Synthesis, characterization and biological activity of mixed ligand chelates of Ni(II) with pyridoxalthiosemicarbazone and dipeptides

Cite this paper: Vietnam J. Chem., 2021, 59(1), 57-68 Article DOI: 10.1002/vjch.202000100 57 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Synthesis, characterization and biological activity of mixed ligand chelates of Ni(II) with pyridoxalthiosemicarbazone and dipeptides A Saritha1, Ch Venkata Ramana Reddy2*, B Sireesha3 1Department of Chemistry, St. Francis College for Women, Hyderabad, India 500016 2Department of Chemistry, Jawaharlal Nehru Technological University Hyderabad, Hyderabad, India 500085 3Department of Chemistry, Osmania University, Hyderabad 500001 Submitted April 30, 2020; Accepted August 18, 2020 Abstract The synthesis, characterization of mixed ligand chelates of Ni(II), [NiAL] involving Pyridoxalthiosemicarbazone (A) and dipeptides (L) viz., glycyl-glycine (gly-gly), glycyl-L-leucine (gly-leu), glycyl-L-tyrosine (gly-tyr) and glycyl- L-valine (gly-val) and their biological activities have been studied. The complexes were characterized based on their elemental analysis, LC-MS, IR, UV-Vis spectral studies, magnetic moment, molar conductance and thermal analysis. The mixed ligand complexes were formed with 1:1:1 (Ni:A:L) ratio. The molar conductance data reveal the non- electrolytic nature of the metal chelates. IR spectra show that the ligands are coordinated to the metal ion in a tridentate manner, involving O,N,S and O,N,N donor sites of ligands, A and L respectively. Based on the analytical data, octahedral geometry has been proposed for the metal complexes. DNA binding properties of the complexes have been investigated by UV-Vis and fluorescence spectroscopy and also by viscosity measurements. The obtained results indicate that the complexes bind to DNA through intercalation mode, which is further validated by molecular docking studies. The hydrolytic cleavage of the pBR322 DNA from supercoiled to nicked form, by the metal complexes was investigated by gel electrophoresis technique. The metal complexes were also screened for their antioxidant, anti- inflammatory and antibacterial activities and the findings have been reported. Keywords. Mixed ligand chelates, DNA interaction, antibacterial, anti-inflammatory, antioxidant, molecular docking. 1. INTRODUCTION Polydentate Schiff base ligands and their transition metal complexes are of great interest in coordination chemistry,[1,2] due to their structural features and biological activities. Pyridoxalthiosemicarbazone (PLTSC), a tridentate Schiff base ligand with O, N, S donor atoms, forms stable metal chelates.[3] Binary complexes of PLTSC with the transition metal ions have been reported by various researchers.[4-7] A major interest in the metal complexes of PLTSC derives from their biological and chemotherapeutic activities, such as suppressive effect on Friend erythroleukemia cells (FLC), inhibition of reverse transcriptase and cytotoxicity.[8-10] Similarly, dipeptides are also versatile ligands for complexation with many metal ions. The solution chemistry and synthesis of binary complexes of dipeptides are reported, where in the common binding sites of dipeptides include amino nitrogen, peptide oxygen or peptide nitrogen and carboxylate oxygen.[11,12] The metal chelates of dipeptides were found to show various biological activities as antibacterial, anti-inflammatory and antitumor activities.[13-16] The role of mixed ligand chelates in biological processes such as activation of enzymes, storage and transport of substances across the membranes is well established. The potential ligands compete for the metal ions in vivo which results in mixed ligand chelation in biological fluids. They act as antimicrobial, antioxidant, cytotoxic, anticancerous agents[17-20] and also as catalysts in various organic reactions.[21,22] Though there are some reports on binary complexes of PLTSC and dipeptides, there are no significant studies on the mixed ligand complexes involving these ligands. Accordingly, we report herewith the synthesis, characterization and biological activity studies as antibacterial, Vietnam Journal of Chemistry Ch Venkata Ramana Reddy et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 58 antioxidant, anti inflammatory, DNA binding and cleavage of the mixed ligand chelates of NiAL, involving PLTSC (A) and dipeptides (L) viz., glycyl-glycine (gly-gly), glycyl-L-leucine (gly-leu), glycyl-L-tyrosine (gly-tyr) and glycyl-L-valine (gly- val). 2. MATERIALS AND METHODS 2.1. Materials and physical measurements Pyridoxal hydrochloride, thiosemicarbazide, dipeptides, CT DNA and pBR 322 DNA, DPPH, Diclofenac Sodium were purchased from Sigma Aldrich. KH2PO4, K2HPO4 and nickel(II) chloride hexahydrate were obtained from Merck, India. Muller Hinton Agar medium was purchased from Himedia. All other chemicals and solvents were of analytical grade and were used without further purification. PLTSC was prepared by a known procedure.[23] Elemental (C,H,N) analysis was carried out on a Thermo Finnigan 1112 elemental analyzer. Mass spectra of the complexes were recorded on a LCMS 2010A, Shimadzu spectrometer. The other spectroscopic measurements were made using the instruments, IR: Shimadzu IR Prestige-21 Spectrometer (KBr, 4000-250 cm-1); UV-Vis: Systronics UV-Vis Double beam spectrophotometer 2201; Fluorescence: Shimadzu Spectrofluorometer, RF-5301. The molar conductivity of the freshly prepared (10-3 M) solutions of complexes in DMSO was measured using a Digisun digital conductivity bridge. Thermo gravimetric analysis (TGA) was performed using Shimadzu TGA-50H in nitrogen atmosphere in the temperature range from room temperature to 1000 ºC with a heating rate of 20 ºC per min. Magnetic susceptibilities were measured at room temperature on Faraday balance, model 7550 using Hg[Co(NCS)4] as an internal standard. Diamagnetic corrections were made using Pascal’s constants.[24] Molecular docking study was carried on Autodock 4.2 programme. 2.2. Synthesis of mixed ligand Ni(II) complexes 0.4838 g (1.7 mmol) of PLTSC and 0.3293 g (1.7 mmol) of gly-gly/0.3293 g (1.7 mmol) of gly- leu/0.4168 g (1.7 mmol) of gly-tyr/0.3048 g (1.7 mmol) of gly-val was added simultaneously to an aqueous solution containing 0.404 g (1.7 mmol) of NiCl2.6H2O. An immediate color change was observed. The mixture was refluxed over the steam bath for 3 hours. A red colored precipitate was obtained by adjusting the pH to 7-8 with a methanolic solution of ammonium hydroxide. The solid obtained was filtered, washed several times with hot distilled water, followed by petroleum ether and finally air dried. [Ni(PLTSC-H)(gly-gly-H)] C13H18N6O5SNi (1): Anal. Calcd. (%): C, 36.36; H, 4.19; N, 19.58. Found: C, 36.32; H, 4.23; N, 19.55. APCI-MS (m/z): 428 [M+], (Fig. S1). IR (KBr) cm-1: ν(C=N) 1606, ν(Ar.C-O) 1151, ν(C=S) 812, ν(peptide –NH) 1554, ν(-COO- asym) 1618, ν(-COO- sym) 1388. UV-Vis (DMSO) λmax/nm: 259, 336, 400, 486. μeff (BM): 2.99. Λm [Ω-1cm2M- 1,10-3, DMSO]: 07. [Ni(PLTSC-H)(gly-leu-H)]C17H26N6O5SNi (2): Anal. Calcd. (%): C, 42.06; H, 5.36; N, 17.31. Found: C, 42.01; H, 5.40; N, 17.28. APCI-MS (m/z): 486 [M+] (Fig. S2). IR (KBr) cm-1: ν(C=N) 1612, ν(Ar.C- O) 1147, ν(C=S) 815, ν(peptide –NH) 1560, ν(-COO- asym) 1616, ν(-COO- sym) 1388. UV-Vis (DMSO) λmax/nm: 262, 400, 489. μeff (BM): 2.96. Λm [Ω-1cm2M-1,10-3, DMSO]: 06. [Ni(PLTSC-H)(gly-tyr-H)].H2O C20H26N6O7SNi (3): Anal. Calcd.: C, 43.39; H, 4.70; N, 15.19. Found: C, 43.35; H, 4.66; N, 15.22. APCI- MS (m/z): 553 [M+] (Fig. S3). IR (KBr) cm-1: ν(C=N) 1610, ν(Ar.C-O) 1147, ν(C=S) 815, ν(peptide –NH) 1560, ν(- COO- asym) 1616, ν(-COO- sym) 1386. UV-Vis (DMSO) λmax/nm: 259, 400, 489. μeff (BM): 3.01. Λm [Ω- 1cm2M-1,10-3, DMSO]: 10. [Ni(PLTSC-H)(gly-val-H)] C16H24N6O5SNi (4): Anal. Calcd.: C, 40.76; H, 5.09; N, 17.83. Found: C, 40.72; H, 5.13; N, 17.80. APCI-MS (m/z): 472 [M+] (Fig. S4). IR (KBr) cm-1: ν(C=N) 1610, ν(Ar.C- O) 1145, ν(C=S) 815, ν(peptide –NH) 1553, ν(-COO- asym) 1591, ν(-COO- sym) 1388. UV-Vis (DMSO) λmax/nm: 271, 399, 482. μeff (BM): 2.89. Λm [Ω-1cm2M-1,10-3, DMSO]: 07. 2.3. DNA binding studies 2.3.1. UV-Visible absorption titration The DNA binding interaction of the metal complexes 1-4 was measured in potassium phosphate buffer solution (pH 7.2). The absorption ratio at 260 and 280 nm of Calf Thymus DNA (CT DNA) solutions was found as 1.9:1, which shows that the DNA is sufficiently free from protein. The concentration of DNA was determined by UV- visible absorbance at 260 nm, using ε value of 6600 M-1cm-1. The titration experiments were performed by maintaining the concentration of metal complexes constant at 20 µM, while the concentration of CT DNA was varied within 0-20 µM. An equal quantity of CT DNA was also added to the reference solution to eliminate the absorption Vietnam Journal of Chemistry Synthesis, characterization and biological © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 59 by DNA. After each addition of CT-DNA to the complex, the resulting solution was incubated for 10 min and the absorption spectra were recorded in the wavelength range of 200-500 nm. The binding constants (Kb) were calculated from the spectroscopic titration data using the equation: [DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/Kb(εb-εf) (1) where, [DNA] is the concentration of CT-DNA, εa, εb,εf are the extinction coefficients of apparent, bound and free complex respectively. Kb of the complex is calculated from the ratio of slope to intercept in the plot of [DNA] vs [DNA]/(εa-εf).[25] 2.3.2. Competitive DNA binding fluorescence studies Further support for the intercalative mode of binding to DNA was obtained using fluorescence spectral experiments, wherein the ability of a metal complex to displace ethidium bromide (EB) from a DNA-EB adduct was studied. EB displacement experiments were carried out by the addition of metal complex solutions to a DNA and EB mixture in potassium phosphate buffer solution (pH 7.2). The DNA was pretreated with EB at a concentration ratio of [DNA]/[EB] = 1 and incubated for 30 min at room temperature. Then the changes in fluorescence intensities of EB bound to DNA at 605 nm were recorded with an increasing amount of the complex concentration from its 50 µM stock solution. The observed changes of fluorescence intensity with increasing concentration of the quencher (complex) were used to calculate the binding constant or Stern - Volmer quenching constant Kq.[26,27] 2.3.3. Viscosity studies The viscosity measurements were carried out on an Ostwald viscometer, immersed in a thermostatic water bath maintained at 25±1 ºC. Concentration of ternary metal complexes was varied by adding increasing amounts from their 50 µM stock solution to CT-DNA solution (300 µM) in phosphate buffer (pH 7.2). Flow time was recorded using a digital stopwatch in triplicate and an average flow time was calculated. Data are presented as plot of (η/η0)1/3 versus [complex]/[CT-DNA], where, η is the viscosity of CT-DNA in the presence of complex and η0 is the viscosity of CT-DNA alone.[28] 2.4. DNA cleavage studies Interaction between pBR 322 plasmid DNA and the mixed ligand Ni(II) complexes was examined in 5 mM Tris.HCl/50 mM NaCl buffer (pH 7.2), by agarose gel electrophoresis experiments. Freshly prepared complex solutions in DMSO (20, 40 and 60 µM) were incubated with plasmid DNA (300 ng/3 µl) at 37 ºC for 1 h. Then, a loading buffer (1 µl) containing 1 % bromophenol blue and 40 % Sucrose was added and loaded onto a 0.8 % agarose gel containing EB (1 µg/ml). The samples along with the control DNA were subjected to electrophoresis in TAE buffer (Tris-acetic acid-EDTA) at 60 V for 2 h. The bands of DNA have moved on the agarose gel under the influence of electric field. These bands were visualized by viewing the gel on a transilluminator and photographed.[29] 2.5. Antibacterial assay The complexes were screened against Staphylococcu saureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa for their antibacterial activity. The tests were performed using well diffusion method. The stock solutions of the complexes (1 mg/mL) were prepared in DMSO. Petri plates containing 20 ml Mueller Hinton Agar medium were inoculated with 100 µL of 24 hour culture of the test bacterial strain and kept for 15 min for adsorption. Using a sterile plastic borer of 8 mm diameter, wells were bored into the seeded agar plates and were loaded with a 100 µL solution of each metal complex. The diameter of inhibition zone around each well was measured in mm after 24 h. DMSO was used as negative control and amikacin (30 µg) was used as the standard.[30] 2.6. Antioxidant activity The antioxidant activity of the mixed ligand Ni(II) complexes has been evaluated using 2,2-diphenyl-2- picrylhydrazyl (DPPH) radical assay. The DPPH is a stable free radical with a λmax at 517 nm. Stock solutions of the metal complexes were prepared in DMSO, from which different concentrations containing 50, 100, 150, 200 and 250 μg/mL were prepared by diluting with methanol. 1ml of each solution was added to a solution of DPPH in methanol (0.4 mM, 1 mL) and the final volume was made to 3 mL with methanol.[31] DPPH solution in methanol was used as a positive control and methanol alone as a blank. The solutions were thoroughly shaken and incubated at room temperature for 30 min in dark. The decrease in absorbance of DPPH was measured at 517 nm. Ascorbic acid was used as a standard. All the measurements were made in triplicates. The percentage of inhibition (I%) of free radical production by DPPH was calculated using the Vietnam Journal of Chemistry Ch Venkata Ramana Reddy et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 60 formula, (I%) = [(A0 - Ac)/A0]  100 where A0 and Ac are the absorbance in the absence and presence of the metal complexes respectively.[32] 2.7. In vitro Anti-inflammatory activity The in vitro anti-inflammatory activity of the mixed ligand complexes was studied by using inhibition of Bovine Serum Albumin (BSA) denaturation technique as reported by Mizushima et al.[33] and Sakat et al.[34] with minor modifications. Reaction mixtures consisting of 1 % BSA and the complexes in phosphate buffer at pH 7.4 were incubated at 37 ºC for 20 min. Then they were heated at 51 ºC for 30 min. After cooling, the turbidity of the samples was measured at 660 nm. The percentage inhibition of protein denaturation was calculated by using the formula, Percentage inhibition = [(Acontrol- Asample)/Acontrol]  100.[35-37] 2.8. Molecular Docking studies Docking studies of the interaction of metal complexes with DNA was carried out in Autodock 4.2 as reported.[38,39] Crystal structure of DNA was downloaded from protein data bank (www.rcsb.org)pdb id: 1N37,[40] which was prepared by protein preparation wizard applying OPLS 2005 force field in Schrodinger suite. A grid was prepared around the intercalation site by selecting the co- crystallized ligand. Metal complexes were constructed and optimized in ChemDraw. These were docked into DNA intercalation site using Autodock 4.2. Molecular interaction diagrams are obtained from PMV.[41] 3. RESULTS AND DISCUSSION 3.1. Characterization of complexes The mixed ligand complexes, [Ni(II)-PLTSC- dipeptide] obtained were amorphous, colored solids and stable at room temperature. They are soluble in DMSO, DMF and insoluble in common organic solvents. The complexes gave satisfactory C, H, and N analysis. The molar conductivity values (Λm: 05- 15 ohm-1cm2mol-1) of 10-3 M solutions of the complexes in DMSO indicate the complexes to be non-electrolytes. The magnetic moment values (µeff: 2.89-3.01BM) of the complexes suggest the presence of two unpaired electrons in Ni(II) ion. 3.1.1. APCI-MS The mass spectra of the mixed ligand complexes were recorded in APCI-positive mode. The mass spectra provide information regarding the 1:1:1 (M:A:L) behavior of complexes. The complexes 1 to 4 (Fig S1-S4) show peaks at m/z 428[M]+, 486[M+1]+, 553[M]+ and 472[M+1]+ respectively. Complexes 1 and 2 also display peaks at m/z 450 and 508 respectively assigned to [M+23]+. The data are in good agreement with the stoichiometry of the ternary complexes in 1:1:1 (M:A:L) ratio and the proposed molecular formulae. 3.1.2. IR spectra All the complexes exhibited similar IR spectroscopic properties. The comparison of IR spectra of ligands and the complexes (Fig S5-S13) support the coordination of PLTSC (A) and dipeptides (B) to Ni(II) ion. Assignments of the characteristic IR bands of the ligands and the ternary complexes are presented in table 1. The PLTSC is coordinated in tridentate mode, through deprotonated phenolic oxygen, nitrogen of azomethine and sulphur of thioamide group. The band attributed Py-NH+ due to the migration of Py-OH proton to Py-N in PLTSC[42] at 2823 cm-1 has disappeared. The shifts in the stretching vibrations arising from the C=N (1650- 1600 cm-1) agree well with the involvement of the azomethine nitrogen in the coordination. The participation of the phenolic oxygen in coordination is clear from a shift in the stretching frequency of ArC-O from 1126 to 1147 cm-1. A shift in the C=S band in the metal complexes from 841 cm-1 of PLTSC indicate the coordination of sulphur to the metal ion.[43] Dipeptides are coordinated in tridentate mode via nitrogen of free amino group, nitrogen of peptide nitrogen and oxygen of deprotonated carboxyl group. Existence of the free dipeptides as zwitter ions in the solid state is confirmed by a medium intensity band near 2100 cm-1 region, due to δ(NH3+) and ρ(NH3+). This band disappears upon coordination to the metal ion and the N-H stretching frequency of the amino group is shifted to higher frequencies compared to the free dipeptides.[44] These spectroscopic observations indicate the binding of NH2 group to the Ni(II). The peptide -NH coordination is suggested by the shift in the stretching vibration (100-120 cm-1) around 3180 cm-1 and bending frequency by 20-40 cm-1 around 1550 cm-1. The difference in stretching frequencies of carboxylate group [Δ = νas(COO–)-νs(COO–)], for all the complexes (above 200 cm-1) were larger than the Δ values of the free dipeptides (150-185 cm-1) indicating the coordination of carboxylate oxygen.[45] Vietnam Journal of Chemistry Synthesis, characterization and biological 61 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Table 1: IR spectral data of the ligands and ternary complexes (cm-1) Ligand/ complex ν(NH2) ν(NH) ν(C=N) ν(COO-) ν(ArC-O) ν(NH) bend ν(C=S) asym sym PLTSC 3296 3184 3184 1625 --- --- 1126 1184 --- 841 gly-gly 3286 3055 --- 1575 1408 --- 1533 --- 1 3313 3178 1606 1616 1419 1151 1554 812 gly-leu 3257 3068 --- 1556 1406 --- 1514 --- 2 3317 3188 1612 1616 1388 1147 1560 815 ly-tyr 3361 3030 --- 1604 1419 --- 1517 --- 3 3317 3188 1610 1616 1386 1147 1560 812 gly-val 3251 3070 --- 1558 1406 --- 1519 --- 4 3311 3184 1610 1591 1388 1145 1553 815 3.1.3. Thermal analysis The thermal stability of the mixed ligand complexes was studied by recording themogravimetric analysis. The TG curves of t