Heteroleptic C, N- Donor Pd(II) Complexes: Synthesis, Characterization, DNA/BSA Binding Interactions And Biological Studies

A series of trisubstituted pyrazole-based ligands (L 1 -L 6 ) and corresponding palladium(II) complexes were synthesized and characterized by conductivity measurement, 1 H NMR, 13 C NMR, Fourier Transform infrared (FT-IR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Synthesized compounds were screened for various biological activities. UV-Vis spectroscopy, viscosity measurement, uorescence spectroscopy and molecular docking studies were used to determine the binding mode between HS-DNA and complexes, which suggest intercalation mode of binding. The protein binding study of complexes was evaluated by UV-visible spectroscopy. Antibacterial study of the complexes was screened against two Gram (+ve) and three Gram (-ve) bacteria and results show that all complexes are more effective against microorganisms than their respective ligands. The cytotoxicity of the synthesized compounds was tested against brine shrimp and MCF-7 Cells. The LC 50 values of the ligand and complexes were found in the range of 9.24-4.12 µg/mL and 5.68-7.94 µg/mL, respectively. , , 1319 ѵ(C-N) ar. Stretching , 910 ѵ(C-Br) stretching , 794 ѵ(= C-H) bending,


Introduction
All life born on the earth creates something in this world and ends with some reason. In some cases, whenever we analyze the cause of life end at the global level, that analysis states that the cause of life end is cardiovascular disorders followed by cancer [1]. Cancer is a pensive health problem in society. The WHO study says that 9.6 million lives ended in 2018 with stress of cancer and total death expected in 2040 will double due to various types of cancer [2]. Anticancer drug available in the market has limitations like high toxicity, missing target specialty, acquired resistance and heavy dosage, so to work out the e cient anticancer drug, target-speci c and having a minimum side effect is a pivotal in medicinal chemistry [3]. At the global level, many researchers are committed to working out metal coordination compounds that can bind with DNA (by covalent mode or non-covalent mode) to produce an equipotential activity of cisplatin [4][5][6][7][8][9] .
Organometallic Pd(II) coordination compounds show unusual activity in tumor curing agents compared to rst-generation anti-tumor drugs, which show drug resistance, nephrotoxicity, and other problems [10].
Due to the structural similarity of Pd(II) and Pt(II), coordination compounds are ideal as metallodrugs.
The ligand, which is incorporated with metal ions, plays a vital role in bio-activity [11][12][13][14] . Predominantly various bio-activities of the ligands will spank results when it combines with the cation in metal complexes [15].
Due to diverse bio-activities, pyrazole moiety with different derivatives is on the list of studies of many researchers in medicinal chemistry [16][17][18][19][20] . Many researchers reported that coordination compound with pyrazole moiety with the different metal and various substituted pyrazole shows a long bio-activity list [21][22][23][24]. Experimental

Materials and methods
All the chemicals and solvents were of the reagent grade and used without further puri cation. Sodium tetrachloropalladate(II) was purchased from S.D. Fine Chem limited(SDFCL). 5-Bromothiophene-2carbaldehyde and substituted acetophenone were purchased from Sigma Aldrich. Herring sperm (HS)-DNA was purchased from Sigma Aldrich Chemical Co (India). Nutrient broth (NB), agarose, ethidium bromide (EtBr), tris-acetyl EDTA(TAE) and bromophenol blue were purchased from Himedia (India). All chemicals and reagents used for DNA binding were purchased from SRL (Sisco Research Limited India), MCF-7 was purchased from NCCS, Pune, INDIA. The solvents used for spectral measurements were of HPLC grade. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Brucker Avance (400 MHz) spectrometer using deuterated dimethyl sulphoxide (DMSO-d 6 ) solvent with chemical shift ( in ppm) reference to internal TMS. IR Spectra were recorded on an FT-IR ABB Bomen MB-3000 spectrophotometer.

Physical measurements
Melting points were determined on thermocal 10 melting point apparatus (Analab Scienti c PVT.LTD, India) in open capillaries. Electronic spectra were recorded with UV-160A UV-Vis spectrophotometer, Shimadzu, Kyoto (Japan). Conductance measurements were carried out using conductivity meter model number space E-660A. C, H, N-elemental analysis was performed on model Euro vector EA 3000(for ligands) and Perkin-Elmer 240(for complexes) elemental analyzer. Electron spray ionization (ESI) mass spectra were obtained using thermo scienti c mass spectrometer (USA).
2.3 Preparation of thiophene-pyrazole based C, N-donor ligands (L 1 -L 6 ) Substituted thiophene-based enones (α, β-unsaturated ketones) were synthesized by 5-bromothiophene-2carboxyldehyde and substituted acetophenone with the help of base (sodium hydroxide). The ketones were used for further reaction to getting ligands (L 1 -L 6 ). Potassium tertiary butoxide added in an alcoholic solution of substituted thiophene-based enones and substituted phenylhydrazine hydrochloride were added and re uxed at 60 C for 6-7 h in methanol. It gave thiophene-pyrazole-based C, N donor ligands (L 1 -L 6 ). The reaction scheme for the preparation of thiophene-pyrazole-based C, N-donor ligands is depicted in scheme 1.

MIC by broth dilution method
All synthesized compounds were tested for MIC using the broth dilution method. The method used to evaluate MIC value is according to the reported process [25]. Serially two-fold dilution of the test compound added to three Gram(− ve) microorganisms, namely Pseudomonas aeruginosa (MTCC P-09), Escherichia Coli (MTCC 433), Serratia marcescens (MTCC 7103) and two Gram(+ ve) bacteria, namely Bacillus subtilis Staphylococcus aureus (MTCC 3160), (MTCC 7193).

Evaluation of in vitro cytotoxicity
The cytotoxicity of complexes was studied on brine shrimp, Artemia cysts, according to the method reported by Meyer et al. [25].

Cell proliferation assay
Tested compounds were diluted in DMSO with a regular interval in RPMI cell tissue medium in quadruplicate. Cells were sustained in RPMI-1640 implement completed with 10% fetal bovine serum (FBS) without antibiotics. Cells were cultured in monolayer and sustained with 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) at 37°C with CO 2 (5%) for 1 day. At the end of the treatment period, medium was withdrawn from cells and added 0.5 mg/mL MTT, prepared from PBS and incubated in 5% CO 2 for four h. After four h, the MTT implement was discarded from the cell and washed utilizing PBS (300 µL). The formed precipitates were soluble in 100 µL DMSO and yellow dye turned into purple formazan, which color intensity was observed at 570 nm.

Viscosity measurement
Further clari cation of the interaction between the complexes and DNA was carried out by viscosity measurements. Measurement of viscosity is a simple, complementary and excellent method to know the binding mode of metal complexes to DNA. The detailed experimental process was as reported in the literature [27].

Fluorescence quenching analysis
Emission spectral study of EB-DNA complex used to recognize the binding of test complexes to DNA regardless of their binding modes and only judge their capacity impact the EB intensities in EB-DNA complex. Metal complexes quenching constants to HS-DNA were diagnosed by uorescence titration study using EB, which contrives highly uorescent EB-DNA compound with emission property at 610 nm. Fluorescence experiments were performed by addition of synthesized compounds to PB of EB-DNA. In this study, EB concentration was xed at 33.3 µm and 10 µm of HS-DNA added in PB solution. The solution was incubated at R.T for 10 min. After the addition of the above solution, emission spectra were recorded at 500-800 nm in Fluoromax-4, HORIBA spectro uorometer with 1 cm path length quartz cell. Competitive spectral study of EB bound HS-DNA with or without quencher (complex) have been noted for [EB] = 33.3 µm and [HS-DNA] = 10 µm against insertion of increasing quantity of compounds (10-100 µm).

Molecular docking study
Molecular docking is a vital tool in computational drug design [28]. Docking study showed the binding a nity and some hydrogen bonds. It is interesting to note that the binding a nities have negative values. Molecular docking is an approach used in drug design that functions by placing a small molecule into a receptor site and studying the t orientation bind mainly via non-covalent mode. The molecules were allowed to run in chembio3D software to re ne the molecule according to the most stable conformational arrangement. The molecules were formatted as PDB les and were allowed to interact with B-DNA in HEX 8.0 software on CORE i3 processor in windows 8. 1

Fluorescence quenching study
Fluorescence quenching of metal complex and protein is performed according to previously published literature [4,5].

1 H NMR and 13 C NMR spectra
The shifting of the peak in 1 H NMR spectra suggests the involvement of pyridine ring nitrogen as a coordinating atom. In the case of ligands (L 1 -L 6 ) and Pd(II) coordination complexes (I-VI), aromatic protons are observed in the range of 8.49-6.77 δ ppm and 8.50-6.78 δ ppm, respectively. The H 4'a and

In vitro antibacterial activity
The ligands L 2 , L 4, and L 6 exhibit signi cant activity than L 1 , L 3, and L 5 against ve organisms.
Complexes I, III and V exhibit signi cant activity than complexes II, IV and VI against ve organisms. The coordination compounds (45-105 µm) show signi cant activity than ligands (255-375 µM), and are comparable with reported palladium (II) complexes [27]. Graphical representation of MIC value of ligand(L 1 -L 6 ) and Pd(II) complexes(I-VI) is shown in Fig. 1 The higher antimicrobial activity (lower MIC value) of the metal complexes (MIC = 45-105 µM) compared to the metal salt (MIC > 1000 µM) and respective ligand is due to nuclearity of the metal center in the complexes, chelate effects, nature of the ligands, and ion neutralizing the complexes [25].

In vivo brine shrimp lethality bioassay
The BSLB is a valuable method for assurance of lethal nature of synthesized compounds and demonstrates the pharmacological exercises of the compounds. It is widely used in the bioassay. Figure   2 represents the graph of LC 50 value of ligands (L 1 -L 6 ) and Pd(II) complexes (I-VI), respectively. Order of the LC 50 value: I > V > III > II > IV > I > L 3 > L 1 > L 2 > L 5 > L 4 > L 6 , Complexes I and V show potent cytotoxicity activity than complexes II, III, IV, and VI. In ligands, L 1 and L 3 exhibit potent cytotoxicity activity than L 2 , L 4 L 5 and L 6 . LC 50 value of heterocyclic agents and Pd(II) complexes are observed range in range of 10.56-16.63 µg/mL, 5.70-7.58 µg/mL, respectively which is comparable to reported Pd(II) complexes (LC 50 = 5.68-7.94 µg/mL) [29]. The complexes exhibit good cytotoxicity activity than heterocyclic compounds.

Fluorescence quenching analysis
Fluorescence spectroscopy has bene cial applications as a tool to study DNA-complex interactions due to its ability to examine the effects of environmental conditions on various DNA interactions. It is considered a non-destructive and sensitive technique for the characterization of DNA-complex interactions. A competitive binding study of each complex with EB was carried out to determine whether it can displace EB from its HS DNA-EB complex. So, a comparison of the intrinsic emission spectra of the free EB-DNA complex and the metal complex's presence can help to evaluate the mode of DNA interaction with the complexes. Here we have used the more concentrated solution of the EB than the tested compound, giving less gap between two sequential readings and an accurate binding constant.
In emission titration of Pd(II) complexes, emission intensity decrease on insertion of quencher to EB bound HS-DNA was recorded at 610 nm, which preferred intercalative binding. Value of K sv , binding constant (K a ), and binding sites (n) are observed in the range of 2.1-8.2 × 10 3 M -1 , 0.20-6.1 × 10 4 M -1 and 0.904-1.298, respectively, which is comparable with reported palladium complexes [33,34]. The order of binding constant K a of pyrazole-based palladium complexes (I -VI) are: I < V < VI < II < IV < III.
Fluorescence spectra of graph complex I is as shown in Fig. 6.

Molecular docking study
The adequate energy of interaction in which molecule be t with DNA was counted as its docking energy. representative graph of the ligand1(L 1 ) and complex-I is shown in Fig. 8

Fluorescence quenching study
In emission intensity of Pd(II) complexes, emission intensity decrease with a blue shift in maximum emission wavelength (λ em ) of BSA is observed with an increase in quencher concentration at 345 nm, indicating the associative interaction between the BSA and quenchers. The intensity of uorescence regularly decreases with a concentration of complexes increase (Fig. 10) and results suggest that complex quenches intrinsic uorescence of BSA. Value of K sv , binding constant (K a ), and binding sites (n) are in range of 1.07-2.80 × 10 3 M − 1 , 0.10-0.58 × 10 5 M − 1 , and 0.92-1.12, respectively which is comparable with reported literature [36,37]. The order of binding constant K a of pyrazole-based palladium(II) complexes: I < VI < II < V < IV < III.

Conclusion
In this work, thiophene-pyrazole-based C, N-donor ligands (L 1 -L 6 ) and their complexes with Pd(II) were synthesized and characterized by various spectral and analytical techniques. The 1 H NMR and 13 C NMR spectra signals are slightly shifted in complexes (I-VI) than corresponding ligands. Similarly, IR frequency changed upon complexation. The molar conductance value of complexes (I-VI) observed in range 14-21 Ω-1 cm 2 mol − 1 suggests a negative charge of complex and one sodium ion presence in the outer sphere.
The geometry of Pd(II) coordination complexes are four coordinated with dsp 2 hybridization, low spin-d 8 system and diamagnetic. The data suggest that all complexes exhibit square planar geometry. The UVvisible, uorescence titration viscosity measurement and molecular docking studies of Pd(II) complexes reveal that the interaction of synthesized complexes with HS-DNA is speci cally through non-covalent interaction (i.e., intercalation) types of binding with DNA base pairs. The binding ability and binding energy of complex-DNA are higher compared to the corresponding ligand. All Pd(II) complexes exhibit good antibacterial activity with low MIC values against Gram(+ ve) and Gram(-ve) bacteria, which indicates that the complexes show good activity as compared to ligands. The platinum(II) and palladium(II) complexes exhibit higher potency in cellular level cytotoxicity and brine shrimp lethality bioassay than the pyrazole-based ligands. Pd(II) complexes show signi cant cytotoxicity against MCF-7 (human adenocarcinoma cell line) cell line. And comparable with clinically used metallodrugs such as cisplatin, carboplatin and oxaliplatin.

Con ict of interest
The author declares no con ict of interest.      Fluorescence emission spectra of BSA quenching of complex-I

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