INFRARED SPECTRA, THERMOGRAVIMETRIC ANALYSIS AND ANTIFUNGAL STUDIES OF NOVAL Cr(III), Fe(III) AND Cu(II) 2-METHYL-QUINAZOLINONE COMPLEXES

Some new solid complexes [CrCl3(L)3]⋅6H2O, [FeCl3(L)3]⋅6H2O and [Cu(CH3COO)2(L)3]⋅2H2O have been synthesized quantitatively by the interactions of 2-methyl-quinazolinone (L) with CrCl3.6H2O, FeCl3.6H2O and Cu(CH3COO)2.2H2O in a mixture of an ethanol-bidistilled water (1:1), at 60 °C. They were characterized by melting point, molar conductivity, magnetic moment, elemental analysis, infrared spectra and thermal analyses. The results supported the formation of the complexes and indicated that the ligand reacted as a monodentate ligand bound to the metal ion through the oxygen atom. The antifungal activity of the free ligand and their metal complexes were evaluated against several species, such as Fusarium solani, Rizoctonia solani, Sclortium rolfsii and Botryodiplodia and they showed a good antifungal activity to some selected fungal strain as compared with free ligand.


INTRODUCTION
2-Methyl-quinazolinone (Formula 1) is one of the important compounds from the series of quinazolinone derivatives.Quinazolinone and its derivatives are inhibitory to a number of fungal pathogens of plants, including Helminthosporium turcicum, Stagonospora nodorum, Microdochium nivale, Fusarium moniliforme, Fusarium culmorum, and Gaeumannomyces graminis.Some isolates of G. graminis, F. culmorum, F. moniliforme, F. subglutinans, and of a number of other Fusarium species are able to degrade benzoxazolinone compounds to products which are less inhibitory to fungal growth and also important compounds in chemistry and pharmacology [1][2][3][4][5].They have drawn much attention due to their broad range of pharmacological properties, which include anticancer, anti-inflammatory, anticonvulsant and antidiuretic activities [6][7][8][9][10].Consequently, considerable efforts have been made to explore new simple and direct approaches towards the construction of 5(4H)-quinazilinone skeletons such as via amidation of 2-aminobenzonitrile, followed by oxidative ring closure and Pd-catalyzed heterocyclyzation of nitroarenes [11,12].
Benzoxazinone derivatives are also used as antiphlogistic drugs, antifungal and antibacterial agent [13,14].If a vinyl or phosphate functional group is connected to an aromatic ring located at the position two of the heterocyclic, the resulting compounds possess antimuscular contraction properties and can be used as a hypnotic drug [15,16].For many years, quinazolinone derivatives and their metal complexes have been the subject of most structural and mechanical studies due to their potential biological value [17].In recent years the chemistry of heterocyclic compounds are well known for their diverse therapeutic properties and exhibited antibacterial, anticancer, antiulcer, diuretics, anticonvulsant, antihypertensive, antitumor, antifungal, anti-AIDS and antiviral properties [18].
The present study describes the coordination behavior of novel 2-methyl-quinazolinone towards some transition elements, which may help in more understanding of the mode of chelation of them towards metals.For this purpose the complexes of Cr(III), Fe(III) and Cu(II) ions with 2-methyl-quinazolinone are studied in the solid state.The structure of the studied complexes is characterized using elemental analysis, infrared spectra and thermal analyses (TGA and DTG) measurements.The biological activity of the parent, 2-methyl-quinazolinone and its metal complexes has been tested against antifungal screening, Fusarium solani, Rizoctonia solani, Sclortium rolfsii and Botryodiplodia.

Chemicals
All chemicals used for the synthesis of compounds were of the analytical reagent grade and of highest purity available.Cupper acetate dihydrate was purchased from Sigma, chromium chroide and ferric chloride were purchashed from Prolabo.Solvents were dried by the standard procedures [19].

Instruments
Elemental microanalysis of the solid complexes for C, H, N and halogen was carried out on a Perkin-Elmer CHN 2400 and performed at the Micro-analytical Center, Cairo University.The analysis was repeated twice to check the accuracy of the analyzed data.The percentage of the metal ions were determined gravimetrically by transforming the solid products into oxide and also determined by using atomic absorption method.Spectrometer model PYE-UNICAM SP 1900 fitted with the corresponding lamp was used for this purposed.Infrared spectra of the three solid complexes, [CrCl 3 (L) methyl-quinazolinone and the final products of the thermogravimetric analysis were recorded on a Perkin-Elmer FT-IR type 1650 spectrophotometer in wave number region 4000-400 cm -1 .The spectra were recorded as KBr pellets.The thermal analyses (TGA and DTG) were carried out in dynamic nitrogen atmosphere (20 mL.min -1 ) with a heating rate of 10 °C min -1 using Shimadzu TGA-50H thermal analyzer within the temperature range from room temperature to 1200 °C.Molar conductivities of the solution of the ligand and metal complexes in DMSO at 1×10 -3 M were measured on CONSORT k410.All measurements were carried out at ambient temperature with freshly prepared solution.

Synthesis of 2-methyl-quinazolinone
The pale brown of 2-methyl-quinazolinone was prepared according to published method [20,21] by heating of 10 mmol 2-methyl-3,1-(4H)-benzoxazin-4-one and 25 mL of formamide for 3 h in oil bath and the reaction mixture was left aside at room temperature until the precipitation occurs, the solution was filtered under vacuum and the solid product was recrystallized from suitable solvent (ethanol).

Synthesis of metal complexes
The black solid complex [CrCl 3 (L) 3 ]⋅6H 2 O was prepared by adding 2 mmol (0.533 g) of chromium chloride (CrCl 3 .6H 2 O) in 25 mL mixture of an ethanol-water (1:1) drop wisely to a stirred hot solution (60 °C) of 6 mmol (0.966 g) of 2-methyl-quinazolinone in 25 mL of the same mixture in 1:3 molar ratio.The reaction mixture was stirred under heating for four hours where upon the complex precipitated.The black solid complex collected by filtration and purified by washing with an ethanol-water mixture and diethyl ether.The gray and light green solid complexes of [FeCl 3 (L) 3 ]⋅6H 2 O and [Cu(CH 3 COO) 2 (L) 3 ]⋅2H 2 O, respectivly, were prepared in a similar manner described above by using FeCl 3 .6H 2 O or Cu(CH 3 COO) 2 .2H 2 O, respectivly, in 1:3 molar ratio.The three complexes were characterized by their elemental analysis, infrared spectra as well as thermal analyses.

Antifungal activity
The effect of the prepared compound 2-methyl-quinazolinone and its metal complexes on the linear growth of tested soil fungi, such as Fusarium solani, Rizoctonia solani, Sclortium rolfsii and Botryodiplodia on potatoes dextrose agar (PDA) medium was evaluated by a previously reported modified method [22].The medium was prepared as usual using 50 mL of PDA and transferred to a conical flask (150 mL) and autoclaved.
Adequate drops of 25% of lactic acid were added on PDA after sterilization and before pouring in Petri dishes to prevent bacterial growth.Appropriate amounts of each prepared compound were added to flasks containing PDA medium just before solidifying and shacked before pouring to obtain the required concentrations of the prepared compounds (1000 µg/mL).The different preparations of each compound were then poured in Petri dishes.Four dishes were used as replicates for each compound.Agar disks 6 mm in diameter of growth cultures of each tested fungus on PDA medium were seeded on center of each amended PDA plate with tested compounds.Other four dishes that were not treated with any chemical were incubated with fungal disks and used as control.All dishes were incubated at 25±1°C.The linear growth of fungal mycelium was measured in mm when any of the colonies covered the plate.The data of linear growth of fungi as affected by prepared compounds are recorded.

RESULTS AND DISCUSSION
The prepared solid complexes were subjected to elemental analysis (C, H N, halogen and metal content), infrared spectral studies (IR), and thermal analyses (TGA and DTG), to identify their tentative formula in a trial to elucidate their molecular structures.All these complexes were colored and hydrates with a metal to ligand ratio amounting to 1:3 for three complexes and the metal ions complete the coordination number to six for Cr(III) and Fe(III) or five for Cu(II) with chloride or acetate ions, respectively.The structures of the complexes suggested from the elemental analysis agreed well with their proposed formula (Table 1).The magnetic moment of Fe(III) complex at room temperature was found to be at 2.42 B.M., which indicated for low spin paramagnetic Fe(III) in octahedral geometry [23,24].For Cu(II) complex the measured magnetic moment value was 1.77 B.M., which is very close to that of the spin only value (1.71 B.M.) expected for a complex having one Cu(II) with a single unpaired electron [25].Also, the magnetic moment for Cr(III) complex was found at 3.82 B.M. [24].Molar conductance values of 2-methyl-quinazolinone and all complexes were found to be in the range 8.11-12.50S cm 2 mol -1 suggesting their non-elyctrolytic nature.Qualitative reactions revealed the presence of chloride and acetate as ligand (inside the complex sphere for the three complexes).The biological activity of the ligands and their metal chelates were studied against antifungal organisms Fusarium solani, Rizoctonia solani, Sclortium rolfsii and Botryodiplodia.

IR spectral studies
The infrared spectra of 2-methyl-quinazolinone, 2. The infrared spectra of the three complexes were compared with those of the free ligands in order to determine the coordination sites that may involved in chelation.There were some guide peaks, in the spectra of the ligand, which were a good help for achieving this goal.These peaks were expected to be involved in chelation.The position and/or the intensities of these peaks were expected to be changed upon chelation.The presence of the spectral absorption bands in the region 3475-3395 cm -1 indicated the presence of water molecules in the obtained complexes [26,27].The characteristic ν(N-H) stretching frequencies, appeared in the region 3362-3210 cm -1 .The stretching vibrations ν(C-H) of phenyl groups in all complexes occurred as a number of bands in the range 3182-3000 cm -1 , while that the corresponding vibrations ν(C-H) of -CH 3 unit was observed in the range 2981-2914 cm -1 .The assignments of all the C-H stretching vibrations agreed quite well with the expected in literature [28].The ν(C=O) stretching vibration of free L was found at 1670 cm -1 .The shift of ν(C=O) to a lower wave numbers in the three complexes at 1621 cm -1 for Cr(III), at 1664 cm -1 for Fe(III) and at 1659 cm -1 for Cu(II) indicated the participation of 2-methylquinazolinone in coordination and the formation of a bond between the oxygen of L with the central metal ions, and that nitrogen atom was not the donor atom.For [Cu(CH 3 COO) 2 (L) 3 ]⋅2H 2 O complex the stretching asymmetric (ν as ) of carboxylate group for acetate ion found at around 1689 cm -1 and of the symmetric vibrations (ν s ) at 1442 cm -1 confirmed the donation of acetate as monodentate.Unidentate carboxylate complexes exhibited ∆ν values at > 200 cm -1 + ∆ν = ν as ( COO -)ν s ( COO -) ] [29].
The phenyl breathing vibration, ν(C=N) and ν(C=C) in all complexes occurred as a group of medium to very strong bands lying in the range 1601-1401 cm -1 (Table 2).The CH deformation motions of the -CH 3 group occurred as a medium band at 1381 and 1337 cm -1 for L and as a weak or medium bands at 1346 and 1301 cm -1 for Cr(III), at 1395 cm -1 for Fe(III) and at 1383 cm -1 for Cu(II) complexes.The ν(C-O), ν(C-N) and ν(C-C) stretching vibrations in all compounds are assigned to a large number of bands lying in the 1299-1000 cm -1  bending vibrations of the three complexes and free ligand were assigned to the group of bands of varying intensities in the range 976-704 cm -1 .The assignments of these bands agree quite well with the literature [30].
The coordination of the metal ions via oxygen of L is also confirmed by the ν(M-O) bands at 571 and 510 cm -1 for Cr(III), 546 and 525 cm -1 for Fe(III) and at 557 and 485 for Cu(III).Therefore, from the infrared spectra, it was concluded that the 2-methyl-quinazolinone behaved as a neutral monodentate ligand with oxygen donor site involved in the coordination sphere.Key: s = strong, m = medium, br = broad, w = weak, ν = stretching.

H NMR spectrum
The new ligand 2-methyl-quinazolinone was characterized using 1 H NMR spectrum.The 1 H NMR spectrum of 2-methyl-quinazolinone L, was carried out in DMSO-d 6 as a solvent.The 1 H NMR spectrum exhibit peaks in the range 7.4-7.9ppm, which were assigned to -CH of benzene ring and the NH amide found at 8.00 ppm.Also, the methyl group observed at 0.9 ppm.  3 gives the maximum temperature values T max /°C, species lost together with the corresponding weight loss for each step of the decomposition reaction.The data obtained strongly supported the proposed formulas of the ligand and the three complexes and indicate that the decomposition mode of the ligand occurred in one step at two maxima 119 and 209 °C.This step was associated with a weight loss value of 100% in a good agreement with the obtained value 99.33%.Thermogravimetric (TGA) curve for [CrCl 3 (L) 3 ]⋅6H 2 O showed two weight loss events.The first step of decomposition occurred in the range 40-120 °C, with a maximum temperature at 72 °C corresponds to the loss of three water molecules of hydration.The second step of decomposition occurred in the range 120-1200 °C, with two maxima at 230 and 376 °C.The thermal decomposition of [Fe(L) 3 CL 3 ].6H 2 O complex proceeds two degradation steps.The first step of decomposition occurred in the range 30-200 °C, with a maximum temperature at 169 °C corresponds to the loss of five water molecules.The second step of decomposition occurred in the range 200-1200 °C, with four maxima at 268, 321, 747 and 932 °C and was simultaneously decomposed to oxide with intermediate formation of very unstable products which were not identified [31] and is accompanied by a weight loss of 59.24%, corresponding to the loss 2CO+7C 2 H 2 +2N 2 +0.5H 2 O+H 2 +HCl+2NH 4 Cl.The actual weight loss from these two steps was 71.19%, close to the calculated value 71.75%.The TGA curve of [Cu(CH 3 COO) 2 (L) 3 ]⋅2H 2 O complex (Figure 2) and the data were listed in Table 3 exhibits two main degradation steps.The first step of decomposition occurred at maximum temperature of 71 °C and was accompanied by a weight loss of 13.35%, corresponding to the loss of 2H 2 O+2CO+1.5H 2 .The second step of decomposition occurred at one maximum 256 °C and is accompanied by a weight loss of 65.82%, corresponding to the loss of 12C 2 H 2 +4NO+NH 3 +0.5N 2 giving CuO as a final product.Water of crystallization lost for the three complexes at a relatively low temperature may be indicated to a weak H-bonding involving the H 2 O molecule and the complexes.According to the above discussion, the mechanisms for the thermal decomposition of the ligand and the three complexes are as follows:  The decomposition mechanisms were only based on speculation hypothesis and the thermal analysis weren't connected with a complementary technique (gas chromatography) [31].The suggested residues confirmed only on the basis weight loss percent calculation and the infrared spectra, which clearly shows the bands associated to the metal oxides and disappearance of the bands characteristic for the 2-methyl-quinazolinone and acetate ions Figure 2.

Figure 3. Statistical representation for antifungal activity of 2-methyl-quinazolinone and its complexes
The proposed structure formula (II) on the basis of the results discussed in this paper may be as follows:

Biological activities
The fungicidal activity of 2-methyl-quinazolinone and the three prepared heterocyclic complexes were evaluated under laboratory conditions.The tested fungi were Fusarium solani, Rhizoctonia solani, sclerotium rolfsii and Botryodiplodia sp.Data presented in Table 4 reveals the antifungal potency of these compounds.The highest antifungal activity for the tested compounds was observed after 48 h of treatment, with obvious decreasing on their activities with prolongation of incubation time at 72 h or 96 h.However, the Cr(III) complex against the three fungi Fusarium solani, Rhizoctonia solani and Botryodiplodia gave the highest inhibition at 48 and 72 h and then the rate of inhibition was decreased to 96 h except for Botryodiplodia.Also, the 2-methyl-quinazolinone (L), completely inhibited the growth of Sclerotium rolfsii at the three tested incubation times.Friebe et al. [3] found that, benzoxazinones and benzoxaolinones have a strong inhibition to various phytopathognic fungi including Helminthosporium turcicum, Stagonospra nodorum, Microdochium nivale and Fusarium sp.Some isolates of Fusarium sp. are able to transforme benzoxazinone compounds to less inhibitory derivative.Also Niemeyer [32] indicates that the benzoxazinone and aglycones that found as natural compound in plants and their degradation products exhibit fungistatic and bacterostatic activity.Depending on the percent of inhibition at 96 h quinazolinone as a most active compound against sclerotium sp, where as Cl methylbenzoxazinone only was the most active compound against Fusarium solani, Rhizoctonia sp and Botryodiplodia sp.So, 2-methyl-quinazolinone is considered as qundidint compound and was used as ligand to prepare complexes.On contrary, Fusarium sp. was more sensitive to complexes [CrCl 3 (L)   O against Botryodiplodia sp.From the above results, it could be concluded that the conjugation of metallic compounds with heterocyclic compounds in complexes changed the bioactivity of heterocyclic compounds against fungi in two forms: (i) no change in bioactivity and (ii) decrease the bioactivity less than ligand, or increase the bioactivity more than ligand these changes depending on sensitivity of tested fungi and type of metallic compounds that used in complex reaction

The physico-chemical properties of the tested active ingredient (complexes)
All complexes were insoluble in water and xylene where as [FeCl 3 (L) 3 ]⋅6H 2 O complex showed a low solubility in acetone their solubility percentages was 14%.On the other hand the free acidity of all tested complexes were found in two forms (Table 5).From above results it could be concluded that dustable powder formulation is the suitable formulation to these active ingredient because their physico properties are not soluble in water and xylene also it will be use in controlling the soil borne fungi.Calcium carbonate possessed an ionization constant pK a higher than 3.3 (Table 6).Accordingly this carrier has slight acidic surface sites that are safe to mixed with our complexes.However talck has pK a level >1.5 and <3.3.This means that talck has acidic sites less than calcium carbonate.On the other hand both tested carriers were slightly alkaline and varied in their pH values.Talck showed the highest pH value 8.33, while calcium carbonate possessed lower value 7.98 The tested inert varied considerably according to their bulk density.Calcium carbonate showed the highest bulk densities (loss and packed) (0.8, 1.14).According to the specifications of WHO (1979), the bulk density of powder after compacting (packed bulk density) should not exceed the value obtained before compacting by more than 60%, both tested inert confirmed` these specification.On the other hand, calcium carbonate and talck have particle size range less than 40 microns.

CONCLUSION
The new reaction of some transition metals Cr(III), Fe(III) and Cu(II) with 2-methylquinazolinone (L) has been studied.The results of the elemental analysis, molar conductivity, magnetic moment, infrared spectra and thermogravimetric analysis deduced the formation of 1:3 metal/ligand complexes in all cases.Antifungal studies were carried out against several species.The results showed significant increase in antifungal activity of some metal complexes as compared with uncomplexed ligand.
Thermogravimetric (TGA) and deferential thermogravimetric (DTG) were carried out for the ligand 2-methyl-quinazolinone and the three solid complexes [CrCl 3 (L) 3 ]⋅6H 2 O, [FeCl 3 (L) 3 ]⋅6H 2 O and [Cu(CH 3 COO) 2 (L) 3 ]⋅2H 2 O under N 2 flow and heating rates were suitably controlled at 10 °C min -1 and the weight loss was measured from the ambient temperature up to ∼1200 °C.The TGA and DTG curves of the complexes were shown in Figure 1.Table

Table 4 .
Evaluation of fungicidal activity of 2-methyl -quinazolinone (L) and its complexes against different soil fungi under laboratory conditions.