Synthesis, Spectroscopic and Toxicity Studies of Titanocene Chelates of Isatin-3-Thiosemicarbazones

The reactions of bis(cyclopentadienyl)titanium(IV) dichloride with a new class of thiosemicarbazone (LH2), derived by condensing isatin with different N(4)-substituted thiosemicarbazides, have been studied and products of type [Cp2Ti(L)] have been isolated. On the basis of various physico-chemical and spectral studies, five coordinate structures have been assigned to these derivatives. Toxicity studies of titanocene complexes at tbur different concentrations have been carried out against snail Lymnaea acuminata. The effect of most potent compounds on the activity of acetylcholinesterase enzyme, which inhibits the activity of enzyme, possibly by the formation of enzyme-inhibitor complex, was also studied.


INTRODUCTION
The potential antitumour, antibacterial, antiviral, fungicidal and antimalarial activities of thiosemicarbazones and their metal complexes have spurred the study of the coordination chemistry of these ligands /1-22/. Heterocyclic thiosemicarbazones exercise their beneficial therapeutic properties in mammalian cells by inhibiting ribonucleolide reductase, a key enzyme in the synthesis of DNA precursors/5-8/. Their ability to provide this inhibitory action is thought to be owing to coordination of iron via their N-N-S tridentate ligating system, either by a preformed iron complex binding tothe enzyme, or by the free ligand complexing with the iron-charged enzyme. Studies of iron and copper complexes have shown that they can be more active in cell destruction, as well as in the inhibition of DNA synthesis, than the uncomplexed thiosemicarbazones.
Recent developments in the structural nature of metal complexes of heterocyclic thiosemicarbazones, depicted below, are correlated with their biological activities. 2 3 4 ---N--NH--C(S) NR 2 It has been suggested/7,8/that the stereochcmistrics and activities of complexes often depend upon the anion of metal salt used, the nature of N(4)-substituents and on groups attached to N(I).
Synthesis, Spectroscopic and Toxicity Studies of Titanocene

Chelates
The 3-thiosemicarbazones of isatin have been of interest since 1-methyl isatin-3-thiosemicarbazone was found to be active/23-26/in the treatment of smallpox 40 years ago. Substitution at the N(4) position of the thiosemicarbazone moiety was found to reduce anti smallpox activity. Substitution of two butyl groups at the N(4) position yields a compound with demonstrated activity against ectromelia (a vaccina virus) and also against type 2 polio, which is an entrovirus and quite related to the vaccina family. Though biological activity of isatin thiosemicarbazones has been studied, there is little published information /27-30/ on the coordination behavior of this type of ligand. West et al. published/26/a paper on copper (II) complexes with N(4)-substituted isatin-3-thiosemicarbazones. The present paper describes the synthesis, characterization and molluscicidal activity of bis(cyclopentadienyl)titanium(IV) derivatives with isatin-3-thiosemicarbazones.
The structure of the ligands is shown below (I): 80% of LCs0 of [Cp2Ti(IPMET)] Animals were exposed for 24 h. After treatment for 24 h, snails were removed from the aquaria and washed with water and nervous tissue was taken out for the measurement of AChE activity.

AChE Estimation
The AChE activity was measured according to Ellman et al./35/as modified by Singh and Agrawal/36/ Nervous tissue (50 mg) around buccal mass was homogenized in one ml of 0.1M phosphate buffer (pH=8.0) for 5 min in an ice bath and centrifuged at 1000 g for 30 min at -4C. Supernatants were used as an enzyme source. The enzyme activity at 25C was measured in a 10 ram-path length cuvette, using an incubation mixture consisting of 0.10 ml (5x104M) of freshly prepared acetylcholine iodide solution in distilled water, The enzyme activity was expressed at pM "SH" hydrolysed minlmg-lprotein. Each experiment was replicated at least 6 times.

Preparation of complexes
A mixture of Cp2TiCl2 (30 mmol) and appropriate isatin-3-thiosemicarbazone (30.mmol) was dissolved in dry THF (ca. 60 cm3). To the resulting clear solution, Et3N (60 mmol) was added and the mixture was stirred for ca. 6-7 h at room temp. Precipitated Et3N.HCI was removed by filtration and the volume of the solution was reduced to ca. 15 cm under reduced pressure. The colored complex so obtained was recrystallised from a THF/petroleum ether (1:1) mixture.
The details of synthesis, yields and elemental analyses of the isolated complexes are given in Table 1.

RESULTS AND DISCUSSION
A systematic study of the reactions of bis(cyclopentadienyl)titanium(IV) dichloride with isatin-3thiosemicarbazone (molar ratio 1:1)   (62.1) (4.4) (11.1) (6.4) (9.6) IPTH2 Thiosemicarbazone derived from isatin and thiosemicarbazide of aniline IOMTH2 Thiosemicarbazone derived from isatin and thiosemicarbazide of o-toluidine IPMTH2 Thiosemicarbazone derived from isatin and thiosemicarbazide ofp-toluidine IOMETH2 Thiosemicarbazone derived from isatin and thiosemicarbazide of o-anisidine IPMETH2 Thiosemicarbazone derived from isatin and thiosemicarbazide ofp-anisidine Table 2 Infrared spectral bands (cm"1) of bis(cyclopentadienyl)titanium(IV) derivatives The characteristic infrared spectral bands of bis(cyclopentadienyl)titanium(IV) derivatives are given in Table 2. The ligands show bands at ca. 3350-3300, 3230-3200 and 1620-1600 cm -, assignable/26/to D(N (4) H), 19(N(Z)H) and t)(C=N), respectively. In the complex, the first band remains almost at the same position, indicating the (N4H) nitrogen atom is not coordinated to the metal. The second band at ca. 3230-3200 cm" is absent in the complexes; however the third band (ca. 1620-1600 cm) is lowered (ca. 15-20 cml)  Thus, the infra-red spectra reveal that isatin-3-thiosemicarbazones behave as dibasic, tridentate ligands coordinating through thiol sulphur, enolic oxygen and azomethine nitrogen. H n.m.r. Spectra The IH nmr spectra of the [Cp2TiL] type complexes have been recorded in DMSO-d6. (Table 3). Coupling between various groups complicates the spectra, but a comparison of the spectra of ligands with those of the complexes can lead to the following conclusion" (a) The 6 6.65-6.80 signals may be assigned to the cyclopentadienyl ring protons and indicate the rapid rotation of the ring about the metal ring axis.
The spectra of the complexes show the absence of first peak and the presence of second peak almost at the same position.
(c) The chemical shift due to aromatic ring appears at ca. 7.8-8.1 ppm, which slightly shifts downfield in the complexes. This may be due to decrease of electron density after forming the complex.

Chelates
The peak due to N(I)H of isatin ring appears at ca. 11.2 ppm in the spectra of ligands which disappear in the corresponding complexes. (a) The peak due to cyclopentadienyl groups appears at ca. 116 (relative to TMS).
(b) The ligands show amide C(2) and thioamide C(10) at ca. 5160 and ca. 5150, respectively. In the complexes, these signals are at significantly higher field. The significant shift in the position of C(2)   (Fig. 1). The slope values were steep and results were found to be within 95% confidence limits of LCs0. Steep slope value of ldp line indicate that a small increase in the concentration of compound cause large mortality in snail. The t-ratio greater than 1.96 indicate that a regression is significant. Heterogeneity factor values less than 1.0 denote that in the replicate tests of random samples, the concentration response line would fall within 95% confidence limit and thus model fits the data adequately. The index of significance of potency estimation (g) indicates that the value of the mean is within the limits at all probability levels (90%, 95%, 99%) as it is less than 0.5. Sublethal treatment of [Cp2Ti(IPMET)] caused significant change in the AChE activity in the nervous tissue of L. acuminata. 40% and 80% of 24h LC.s0 treatment caused a reduction in AChE activity up to 32% (0.35+0.022 .tm'SH'hydrolysed/min/mg/protein) and 28.85% (0.101+0.007 tm'SH'hydrolysed/min/mg/protein) of control (0.35+0.022 tm 'SH' hydrolysed/min/mg/protein), respectively. ] adversely affect the neurotransmission mechanism in the snail. These compounds are both metabolised in the snail body and transformed into more toxic form than their parent compounds or in due course of time i.e. from 24 h to 96 h exposure, there is an increase in concentration of the compound inside the snail body, which ultimately cause more mortality at higher exposure period.