Synthesis, Characterization, Cytotoxicity and DNA/BSA Interaction of Pd(II) Complexes with Alkyl-Amine and 1,10-Phenanthroline

Abstract A novel series of Pd(II) complexes, [Pd(alkyl-amine)2(phen)](NO3)2, I-V, (where alkylamine is ethyl-, propyl-, butyl-, hexyl- and octyl-amine and phen is 1,10-phenanthroline) were synthesized and characterized based on FT-IR, 1H NMR, COSY-NMR, UV-Vis, molar conductivity, elemental analysis and density functional theory (DFT) approaches. The group, I-V, was screened for their primary in-vitro cytotoxic action against human cancer cell line MOLT-4, showing promising anti-tumor activities. Their interaction activity was tested using various spectroscopic techniques to explain the mode of binding between these compounds with CT-DNA and BSA. Results of UV-Vis studies showed that, at low concentrations, they all interacted effectively with CT-DNA and BSA. Studies of fluorescence emission spectra displayed that the complexes quenched CT-DNA pretreated with methylene blue (MB) and the intrinsic fluorescence of BSA via static quenching. Using thermodynamic parameters (ΔG°, ΔH°, and ΔS°) obtained from fluorescence studies, it was proposed that the hydrogen bonding and van der Waals forces play the main role between metal complexes with CT-DNA and BSA. The binding was spontaneous in all cases due to ΔG° <0. Also, Molecular docking simulation of all five Pd(II) complexes with DNA was performed to determine the compound with the highest binding affinity and investigate its binding mode. Next, molecular docking was utilized for this compound to study its BSA binding mode and affinity. The results obtained from this work may lay the foundation for structural changes exerted on DNA and BSA by Pd(II) antitumor compounds bearing various hydrocarbon chain length as well as structural relationship which are vital for pharmacokinetic and pharmacodynamics.


Introduction
Following the discovery of anti-cancer and biological activity of cisplatin by Rosenberg et al. in the 1960s, many researchers focused on the development of other metal-based drugs with amended efficacy and safety. 1 The Pd(II) analogs are suitable candidates to replace Pt(II) compounds because their structural properties and coordination chemistry are very analogous. Searching for different metal complexes with enhanced effectiveness, less toxicity and ideally noncoordinative interactions is therefore necessary. 2,3 Non-coordinative interactions with DNA include electrostatic force, van der Waals interaction along with hydrogen bonding (groove binding) and intercalation. 4,5 Dispersion and transportation of common substances as well as drugs in the body are related to their affinities toward serum albumin. Therefore, studying the interaction of compounds with serum albumin is essential for the development of new drugs. 6,7 It should be noted that a large number of different multidentate aromatic ligands possessing possible binding ability toward metal ions are the most investigated compounds due to their DNA-binding affinity and cytotoxicity properties. 8 Likewise, the bidentate nitrogen ligands i.e., 1,10-phenanthroline(phen) and their derivatives are the subject of interest in the recent investigations. [9][10][11] Recently, G. Marverti et al. 12 tested the toxicity of 16 new DNAintercalating agents on human ovarian carcinoma cell lines and their resistance counterparts. They found the Pd(phen) invariably showed the highest antiproliferative efficacy. Barra et al. 13 addressed bioactivity of [Pd(phen)(tu) 2 ] 2þ and suggested that the ancillary thiouria ligand does not affect the intercalating ability of these agents, but different effects have already been observed by other workers. [12][13][14][15][16][17] Because of the above considerations, in this work, five new palladium complexes, [Pd(alkylamine) 2 (phen)](NO 3 ) 2 , were designed, synthesized and characterized by experimental and density functional theory (DFT) approaches. The purpose of present work is therefore to analyze the effect of ancillary alkyl-amines having different hydrocarbon chain lengths coordinated to the Pd(II) center ( Figure 1) on the biological properties of new complexes. For this purpose, an introductory anticancer activity test was conducted and the synthesized complexes were subjected to human acute lymphoblastic leukemia cells line MOLT-4. Besides, the interaction of these complexes with calf thymus DNA and BSA were studied using fluorescence emission spectroscopy and UV-Vis experiments. Additionally, molecular docking studies monitored the interaction mode between these metal complexes and DNA/BSA.

Materials and methods
PdCl 2 , ethyl-, propyl-, butyl-, hexyl-and octyl-amine, 1, 10-phenanthroline, methylene blue (MB), ethidium bromide (EB) and Trisbuffer were purchased from Merck company. Highly polymerized calf thymus DNA type 1 and bovine serum albumin (BSA) were bought from Sigma company. Other material used were of analytical grade purity and solvents were purified by standard methods.
Methods to characterize the synthesized complexes I-V (CHN analysis, FT -IR 1 H-NMR, COSY -NMR UV-Vis, conductivity measurements, DFT approach and decomposition temperature) were described earlier. 18 Different absorption and fluorescence techniques and molecular docking were used to study the mode of interaction between Pd(II) complexes with CT -DNA and BSA. 19 The procedure followed to study cytotoxicity of the metal complexes I-V was similar to that reported earlier. 20 2.2. Synthesis of [Pd bis(alkylamine)(phen)](NO 3 ) 2 , a series denoted by I-V (alkylamine 5 ethyl-, propyl-, butyl-, hexyl-and octyl-amine) [Pd(alkyl-amine) 2 (phen)](NO 3 ) 2 complexes were prepared in the following steps: Step 1: Synthesis of Cis-[Pd bis(alkyl-amine)Cl 2 ] Cis-[Pd bis(alkyl-amine)Cl 2 )] complexes were synthesized according to the method reported by our group. 21 Step 2: Synthesis of Cis-bis(alkyl-amine)(1,10-phenanthroline)palladium(II) nitrate In a balloon, 1 mmol of the Cis-[Pd bis(alkyl-amine)Cl 2 )] complex was stirred in 40 mL of a mixture of methanol (25 mL) and acetonitrile (15 mL) solvents at the temperature of 45-50 C until it was completely dissolved and a clear solution was obtained. The solution was then cooled to the ambient temperature and while being stirred, 1 mmol of 1,10-phenanthroline monohydrate ligand was gradually added as powder. The reaction mixture was stirred for 3 hours at 45 C. It was then cooled to the ambient temperature and 2 mmol of silver nitrate was added and stirred for 6 hours without heating. Then, the silver chloride precipitates were filtered. The weight of the silver chloride precipitates matched the calculated value. The obtained yellow filtrate was evaporated under vacuum condition to the volume of 10 mL and exposed to diethyl ether diffusion. Some data about these complexes are gathered in Table 1 and the proposed structures are demonstrated in Figure 1.

Results and discussion
In the present work, the two chloride coordinated ions in the cis-[Pd(alkyl-amine) 2 Cl 2 ] complexes 21 were substituted with a 1, 10phenanthroline ligand ( Figure 1) and promising cytotoxicity properties of them were observed. Thus, following sections include characterization and indetailed experimental and docking studies on the interaction between these synthesized compounds with CT -DNA and BSA as target and carrier biomolecules, respectively.

Conductometry
The molar conductivity (k) of the complexes I-V was measured with the concentration of 10 À4 M in double-distilled water. The results indicated the molar conductivity range and the type of electrolyte or number of ions. In fact, molar conductivity values for the complexes I-V in double-distilled water were obtained in the range of 158-161 Ohm À1 cm 2 mol À1 ) Table 1). These results were consistent with the structures of complexes I-V and electrolytes having 2:1 ion ratio. 22,23 3.1.2. FT-IR spectra of Pd(II) complexes (I-V) FT-IR spectra of complexes I-V were recorded on KBr pellet in the range of 400-4000 cm À1 . The main IR active functional groups of these molecules were: ÀNH 2 (stretching), CÀH (aliphatic), NÀH (bending), C ¼ N, CÀN, PdÀN and NO À 3 (uncoordinated). The frequencies corresponding to these groups are presented in Table 2. The data in Table 2 are matched with our previous report 21 as well as 1,10-phenantroline complexes reported by other workers. 24,25  3.1.3. The UV-Vis absorption spectra of Pd(II) complexes (I-V) Absorption spectral pattern of the complexes I-V are similar. UV-Vis spectrum of a solution with the concentration of 2 Â 10 À6 from each of these complexes showed two intense and broad bands at $ 230 and $ 275 nm (see Figure 2 for the complex I and Table S1 for II-V). These two bands are related to p ! p Ã and n ! p Ã transitions, respectively. 14 Also Figure 2 shows a blue shift of the band at 275 from less polar ethanol to more polar water solvent. This shift is the reason for the metal to bind (Pd ! phen) charge transfer. 26,27 While increasing the concentration of the complex solutions to 5 Â 10 À3 , a broad band in the range of 350 À 450 nm is appeared which might be due to d ! d transition since the expected geometry of these Pd(II) complexes is square planar. Moreover, another medium intense peak observed at $ 390 nm as shoulder, expected to be due to the absorption of NO À 3 counter ion. This peak was cross checked with the absorption spectrum of NaNO 3 solution which has two peaks in the region of 235 À 310 nm. That may overlap with the n ! p Ã band of complexes. 28

1 H NMR spectra of Pd(II) complexes (I-V)
To extend the characterization of the complex series I-V ( Figure 1) and to clarify the proposed structures of them, three experiments were carried out: (i) 1 H NMR spectrum of complex I in D 2 O to check the exchangeable protons with deuterium, (ii) 1 H NMR spectra of complexes II-V in DMSO-d6 to find out chemical shifts of protons presented in all these compounds, and iii) 2 D (COSY) of complex III having medium sized hydrocarbon chain length in these series to observe coupling of protons in the aliphatic as well as aromatic moieties of them. Findings of chemical shifts (ppm) of all the complexes are compiled in Table 3 and explained below In proton NMR spectrum of complex I (in D 2 O), signals appearing at 1.1 and 2.1 ppm are attributed to F (triplet) and e (quartet) protons, respectively. The protons associated to the amine Table 2. FT-IR data (cm À1 ) of the Pd(II) complexes I-V. 24,25,28,38,46,47   groups have been exchanged with deuteron owing to the use of D 2 O as solvent. 29 The signals appearing with the chemical shift of 7.73, 7.82, 8.38, and 8.57 ppm are assigned to b, c, d and a protons, respectively (see Figure 1 and Table 3). 26 The 1 H NMR spectra of complexes II-V were recorded in DMSO-d6 in the presence of TMS as internal standard. The spectrum of complex III ( Figure S1) shows five signals at 0.94, 1.42, 1.79, 2.93 and 5.36 ppm assigned to h (triplet), g (multiplet), f (multiplet), e (multiplet) and k (singlet broad) protons of aliphatic moiety, respectively. The aromatic proton signals (i.e., 1,10phenantroline) resonate at 8.27, 8.36, 8.91 and 9.13 ppm as assigned to b, c, d and a protons, respectively. Other three complexes (II, IV and V) showed similar pattern of chemical shifts like III. Data corresponding to all complexes for their aliphatic as well as aromatic protons are presented in Table 3.

2D-1 H-NMR spectrum of [Pd(Bu-am) 2 (phen)](NO 3 ) 2 complex (III) COSY type
To further characterize the structures of the new Pd(II) complexes, a twodimensional (2 D) correlated spectroscopy (COSY) of [Pd(Bu-am) 2 (phen)] (NO 3 ) 2 (complex III) was recorded (Figures S2 and S3, as expanded in two parts). In these spectra, the contours shown on the diagonal line (the line that connects the top right corner to the bottom left corner of the spectrum) are called the main contours, which indicate the chemical shift of the signals. The ones on the either side of diagonal line are called cross contours, representing the protons whose spin moments have been coupled to each other. In the spectrum of the above complex III, the most shielded are h protons of alkyl amine ligand and the most deshielded ones are a protons of 1,10phenantroline ligand. In the COSY spectrum of complex III, two sets of contours (prominent designs) are observed which are interpreted here: First set ( Figure S2) represents main contours of h (ÀCH 3 , 0.94 ppm), g (ÀCH 2 À, 1.42 ppm), f (ÀCH 2 À, 1.79 ppm), e (ÀCH 2 À, 2.93 ppm) and k (ÀNH 2 À, 5.36 ppm) protons. The main contours assigned to h protons shows cross contours with g protons, g with f, f with e and e with k protons. Therefore, the chemical shifts and coupling of h, g, f, e and k protons of the coordinated butyl amine ligands to Pd(II) ion are identified.
Second set ( Figure S3) represents the main contours which are assigned to a (9.13 ppm), b (8.36 ppm), c (8.91 ppm), and d (8.27 ppm) protons of coordinated 1,10-phenantroline. In these main contours, the "a" protons show cross contour with b, b with c and c with d protons. Thus, spin coupling of these protons as well as the chemical shifts of protons on coordinated 1,10-phenantroline ligand are identified.

Density functional theory (DFT) analysis
Structural optimization of all five Pd(II) complexes were performed by DFT method. The obtained structure of complex I along with the selected bond lengths and angles calculated by DFT (B3LYP/GENECP) approach and aug-cc-pVTZ-PP basis set for Pd atom and 6-311 G(d,p) for the other atoms are shown in Figure 3. The same approach and basis set were used for other four complexes and the magnitudes of selected bond lengths and angles for all metal complexes are listed in Table 4. It is observed from this table that all the similar bond lengths are equal in five compounds but bond angles differ slightly.

Stability test
The stability of metal complexes is an important parameter that affects their applications in biological studies. So, the stability of the complexes in water as well as DMSO was checked following the procedure reported earlier 18 via electronic absorption approach. As seen in Figure 4(A,B), the spectral pattern of complex I (3Â10 À6 M) did not vary after different incubation times (0, 24,

Compound
Bond lengths(Å) Bond angles( ) 48, and 72 h) which shows the stability of this Pd(II) complex in aqueous and DMSO media. Similar experiments were carried out for the other four Pd(II) complexes.

Evaluating cytotoxicity of the synthesized complexes I-V
The inhibitory effect of palladium(II) complexes (I-V) against human lymphocyte cancer cell line, MOLT-4, was investigated. The results of cytotoxicity of palladium(II) complexes, doxorubicin and cisplatin (as the positive controls) against MOLT-4 are collected in Table 5. These results show that complexes I-V have deterrent effect on the growth of MOLT-4 cancer cells, which is comparable to cisplatin. As the IC 50 diagram of synthesized complexes shows ( Figure S4), the ability to inhibit the growth of MOLT-4 cancer cells enhances slightly by increasing the length of aliphatic amine chains present in the structure of synthesized complexes. 30 The inhibitory effect against MOLT-4 cell line increases in the following order II < I <III < IV < V.

UV-Visible binding studies for interaction with CT-DNA
The electronic absorption spectra of interaction of complexes I-V with CT-DNA in Tris buffer solution was recorded at two temperatures of 298 and 310 Kelvin. The obtained results show that the gradual addition of complexes I, II, III, IV and V to the CT-DNA solution leads to the hypochromic effect i.e., reduction in absorption. As an illustration, Figure 5 shows that the absorption of CT -DNA at 298 and 310 K decreases with the addition of metal complex III. The reason behind this reduction is the refolding of CT-DNA strands and hiding its bases from the UV light.
According to the reports, 31 a red displacement of more than 10 nm at the maximum absorption intensity (k max , DNA ¼ 260 nm) along with hypochromic effect indicates that the interaction of metal complex with DNA is of intercalation type, whereas the observed hyperchromic effect ( Figure 5) does not show unexpected shift and supports the groove binding of metal complex III.  Considering other reports, 31 the red shift of less than 10 nm at k max , DNA ¼ 260 nm confirms the insertion of complexes I-V in DNA grooves. Furthermore, the plot of 1/(A obs -A 0 ) versus 1/[L] was drawn for all complexes I-V and the corresponding graph is shown for the compound III in Figure S5. From these plots, the magnitudes of K app for the new complexes (I-V) were calculated and summarized in Table 6. It is observed from this table that K app decreases with rising temperature from 298 to 310 K for the compound II and IV and increases for the complexes V, I and III. This might be due to the effect of different space orientations of hydrocarbon chains present in the structure of these complexes on DNA site/sites being favorable or unfavorable. The values of K app (% Â10 3 M to %Â10 5 M) indicate the affinity of all complexes to interact with DNA. These values are all lower than the reported value for the classical intercalator, ethidium bromide, 32 supporting the groove binding mode of the metal complexes (I-V) with CT-DNA.
In a second experiment, from a plot of (A 260 -A 640 ) against complex concentration, the L1/2 was calculated for all compounds at 298 and 310 K. This diagram is shown for compound III in Figure S6. Using these studies, the values of L 1/2 were calculated and collected for the complexes I-V in Table 6. Recall, L 1/2 is the concentration of metal complex at which 50% of the biomacromolecule structure is denatured. Small values of L 1/2 indicate that low concentrations of Pd(II) complexes are required if they are administered as chemotherapy agent, which is important in drug design and pharmakokinetic. Moreover, in the spectra obtained from the interaction of complexes  I, III, and IV with DNA, some isosbestic points are also observed ( Figure 5 and Table 6). The existence of these points confirms that the ensemble of metal complex-biomacromolecule is formed and there exists equilibrium between free DNA and DNAmetal complex.

UV-Visible binding studies for interaction with BSA
In this study, BSA titration with the above complexes was performed according to the reported method. 33 Since BSA absorbs the UV light in the 280 nm region, the absorption spectrum of BSA in the presence of complexes I-V in this region at the temperatures of 298 and 310 K was examined and recorded. These spectra for complex II (as an illustration) are shown in Figure 6. The results after overlying the spectra indicate that the gradual addition of complexes I-V to the BSA solution leads to a reduction in the maximum absorption (k max ¼ 280 nm). This hypochromic effect for BSA in presence of metal complexes is due to the conformational alteration in BSA structure. As a result, the UV-active residues (mainly tryptophan amino acids) of BSA get hidden from the light and the reduction in the intensity of spectrum is observed. 34 To determine K app for BSA binding and according to the findings of the above experiments, the plot of 1/(A obs -A 0 ) was drawn in terms of 1/[complex]. This diagram is shown for the complex II in Figure S7. In these diagrams, K app values were obtained by dividing the intercept by the slope and collected in Table 7. 35 From the above experimental data, (A 260 -A 640 ) were plotted against concentration of each metal complex at 298 and 310 K. These curves are shown for complex II in Figure S8 and the values of L 1/2 are collected in Table 7 for all complexes I-V. In summary, the value of K app for the metal complexes II, III and V shows an increase by rising temperature from 298 to 310 K. Considering this, the stability of the BSA-metal complex ensemble increases at higher temperatures, which confirms the interaction of the complex with BSA, is endothermic. The value of L 1/2 increases from 298 to 310 K for the complexes I and IV, meaning the complex with longer hydrocarbon chain length probably show better interaction. However, there is no proper trend in the series of complexes I-V. The values of L 1/2 obtained from the interaction of new complexes I-V with BSA are in the range of 4À8 mM which are smaller than the same results obtained from our previously reported metal complexes. 21 Figure 6. BSA titration spectra with complex II at the temperature of 298 K (the spectra in the right of the figure were obtained at 310 K).

Fluorescence emission studies using methylene blue (MB) intercalator in the interaction with CT-DNA
The ability of synthesized complexes to intercalate between DNA-base pairs was tested using fluorescence technique. In these studies, methylene blue (MB) intercalator was used. This molecule has the following characteristics: (i) it is a fluorescence-active compound in the free form, which means that should it be excited at the wavelength of 285 nm, it releases fluorescence emission at the wavelengths of 583 nm and 682 nm. Between these two emissions, the bandwidth intensity at $682 nm is very high and suitable for monitoring and (ii) if the MB solution is titrated with DNA, its emission will increase at $583 nm and decrease at $682 nm. In this process, DNA is a quencher. Thus if MB -DNA solution get titrated with another molecule (say, A) and emission intensity at $ 682 nm abruptly increases, it means interacted MB is displaced by A and the A itself is an intercalator.

Determining the Stern-Volmer quenching mechanism
The emission spectrum of MB-CT-DNA resulting from the interaction with complexes I, II and V were recorded at three temperatures of 298, 310, and 315 K. We selected these compounds among the five Pd(II) complexes because I and V have the smallest and largest chain lengths for the amine ligand, respectively and II has the highest K app for DNA binding from UV-Vis test. In these studies, first, the emission of methylene blue solution with the concentration of 1 Â 10 À5 mol/L at 690 nm was recorded, then 130 mL of stock DNA solution was added to the MB solution and its emission was recorded on the MB spectrum. As shown in Figure 7, the emission intensity of the MB-CT-DNA solution is much lower than the MB solution at 690 nm because CT-DNA quenches the MB emission. In the next step, the MB-CT-DNA solution is titrated with the various amounts of Pd(II) complexes (the stock concentration of 1 Â 10 À4 mol/L). 34 As shown in Figure 7, with the gradual addition of complex V solution to the MB-CT-DNA solution, the emission at 690 nm has a gentle increasing trend. 36 Furthermore, the maximum wavelength shows a displacement of less than 15 nm toward longer wavelengths (red-shift). This increase in the emission intensity can be interpreted in several ways as follows: 1. considering the above conditions that all CT-DNA sites are occupied by MB, the metal complex can compete with MB and separate it from the intercalation sites of CT-DNA (between base pairs), as a result, an enhancement will be observed in the emission spectrum at 690 nm due to the presence of more free MB molecules in the solution, (ii) the emission intensity of MB-CT-DNA-Pd(II) complex ensemble is more than MB-CT-DNA, which means that the complex could change the conformation of CT-DNA, hence fewer locations for MB molecules are presented on CT-DNA and (iii) the metal complex interacts with CT-DNA to change its conformation such that the denatured CT-DNA can emit more than natured CT-DNA. These observations show that the complexes I-V, despite having a flat aromatic ligand (phen), cannot fully compete with MB because if they do so, the emission intensity would have increased sharply due to the release of free MB molecules into the medium.
In the case of fluorescence enhancement, the Stern -Volmer equation becomes as F 0 /F ¼ 1 - where K E is dynamic enhancement constant, analogous to quenching constant (K SV ), and K D is the bimolecular enhancement constant, analogous to k q. 37 Based on this equation, the values of K E and k D were obtained by drawing the F 0/ F against complex concentration [E] at 298, 310, and 315 K and the results are collected in Table 8. This plot is shown in Figure S9 for the complex V. Considering the equivalency of the bimolecular quenching and enhancement for the complexes I, II and V, the values of K E and k D decrease with increasing temperature, so the enhancement mechanism is probably static for this group of complexes.  Table 8. This diagram is shown in Figure S10 for complex V at three temperatures of 298, 310, and 315 K.
As the findings in Table 8 show, there is no noticeable trend in the K b values or tendency of these complexes to bind with CT-DNA. This might be due to different space orientation of alkyl   chains of alkylamines when these complexes interact with single binding site on CT-DNA. The single binding site comes from the n values, which are around one (Table 8). However, the values of K b are in the range of 10 3 -10 4 for these complexes which are lower than the classical intercalator ethidium bromide 4 indicating no intercalation occurs in these processes.

3.4.3.
Determination of the thermodynamic parameters (DG , DH , and DS ) for CT-DNA binding with complexes I-V To further identify and understand the effective forces in the interaction of synthesized complexes with CT-DNA with the help of fluorescence studies, the values of thermodynamic parameters, i.e. free energy changes (DG ), enthalpy changes (DH ) and entropy changes (DS ) were calculated. These values were obtained for the interaction of complexes I, II, and V with CT-DNA at the temperatures of 298, 310 and 315 K using the slope, intercept of van't Hoff equation, as collected in Table 9. The van't Hoff diagram for the complex V is shown in Figure S11. According to the data in Table 9, the values of DH˚are negative for the complexes I, II, and V which means their interactions with CT-DNA are exothermic.
Based on the reports, due to the negative signs of DH and DS for complexes I, II and V, the driving forces for the interaction of these compounds with CT-DNA are of hydrogen bond and van der Waals type. 38,39 Therefore, the new palladium complexes (I, II, and V) are likely able to bind in the grooves of CT-DNA. 40 The values of Gibbs free energy changes (DG ) were obtained at the temperatures of 298, 310, and 315 K. DG <0 confirms the spontaneity of interaction between CT-DNA and studied metal complexes. Finally, the negative values of DH , DS and DG imply that the interaction between CT-DNA and new Pd(II) complexes is enthalpy driven.

Investigating the quenching of BSA emission in the interaction with complexes I, II, III and V
Studying the fluorescence emission of BSA in the interaction with complexes I, II, III and V was performed at the temperatures of 298, 310, and 315K. The fluorescence emission spectrum of BSA solution was recorded in the range of 300-500 nm. The emission spectra in the presence of various amounts of each complex was obtained separately and for the complex V is demonstrated in Figure 8. Examining the recorded emission spectra of BSA shows that gradual increase in the concentration of the complex has led to the quenching of BSA emission and decreasing emission intensity at 350 nm. A slight displacement is also observed in the position of maximum wavelength. According to the reports, 41 if no displacement is observed in the wavelength of maximum emission, then the dielectric of micro-environment of BSA has not changed. Furthermore, the decrease in the intensity of emission can be due to the binding of the metal complex to this protein, which leads to the change in the conformation of tryptophan environment. Therefore, by  Table  10. In addition, the Stern-Volmer diagram is shown for the complex V in Figure S12. It is seen from Table 10 that by raising temperature from 298 to 315 K, a decrease in K SV and kq is observed, depicting the possibility of static quenching during BSA binding. 43 3.5.2. Calculation of binding constant (K b ) and number of binding sites (n) for BSA binding The values of K b and n were obtained for BSA binding with complexes I, II, III and V by plotting the log(F 0 -F)/F in terms of log[L] t at three different temperatures and the data are tabulated in Table 10. This plot is also shown in Figure S13 for complex V at three temperatures of 298, 310 and 315 K. As the findings in Table 10 show, the values of K b and n for the above mentioned complexes show a decreasing trend with increasing temperature and K b is in the order of    Table 11. The corresponding diagram is seen in Figure S14 for the complex V. According to the data in Table 11, the value of DH is negative for the complexes I, II, III and V. Therefore, the interaction of these complexes with BSA releases energy. These data are consistent with the results obtained for K b in Table 10 where by increasing temperature, the magnitude of K b decreases. The negative value of DG˚indicates the interaction of studied compounds with BSA is a spontaneity process. Negative values of DH and DS suggests (i) hydrogen bond and van der Waals interaction play the main role in the interaction of studied compounds and protein and (ii) their interaction is driven by favorable enthalpy changes.

Calculation of FRET parameters
Using the overlap of the fluorescence spectrum of metal complex-BSA ensemble with the selectronic absorption spectrum of each of the (alkyl-amine) 2 (1, 10-phenanthroline) palladium (II) nitrate complexes, the magnitudes of J, E, R 0 and r were calculated for the above complexes I, II, III and V. The obtained values are compiled in Table 12 and the overlap diagram for the complex V is shown in Figure 9. 44 As seen in Table 12, the values of r are in the range of 3.41-4.43 nm (less than 8 nm). The results can be summed up as follows: (i) BSA has the ability to transfer energy to the synthesized complexes I, II, III and V, (ii) the transfer of energy can confirm the interaction between the BSA and the above complexes and (iii) according to the reports, 45 since the values of r are greater than R 0 , it is suggested that the quenching mechanism of BSA is by non-reflective energy transfer and static quenching. This is quite consistent with the results observed from fluorescence studies.

In-silico binding study
Molecular docking was carried out for all new metal complexes to find the superior compound having the highest free energy of binding with DNA and the results are listed in Table 13. As can be seen, the compound II has the most negative value for free energy of binding, DG docking ¼ À7.02 (kcal/mol), which is in accordance with the results obtained from UV-Vis experiment, DG UV-Vis ¼ À 7.33 (kcal/ mol). It should be noted that the metal complexes with higher alkyl length than compound II have lower DNA binding affinity that could be due to steric hindrance of alkyl chain. The best-docked pose of compound II insides DNA is shown in Figure 10 along with their significant interactions. As seen in this figure, the metal complex prefers the groove-binding mode inside DNA, which is consistent with the results of, fluoresce finding. The main interactions between II and DNA are shown in the right side of Figure 10. It is observed that there exist two hydrogen bonds between C5 and complex II with the lengths of 1.94 and 2.01 Å, respectively. Furthermore, there are two hydrogen bonds between A4 and A6 with the complex II with the lengths of 3.25 and 1.84 Å, respectively. As mentioned above, the compound II has the highest DNA binding affinity in compare to the other studied Pd(II) complexes. So, docking simulation was performed for this compound with both drug binding sites of BSA. The values of DG docking,site I ¼ À6.31 kcal/mol and DG docking,site II ¼ À6.98 kcal/mol indicate that this metal complex prefers the drug binding site II. Therefore, we depicted the docking results of site II in Figure 11. As seen in this figure, this Pd(II) complex has one hydrogen bond with Ser488 residue and six van der Waals interactions with the following amino acid residues: Cys391, Val432, Leu406, Leu429, Leu452, Ile387 amino acids. There are also two psulfur interactions with Cys391 and Cys437 amino acid residues.

Conclusion
Five new complexes (I-V) with the general formula of [Pd(alkyl-amine) 2 (phen)](NO 3 ) 2 , where alkylamine includes ethyl, propyl, butyl, hexyl, and octylamine were synthesized and characterized with the help of spectroscopic and nonspectroscopic techniques. Cytotoxicity assay demonstrated that all five compounds have antitumor effect and IC 50 values comparable to that of cisplatin. Therefore, DNA and BSA binding ability of these complexes were investigated through UV-Vis and fluorescence experiments. The results showed that the compounds I-V interact with  both biomacromolecules mainly via hydrogen bond and van der Waals interactions. To better understand the effective forces in the interaction, molecular docking simulation of all compounds with DNA was performed and the compound II had the highest free energy of binding with DNA. The same finding was observed from UV-Vis experiment. Then, docking simulation was accomplished for this complex with BSA. It was observed that this compound prefers site II of BSA via hydrogen bond and van der Waals forces. Docking results also confirmed the groove binding of these complexes with DNA. Another noteworthy observation of this study was that despite the presence of aromatic and flat ligand, 1,10-phenanthroline, the new complexes were not intercalated in DNA, due to the steric hindrance effects of hydrocarbon chain.