Synthesis , Characterization , Antifeeding and Insect Growth-Regulating Activities of Cr ( III ) , Mn ( II ) , Fe ( III ) , Co ( II ) , Ni ( II ) and Cu ( II ) Complexes with N-Acetylacetonyl-3-Aminocoumarin

Metal complexes of Cr(III), Mn(II), Fe(III), Co(II), Ni(II) and Cu(II) have been synthesized using N-accetylacetonyl-3-aminocoumarin as chelating agent. The ligand behaves as a monobasic tridentate ONO donor towards Ni(II) and as monobasic bidentate ON donor towards other cations. All the complexes have been proposed to have octahedral geometry on the basis of analytical, thermal conductivity, spectral and magnetic data. The complexes have been screened against Spodoptera litura; F (Lepidoptera: noctuiidae) for antifeeding and insect growth-regulating activities The results show appreciable antifeeding and insect growth-regulating activities associated with metal complexation at higher and lower doses respectively.


Introduction
Several vital functions of living organisms are thought to be due to the chemistry of metal-bound biomolecules.Investigations involving synthesis, stability, structure and reactivity of metal-containing compounds are well advanced.When a metal ion is bound to a biologically active organic molecule, it may result in apparent modification of the activity, generally depending on the specificity of the binding site and the stereochemistry of the metal complex.
Although coumarin is a plant product known to possess insecticidal effect, no significant attempt has been made so far to study the antifeeding (AF) and insect-growthregulating (IGR) activities with azomethine derivatives of 3-aminocoumarin and their metal complexes.The present paper reports AF and IGR activities of newly synthesized transition metal complexes of N-acetylacetonyl -3-amino coumarin (H-acacAc) against Spodoptera litura with the aim of providing knowledge that may be of use in the development of new agrochemical products.

Materials
The reagents and chemicals used were of BDH(AR) or E. Merck (AR) grade.All solvents were distilled under anhydrous conditions before use.

2.2.
Analytical and physical measurements.
The mass spectrum of the ligand was recorded on a VG-Micromass 7070-H spectrometer.
Elemental analyses were carried out by micro analytical methods.Metal contents were determined using a Varian Spectra 30/40 atomic absorption spectrometer, employing the necessary standards.DTA studies were carried out on a Leeds and Northrup, U.S.A. instrument.Thermal decompositions were effected at normal atmosphere in Robert-Grimshaw ceramic sample holders at a heating rate of 10°C/min using α-alumina as reference.Conductance measurements were carried out on a Digisun D1-909 digital conductometer.IR spectra were recorded on a Perkin-Elmer-783 spectrophotometer using KBr pellets.Reflectance electronic spectra were recorded in Nujol mulls on a Cary 2390 UV spectrometer.Magnetic susceptibilities were measured on a vibrating sample magneto meter of EG & G Princeton, research model.

Synthesis of metal complexes.
A general method was adopted for the preparation of all the complexes.A hot methanolic solution of the ligand was added to a methanolic solution of the metal chloride (ligand/metal ratio 1:1).A distinct reduction of the pH was observed.The pH of the solution was then increased to 7 using a methanolic ammonia solution and the contents were refluxed on a hot water bath for 6-8 h.The separated solid complex was filtered, washed with methanol, petroleum ether and dried in vacuum desiccator over P 2 O 5 .

Pharmacology
To trace antifeedant and IGR activities, the compounds were primarily screened on Spodoptera litura ;F (Lepidoptera: noctuiidae).The insects were reared on a natural diet of mulberry leaves in the larval stages and sugar syrup was supplied for the adults.The total rearing period comprised 12 h of photoperiod and 12 h of dark period.The experiment was carried out at a temperature of 27°C +/-1°C.The insects were in various stages of life cycle viz egg, larval, pupal and adult.The long duration period being the larval stage is also of voracious feeding habit.Proper feeding of the larvae was taken care by the regular supply of mulberry leaves.1% and 5% acetone solutions of the ligands, metal salts and their corresponding metal complexes and related compounds were prepared.These solutions used as media for transporting the chemical to the insect body or on to the leaf surface on which the insects/larvae reared.
Antifeeding test: The fifth instar larvae was selected for testing the antifeedant activity.Leaf discs of uniform area of 78 cm 2 were punched out from mulberry leaves with their petioles intact.The leaf discs to be treated were dipped in the prepared acetone solutions of the various test compounds for 2 min.The leaf discs were air dried and placed in a conical flask containing small amount of water to facilitate water translocation and thus enable to keep the leaf disc fresh throughout the duration of the experiment.Five fifth instar larvae of the test insects which have been pre-starved for 6 h were introduced on the leaf discs and allowed to feed on them for 2 h.One set of the control leaf discs was dipped in pure acetone and another set was kept without any chemical.The results were recorded after 2, 12 and 24 h.The area of the leaf disc consumed was measured by Dethiers method [17].
The fifth instar larvae were topically treated leaves with the ligand and their corresponding metal complex solutions at different dosages.A continuous observation on the larvae was maintained from the time of the treatment with the ligand and complex solutions through the formation of the adult until its death.In each experiment, 10 insects were employed and the experiments were repeated thrice for IGR juvenile hormone activity test [18].

Characterization of the ligand
Elemental analysis indicated that the ligand has the molecular formula given in Table 1.The mass spectrum shows parent peak at m/e 243 which is consistent with the molecular formula (Fig. 1).
The IR spectrum of the free H-acacAC ligand shows a broad band in the range 3300 cm -1 to 3500 cm -1 centered at 3400 cm -1 .This band has been assigned to the presence of enolic hydroxyl group [19].The band located at 1700 cm -1 with shoulders at 1720 cm -1 and at 1740 cm -1 attributed to ν (C=0) moiety of the coumarin [20].This band structure is indicating the involvement of the lactone carbonyl in multiple inter and intra molecular hydrogen bonding.These observations suggest that the free H-acacAC is predominantly in the form of the enolic tautomer.The band observed at 1630 cm -1 has been attributed to the ν (C=C) stretch [21].The high intensity band at 1600 cm -1 [22] is assigned to the azomethine stretching.The enolic stretching, ν (C-O) is observed at 1130 cm -1 .The ethereal C-O-C stretch is identified at 1020 cm -1 .The band at 1380 cm -1 is assigned to the ν (C-N) .
The electronic spectrum of H-acacAC reveals a multiple band in the region of 40000 cm -1 to 35710 cm -1 .This has been attributed to the π -π* and n -π* transitions which are possible mainly due to the lactone oxygen of the coumarin moiety [23].The broad band identified in the region of 25000 cm -1 to 22220 cm -1 is attributed to the n -π* transition of the azomethine group.
The NMR spectrum of H-acacAC reveals the presence of two -CH protons at 5.38 ppm.The methyl protons are indicated in the range of 2.19 ppm to 2.31 ppm.The ring protons are identified in the range of 7.38 ppm to 7.41 ppm [24].The signal expected for enolic hydroxyl proton is recorded at 12.1 ppm, substantiated by D 2 O exchange studies.The absence of the signals due to methylene protons and the presence of two -CH protons confirmed enolization.

Characrerisation of metal complexes
The complexes are soluble in acetone, chloroform, 1,4-dioxane, THF, DMF and DMSO.They melt in the temperature range of 120°C to 264°C.Some of them melt at temperatures lower than the melting point of the free ligand.

Elemental analysis
It appears from the analytical data (Table 1) that the ligand undergoes deprotonation of its enolic proton during the complex formation with metal chloride.The analytical data reveals M:L ratio of 1:2 in Cr(III) and Co(II) and 3:4 in Cu(II) and Mn(II) complexes.In Fe(III) and Ni(II) complexes, the M: L ratios are 2:5 and 2:3 respectively.In Cr(III) and Co(II) complexes, the data are indicative of the presence of coordinated ammonia molecules (which are confirmed by spectral data).The absence of chloride in the cobalt complex and the presence of chloride in other complexes are noted.

Molar conductance studies
The conductance measurements, recorded for 10 -3 M solutions of the metal complexes in DMSO are listed in Table 1.The data reveals that the Cr(III) and Fe(III) complexes are conductors and the Mn(II), Co(II), Ni(II) and Cu(II) complexes are non conductors.The Cr(III) complex exhibits a molar conductance of 130 mhos and the Fe(III) complex, a value of 65 mhos.These values reveal that the former is a 1:2 electrolyte and the later is a 1:1 electrolyte [20].

Infrared spectral studies.
The characteristic infrared frequencies of metal complexes are given in Table 2. Nonligand bands have been identified at 3350 cm -1 and 3450 cm -1 in the Cr(III) and at 3000 cm -1 and 3400 cm -1 in the Co(II) complexes.These bands are characteristics of N-H stretching vibrations and thus have been assigned to the ν (N-H) (stretch) arising as a result of coordinated ammonia molecules [25].The broad trough of medium intensity observed in the free ligand spectrum in the range of 3300 cm -1 to 3500 cm -1 was absent in all the complexes [26].This clearly indicates that the deprotonation of the enolic hydroxyl group has taken place.
In the IR spectra of all the complexes except that of Ni(II), a sharp peak has been identified in the range of 1700 cm -1 to 1720 cm -1 .This has been assigned to the ring carbonyl stretching frequency.As compared with the corresponding band in the free ligand, this band has not undergone any apparent shift.It is therefore, concluded that the ring carbonyl did not participate in coordination in all these complexes [27].In the case of Ni(II), this band has undergone a downward shift by 70 cm -1 [28] and has been identified at 1650 cm -1 .This is indicative of ion of the ring carbonyl in coordination.
ν (C=C) has been identified in Cr(III), Mn(II), Fe(III) and Co(II) complexes at 1630 cm -1 , in the Cu(II) complex at 1660 cm -1 and in Ni(II) complex at 1610 cm -1 .The corresponding free ligand band was observed at 1630 cm -1 .
The sharp peak at 1600 cm -1 in the free ligand is observed at 1560 cm -1 in the Ni(II) complex and at 1570 cm -1 in the rest of the complexes.This band has been assigned to the azomethine stretching frequency.In comparison with the free ligand, this band has experienced a downward shift by 30 cm -1 to 40 cm -1 which indicates the participation of the azomethine nitrogen in coordination [29].In place of ν (C-O) (enolic) at 1130 cm -1 in the ligand, the spectrum of each complex shows two bands in the range of 1140 to 1200 cm -1 .These bands indicate a positive shift of ν (C-O) suggesting coordination through enolic oxygen.One of these bands shows a large shift in the range of 30 cm -1 to 70 cm -1  which can be attributed to bridging phenomenon [30].It is interesting to note that each complex shows the presence of two types of enolic oxygens, one non-bridging and the other bridging.The ligand H-acacAC may be mentioned as a unique example indicating the flexibility of chelating agent in metal binding interactions.There is no change in the ring ν (O-C-O) frequency suggesting that there is no bonding between the oxygen atom of coumarin ring and the metal atom in each of the complexes [31].
The new bands identified in complexes at 340 cm -1 , 320 cm -1 , 260 cm -1 and 240 cm -1 have been assigned to the metal chloride stretching frequencies.New non-ligand bands have been observed at 560 cm -1 , 640-650 cm -1 and at 800 cm -1 in all complexes.In Cr(III) and Co(II) complexes, additional non-ligand bands have been identified at 450 cm -1 , 580 cm -1 , 730 cm -1 , 850 cm -1 and 970 cm -1 respectively.These new bands can be attributed to M-N, M-O stretching vibrations [32].The greater number of non-ligand bands in the Cr(III) and Co(II) complexes may be attributed to the presence of coordinated ammonia molecules [33].
From the IR data, it has been concluded that the ligand H-acacAC behaves as a monobasic tridentate ONO donor in the case of Ni(II) complex, coordinating through the ring carbonyl, azomethine nitrogen and the enolic oxygen atoms.In all the other complexes, the ligand is found to coordinate through the azomethine nitrogen and enolic oxygen, thereby acting as a monobasic bidentate ON donor.The enolic oxygen acts as a bridge between the two metal centers.

Electronic spectral studies.
The multiple band observed in the region of 40000 cm -1 to 35710 cm -1 of the free ligand electronic spectrum has undergone considerable change.The multiplicity of the band is absent in all the complexes.The broad band assigned to the azomethine group in the free ligand spectrum in the range of 25000 cm -1 to 22220 cm -1 has also undergone considerable modification.This supports the possible involvement of azomethine group in coordination.
The electronic spectra of Cr(III) complex has three distinct bands at 35710 cm -1 , 25000 cm -1 and 16670 cm -1 respectively.These bands are assigned to 4 A 2g → 4 T 1g (P), 4 A 2g → 4 T 1g (F), 4 A 2g → 4 T 2g transitions respectively.These transitions are indicative of an octahedral geometry [33].The electronic spectrum of the Mn(II) complex exhibits weak absorption bands at 26320 cm -1 and 33330 cm -1 .It is known [34] that d-d bands of hexa coordinated high-spin Mn(II) complexes have extremely low intensity and in the presence of organic ligands they could rarely be detected.Therefore, an octahedral geometry is proposed.In the Fe(III) complex, a number of transitions are observed at 28570 cm -1 , 21740 cm -1 , 20000 cm -1 , 16670 cm -1 , 12200 cm -1 , 8550 cm -1 and 7259 cm -1 respectively.These are attributed to several spin forbidden and charge transfer bands.The transitions may involve 4 π g , 4 T 2g , 4 A lg , 4 E g , 4 T g (D), 4 E g (D), 4 T lg (P), 4 A 2g (F), 4 T lg (F) etc. in octahedral stereochemistry .Due to the doubly forbidden nature, no specific assignments are made [35].Thus, for the Fe(III) complex, an octahedral geometry has been proposed.
The electronic spectrum of Co(II) shows bands at 20000 cm -1 (multiplet), 14290 cm -1 and at 6800 cm -1 assignable to 4 T lg → 4 T 1g (P), 4 T lg → 4 A 2g , 4 T lg → 4 T 2g (F) transitions respectively [36].Additional bands observed at 27030 cm -1 and at 29410 cm -1 have been assigned to charge transfer transitions.The ν 2 /ν 1 ratio is equal to 2.1.The transition patterns and the ν 2 /ν 1 ratio are both supportive of octahedral geometry.In the spectrum of Ni(II), electronic transitions have been observed at 11760 cm -1 , 20830 cm -1 and 25000 cm -1 which are assigned to 3 A 2g → 3 T 2g , 3 A 2g → 3 A lg (F), 3 A 2g → 3 T lg (P) respectively [17].The Charge transfer bands are identified at 35710 cm -1 and the spin forbidden bands in the range of 16670 to 15380 cm -1 .Thus, Ni(II) complex is assigned an octahedral geometry.In Cu(II), a multiple band is identified in the range of 18180 cm -1 to 22220 cm -1 and charge transfer bands at 26320 cm -1 , 28570 cm -1 and at 35710 cm -1 respectively.The multiple bands have been assigned to 2 E g → 2 T 2g transition.The nature of the band (broadness and multiplicity) suggests tetragonal distortion from the regular octahedral geometry [37].

The ESR studies
The ESR spectrum of the Cu(II) complex was recorded at liquid N 2 temperature in the polycrystalline form using DPPH as a reference standard.The g ║ and g ┴ values have been found to be 2.12 and 2.16.The complex shows axial symmetry.The difference in g ║ and g ┴ values indicates the octahedral distortion in the complex [38].Absence of hyperfine interaction reveals that the M-L interaction is far higher and more prominent in comparison with the possible hyperfine interaction in free Cu(II) ion.
On the basis of above spectral analyses the structures of Cr(II), Mn(II), Cu(II), Fe(III), Co(II) and Ni (II) complexes can be predicted as illustrated in Figs.2a-e.

Antifeeding activity
It was observed that during the total experimental period, the insects did not show any repellant behaviour towards the treated leaf surfaces.It was observed that the ligand H-acacAC and its metal complexes did not exihibit any antifeeding activity at lower dosage of 1% (Table 3).The insects were feeding on the leaves normally.At higher dosage of 5%, a gradational drop in the feeding was observed.The metal salts, acac and AC did not exhibit any antifeeding activity either at a lower or at a higher dosage.At 5% dosage: Ni >> Co = Cr > Fe > Cu = Mn > H-acacAC

IGR activity
IGR activity was not recorded with metal salts, AC, acac.The metal complexes of H-acacAC were found to exhibit IGR activity at lower dosages of 1% alone (Table 3) The activity order of the metal complexes of H-acacAC is as follows: larvae from developing into adult insects.At high levels of Juvenile hormones, larva can still molt, but the result will only be a bigger larva, not an adult.Thus the reproductive cycle is broken.Similar results were also noticed when the plant originated antifeedant azadirhychtin was tested for juvenile hormone activity [44].
Thus it can be concluded that the metal complexes used in the present study have AF properties at higher dosages and IGR juvenile hormone activity at lower dosages.However, further detailed study is required regarding the activity enhancement of the test compounds with structural modifications, identification of the site of action of the compound, bioassay against various insects and their toxicity evaluation against various insects at laboratory and field levels.

Conclusions
The ligand H-acacAC acted as a monobasic tridentate ONO donor in the case of Ni(II) complex, coordinating through the ring carbonyl, azomethine nitrogen and the enolic oxygen atoms.In all the other complexes, the ligand was found to coordinate through the azomethine nitrogen and enolic oxygen, thereby acting as a monobasic bidentate ON donor.The enolic oxygen also acted as a bridge between the two metal centres.On the basis of analytical, conductance, thermal, spectral and magnetic data, octahedral geometry has been proposed and the following formulae have been concluded.From the pharmacological studies, it is clear that these metal complexes may be used as a new group of pesticides.However, further detailed study is required regarding the activity enhancement of the test compounds with structural modifications, identification of the site of action of the compound.

Table 1 .
Elemental analyses and molar conductance data of metal complexes of HacacAc

Table 2 .
The IR absorption frequencies (in cm -1 ) of metal complexes.