Syntheses, characterization, in vitro antiglycation and DPPH radical scavenging activities of isatin salicylhydrazidehydrazone and its Mn (II), Co (II), Ni (II), Cu (II), and Zn (II) metal complexes

2.1 Physical parameters

Carbon, hydrogen and nitrogen were estimated by using Perkin Elmer 2400 Series II CHNS/O Elemental Analyzer. The IR spectra and Uv–Vis spectra of isatin hydrazone and its metal complexes were recorded on a HITACHI-270 IR spectrophotometer in the 4000–250 cm⁻¹ region in KBr disc and Perkin Elmer Lambda 35 UV–Vis spectrophotometer in the region of 200–800 nm respectively. The ¹H NMR spectrum of the ligand was recorded on a BRUKER 300 MHz spectrometer at room temperature in DMSO-d₆ using TMS as an internal reference. Electron impact mass spectra (EIMS) were recorded on a Finnigan MAT-311A Germany, metal content analyses were made on Perkin Elmer Analyst 800 atomic absorption spectrophotometer. The magnetic moments were carried out at 25 °C on the solid states by Faraday method using Bruker BM6 instrument. Germany. Pre-coated silica gel aluminium plates (Kieselgel 60, 254, E. Merck, Germany) were used for thin layer chromatography (TLC) and 254 and 365 nm UV wavelengths used for visualization of chromatograms. Molar conductance was measured by Thermo scientific Orian 5 Star conductivity meter. Thermograms were evaluated by Perkin Elmer Diamond TG/DTA instrument.

2.2 Materials and methods

Reagent grade chemicals were used throughout the studies. Bovine serum albumin (BSA) was purchased from the Research Organics (Cleveland, USA), while other chemicals isatin, salicylic acid, manganese, cobalt, nickel, copper, and zinc acetates, glucose, trichloroacetic acid (TCA), sodium azide (NaN₃), dimethyl sulfoxide (DMSO), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH₂PO₄), potassium dihydrogen phosphate (KH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), potassium chloride (KCl), and sodium hydroxide (NaOH) were purchased from Sigma–Aldrich, USA. Sodium phosphate buffer (pH 7.4), was prepared by mixing Na₂HPO₄ and NaH₂PO₄ (67 mM) containing sodium azide (3 mM). Phosphate buffer saline (PBS) was prepared by mixing NaCl (137 mM), Na₂HPO₄ (8.1 mM), KCl (2.68 mM), and KH₂PO₄ (1.47 mM) and pH 10 was adjusted with NaOH (0.25 mM). BSA (10 mg/mL) and glucose anhydrous (50 mg/mL) solutions were prepared in sodium phosphate buffer. Standard test solutions were prepared in DMSO (1 mM/mL).

2.3 Synthesis of 2-hydroxy methyl salicylhydrazide and isatin hydrazone

2-Hydroxy methyl salicylate and isatin hydrazone were synthesized according to previously reported methods and their spectroscopic results are in close agreement with reported compounds (László, 2001; Buu et al., 1953; Ali et al. 1989).

2.4 Synthesis of Mn (II), Co (II), Ni (II), Cu (II), and Zn (II) metal complexes

Methanolic solution of isatin hydrazone (1 mmol) and ammonium acetate was mixed with different metal acetates (0.5 mmol), in 2:1 ligand:metal ratio and was refluxed for 6 h. The solvent was evaporated and coloured precipitate was obtained (Table 1), washed with water to eliminate any unreacted metal salt, dried and characterized by UV–Vis, IR, CHN and TG/DTA analysis.

2.5 Antiglycation activity assay

This test was used to evaluate the ability of the candidate compounds to inhibit the methyl glyoxal mediated development of fluorescence of BSA (see Supplementary Information).

2.6 DPPH radical scavenging assay

The free radical scavenging effects of all the metal complexes as well as legend with the DPPH radical were evaluated by the approach of Blois (Navnath et al., 2010) under the same condition (see Supplementary Information).

3.1 Chemistry

The literature reveals that these metal complexes were not so much studied so it is needed to synthesize and discussed its various parameters as well as their biological potential. The metal complexes of isatin hydrazone with Mn-II, Co-II, Ni-II, Cu-II, and Zn-II were synthesized by mixing metal acetates with ligand in 1:2 M ratio in methanol in the presence of ammonium acetate. During refluxing colour of the solution was changed and it was taken as indication for complexation. After 6 h solvent was evaporated and coloured precipitates were obtained which were washed with water as well as methanol in order to remove any unreacted material. These precipitates were dried and subjected towards UV–Vis, IR spectroscopy, CHN/S and TG/DTA analysis for structural confirmation.

3.2 Colour, solubility and melting point

All the synthesized metal complexes were found to be coloured, nonhygroscopic in nature, stable in air. The metal complexes were found to be soluble in DMSO, while they were insoluble in other common organic solvents such as DCM, Hexane, and partially soluble in ethanol and methanol. The melting points of the complexes were found to be in the range of 335–367 °C Table 1.

3.3 Molecular formula of metal complexes

Elemental analyses (CHN/S) give satisfactory results for all the compounds. CHN/S values are in close agreement with expected molecular formulae assigned to these complexes Table 1, suggesting 1:2 metal–ligand stoichiometric ratios. This 1:2 metal–ligand relationship, indicating the tridentate nature of the ligand. The IR and TG/DTA data confirm that there is no role of water molecule in coordination. The possible reaction between metal salt and ligand is given below (see Fig. 1).

Close

Fig. 1 Reaction scheme for the synthesis of metal complexes.

Export to PPT

3.4 Molar conductance

The molar conductance values of 1 × 10⁻³ M solution of metal complexes in DMSO are in the range of 5.90–11.65 Ω⁻¹ cm⁻² mol⁻¹ for all the metal complexes. The low conductance values indicated the non-electrolytic nature of the complexes Table 1.

All the data have been summarized in Table 1.

3.5 Electronic spectra

The electronic spectral data of the complexes are presented in Table 2. The electronic spectrum of free Schiff base showed a band around 341 nm characteristic of π–π^∗ transitions. In the metal complexes, this band is shifted to a longer wave length with increasing intensity. This shift may be attributed to the donation of the lone pair of electrons of nitrogen of Schiff base to metal ion, i.e. ligand to metal charge transfer (LMCT) bands. In the view of previous literature that bathochromic shift is a sign of complexation, so, the bathochromic shifting of peak from 341 nm to 413, 418, 410, 420, 405, for Mn, Co, Ni, Cu and Zn metal complexes respectively was taken as evidence for the complex formation Table 2.

3.6 Magnetic moment

Magnetic studies have been used for the confirmation of the geometry of complexes. The magnetic moment values 5.85 B.M for the MnL₂ were found to be in close agreement with the octahedral structure with five unpaired electrons. The magnetic moment of CoL₂ complex was 4.13 B.M indicating an octahedral environment around the metal ion. NiL₂ complexes showed magnetic moment values 3.09 B.M higher than that of normal value 2.87 B.M. These higher values were due to orbital contribution. Cu (II) complex showed μeff 1.77 B.M suggesting an octahedral structure with one unpaired electron. ZnL₂ complex was found to be diamagnetic as expected (Fuxin et al., 2004) Table 2.

3.7 IR spectroscopy

In IR spectra of ligand, peaks were observed at 1731 cm⁻¹ —C⚌O and 1667 cm⁻¹ for —C⚌N starching respectively. A sharp band at 1516 cm⁻¹ might be due to imin-ol tautomerism found in the target compound. The absorption patterns of complexes look quite similar to that of the free ligand which is in agreement that coordination occurs through tautomeric forms with some alteration in peak values. In metal complexes spectra —C⚌O peak was not observed due to complete enolization comes chelating effect or shifted towards lower wavelength. The IR peaks for Mn, Co, Ni, Cu and Zn complexes appeared at 1710, 1683, 1675, 1685, 1683 and 1705 cm⁻¹ respectively, this is a sign of an indication that —C⚌O group involved in coordination while peak for C⚌N at 1667 cm⁻¹ underwent 68–44 cm⁻¹ suggesting azomethine group also playing part in coordination. N—N stretching peaks at 1009 cm⁻¹ incremented 10–15 cm⁻¹ due to C⚌N—N⚌C— formation in case of metal complexes. Peaks at 1606 cm⁻¹ Mn, 1588 (Co), 1593 (Ni), 1588 (Cu) and 1606 (Zn) for C—O also supporting the coordination effect of C⚌O after enolization. Peaks in the range of 512–526 and 420–480 cm⁻¹ are due to M—O and M—N respectively Table 3.

3.8 Thermogravimetric analysis

Thermal behaviour and decomposition pattern of the metal complexes was established by recording thermo gravimetric analysis (TGA), differential of thermogram (DTG) and differential thermal analysis (DTA) curves. The thermo gravimetric (TG) curves of representatives Schiff base and its respective metal complexes (see Supplementary Information). No weight loss was observed upon heating till 300 °C, and thus, ruling out the presence of water molecules. Isatin hydrazone undergoes decomposition in single stage between the temperature range 320–350 °C and 55.38% weight loss was observed with DTG peak at 341 °C, while an endothermic peak at 333.01 °C, and an exothermic peak at 344.71 °C were observed in DTA thermogram.

MnL₂ complex decomposed into two stages (Stage I: 250–280 °C with 6% and Stage II: 300–350 °C with 19% weight loss). DTG peaks were observed at 271 °C and 339 °C. An endothermic at 257 °C and exothermic at 343 °C were observed in DTA. TGA Curve of Co-complex indicates that weight loss occurred in 3-stages, (Stage I: 325–335 °C that is 9.7%, Stage II: 390–400 °C 29.3%, and Stage III, 410–510 °C and loss is 40.4%), the residue about 10% remained which may be the metal oxide. The overall 90% weight loss may be due to the decomposition of both the ligand molecules. DTG peaks were observed at 335, 390, and 410 °C for decomposition in 3 steps. An endothermic peak was observed at 333 °C which may be due to melting of one molecule of hydrazone, an exothermic peak was also observed at 435 °C due to some chemical or physical phenomenon. TGA study of Ni-complex indicates that weight loss in two stages (Stage I: 360–390 °C, weight loss 30.00% and Stage II: 392–430 °C, weight loss 13.00%), and maximum loss is observed i-e DTG at 395 °C .While its DTA thermogram showed one endothermic peak at 390 °C. TG Curve of Copper Complex indicated that weight loss occurred in 2 stages, (Stage I: 318–350 °C, 35.91%, Stage II: 352–500 °C that is 14.26% weight loss) with DTG peaks at 318 °C and 327 °C. In DTA thermogram two exothermic peaks 319 and 331 °C were observed. In ZnL₂ complex 41% weight loss was observed in two stages, (Stage I: 300–345 °C that is 10.5%, Stage II: 360–400 °C 29.37%) with DTG peaks at 333 and 391 °C. DTA thermogram showed two endothermic peaks at 331 and 390.58 °C. The above study revealed that all the complexes are thermally stable up to 300 °C. Mostly complexes were decomposed into 2 stages although Co II complex showed decomposition in 3 steps, while ligand degradation occurred in a single step. Only Mn II complex showed minor loss of 6% in the range of 250–280 °C. The thermal stability profile of metal complex as Co > Ni > Cu,Zn > Mn, While according to total weight loss thermal stability found to be as Mn > Zn > Ni > Cu > Co (see Table 4).

3.9 Structural interpretation

On the basis of the above spectroscopic, elemental analysis, molar conductance, magnetic moment and thermal analysis results the proposed structures for the metal (II) complexes are shown (Fig. 2). It is concluded that the ligand and metal are in 2:1 ratio and metal coordinated via N and O atoms of the ligand in such a manner that they form octahedral complexes and these complexes are non-electrolyte in nature.

Close

Fig. 2 Possible structure of metal complexes.

Export to PPT

4.1 Antiglycation activity

Isatin hydrazone and its metal complexes were screened for their anti-glycation potential Table 5. Ligand (IC₅₀ = 413.18 ± 6.21 μM) itself found to be weaker active, although; all the metal complexes CoL₂ (IC₅₀ = 168.23 ± 2.37 μM), ZnL₂ (IC₅₀ = 234.27 ± 4.33 μM), MnL₂ (IC₅₀ = 257.1 ± 6.43 μM), CuL₂ (IC₅₀ = 267.7 ± 8.43 μM), NiL₂ (IC₅₀ = 269.0 ± 8.56 μM) were showed significant antiglycation activity and found to be better active than the standards Rutin (IC₅₀ = 294.46 μM), used in antiglycation activity. Among these complexes CoL₂ (IC₅₀ = 168.23 ± 2.37 μM), complex showed higher activity and it is far better active than the standard routine. However, ZnL₂, MnL₂, CuL₂, and NiL₂ comparatively less active than CoL₂ complexes, although they were found to have better activities than the standard rutin. By portraying the activity pattern of theses complexes, i.e. CoL₂ > ZnL₂ > MnL₂ > CuL₂ > NiL₂, it is concluded that these complexes may have competent capability to bind protein or glucose and inhibit the further progression of glycation. Among these CoL₂ has protein or glucose binding ability in greater extent. It is recently reported that Co Salen complex can efficiently bind with the BSA by axial coordination (Li et al., 2013). The HOSalenCo was inserted into the hydrophobic cavities of the BSA and formed the BSA/HOSalenCo conjugation. So, in the same manner our synthesized ligands may bind with the BSA and slot into hydrophobic cavities which might be responsible for suppressing further glycation phenomenon, while other complexes also capable, but could not bind as much efficiently as CoL₂, So they also bind with the BSA protein but in lesser extent than CoL₂.

4.2 Antioxidant assay

Isatin hydrazone and its metal complexes were evaluated for DPPH radical scavenging activity Table 6. All the complexes showed remarkable antioxidant potential with IC₅₀ values 29.63 ± 2.76, 31.13 ± 1.41, 35.16 ± 2.45, 43.53 ± 3.12, 57.71 ± 2.61 μM Cu, Zn, Mn, Co and Ni respectively, and almost all the complexes found to have better activities than standard tert-butyl-4-hydroxyanisole (IC₅₀ = 44.7 μM). The ligand isatin hydrazone (IC₅₀ = 54.14 ± 2.43 μM) was found to be lesser active than standard as well as its respective complexes except Ni complex. Copper (IC₅₀ = 29.63 ± 2.76), and zinc (IC₅₀ = 31.13 ± 1.41) complexes showed remarkable antioxidant activity while manganese complex (IC₅₀ = 35.16 ± 2.45 μM) was found to be a good antioxidant too. The Cobalt complex also showed significant activity (IC₅₀ = 43.53 ± 3.12 μM). Nickel complex was found to be active in the DPPH radical scavenging activity, but surprisingly it was found to be lesser active than the standard and it looks like complexes as well as ligand. So, the order of activity of these complexes is as Cu > Zn > Mn > Co > Ni. This significant activity of the complexes might be due to the stabilization of free radical by the metal ion with phenolic moiety.