Txicological Aspects of Newly Designed Macrocyclic Complexes of Iron(II)

Brine shrimp lethality of a new series of 16 to 26-membered macrocycles of iron(II) containing tetraaza groups and prepared by the template condensation reaction of diacarboxylic acids (malonic, succinic, glutaric or adipic) with 2,6-diaminopyridine and diethylenetriamine in 1:2:2 molar ratios have been studied. Structures and bonding of the macrocyclic complexes have been proposed based on elemental analyses, IR, electronic, X-ray and mass spectral studies. An octahedral geometry for these complexes has been proposed as the binding sites are the nitrogen atoms of the macrocycles. The formation of the complexes as [Fe(Ln)Cl2] has been established on the basis of the chemical composition. The complexes have also been screened against several microbes.


INTRODUCTION
Studies on macrocyclic complexes have shown that some of them are involved in important biological processes, such as photosynthesis and dioxygen transport in addition to their catalytic properties 2 which may lead to important industrial applications. Their enhanced kinetic and thermodynamic stabilities led to a widespread study of the features which also influence their potential applications as metal extractants and as radiotherapeutic 4 and medical imaging agents5. Macrocyclic complexes are best prepared with the aid of metal ions as templates to direct the steric course of the condensation reaction which 6g ultimately results in ring closure'. Different ring sizes of macrocycles are now readily available by convenient methods as already reported9"6. In most of the cases, high dilution techniques are employed for the cyclization process 12 when template is not operative. Macrocyclic polyamines have attracted increasing attention because of their unique property to form a very stable chelates with various heavy metal ions7.
Many of new organic chemicals are prepared annually throughout the world and many of them entered into pharmacological screening to determine if they have useful biological activity 18 With this intention, we have selected four synthesized compounds of iron(II) having a tetraoxotetraaza ligand, formed by the template condensation of dicarboxylic acids (malonic, succinic, glutaric or adipic) with 2,6diaminopyridine or diethylenetriamine in the presence of ferrous chloride. Brine shrimp lethality bioassay is a recent development in the assay procedure 19 for the bioactive compounds, which indicates cytotoxicity as well as a wide range of pharmacological activity e.g., anticancer, antifungal, pesticidal, etc. Bioactive compounds are almost always toxic in high dose. Pharmacology is simply toxicology at a lower dose or toxicology is simply pharmacology at a higher dose. Thus in vivo lethality a simple zoological organism can be used as a convenient monitor for the screening of bioactive synthetic compounds.
The purity of the compounds was checked by T.L.C. on silica gel-G using anhydrous methanol and dimethylformamide (1:2) as a solvent. Each of the compound moves as a single spot indicating the presence of only one component and hence their purity. Analytical Methods and Physical Measurements Conductivity measurements were made with a systronic model 305 conductivity bridge in dry dimethylformamide. Molecular weights were determined by the Rast camphor method. IR of the soild samples were recorded as KBr discs on a Nicolet magna FT-IR 550 spectrophotometer. Electronic spectra in dimethylsulphoxide were recorded on a UV-160A, Shimadzer spectrophotometer in the range 200-600 nm using methanol as the solvent. X-Ray powder diffraction spectra of the compound was obtained on the philips model P.W. 1840 automatic diffractometer using Fe (Kct) target with Mg filter. The wavelength used was 1.9373 A and the reflection from 5-65C was recorded. The mass spectra of the compound was recorded on a JEOL FX 102/DA-6000 mass spectrometer/data system using Argon/Xenon (6 KV, 10 mA) as the FAB gas. The accelerating voltage was 10 KV and the spectra recorded at the room temperature. M-Nitrobenzyl alcohol was used as the matrix. Nitrogen and chlorine were estimated by Kjeldahl's and Volhard's method, respectively. Iron was estimated gravimetrically.

RESULTS AND DISCUSSION
The physical properties and analytical data of the complexes are given in Table I SPECTRAL STUDIES

Infrared Spectra
The infrared spectra of the starting materials and their metal complexes were studied and some important features may be summerized as follows The IR spectra of 2,6-diaminopyridine or dietylenetriamine and dicarboxylic acids show the bands due to hydroxyl and amino group, which disappear in corresponding metal complexes, indicating the condensation of amines with the dicarboxylic acids and formation of the proposed macrocyclic frame work. A broad band due to NH in case of diethylenetriamine appears almost at the same position -3058 cm in metal complexes indicating that the grouPlremains uncoordinated. The spectra of all the complexes show a medium intensity band at 3244-3271 cmwhich is assigned to v(NH) mode of the amide group 2 or the secondary amino group of the diethylenetriamine. The amide I, amide II, amide III and amide IV bands appear at 1672-1704, 1524-1570, 1212-1263 and 650-677 cm -, respectively2. Strong and sharp absorption bands appearing in the regions 2895-2923 and 1400-1442 cmin the complexes are assigned to the C-H stretching and C-H bending vibrational modes, respectively. The aromatic ring stretching appeared at 1648, 1527 and 1446 cm-. The presence of aromatic C-N bands in the complexes appeared in the region of 825-839 cm-. The spectra of the complexes derived from 2,6-diaminopyridine do not show any change in the pyridine ring which confirms that the nitrogen does not participate in the coordinaton22. The bands in the region 350-452 cmin the spectra of all the complexes may be attributed to the Fe-N stretching vibrations23.
The Fe-CI stretching vibrations have been assigned at 265-340 cm as reported by others also4 The infrared spectral data of the complexes are given in Table-II.   Table II   Electronic Spectra The electronic spectra of iron (II) tetraazamacrocyclic complexes exhibit a weak intensity band in the region 840-892 nm, which may be assigned to the 5T.g--->5Zg transitions consistant with an octahedral eometry 25.

Fe M6ssbauer spectra
The m6ssbauer spectra of the iron (II) complexes have been recorded. The chemical isomer shift values (6) relative to natural iron foil, which are sensitive to both the oxidation and spin states of iron, are in agreement with the structural assignments made on the basis of above s,ectral evidences. The value of isomer shift (0.25-0.30 mm sl) and quadrupole splittings (0.65 mm s) at the room temperature are characteristic of sx coordinated low spin iron (II) complexes Mass Spectra In the mass spectrum of the compound CtsH3404N6CI2Fe, the molecular ion peak appeared at m/z The antifungal activity of the compounds have been evaluated against Macrophomina phaseolina, Fusarium oxysporum and Aspergillus niger by the Radial Growth Method 27 using czapek's agar medium. The compounds were directly mixed with the medium in 50, 100 and 200 ppm concentrations. Controls were also run and three replicates were used in each case. The linear growth of the fungus was obtained by measuring the diameter of the fungal colony alter four days. The amount of growth inhibition in all the replicates was calculated by the equation, Percent inhibition C-T/Cxl00, where, C is the fungal colony in the control plate and T is the diameter of the fungal colony in the test plate.

Mode of Action
The chelation theory 9 accounts for the increased activity of the metal complexes. The chelation reduces the polarity of the metal atom mainly because of partial sharing of its positive charge with the donor groups and posible n-electron delocalisation within the whole chelating ring. The chelation increases the lipophilic nature of the central atom which subsequently favours its permeation through the lipid layer of the cell membrane.
The degradative enzymes produced by the microorganism are important in host infection. For food deterioration and break down of organic matter. The enzyme production is here intended to mean both synthesis of the enzyme by the microorganisms and activity of the enzyme in the medium after it is produced. Since the metal complexes inhibit the growth of microorganism it is assumed that the production of enzyme is being affected and hence the microorganism is unable to utilize the food for itself or the intake of nutrients in suitable forms decreases and consequently the growth of microorganism is arrested, while higher concentration proves fatal. The higher concentration destroys the enzyme mechanism by blocking any of the metabolism path way and due to the lack of availability of proper food, the organism dies.
The results of biological activity have been compared with the conventional fungicide, Bavistin and the conventional bactericide streptomycin used as standards. The results achieved out of these studies have been enlisted in Tables IV and V in which the antifungal activity indicated that the metal chelates are more active than their parent amines and dicarboxylic acids. Similar trends were observed, in case of antibacterial activity. Brine Shrimp Lethality The eggs of Brine Shrimp, Artemia Salina (Leech) were hatched in a small tank divided by a net containing brine water. One part of the tank contained the eggs and on the other part, a light source was placed in order to attract the nauplii. Two days were allowed to hatch all the eggs and the nauplii were sufficiently matured for experimenta9.
The test compounds dissolved in DMSO were applied at five concentrations 5, 10, 20, 40 and 80 gm/ml. However not more than 50 lal of DMSO was added to nauplii in each vial. For each concentration, one vial containing the same value of DMSO plus brine water was used as a control group. After 24 hours of incubation the vials were observed with the help of a magnifying glass and the number of survivors in each vial were counted and noted. From this data the mean percentage of mortality of the nauplii was calculated for each concentration.   Table VI. The rate of mortality of the nauplii was increased with the increase in concentration of each sample.
A plot of Log of sample's concentration versus percentage of mortality showed a linear correlation. From the graph, the LCs0 values of the samples were calculated and they were found 29.51, 14.51, 9.12 and 5.75 la gm/ml, respectively. Drug activity depends on the size, shape and degree of ionization of the drug molecule normally. It is found that the specific type of biological activity of a molecule is dependent upon more than just one functional group. Consequently, the addition of a single functional group to an inert organic substance doses not ordinarily imbue a molecule with a specific biological activity since more than one functional group normally is required for potent activity. highly toxic for brine shrimp nauplii.
The compound [Fe(L2)CI2] got significant antimicrobial activity but showed less cytotoxicity, therefore, the compound has potentiality to be a safe and effective antibiotic. But further extensive investigations on higher animal model is necessary to study it's other toxic effects.