Fertility Inhibitors Macrocyclic Complexes of Bivalent Manganese: Synthetic, Spectroscopic and Medicinal Approach

The modern physico-chemical, spectroscopic and biochemical methods have proved an important tool to elucidate the constitution of transition metal complexes. This paper presents a brief account of the synthesis, spectroscopic and medicinal aspects of tetraazamacrocyclic compounds of manganese(II). Sixteen to eighteen membered tetraamide macrocyclic ligands DTTD1 and DTTD2 have been synthesized by the condensation of 1,2-diaminoethane and 1,3-diaminopropane with phthalic acid in the presence of condensing reagents dicyclohexylcarbodiimide and 4-dimethylaminopyridine. On reduction these macrocyclic ligands give new tetraazamacrocycles TTD1 and TTD2 which form complexes with manganese (II) nitrate and manganese (II) acetate. Based on chemical analyses, molecular weight determinations, conductance measurements, magnetic moment, IR spectra, 1H NMR, 13C NMR spectra, electronic spectra, mass spectra and X-ray spectral analysis, an octahedral geometry has been assigned to the newly synthesized products. The formulation of the complexes of the type [Mn(TTDn)X2] [where, n or 2, X (NO3) or (CH3COO)] has been established on the basis of chemical composition. The possibilities of potential uses of these complexes as fungicides and bactericides, studied in vitro, are also discussed. The testicular sperm density, sperm morphology, sperm mortality, density of cauda epididymis, spermatozoa and fertility in mating trials and biochemical parameters of reproductive organs of rat have been examined and discussed.


INTRODUCTION
The chemistry of macrocyclic complexes has received much attention and such compounds have been extensively studied in recent years /1/.Dramatic progress has been achieved in the field of macrocyclic chemistry on account of its various applications/2/in bioinorganic chemistry.Catalysis, extraction of metal ions from solution and the activation of small molecules gave impetus to this endeavor.In view of the *Author for Correspondence E-mail kudiwal@datainfosys.net; Fax +91 (141) 2708621 presence of two possible potential donor atoms, viz nitrogen and oxygen, the coordination chemistry of amide macrocyclic deserves special attention.It has been shown that amide macrocyclic compounds bear the dual structural features of macrocyclic tetraamines and oligopeptides and can stabilize higher oxidation states in some of the metal ions/3/.There are many examples of macrocyclic synthesis, mixtures of two or more donor sites have also been employed to tune of the selectivity and stability/4/.The current interest is often inspired by some other applications/5/and importance in the development of industrial area/6/.
Transition metals have aroused great interest in researchers, due to their biopotency, unusual stereo-and magneto-chemistry and ability to form multiple complexes.The eagerness to reveal the characteristics of transition metals has led to innovations in the area of model compounds/7-9/, in enzymatic mechanism/10-11/, noble stoichiometric /12/ and catalytic oxygen transfer systems.The complexes of manganese play an important role in photochemical reactions /13,14/.Several manganese complexes are known to exhibit antifungal /15/ and antileukemic activities.In view of these interesting aspects we have synthesized and characterized the tetraazamacrocyclic complexes of manganese(If) in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine as condensing reagents for the condensation of 1,2- diaminoethane, 1,3-diaminopropane and phthalic acid.The present paper deals with the striking structural features, synthesis and appreciable biological applications of these complexes.
Synthesis of the Ligands: 3,4 11, 12-Dibenzo-2,5,1 O,13-tetraoxo-l,6,9,14-tetraazacyclohexadecane (D TTDI) The reaction is carried out in 2 2 molar ratios of 1,2-diaminoethane and phthalic acid.A catalytic amount of 4- dimethylaminophyridine and an appropriate amount of dicyclohexylcarbodiimide (1.3274g;6.43mmol) in a minimum amount of dichloromethane at 0C were kept magnetically stirred in a two-necked round bottom 100 mL flask.The reaction was followed by the addition of 1,2-diaminoethane (0.3866 g; 6.43 mmol) and phthalic acid (1.0681g; 6.43 retool) in dichloromethane.The resulting reaction mixture was stirred for about 10-12 hours at 0C.The solid product was obtained by filtration and washing with dichloromethane.The white product thus obtained was recrystallised from benzene and dried in vacuo.

Synthesis of the Complexes
Dinitrato/acetato(1,6,9,14-tetraazcyclolteradecane)manganese(ll)ln(TTDl)X/ The reaction is carried out in a molar ratio of ligand TTD and metal salt.To a methanolic solution of metal salt (0.9467 g; 3.77 mmol) was added a methanolic solution of TTD (corresponding to metal salt).
The reaction mixture was stirred for about 15-18 hours.The resultant solid product obtained was filtel'ed off and washed with methanol and dried in vacuo.

Physical Measurements and Analytical Methods
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 Nicolet Magna FT-IR 550 spectrophotometer in KBr pellets.H NMR and 13C NMR spectra were recorded in DMSO-d6 using TMS as standard on a JEOL FX-90Q spectrometer.Electronic spectra in dimethylsulphoxide were recorded on a UV-160A Shimadzu spectrophotometer.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 -65A 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 was estimated gravimetrically.Nitrogen was estimated by Kjeldahl's method.Carbon and hydrogen analyses were performed at Central Drug Research Institute, Lucknow.

Fertility inhibitors .Macrocyclic Complexes of Bivalent Manganese
Synthetic, Spectroscopic and Medicinal Approach

RESULTS AND DISCUSSION
A series of sixteento eighteen-membered tetraazamacrocyclic ligands and their manganese (II) complexes were derived by the condensation of phthalic acid with 1,2-diaminoethane or 1,3-diaminop'opane in the presence of condensing reagents dicyclohexylcarbodiimide and 4-dimethylaminopyridine.The reactions proceeded as given in equation 1.
All the complexes are coloured solids.They are soluble in most of the organic solvents.The experimental conductivity values measured for 103M solutions in anhydrous dimethylformamide are in the range 16-32 ohln -I cm tool 1 showing that they are non-electrolytes.This indicated that the anions are coordinated to the manganese metal in these complexes.The physical properties and analytical data of the complexes are given in Table 1.
Bioinorganic Chembstry and Applications >., >-, Fertility Inhibitors Macrocyclic Complexes of Bivalent Manganese Synthetic, Spectroscopic and Medicinal Approach Infrared Spectra: A comparative study of the IR spectra of the tetraamides, tetraazamacrocyclic ligands and their corresponding manganese (II) complexes confirmed the formation of the macrocyclic complexes with the proposed coordination pattern.The ligands DTTD and DTTD show the absence of bands corresponding to primary amino and hydroxy groups, which confirms their involvement in the formation of tetraamide macrocycles.The presence of four bands in the regions 1640-1648, 1485-1489, 1230-1239 and 620-632 cm 1 is due to amide I, amide II, amide III, and amide IV bands respectively/16/.This provides strong evidence for the presence of a closed cyclic product.A single sharp band observed for amide ligands DTTD and DTTD in the region 3275-3289 cm 1 is due to v (NH) of amide group.Strong and sharp absorption bands that appear in the regions 2800-2925 and 1400-1450 cmin all the complexes may be ascribed to the C-H stretching and bending vibrations, respectively/17/.In the IR spectra of tetraazamacrocyclic ligands TTD and TTD2, it is observed that the amide bands have been found absent on comparing with DTTD and DTTD2.
In the spectra of macrocylic complexes of manganese (II) as compared to their analogous metal free tetraaza ligands a slight negative shift in the v (N-H) band that appeared in the region 3210-3235 crnwas observed.It is ascribed to the coordinated N-H stretching vibration.The shift to the lower frequency of v (N-H) mode, along with the appearance of a new band in the region 303-329 cm 1 assignable to the v(Mn-N) vibrations, suggested that the amide nitrogen is coordinated to the manganese/18,19/.The infrared spectral data of the ligands and their manganese(If) complexes are reported in Table 2.The nitro complexes exhibit a band in 243-258 cm region assignable to v (Mn-O) of ONO2 group/20,21/.
H and 13C NMR Spectra The bonding pattern in the ligands has also been sustained by the proton magnetic resonance spectra.The ligands DTTD and DTTD do not show any signal corresponding to primary amino and alcoholic protons suggesting that the proposed macrocyclic skeleton has been formed through condensation reaction.Broad signals exhibited by the ligands DTTD and DTTD at 6 8.44 and 8.47 ppm, respectively, are due to the amide proton (CO-NH).The signals due to the methylene protons 5 3.39-3.68ppm were also assigned for the ligands.A multiplet in the region 6 1.98 2.04 ppm may be attributed to the central methylene protons, -C-CH-C-.A multiplet ofaromatic protons was observed at 5 7.28 8.05 ppm in the spectra of all the ligands.The conclusions drawn from the IR and H NMR spectra are parallel with the carbon-13 spectral data regarding the authenticity of the proposed skeleton.H NMR and 13C NMR spectral data of the ligands are listed in Table 3.

Electronic Spectra
The electronic spectra of the complexes display weak absorption bands in the regions 17245-16930, 23827-23204 and 27020-26035 cm for 6Aig 4ylg 6A lg 4T2g and 6Alg 4Tlg respectively.These data are in fair agreement with the octahedral geometry for the Mn(II) complexes/22/.R. l,: Singh et al.

Magnetic Moment"
The lab values for the complexes observed at 5.82-5.89BM are within the range required for an octahedral geometry/23/.

Mass Spectrum
The mass spectrum of the compound [Mn(TTDZ)(CH3COO)2] shows the molecular ion peak at m/z =526.
The prominent fragments are at 454 for [Mn(C23 H30N204)]+,422 for [Mn(C18H30N404)] + and 382 for [Mn(C20H2204)] + due to the loss of C3HsN2, CH and C6HI6N4, respectively.Another peak appeared at m/z 408 due to the loss of two acetate groups from the parent ion.

X-ray Diffraction Studies
The X-ray diffraction analysis of the compound [Mn(TTD)(NO3)2] confirms the orthorhombic crystal system for this derivative having unit cell dimensions, a=13,.To assess the biological potential of the ligands and their macrocyclic complexes, laboratory and field experiments have been conducted.The following techniques have been used for the antimicrobial activities of these compounds.
Antifungal Activities 1. Poisoned Food Technique: The antifungal activity was evaluated by the food poisoned technique/24/.
2. Spore Germination Method/25/: The spore germination test includes depositions of chemicals on a slide, evaporation to dryness and addition of a drop of water containing spore of the test fungus.
A drop of spore suspension was placed on a clear slide.The number of spores in the drop under low power magnification was determined and the suspension was adjusted to obtain nearly ten spores per microscopic field.These slides were incubated for 12 hours at 25C in petri dishes working as moisture chambers.Slides were put in such a position that spore drops keep hanging.After incubations of 12 hours, spore germinations were counted.Each treatment was replicated thrice.In this technique total number of spores, number of germinated spores and number of ungerminated spores were counted.LDs0 values were calculated to compare the activity of the ligands along with their chelates by plotting graphs between the concentration of ligand vs complex with the number of germinated spores.The organisms selected fir this method are; Alternaria alternata and Helminthosporium gramineum.

Antibacterial Activity
Inhibition Zone Technique/26/: In this technique sterilized hot nutrient agar medium and 5 mm diameter paper discs of Whatman No. were used.The agar medium was poured into the petri plates.After solidifications, the petri plates were stored in inverted position so that there was condensation of water in the upper lid.Solutions of test compounds in methanol in 500 and 1000 ppm concentrations were prepared in which discs were dipped in solution of the test sample placed on seeded plates.The petri plates having these discs on the seeded agar should first be placed at low temperature for two or four hours to allow for the diffusion of chemicals before being incubated at suitable optimum temperature 28 + 2C for 24-30 hours.After the expiry of their incubation period, the zone of inhibition associated with the treated disc was measured in mm.The compounds were tested against Pseudomonas cepacicola (-), Xanthomonas compestris (-), Escherichia coli ('-) and Staphylococcus aureus (+).

Antlertility Activity
The antifertility activity of the ligands and their complexes was studied on male albino rats.ttealthy, adult male albino rats of sprague dawley strain were used in the present investigations.The rats were divided into seven groups of eight animals each.The group A served as vehicle (olive oil) treated control.In the group B, iigand D'FTD 25 mg kg body weight suspended in 0.2 mL olive oil was given Fertility Inhibitors Macrocyclic Complexes of Bivalent Manganese A),nthetic, Spectroscopic and Medicinal Approach orally for a period of forty five days.The animals of groups D and G received the same dose of compounds, respectively, for a similar period.The animals were screened for fertility test and autopsied for detailed pathological and biochemical studied.Reproductive organs were excised, blotted free of blood, weighed and fixed in Bouin's fluid for histological studies.
The sperm motility and density of Cauda epididymal spermatozoa was assessed.The protein, sialic acid /27/and fructose were determined/27/.The activity of acid phosphate was estimated.The vaginal smear or vaginal plug confirmed the positive mating.The females were checked for implantation after 16 days of pregnancy.The results were analysed with the help of student 't' test.

RESULTS AND DISCUSSION
Antifungal and Antibacterial Screening Antifungal and antibacterial activities of representative ligands and their manganese complexes have been screened in Tables 5, 6 and 7.The results howed that there is considerable increase in the toxicity of the complexes as compared to the ligands.
On giving a closer look at these results a common feature which appears is that the bioactivity enhances undergoing chelation.This can be well ascribed to Tweedy's chelation theory/28/.According to this, the chelation reduces the polarity of the central atom mainly because of partial sharing of its positive charge with the donor groups and possible -electron delocalisation over the whole chelation ring.This increases the lipophilic nature of the central manganese atom, which subsequently favours its permeation through the lipid layer of the cell membrane.
Solubility and concentration of the compounds play a vital role in ascertaining the extent of inhibition.The biological potency of these compounds may be attributed to their ability to inactivate various cellular enzymes which play a vital role in different metabolic pathways of these microorganisms.It has been proposed that the ultimate action of these compounds is the denaturation of one or more proteins of the cell as a result of which normal cellular processes are impaired.It may also be postulated that these complexes might act as uncoupling agents of oxidative phophorylation of ADP to ATP /29/.These agents are less effective for bacteria.
Representative ligands and their manganese complexes have been screened in vivo to study their potency on crop plants guar (guar blight by Alternaria cymopsidal) mung (Cercospora leaf spot) using l:'ercent Disease Incidence (PDI) technique.
The Percent Disease Incidence (PDI) is the area covered on foliage by specific disease and was calculated by using the following formula:  Antifungal Screening Data of the Ligands and their Macrocyclic Complexes of Manganese(II).Comparison of LDso Values (ppm) of Ligands and their Macrocyclic Complexes of Mangnese(ll).Vol. 1, Nos. 3-4, 2003   FertiliO Inhibitors Macrocyclic Complexes of Bivalent Manganese Synthetic, Spectroscopic and Medicinal Approach Field experiments were laid out in randomized block design with three replications.The crops were raised in each plot.Compounds with a standard fungicides, bavistin [2-methoxycarbamyl) benzimidazole] were tried in addition to check (water spray).
After sowing of 25 days, the plants were inoculated artificially by spraying the conidial suspension.The conidial suspension was prepared by crushing the infected leaves in water.The inoculation was done late in the evening.
The first spray of the respective fungicide was given when lesions were first seen and spraying was repeated after 10 days.Disease intensity was recorded 10 days after the second spray.The data was analysed statistically and Percent Disease Control was worked out.It was observed that, although all the fungicides were significantly at par with each other and different from check, yet the performance of the complexes are higher than their respective ligands.

Antifertility Activity:
The testicular morphology, testicular sperm density, sperm motility, density of cauda epid!dymal spermatozoa and fertility in mating trials and biochemical parameters of reproductive organ.,;with tetraamides and their manganese complexes in vivo were examined and discussed.
The results show that the ligands alone were able to inhibit fertility but due to the added synergistic effects of the manganese complexes, their activity becomes enhanced.The results are grouped under the following headings: Bo@ and Organ Weights Body weights of rat were not affected after their manganese complexes were administrated.However, the weights of testes, epididymis, seminal vesicle and ventral prostate were significantly decreased [Table 8].

Fertility Test
The sluggish motile spermatozoa was unable to fertilize normal cyclic females.The test was 68 to 92% negative in rats treated with the compounds.

Sperm Motility
The sperm motility declined significantly after treatment with compounds.

Sperm Density
The sperm density in testes and cauda epididymis declined significantly after treatment [Table 9].
Biochemical Changes Total protein and sialic acid contents of testes, epididymis, ventral prostate and seminal vesicle were depleted significantly after treatment with ligands and their complexes.The acid phosphate levels of testes, epididymis and ventral prostate were also reduced significantly.A significant decrease in seminal vascular Fertility hhibitors Macrocyclic Complexes of Bivalent Manganese Synthetic; Spectroscopic and Medicinal Approach "Jl-V!VI Bioino1Lganic ChemistIy and Applications fructose contents was also noticed whereas the testicular cholesterol contents were increased significantly after the treatment with various compounds [Tables 10 and 11 ].

Discussion
The present study revealed that administration of ligands and their manganese complexes caused a significant reduction in the weights of testes and other sex accessory glands.The prostate and seminal vesicle are well documented androgen dependent processes /31/.Sperm motility is considered as an important parameter in evaluating the fertility potential/32/.The tetraamide ligands and their manganese complexes significantly reduce the fertility of male rats.Since a number of androgen sensitive parameters (protein, sialic acid, fructose, acid phosphatase and total cholesterol) in target organs were found to be altered by these complexes, it is probable that the structure and function of epididymis and other sex accessory organs are changed.
Manganese chloride causes testicular germ cells in rats and rabbits /33/ and decreased libido and impotency in a man occupationally exposed to manganese/34/.Our findings indicate that the ligand TTD and its manganese complex have a more pronounced effect of fertility on various biochemical parameters of reproductive organs as compared to other ligands and complexes discussed in this paper.oI. 1, Nos. 3-4, 2003   Fertility bhibitors Macrocyclic Complexes of Bivalent Manganese S),thetic, Spectroscopic and Medicinal Approach VI VI Vl Fig.