SYNTHESIS AND CHARACTERIZATION OF TWO HYDRAZONES OF ISATINE AND THEIR SOME TRANSITION METAL COMPLEXES.

1. Introduction

Isatin and 1-alkylisatins furnish condensation products at the C-3 position when reacted with: hydrazine, alkyl and arylhydrazines [1, 2], heteroarylhydrazines derived from pyrimidine, pyrazine [3], thiazole, 1,2,4-triazine [4], quinazoline [5], benzimidazole, benzo-thiazole [6], phthalazine, triazines, as well as acylhydrazides of oxalic, benzoic, phenoxyacetic and oxanilic acids, arylsulfonylhydrazides, guanylhydra-zones, semicarbazines and thio-semicarbazides [8].

The reaction of 1-methylisatin and semicarbazone yielded methis-azone, a compound that found use in the treatment of smallpox, a viral disease that has now been eradicated [9]. Isatin-3-imines also react with hydrazine derivatives such as heteroarylhydrazines [10], thiosemicar-bazides and acylhydrazides, resulting in a substitution reaction at the C-3 position. Substitution reactions are also described to occur when O-methylisatin is treated with thiosemicarbazines, furni-shing isatin-2-thiosemicarbazones [11].

Isatin, due to its cis α-dicarbonyl moiety, is a potentially good substrate for the synthesis of metal complexes, either alone or with other ligands. Their derivatives, mostly those substituted at C-3, such as isatin-3-hydrazones and isatin- 3-imines bearing an extra heteroaromatic ring are also generally employed as ligands. In this manner, Schiff bases formed from isatin and amino silica gel are useful sorbents for divalent cations and for Fe (III) [11-14].

Due to its ability to bind ferric ions, isatin-3-thiosemicarbazone can be used to form magneto-polymer composites with poly (vinyl chloride) [15]. Here we report the synthesis and characterization of two Isatin hydrazones, also to report the structures of the metal complexes with hydrazones derived from the Isatin, possessing donor sites oxygen of carbonyl oxygen, hydroxyl group and azomethine nitrogen.

2. Experimental.

(A) MATERIALS

Isatin (Fluka), salicylaldehyde (Fluka), Hydrazine monohydrate (Fluka), Absolute 99/100% and Methanol (Fluka), and  2,4-dinitrophenyl hydrazine (2,4DNPH) (Fluka), were used without further purification. Cobalt acetate tetrahydrate (Fluka),Copper acetate monohydrate (BDH), Nickel acetate tetrahydrate (Riedel-de Haen),  zinc acetate dihydrate (Fluka), Cadmium acetate (Fluka) were reagent grade .

(B) SYNTHESIS OF THE LIGANDS :

(I)  Synthesis of 3-[(2- Hydroxy– benzylidene )-hydrazono]- 1,3-dihydro- indol- 2-one Ligand(HL_a)      

      Add (2.943g, 20 mmol ) of Isatin, in 100 ml absolute ethanol drop wise with stirring to Salicylaldehyde hydrazone (2.72g, 20 mmol) in 100 ml absolute ethanol in 250 ml round flask. The mixture was heated to reflux for 48 hours, during which the color of the solution changed to dark red. The formed dark red solid product was left to coagulate, then filtered off and recrystallized from absolute ethanol. The yield was (2.6515g, 50.03%)

(II) Synthesis of 3-[(2,4-dinitro-phenyl)-hydrazono]-1,3-dihydroindol-2-one Ligand (L_b)

Add (1.4714 g, 10 mmol) of Isatin, in 100 ml absolute ethanol drop wise with stirring to (1.9814 g, 10 mmol) of 2,4-dinitro phenyl hydrazine (2,4DNPH) in100 ml absolute ethanol in 250 ml round flask. The mixture was heated to reflux for 20 hours, during which the color of the solution changed to orange reddish. The former orange reddish solid product was left to coagulate, then filtered off and recrystallized from n,n-dimethyl-formamide. The yield was (2.6822 g, 57.37 %).

(III) Complexes of the Hydrazone HL_a, and L_b Ligands

The HL_a and L_b ligands reacted with Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) ions to yield the corresponding metal complexes. Table 1 lists the physical and analytical data of the hydrazone, HL_a and L_b ligands and their transition metal complexes. The complexes were investigated by Elemental analyses, FTIR UV. Visible, mass, spectral analysis and ¹H-NMR for Zn(II) and Cd(II) complexes, electronspin resonance (ESR) spectra for Cu(II) complexes, molar ratio (1:1), thermal gravimetric analyses (TGA) & (DTA) for Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) Complexes, magnetic and molar conductivity measurements.

(D) Physical Measurements

(I) Melting point

All melting points reported for the compounds are uncorrected and measured on a MEL-TEMP [SMP₃] melting point apparatus.

(II) FT-IR Spectra

The FT-IR Spectra (400-4000 Cm^-1) of the compounds were measured as KBr discs using FTIR Nicolet 6700 Thermo Scientific at microanalytical Unit, Ain shams University , Cairo, Egypt; (200-400 Cm^-1) using FT-IR 1650 ( PERKIN, Elmer cairo  University, Egypt and (200-600 Cm^-1) using Nexus 670 spectrometer at Micro-analytical Unit, at the National Research Center Dokky, Giza, Egypt.

(III) UV-Visible

The UV-Visible spectra of the investigated compounds were obtained using 8-650UV/Vis. Spectrophotometer (UK-Tenway) with one centimeter quartz cell.                                                                                                                                                                                            

(IV) Elemental Analysis

Elemental analysis for C,H,N was performed by elemental analyzer 2400 at Ain shams university , Cairo, Egypt and the determination of the metal cations was performed using Atomic Absorption spectrophotometer, Perkin Elmer 3100(U.S.A).

(V) ¹H-NMR Spectra

The ¹H-NMR  measurements  were Carried out on a varian GEMINI -200 "NMR".The deuterated Dimethyl sulfoxide (DMSO-d₆) solvent is indicated in brackets and chemical shift (ઠ) are given down field  relative to tetra methyl silane (TMS), as internal stander Egypt .

(VI) Mass Spectra

Mass Spectra were carried out On GCQ  Finnigan at microaalytical Unit,Ain shams University, Cairo, Egypt.

(VII) Thermal Gravimetric (TGA) and (DTA) Analyses :

TGA and DTA Curves were obtained using Shimadzu , DTG 60 H at the Microanalytical  Center,  Cairo  ,  University . Thermal analyzer equipped with a thermo balance . Samples (~ 50 mg) were heated at a programmed rate 20 ml/min in a dynmic N₂ atmospher . The sample  was  contained in aboat shaped platinum pan suspended in the center of furnace .  

(VIII) Electronic Spin Resonance (ESR) Spectra

ESR spectra recorded on the Brucker ELXSYS 500E. X-band with auto detection for peak without needs any calibration.

( IX ) Magnetic Measurements

Magnetic susceptibiliies were measured by the Gouy method at room temperature using magnetic susceptibility balance (Johnos Matthey alfa product , Model No.MKI). Diamagnetic corrctions calculated from pascals' constants.

(X) Biological activity

Antibacterial activities of the ligands were studied against two types of bacteria Escherichia coli (gram –ve) and staphylococcus aureas (gram +ve). The nutrient agar media wich used were constituent as gram/liter. The media contents from peptone 5.0 g/L, Beef 3.0 g/L, Sodium chloride 8.0 g/L & Agar No.2 2.0g/L at pH 7.3±0.2 in DMF as solvent.

3. Results and discussion

3.1 HL_a and L_b ligands

Infrared spectra of the reaction products between Isatin and Salicylaldehyde hydrazone (HL_a) or 2,4-dinitrophenyl hydrazine (L­_b) ligands assignments were listed in Table 1. The disappearance of both stretching frequency, the amino group, υ(NH₂) fromthe infrared spectra of the hydrazone (HL_a) and (L_b) ligands which were appeared as broad bands at 3286 cm^-1 and 3105 cm^-1 in the free salicylaldehyde hydrazone (SH) and 2,4- dinitrophenyl hydrazine (2,4DNPH); respectively, and the stretching frequency, υ(C=O) of the keto groups C=O, were appeared at 1728 cm^{-1 in the free Isatin, were shifted to low frequencies at 1720cm-1} and 1636cm^-1 for HL_a and L_b respectively. The appearance of most intense band at 1620 and 1595cm^-1, which was assigned to the stretching frequency, υ(-C=N), of the azomethine (-C=N) group for the HL_a and L_b respectively. These bands were characterized for the azomethine moiety of most the azomethine compounds [16].

Table:1. Characteristicbands of the hydrazone, HL_a and L_b ligands  Isatin (IS), salicylaldehyde hydrazone (SH) and2,4- dinitrophenyl hydrazine (2,4 DNPH) and their assignments.

*  s = strong, m = medium, w = weak, vs = very strong, sh = shoulder,

    br = broad, and oop = out of plane.

Continued:

*  s = strong, m = medium, w = weak, vs = very strong, sh = shoulder,

    br = broad,and vw= very weak.

¹H-NMR spectra of the hydrazone: The ¹H-NMR spectra of HL_a and L_b ligands in DMSO-d₆ and chemical shifts (ppm) of the ligands were listedin Table 2. The chemical shifts of proton signals of the phenolic OH of HL_a ligand at 11.013 ppm and the chemical shifts of the protons N-H groups of isatin moieties for HL_a and L_b were assigned at 10.674 ppm and 10.294 ppm respectively.

The mass spectra of HL_a and L_b ligands, revealed the molecular ion peak at m/e 265 of the HL_a and 327 for the L_b ligands which coincident with the formula weight (265) for the HL_a ligand and (327) for the L_b ligand and supported the identity of the proposed structures. The base peak corresponds to the loss of the (o-ph-CN) at m/e (118). A peak corresponding to the loss of CO (ion a) can also be observed at m/e (237), whose intensity decreases to 20.61%. Ion a usually looses HCN, leading, to a fulvene ion (ion b). An arene aziridine is also observed (ion c), which arises from a second loss of CO [17-23]. The ions b and c are also observed in the gas-phase pyrolysis of isatin [24]. In the mass fragmentation pattern of the L_b ligand, a peak corresponding to the loss of CO (ion a) can also be observed at m/e (299),with decreased intensity (0.94%). Ion (a) usually looses HCN, leading to ion (b) at m/e (272) and ion (c) at m/e (91).

Table 2: ¹H-NMR chemical shifts (ppm) for the HL_a and L_b ligands and their assignments.                                                                                 

                                           L_b                                                                       HL_a    

(1) s = singlet

(2) Chemical shift (ppm)were referenced internally at 25⁰C with respect to TMS.

(3) Notations are illustrated in the above structure .

(4) Signals disappeared after the addition D₂O.

Electronic spectral analyses of the HL_a and L_b ligands and their transition metal complexes were shown in Figures 1 and 2; recorded in DMF solutions and listed in Table 3. The HL_a ligand exhibited five absorption bands at 272.5, 309, 340, 360, and 420, also the ligand L_b exhibited at 272.5, 309, 339, 364, and 425.5 nm. The first and third bands correspond to ¹L_a→¹A₁and ¹L_b→¹A₁ transitions of the phenyl ring [25]. The band 309 nm for each HL_a and L_b is attributed to the π→π^* transition of the C=O group of the isatin moieties. The bands observed at 365 nm for HL_a and 364 nm for L_b ligands were corresponded to the π→π^*transition of the azomethine group (C=N), and the last band at 420nm (HL_a) and 425 for (L_b) was corresponded to the n→π^*transition due to the lone pairs electron of the oxygen and nitrogen [26]. However, from the elemental analysis, infrared, mass spectra, ¹H-NMR and electronic spectra, it is expect that, the hydrazone, HL_a ligand acts as monobasic ligand with NO₂ tridentate sites, while the L_b ligand acts as monobasic ligand (Enol) with NO₂ tridentate sites.

3.2 HL_a and L_b ligands complexes      

                IR spectra of the complexes were recorded to confirm their structures. The vibration frequencies and their empirical assignments for HL_a and L_b ligands with their transition metal complexes were listed in Table 4 and shown in Figures 3, 4. The vibrational mode assignments of the metal complexes were supported by comparison with the vibrational frequencies of the free ligand and other related complexes; such as the metal complexes of 2-hydroxy-1-naphtalideneisatinhydrazone [27]. There are main features in the infrared spectra of the investigated complexes. The first feature is the disappearance of the phenolic (OH) band of HL_a ligand and (OH) of the enolic form of isatin moiety of L_b ligand which were observed at 3498 cm^-1 and 3456 cm^-1 respectively, from their complexes which indicate the deprotonation of this group, and the participation of the oxygen atom in the coordination to metal. As the consequent involvement of the oxygen atom in the coordination to metal in all the investigated complexes of HL_a and L_b ligands the band assigned to the vibration frequency of both the phenolic and enolic (C-O) groups undergoes positive shifts, confirming that the two ligands HL_a and L_b were bonded to the metallic ions through the phenolic and enolic oxygen atoms respectively. The second feature is the shiftof the bands corresponding to υ(C=N) to lower frequencies (∆υ =3-22 cm^-1) in the spectra of the HL_a and L_b complexes which indicated the participation of the nitrogen atom of the (C=N) group of azomethine of

Figure 1: Electronic absorption spectra of the HL_a , ligand and its  complexes with Co⁺²,Ni⁺²,Cu⁺²,Zn⁺²,andCd⁺²ion.

Figure 2: Electronic absorption spectra of the L_b , ligand and itscomplexes withCu⁺²and Zn⁺² ion.

Figure 3. Infrared spectra of the: (a) HL_a ligand, (b)[Zn(L_a)(CH₃COO)]CH₃OH,  (c) [Cu(La)(CH3COO)].CH₃OH complexes.

Figure 3. Infrared spectra of the: (a) HL_a ligand,                             

(b) [Zn(L_a)(CH₃COO)]CH₃OH, 

(c) [Cu(La)(CH₃COO)].CH₃OH complexes.

Figure 4. Infrared spectra of the :(a) L_b ligand, (b)[Cu(L_b)(CH₃COO)] (c)[Zn(L_b)(CH₃COO)]complexes.

the Isatin moiety in the coordination. Unfortunately the participation of the oxygen of keto form of HL_a ligand in the coordination with metal ions did not identify because of the appearance of υ(C=O) of the coordinated acetate group at the same region. The participation of the acetate groups in coordination with metal ions in all the complexes of HL_a and L_b ligands, as a monodentate acetate group, show two bands at (1534–1549 cm^-1) and (1426 – 1433 cm^-1) ranges for HL_a complexes and  (1573, 1576 cm^-1) and (1417, 1428 cm^-1) for Cu(II) and Zn(II) complexes of L_b which were assigned to υ_as acetate and υ_s acetate vibrations respectively [28].

The electronic spectrum of the Cu(II) complexes (HL_a, L_b) exhibited two broad bands for each at 19350 and 13760 cm^-1 for copper complex of HL_a and 22321 and 13860 cm^-1 for L_b copper complex. They were assigned to ²B_1g → ²A_1g and ²B_1g → ²E_g, respectively [29]. These transitions, as well as the measured magnetic moments ( µ_eff ) 2.02 and 1.87 BMfor HL_a and L_b copper complexes respectively, suggest a square–planar geometrical structure for (3) and (6) complexes. The molar conductance of Cu(II) complexes (3) and (6) were 15.6 and 2.73 ohm^-1cm²mol^-1 respectively, which indicate the neutral nature of the complexes.

X-Band ESR spectra of Cu(II) complexes(3) and (6), were recorded in the solid state at 25⁰C and shown in Figures 5 and 6. The spectra showed two bands with g = 2.0523 and g = 2.207for complex (3) and g = 1.998 and g = 2.159 for complex (6), which attributed to large component of low symmetry in the ligand field. The shape of the spectra was consistent with the square planar geometrical structure around the Cu(II) environment in the complexes [30].

Figure 5:  ESR spectrum of the: [Cu(L_a)(CH₃COO)].CH₃OHcomplex

Figure 6:  ESR spectrum of the: [Cu(L_b) (CH₃COO)] complex

The mass spectrum of [Cu(La)(CH₃COO)].CH₃OH complex (3), revealed the molecular ion peak at m/e 418.32 which coincident with the formula weigh [418.54] and support the identity of the structure.

 Thermal gravimetric analysis for [Cu(La)(CH₃COO)].CH₃OH complex (3), was obtained to give information concerning the thermal stability of the complex to decide whether the Methanol molecule was in the inner or outer coordination sphere of the central metal ion. From TGA data methanol molecule was lost within the temperature range 25-100 ⁰C (weight loss, Found /calc., 7.65/7.64) which indicate that methanol molecule was uncoordinated. The thermal gravimetric analysis of the [Cu(La)(CH₃COO)].

The electronic spectrum of the  Zn(II) complexes and Cd(II) complex exhibited shift to lower and higher frequencies, for the n→π* and π→π* transitions, compared to those of the ligands as shown in Table 3.4, indicating the coordination of the ligands HLa and Lb to the Zn(II) and Cd(II) ions. The Zn(II) and Cd(II) complexes (4), (7), and (5) are diamagnetic and there is no either electronic d-d transition or significant magnetic moment. The absorption bands observed at 29481cm^-1, 29412cm^-1, and 29550cm^-1 for Zn(II) and Cd(II) complexes (4), (7) and (5) respectively were assigned  as charge transfer transitions [31, 32]. On the base of the spectra and the magnetic moments measurements of the complexes (4), (7) and (5), a tetrahedral geometrical structure could be suggested for the investigated complexes of Zn(II) and Cd(II) ions with HLa and Lb ligands.

Molar conductance values of Zn(II) and Cd(II) complexes in DMF solution were 4.33, 19.79 and 4.01 ohm^-1.cm².mol^-1, for the complexes (4),(5) and (7) respectively, which indicated the non electrolytic  nature of the complexes .

Thermal gravimetric analysis for the [Zn(La)(CH₃COO)]. CH₃OH, H₂O complex (4) was obtained to give information concerning  the thermal gravimetric stability of the complex and to decide whether the methanol and water molecules were in the inner or outer coordination sphere of the central ion. From the TGA data methanol and water molecules were lost at the temperature range 25-170⁰C (weigh loss; Found /Calc; 12.44/11.48) which indicate that methanol and water molecules were uncoordinated.

The electronic spectrum of Ni(II) complex (2) of HLa ligand showed several bands. Generally, three spin allowed transition are expected, because of the splitting of the free ion, ground ³F term and the presence of the ³p term. Tanab-sugano diagram can be used to interpret the spectra, usually, the spectra of tetrahedral Ni(II) complexes consist of bands which are accordingly assigned as charge transfer like that observed  at  30902 cm^-1, 18315 cm^-1 (⁴A₂ → ⁴T₁(p) ) transition, 40650 cm^-1 and 34482 cm^-1 which were found in the spectrum of the HLa ligand of the ππ*  transitions but they were shifted to lower and higher frequencies, confirming the coordination of the ligand to the metallic ion.

The measured value of the magnetic moment µ_eff, was 4.45 BM, which indicate the tetrahedral geometrical structure of the complex (2). The molar conductance of Ni(II) complex (2) was measured in  DMF  solvent  and  the  measured value was 0.4 ohm^-1cm²mol^-1 which indicate the non electrolytic nature of the complex (2). Thermal gravimetric diagram for the [Ni(La)(CH₃COO)].H₂O complex (2) was measured to obtaine information concerning the thermal gravimetric stability of the complex and decide whether the water molecule was in the inner or outer coordination sphere of the central ion. From the TGA data water molecule was lost with the temperature rang 25-100⁰C (weigh loss; Found/calc.; 4.3/4.5 which indicated that water molecule was uncoordinated.

The electronic spectrum of the Co(II) complex of HLa ligand (1) is expected for the octahedral structure. According to Tanade – sugano diagram [33] the possible transitions of the Co(II) octahedral complex can be interpreted. Three bands are usually associated with the spectrum of the Co(II) octahedral complex. The first band, which is assigned to ⁴T_2g(F) ← ⁴T_1g(F) transition, which occurs in the near infrared region, was not observed because it is out of the range of the used instrument. The second band was not observed which is due to ⁴A_2g(F) ← ⁴T^1g(F) transition. The third band observed at 635 nm is due to ⁴T_1g(P)←⁴T_1g transition. This transition may be overlapped by the ligand n→π* transition which was observed at 420 nm for HLa ligand.

The magnetic moment measured, 1.82 B.M. indicated the octahedral geometrical structure for the complex (1) [34]. The molar conductance of Co(II) complex (1) was measured in DMF solvent and the measured value was 0.99 ohm^-1cm²mol^-1 which indicate the non electrolytic nature of the complex (1).

Thermal analysis diagrams of the [Co(La)(Ac)] complex (1) was measured to obtain  information concerning the thermal gravimetric stability of the complex and to decide whether the ethanol and water molecules were in the inner or outer coordination sphere of the central  ion. From TGA and DTA one endothermic with loss in weight, due to the elimination of water molecule with the temperature range over 190 ⁰C to 210 ⁰C (weight loss; Found/calc.; 4.01/4.03 which indicated that water molecule was coordinated to the central metal ion Co(II). Also, the ethanol molecule was lost after awhile to at 255 ⁰C (weight loss; Found/calc.; 14.45/14.35 %).

From the results and Discussion given above, one can expect the metal chelates of the ligands HLa and Lb would acquire structures.

The Biological activity

The effect of the HLa ligand on germination of the types of bacteria was studied against two types of bacteria: Escherichia Coli (g –ve) and staphylococcus aureas (g +ve). The effect of the ligand on the growth of E.coli (g -ve) there was no inhibition zone formed in the petridish which was contained the media with bacteria but the effect on staphylococcus aureas (gram +ve) there was presence of inhibition zone surrounded the ligand HLa. The resulted were presented here indicated that, generally the ligand HLa inhabited and killed the cells of bacteria (staphylococcus aureas, g +ve) may be by a decline in the mitotic division and destroy all bacteria, and stopped the growth, finally killed all the bacteria (staphylococcus aureas, g +ve) with used HLa ligand.

Conclusion  

  The hydrazone, HLa and Lb, ligands and their corresponding transition metal complexes were prepared by the condensation of isatin with salicylaldehyde hydrazone or 2,4-dinitrophenyl hydrazine, respectively, in the molar ratio 1:1. These compounds were characterized by elemental analyses, IR, 1H-NMR, Electronic and Mass spectra. From the obtained data, the physical, analytical data and the structures were proposed, which are were octahedral (Co(II)-HLa), tetrahedral for Ni(II), Zn(II), Cd(II)-HLa complexes and square planar for (Cu(II)-HLa, Lb Zn(II)-Lb complexes. We can used the ligand HLa as antibacterial for type of bacteria (staphylococcus aureas, g +ve).