL).

To determine the values of stepwise formation constants of the complexes formed between metal ions and the ligand, the average number of ligand molecules attached per metal ions (n') and free ligand exponent (PL) are calculated using the equations:

(2)

(3)

Where TC_Mo the total concentration of metal ion presents in solution, ᵝnH the overall stability constant, V₁, V₂ and V₃ are the volumes of NaOH required to reach the same pH in titration curves in acid mixture, organic ligand and the complex respectively. On plotting the values of n' against PL and interpolating at the half-integrals, the stepwise stability constants of the complexes can be evaluated (Figure 5).

Figure 5: Formation curves of Mn²⁺, Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺ complexes with ligand (HL).

The values of log K_n^H and log K_n^f gathered from potentiometric titration along with the effective charge (e/r) values are listed in Table 1.

Complex	logK_n^H	Step-wise formation constants; logKnf
		Mn²⁺	Fe³⁺	Ni²⁺	Cu²⁺	Zn²⁺
		(e/r)	(e/r)	(e/r)	(e/r)	(e/r)
HL	logK₁^H=3.1	4.11	4.64	4.22	4.52	4.31
HL	logK₂^H=5.5	0.026	0.043	0.026	0.029	0.023

Table 1: Proton-reagent stability (log K_n^H) and stepwise formation (log K_n^f ) constants of Mn²⁺, Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺ complexes with ligand (HL).

Part B: Studies on the metal complexes in solid state

All the prepared complexes are nonhygroscopic, having different colours and of high melting points (≥ 180ºC). Elemental analysis measurements showed satisfactory agreement between the proposed and obtained formulae, while molar conductance values indicate their nonionic nature except those prepared from copper sulfate, lead sulfate (Table 2).

No.	Tentative formula	FW	M.P (ºC)	Anal./Found (Calc.) (%)					Conductivity Λ
				Anal./Found (Calc.) (%)
				C	H	N	S	M
HL	C₂₀H₁₆N₂O₈	412	240	58.47	4.15	7.07	-	-	-
HL	C₂₀H₁₆N₂O₈	412	240	(58.25)	(3.91)	(6.79)	-	-	-
I	((HL)Mn (OAc)₂(H₂O)₂).2H₂O	621	180	46.39	4.22	4.51	-	8.84	13.5
I	C₂₄H₂₆MnN₂O₁₄	621	180	(46.15)	(4.2)	(4.4)	-	8.84	13.5
II	((HL) Ni (NO₃)₂ (H₂O)₂).2H₂O	631	210	38.42	3.05	9.17	-	9.3	14
II	C₂₀H₂₀N₄NiO₁₆	631	210	(38.06)	(3.19)	(8.88)	-	9.3	14
III	((HL)Cu (OAc)2(H2O)2).2H2O	630	220	47.9	4.18	4.86	-	10.09	11.7
III	C₂₄H₂₆CuN₂O₁₄	630	220	(45.75)	(4.16)	(4.45)	-	10.09	11.7
IV	((HL)Cu (SO₄) (H₂O)₃).2H₂O	625	301	38.75	3.4	4.75	5.23	10.15	48
IV	C₂₀H₂₂CuN₂O₁₅S	625	301	(38.37)	(3.54)	(4.47)	(5.1)	10.15	48
V	((HL)₂Ni (OAc)₂) .3H₂O	1001	250	53.2	3.62	5.89	-	5.86	13
V	C₄₄H₃₈N₄NiO₂0	1001	250	(53.77)	(3.82)	(5.59)	-	5.86	13
VI	((HL)₂Cu (OAc)₂) .3H₂O	1022	320	52.87	4.14	5.48	-	6.22	11.5
VI	C₄₅H₄₂CuN₄O₂₀	1022	320	(52.94)	(4.17)	(5.36)	-	6.22	11.5
VII	((HL)₂Zn (SO₄) (H₂O)).2H₂O	1004	300	47.27	3.18	5.81	3.34	6.51	14.5
VII	C₄₀H₃₄N₄O₂₁SZn	1004	300	(47.84)	(3.41)	(5.58)	(3.2)	6.51	14.5
VIII	((HL)Ca (OAc)₂(H₂O)₂) 3H₂O	606	200	47.52	4.32	4.62	-	6.61	7
VIII	C₂₄H₂₆CaN₂O₁₄	606	200	(47.45)	(4.25)	(4.55)	-	6.61	7
IX	((HL)Ca (Cl)₂ (H₂O)₂).2H₂O	575	190	43.83	4.2	4.87	-	6.97	9.5
IX	C₂₁H₂₄CaCl₂N₂O₁₀	575	190	-43.75	-4.18	-4.77	-	6.97	9.5
X	((HL)Pb (SO₄) (H₂O)₃).2H₂O	769	210	31.21	2.88	3.64	4.17	26.92	30
X	C₂₀H₂₂N₂O₁₅PbS	769	210	-31.19	-2.85	-3.6	-4.1	26.92	30

Table 2: Analytical and physical data of ligand (HL) and its metal complexes.

Thermal analysis

The thermo gravimetrical behavior of some selected solid complexes is studied as further support for chemical structure. From the TG curves percent of physically adsorbed and coordinated water molecules were detected and the percent of metal ion in complexes was calculated from the percent of metallic residue remained after complete degradation. Table 3 summarizes the sequence and assignment of degradation steps and show representative thermo gram (Table 3), (Figure 6).

chemical-pharmaceutical-research-complexes

Figure 6: TGA thermal charts of complexes (III) and (IV).

Complex	Temp. (ºC)	% Wt. loss	Assignment
III	44.27	57.317	Loss of (H₂O) hydrated water molecules.
	430.58	20.065	Loss of (H₂O) coordinated water molecules.
	666.31	12.686	Beginning decomposition with evolution of N₂ and CO₂
			Final decomposition of complex leading to CuO
IV	47.55	23.256	Loss of (H₂O) hydrated water molecules.
	322.9	45.361	Loss of (H₂O) coordinated water molecules.
	567.06	25.907	Beginning decomposition with evolution of N₂ and CO₂
			Final decomposition of complex leading to CuO
VIII	42.39	12.687	Loss of (H₂O) hydrated water molecules.
	235.22	15.664	Loss of (H₂O) coordinated water molecules.
	313.01	31.615	Beginning decomposition with evolution of N₂ and CO₂
	605.28	29.273	Final decomposition of complex leading to CaO
IX	42.11	16.04	Loss of (H₂O) hydrated water molecules.
	333.45	54.15	Loss of (H₂O) coordinated water molecules.
	580.78	21.772	Beginning decomposition with evolution of N₂ and CO₂
			Final decomposition of complex leading to CaO

Table 3: Thermal analysis data for some metal complexes.

Infrared spectra

To through light on the mode of bonding and functional groups participating in complex formation, the IR spectra of the solid complexes were compared to that of the free ligand using KBr disc technique. IR spectrum of the ligand (HL) showed broad band located at 3442 cm^-1 and weak band at 3290 cm^-1 due to the stretching vibrations of intra-molecular hydrogen bonded OH groups and the stretching vibration of NH₂ groups, respectively [26]. The latter band is strongly shifted to lower frequency in the spectra of complexes indicating its contribution in coordinate bond formation. On the other hand, the strong band present at 1604 cm^-1 in the spectrum of the free ligand due to the υ (C═O) amide is, more or less, unaffected on the spectra of the solid complexes indicated that this group is not involved in the coordination to the metal ion. A new set of two bands located at 432-635 cm^-1 and 533-750 cm^-1 appeared in the spectra of solid complexes and were assigned to υ(M-N) and υ (M-O), respectively. Other frequencies (cm¹) of different anions of metal salts are cited in (Table 4).

Complex	ν (H₂O)	ν(OH)	Υ NH₂	δOH	ν (OAc/SO₄/NO₃)	ν(M-O)	υ(M-N)
Complex	ν (H₂O)	ν(OH)	Υ NH₂	δOH	ν (OAc/SO₄/NO₃)	ν(M-O)	υ(M-N)
HL	-	3442	3418	1385	-	-	-
I	3439	3481	3143	1319	1384, 1274	573	459
II	3520	3402	3150	1457	742	556	424
III	3679	3399	3257	1325	1400, 1325	750	635
IV	3678	3448	3141	1566	1394, 1277,667	550	458
V	3510	3450	3270	1504	1382,1311,882	590	452
VI	3452	3384	3175	1225	1,40,51,225	612	419
VII	3450	3415	3275	1459	1,27,71,107	544	458
VIII	3473	3430	3107	1272	1,45,61,425	586	461
IX	3471	3447	3068	1386	-	669	611
X	3505	3428	3205	1240	1035,879, 859	433	418

Table 4: IR Frequencies (cm-1) of some important band the ligand (HL) and its metal complexes.

Mass spectra

In combination with elemental and thermal analysis, mass spectra of the solid complexes were measured to ascertain their chemical structure. The technique is accurate and give confidential results. The molecular peaks (m/z; amu) of different fragments are cited with assignment in (Table 5).

Complex	Mole. ion peak m/z	Assignment	Mole. ion peak m/z	Assignment
Free ligand	76	C₆H₄,	335	C₁₄H₁₁O₈N₂
	181	C₁₂H₅O₂	374	C₁₇H₁₆O₈N₂
	304	C₁₄H₈O₈	400	C₁₉H₁₆O₈N₂
			412	C₂₀H₁₆O₈N₂ (F.Wt
((HL)Mn (OAc)₂(H₂O)₂).2H₂O (I)	171	C₆H₇O₄N₂, C₁₀H₁₃O₆N₂MnC₁₀H₁₆O₆N₂MnC₁₄H₂₁O₇N₂Mn	418	C₁₅H₂₃O₈N₂Mn, C₂₁H₂₃O₉N₂Mn, C₂₂H₂₅O₁₁N₂Mn, C₂₄H₂₅O₁₃N₂Mn C₂₄H₂₆O₁₄N₂Mn
	257		505
	319		552
	388		604
			621
((HL)Cu(OAc)₂ (H₂O)₂).2H₂O (III)	444	C₁₄H₉O₁₁N₂Cu, C₁₄H₁₄O₁₁N₂CuC₁₆H₁₆O₁₁N₂Cu C₁₇H₁₆O₁₁N₂Cu	535	C₁₈H₂₀O₁₃N₂Cu, C₂₀H₂₆O₁₄N₂Cu C₂₄H₂₆O₁₄N₂Cu
	449		581
	475		629
	487
((HL)Cu (SO₄) (H₂O)₃).2H₂O (IV)	192	C₄H₄O₅N₂S, C₆H₁₀O₅N₂S, C₇H₁₂O₅N₂SCuC₇H₁₅O₆N₂SCu	398	C₁₁H₁₅O₈N₂SCu,
	222		433	C₁₁H₁₈O₁₀N₂SCu,
	299		519	C₁₄H₂₀O₁₃N₂SCu,
	318		601	C₁₈H₂₂O₁₅N₂SCu,
			626	C₂₀H₂₂O₁₅N₂SCu
((HL)₂Ni (OAc)₂) .3H₂O (V)	276	C₇H₆O₉N₃, C₇H₆O₉N₃Ni, C₁₈H₁₄O₉N₄Ni, C₁₉H₁₄O₁₀N₄Ni	683	C₃₀H₁₆O₁₂N₄Ni, C₃₄H₁₆O₁₄N₄Ni, C₃₉H₃₄O₁₉N₄Ni C₄₄H₃₉O₂₀N₄Ni
	335		762
	488		920
	516		1000
((HL)₂Cu (OAc)₂) .3H₂O (VI)	121	C₄H₁₁O₃N, C₄H₂₀O₃N, C₅H₂₄O₅N, C₇H₂₆O₅N₂Cu,	381	C₁₄H₂₆O₆N₂Cu, C₂₅H₂₈O₁₂N₂Cu, C₃₆H₃₄O₁₈N₂Cu, C₄₂H₄₂O₂₀N₄Cu C₄₅H₄₂O₂₀N₄Cu
	130		611
	178		845
	281		986
			1021
(((HL)₂Zn (SO₄) (H₂O)) .2H₂O (VII)	671	C₂₂H₂₈O₁₅N₃SZn, C₂₄H₂₈O₁₅N₃SZn, C₂₇H₂₈O₁₅N₄SZn, C₃₅H₃₂O₁₅N₄SZn,	890	C₃₆H₃₃O₁₇N₄SZn, C₄₀H₃₄O₁₈N₄SZn C₄₀H₃₄O₂₁N₄SZn
	695		955 \1002
	744
	845
((HL)Ca(OAc)₂ (H₂O)₂).3H₂O (VIII)	477	C₁₈HO₁₂N₂Ca, C₁₈H₄O₁₂N₂Ca, C₁₉H₇O₁₂N₂Ca, C₂₀H₈O₁₃N₂Ca, C₂₀H₁₅O₁₃N₂Ca	544	C₂₁H₁₆O₁₃N₂Ca, C₂₃H₁₉O₁₃N₂Ca, C₂₃H₂₁O₁₄N₂Ca, C₂₃H₂₆O₁₄N₂Ca C₂₄H₂₆O₁₅N₂Ca
	480		571
	495		589
	524		594
	531		606
((HL)Ca (Cl)₂ (H₂O)₂) .2H₂O (IX)	102	C₃H₄O₃N, C₉H₁₁O₅N, C₁₂H₁₇O₇N₂, C₁₈H₁₇O₇N₂,	454	C₂₀H₁₈O₈N₂Ca, C₂₀H₂₃O₉N₂Ca C₂₁H₂₄O₁₀N₂Cl₂Ca
	213		475
	301		575
	413
((HL)Pb (SO₄) (H₂O)₃).2H₂O (X)	365	C₅H₄O₃NSPb, C₆H₆O₄NSPb, C₉H₆O₄N₂SPb, C₉H₁₀O₁₀N₂SPb	596	C₁₃H₁₃O₁₀N₂SPb, C₁₄H₁₇O₁₂N₂SPb, C₁₈H₂₂O₁₅N₂SPb C₂₀H₂₂O₁₅N₂SPb
	394,		644
	445		745
	545		769

Table 5: Mass spectral data (Molecular ion peak; m/z; amu) for some selected complexes.

Based on the above mentioned analytical and physical techniques, the mode of bonding and structure of the prepared complexes can be formulated (Figure 7).

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Figure 7: The mode of bonding and structure of the prepared complexes.

Electron Spin Resonance (ESR)

The ESR spectra for the two copper complexes (III), and (IV) were measured and their spectral data are presented in Table 6. The spectra are characteristic for those of d⁹ configuration having axial type of a d_(x2-y2) ground state which is the most common for Cu (II) complexes [27]. The complexes showed g_||>g┴>2.0023, indicating octahedral geometry around Cu (II) ion [28]. The G values of the complexes are 2.75 and 3.80 i.e G<4.0 indicating that spin-exchange interactions took place between Cu (II) ions [29]. This phenomenon is further confirmed by the magnetic moments values (1.45 and 1.65 B.M.). On the other hand, the ratio of g_||/A_|| value is also considered as a diagnostic term for stereochemistry [30]. The g_||/A_|| values for the copper complexes are 170.8 and 223 cm^-1 which lie just within the range expected for the tetragonal distorted octahedral Cu(II)complexes. The values of α² (Cu) for the two complexes calculated as: α² (Cu) = (A_||/0.036) + (g_||-2.002) + 3/7 (g-2.002) + 0.04 are 0.6 and 0.58 suggesting a covalent bonding [31].

Complex	g\|\|	g^	g_iso^a	A_\|\|	A_^	A_iso^b	G ^c	g_\|\|/A_\|\|	α ²	ß ²
Complex	g\|\|	g^	g_iso^a	(G)	(G)	(G)	G ^c	g_\|\|/A_\|\|	α ²	ß ²
(III)	2.22	2.08	2.03	115	10	45	2.75	170.8	0.61	1.65
(IV)	2.23	2.06	2.12	95	5	35	3.8	223	0.58	1.34

Table 6: ESR data for Cu²⁺ complexes.

Transmission Electron Microscopy (TEM)

Study of the TEM images of some selected complexes reveals that the average diameter of particles sizes ranges from 43 to 58 nm that are lying in the nano-sized range. Illustrative example is shown in Figure 8 for Cu complex (III) (Figure 8).

Figure 8: TEM images for Cu Complex (III).

Antimicrobial activity assay

The antimicrobial profile was tested against two Gram positive bacterial species (Staphylococcus aureus, (Staph. Aureus) and Bacillus subtilis, (Bacillus subtilis)), two Gram negative bacterial species (Escherichia coli, (Escherichia coli), Proteus vulgaris), one filamentous fungus (Aspergillus fumigatus, (Asp. fumigatus)) and one yeast species (Candida albicans, (Cand. albicans)) using a modified well diffusion method. The resulting inhibition zone diameters were measured in millimeters and used as criterion for the antimicrobial activity with addition to the % Activity Index.

DMSO, used for dissolving the test compounds, was included in every experiment as negative controls and showed no inhibition zones. Positive controls were also performed using gentamycin as standard antibacterial drugs (4 μg/ml) and ketoconazole as standard antifungal drug (100 μg/ml). The antibacterial and antifungal results (Table 7) showed that the organic free ligand showed high activity towards the tested organism compared to the positive controls used as standard drugs and most of the complexes exhibit higher activities than the free ligand except for the Gram positive bacteria, Staph. Aureus, where the solid complexes showed less activity than the organic ligand. The increased activity of metal complexes is explained by the overtone's concept and the Tweedy's theory according to which chelation reduces the polarity of the ligand due to partial sharing of its negative charge with the metal favouring the transportation of the complex across the lipid layer of the cell membrane. On the other hand, the negative results observed in some cases can be attributed to the inability of the complex to diffuse into bacterial cell membranes or it can diffuse by inactivited rout by any cellular mechanism [32-35]. The test was done using the diffusion agar technique, well diameter: 6.0 mm (100 μl was tested), at RCMB: Regional Center for Mycology and Biotechnology.

	Staph. aureus		Bacillus subtilis		Escherichia coli		Proteus vulgaris		Asp.fumigatus			Cand. albicans
Com.	Inh. zone	% Ac.Ind	Inh. Zone	% Ac.Ind	Inh. zone	% Ac.Ind	Inh. Zone	% Ac.Ind	Inh. zone	% Ac.Ind		Inh. Zone	% Ac.Ind
HL	21	87.5	13	50	12	40	18	72	14	82.4		10	50
2	16	66.7	15	57.7	18	60	18	72	15	88.2		12	60
3	9	37.5	17	65.4	12	40	22	88	17	100		14	70
4	13	54.2	20	76.9	NA	NA	12	48	17	100		14	70
5	15	62.5	19	73.1	12	40	18	72				12	60
6	17	70.8	15	57.7	11	36.7	17	68	20	117.6		NA
7	15	62.5	17	65.4	15	50	20		NA			NA
8	14	58.3	12	46.2	12	40	17	68	NA			NA
9	16	66.7	20	76.9	15	50	22		16	94.1		NA
10	10	41.7	22	84.6	12	40	NA	NA	13	76.5		NA
11	12	50	23	88.5	18	60	21		16	94.1		18	90
	Gentamycin (4mg/ml)				Gentamycin (4mg/ml)				Ketoconazole (100mg/ml)
	24	100	26	100	30	100	25	100	17		100	20	100

Table 7: Antibacterial activities of studied ligand and its tested complexes in terms of inhibition zone diameter (mm) and % activity index.

• Positive control for fungi: Ketoconazole 100 μg/ml.

• Positive control for bacteria: Gentamycin 4 μg/ml.

• % Activity index (%Ac. Ind.) = (inhibition zone of sample/inhibition zone of control) × 100.

• *NA: No activity.

• The sample was tested in 20 mg/ml concentration.

Conclusion

Some metal complexes of transition and non-transitiono metal ions with new polyhydroxyl amide ligand derived from Gallic acid viz: N, N'-(1,2-phenylene) bis (3,4,5-trihydroxy benzamide, expected to have biological activity, had been synthesized and characterized by different spectroscopic and physical techniques. The stoichiometry and stability constants of some complexes were determined by electrometric techniques (conductometric and potentiometric titrations). The biological activity of the prepared complexes towards the Gram positive bacterial species; Staphylococcus aureus and Bacillus subtilis, Gram negative bacterial species; Escherichia coli, Proteus vulgaris, as well as one filamentous fungus (Aspergillus fumigatus) and one yeast species (Candida albicans) was studied using a modified well diffusion method. Gentamycin antibiotics (4 μg/ml) was taken as positive control for bacteria and ketoconazole antifungal (100 μg/ml) as positive control for fungi. It was found that the majority of metal complexes showed high activities compared to the free ligand except that for the Gram positive bacteria, Staph. Aureus, where the solid complexes showed less activity.

Journal of Chemical and Pharmaceutical Research (ISSN : 0975-7384)