Characterization and Antimicrobial Activity of Erbium(III) Complexes of C-3 Substituted 2-hydroxy-1,4-naphthalenedione-1-oxime Derivatives

Erbium(III) complexes of 2-hydroxy-l,4-naphthalenedione-1-oxime and its C-3 substituted derivatives are synthesized and characterized by elemental analysis, thermogravimetric analysis, infrared spectroscopy, magnetic susceptibility measurements 2-hydroxy-1,4-naphthalenedione-1-oxime derivatives are analysed using 1H and 13C NMR spectroscopy. The molecular composition of the synthesized complexes is found to be [ML3(H2O)2]. The antimicrobial activity of these complexes is determined by well diffusion method against the target microorganisms- Staphylococcus aureus, Xanthomonas campestris, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger. The antimicrobial activities of 2- hydroxy-1,4-naphthalenedione-1-oximes and their complexes are compared. It is observed that 2-hydroxy-1,4-naphthalenedione-l-oximes exhibit higher antifungal activity as compared to antibacterial activity. These activities are reduced upon complexation of these oximes with Erbium.


INTRODUCTION
The utilization of the lanthanides and their complexes in biological and biochemical studies has been reviewed by Williams[l]. The application for localization of tumors using appropriate lanthanide complexes was studied by Hider et a/. [2] and Lauffer [3]. Yam et al. have reported that lanthanide complexes play a key role in various dignostic areas [4]. Gaikwad [5] and Dandawate [6] have studied the complexes of some lanthanide metals with hydroxynaphthoquinone-oximes. In continuation of studies on lanthanide complexes as antimicrobial agents [5][6][7], we are reporting antimicrobial activity of Erbium(III) complexes by well diffusion assay method. General structure of the ligating system( Fig. 1) is as reported earlier [8].

Synthesis of complexes
To a hot solution of 3 mmol of oxime derivative [0.568 g of (I), 0.60 g of (II), 0.671 g of (III), 0.804 g of (IV) and 0.945 g of (V)] in 25 mL of ethanol, an aqueous solution of mmol of erbium trichloride hexahydrate (0.381 g was added. The pH of the mixture was kept around 6 using aqueous ammonia (1:20 v/v). It was refluxed for 3 h and then cooled overnight. The precipitate was filtered off, washed with water, followed by hot methanol and dried in vacuum over fused CaCI2 at ambient temperature. The solubility of these complexes was tested in H20, CH3OH, CH3CN, DMF, DMSO and inert solvents. B.P. Kapadnis et al.
Characterization and Antimicrobial Activity of Erbium(lll) Complexes Of C-3 Substituted 2-Hydroxy-1, 4-Naphthalenedionel-Oxime Derivatives The elemental analysis was carried out using a Hosli-Holland C, H Analyzer. The magnetic studies were carried out at room temperature by the Faraday technique using mercury(ll) tetrathiocyanatocobaltate as calibrant. The Infrared soectra were recorded in nujol mulls on a Perkin-Elmer FTIR spectrophotometer (Model 1600,4000-450cm'a). H NMR and 3C NMR spectra were recorded on Varian Mercury-300 NMR spectrophotometer.

Media
The dehydrated plate count medium (g/100 ml distilled water glucose 0.1, yeast extract 0.25, tryptone 0.5) and Sabouraud's dextrose agar g/100 ml distilled water glucose 4, peptone) purchased from Hi-Media Laboratories, India were used respectively for antibacterial and antifungal activity.

Microorganisms and their maintenance
The target microorganisms included Staphylococcus aureus NCIM2079, Xanthomonas campestris NCIM 2954, Pseudomonas aeruginosa NCIM2036, Candida albicans NCIM3471 and Aspergillus niger NCIM 545. These were obtained from NCIM, NCL, India. These strains were selected because they are routinely used in testing of disinfectants [10]. The stock cultures of these microorganisms were maintained at-20C in 15% glycerol [11]. The inoculum was prepared from stock cultures by streaking onto the plate count agar for bacteria and on Sabouraud's dextrose agar for fungi. After an overnight incubation single colony was used to inoculate sterile liquid media. The 5ml broth was dispensed in test tubes and sterilized in the autoclave at 121C for 15 min. The broths were then inoculated with respective cultures and incubated on an orbital shaker (150 rpm) overnight at 30C A540 of bacterial cultures and Candida albicans. was adjusted to 0.12 and 0.20 respectively. This corresponds to 106-10 colony forming unit (cfu/ml). The spore inoculum of A. niger containing 10 spores per ml was used.

Determination of Minimum tidal concentration(MCC) by the well diffusion assay method
The solutions of ligands and complexes prepared in DMSO [12] were diluted in DMSO and added to tubes containing 3 mL liquid medium and inoculated with 30 laL of the cultures. Incubation was done for 18 h at 37C. The minimum cidal concentration was then found out. This concentration was used for well diffusion assay method [13].

RESULTS AND DISCUSSION
The reaction involving Er(IIl) and 2-hydroxy-l,4-naphthalenedione-l-oximes in ethanolic medium with 1"3 metal-ligand(M:L) ratio resulted in the complexes having molecular composition as ErL3.(l-lzO)_, (where L anion of the corresponding oxime derivatives) which is supported by elemental analysis. All the oxime derivatives are soluble in water, methanol, DMF, DMSO and acetonitrile, while their complexes are soluble in DMF, DMSO, methanol and acetonitrile and are insoluble in inert solvents like n-hexane, benzene, 1,4-dioxane etc ( Table 1).
The elemental analysis of ligands is already reported [8].  Table 2. A medium broad band at 3100-3600 cm exhibited by ligands, is due to the (O-H) vibration of oximino and phenolic hydroxyls functions. Due to overlapping of this stretching frequency with coordinated water molecules, this band is further broadened upon complexation [6]. The redistribution of electron density in the quinonoidal ring is indicated by shifting of (C=O) stre, tching frequency (1600-1630 cm for ligands towards lower frequency region by [20][21][22][23][24][25][26][27][28][29][30] cm" is suggestive of complexation. The coordination through the oximino nitrogen reflected by shifting of C=N vibration band (1570-1590 cm) at lower frequency by -40-70 cm is due to chelation. The quinone absorption is found at 1285-1296 cm". The shifting of about 10-35 cm for (C-O) stretching frequency (1210 cm) of ligand is indicative of phenolato oxygen as the other coordinating center for oxime derivatives.  The coordination through oximino nitrogen is further confirmed by the increase in the (N-O) s, tretching frequency (the increase in the 1050 cmt) by 5-20 cm [14]. Absorption at 688-695 cm" is assigned to (C-X); this band is shifted to lower region by 3-10 cm . For complexes, a medium intensity band at 465-475 cm "t is assigned to (Er-O) stretching vibration. 13 b) Hand C NMR studies lH and 3C NMR data for (I)-(V) is depicted in Table 3and 4 respectively. The syn-amphi isomers of 2-hydroxy-l,4-naphthalenedione-l-oxime derivatives are shown in Fig.2. It has been reported earlier that C 2-hydroxyl signal for lawsone is observed as a broad signal (in CDCI3) at 7.42 ppm indicating a dimeric associations [15]. However, such a dimeric nature was found to be destroyed when it's spectrum was recorded in DMSO-d6; wherein this band appeared at 11.63 ppm as a broad signal is typical of intramolecular hydrogen bonding [16]. B.P. Kapadnis et al.
Characterization and Antimicrobial Activity of Erbium(lll) Complexes Of C-3 Substituted 2-Hydroxy-1, 4-Naphthalenedione-1-Oxime Derivatives The C2-OH signal for (II), (IV) and (V) appeared in the region 12.72-13.61 ppm is suggestive of its involvement in intramolecular hydrogen bonding. However, the spectrum for (I) and (III) do not exhibit any signal originating from this C2-hydroxyl group probably indicating the increased stability of intramolecular hydrogen bonding.
Quinone oximes [17] are found to possess syn-amphi isomers (Fig.2); similar to nitrosophenol tautomers [18]. Rane et al have suggested that amphi form is predominant for quinone oxime derivatives mainly due to the steric effects of C-3 substituents as well as their inductive nature. The signal at-9 ppm is assigned to oximino proton(Hl) which is coupled with H9 in equal intensity suggesting the predominance of amphi form of oxime (Fig. 2). Also, the doublet at-7.9-8.3 ppm for these oxime derivatives supplemented such an observation. H6,7 protons appeared as a doublet of doublet at-7.57-7.79 ppm indicating the spin-spin coupling with H5 and H8 protons, while H5 signal is found to be a doublet. Signal originating from H3 proton for (I) appeared at 6.16 ppm as a singlet same that of lawsone [16].  9.01(d) 9.02(d) 9.06(d) 9.04(d) C2-OH 11.66(br) 12

8.28(d)
The 3C NMR data of (I)-(V) is summarized in Table 4. Although, 3C NMR of quinone oximes has not been fully explored, we have assigned the resonance signals based on the earlier reports on 2methoxy-1,4-naphthoquinone [ 19] and lawsone [ 16]. It is significantly observed that the C signal is substantially shifted towards higher field (by -18-25 ppm) indicating the influence of basic nitrogen atom of oximino function [20]. Moreover, the adjacent C_ position has also undergone upward shift by-16-21 ppm. This clearly indicates that C2-hydroxyl group is involved in intramolecular hydrogen bonding leading to the dominance of amphi-form of oxime derivatives. The resonating structure of quinonoidal part of naphthoquinones seems to be disturbed due to oximation which is clearly reflected in the increased energy of C4 resonance for (I)-(V) as compared to lawsone. However, other signals originating from remaining carbon atoms are only slightly affected. c) Antimicrobial activity of the compounds The extent of inhibition of Staphylococcus aureus by ligands is found to be more pronounced than their complexes [21][22][23][24]. However the activity (I), (II)is more inhibitory to Staphylococcus aureus as compared to dichlone. (I)-(V) inhibited Xanthomonas. campestris significantly than their metal complexes. The extent of inhibition for ligands is more than that of dichlone [25]. The Metal Based Drugs Vol. 8, Nr. 3,2001 extent of inhibition of Pseudomonas aeruginosa by ligands and their complexes is not much altered. However (I), (II), E, (III) exhibit increased inhibition against Pseudomonas aeruginosa. Erbium complexes in general of (I)-(V); exhibit lesser antimicrobial activity against most of the organisms studied as compared to their oxime derivatives [23,24,26]. Pseudomonas aeruginosa are relatively less sensitive which is an indication of their inherent resistance for these compounds. All the compounds, especially the ligands, have better antifungal activity than that of antibacterial as indicated by the highest sensitivity of Candida albicans and Aspergillus niger [27,28]. It can be concluded that complexes, in general possess lower antimicrobial activity than the ligands. Staphylococcus atreus is more sensitive to (I) and (II). The Candida albicans and Aspergilhts niger are highly sensitive to (II), (III), (IV) and (V). The sensitivity of all these fungi is comparable to that of Staphylococcus aureus.