INTRODUCTION:

The study of pyrazole and its derivatives have been presented as a promising research area due to their rich and extensive coordination chemistry and large area of application [1]. It is evidenced that this class of compounds possess a broad spectrum of important biological and pharmaceutical activities such as antimicrobial, antihypertensive, antitumor, anti-inflammatory and antidepressant activities [2]. It was found that some of these activities may be present as well as enhanced upon metalation of these type of ligands to an appropriate metallic centre. For example, Azan and co-workers have demonstrated that Pd(II) complexes containing 1-thiocarbamoyl-pyrazole derivatives have shown better antiamoebic activity than their corresponding free ligands [3].

Morbidity and mortality due to enteric protozoan infection remains an important health problem worldwide mainly in developing countries and regions such as the Indian subcontinent, parts of South America and tropical part of Africa [4]. Invasive amoebias is caused by Entameoba histolytica is one of the world’s most prevalent and fatal infectious diseases. Patients usually suffer from diarrhea or dysentery together with a wide range of symptoms such as stomachache, cramps, bloating or tenderness [5]. It was found that some of these activities may be present as well as enhanced upon metalation of these type of ligands to an appropriate metallic centre. For example, Azan and co-workers have demonstrated that Pd(II) complexes containing 1-thiocarbamoyl-pyrazole derivatives have shown better antiamoebic activity than their corresponding free ligands [5]. A similar behaviour of this family of complexes was reported by Netto and co-workers [6-8]. Quite serious damage to plants and animals has been observed due to various pathogenic fungal species. Therefore, in view of the emergence and diversity of drug-resistant fungal strains, the demand for novel antifungals has increased markedly in recent years [9,10]. There is a growing interest to produce and screen large number of antimicrobial agents; success of those will largely affect the discovery of cytotoxic compounds. The leads of them might become future antineoplastics as most of the current antineoplastics have had been long used as antifungals [11,12].

EXPERIMENTAL:

All material and solvents used in the research of the two companies form BDH and Fluka and used without any modification. The measurements were done at Mustansiriya University laboratories and Technology University laboratories. The melting points were recorded in Coslab melting point apparatus. Elemental analysis carried out using EA-Elemental analyser, Electronic spectra using Varian UV-visible spectrophotometer and molar conductivity measurements using Philips conductivity meter. The contents of metal in solid complexes were determined via atomic absorption using Shimadzu model 6809 and Elemental C, H, N, and S analysis were carried out with a Fison EA 1108 analysis in Ibn –Sina company. The Infrared (FTIR) spectra were recorded by using FTIR 8300 Shimadzu spectrophotometer in the frequency range of 4000-400 cm^-1 . The magnetic susceptibility of the solid complexes were obtained at room temperature using Magnetic Susceptibility Balance Johnson Matthey . The spectra of ¹H and ¹³C NMR spectra were recorded on Bruker300 MHZ in Jordan in d6-DMSO solutions.

Synthesis

The Vilsmeier- Haack reaction was used for ring closure of substituted benzoyl hydrazone in DMF/POCl₃ reagent according to the modified procedure [15]. The formylation reaction of benzaldehyde hydrazones with DMF/POCl₃ at ⁰C temperature afforded A derivative A, Scheme 1. According to the method described in literatures [7,9], followed by stirring reaction mixture at 60-65⁰C for 6 hours and neutralization with sodium bicarbonate to afford pale yellow precipitate of 1,4-diphenyl-1H-pyrazole-3-carbaldehyde.

Scheme 1.Structure of 1,4-diphenyl-1H-pyrazole-3-carbaldehyde.

Colour: Pale Yellow.

M.P C: 155-157.

MS (EI, m/z (%)): 249 (M⁺, 100), 248(M-H).

Anal. calcd. for C₁₆H₁₂N₂O: C, 77.10; H, 4.19; N, 11.00, Found: C, 76.33; H, 4.00; N, 10.88 %.

Synthesis of LH ligand

An ethanolic solution of 1,4-diphenyl-1H- pyrazol -3-carbaldehyde (0.001mol, 0.248 gm) was added to ethanol solution of 2-amino-thiazol-4-ol (0.001 mol, 0.116gm) followed acidification with drops of glacial acetic acid then refluxed for 8 hours on water bath. During refluxing the colour of the solution turned to yellow .the reaction mixture was transferred to a beaker and cooled in freezer for 2 hours to afford a solid product , the precipitate was separated, filtered, washed with ethanol twice and then dried at room temperature (Scheme 2). Recrystallization from dry methanol afford 2.73 gm (yield 61%) ; Elemental analysis cala. (found ). % N 14.96 (14.11) , % C 67.36 (66.80) , % H 4.85 (4.22), % S 8.56 (8.50)

RESULTS AND DISCUSSION:

The synthesis route for the free ligand are shown in Schemes 1 and 2. The complexes are stable solids in air, with varying shades of green colouration and their structures were established from their elemental analyses, infrared and electronic and H NMR spectra . The results of the elemental analysis are in good agreement with the calculated values of 1:1 metal to ligand combination for the metal complexes. The complexes are completely soluble in DMF, DMSO and acetonitrile, partially soluble in other polar solvents such as water, ethanol and methanol but are completely insoluble in non polar organic solvents. Low molar conductance values between 10.0 and 30.22 Ω–1 cm² mol^–1 obtained for the complexes in DMF indicates they are non-electrolytes [21] and the nature of chlorine to metal bonds can be described as coordinative. The summary of the analytical data and other physical properties of the complexes are recorded in Table 1.

H NMR spectra

The free ligand in d6-DMSO solution exhibited deshielded peaks at 11.26 ppm and 7.21-7.27 ppm that are remarkably belonged to acidic –OH attached to thiazole ring and aromatic Ar-H protons [13]. However the overlapped absorption of imine –CH=N- may be observed in the region 8.65 ppm and this will support the imine –HC=N- formation uo on condensation the amino moiety of thiazole with carboxaldehyde compound of pyrazole derivative A. [14]. The shielded protons of sovent was observed at 3.4 ppm due to moisturizing the sample . As well as the carbon-13 NMR spectra agree well with the number of carbon atoms in the formed LH ligand and this is observed from deshielded resonance of carbons related to HC=N-,HO-C=C- and –C=C-Ar respectively. The H NMR of Cd(II) complex exhibitsremarkable shift in absorptions toward deshielding region revealing the effect of electronic environment of metal d¹⁰ configuration on the position of the groups [13,14].

FTIR Spectra

The FTIR spectrum of this ligand (LH) showed some characteristic stretching bonds at : 3220, 1620, 1500, 1608 and 1159assigned to O-H , C=N , Ar , C=C , C-O and the last one is for stretching C-S bond , respectively which could found in complexes. the infrared spectrum of the ligand shows a broad bond in the range 3699 - 3552 cm-1 which is assigned to intra-molecular hydrogen -bonded O-H. The strong vibration mode at 1620 cm-1 could be attributed to isomethine group which confirms the Schiff base formation [15-17].Further confirming the in involvement of protonated phenolic oxygen in complexation .A positive (except Mn complex) shift in C=Nof in all complexes indicates the participation of azomethine nitrogen in coordination. The presence of broad absorptions in the region 3500-3430 cm-1 in the spectra of complexes suggests the presence of coordinated water which is further confirmed by the appearance a non - ligand band in region 825-813 cm-1 assignable to rocking mode of coordinated water [18,19] .The exceptional case is that the (C=N) complexes (S1 – S6 ) were found to be shifted to lower (1550-1605) wave length number to the ligand suggesting a participation of this moiet in bondingwith the metal ions [20].Another new bound was appeared which were supported by the appearance frequencies of M-N , M-O [21-22] . The major FTIR bounds and their probable assignment are given in table 2.

Table 2. Distinct vibrational modes (cm^-1) for ligand and metal complexes

ν C=C	ν M-N,M-O	ν N-N	ν C=N	ν C-O	ν OH\H₂O	Compound
1500 (s)	---	1280 m	1620 m	1159 m	3220(br)	LH
1584(m)	528 w 466	1278 m	1630 s	1170 s	3425 m	S_1(Mn)
1583(m)	592 w 433	1283 m	1610 sh	1188 s	3518 m	S_2(Co)
1590 (s)	572 m 460 w	1286 m	1570sh	1190 m	3450 m	S_3(Ni)
1588 (s)	553 m 444 w	1279 m	1533sh	1186 m	3520 m	S_4(Cu)
1587 (s)	525 w 480 m	1270 m	1605s	1180 s	3510 m	S_5(Cd)

*br-broad , s- strong , m –medium ,sh- shoulder , w- weak

Mass spectra:

Mass spectrometry (MS) is an analytical technique which identifies compounds based on the atomic sample composition of the molecules and their charge state [23]. The molecular ion at 347 and 330 may be prove the base peak of LH in the ionized form of mass spectra and agree with the proposed formula of ligand C₁₉H₁₄N₄SO. However the mass spectra of A derivative shows 100% intensity peak at 249 and other at 248 of 55% abundance due to presence of A compound in the ionization then confirm the structure [23,24].

Table(4)-GC-mass spectra of the template metal complexes.

Comp.	Peak data	Assignment peak
LH	347, 330, 298	*M⁺,M-OH
A	239, 238,190	*A⁺-H,M- ,A-Thiazoilinre

Where * refers to the molecular ion peak of the complex in gm./mole

Magnetic Moment and UV. Visible Spectra

Practically UV/Vis. Spectra of ligand in free state with ethanol solvent showed absorption peaks at (206 and 348)nm, the first peak to (π-π*) transition while the second peak present (n-π^*) transition. The peaks that related to (n-π^*) transition in the ligand dislodged toward different frequency pair of metal ion and this shift due to coordinate electrons for N and O of the ligand to the metal ion [25].The tetrahedral Mn(II) complex was generally yellow this color is an effective, simple and reliable criterion to confirm that the formed manganese(II) complex has tetrahedral geometry rather than octahedral [26] ,otherwise shows intense absorption bands in the visible region at 380nm assigned toT₁⁴←A₁⁴ transition..It also shows two strong charge transfer bands at 347 and 413 nm .The tetrahedral Mn(II) complex gives an essentially spin-only magnetic moment value of 6.0 B.M. which does not vary much since the magnetic moment is temperature independent. The observed magnetic moment value for Co(II) complex is appreciably close to the calculated spin value only at 4.00 BM suggesting the orbital contribution of high spin-tetrahedral cobalt(II) complex. The solution of cobalt(II) complex in DMF exhibits weak bands in the regions 690 nm that are assigned to⁴A₂→⁴T₁(P) represent (₃υ), while extraction the (₁υ) of Note infrared spectrum of a complex record. The complexes of Cu (II) show an absorption band in the region 540-600 nm The envelopes of these bands are generally unsymmetrical, seeming to encompass several overlapping transitions of distorted tetrahedral complex due to effect of solvent on z-axis [26-28].The cadmium(II) complex did not show any dd transition as they were d¹⁰.

Table 3.Electronic spectral, molar conductance and magnetic moments for the prepared complexes.

Comp.	nm	Molar conductivity (ohm^-1. cm^-2. Mole ^-1)	μeff BM	Geomtry
L	318, 354	……	-------	…………..
S₁	380, 347, 413	22.0	6.0	T.h
S₂	14084, 42016	10.0	4.00	T.h
S₃	690,400,290	18.22	3.22	T.h
S₄	600,540,360	30.11	2.10	T.h
S₅	390,280	12.70	D.	T.h

D=diamagnetic.

Figure 1. Biological activities for the ligand and their complexes against isolated bacterial.

Table 5. Biological activities for the ligands and their complexes and inhibition zones.

Diameters of inhibition zones (mm)							Bacterial isolates
Cd-ligand/ DMSO	Cu-ligand/ DMSO	Mn-ligand/ DMSO	Co-ligand/ DMSO	Ni-ligand/ DMSO	Ligand/ DMSO	DMSO	Bacterial isolates
16	17	15	0	11	15	-	*Staphylococcus aureus*
18	21	20	0	19	17	-	*Streptococcus mutans*
15	12	17	14	17	11	-	*Klebsiella*
17	12	20	10	15	13	-	*E. coli*

C₂₁H₁₈N₄O₅=Ligand , Co= Cobalt, Cu - Copper, Ni=Nickel, Cd = Cadmium , Mn-Magnesium, Control: DMSO

Biological activity

Biological activities of these complexes against a different bacterial isolates were experienced in the present studies and results are shown in Tables(5).The Biological activities of the test compounds were evaluated by the well diffusion method against Staphylococcus aureus, Streptococcus mutans, Klebsiella pneumonia and E. coli.

In this method, pure isolate of 24hrs growth was cultured in Muller-Hinton Agar plate (Hi Media, Mumbai, India) by using sterile swab so as to achieve a confluent growth. The plates were allowed to dry and a sterile cork borer of diameter 8.0mm was used to bore four wells in each agar plates. A 10μL volume of each complex was applied by micropipette in the wells into Muller-Hinton Agar plate. Distilled water served as control. The plates were allowed to stand for 1h or more for diffusion to take place and then incubated at 37ºC for 24hrs. The zone of inhibition was recorded [30].

Several research have shown that coordination of organic compounds to a metallic element causes significant changes in the biological activity of both the organic ligand and the metal [31-33],figure 1.The ligand showed antimicrobial activity against both kinds of bacteria. The complex Mn- ligand/ DMSO has higher inhibition zone that than the others. The complex Cd- ligand/ DMSO, Cu-ligand/ DMSO and Ligand/ DMSO also has showed significant antimicrobial activity effect except complex Co-ligand/ DMSO, which does not have antibacterial activity against Staphylococcus aureus and Streptococcus mutan shown in figure(3) .The higher inhibition zone of metal complexes than those of the ligand can be explained on the basis of Overtone’s concept and Chelation theory. On chelation, the polarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups [33] .The weak antibacterial activity for Cu-ligand/ DMSO and Co-ligand/ DMSO against gram negative bacteria was ascribed to the presence of an outer membrane which poses hydrophilic polysaccharides chains as a barrier to these complexes [32,33] .Cobalt is generally not considered to be a very toxic element [33] .A big amount of reports on the antibacterial properties of cobalt complexes have show, with Co (II) complexes being the most studied probably due to their aqueous stability, accessibility, and ease of synthesis. However, only a small number of cobalt (III) complexes have biochemical roles. Vitamin B12 is a cobaloxime, a cobalt complex having a glyoxime ligand, and is one of the unusual examples of a naturally occurring organometallic complex i.e. possess a metal carbon bond [32,35].

CONCLUSION:

The ligand 5-{[(E)-(1,4-diphenyl-1H-pyrazol-3-yl) methylidene] amino}-1,3-thiazol-4-ol was successfully synthesized by condensation of formyl pyrazole derivative with amino thiazole .The new Schiff base wasused as bi dentate ligand to prepare complexes with manganese, cobalt, nickel, copper and cadmium(II) ions. The IR spectra suggest the coordination of hydroxyl amd imine groups to link with the selkected metal ions forming tetrahedral chelates. Studies conducted on the effect of differentions on the effectiveness of the different types of bacteriapositive and negative show that the complexes due to the effects of different Biological reflect the different interactions generated by ion metalon the nature of ligand the nature of life system components Obviously complexes metallic can work in many ways shown; it canof a compound that inhibits the bacteria through the occupancy of sensitive sites on the surface of bacteria that can be used to effect in the host cells, it can also be complicated for his state of neutral or ionic that penetrates the cell wall and prevents the proliferation of bacteria[33] .

Figure2. Tetrahedral structures of the prepared complexes.

M= Mn⁺² , Co⁺², Ni⁺², Cu⁺²and Cd⁺²

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M % Calc. (found)	M.p ˚c	Color, yield %	Molecular formula	Symb.
------	180 -182	Dark yellow, 83	C₂₁H₁₈N₄OS	LH
14.40 (13.22)	290 d	Brown, 90	C₂₁H₁₉ Cl MnN₄O₂S	S₁
12.13 (12.11 )	310 d	Olive, 79	C₂₁H₁₉ Cl CoN₄O₂S	S₂
13.09 (12.93 )	315 d	Brown, 93	C₂₁H₁₉ Cl Ni N₄O₂S	S₃
15. 96 (14..96)	296 d	Deep blue, 95	C₂₁H₁₉ Cl CuN₄O₂S	S₄
20.08 (20.11)	305 d	White off, 70	C₂₁H₁₉ Cl CdN₄O₂S	S₅