Synthesis and Characterization of Coordinatively Unsaturated Copper (II) Complexes of 1,3-Bis(2'-Pyridyl)-1,2-Diaza-2-Butene and Their Antityrosinase Activity

The coordinatively unsaturated copper (II) complexes of 1,3-bis(2'-pyridyl)-1,2-diaza-2- butene with different ancillary anions were synthesized which can bind to copper centers of tyrosinase enzyme. The compounds were found to exhibit inhibitory activities against mushroom tyrosinase and the nature and extent of inhibition is modulated according to the type of ancillary anions.


Introduction
The observation of Prabhakaran and Kircheimer [1] regarding Mycobacterium leprae being unique among mycobacteria in possessing a Dopa (dihydroxyphenylalanine) uptake and tyrosinase (3,4-diphenoloxidase) enzyme system has remained controversial while some ascribing it as simple amino acid uptake of no special significance [2] and others ascribing the dopa-dopaquinone system as part of the possible respiratory chain [3].The intracellular accumulation of radiolabelled pigments following incubation with 4C-Dopa, however, indicates some selective oxidative process operative in the organism [4]. The role of such an enzyme-uptake-respiratory system, although, is still not confirmed, this putative enzyme provides a possible selective target for the design of novel antileprotic compounds which wouldpossess altogether different structures from the conventional antileprotic drugs [5].
Based on this approach a series of indole analogues having general structure (1) were designed as possible inhibitors of tyrosinase [6] while in anot.her study substituted aromatic and heterocyclic carboxylic acids were tested as putative inhibitors against mushroom tyrosinase as a model system [7].
The fact that the successful inhibitors in this model system showgood antileprotic activity in vitro when tested against M. leprae [5] motivated us to consider it as a preliminary rapid screen for evaluating further putative compounds belonging to imidazole series of ligands and their metal complexes.
The present work deals with the synthesis and characterization of three water soluble copper complexes of the hgand 1,3-bls(2-pyrldyl)-l,2-dlaza-2-butene (PPHY, 2), wh ch is a neutral, tridentate Schiff base derivatNe obtained by the condensation reaction of 2-acetylpyridine with 2-hydrazinopyridine, and their tyrosinase inhibitory activities. Analogous heteroc,clic hydrazone derivatives have been proposed as acceptable replacements for the active tut cytotoxic thiosemicarbazone antileprotic compounds. These compounds have been found to be effective against M. lufu independently or n combination with dapsone or trimethoprim [8]. Our studies indicate that the copper compounds of PPHYexhibit inhibitory activities against mushroom tyrosinase and the nature of their inhibition can be modulated according to the nature of ancillary anions.

Materials and Methods Chemicals and Instrumentation"
All solvents and reagents used were of AR grade. 2acetylpyridine was obtained from Aldrich while 2-hydrazinopyridine, L-DOPA (L-3,4-dihydroxyphenylalanine) and mushroom tyrosinase (EC 1.14.18.1) were products of Sigma Chemicals Company.
The elemental analyses were carried out in the Microanalytical Laboratory of University of Pune. The conductance measurements were made on a Phillips GM 4144 Conductivity bridge. Magnetic susceptibilities of the complexes were determined at 300 K on a Faraday type magnetic balance with a permanent magnet having field of 7000 KG. Absorption spectra were recorded as nujol mulls on the Shimadzu UV-160 spectrophotometer. Infrared Spectra were recorded as nujol mulls on a PYE-UNICAM 951 IR spectrometer. ESR spectra of the polycrystalline samples were recorded on a Varian 109 ESR spectrophotometer at 300K with 100 KHz field modulation amplitude.
The enzyme inhibitory studies were made using L-dopa as the substrate following literature protocols [9].
Preparation of 1,3-bis(2'-pyridyl)-l,2-diaza-2-butene (PPHY, 2) and its metal complexes PPHY was synthesized by refluxing 5 ml of ethanolic solution of 2-acetylpyridine (0.005 tool) with an ethanolic solution of 2-hydrazinopyridine (0.005 tool in 10 ml ethanol) for 10 minutes. A slow addition of water to the cooled reaction mixture separated out the ligand 2 (PPHY) as a white solid which was filtered, washed with cold ethanol and diethylether and dried under vacuum.

Results and Discussion
The synthesized copper complexes are shades of yellow to green and are stable at room temperature. They are readily soluble in water and other organic polar solvents. The compounds are coordinatively unsaturated as indicated by the conductivity data in ethanol which indicate them to have a metal to ligand ratio of 1:1 with a general formula of [M(HL)X)]Y, where HL is the neutral ligand and X and Y are the ancillary anions. All of the synthesized complexes exhibit a broad IR absorption around 3350 cm-I corresponding to the protonated hydrazino group which confirms the neutral nature of the hydrazone ligand in these compounds. In the IR spectra of the ligand 2, a sharp band observed at 1600 cmis ascribable to the imino (C=N) stretch which is found to undergo a shift to the higher wavenumbers upon complexation with copper ions (Table 1). Such upward shifts of the imino functionality have been interpreted-as indication of its involvement in the metal coordination [10]. Similar upward shifts can also be noted for the medium intensity band near 1000 cmascribable to the N-N stretching frequency. Five sharp and characteristic pyridine stretches can be observed for the ligand as well as their copper compounds between 1350 and 1600 cm-I similar to the metal complexes of an analogous ligand, viz. pyridine-2-carboxaldehyde-2-pyridyl hydrazone 11]. It has been estabhshed that the complexes of the hydrazino ligands behave much like the complexes of the pyridine ligands [12]. Finally, the perchlorate anions have been shown to exhibit two bands in the IR spectra at 1095 and 620 cmcorresponding to asymmetric stretching and bending frequencies respectively [13].These can be seen in the present B and C complexes around 1090-1095 and 628-630 cm-l respectively which confirm the presence of anionic perchlorate ions associated with these compounds. A low energy band observed in the dimeric complex B around 310 cmmay be assigned to the Cu-C1 bridging mode 14]. Additionally ligand to metal charge transfer band can be seen around 25000 cm-1 while the intra-ligand n--rc* bands involving azomethine nitrogen lone pair are observed at ca. 29000 c m-. The latter are found to undergo a shift to the lower energy side upon metal complexation.   Table 2. PPHY showed 13 % non-competitive inhibition at a concentration of 5 x 10 -4 M similar to thiourea which showed a non-competitive 28 % inhibition (Fig.l).
It is observed that complexation of copper ions with different ancillary anions seem to influence not only the extent of inhibition (from 10 to 25 %) but also the nature of inhibition from non-competitive to competitive one (Fig. 2 a-c) suggesting that binding of these coordinatively unsaturated complexes at the active site of the enzyme is perhaps influenced by the nature and bulk of ancillary ligands. This observation is important as it not only establishes the usefulness of copper complexes as tyrosinase inhibitors but also indicates that their antityrosinase activity can be modulated through choice of appropriate ancillary anions.