Fertility Inhibitor Heterobimetallic Complexes of Platinum(II) and Palladium(II): Synthetic, Spectroscopic and Antimicrobial Aspects

Synthetic, spectroscopic and antimicrobial aspects of some fertility inhibitor heterobimetallic complexes have been carried out. These heterobimetallic chelates [M(C5H5N3)2M2'(R)4]Cl2 (M = Pd or Pt and M' = Si, Sn, Ti and Zr) have been successfully synthesinzed via the reaction of M(C5H7N3)2Cl2 with group four or fourteen dichlorides in 1:2 stoichiometric proportions. The products were characterized by elemental analyses, molecular weight determinations, magnetic susceptibility measurements, conductance, and IR multinuclear NMR and electronic spectral studies. A square planar geometry has been suggested for all the complexes with the help of spectral data. Conductivity data strongly suggest that chlorine atoms are ionic in nature due to which complexes behave as electrolytes. All the complexes have been evaluated for their antmicrobial effects on different species of pathogenic fungi and bacteria. The testicular sperm density, testicular sperm morphology, sperm motility, density of cauda epididymal spermatozoa and fertility in mating trails and biochemical parameters of reproductive organs have been examined and discussed.


INTRODUCTION
The complexes of metal ions with macrocyclic ligands are significant because of their resemblance with many natural systems, such as porphyrins and cobalamines2. Many of the transition metal ions in the living systems work as enzymes or carriers in macrocyclic ligand field environment. Therefore, meaningful research in this direction might generate simple models for biologically occurring metallo enzymes and thus will help in developing our understanding of biological systems. These ligands are also of theoretical interest as they are capable of furnishing an environment of controlled geometry and ligand field strength4"7. A literature survey disclosed that a number of polydentate macrocyclic ligands and their metal complexes have been reported8. Their electronic properties and reactivities are ressemble those of porphyrins.
On the other hand, the discovery of cisplatin, an important cytotoxic drug used in the treatment of a variety of human cancer, has given a new dimention to the coordination chemistry of platinum and palladium. It may be possible that useful anticancer drugs might be found among palladium complexes. A wealth of data has been accumulated in the search of novel potent antitumor agents. Transition metal chelates of nitrogen donor ligands have been extensively screened for their antitumor activity. Most of them showed some activity but significant activities were exhibited by palladium and platinum complexes. In principle, coordination compounds offer a great variety of shapes and reactivities for use in drug design, the most detailed advances have been in understanding how cis-DDP binds to DNA. A clear knowledge of how platinum antitumor drugs work would have major implications for the further design and improvement of these inorganic drugs. The use of metal as template in condensation reactions has led to the synthesis of a large number of metal complexes of macrocyclic ligands. Macrocyclic ligands are relatively rigid and thus impose a specific coordination geometry on the metal ion9. Macrocyclic ligands display a number of chemical interesting features which include unusual structures. The occurrence of metal exchange (transmetallation) reactions is used for the preparation of new metal complexes not accessible by direct synthetic procedure A wider range of organosilicon compounds is being studied due to their importance in resin and liquid polymer Chemistry 1'3 Well known exemplary series of di and triorganotin halides with various nitrogen and oxygen/sulphur containing ligands have been found to possess significant biological and pharmacological activities. Screening data for tin derivatives 16,17 have revealed that many more diorganotin compounds exhibit antitumour activity than the corresponding mono-,tri-,and tetra-organotins or the inorganic tin chemicals, while within the diorganotin class, the highest activity was given by the diethyl tin derivatives 8. Similarly, some of the organotin have been evaluated as potential mosquito larvicides 19.
Transition metal complexes of nitrogen donor ligands have been studied in detail on account of their 20 21 interesting stereochemistry and wide practical utility.
Transition metals and their complexes have evolved great interest due to their biological potential 22'2 unusual structural aspects, unique stereo and magneto chemistry 24 and ability to form multiple complexes. Fertility Inhibitor Heterobirnetallic Complexes of Platinum(II)and Paladium(II) Synthetic, Spectroscopic and Antimicrobial Aspects Several reports have appeared on multimetallic complexes 25 associated with electrochemical, magnetic and spectroscopic studies and their stable complexes which are of biological interest but studies on the heterobimetallic complexes of palladium and platinum seem to be comparatively limited. Therefore, in view of the above facts it was considered as useful to synthesize such a type of compounds with an aim to characterize them structurally, electrochemically and biologically.

MATERIALS AND METHODS
All the chemicals and solvents used were dried and purified by standard methods. The reactions were carried out under strictly anhydrous conditions. Preparation of M(C5H7N3)CI2 The solution of MCI2 (1 mmol) was mixed with the methanolic solution of 2,6 diaminopyridine (2 mmol). The reaction mixture was stirred for 6-7 hours. The resulting precipitate was recovered by filteration washed with methanol and dried in vacuo. The reactions proceed as: MCI2 + 2CsH7N3 -M(CsH7N3)2CI2.

Preparation of Heterobimetallic Complexes
Heterobimetallic complexes were synthesized by stirring a methanolic solution of M(CsH7N3)2CI2 with methanolic solution of group four or fourteen metal dichlorides CpEM'CI2, Ph2MCI2 and MeEMCI2. The reaction mixture was kept at room temperature for overnight and stirred for 6-7 hours to complete the reaction. The coloured solid compound separated out. The excess of the solvent was removed under reduced pressure and the complexes were dried for 3-4 hours in vacuo. The complexes were purified by crystallization. The yield was 60-70%. The analytical and physical data of the isolated precursor and their complexes are given in Table I. Electronic spectra were recorded on a Varian Cary/2390 spectrophotometer. H NMR and metal NMR spectra were recorded in DMSO-d6 versus TMS as standard on a JEOL FX-90Q spectrometer. Magnetic measurements of powdered samples at the room temperature were recorded on a vibrating sample magnetometer Model 155 at the RSIC, IIT, Madras. Platinum, titanum, zirconium, tin and silicon were estimated gravimetrically as their oxides. Nitrogen was estimated by Kjeldahl's method, and chlorine by Volhard's method.

RESULTS AND DISCUSSION
The elemental analysis and spectral data are consistent with the formulations of the compounds as M(CsH7N3)2CI2 and [M(CsHsN3)2M'2(R)4]. The reactions of MCI2 with CH7N3 have been carried out in 1:2 molar ratios in methanol to yield M(CsHTN3) 2C12. This complex reacts with group four or fourteen metal dichloride to yield The reactions proceed easily at room temperature. The coloured solid products so obtained are soluble in DMF and DMSO. The complexes are monomeric in camphor as indicated by the molecular weight determinations. Magnetic measurements showed them to be diamagnetic. However, the complexes are 1:2 electrolytes in DMSO as indicated by their molar conductance values (200-225 ohm "l cm mol).

IR Spectra
The IR spectra of bimetallic complexes compare well the spectra of M(CsHTN3)2Cle. The primary stretch is located at higher frequency than that of the corresponding secondary amine26. The spectra of M(CsH7N3)eCI2 exhibit a broad and strong band in the range 3280-3140 cm" assigned to v(N-H). This band is found at lower frequency (3098 cm) 27 in the complex. This may be taken as an evidence for the coordination of secondary nitrogen to metal. Aromatic ring stretching (c---c) 28 are present at 1646, 1520 and 1468 cm". The presence of aromatic C-H and C-N bonds in the complex have been confirmed by the appearance of two bands at 3055 and 846 cm , respectively.
The coordination of nitrogen to the metal is further supported by the appearance of new bands of medium to weak intensity in the regions 581 cm "1 and 410 cm "1 attributable to (Si <---N) 29 and ag(Sn <--N) vibrations, respec3tvely. Apart from this, the band at 447 cm "l and 442 cm " ma2aY be assignedaatO v(Ti-CsHs) 3 Table II.  The four coordination number of tin in these complexes were further get support by the appearance of sharp signals at -31.23ppm in ll9Sn NMR spectra34.
In the cases of the silicon complexes, signals appeared at 5-50.99ppm are assigned for tetra coordinated state 35 around the silicon atom.

C NMR Spectra
In the 3C NMR spectra of the precursors and their complexes, considerable shifts in the position of carbon atoms adjacent to atoms involved in complex formation clearly indicate the bonding of metal to the imino nitrogen atoms. Spectral data are given in Table Ill. Synthetic, Spectroscopic and Antimicrobial Aspects The formation of heterobimetallic complexes is further supported by the electronic spectra. Palladium (II) and platinum(II) complexes display d-d spin-allowed transitions due to three lower lying 'd' levels to the empty dx2.y2 orbitals. Transitions are located from ground state 1Alg to the exited states rA2, lBlg and lEg in order of increasing energy. Three d-d bands are assigned in the ranges 535 560nm, 455-475nm and 440 -451nm in the palladium complexes and 519-530nm, 440-465nm and 339-376nm in the case of platinum complexes. These bands are attributed to 1Alg )lAEg, 1Alg--1Big and 1Alg--) lEg transitions, respectively. These spectral values support the square planar geometry (Fig.l) around Pd(II) and Pt(II) and are in agreement with those reported earlier for square planar complexesav.

Antibacterial Activity
The antibacterial activity was determined by the inhibition zone technique. All the compounds were dissolved in methanol, paper discs of Whatman No. paper with a diameter of 5mm were soaked in these solutions. These discs were placed on the appropriate medium previously seeded with organisms in Petri discs and stored in an incubator at 30+1C. The inhibition zone thus formed around each disc was measured in mm after 24 hours. Data ofthese activities are summarised in Tables IV and V.  15 16 The results reveal that the activity increases on complexation. The newly synthesized complexes have indeed been found to be more active in inhibiting the growth of fungi and bacteria than the precursors themselves. It may be postulated that these complexes might act as uncoupling agents of oxidation 39 posphorylation. The first uncoupling agent to be described, by Loomis and Lipmann was 2,4-dinitrophenol. Today many different uncoupling agents are known. Most are lipid soluble substances containing an acidic group and usually an aromatic ring. These agents allow electron transport to continue but prevent the phosphorylation of ADP to ATP. They uncouple the energy-yielding fromthe energy conserving reactions. Uncoupling agents function by breaking down a high-energy intermediate or state generated by electron transport. They can promote the passage of H + ions through the cell membrane, which is normally impermeable to them4. However, these agents are less effective for bacteria. The greater toxicity of metal complexes than the precursors can also be explained on the basis of the chelation theory4'4. Chelation reduces the polarity of metal ion mainly because of partial sharing of its positive charge with the donor groups and possible r-electron-delocalisation over the whole chelation ring. This increases the lipophilic character of the metal complex, which subsequently favours its permeation through the lipid layers of the organism cell membrane, and the normal cell process being impaired. Synthetic, Spectroscopic and Antimicrobial Aspects were removed, fat and connective tissue cleared off and kept at -20C until assayed for total protein, sialic acid, cholestrol, fructose and glycogen by standard laboratory techniques.
Group C compared with group B Group G compared with group F The mating exposure tests of all the animals were performed from day 55 th to 60th. They were cohabitated with proestrous females in the ratio 1:3. The vaginal plug and the presence of sperm in the vaginal smear were checked for positive mating. The mated females were separated to note the implantation sites on day 16 th of pregnancy through leprotomy. Body and Organ Weight No significant change was observed in the body weight after treatment with the compounds. A significant reduction in the weight of testes, epididymis, seminal vesicle and ventral prostate was observed after treatment with both the precursors and the compounds (Table VI). Sperm Dynamics Sperm motility in cauda epididymis and sperm density in testes and cauda epididymis were significantly reduced after treatment with both the precursors and their compounds (Table VII).

BIOCHEMICAL PARAMETERS LEADING TO INFERTILITY Total Protein
Treatment with both the precursors as well as their complexes resulted in a significant reduction in the total protein contents of testes, epididymis, seminal vesicle and ventral prostate (Table VIII).
See footnote of Table VI Sialic Acid A significant reduction in sialic acid contents of testes, epididymis, seminal vesicle and ventral prostate was observed after the treatment in all experimental groups (Table IX).   Table VI Cholesterol Cholesterol contents of testes were decreased significantly in all experimental groups (Table X). Fructose A significant decrease in the seminal vesicular fructose was noticed in all experimental groups (Table IX).  (Table IX).
Present study showed that oral administration of PdClz, PtCI2, precursors and their complexes resulted in the reduction Of weights of testes, epididymis, seminal vesicle and ventral prostate. The weight, size and secretory activities of sex accessories are closely regulated by androgen levels43. Reduction in sperm density and motility in cauda epididymis is of importance with regards to fertilization44. Significant reduction in the sperm motility and sperm density was observed in treated animals. This may be due to inhibitory effect of these compounds on the enzyme oxidative phosphorylation45. In our study various androgen dependent parameters that is total protein, sialic acid, fructose, cholestrol and glycogen revealed a significant decrease indicating that administration of these compunds resulted in the fall of circulating androgen46'47. It is inferred that compounds of Pd and Pt are found to be more effective than the starting materials in inhibiting the fertility.