Synthetic, Biochemical, Antifertility and Antiinflammatory Aspects of Manganese and Iron Complexes

Manganese(II) and iron(II) macrocyclic complexes of polyamide groups have been synthesized by the template codensation of diamines (2,6 diaminopyridine, 1,2 phenylenediamine and 1,3 phenylenediame) and triamine (diethylenetriamine) with phthalic acid in 1:2:2 molar ratios. On the basis of elemental analysis, IR, electronic, magnetic moment, Mössbauer, mass and X-ray spectral studies, octahedral structure has been assigned to [ M ( N 4 mac n ) Cl 2 ] (M = Mn(II) and Fe(II), n = 1 to 4) complexes. The complexes have been screened in vitro against a number of fungi and bacteria to assess their growth inhibiting potential. An attempt has been made to correlate the structural aspects of the compounds with their antiinflammatory and antifertility activities.


INTRODUCTION
The field of macrocyclic compounds is developing very fast due to their variety of applications/1/and importance in the area of coordination chemistry/2/. The recognition of a metal ion by a maeroeyclie ligand and modification of the properties of the resulting complexes is controlled to a large extent by a match between the size .of the ligand hole and that of the metal ion/3/. The very high thermodynamic stability and extreme kinetic inertness of transition metal complexes of polyazamacrocyclic ligands are significant since they enhance a number of industrial applications/4/, A review on maeroeyles has revealed the importance of macrocyclic complexes in biological processes such as photosynthesis and dioxygen transport/5/, their catalytic properties /6/, their potential applications as metal extraetants and as radio-therapeutic /7/ and medical imaging agents. Macrocyelic polyamines have attracted increasing attention because of their unique property, namely to form very stable chelates with various heavy metal ions /8/. Studies on the t"ol. 9. Nos. [1][2]2002 ,Svnthetie, Biochemical, Ant/.rtilio., and Antiinflammatory Aspects of Manganese and Iron Complexes polyazamacroeyles, particularly the ttraaza groups have also been published frequently, particularly in view of their potential for binding mor than one mtal ion/9/.
Macrocyli complexes of mangans and iron ar used as antimierobial, antifertility, antiinflammatory and analgesic agents, Manganese, together with copper and iron, ar essential metallic elements and exhibit sufficient biological activity, when associated with certain metal protein complexes participating in th storage of ions/10/, to create enormous interest in the study of systems containing thes metals/11/. A detailed study of the interaction of Mn(II) and Fe(II) with diclofenae sodium (non-steriodal antiinflammatory drug was undertaken by Singh et al. /12/, Chondhekar et al. /13/have reported the fungitoxie studies of Mn(Ii) with hcterocyli Schiff base ligand, due to its wide applications in the food industry and agrochemical activity. Recently, Devereux /14/ and oworkers have shown that a range of carboxylat complexes incorportating manganese and cobalt metal centres inhibit the growth of Candida albicans/15/. Fe(II) complexes with 1,10-phenanthroline ar known to have a wide spectrum of antimicrobial actions and to produce negligible toxicity to skin subcutaneous tissues and mucocus membranes/15/.
Mn(Ii) complexes of 3,4,7,8 tetramethyl-l, 10-phenthroline were used topically to treat patients suffering from a variety of skin conditions, many of whom had chronic dermatological infections due to dermatophytes (e.g. malassezia furfur, trichophyton rubrum) or candida species/16/. The complexes produced a significant decrease in microbial infection in approximately 50% of cases, with infection due to gram positive bacteria generally responding much more rapidly and readily to treatment than infection due to gram negative bacteria. Manganese chloride causes loss of testicular germ cells in rats and rabbits/16/and decreased libido and impotency were noted in men occupationally exposed to manganese, but the data are inconclusive. The aim of the antifertility activity was to assess the effect on fertility and to contribute to a better understanding of the reproductive function of male albino rats. Hence, it was thought of considerable interest to synthesize macrocyelie complexes of manganese and iron with a view to evaluate their antimicrobial, antiinflammatory and antifertility activities.

EXPERIMENTAL
All the chemicals used in the synthesis of the complexes were of AR grade. MnCI2.4H20 and FeCI2.4H20 The weighed amount of MnC12.4H20 (0.99 g/5.0 mmol) was dissolved in a minimum amount of methanol at 0C and put in a magnetically stirred 100 mL round botton flask. The stirred solution of MnCI2.4H20 was reacted with diethylenetriamine (1.04 g/l 0.0 mmol) dissolved in methanol. This was followed by the addition of a methanolic solution of phthalic acid (1.66 g/10 mmol). The reaction was carried out in 1:2:2 molar ratios. The resultant solid products were filtered, washed several times with methanol and dried. These compounds were recrystallized in benzene and dried again in vacuo.

Analytical Methods and Physical Measurements
The molecular weights were determined by the Rast camphor method. Conductivity measurements in dry dimethylformamide were performed with a conductivity bridge type 305. Infrared spectra were recorded on a Nieolet Magna FT.IR 550 spectrophotometer in KBr pellets. Electronic spectra in dimethylsulphoxide were recorded on a UV-160 A, Shimadzu spectrophotometer in the range 200-600 nm using methanol as a solvent. X-ray powder diffraction spectra of the compounds were obtained on the Philips model P.W. 1840 automatic diffractometer using Fe(K,) target with Mg filter. The wavelength used was 1.9373 A and the reflections from 5-65 were recorded. The mass spectra of the compounds were recorded on a JEOL FX 102/DA-6000 mass spectrometer/data system using Argon/Xenon (6 KV, 10 mA) as the FAB gas. m-Nitrobenzyl alcohol was used as the matrix. Manganese and iron were estimated gravimetrically. Carbon and hydrogen analyses were performed at Central Drug Research Institute, Lucknow.

Aspects of Manganese and Iron Complexes
The resulting complexes are coloured solids, having sharp melting points. These are soluble in common organic solvents. The molar conductances of 10 -3 M solutions of the compounds in anhydrous dimethylformamide lie in the range 5-29 ohm -I cm mol-i, which shows that the complexes are nonelectrolytes and thus indicates that the anions are coordinated to the metal in these complexes.
The physical properties and analytical data of the complexes are listed in Table 1.

IR Spectra
The characteristic infrared frequencies of the complexes are summarized in Table 2  On the basis of spectral studies it seems that the ligands act as tetradentate chelating agents and the CIanions remained bonded with the metal atom having four coordination sites. Hence a hexa-coordinated environment around the metal atom, assigned in these complexes, is justified.

Metal-Based Drugs
Biochemical studies Antifungal Activities The fungicidal action of these complexes has been studied against Macrophomina phaseolina and Aspergillus niger by spore germination method/29/and compared with a commercial fungicide bavistin ( Table 4). The metal salts have negligible activity, but on complexation are found to be active.

Antibacterial Activity
The title compounds were screened for their antimicrobial activity against gram negative as well as gram positive microorganisms such as E. coli, S. aureus, S. typhi, B. sublitis, A. aerogenes and B. megatherium.
The solvent used was dimethylformamide. Sensitivity plates were seeded with a bacterial inoculum of x l06 ciu/mL and each well (diameter 10 ram) was loaded with 0.1 mL of test compound solution of variable concentration in DMF. The zones of inhibition were recorded after incubation for 24 h using Vernier callibers (Table 5). The comparative activity data of these compounds may be interpreted in term of phenyl group content in the compounds, i.e. the molecule having a four phenyl ring has a higher effect as compared to that which has a two phenyl ring. Further, the biocidal function of these compounds can also be described in terms of chelation theory/22/. Secondly, the activity increases as the concentration increases.
Iol. 9, Nos. I-2, 2002 ,vntheti; Biochemical, Antifertility and Anti#ammatory Aspects of Manganese and h'on Complexes  (17-20 ram) and (+) weakly active (12-16 ram), (+++) highly active (21-30 ram) Antiinflammatory Activities A freshly prepared 1% suspension of carragenine in 0.9% saline was infected under plantar aponeurosis of the right paw of the mice by the reported method/29/. One group of six mice was kept as control and animals of other groups of six each were treated with the test compound in a dose of 50 mg/kg. One group received the standard phenyl butazone. The volume of foot was measured by the micropipette method/30/ and percentage reduction of oedema was calculated at h, 8h and 24h.
The anti-inflammatory effect of these compounds was measured against simultaneously run controls using the method of Winter et al. /29/. The compounds were found to have significant anti-inflammatory activity at 50 mg/kg dose (Table 6). Metal-Based Drugs oedema was highly significant (P > 0.001) as comparable to phenyl butazone (P > 0.005) while in the test compound [Mn(N4macl)CI2] the reduction was also significant (P > 0.001), but less than phenyl butazone (P > 0.005). After 24 hours the reduction in oedema in most of the compounds was not very significant except phenylbutazone. The onset of action was found to be after hour and the activity reached its peak after  have more antiinflammatory activity than standard phenyl butazone.

Antifertility Activity
The reactivity of synthetic products towards biological systems is an important feature of current research and macroocyclic compounds of transition metals play a significant role in this direction. A large number of manganese compounds have been shown to cause atrophy of the testis, prostate and epididymis in male mice /3 I/. in view of the potential interest in these biologically active compounds, the antifertility activity of some selected compounds has been studied (Table 7) in male mice. The colony-bred adult mice were used and 45 male mice (body weight 40-50 g) were divided randomly into three groups of 15 animals each. The animals were kept in plastic cages measuring 25 cm. 20 cm and only five animals were housed in a cage. The animals were maintained on mice feed pallets (Hindustan Lever ltd., India) and water was provided ad libitum. Only two compounds were used separately and each compound was administered at a dose level of 10 mg/kg wt/day, orally by garage tube for twenty five days. One group served as control and olive oil was used as the vehicle. After 24 h of the last administration, five animals from each group were autopsied and the reproductive organs were removed by dissection, freed from adherent tissues and weighed up to the nearest milligram. The sperm motility and sperm count in cauda epididymis were measured by using Neubaur's hemocytormeter according to the reported method/32/. It was observed that the motility and count of sperm decreased after the administration of the complexes and the spermatogenia and the accessory sex organs were also affected in treated mice. The prostate gland became swollen and the effects did not become normal even after 30 days of recovery, showing the irreversible nature of the effects. A highly significant decline (P < 0.001) in the motility of sperm was observed in the case of [Mn(N4MaC3)CI] complexes. The sperm count was also found to decrease sig:nificantly in the treated IJd. 9. Nos. [1][2]2002 Synthetic, Biochemical, Ant(fertility and Antiinflammatory Aspects of Manganese and Iron Complexes animals. The antifertility activity data indicate that the complexes affect the motility as well as the count of sperm in male mice. Further studies concerning other tests for these complexes are in progress.