SYNTHESIS, CHARACTERIZATION, ANTIMICROBIAL AND CYTOTOXIC STUDIES ON SOME NOVEL TRANSITION METAL COMPLEXES OF SCHIFF BASE LIGAND DERIVED FROM SULFADIAZINE WITH MOLECULAR ORBITAL CALCULATIONS

Some selected solid complexes of the Schiff base ligand HL derived from Sulfadiazine with Co(II), Ni(II) and Cu(II) ions were synthesized and characterized by Micro-analysis, FTIR, Electronic, Mass, and ESR Spectral Analyses, Magnetic susceptibility and Molar Conductance Measurements. The disappearance of ν(O-H) hydroxyl band of the phenolic and the lowering shift of the stretching frequency of the ν(CH=N) azomethine band in the ligand after complexation, indicated the coordination through the phenolic oxygen atom (after deprotonation) and azomethine nitrogen atom respectively of the Schiff base ligand HL. The lower values of molar conductance indicate the non-electrolytic nature of these complexes. The ESR spectrum of the HL copper complex has octahedral geometry. The molecular structures of the investigated compounds were studied by PM3 method, also the heat of formations, HOMO, LUMO and dipole moments were calculated to confirm the geometry of the ligand and the it's complexes. The antimicrobial screening of the synthesized compounds HL and its complexes 1-3 were investigated. The Schiff base ligand HL showed weaker to significant activity against one or more bacterial and fungal strains. In most of the cases higher activities were exhibited upon coordination with metal ions(II). In addition, calculations in silico, the Pharmacokinetic parameters have promising features for applying the ligand as drug.


Synthesis of the Complexes 1-3 2.3.1. General Procedures for Synthesis of the Metal Complexes
The following general procedures were used for preparing all of the complexes under investigation like that of Cu(II) complex: A solution of the Cu(NO3)2 .3H2O(1.208 g, 5 mmol) in 50 mL absolute ethanol was added dropwise to a hot solution of the HL (1.77 g, 5 mmol) in 50 mL absolute ethanol with the molar ratio 1:1.The reaction mixture was heated to reflux for 24 hours.The color of solution was dark green in the beginning of the reaction, and then became pale green at the end of the reaction.On cooling, the reddish brown solid formed, which separated out, was filtered, washed with ethanol and then air-dried.The yields were between (70-78%), with melting points between 309-319 o C.
The solid complexes were kept in a desiccator.

Physical Methods
All melting points reported for the compounds were measured on a Melting point SMP.The FT-IR spectra (350-4000 cm-1) of the investigated compounds were reported as KBr discs using

FT-IR 8400S FOURIER TRANSFORM INFR ARED SPECTROPHOTOMETER (Shimadzu).
The Electronic spectra were recorded on Shimadzu UV-Vis (1601) PC Spectrophotometer equipped with a 10 mm quartz cells, Personal Spectroscopy Software Version 3.6 Shimadzu, Tcc-240 A controller-stability± 0.1 o C Shimadzu.The Conductance mesurments were carried out in DMF solutions for HL complexes using a conductivity meter Metrohm-712 at 25 o C ± 0.1 o C.
Elemental analysis for C, H, and N was performed by elemental analyzer and the metal determination was carried out using (Perkin Elmer 3100(U.S.A).The 1 H-NMR measurements was carried out on a Varian Gemini-200,using deuterated dimethyl sulfoxide ( DMSO-d6 ) solvent.The chemical shifts ( δ ) were given down field relative to tetramethylsilane ( TMS ), as internal stander.Mass spectra were carried out using MSQP 1000 EX Shimadzu.TGA and DTA curves were obtained using NETZSCH-gerateban Bestell-Nr 348472 C. Electronic Spin Resonance (ESR) Spectrum was recorded on the Brucker ELXSYS 500 E, X-band, detection for peak without need any calibration and Magnetic measurements were measured by the Gouy method at room temperature using a magnetic susceptibility balance (Johnson Matthey alfa product, Model No.MKI ).Diamagnetic corrections calculated from Pascal's constants.

Paper Disc Diffusion Technique
The sterilized (autoclaved at 120 °C for 30 min) medium at (40-50° C) was incubated (1 ml/100 ml of medium) with the suspension (10 5 cfu ml -1 ) of the micro-organism (matched to McFarland barium sulphate standard) and poured into a petridish to give a depth of 3-4 mm.The paper impregnated with the test compounds (mg\ml -1 ) was placed on the solidified medium.The plates were pre-incubated for 1 h at room temperature and incubated at 37 °C for 24 and 48 hrs for anti-bacterial and anti-fungal activities, respectively.Cefepime (mg/disc) was used as a standard for antibacterial and antifungal activity respectively.

Minimum Inhibitory Concentration (MIC)
MIC's. of the compounds were determined by agar streak dilution method.A stock solution for each the synthesized compound (100 mg\ml -1 ) in dimethyl formamide was prepared and graded quantities of the test compounds were incorporated in specified quantity of molten sterile agar (nutrient agar for anti-bacterial activity and sabouraud dextrose agar medium for anti-fungal activity).A specified quantity of the medium (40-50 °C) containing the compound was poured into a petridish to give a depth of 3-4 mm, and allowed to solidify.Suspension of the micro-organism was prepared to contain approximately (10 5 cfu ml -1 ) and applied to plates with serially diluted compounds in dimethylformamide to be tested and incubated at 37°C for 24 and 48 hrs for bacteria and fungi, respectively.The MIC was considered to be the lowest concentration of the test substance exhibiting no visible growth of bacteria or fungi on the plate.

Molecular Modeling
The structural model was built using the BUILDER module of MOE, Optimization Conformational analyses of the built molecules were performed in a two-step procedure.First, these compounds were submitted to energy minimization tool using the included MOPAC 7.0, the geometry of the compounds was optimized using the semiemperical PM3 Hamiltonian with Restricted Hartree-Fock (RHF) and RMS gradient of 0.05 Kcal/mol.Then, the obtained model was implemented to the 'Systematic Conformational Search' of the MOE.All items were set as default with RMS gradient of 0.01 Kcal/mol and RMS distance of 0.1 Å.

The Schiff base Ligand HL and its Complexes
The Schiff base of sulfadiazine ligand HL was prepared by the condensation of salicylaldehyde (SALD) with sulfadiazine (SD) in the molar ratio 1:1 (Scheme1).The ligand HL was reacted with Cu(II), Co(II) and Ni(II) ions to yield the corresponding metal complexes (Scheme 2), the physical and analytical data of HL and corrspponding transition metal complexes 1-3 were listed in (Table 1).

Infrared Spectra
The vibrational modes of Infrared spectrum of the ligand HL, the empirical assignments was listed in (Table 2).The disappearance of both stretching frequencies, ν (NH2) of para position of the amino group, NH2 in the infrared spectrum of the HL ligand which occurs as broad bands at 3422s cm -1 , 3260s cm -1 and 3356m cm -1 in the free Sulfadiazine (SD), and the stretching frequency of the C=O group of aldehyde, which occurs at 1664 v.s.cm -1 in the free salicylaldehyde (SALD),confirmed the formation of the ligand.On the other hand, the appearance of the most intense band at 1620 s. cm -1 which was assigned to the stretching frequency of the azomethine (-CH=N) group the ligand HL, which confirmed the proposed structure of ligand HL (Scheme 1) [16].FTIR spectra of the complexes were recorded to confirm their structures.The vibrational modes frequencies and empirical assignments for the ligand and its transition metal complexes were listed in Table 3 and shown in Figure 1.The vibrational modes assignments of the metal complexes were compared with those of the free ligand HL.There are some main features in the infrared spectra of the complexes.The first feature is the lowering shift of the stretching frequencies of the ν( CH=N ) azomethine band occurs at 1620 cm -1 in the Schiff base free ligand HL by 38 cm -1 after complexation, indicating the coordination of azomethine nitrogen atom to metal ions [17], The second feature is the disappearance of the ν(OH) phenolic band occured at 3478 cm -1 in the free ligand HL, in all the complexes suggest the coordination of phenolic oxygen after deprotonation [18] .This result is further supported by the shift of ν(C-O) to 1362 cm -1 ( compared to that of the HL ligand) which was observed at 1281 cm -1 , after complexation.Also, the observed of two non ligand bands at range 457-497 cm -1 and 375 -395 cm -1 due to ν(M-O) [19], and ν(M-N) [11], respectively, in all the investigated complexes supported the coordination of phenolic oxygen atom and azomethine nitrogen atom.The ν(NH) bands due to amino group in the uncoordinated Schiff base free ligand HL remains unchanged in the spectra of it's complexes.This confirmed that the amino nitrogen atom were not contribute in coordination.

Scheme-2. Synthesis of metal complexes
The band observed at ~ 1339 cm -1 of the νas (O=S=O) of the ligand HL, were remained almost at the same positions in its complexes which mean that the sulfonamide oxygen atom did not contribute in coordination with the metal ions, the coordination of the water molecule was indicated by the appearance of a broad at 1529-1574 cm -1 due to δ(H2O) of coordinated water [20].

1 H-NMR Spectra
1 H-NMR chemical shifts (ppm) of the Schiff base ligand HL listed in (table 2).From (table 2), the signal observed at 12.524 ppm was due to phenolic OH proton of the ligand HL.The signal (δ) at 8.963 ppm was assigned to the azomethine group (CH N ) .The signals of H-protons of the phenyl group of salicylaldehyde (SALD) moiety were appeared at 7.413, 7.567 ppm while the H-protons for the phenyl signals of the para substitution moiety were assigned at 6.590, 7.709 ppm.The chemical shift of the amino NH proton of the sulfadiazine (SD) moiety was observed at 6.961 for the ligand.

Mass Spectra
The mass spectrum of the ligand HL, revealed the molecular ion peaks at m/e 354 and base peak at m/e at 290 which support the identity of the proposed structure, and the fragmentation patterns was shown in (Scheme 3).

Electronic Spectra
Electronic spectral data of the ligand HL were recorded in DMF, and exhibited absorption bands at 291, 330, 362 and 370 nm.The first and second bands were correspond to 1 L → 1 A transitions of the phenyl rings [21].The third and fourth bands correspond to the π → π * 's of the azomethine groups -CH=N-.The last band corresponds to the n → π * due to the non-bonding electrons on the oxygen and nitrogen atoms [22] The electronic spectrum of the Cu (II) complex 1 dissolved in DMF (Table 4), which exhibited characteristic bands assigned to π → π* and n → π* as well band due to 2 Eg ← 3 T2g (G) transition was observed at 712 nm.The electronic spectra of the Co(II) complex 2 dissolved in DMF, exhibited characteristic bands assigned to π →π* and n → π* transition.As well band at 525 nm was assigned to 3 T1g(F) ← 3 A2g(F), These bands associated with the octahedral structures [23] (Table 4).Furthermore, the electronic spectra of the Ni(II) complex 3 dissolved in DMF, exhibited the characteristic bands for π → π* and n → π* as shown in Table 4.
However, from the obtained date of the elemental analysis, infrared, mass spectra, 1 H-NMR and Electronic spectra, one can suggest, that, Schiff base HL act as monobasic ligand with NO bidentate sites.

Magnetic and Molar Conductivity Measurements
The measured values of the magnetic moment μeff for complexes 1-3 (Table 1), were 1.36, 1.46 and 1.28 B.M. respectively, which indicate the octahedral structure [24].The magnetic moment values measured were suggesting the possibility of the octahedral structures for the investigated complex with low spin.The molar conductance measured for complexes 1-3 were 3.88, 5.83 and 5.18 Ohm -1 cm -2 mol -1 respectively, which indicate the non electronic nature of the complex (Table 1) .

ESR Spectrum Measurement
The X-Band ESR spectrum of Cu (II) complexes 1 was recorded in the solid state at 25 o C and was shown in figures 2. The spectrum show the g|| = 2.106 and g ┴ = 2.044.These values indicate that the ground state of Cu(II) is predominately dx2_y2, which supports octahedral geometry around the Cu (II) environment in the complex 1 [25].The observed g|| value for 1 is less than 2.3, which indicating that the bond between the organic ligand and copper ion have a covalent character more than the ionic one.Furthermore, the exchange interaction between the copper centers in a polycrystalline solid was calculated (2.40) according to Hathway and Billing [26,27], it's value less than 4.0, which indicates a considerable exchange interaction in solid complex.

Thermal Analysis
Thermal gravimetric and differential Thermal analysis for Ni(II) complex 3 were obtained to give information concerning the thermal stability of the complex and to suggest whether the water and ethanol molecules in the inner or outer coordination sphere of the central metal ion.

Molecular Modeling
In trying to achieve better insight into the molecular structure of the most preferentially stereoisomer tautomeric ligand forms and complexes, the conformational analysis of the target compounds has been performed using the MMFF94 force-field [28,29], (calculations in vacuo, bond dipole option for electrostatics, Polake Ribiere algorithm, RMS gradient of 0.01 kcal/A mol) implemented in [30] (Chemical Computing Group).The most stable conformer was fully geometrical optimized by PM3 [31] semi-empirical Hamiltonian molecular orbital calculation MOPAC package.The computed molecular parameters, total energy, binding energy, heat of formation, the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) energies, and the dipole moment for investigated compounds were calculated (Table 5   and 6).It is obvious that, there is a possibility of existence the prepared ligand in both E and Z stereoisomer forms (Structure 1, Scheme 4), and more than one tautomeric forms HL-HLa ligand (Structure 2, Scheme 4) .The enhancement the computed energies of calculated molecular parameters, illustrated the Z-form is the most stable isomer form of the prepared HL ligand, and Z-HL tautomeric form (table 5, Fig. 4).The lowest minimization energy of the E-form ligand structures exhibited a common arrangement of two phenyl ring in plane with each other HL (Fig. 4), while phenyl ring of sulfa guanidine perpendicular with each metal ring and phenyl ring of salicaldehyde 1-3 (Fig. 4).

Antimicrobial Activity
The main target of the production any antimicrobial compound is inhibiting the causal microbe without or lowest any side effects on the patients.In addition, the basic idea of applying any chemotherapeutic agent, is depending on specificity of only one biological function control not multiple ones, which is the common in chemotherapeutic agent in anticancer treatment field.
In the present time, the most anticancer agents used, was affected in both cancerous diseased cells and healthy ones.So, there is urgent need for having a chemotherapeutic agent, which controls only one function.the antibacterial activity of these compounds we used more than one test organism in vitro at 37 °C, which increase the chance of detecting antibiotic.The synthesized HL and corresponding complexes 1-3, were tested using paper disc diffusion technique [32][33][34][35][36].The  Many scientists working in the new antitumours field search, depend basically on the line of antibiotics affecting Gram-negative bacteria [37][38][39] also, there are some organisms have proved to be difficult treat, and most of them are Gram-negative rods.It is therefore believed that, most the complexes are biologically active against the Gram negative strains may affecting on barrier function of the envelope of these Gram-negative strains activity, which acting in similar way described earlier [37][38][39].Since, Gram-negative bacteria are considered a quantitative microbiological method testing beneficial drugs in both experimental and clinical tumour chemotherapy [40].Therefore we claimed that, the synthesis of these complexes might be established a new line for search to new antitumour agents.

ADMET Factors Profiling
Oral bioavailability was considered playing an important role for the development of bioactive molecules as therapeutic agents.Many potential therapeutic agents fail to reach the clinic, because of ADMET (absorption, distribution, metabolism, elimination and toxic) Factors.
Therefore, a computational study for prediction of ADMET properties of the molecules was performed for the compounds HL and (SD), by determination of topological polar surface area (TPSA), a calculated percent absorption (%ABS) which was estimated byZhao, et al. [41].
Equation [41] ,and ''rule of five'', which have been formulated by Lipinski, et al. [42], which established that, chemical compound could be an orally active drug in humans, if no more than one violation of the following rule: i) ClogP (partition coefficient between water and octanol) < 5, ii) number of hydrogen bond donors sites ≤ 5, iii) number of hydrogen bond acceptors sites ≤ 10, iv), molecular weight <500 and molar refractivity should be between 40-130.In addition, the total polar surface area (TPSA) is another key property linked to drug bioavailability, the passively absorbed molecules with (TPSA>140) have low oral bioavailability [43] .All calculation descriptors were performed using MOE Package, the results were disclosed in (Table 9).Our results of HL revealed that, the CLogP (factor of the lipophilicity [44]less than 5.0, the molecular weight (MW> 500), hydrogen bond acceptors (6), hydrogen bond donors (2) and molar refractivity values approximately 96.81 more than (SD) drug which fulfill Lipinski's rule.Also, the percent absorption of compounds HL more than (SD).These data may suggest that, the ligand HL good oral absorption as antimicrobial compounds than SD as reference drug.

CONCLUSIONS
The synthesized Schiff base ligand derived from sulfadiazine has been coordinated with the Cu(II), Co(II) and Ni(II) metal ions through azomethine-N and phenolic oxygen atoms and their structures have been confirmed by analytical and spectral data.The molecular structures of the investigated compounds were studied by PM3 method, also the heat of formations, HOMO, LUMO and dipole moments were calculated to confirm the geometry of the ligand and the it's complexes.Antimicrobial studies against most tested strains showed, the metal complexes have moderate to significantly activity, and exhibited more potency than Schiff base of sulfadiazine.Furthermore, the pharmakinetic screening showed the HL is good oral absorption than sulfadizine.

Scheme 3 :
Scheme 3: Fragmentation pattern of the mass spectrum of ligand (HL).
From the TGA and DTA data water molecules and ethanol molecule were lost within the temperature rang 25-190 o C (weight loss, Found/Colc ; 14.72 / 14.75 %) whit indicate that two water and ethanol molecules were coordinated.The thermal gravimetric analysis of the complex 3 was shown in Scheme 3 and Figure 3. Finally, from the elemental analyses, molar conductivity, UV-visible spectral data, magnetic measurements, IR spectral data and TGA, it is possible to determine the type of coordination of the Schiff base HL higands in their metal complexes, the ligand behave as monobasic ligand with NO bidentate sites.All complexes of 1-3 were on the octahedral structures as shown in Scheme 2.

Figure- 4 .
Figure-4.Ball and stick rendering for the most stable isomer and \ or tatoumer form of the ligand and complexes as calculated by PM3 semi-empirical molecular orbital calculations.
tested bacterial strains were: (Gram-negative bacteria: Escherichia coli (NCTC-10416), and Gram-positive bacteria: Bacillus subtilis (NCIB-3610).The tested fungus strains were: Trechodenma viride (IMRU-3669), and Aspergillus niger (ATCC-22019).Cefepime was used as standard drug.The results of antimicrobial activity taken as inhibition zone diameter and minimum inhibitory concentration (MIC.) were depicted graphically in (Fig 5 a -d).a. Against Escherichia coli(G -) The biological activity of Cu (II), Co(II) and Ni(II) complexes are the same of free ligand HL, except of Cu (II) 1 and Co(II) 2 have higher potency than HL with concentration (3mg/ml), and Ni(II) 3 at concentration (5mg/ml) while, all complexes (1-3) have low and\or same potency compared with standard drug.b.Against Bacillus subtilis (G + ) The biological activity of Cu(II), Co(II) and Ni(II) complexes is higher than free HL ligand, except Ni(II)3 has lower activity than HL at (5mg/ml) , the Cu(II)1 and Ni(II) 3 have no activity with concentration (1mg/ml).While, all complexes 1-3 have low and\or same potency compared with standard drug.

Table - 5
. Calculated energies of possible steroisomer and tatoumaric forms of HL ligand: