Biheterocyclic ligands : synthesis , characterization and coordinating properties of bis ( 4-amino-5-mercapto-1 , 2 , 4-triazol-3-yl ) alkanes with transition metal ions and their thermokinetic and biological studies

The Co(II), Ni(II) and Cu(II) metal ions complexes of Bis(4-amino-5-mercapto-1,2,4-triazol-3-yl) alkanes (BATs) have been prepared and characterized by elemental analysis, conductivity measurements infrared, magnetic susceptibility, the electronic spectral data and thermal studies. Based on spectral and magnetic results, the ligands are tetradentate coordinating through the N and S-atoms of BATs; six-coordinated octahedral or distorted octahedral and some times four-coordinated square planar were proposed for these complexes. Activation energies computed for the thermal decomposition steps were compared. The ligands and their metal complexes were tested in vitro for their biological effects. Their activities against two gram-positive, two gram-negative bacteria and two fungal species were found to vary from moderate to very strong.


Introduction
In recent years, considerable attention has been paid to the synthesis of biheterocyclic compounds that play a significant role in many pharmacological, biological activities, therapeutic effect and plant growth [1][2][3][4][5][6][7].Antimicrobial activities of some new biheterocyclic compounds were reported recently [5].Antibacterial activities of biheterocyclic compounds are much better than that of heterocyclic [8].Biheterocyclic compounds act as a powerful chelating agent, forming stable coordination polymers, complexes with various transition metals, trace elements, lan-thanide(III), uranium(VI) and thorium(VI) ions [9][10][11][12] Based on spectral and thermal studies, a coordination number of seven was proposed for niobium(V) complexes with biheterocyclic ligand [13].Six-coordination octahedral structures for some transition metal ions complexes of biheterocyclic ligands were determined by magnetic susceptibility measurements, and infrared and electronic spectral data [14].
In 1999, the synthesis of the biheterocyclic compounds of interest, Bis(4-amino-5mercapto-1,2,4-triazol-3-yl) alkanes, (BATs) were first reported [7] by P. F. Xu et al .The characteristic structure of the BATs (i.e.BAT1, BAT2, BAT3 and BAT4) is apparent in the existence of the sulfur of thione and nitrogen of amino groups on both sides of a molecule.This makes the compounds (BATs) electron-rich ligands.However, literature survey has revealed that no attempt has been made to study the complexes of some transition metal ions with the above mentioned ligand compounds.It is a thought of interest to study the synthesis and characterization of the Co(II), Ni(II) and Cu(II) complexes of BATs ligands.The processes of thermal degradation of these metal complexes have been investigated by thermoanalytical (TG, DTG) and Coats-Redfern integral method [15] has been used to determine the associated kinetic parameters for the successive steps in the decomposition sequence.
Some literatures [16][17][18] indicated that in some transition metal complexes with heterocyclic or biheterocyclic are biologically more active than the free ligands.Therefore, the biological activities of the ligands BATs and their metal complexes of the Co(II), Ni(II) and Cu(II) are tested against four strains of bacteria (i.e. two gram-positive and two gram-negative bacteria) and against two fungal species.The effect of the methylene group number between two heterocyclic rings on the biological activity [19] is considered.

Materials
All chemicals, solvents, indicators, metal(II) chlorides, and starting materials required and used as appropriate, were commercially available from BDH and Aldrich chemical Co.

Preparation of the complexes
For metal ion of Co(II), Ni(II) and Cu(II), complexes with the various ligands of interest (BAT1, BAT2, BAT3 and BAT4) were synthesized and collected pure after recrystallization.
In general, 0.01 mol of each ligand (BAT1=2.44 g, BAT2=2.58g, BAT3=2.72 g and BAT4=2.86 g) was dissolved warm in 100 ml ethanol by warming up the solution.As 1: 2 mole ratio was adopted, a 0.02 mol metal chloride (CoCl 2 .6H 2 O=4.758 g, NiCl 2 .6H 2 O=4.754 g and CuCl 2 .2H 2 O= 3.4108 g) in 50 ml ethanol was added dropwise to the 0.01 mol ligand solution.The solution mixture was refluxed (3-4 hours) until precipitation occurred.The precipitates obtained were filtered off and recrystallized from ethanol and their yield percent are given in Table 1.

Chemical and physical measurements
Stuart scientific electrothermal melting point apparatus was used in measuring the melting point of the ligands and their complexes.Metal content was estimated using Perkin-Elmer 2380 atomic absorption spectrophotometer, and chloride was determined gravimetrically using standard method [20].Carbon, hydrogen, nitro-gen and sulfur analysis were determined microanalytically using a Vario EL Fab.CHNS Nr.11042023 instrument.Thin Layer Chromatography (TLC) was carried out on Silica Gel GF 254 plates (mn-kieselgel G., 0.2 mm thickness) with a 3:1 v/v ethylacetate / petroleum ether solution as eluent.The plates were scanned under ultraviolet light 254 nm lamp [21].
The molar conductance of 10 -3 M solutions of the ligands and their metal complexes in DMF solvent were measured on a HACH conductivity meter model sens ion 5.All the measurements were taken at room temperature on freshly prepared solutions.
The electronic spectra of the ligands and their complexes were recorded in nujol mulls in the range 200-1100 nm on a Perkin Elmer Lambda 35 UV/Vis spectrometer.IR spectra of these compounds were recorded on Shimadzu DR-8001 infrared spectrophotometer in KBr pellets.
Magnetic susceptibility measurements of the solid complexes were carried out at room temperature using a Johnson Metthey and Sherwood magnetic susceptibility balance, and the data collected were processed in accordance with an earlier report [22].Diamagnetic corrections were carried out using standard Pascal's constants.
Thermogravimetric Analysis (TGA) experiment was conducted using TGA-50H thermal analyzers.All experiments were performed using a single use top loading platinum sample pan under nitrogen atmosphere at a flow rate of 30 ml/min and a 10°C/min heating rate in the temperature range 25-800°C.The data were extracted from the TG curves for each decomposition step with the assistance of the DTG curves and hence kinetic parameters of decomposition were evaluated using the Coats-Redfern method [15], as appropriate.

Biological screening
The ligands and their metal complexes were tested for their antimicrobial activity against four species of bacteria (Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae, Pseudomonas aeruginosa), and two fungal species (Aspergillus flavus and Candida albicans) using filter paper disc method [23].The diameters of inhibition zones (mm) were measured at the end of an incubation period of 24 hours at 37 °C for bacteria, and 4 days at 28 °C for fungi.Discs saturated with DMSO are used as solvent control.Ampicillin 25 µg/ml was used as a reference substance for bacteria and 30 µg/ml Mycostatin for fungi [6,21].

Results and discussion
Table 1 compares the elemental analysis, molar conductance and some other physical properties of the BATs ligands and their metals complexes under investigation.The prepared metals complexes show single spots on their thin layer chromatography with satisfactory R f values (Table 1), indicating their purity [24].All the complexes are colored, quite stable in atmospheric conditions and insoluble in water and ethanol, but most of them are partially soluble in acetone, Chloroform, Carbon tetrachloride, DMF and DMSO.Most of the complexes have high melting points (>350°C) and do not melt but decompose at higher temperature.However, few of them melt and their melting points appear at the 242-320°C range.
The proposed empirical formulae of the complexes are in good agreement with the stoichiometry of 2:1, metal : ligand concluded from their analytical data (Table 1) and the thermogravimetric analysis results (Tables 4 and 5).The molar conductance values (Table 1) of 10 -3 M solutions in DMF solvent are in the range of 3.7-10.9mS cm 2 mol -1 , indicating a non-electrolytic behavior of these metal complexes and clearly suggest the coordination of the anions with the metal ions [25].The ligands and their metal ions complexes are characterized by elemental analysis, molar conductance, IR spectra, electronic spectra, magnetic susceptibility and with assistance of thermal analysis (Tables 1-5).

Infrared spectra studies
Table 2 shows the significant IR absorp- tion bands of the BATs ligands and their twelve complexes.The IR spectra of BAT1, BAT2, BAT3 and BAT4 accord with the literature [7].A broad IR band in the range 3142-3474 cm -1 in the complexes is attributed to the overlapping vibration of νNH(NH 2 ) with that of νOH(H 2 O) of coordinated water [26].Coordinated water shows metal-oxygen ν(M-O) stretching vibration in the 426-480 cm -1 [27].
The appearance of the four thioamide bands (I-IV) at 1570 -1581, 1302 -1327, 920 -982 and 745 -781 cm -1 [27], refutes the presence of the bands in the 2400-2600 cm -1 regions and at 606 cm -1 due to ν(SH) and ν(C-S), respectively [28] confirming that the ligands of BATs exist mainly in the thione form [5].These four thioamide bands of the ligands are compared with those of their complexes in Table 2, indicating the shift due to complexation.The bands at 1240-1253 cm -1 in the spectra of the ligands assigned to ν(C=S) is lowered by 8-88 cm -1 in the spectra of the complexes, indicating involvement of thione groups sulfur in the coordination.This leads to new bands in the region 350-385 cm -1 tentatively assigned to M-S vibration [27].
The shift to lower frequency of the amino group by 20-124 cm -1 may suggest the participation of the nitrogen atom in the complexation.The bands at 1027-1044 cm -1 due to ν(N-N) in the free ligands are shifted to lower frequencies by 27-120 cm -1 in the complexes formation spectra [29], confirming the coordination through the nitrogen atoms .In addition, the shift in ν(C=N), for C=N being adjacent to N-N in the molecular structure, is to lower frequency by 4-108 cm -1 [13].Coordination through the amino groups nitrogen is consistent with new bands in the 400-406 cm -1 , assignable to the ν(M-N) vibration [23,27,30].The new band at the low frequency 325-359 cm -1 is assigned to ν(M-Cl).The above findings indicate the tetradentate (N and S donor atoms) nature of ligands, enhancing the coordination of two metal ions with one ligand molecule.

Electronic and magnetic studies
Table 3 compares the magnetic moments, electronic spectral data and ligand field parameters of the Co(II), Ni(II) and Cu(II) complexes with the ligands of BATs.The electronic spectrum of the ligands BAT2 and BAT3 exhibit their two absorption maxima in the ranges 42194 -33333 cm -1 and 42373-33112 cm -1 , assignable to π→π* (K-band) and n→π* (R-band) transitions [27], respectively, (Table 3).
From the electronic spectra and magnetic moment data the structures and geometries of these Co(II), Ni(II) and Cu(II) complexes are identified and interpreted, as illustrated in structures I and II and given in Table (3).The electronic spectrum of the Co(II) complexes, Table 3. Magnetic moment, electronic spectral data in Nujol mull and ligand field parameters for the BATs and their Complexes.
Due to instrumental limitation, the values of ν 2 and ν 3 obtained from the electronic spectra are used to calculate the third transition, 4 T 1g → 4 T 2g (F) (ν 1 ) band by the equations used for the d 7 system [32] and found to be 6948 cm -1 .From these transition bands the ligands field parameters (Table 3) calculated for the Co(II) complex of BAT3 ligand are B = 889 cm -1 , β = 0.92, 10Dq = 7889 cm -1 and ν 2 /ν 1 = 2.14 , respectively.Reduction of the B value of the free ion from 971 cm -1 to 889 cm -1 (i.e.β from 1 to 0.92) on the complexation indicates the covalent character of metal-to-ligand bonds [33].The lower is the value the greater is the covalency.
The  [34] for an octahedral geometry around the Co(II) ion (structure I).However, the subnormal magnetic moments of the Co(II) chelates reflect a behavior of the binuclear complexes [30].The 4.27 B.M. value is lower than that previously reported [35].However, magnetic moments lower than 4.80 B.M for the octahedral Co(II) complexes has been observed [36].The anomalous magnetic moment suggests a lowsymmetry around the cobalt (II) ion [37].Moreover, the gray and brown colors of the complexes are in good agreement with those reported for octahedral Co(II) complexes [33].
The observed charge transfer band recorded for the complex of BAT3 at 25,000 cm -1 is mainly due to S→Co [27,32].This charge transfer transitions probably from the π-orbitals of the donor atoms to the metal ion.The UV bands, π→ π* and n→π*, of the ligand most likely moves to lower energy on complexation [29].
The other Co(II) complexes, [Co 2 (BAT1) OH have a magnetic moments (2.67 and 2.10 B.M, respectively) which fall in the region reported for one unpaired electron existing in both square-planer and low spin octahedral geometries [27].The absence of octahedral characteristic bands spectra excludes the low spin octahedral configuration, whereas the existence of a broad band at 20,000 cm -1 in the [Co 2 (BAT2)Cl 4 ]•3/4H 2 O•1/2C 2 H 5 OH electronic spectrum may suggest a square-planer geometry (structure II).Also, the existence of the three bands at 12,255, 14,881 and 16,779 cm -1 confirms the proposed geometry [27,32].These observations together with the magnetic moment value (2.10 B.M.) support the presence of a square planar environment around the cobalt (II) ion [32].
The [Ni 2 (BAT1)Cl 4 ]•H 2 O complex is diamagnetic, which factually supports (structure II) square-planar geometry [30].The commonly square-planar Ni(II) complexes are orange or red but a few have purple or green colors [32].

Copper(II) complexes
The  O possess magnetic moments (1.45 and 1.58 B.M., respectively) found to be within the range reported for the d 9 -system containing one unpaired electron [38], and assignable to a distorted octahedral geometry (structure I).The dark brown color of these complexes supports the proposed geometry [38].
The electronic spectrum of show weak shoulders at 18,797 cm -1 and 13,736 cm -1 .These bands are due to 2 B 1g → 2 E g and 2 B 1g → 2 A 1g transitions in a distorted octahedral geometry [30,32].The broadness of the observed band may be due to Jahn-Teller effect [39], which enhances the distortion of the octahedral geometry.[39] for a squareplanar geometry (structure II).The lower value of µ eff (1.45, 1.58 and 1.41 B.M.) than that calculated (1.73B.M.) for one unpaired electron is normal and expected if binuclear complexes are considered [30].Thus, in general a decrease in the magnetic moment below the spin-only value (1.73B.M.) may be attributed to molecular association taking place to form bi-or poly nuclear molecules [40].Little changes in the ligands bands (π→π* and n→π*) energies are detected on complexation [29].It is proved that the bands observed at 20661 and 21736 cm -1 are assigned to S→Cu(II) transition [27], and that at 28421 and 28409 cm -1 may be assigned to d→π* [41].

Thermal analysis
Tables 4 and 5 compare the characteristic thermal and kinetic parameters determined for each step in the decomposition sequence of the complexes.It can be seen clearly (Tables 4 and 5) that the mass losses (∆m%) obtained from the TG curves and that calculated for the corresponding molecule or molecules are in good agreement as is the case for all of these complexes.However, as the compositions of the final decomposition products (i.e.final residues) are not proved, illdefined final states are considered for the thermal decomposition of these six complexes.The integral method used is the Coats-Redfern equation [15] for reaction order n ≠1, which when linearised for a correctly chosen n yields the activation energy from the slope; where: α = fraction of weight loss, T = temperature (K), n = order of reaction, Z = pre-exponential factor, R = molar gas constant, E a = activation energy and q = heating rate.
The TGA (TG and DTG) curves recorded for the BAT2 and BAT3 ligands are given in Figure 1.The BAT2 and BAT3 ligands exhibit almost similar thermal decomposition process and the resulting mass loss (∆m %), order reaction (n), activation energy (E a ), initial (T i ), final (T f ) and maximum rate (T DTG ) temperatures of mass loss are given in Tables 4 and 5.The ligands BAT2 and BAT3 start losing 43% and 49% of their masses in two rapid and consecutive steps in the 130-414°C range, followed by slow and continuous bleeding of 41.5% and 29.8% mass losses in the 262-800°C range with 15.5% and 21.2% residual mass at the end of the reaction (800°C), respectively.In the two apparent consecutivesteps, the strongly sharp peaks (T DTG ) at 249°C and 324°C due to the rapid and consecutive 19% and 24% mass losses with activation energies 234 kJ mol -1 and 201 kJ mol -1 observed for the BAT2 ligand are more or less comparable with that at 230°C and 295°C with 19% and 30% mass losses and activation energies of 186 kJ mol -1 and 184 kJ mol -1 observed for BAT3 ligand.
The TG/DTG curves of the metal-complexes in Figures 2 and 3 reveal that with overlapping steps being various, three, four or five steps in the sequential decomposition of BAT2 and BAT3 metal-complexes are observed.The The second-step of decomposition in the 112-400°C range indicates the mass losses between 5.57 and 27.70% may be due to either the release of various ratio of coordinated water molecules The third-step of these complexes occurs at the 315-608°C range with a gradual mass loss ranging from 10.60 to 35.30%.This corresponds to either the release of two chloride atoms, one chloride atom along with various percent of backbone components or various percent of backbone components (cal.10.70 to 35.29%) with activation energies between 109-282 kJ mol -1 .The third-step (400-608°C) is an end of reaction step for the decomposition of Ni-BAT2 complex leaving a 27.90% mass loss that is in consistent with the final product (cal.28.04%) of an ill-defined final state.
The fourth-step at the 414-648°C range is assigned to the removal of one chloride atom along with 15.5% of the backbone components (found 12.60%, cal.12.50%), one chloride atom along with 16.2% of the backbone components (found 12.63%, cal.12.61%) and 23% of the backbone components (found 10.80%, cal.10.70%) in the decomposition process of Co-BAT3, Ni-BAT3 and Co-BAT2 complexes with activation energy of 233, 314 and 237 kJ mol -1 , respectively.In the contrary, this fourth-step in the 499-800°C range is the end of decomposition of Cu-BAT2 and Cu-BAT3 complexes for eliminating the remaining 52% (found 23.40%, cal.23.50%) and 58% (found 24.20%, cal.24.22%) of the backbone components with the deposition of illdefined final residues 32.70% (cal.32.10%) and 28.80% (cal.28.70%) at the end of the reactions, respectively.Their activation energies found to be 98 and 242 kJ mol -1 , It is found that release of the coordinated water molecules and chloride atoms fairly accord with literature [42,43].
It can be concluded that the thermal decomposition of these six complexes is of a variety of degrees of similarity (Table 4 and 5).For instant, the first, second and third steps in the sequential decomposition of the square-planar complexes of Co-BAT2 and Cu-BAT2 are of very close thermal and kinetic parameters.This is, maybe, due to similar nature of decomposition process, i.e. similar mechanism and kinetics of the decomposition reaction that took place in the first three steps of the Co-BAT2 and Cu-BAT2 complexes.Acceptable similarities in the thermal and kinetic parameters are observed for the five steps in the decomposition sequence of the Co-BAT3 and Ni-BAT3 octahedral complexes.However, this is inconsistent with the thermal decomposition of the octahedral complexes of Ni-BAT2 and Cu-BAT3 with their three and four steps in the decomposition process.

Biological activity
The ligands BAT1, BAT2, BAT3 and BAT4 under investigation and their Co(II) Ni(II) and Cu(II) complexes were screened in vitro for their antibacterial activity against four strains of bacteria, i.e. two gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes) and two gram-negative bacteria (Haemophilus influenzae, Pseudomonas aeruginosa), and two fungal species (Aspergillus flavus and Candida albicans).Discs saturated with DMSO solvent, which show no zone of inhibition (-) were used as control and the results of the antibacterial and antifungal activities were summarized in Table 6.
The ligands BAT1, BAT2, BAT3 and BAT4 and their Co(II), Ni(II) and Cu(II) complexes are found to possess various antibacterial and antifungal activities towards the strains of the bacteria and the fungal species used.However, no inhibition zone is observed for the ligands BAT3 and BAT4 against the gram-posi- tive bacteria and only for the complex Cu-BAT3 against Streptococcus pyogene.The highest activity observed is for the BAT2 against Staphylococcus aureus and for the Co(II) and Ni(II) complexes of BAT2 towards Streptococcus pyogene.Apart from the above-mentioned ligands and complexes, the remaining ligands and metal complexes exhibit moderate to high antibacterial activity.
Apart from the ligands and metal complexes that possess a high activity against Aspergillus flavus and Candida albicans, all the rest of the ligands and the complexes exhibit moderate antifungal activities against the fungi tested.
It has been reported [16][17][18] that complexes of some heterocyclic ligands with transition metals increase their biological activities.This can be seen relatively more in the higher activity of the metals complexes towards Staphylococcus aureus than the free ligands, but slightly less against Streptococcus pyogene.In contrast, increasing the number of methylene groups between two heterocyclic rings may decrease the antibacterial activity of these compounds [19].Close examination of the antibacterial activity of the ligands and their metal complexes reveals that the ligand BAT4 with the highest methylene groups and its metal complexes possess the lowest antibacterial activity against gram-positive bacteria than the others.

Conclusion
Based on the analytical data and the TGA results, the formulae and the stoichiometry of the prepared BATs ligands and their Co( II) , Ni(II) and Cu(II) metal complexes are suggested.The IR spectral studies indicate neutral tetradentate behavior of the ligands coordinating through the amine group nitrogen and thione form sulphur.The molar conductance values show the nonelectrolytic nature of the complexes.The spectral and magnetic results reveal an octahedral geometry (structure I) for some of the complexes and a square-planer (structure II) for the others.
Generally, the metal-complexes of a particular ligand with various metals exhibit various thermal decomposition behavior, and the kinetic parameters for the successive steps in the decomposition sequence are determined.The inconsistent biological activities observed again the four strains of bacteria and the two fungal species reflect various degrees effect nature of these metal-complexes.

Table 4 .
Characteristic parameters of thermal decomposition (10°C min -1 ) for Co(II), Ni(II) and Cu(II) complexes of BAT2.first decomposition steps of both ligands combetween 23-275°C with mass losses rangfrom 5.60 to 15.70% correspond to the coevolution of solvent with various number of adsorbed or/and coordinated water molecules (cal.5.70-15.80%),and as a result broad peaks either due to peaks overlapping or a gradual extending end (T f ) is observed.The corresponding activation energy values of these steps range between 53 to 94 kJ mol -1 .

Figure 1 .
Figure 1.The TG and DTG thermograms of the BAT2 and BAT3 ligands in nitrogen at the heating of 10°C min -1 .

Figure 2 .
Figure 2. The TG and DTG thermograms of the Co, Ni and Cu complexes with BAT2 ligand in nitrogen at the heating of 10°C min -1 .

Figure 3 .
Figure 3.The TG and DTG thermograms of the Co, Ni and Cu complexes with BAT3 ligand in nitrogen at the heating of 10°C min -1 .

Table 6 .
Effect of the ligands and their Co(II), Ni(II) and Complexes on the growth of Bacteria and Fungi (Zone of in mm).

Table 1 .
Analytical and Physical data of the BATs ligands and their Co(II), Ni(II) and Cu (II) complexes.