1. Introduction

Hyperglycemia has been identified as a major risk factor for the development of diabetic complications[1]. Aminoguanidine (AG) has been extensively studied as one of the most promising compounds for the treatment of diabetic complications, because the compound has both advanced glycation inhibitory activity[2] and antioxidant activity[3,4]. It has been reported that a pyridoxal- aminoguanidine (PL-AG) Schiff base adduct exhibits advanced glycation inhibitory activity comparable to that of AG, while causing no decrease in the liver pyridoxal phosphate content of normal mice[5,6]. Also, PL-AG is more potent than AG in preventing nephropathy in streptozotocin-indluced diabetic mice[7]. These findings suggest that PL-AG is superior to AG for the treatment of diabetic complications because not only it prevents vitamin B6 deficiency, but it is also superior at controlling diabetic nephropathy. The preventive effect of this adduct against diabetic nephropathy is mediated via inhibition of both oxidation and glycation[8]. The inhibition of advanced glycation end products (AGEs) at millimolar concentrations of AGEs inhibitors, used in many in vitro studies, results primarily from the copper chelating or antioxidant activity of the AGEs inhibitors, rather than their carbonyl trapping activity[9]. Defining the chelating activity of AGEs inhibitors is essential for understanding the mechanism of action of drugs and possible benefits of chelation therapy in diabetes, as well as for developing more effective clinically useful inhibitors of diabetic complications. Bearing in mind the biological and medicinal importance of AG and PL-AG adduct and the influence of copper (II) ions on their inhibitory activity, in the present study evaluation of the chelating ability of PL-AG inhibitor was attempted in order to cast more light on the role of copper chelation in the mechanism of action of AGEs inhibitors.

2. Experimental Section

2.1. Reactants

All commercially obtained reagent-grade chemicals were used without further purification, except for the ligand, which was prepared according to the previously described procedure[10^a,b].

2.2. Physical Measurements

Elemental (C, H, N) analysis of air-dried samples was carried out by standard micromethods inthe Center for Instrumental Analysis, Faculty of Chemistry, Belgrade. The X-ray analysis was performed in the Oxford Chemical Crystallography Service.

2.3. Crystal Data Collection and Processing

A single crystal was mounted on a glass fibberin random orientation. Data collection was performed at temperature 150 K on a computing data collection Nonius Kappa CCD diffractometer using graphite mono-chromated MoKα radiation (λ = the data can be obtained free of charge by writing to 0.71073 Ǻ). Data collection and cell refinement we details concerning data collection and structure re carried out using DENZO and COLLECT[11,12]. The structures were solved with SIR-92 and refined using CRYSTALS [13,14]. In general, the hydrogen atoms were visible in the difference map. Therefore, they were positioned geometrically and refined in a separate hydrogen cycle (with soft restraints) before inclusion in the refinement using a riding model. For more information see the Supplementary information. Crystallographic data has been deposited at the Cambridge Crystallographic Data Centre. Copies of thedata can be obtained free of charge by writing to CCDC, 12 Union Road, Cambridge CB2 IEZ, UK; e-mail:deposit@ccdc.cam.ac.uk. The CCDC deposition numbers are 828036. Crystal data anddetails concerning data collection and structure refinement are given in Table 1.

Table 1. Crystal data and structure refinement details of the complex

2.4. Preparation of the Ligand and Complex [Cu(PLAG)(NCS)₂]₂

2.4.1. PLAG•HCl•H₂O

The ligand (PL-AG•HCl•H₂O), synthesized in a similar manner as described for PL-AG[6], is a yellow microcrystalline compound which is moderately soluble in water and sparingly soluble in MeOH, EtOH and DMF[10].

2.4.2. [Cu(PLAG)(NCS)₂]₂

To a warm methanol (20cm³) solution containing 0.28g (1 mmol) of PLAG 0.26g (1.2 mmol) of CuCl₂ in 10cm³MeOH was added followed by 0.12 g (1.6 mmol) of NH₄NCS. Green mixture was formed which was left behind at room temperature for about 50 hours. Green crystals started to form after a couple of hours. After the sedimentation has completed the product was isolated by vacuum filtration and was then washed with methanol. Yield: 0.18 g.

3. Results and Discussion

3.1. Synthesis and Characterization of the Ligand and the Complex

Neutral form of the ligand pyridoxal-aminoguanidine (PLAG), is synthesized according to a previously described procedure[10], involving a reaction of water mixture between PL•HCl and AG•H₂CO₃[1:1] in the presence of Na₂CO₃•10H₂O (Scheme 1).

PLAG is a yellow microcrystalline compoundvery soluble in MeOH and DMF, but slightly less in H₂O and EtOH. The resulting compound is stable in air and at high temperatures. The elemental analysis data are given in Table 2.

By using reaction of a warm MeOH mixture of CuCl₂ and PLAG, the mole ratio of 1:1, with the addition of NH₄NCS a dark-green monocrystaldimeric complex[Cu(PLAG) (NCS)₂]₂ (Scheme 2) was obtained.

Scheme 1. Synthesis of pyridoxal-aminoguanidine (PLAG)

Scheme 2. Syntheses of the complex[Cu (PLAG)(NCS)2]2

The resulting complex is crystalline solid that is stable in air and at high temperatures (melting point of the complex is 330ºC). It is very soluble in DMF, less in H₂O, MeOH and EtOH. The elemental analysis data are given in Table 2.

Table 2. The elemental analysis data for the ligand and complex

Comparison of the ν(C-O) signals in the IR spectra of the ligand (ν(C-O) ~ 1290 cm^-1) and the complex (ν(C-O) ~ 1330cm^-1) indicated coordination of oxygen and deprotonation of the phenolic hydroxyl moiety (Figures 1 and 2). Intense peak in the IR spectrum of the complex at 1650 cm^-1 originated from ν(C=N) or the coordinated azomethine group and the value has been moved to a lower energy with respect to the corresponding value observed for the free ligand.

The bands that could be assigned to the guanido group in the complex are located at ~1552 cm^-1 in the spectrum of the complex, and ~1631 cm^-1 in the spectrum of the ligand.

Figure 1. IR spectra of complex

Figure 2. IR spectra of PLAG

3.2. X-ray Crystallography

As shown in the empirical formula, the complex contains a neutral form of PLAG ligand. The neutral form represents a proton transfer from the phenolic hydroxyl to the pyridine nitrogen atom. The doubly deprotonated form occurs from deprotonation of the phenolic oxygen and hydrazine nitrogen atoms, similar to a series of ligands withpyridoxalcarbazones[15].

The complex[Cu(PLAG)(NCS)₂]₂ possesses a hexacoordi-nated copper ion formed with the usual tridentate ONN coordination of ligand PLAG, coordination of two NCS-groups and a bonding interaction with the nearby Cu atom from a different molecule (Figure 3). Chelating ligand coordination is achieved through the oxygen atom of deprotonated phenolic hydroxyl, azomethine group nitrogen atom and guanido group nitrogen atom. As it can be seen from Table 1, the complex crystallizes in the monoclinic system, with four dimer molecules (Z=4) in the unit cell. Coordination sphere of the complex was built from the neutral form of the ligand molecule PLAG and two NCS groups. In this complex, as already mentioned, the tridentate ONN ligand in its neutral form is coordinated by building two metallocycles: one six-membered (pyridoksilydene) and one five-membered (aminoguanidine).

The bond lengths of for the metal-ligand distances and the corresponding bond angles are given in Table 3.

Table 3. Bond lengths[Å] and angles[o] for the complex

Figure 3. Crystal structure of [Cu(PLAG)(NCS)2]2

The bond lengths of Cu-ligand in the equatorial plane have very similar values: Cu(1)-O(5) 1.915(3)Å, Cu(1)-N(13) 1.963(4) Å and Cu(1)-N(16) 1.947(4) Å. The Cu(1)-O(5) bond distance is the shortest one, which was expected, be-cause of the negative charge present on the oxygen atom of deprotonated phenolic hydroxyl. On the other hand the central copper forms the the longest bond distance with nitrogen atom of azomethine group in equatorial plane (Cu(1)-N(13)1.963(4) Å). The longest bond distance in the Cu coordination sphere is apical Cu(1)-S(2) bond formed by the central atom with the sulfur atom NCS-group and its value is 2.881(14) Å. The N(13)-Cu(1)-N(21) bond angle of 171.58(17)° is close to the theoretical 180°, but the O(5)-Cu(1)-N(16) bond angle is 169.59(15)º due to the chelation rings strain.

4. Conclusions

The synthesis, characterization and structural analysis of a Cu(II) complex incorporating PLAG ligand is described. The Cu II environment is best described as octahedral. The equatorial plane is formed by the tridentate ONN-coordinated PLAG ligand and one NCS molecule. This compound crystallizes in a monoclinic crystal symmetry with the space group of P 1 21/c 1.

5. Supplementary Information

Crystallographic data (excluding structure factors) for the structure (1) reported in this paper have been deposited with the Cambridge Crystallographic Data Center, CCDC No. 828036. Copy of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK(fax:+441223336033;e-mail:deposit@ccdc.cam.ac.uk http://www.ccdc.cam.ac.uk).

ACKNOWLEDGMENTS

The author acknowledge the Oxford Chemical Crystal-log-raphy Service for the use of the instrumentation and many thanks DJWatkin research group for help.