Synthesis, spectral, DFT calculation, antimicrobial, antioxidant, DNA/BSA binding and molecular docking studies of bio-pharmacologically active pyrimidine appended Cu(II) and Zn(II) complexes

Abstract A new pyrimidine derivative Schiff base (HL) [HL = 2-((4-amino-6-chloropyrimidin-2-ylimino)methyl)-4-nitrophenol] has been synthesized using 2,6-diamino-4-chloropyrimidine and 5-nitrosalicylaldehyde. Transition metal complexes of Cu(II) and Zn(II) complexes [CuL(OAc)] (1), [ZnL(OAc)] (2) are prepared with HL/metal(II) acetate with molar ratio of 1:1. The Schiff base (HL) and the complexes 1 and 2 are evaluated by UV-Visible, 1H-NMR, FT-IR, EI-MS and ESR spectral techniques. Complexes 1 and 2 are confirmed as square planar geometry. Electrochemical studies of the complexes 1 and 2 are used to analyse the quasi reversible process. Density Functional Theory (DFT) using the B3LYP/6-31++G(d,p) level basis set was used to get the optimised geometry and non-linear optical properties. The complexes 1 and 2 are good antimicrobial agents than Schiff base (HL). The interactions of the HL and complexes 1 and 2 with Calf Thymus (CT) DNA are investigated by electronic absorption methods and viscosity measurements. Various molecular spectroscopy techniques, such as UV absorption and fluorescence, were used to explore the mechanism of interaction between the BSA and the ligand HL and complexes 1 & 2 under physiological settings. Complexes 1 and 2 are act as potential antioxidants than free Schiff base (HL) by DPPH radical scavenging assay. Furthermore, the purpose of the molecular docking studies was to better understand how metal complexes interact with biomolecules (CT-DNA and BSA). From these biological analyses, complex 1 acts as good intercalator with CT DNA & BSA and potent antioxidant with DPPH radical than complex 2. Communicated by Ramaswamy H. Sarma


Introduction
Heterocycles are in abundance in nature and are very significant in our lives because of their existence in many naturally occurring molecules such as hormones, antibiotics and caffeine, etc. (Nagaraj & Reddy, 2008).Over the years, the heterocyclic compounds have attracted numerous attentions due to their wide applications in medicinal chemistry research.Among these, nitrogen containing heterocycles play an important role to the society in different life processes (Kerru et al., 2020).Pyrimidine which is nitrogen containing heterocycle occurs naturally in substances such as vitamins like thiamine, riboflavin (found in milk, egg and liver), folic acid (from liver, yeast), barbituric acid (2,4,6-trihydroxy pyrimidine), nucleic acid components (uracil, cytosine, thymine), coenzymes, purines, pterins, nucleotides, alkaloids obtained from tea, coffee, cocoa and essential components of many drug molecules (Gupta et al., 2010).Pyrimidine moiety is of great interest because they constitute nucleic acid components such as uracil, cytosine and thymine to their current usage in the chemotherapy of AIDS (Jain et al., 2006).Due to its probiotic nature to living cells in biodiversity, it is a highly privileged moiety for the development of pyrimidine derivative Schiff base and its metal complexes in pharmaceutical research (More et al., 2019).Pyrimidine based derivatives act as potential agents due to their resistance to microorganisms and for inventing new chemotherapeutic drug (Zhuang & Ma, 2020).Vicine may be the first simple pyrimidine derivative found to occur in nature (Ajani et al., 2015).2,6-diamino-4-chloropyrimidine is applied as an internal standard for the quantitative analysis of melamine (Miao et al., 2009).A 2,6-diamino-4-chloropyrimidine derivative such as aronixil is used as anti-atherosclerotic in medicinal field.The interaction between DNA with various drug molecules acts a prime role in the pharmaceutical field and drug design (Sirajuddin et al., 2013).Now a day the interaction between DNA and pyrimidine derivative is an important role to analyse the action of drugs (Sharma et al., 2014).
In biological systems, serum albumins are in charge of balancing the pH of the blood (Carter & He, 1992).About 60% of the protein in a cow's blood plasma is in the form of bovine serum albumin (BSA) (Cheng & Zhang, 2008).The BSA serves as a transporter for physiologically active compounds to serum albumins in vivo, which is a requirement for pharmacokinetics.The availability of the free drug and the drug's binding constant to serum albumin are inversely correlated.That is, if the medication and protein have a stronger affinity, there will be less free drug in the circulation.The amount of free drug that is present in the circulation will increase if the protein and drug's binding is weaker.The protein BSA has a spherically shaped structure with 583 amino acids (Majorek et al., 2012).The BSA is primarily composed of three domains, namely domains I, II, and III.Additionally, there are two subdomains-subdomain A and subdomain B-within each domain.According to the literature, aromatic and heterocyclic compounds tend to bind to subdomains IIA and IIIA of site I and site II, whereas fatty acids prefer to attach to IB, IIIA, and IIIB (Ghosh & Dey, 2015;Guo & Zhou, 2019;Szkudlarek et al., 2017;Tantimongcolwat et al., 2019).The major components of BSA's intrinsic fluorescence are the two tryptophan residues, tryptophan-134 and tryptophan-213 (Siddiqui et al., 2019).Because BSA and Human Serum Albumin (HSA) resemble each other around 76% and are both inexpensive and widely available, we chose BSA for the current investigation instead of HSA (Shaikh et al., 2007;Wang et al., 2019).In our on-going research, we here in report synthesis of Schiff base HL and its complexes 1 and 2. Further spectral and biological evaluation of pyrimidine derivative Schiff base HL and its complexes 1 and 2 is also carried out.

Materials and methods
All the chemicals are Sigma Aldrich products and used as received.Bovine Serum Albumin (BSA, Fraction V) was purchased from Sigma Chemical Company, St Louis, USA.Solvents are purchased and purified according to the standard procedure.A Bruker Avance DRX 300 FT NMR spectrometer is used to record the sample's 1 H NMR spectra at 300 MHz.Using a Varian E1/2 spectrometer, the sample's X band ESR spectra are recorded.The elemental analysis of the sample is determined by Elementar Vario EL III CHNS.EI mass spectra are obtained using the JEOL GCMATE II spectrometer.Electrochemical studies are carried out using EG and E Princeton Applied Research Potentiostat/galvanostat model 273 A.A Shimadzu FT-IR spectrometer is used to measure the IR spectrum of KBr pellets.A Shimadzu UV-Visible 1800 spectrophotometer is used to measure electronic absorption spectra, DNA binding and antioxidant activity.

Synthesis of pyrimidine derivative Schiff base (HL)
Separately, 50 mL of methanol is used to dissolve 0.1445 g of 2,6-diamino-4-chloropyrimidine and 0.1671 g of 5-nitrosalicylaldehyde.The two solutions were then combined and they are refluxed continuously for 6 hours.By the slow evaporation, yellow solid (HL) is obtained and recrystallized using ethanol (Scheme 1).

Preparation of metal(II) complexes 1 and 2
The complexes 1 and 2 are prepared with molar ratio 1:1of Schiff base HL (1 mM) and metal(II) acetate in methanol.The mixtures are refluxed for three hours.The resulting solutions are cooled to room temperature once the reaction is finished.The products are separated from the solvent by slow evaporation.Then, it is recrystallized with ethanol.

Antibacterial activity
The newly synthesized Schiff base (HL) and its complexes 1 and 2 are analysed in vitro for their antibacterial screening against two gram positive bacteria such as Bascillus sonorensis (B.sonorensis), Staphyllococcus aureus (S. aureus) and five gram negative bacteria like as Escherichia coli (E.coli), Klebsiella pneumonia (K.pneumonia), Shigella sonnei (S. sonnei), Proteus mirabilis (P.mirabilis) and Pseudomonas aeruginosa (P.aeruginosa) (Revathi et al., 2021).The bacterial culture is swabbed in nutrient agar medium in petri dishes.The samples in DMSO injected in bacterial cultures and Scheme 1. Synthesis of 2-((4-amino-6-chloropyrimidin-2-ylimino)methyl)-4nitrophenol incubated at 37 � C for 24 h.The DMSO is non-toxic and did not affect the bacterial culture.The resistance of the samples against bacterial cultures are measured as zone of inhibition (in mm) and compared with standard drug streptomycin.

Antifungal activity
The Schiff base (HL) and complexes 1 and 2 are investigated by in vitro anti-fungal screening against the various fungi like as Aspergillus niger (A.niger), Candida albicans (C.albicans), Candida tropicalis (C.tropicalis) and Mucor campestris (M.campestris).This method includes the calculation of reduction percentage in the radial growth diameter in mm over DMSO as control is calculated and compared with standard drug nystatin (Kalarani et al., 2020).

Studies on DNA interaction with HL and complexes 1 and 2
The stock solutions of Schiff base (HL) and complexes 1 and 2 are prepared using DMSO and Tris HCl/NaCl (5:50 mM) buffer at pH 7.2 and diluting them with the same buffer solution to the required concentrations for all experiments.

Electronic absorption method
The interaction of Schiff base (HL) and complexes 1 and 2 with CT DNA are performed using Tris HCl/NaCl buffer at pH 7.2 at room temperature.The CT DNA spectrum has a band at 260-280 nm with an absorbance ratio of 1.8-1.9:1,indicating that the DNA is sufficiently free of protein.At 260 nm, the molar extinction coefficient of CT DNA is estimated using a value of 6600 M À 1 cm À 1 .By utilising Tris HCl buffer to change the concentration of CT DNA while keeping constant concentrations of the Schiff base (HL), complexes 1 and 2, absorption titration studies are carried out (Sudhakar et al., 2018).Until there is no longer any change in the spectra, indicating binding saturation has been attained, the titration procedure is repeated.The Wolfe Shimmer equation is used to calculate the intrinsic binding constant (K b ) from the absorption data: where [DNA] is the concentration of CT DNA in base pairs.The apparent absorption coefficients e a , e f , and e b relate to A obs. /M, the extinction coefficient for the Schiff base (HL) and free complexes 1 and 2 and extinction coefficient for the Schiff base (HL) and complexes 1 and 2 in the fully bound form respectively.K b can be calculated using a plot of [DNA]/(e a À e f ) vs [DNA] and the slope-to-intercept ratio.The Van't Hoff equation can be used to determine the standard Gibb's free energy change (G) for DNA binding: where R is gas constant and T is temperature (T ¼ 298 K).

Viscosity measurements
In viscosity measurement, for minimizing the complexities of CT DNA samples are subjected to sonication and then prepared to desired concentration.The sample solution is filled in Ostwald viscometer and flow times are measured by triplicate (Karekal et al., 2013;Phadte et al., 2019).The viscosity of the samples is calculated using the average of three duplicate measurements.Using the following relation where g is the viscosity of the Schiff base (HL) and complexes 1 and 2 with CT DNA, t is the flow time of the Schiff base (HL) and complexes 1 and 2 with DNA in seconds and t 0 is the flow time of CT DNA only in seconds.Likewise, g 0 can be calculated using CT DNA and Tris HCl buffer by using the above equation.Then, the graph is plotted between (g/g 0 ) 1/3 Vs [Complex]/[DNA], where g/g 0 is the relative viscosity.
Studies on BSA interaction with HL and complexes 1 and 2 BSA interactions.Initial testing was done to determine the ideal BSA concentration and 2.5 M was discovered to be the best value.The chemicals' concentrations were preserved in the range of 0 to 100 M. The fluorescence spectra were recorded in the 290-500 nm wavelength range.
Absorption measurements.At a temperature of 300 K, the UV-visible spectral data were scanned in the 300-500 nm region.The compounds were held at a range of concentrations between 0 and 100 M while the BSA solution was used at a concentration of 2.5 M for the absorption investigations.

Molecular docking
The two-dimensional (2D) chemical structures of the title compounds were sketched using Chem Draw Ultra and converted to pdb files using Chem3D Ultra.The CT-DNA (PDB ID: 355D) and BSA (PDB ID:4F5S) (Sankarganesh et al., 2019a) was retrieved from the RCSB protein data bank (http://www.rcsb.org/pdb) for molecular docking study.Protein and grid preparation were made using the AutoDock Tool 4.2 (Morris et al., 2009).Polar hydrogen atoms were added to the protein kinase receptor structure and then the Gasteiger charge was computed and added, accordingly.Molecular docking study of complexes was performed with the AutoDock Vina (Trott & Olson, 2010).The DNA was loaded into Auto Dock Tool 4.2, creating a PDBQT file that contains a protein structure with hydrogens in all polar residues.The docking site on the protein target was defined by establishing a grid box with the dimensions of X: 65 Y: 68 Z: 73 Å, with a grid spacing of 0.375 Å, centered on X: 52.84 Y:0.135 Z: 21.80 Å.The interactions of complex with DNA/BSA, including hydrogen bonds and hydrophobic interactions, were analysed using Discovery studio 4.0 client (Herowatia & Widodo, 2014) and PyMol (Jahanshahtalab et al., 2019).

Antioxidant activity using DPPH assay
DPPH radical is used to evaluate the antioxidant activity of Schiff base (HL) and complexes 1 and 2 according to the method of Blois (1958).A solution of DPPH in DMSO is mixed with a variable concentration of the Schiff base (HL) and complexes 1 and 2, and the final volume is created up to 3 mL.The mixture is forcefully mixed and left to stand for 0-20 minutes at room temperature.At 517 nm, the absorbance drop is quantified.The identical experiment is run without using a complex as a control.This equation is used to determine the proportion of radical scavenging activity: where A 0 is control absorbance and A 1 is sample absorbance.

H NMR spectral analysis
Figure S1 displays the 1 H-NMR spectra of the ligand and its complex 2. The azomethine proton (-CH ¼ N-) exhibits a singlet peak in the 1 H NMR spectra of Schiff base (HL) at approximately 8.31 ppm (s, 1H), which shifts to approximately 8.70 ppm during complexation, showing the coordination of the N atom of the group (-CH ¼ N-) with the metal ions (Sankarganesh et al., 2019b).At about 5.75 ppm (s, 1H), a singlet caused by the -OH proton was seen in the ligand.In complexes that showed deprotonation and oxygen-based bonding is not appeared.At 1.81 ppm, a coordinated acetate group singlet was seen, confirming the 1:1 M:L ratio (Senthilkumar et al., 2022).The broad proton peak was seen at about 6.80 ppm (s, 2H, NH 2 ), confirming the ligand's 2amino location.Additionally, the 2-amino proton demonstrates that it is not necessary for the azomethine bond to develop.The proton that is present in the ligand's pyrimidine ring's fifth position is what causes the singlet peak that can be seen at 7.20 ppm.The 5-nitrosalicylaldehyde's aromatic protons exhibit peaks at 6.8-7.2 ppm (m, 5H, Ar-H), indicating that the protons were not involved in the coordination of the ligand.In the complex 2, the Schiff base functions as NO donor type bidentate nature.

IR spectral analysis
FTIR spectra of HL and its complexes 1 and 2 are shown in Figure S2.The Schiff base's (HL) IR spectra shows a band at 1570 cm À 1 that is permitted to the imine group m(-CH ¼ N-).
The complexes' shifting of the azomethine group at 1550 cm À 1 (1) and 1557 cm À 1 (2), respectively, confirms the participation of the imine group in complex formation.The wide band at 3392 cm À 1 indicates the Schiff base -OH group, which is no longer present in complexes 1 and 2. The -OH peak absence proves that oxygen was coordinated to the metal(II) ion through deprotonation.The existence of a chlorine atom in the pyrimidine ring is confirmed by the C-Cl band, which is observed at 798 cm À 1 .At 443 cm À 1 (1), 447 cm À 1 (2), and 515 cm À 1 (1) and 517 cm À 1 (2), respectively, two additional bands are observed, confirming the creation of the M-N and M-O stretching frequencies.All evidence points to the Schiff base (HL) serving as a -NO bidentate donor site for the synthesis of metal(II) complexes (Jeevitha Rani et al., 2022).The carboxylate of the acetate group absorbs substantially at 1527 cm À 1 (1) and 1547 cm À 1 (2) (m asymmetry ) in the FTIR spectra of the complexes 1 and 2, and less strongly at 1411 cm À 1 (1) and 1423 cm À 1 (2) (m symmetry ).The m as -m s values of the carboxylate are (116-124 cm À 1 ) and are a strong indicator of the carboxylate group's bidentate behaviour toward metal(II) ions (Mehrotra & Bohra, 1983;Nakamoto, 1986;Ye et al., 2002).The complexes 1 and 2 have four verified coordination sites: -CH ¼ N-(azomethine), -O (-OH), and two oxygen atoms from -OAc (acetate).

UV-visible spectral analysis
The UV-visible spectra of HL and its complexes 1 and 2 were given in Figure S3.Due to p-p � and n-p � transitions, the Schiff base (HL) produces the absorption bands at 23584 and 31152 cm À 1 respectively.The bond formation of Schiff base (HL) to complexes 1 and 2 is confirmed by the shifting of these transitions at 20684 and 31024 cm À 1 .This band in the complexes is correctly identified as a metal to ligand charge band (MLCT).It is known that for square planar geometry that the d-d transitions are observed in the complex 1 at 20721 cm À 1 ( 2 B 1g ! 2 A 1g ) and 12748 cm À 1 ( 2 B 1g ! 2 E 2g ) (Adwin Jose et al., 2022).The absence of a d-d transition is confirmed by the electrical configuration for the complex number 2, which is d 10 .The only INCT bands on the complex 2 are at 30258 and 27152 cm À 1 .

Mass spectral analysis
The Schiff base's molecular ion peak (HL) is located at m/z 293.The molecular ion peaks for the complexes 1 and 2 are at m/z 415 (1) and 417 (2) (Figure S4).The results mentioned above, which support [ML(CH 3 COO)] as the stoichiometry of metal complexes (1 and 2).The postulated structure as square planar (1 and 2) geometry for the complexes is further supported by these arguments (Ross et al., 1998).

EPR spectral analysis
Cu(II) complex 1 has an EPR spectrum that was captured in DMSO at a low temperature and that displays their characteristic derivative peaks (Figure S5).These spectra yield g | and g ?values of 2.31 and 2.07, respectively.The Cu(II) complexes should have an unpaired electron in their d x2-y2 molecular orbital, according to the g | values, which are higher than the corresponding g ?values.This is supported by the magnetic moment values of the Cu(II) complexes, which are 1.86 BM.
The complex 1 value of g | =A | is discovered to be 155, indicating square planar geometry (Yallur et al., 2021).
Based on the analytical and spectral of the Schiff base and complexes 1 and 2, proposed structure of the complexes has been given in Figure 1.

Cyclic voltammetry
The reduction peaks at Ep c ¼ 0.6 V and 0.9 V, as well as the oxidation peaks at Ep a ¼ 0.4 V and À 1.

DFT studies
The structure of the HL and its complexes 1 and 2 optimized by DFT/B3LYP (Becke 3-parameter exchange functional together with the unrestricted Lee-Yang-Parr correlation functional) level of theory and 6-31 G � (non-metal atoms) and LANL2DZ (metal atoms) basis set using Gaussian 09 programme package (Sankarganesh et al., 2019a).The B3LYP functional has been successfully used in some previous reports for geometry optimization of transition metal complexes (Senthilkumar et al., 2021).The optimized structure of HL and complexes 1 and 2 were shown in Figure 2. The complexes, has distorted square planar geometry around the central metal ions.The IR frequencies of the metal complexes were obtained theoretically by B3LYP calculations employing the standard basis set for optimized geometries and compared within the region of 400-4000 cm À 1 .Using Gauss View 5.0 molecular visualization program, the vibrational frequency assignments and other parameters are made, which are very closer to the experimental values.
From the FMO analysis, in Ligand, both the HOMO and LUMO is located all over the molecules (Figure 3).In Copper complex, both the HOMO and LUMO is present all over the molecule except acetate molecule.LUMO of zinc complex is spread over the entire ligand except metal atom and acetate molecule.We have found energy gap between the HOMO and LUMO of the metal complexes and ligand.In ligand, LUMO is more destabilised and HOMO is highly stabilized so that ligand (4.06 eV) is more stable than complex.Cu complex (3.87 eV) is highly stable than the zinc complex (3.80 eV).Chemical reactivity descriptors of ligand (HL) and metal complexes 1 and 2 were calculated using standard formula and parameters were displayed in Table 2 (Gurusamy et al., 2022b).

Antibacterial activity
The potentiality of the complexes 1 and 2 is assessed by measuring the diameter of the zone of inhibition in millimetres for the Schiff base (HL) and complexes 1 and 2 against diverse bacterial strains (Figure 4 and Table 3).Complexes 1 and 2 possess better biological activity than Schiff base (HL), according to antibacterial studies.It is possible to attribute the increased activity of the complexes 1 and 2 to the chelation of metal ions with Schiff base (HL) and greater lipophilicity as a result of p-electrons delocalizing across the chelating ring.This increased lipophilicity improves complexes 1 and 2's ability to penetrate lipid membranes and prevents the metal binding sites in microbial enzymes.These complexes also interfere with how cells breathe, which prevents the creation of proteins and limits the organism's ability to expand further (Gurusamy et al., 2021).The data showed that the Schiff base's (HL) activity increased during complexation, but less than reference compound.Comparing all of the collected data reveals that complexes 1 and 2 are more effective against all bacterial strains than free Schiff base (HL).

Antifungal screening effect
According to the findings reported in Table 4, complexes 1 and 2 had high fungal activity (Figure 5).The coordination of the metal atom with the oxygen in the ligand may be the cause of the increased toxicity of organo-metals.The influence of metal ions on the regular cell function may also be the cause of the increase in activity of metal complexes 1 and 2. The partial sharing of the metal ion's positive charge with a donor group and potential pi-electron delocalization throughout the entire molecule is the main causes of the metal ion's polarity being significantly diminished after chelation.Such chemicals make metal complexes more lipophilic, which likely causes the permeability barrier of the cells to break down and interfere with normal cell function (Sukuur Saleem et al., 2021).The promising activities of the metal complexes as compared to the free ligand can be well explained in terms of chelation theory.It explains that a decrease in polarizability of the metal enhances the lipophilicity of the complexes.

UV-visible spectroscopic method
Figure 6 depicts the complexes 1 and 2's absorption spectra in both the presence and absence of CT DNA.For complexes 1 and 2, the hypochromism with blue shift is seen when CT DNA concentration rises.These spectrum properties unmistakably imply that the complexes 1 and 2 interact with DNA most likely via a mode involving an aromatic chromophore stacking interaction with the DNA base pairs.After intercalating DNA base pairs, the intercalated ligand's p � orbital may couple with the base pairs' orbital, lowering the p-p � transition energy and producing bathochromism.On the other hand, the coupling orbital is only partially filled with electrons, which lowers the likelihood of a transition and causes hypochromism (Kalanithi et al., 2020).The slope to intercept ratio of the plots of [DNA]/(e a À e f ) vs [DNA] can be used to calculate the intrinsic binding constant K b .The intrinsic binding constant K b of the manufactured complexes to CT DNA is displayed in Table 5 to allow for quantitative comparison of the affinity of the complexes for DNA.Complex 1's binding efficiency is greater than Complex 2's.

Viscosity measurements
The viscosity analysis further supports the binding of Schiff base HL and complexes 1 and 2 to CT DNA, since the relative viscosity of CT DNA continuously increased after each addition of Schiff base HL and complexes 1 and 2 (Figure 7).This phenomenon can be studied by plotting the (g/g 0 ) 1/3 Vs [complex]/[DNA].The increased degree of viscosity for complex 1 is larger than complex 2 and HL ( Gurusamy et al., 2022a).From this we have concluded that complex 1 has high relaxation of the double helix than complex 2 and HL.These results indicate that a considerable relaxation of the double helix is possible in the presence of metal(II) complexes 1 and 2.

Fluorescence study
Tryptophan, Tyrosine, and phenylalanine are only a few of the amino acid residues that give proteins their intrinsic fluorescence.When compared to the other two residues, tryptophan exhibits the strongest fluorescence (Joshi et al., 2011;Skrt et al., 2012).Tryptophan's fluorescence property is extremely sensitive to its surroundings.As a result, this characteristic is often used as an endogenous fluorescent probe to study how albumin interacts with bioactive chemicals (Wang et al., 2019).
The fluorescence spectra of all BSA after the addition of chemicals such ligand HL and complexes 1 and 2 are shown in Figure 8.It was discovered that the fluorescence intensity of BSA consistently reduced as the ligand concentration was raised gradually from 0 to 100 M. Additionally, it was noted that the fluorescent intensity of BSA reduced as the emission of wavelength (k em ¼ 341-338 nm (HL), 340-334 (1), 341-335 nm (2)) increased with blue shift, suggesting that the compound can quench BSA's fluorescence and also change Tryptophan's environment.
Fluorophore and quencher interactions involve three different types of quenching mechanisms: static, dynamic and mixed static and dynamic mechanisms (Akram et al., 2019;Lakowicz, 2006;Vaden et al., 2007;Wang et al., 2016).The sort of relationship between the quenching constant (K sv ) and the temperature or life of the excited state can be used to identify the static and dynamic quenching methods (Akram et al., 2019;Wang et al., 2016).That is, for static quenching, the values of K sv and temperature are inversely proportional, whereas for dynamic quenching, they are directly proportional to one another.In the current study, the quenching process was clarified through analysis of the    fluorescence intensity data of BSA following the addition of the ligand HL and complexes 1 and 2. The Stern-Volmer Eqs.
where F 0 is the fluorescence intensity of BSA alone, F is the fluorescence intensity of BSA in the presence of compounds, K sv is the Stern-Volmer quenching constant, K q is the quenching rate constant, [Q] is the concentration of compound and s 0 is the average life time of BSA in the absence of the compound (s 0 ¼ 10 À 8 s).
The obtained K sv and K q values are listed in Table 6.K SV and K q are determined using the regression equation's slope.The findings showed that K sv values rose with temperature, suggesting a dynamic sort of quenching mechanism.However, the values of K q are less than 2.0 � 10 10 M À 1 s À 1 , indicating a dynamic quenching process (2.0 � 10 10 M À 1 s À 1 is the maximum dynamic quenching rate constant).Therefore, it can be concluded that there is quenching mechanism at work when a chemical interacts with BSA.

Analysis of binding and thermodynamic parameters
The fluorescence readings were exploited to calculate the binding constant K and the number of binding sites n for compound-BSA complex by the following relation (Ge et al., 2010): For the compound-BSA complex, we discovered a strong correlation between log (F 0 À F)/F and log[Q] and the values of K and n may be determined from the intercepts and slopes, respectively.At all three temperatures, the n values were approximately equivalent to one, indicating that the compound-BSA complex forms a 1:1 ratio.The binding affinity of the complex with BSA was exemplary as evidenced by the K values, which were in the 2.24 � 10 5 M À 1 to 2.94 � 10 5 M À 1 range (Wang et al., 2019).However, the K values rose as the temperature rose, indicating that the compound-BSA complex was more stable at higher temperatures.The following Van't Hoff and thermodynamic Eqs. ( 5) and ( 6) are used to analyze the thermodynamic parameters in the binding phenomenon between the compound and BSA.
The plot clearly shows a straight correlation between log K vs 1/T, suggesting that the H 0 and S 0 can be thought of as constants.The estimated G 0 values (Table 7) were discovered to be negative values, which indicates that the chemical binds to BSA by its own volition.A positive S 0 is commonly interpreted as evidence for hydrophobic interaction in light of the water structure.According to the computed values of H 0 and S 0 , which were 1.64 � 10 5 J mol À 1 K À 1 and 6.51� 10 2 J mol À 1 K À 1 , respectively, the binding of the molecule to BSA is mostly entropy motivated and the enthalpy is unfavourable for it, with the hydrophobic forces playing a substantial role in the binding process (Kandagal et al., 2007;Ross & Subramanian 1981).

Energy transfer studies
Fluorescence spectrum analyses revealed that BSA and chemical formed a complex.On the basis of Fluorescence Resonance Energy Transfer, the distance r between the protein residue and the chemical was calculated (FRET) (Sharma & Schulman, 1999).The amount of energy transmission is estimated by the separation space and spectral intensity.Figure 9 depicts how the chemical absorption spectrum and protein fluorescence spectrum overlap.Following is the relationship between r and the efficiency of energy transfer E: where R 0 is the critical distance when the energy transfer efficiency is 50%, which can be calculated using the below mentioned equation: where k 2 is the spatial direction factor of the dipole, g is the refractive index of the medium, / is the fluorescence quantum yield of the BSA and J is the overlap integral of the protein fluorescence spectrum and acceptor absorption spectrum.The value of J was calculated by the below formula: where F(k) is the fluorescence intensity of BSA at wavelength and eðkÞ denotes the compound's molar absorption coefficient at wavelength.In this instance, the BSA-compound system has k 2 ¼ 2/3, g ¼ 1.336, and / ¼ 0.15 (Cui et al., 2004).By using Eqs.( 11) and ( 12), we estimated the following values, and the results were listed in Table 8.These findings show that the BSA to compound space is less than 8 nm,   indicating that there is a high possibility of energy transfer from BSA to compound (Hu et al., 2005).

UV-visible spectral studies
A straightforward yet effective technique for determining the complex formation between serum albumin and a bioactive chemical is UV-visible absorption spectroscopy (Bi et al., 2005).Figure 10 shows the absorbance spectra of BSA both by itself and with various chemical concentrations.The pp � transition caused by the BSA's amino acids is represented by the peak at 280 nm that was obtained by the BSA.When the chemical was added, BSA's absorbance increased, indicating that a compound-BSA complex had formed as a result of the BSA molecules being linked to the compound.The wavelength of the ligand and complexes are observed in the range of 440-426 nm (HL), 332-335 nm   (1) and 275-282 nm (2) by the addition of the BSA.The maximum absorbance peak has also slightly migrated upward in wavelength.Together, these two findings -a shift in the maximum absorbance and an increase in the intensity of the absorption -clearly implied the existence of an interaction between the chemical and the BSA and also pointed to conformational changes in BSA (Tanveer et al., 2017).

Selection of binding site
According to published studies, BSA has three main binding sites for various types of ligands: Site I, Site II, and Site III (Sjoholm et al., 1979;Sudlow et al., 1976).Many ligands, including phenyl butazone, oxyphenbutazone and warfarin, bind to site I of the protein.Diazepam, ibuprofen, indoxyl sulphate, and diflunisal bind to site II (Wang et al., 2019) and Digitoxin bestows to the protein's site III (Umesha et al., 2012).To confirm the compound's binding position on BSA, a displacement assay using site-specific markers such Warfarin, Ibuprofen, and digitoxin was performed.Using Eq. ( 7), the resulting binding constants or K values were determined in the presence of site-specific probes and the results are listed in Table 9.The K values show that the Digitoxin and BSA combination is primarily less effective than the native BSA system, although the K values of the warfarin-BSA system and the ibuprofen-BSA complex have not significantly changed.These findings support the hypothesis that the chemical substitutes for digitoxin in the digitoxin-BSA system.Therefore, the compound's binding site, site III, is the same as that of digitoxin.

DNA docking
To find the binding ability of ligand and complexes with CT-DNA, molecular docking studies were carried out (Figure 11).In free ligand hydrogen bonding interactions are between DC9, DC15, DG14 with O10, O9, O6. p-p stacking interactions are observed between DG10, DC9 and the aromatic centers with affinity of À 6.7 kcal/mole.In complex 1, conventional hydrogen bond interactions are between DG10, DG12, DC13 with O10, N16, O2 of the complex.Hydrophobic interactions are observed in DG14, DA17 with O4 and O9 with the binding affinity of À 8.2 kcal/mole.In complex 2, DA18, DC9, DG16 nucleotides are involved in interactions through hydrogen bonding, hydrophobic and stacking interaction.The binding affinity for complex 2 was found to be À 8.4 kcal/mole.The order of DNA binding as follows: 2 > 1 > HL.

BSA docking
The molecular docking method was used to get the preferred binding location and help the understanding of the complexes-protein interaction.In the presence of free ligand, three interactions are observed with the protein.Hydrogen bonding interactions are observed between Glu564, Gln403, Arg427, Lys431 with the distance of 2.05 � Å.It has electrostatic  and hydrophobic interactions are observed with Glu399, Tyr400 with the overall binding affinity of À 7.2 kcal/mole.In complex 1, p-p stacking interactions are observed with the Tyr400 amino acid residue with the aromatic moiety of the complex (Gurusamy et al., 2022c).It also shows hydrogen bonding interactions with Arg427, Lys431 with the binding affinity of À 9.1 kcal/mole.Complex 2 shows higher binding than 1 with the energy of À 9.4 kcal/mole.The major interactions of complex 2 are between Gln521, Glu399, Asp562 with heteroatoms present in the complex.The order of BSA binding of complexes as follows; 2 > 1 > HL (Figure 12).
Antioxidant activity DPPH assay.The stable organic radical 1,1-diphenyl-2-picryl- hydrazyl (DPPH) is frequently employed in oxidative assays to count substances' potential to act as hydrogen donors or radical scavengers.The complexes 1 and 2 have significantly better DPPH scavenging activity than free Schiff base (HL), indicating that they are a more effective antioxidant and free radical scavenger than Schiff base (HL) (Figure 13).The complexes 1 and 2 and standards both exhibit improved dosedependent radical scavenging activity.Antioxidant ability of Schiff base (HL) is increased after chelation of transition metal ions.The complexes' oxidising potentials correspond to their ability to break the free radical chain by donating hydrogen atoms.Therefore, the results found from this study are responsible for the treatment of pathological diseases arising from oxidative stress (Sankarganesh et al., 2022).

Conclusion
Schiff base (HL) and complexes 1 and 2 are prepared.Structural characterization of the complexes 1 and 2 indicates the square planar geometry.Electrochemical behavior of the complexes 1 and 2 shows quasi reversible nature.
Complexes 1 and 2 acts as good antimicrobial agent than Schiff base (HL).DNA binding study shows that complexes interact with CT DNA through intercalation mode.The synthesized heterocyclic compound quenched the fluorescence intensity of BSA, according to the study's state-of-the-art fluorescence spectral results and the quenching mechanism was discovered to be a combination of both dynamic and static type.According to the thermodynamic data, chemical binding to BSA is predominantly entropy motivated, with enthalpy being unfavorable and hydrophobic interactions predominating in the binding process.Additionally, it was discovered that the compound was attached to BSA's site III and that interaction with the compound caused BSA to  modify its conformation.In summary, we find that this work provided insightful data on BSA's interactions and binding studies with pyrimidine derivatives.The pharmacokinetics, biological activity, and mechanism of action of the chemical need to be investigated further.Complexes are good antioxidant character which is confirmed by DPPH assay.
0 V, respectively, on the complex 1's cyclic voltammogram demonstrate the redox process.The complexes 1 and 2 exhibit a quasi-reversible mechanism (Abo El-Maali et al., 2005).The two cathodic peaks correspond to the reduction of Cu(II) to Cu(I) and Cu(I) to metallic Cu deposition respectively.Similarly in the reverse sweep, two anodic peaks correspond to the oxidation of Cu(0) to Cu(I) and Cu(I) to Cu(II) respectively.It is established that Cu(II)/Cu(I) electron transfer couple is very distinctive.Electron transfer between Cu(II) and Cu(I) would therefore be accompanied by major structural and stereo chemical changes.The stability of Cu(II) and Cu(I) ions depends highly on the environment in solution.

Figure 1 .
Figure 1.Proposed structure of the complexes 1 and 2.

Figure 4 .
Figure 4. Antibacterial activity of HL and complexes 1 and 2.

Figure 6 .Figure 7 .
Figure 6.Absorption spectra of the complex 1 in the absence and presence of increasing amount of CT DNA.Arrow indicates the absorbance changes upon increasing concentration of CTDNA.

Figure 10 .
Figure 10.UV-visible spectra of BSA in the absence and presence of the compounds (a) HL; (b) 1; (c) 2.

Table 2 .
Chemical reactivity descriptors of ligand (HL) and its complexes 1 and 2.

Table 3 .
Antibacterial data for the HL, complexes 1 & 2 and standard drug.Compound Zone of Inhibition (mm)

Table 6 .
Stern-Volmer parameters of the compounds.

Table 8 .
FRET studies parameters for compounds.

Table 9 .
Binding constant (K) values for BSA-complex system in the presence and absence of site probes at 300 K.