HSA interactions and in vitro cytotoxic activity

Accepted Manuscript Synthesis and characterization of planar chiral cyclopalladated ferrocenylimines: DNA/HSA interactions and in vitro cytotoxic activity Yangyang Zhou, Ting Song, Yuan Cao, Guidong Gong, Yanjin Zhang, Haihang Zhao, Gang Zhao PII:

S0022-328X(18)30402-9

DOI:

10.1016/j.jorganchem.2018.06.027

Reference:

JOM 20486

To appear in:

Journal of Organometallic Chemistry

Received Date: 28 May 2018 Revised Date:

22 June 2018

Accepted Date: 26 June 2018

Please cite this article as: Y. Zhou, T. Song, Y. Cao, G. Gong, Y. Zhang, H. Zhao, G. Zhao, Synthesis and characterization of planar chiral cyclopalladated ferrocenylimines: DNA/HSA interactions and in vitro cytotoxic activity, Journal of Organometallic Chemistry (2018), doi: 10.1016/j.jorganchem.2018.06.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Synthesis and Characterization of Planar Chiral Cyclopalladated Ferrocenylimines: DNA/HSA interactions and in vitro cytotoxic activity a

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A series of planar chiral cyclopalladated ferrocene compounds were synthesized and characterized. The absolute configurations of three compounds were determined by single-crystal X-ray analysis. The binding of the compounds with Native Calf Thymus DNA (CT-DNA) was monitored using UV-visible absorption spectrophotometry, fluorescence spectroscopy and circular dichroism (CD) studies. The results indicate that these compounds can interact with DNA via intercalation mode. In addition, the HSA interactions of these compounds were investigated using UV-visible absorption spectrophotometry and fluorescence spectroscopy. The results of fluorescence spectroscopy show that the fluorescence quenching mechanism of HSA is a static process. The cytotoxic activities of the synthesized compounds and cisplatin exhibited different inhibition potencies on the viability of MCF-7, HCT-116, MDA-MB-231 and Hela cancer cell lines. Compound (Rp, S) - 6 was 17-fold more potent than cisplatin in breast cancer cells.

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Keywords: Cyclopalladated compounds CT-DNA HSA cytotoxic activities

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Yangyang Zhou , Ting Song , Yuan Cao , Guidong Gong , Yanjin Zhang , Haihang Zhao ,Gang a, Zhao *

1. Introduction

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Cancer is a major cause of death worldwide[1]. Cisplatin and its analogues are important medical treatments for cancer[2]. However, the clinical application of these compounds is severely hampered by the side effects and inefficiency of cisplatin-resistant tumors[3]. Therefore, in order to overcome the disadvantages of cisplatin, many new compounds have been synthesized. Considering the similar structure, chemical properties, and hybridization of palladium (II) and platinum (II), there is great interest in the design of palladium (II) derivatives[4]. Among the palladium (II) compounds, particular attention has been given to nitrogen-containing donor ligand compounds such as various alkyl and aryl substituted amines and imines, azo compounds and heterocyclic compounds. Chelating rings usually have three to seven members, of which the five-membered ring is the most stable. These compounds have been successfully applied in organic synthesis, asymmetric synthesis and potential bioactive substances [5-7]. In the development of new metal-based therapies, the interactions between DNA and transition metal compounds need to be studied in detail[8]. Depending on the exact nature of the ligand and metal, compounds may be covalently or non-covalently binding nucleic acids.[9, 10]. There are three main modes of non-covalent

YY Zhou, T Song, Y Cao, GD Gong, YJ Zhang, HH Zhao, Prof. G Zhao College of chemical engineering Sichuan University Chengdu 610065. China E-mail: [email protected]

interactions between compounds and DNA: intercalative binding, electrostatic attraction, and groove binding[9]. Therefore, the study of the interaction between transition metal compounds and DNA is of great significance for the design and application of drugs. The study found that certain metal compounds can bind to DNA through interaction[11]. It has also been reported that some planar structures of cyclopalladated compounds have cytotoxic effects on certain tumor cells that interact to damage DNA [12].On the other hand, studying the effects of metal ions on drug-protein binding is helpful for understanding the transport and mechanism of drugs in the body. Plasma is the most abundant protein in serum protein, whose main function is to carry a variety of physiological ligands to a specific target organ. Many transition metal compounds and drugs are transported to albumin in blood [13]. Therefore, in order to find out the mechanism of drug transport in the body, it is important to study the formation of compounds between proteins and drugs. It is necessary to design and synthesize new palladium compounds to determine their reactivity to DNA and proteins and cytotoxicity. Recently, as a potential drug with excellent anti-tumor activity, cyclopalladated compounds have attracted attention. [14, 15]. However, studies on the interactions between cyclopalladated compounds and DNA/HSA are very rare.

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Scheme1. Synthesis of the compounds ((Rp, R, R, Rp)-4, (Sp, R, R, Sp)-4, (Sp, S, S, Sp)-4, (Rp, S, S, Rp)-4, (Rp, R)-6,(Sp, R)-6,(Sp, S)-6,(Rp, S)-6)

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Many applications of ferrocene derivatives have been reported recently [41-44]. Ferrocene derivatives are an increasingly important class of complexes with possible applications in biology, material sciences. In some antitumor drugs, ferrocene and its derivatives are used as substitutes [16, 17]. Previously, it has been found that cyclopalladated compounds with different planar chiral ferrocene show different anti-tumor activities [19-21]. Murphy and Smith published a review of mitochondrial-targeted antioxidants formed by combining lipophilic triphenylphosphine cations with an antioxidant material[18].The main feature of triphenylphosphonate compounds is that they can easily pass through all biofilms. Herein, we synthesized eight ferrocene cyclopalladated compounds (Scheme 1). A variety of spectroscopic methods are used to study the interaction between compounds and DNA, such as UV-Vis absorption spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy. In addition, The HSA binding ability has also been measured by UV-Vis absorption spectroscopy, fluorescence emission spectroscopy, to monitor and calculate the binding constant(kb), binding site(n). In addition to studying the interaction of compounds with DNA and HSA, The cytotoxic activity of the compounds against Hela (human cervix carcinoma), HCT-116 (colorectal cancer), MCF-7 (breast cancer )and MDA-MB-468 (human breast carcinoma) cell lines was evaluated by MTT assay.

2. Results and discussion

2.1. Synthesis and Characterization of the Compounds The compound 3 were obtained by a condensation reaction between ferrocenecarboxaldehyde and chiral amines (R)-2 or (S)-2 in dry toluene, as orange plates at a good yield. (R)-3 or(S)-3 was mixed with Na2PdCl4 and NaOAc·3H2O in a 1:1:1 molar ratio in dry methanol at room temperature for 24 h, obtaining the corresponding planar chiral cyclopalladated compounds (Rp,R,R,Rp)-4, (Sp, R, R, Sp)-4, (Sp, S, S, Sp)-4 and (Rp, S, S, Rp)-4 in 1 13 moderate yields .They were fully characterized by HNMR, CNMR (Figures S1-S8), ESI-MS, elemental analysis (EA), melting point and optical rotation. The compounds((Rp, R)-6,(Sp, R)-6,(Sp, S)-6 and (Rp, S)-6) were obtained by the compounds ((Rp, R, R,Rp)-4, (Sp, R, R, Sp)-4, (Sp, S, S, Sp)-4 and (Rp, S, S, Rp)-4) with PPh3 in a 1:5 molar ratio at room temperature for 2 h. All compounds were fully characterized by 1 13 31 HNMR, CNMR, PNMR (Figures S9-S20), ESI-MS, elemental analysis (EA), melting point and optical rotation. 2.2. X-ray diffraction analysis. Single crystals of compounds (Rp, R, R, Rp)-4(CCDC 1836282）, (Sp, R)-6(CCDC 1836283),(Rp, S)-6(CCDC 1836284) (Figure 1 and 2, thermal ellipsoids) were obtained by the slow evaporation of the

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Figure.2. An ORTEP drawing of the molecular structure of the compounds ((Sp, R)-6 and (Rp, S)-6);

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The cyclopentadienyl ring is in a plane, almost parallel to each other, and the dihedral angle of (Sp, R)-6 is 3.81°and a dihedral angle of (Rp,S)-6 is 3.5°. The bridged ring composed of cyclopentadienyl and metal rings of Palladium is almost coplanar. (Sp, R)-6 has a dihedral angle of 2.59°and a dihedral angle of (Rp, S)6 is 2.61°. Also, It is not difficult to deduce the absolute configuration of the corresponding ferrocene chloride-bridged palladium dimer (Rp, S, S, Rp)-4 and (Sp, R, R, Sp)-4 according to the crystal structure of the compounds ((Rp, S)-6, (Sp, R)-6).

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solvents from CH2Cl2. Crystal data, selected dihedral angles in compounds are shown in Tables (S1, S2) ,separately. The crystal structure of (Rp, R, R, Rp)-4 shown in Figure 1. The two ferrocene groups are on the same side of the molecule and are all arranged in a cis form. Pd2Cl2 ring has obvious folding, The angle between the two planes defined by Pd (1), Cl (1), Cl (2) and Pd (2), Cl (1), Cl (2) is 29.41°,The angle between the two planes defined by Pd (1), Cl (1), Cl (2) and Pd (1), C (1), N (1) is 2.52°,The angle between the two ring palladium planes N (1) -Pd (1) -C (1) and N (2) -Pd (2) -C (17) is 32.01°. The palladium atoms in the metal ring are in a slightly deformed square coordination environment. Each cyclopentadienyl ring is in a plane, almost parallel to each other, the dihedral angle of 2.6°. The two planes of the bridged ring in which the cyclopentadienyl group of the palladium is substituted are almost coincident with a dihedral angle of 1.3°.Pd-C bond length is 1.949Å, and the distance between Pd (1) ... Pd (2) is 3.338Å, which is much larger than the covalent radius of Pd (II) by 1.31Å. Among the four atoms connected to the same Pd, The Pd-Cl bond will bond at the trans position of Cl is 2.329 Å, which is shorter than the Pd-Cl bond length of the C atom in the ferrocene, originating from the transposition of the oxygen atom. According to the crystal structure of (Rp, R, R, Rp)-4, it is easy to deduce the absolute structure of compound (Rp, R)-6. The crystalline structure of the compounds ((Sp, R)-6 and (Rp, S)-6) shown in figure 2, respectively, which is a pair of enantiomers. The central chirality of the tetrahydrofurfurylamine unit is still consistent with the raw amine of the chiral amine, which is fully consistent with the speculation above. In the (Sp,R)-6, the four atoms adjacent to the two atoms of the bond angle range of 81.0895.8° . In the (Rp, S)-6,

Figure1. An ORTEP drawing of the molecular structure of the compound (Rp,R,R,Rp)-4;

2.3. DNA binding studies

2.3.1. Electronic absorption titration. Electron absorption spectroscopy is an effective way to study the interaction between CT-DNA and metal compounds. The absorption spectra of the eight compounds in the presence of CT-DNA are given in Figure (3, S21, S22). The intense absorption bands around 275 nm reveal the intraligand π-π* transition of the coordinated groups. Typically, compounds and DNA interact via an intercalation mode resulting in hypochromism with or without a small red or blue shift due to strong stacking interactions between compounds and DNA base pairs[22, 23]. Increased amount of CT-DNA leads to a decrease in absorbency (hypochromism) without any shift in the maximum absorption wavelength of the compound ((Rp, R)-6, (Sp, R)-6, (Sp, S)-6 and (Rp, S)-6). The results clearly show that as the DNA is constantly added to the compound ((Rp, R, R, Rp)-4, (Sp, R, R, Sp)-4, (Sp,S,S,Sp)-4 and (Rp, S, S, Rp)-4), it produces a significant red-shift and significant hypochromism. Therefore, the observed hypochromic effect suggests that the cyclopalladated compounds maybe bind to CT-DNA via intercalation mode. To further study the binding ability of the interaction between compounds and DNA, Calculate the intrinsic binding constant Kb from eqn [24] [DNA]/(εa-εf) =[DNA]/(εb-εf) +1/(Kb(εb-εf)

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[DNA] is the concentration of DNA, and ɛa, ɛf and ɛb correspond to Aobs/[compound], the extinction coefficient for the free compound and the extinction coefficient for the compound in the fully bound form, respectively. The intrinsic binding constant Kb is determined by the ratio of slope to the Y intercept in plots of [DNA]/(ɛa-ɛf) versus [DNA] (Figure S21-22). From the values of the binding constant (Kb), the free energy(ΔG) of the compound-DNA compound was calculated using eqn ΔG= -RT ln Kb

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Figure 3, Electronic spectra of the compound in buffer solution (5 mM TrisHCl/50 mM NaCl at pH 7.4) upon addition of CT-DNA. C((Rp,R,R,Rp)-4) = 50uM, C(CT-DNA) = 0-40uM. Arrow shows that the absorption intensities decrease upon increasing DNA concentration. Inset: Plots of [DNA]/[εa -εf] vs. [DNA] for the titration of the compound with DNA .

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It is well known that EB emits strong fluorescence in DNA because it has a strong interaction between DNA base pairs. The ethidium bromide (EB) displacement experiment was conducted to obtain support for the combination of the compound with DNA. According to reports, enhanced fluorescence can be weakened by adding a compound that can bind to DNA by competing with EB. This is a proof the compound intercalate to base pairs of DNA [22, 25]. The degree of quenching of EB-DNA fluorescence was used to determine the degree of binding between the compound and the DNA. In the absence and presence of compounds, the emission spectra of EB bind to DNA shown in figure (4, S23 and S24). Adding these compounds to the DNA used with EB results in a significant decrease in fluorescence intensity. The decrease in fluorescence intensity can be clearly observed. The Compounds replace the binding sites of EB molecules with DNA.

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2.3.2. EB displacement experiment.

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Binding constants are a measure of stability of compound-DNA compounds, whereas free energy represents the spontaneous / non spontaneous combination of compound-DNA. The free energy of -1 (Rp, R, R, Rp)-4 was evaluated as negative value (-27.0 kJ mol ), indicating the spontaneous nature of the compound-DNA interaction. The same is true for (Sp, R, R, Sp)-4, (Rp, R)-6, (Sp, R)-6, (Sp,S,S,Sp)-4, (Rp, S, S, Rp)-4, (Sp, S)-6 and (Rp, S)-6(Table 1).The compounds ((Rp, R, R, Rp)-4, (Rp, R)-6, (Sp, S, S, Sp)-4, (Sp, S)-6) 5 -1 4 -1 exhibit stronger binding interactions (10 M ) than others (10 M ) due to its specific spatial structure.

Table1 Quenching constant (Ksv), (Kq), binding constant (Kbin), free energy (ΔG) for the interactions of compounds with DNA. Ksv/M

(Rp,R,R, Rp)-4

2.6×10

5

3.5×10

13

2.6×10

13

3.5×10

4

4.3×10

4

5.8×10

(Sp, S, S, Sp)-4

4.3×10

(Rp, S, S, Rp)-4

5.8×10

12 12

5

3.4×10

5

2.4×10

(Rp, R)-6

3.4×10

(Sp, R)-6

2.4×10

13 13

4

4.3×10

4

5.9×10

4.3×10

(Rp, S)-6

5.9×10

-ΔG(kJ· M )

5

27.0

4

23.0

5

27.2

4

24.6

5

29.4

0.6×10 1.1×10 0.6×10 2.1×10 1.4×10

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4

23.3

5

26.3

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20.7

1.2×10

12

0.4×10

12

4.3×10

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Kq/M S

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Therefore, all compounds are like to intercalation mode. The compounds are like to intercalation mode as the same result of the UV experiment. Fluorescence quenching was described by the linear Stern-Volmer [26]. I0/I=KSVQ+1

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I0 indicates the measured fluorescence intensity value when no quencher is added, I represents the fluorescence intensity measured after adding the quencher; Q represents the concentration of the quencher; Ksv is the Stern-Volmer quenching constant, The Ksv value is obtained as a slope from the plot of I0/I versus [Q](Sterne-Volmer plot). (Figure 4 and S23-S24) The experimental results data (Table1) Indicates that this compound has a large degree of binding to DNA and has a high quenching efficiency [27]. 2.3.3. Circular dichroism studies. The circular dichroism spectrum of the DNA-type substance provides diagnostic information of DNA morphological changes through the interaction with the transition metal compound and the instability of the DNA helix [28]. The CD spectrum of CT-DNA is positively banded at 275 nm based on base stacking, and the negative band at 245 nm is due to DNA helicity [29]. It is known that the secondary structure of DNA is disturbed by the intercalation of compound. Therefore, it increases the intensity of two bands and stabilizes the right-handed B conformation of DNA. Whereas, the simple groove binding intensities of both the negative and positive bands decrease and show less or no perturbation on the base stacking and helicity bands.

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Figure.4. The emission spectra of the DNA-EB system, in the presence of (Rp, R, R, Rp)-4. [DNA] =50 uM, [Compound] =0-10 uM, [EB] = 10uM. The arrow shows the emission intensity changes upon increasing compound concentration. Sterne-Volmer plots of the EBDNA fluorescence titration data of (Rp,R,R,Rp)-4.

visible absorption spectroscopy is a common method for exploring changes in protein structure and studying the formation of protein compounds. HSA has two major absorption bands in the UV spectrum, one at 278 nm which is the absorption band of aromatic amino acids(Trp, Tyr, and Phe) and the other within the range of 220-240 nm which is the skeleton absorption peak (a-helix structure) [31].It is well known that the absorption of chromophores depends on whether they are transferred to a more hydrophobic or more hydrophilic environment in the direction and magnitude. These shifts are attributed to changes in solvent polarizability caused by changes in the π-π‫ ٭‬transition[27]. As shown in Figure (6 and S31S32) , The results showed that the HSA skeleton absorption intensity decreased in the 220-240 nm range and a red shift occurred during the addition of the compound, which can be attributed to the induced perturbation of the protein helix caused by the specific interaction with the ligand.[32]. In addition, the maximum absorption at 278 nm is enhanced, showing that more aromatic acid residue is extended into the water environment. These results indicate that the interaction between the compound and HSA is mainly a static quenching process [33]. These observations indicate that the specific interaction between HSA and the compound causes a perturbation of the helix and changes the polarity of the microenvironment surrounding the Trp, Tyr and Phe of HSA.

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2.4.2. Fluorescence spectroscopy of HSA

Fluorescence spectrometry is a common and effective method to study the conformation of protein molecules. It is widely used in the study of the interaction between compound and proteins[34]. The endogenous fluorescence of proteins is mainly derived from tryptophan, tyrosine, and phenyl alanine residues[23]. The research shows that when the excitation wavelength is 280 nm, the protein fluorescence comes from the contribution of tryptophan and tyrosine, but mainly the contribution of tryptophan. Quenching can occur either dynamically or statically. Dynamic quenching refers to the process of contact between the fluorophore and the quencher

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Figure 5.CD of CT-DNA (100uM M) in the absence and presence of (Rp,R, R,Rp)-4 (5 uM ) in 5 Mm Tris HCl with 50 Mm NaCl (pH=7.4).

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As shown in Figure (5, S25 and S26), the CD spectra of DNA were monitored in the presence of compounds. The decrease of negative bands and increase of positive bands showed that (Rp, R, R, Rp)-4 inserted into the DNA base pairs, resulting in pairs of vertical separation, thus distorting the phosphate backbone and change the rotation degree of continuous base pairs and DNA into a A- like conformation. The similar phenomenon were also observed for (Rp, S, S, Rp)-4, (Sp, R, R, Sp)-4 (Sp, S, S, Sp)-4, (Rp, S)-6, (Sp, R)-6 and (Sp, S)-6.The positive band for (Rp, R)-6, red-shifted and then gradually negative increases are stabilizing the right handed B conformation of DNA, showing the intercalative binding mode. 2.4. HSA binding study 2.4.1. UV-visible absorption spectra of HSA Since about 55% of serum albumin accounted for total plasma protein plays an important role in drug distribution, metabolism and excretion in recent years, the interaction of serum albumin with chemicals has attracted increasing research interests[30].UV-

Figure 6.UV absorption spectra of C(HSA) = 6 uM in the absence and presence of C((Rp,R,R,Rp)-4) = 0-3 uM in 5 Mm Tris–HCl with 50 Mm NaCl. arrow shows the emission intensity changes upon increasing (Rp,R,R,Rp)-4 concentration.

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Figure.7. Emission spectra of HSA upon the titration of (Rp,R,R,Rp)4.[HSA] = (20 uM ), [compound] = (0–3.2 uM ). Arrow shows the change upon the increasing compound concentration. Inset: Plots of I0/I vs [Q].

log[(I0-I)/I] =logKb+nlog[Q]

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I and I0 represent the fluorescence intensity in the presence and absence of compounds, respectively. Ksv is a linear Sterne-Volmer quenching constant.τ0 is the average lifetime of the fluorophore -8 without quencher, the value of τ0 of the biopolymer is 10 s, Kq is the quenching rate constant of biomolecule, and [Q] is the concentration of quencher. Ksv can be obtained from the slope of a plot of I0/I versus [Q]. The calculated values of KSV and Kq for the interaction of the compounds with HSA are given in Table2 exhibiting the (Sp, R, R, Sp)-4 is the highest protein-quenching ability. The Kq value is higher than the different kinds of quencher 10 -1 -1 used for biopolymer fluorescence(2×10 M S ), indicating the existence of the static quenching mechanism[37]. For static quenching interactions, assuming that similar and independent binding sites exist in macromolecules, the binding constant (Kb) and the number of binding sites (n) can be determined based on the use of the following equa[38].

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activity on HCT-116, MDA-MB-231 and Hela, which were 17 times, 8 times and 6 times that of cisplatin respectively. The activity of the compounds on MCF-7 also shows a strong universal.

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in the transient excited state, while static quenching refers to the formation of fluorophore compounds in the ground state Fluorescence quenching of biological macromolecules can be divided into dynamic quenching, static quenching and energy transfer quenching[15].So it is necessary to seriously distinguish between dynamic quenching and static quenching. According to Figure (7, S27 and S29), With the increase of the concentration, the fluorescence emission intensity of HSA decreased at 343 nm, indicating that the interaction between (Rp, R, R, Rp)-4 and HSA could lead to the change of protein secondary structure, leading to the change of HSA tryptophan environment [35]. Other compounds are the same result To better understand the emission intensity of HSA quenched by these compounds, the linear Sterne-Volmer equation is also employed[36].

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Where n is the average number of binding sites per albumin molecule, and Kb is the binding constant of the compound-protein interaction in the present case. The double logarithmic plot of log[I0-I/I] vs. log [Q] is shown in Figure (8 and S28, S30) according to Table2, The Kb value shows the interaction and binding ability between the compound and HSA. The binding propensity of compounds with HSA follow this order: (Rp, S, S, Rp)-4 >(Rp, R, R, Rp)-4>(Sp, R, R, Sp)-4>(Sp, S, S, Sp)-4>(Sp, R)-6>(Rp, S)-6>(Rp, R)6>(Rp, S)-6. The negative values of ΔG by fluorescence results also support free energy variations that indicate the spontaneous of the compound-HSA binding. 2.5. Cytotoxic activity against human tumor cell lines The inhibitory activity of the compounds on MCF-7, HCT-116, MDA-MB-231 and Hela was studied by MTT assay. As an approved anticancer drug, the cytotoxic activity of cisplatin was compared under the same experimental conditions. The IC50 values are shown in Table3. The results showed that the eight kinds of ferrocene palladium compounds had strong inhibitory activity against four kinds of candidate cancer cells compared with the traditional anticancer drug cisplatin, in which the (Sp, S)-6 had the highest inhibitory

Figure.8 Scatchard plots of log [(I0 -I)/I] vs log [Q] for determination of the compound-HSA binding constant and the number of binding sites on HSA for (Rp,R,R,Rp)-4. Table 2 Quenching constant (Ksv), binding constant (Kbin), free energy (ΔG) and number of binding sites (n) for the interactions of the Compounds with HSA. Compounds (Rp,R, R, Rp)-4

Ksv/M-1 3.2 ×10

5

5

Kq/M-1 S-1

Kbin/M-1

13

3.2 ×10

n

6

39.4

1.2

6

8.1 ×10

(Sp, R, R, Sp)-4

3.7×10

38.4

1.2

(Sp, S, S, Sp)-4

2.9×105

2.9×1013

1.9 ×106

35.8

1.1

(Rp, S, S, Rp)-4

3.3×10

5

13

1.66×10

7

41.1

1.3

(Rp, R)-6

3.5×104

3.5×1012

4.5 ×105

32.2

1.2

(Sp, R)-6

4

12

9.1 ×10

5

34.0

1.2

2.2 ×10

5

30.4

1.2

6.5 ×105

33.2

1.2

4.4 ×10

4

(Sp, S)-6

3.5×10

(Rp, S)-6

3.5×104

3.7×10

13

-ΔG(kJ· M-1)

3.3×10

4.4 ×10 3.5×10

12

3.5×1012

5.4×10

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MCF-7

HCT-116

MDA-MB-231

Hela

2.7±0.5

5.1±0.1

7.5±0.3

9.1±0.1

(Sp, R,R, Sp)-4

2.2±0.1

3.0±0.1

6.4±0.4

6.7±0.1

(Sp, S, S, Sp)-4

1.3±0.1

3.7±0.1

9.2±0.1

4.6±0.2

(Rp, S, S, Rp)-4

3.2±0.1

4.6±0.1

9.2±0.1

8.9±0.1

(Rp, R)-6

0.9±0.1

4.3±0.1

7.3±0.3

6.9±0.1

(Sp, R)-6

0.5±0.1

3.1±0.1

4.0±0.1

4.8±0.3

(Sp, S)-6

0.6±0.1

1.6±0.1

4.0±0.4

3.6±0.1

(Rp, S)-6

0.4±0.1

3.7±0.2

8.0±0.2

6.4±0.4

cisplatin

6.5±0.4

27.6±0.8

33.7±5.2

22.2±0.9

IC50 refers to the concentration of the drug when the growth inhibition rate reaches 50%

2.6 Assessment of ROS After incubation with compounds (15μM) for 6 h, intracellular ROS generation of monocytes was increased (Figure.9). Excessive production of ROS leads to indicators of oxidant exposure, damage of intracellular molecules and organelles, and ultimately cell death [45]. ROS are known to affect mitochondrial membrane potential and trigger a series of mitochondria associated events [46]. ROS may also serve as common mediators of cell death in response to many toxicants and pathological conditions [47]. In the previous report [48], These results suggest that DNA may not be the primary target for the planar chair ferrocene cyclopalladated compounds. That may induce reactive oxygen species (ROS)-mediated apoptosis of cancer cell lines.

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Compounds (Rp,R,R, Rp)-4

presence of bulky PPh3 groups which facilitate transport through the cellular membranes [26]. The planar chirality of the C, Npalladacycle the central chirality of the N-C-substituent was influences of these compounds on cytotoxicity, but the relationship requires further study.

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Table 3 IC50 (μM) values against the selected cancer cell lines for the compounds under studya

2.7. Conclusions

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In this study, some cyclopalladated compounds were synthesized and characterized by NMR, optical rotation, and ESIMS. The single crystal X-ray crystallographic study of the compounds ((Rp, R, R, Rp)-4, (Sp, R)-6, (Rp, S)-6) show the planar geometry around the compound. Using electron absorption, fluorescence spectroscopy, and circular dichroism (CD) measurements, the DNA binding patterns of eight compounds were explored. This indicates that the compound can interact with DNA through the intercalation mode. The reactivity towards HSA revealed that the quenching of HSA fluorescence by the eight compounds are static quenching .Follow the binding affinity order of (Rp, S, S, Rp)-4 >(Rp, R, R, Rp)-4>(Sp, R, R, Sp)-4>(Sp, S, S, Sp)4>(Sp, R)-6>(Rp, S)-6>(Rp, R)-6>(Rp, S)-6. The cytotoxicity studies have shown that these compounds have a high cytotoxic effect on the cytotoxic activity of different cell lines. Our research team is currently further investigating cyclopalladated compounds in vivo and in vitro against cancer.

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Figure.9. Effect of compounds((Rp,R,R,Rp)-4, (Rp,R)-6) on intracellular reactive oxygen species (ROS) generation. **P < 0.01 vs. control, *P < 0.05 vs. control. Data are represented as means + standard error of the means

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For MCF-7, these 8 cyclopalladated compounds exhibit high anticancer activity. The IC50 values of (Sp, S)-6, (Rp, R)-6, (Sp, R)-6, (Rp, S)-6 were all lower than 1uM, the inhibitory activity of (Rp, S)-6 was the highest, which was 18 times that of cisplatin. Its activity is about 9 times that of (Rp, S, S, Rp)-4, 7.5 times of (Rp,R,R, Rp)-4, 6 times of (Sp, R,R, Sp)-4 ,3.5 times of (Sp, S, S, Sp)-4,3 times of (Rp, R)-6. For HCT-116, the cell activity of (Sp, S)-4 is 2 to 3 times that of several other compounds. In the case of MDA-MB-231, the activity of (Sp, S, S, Sp)-4, (Sp, S)-6 is about Cisplatin 8 times the activity of the remaining compounds 1-2 times. For Hela, (Sp, S)-6 had the highest inhibitory activity, which was 6 times that of cisplatin and 1 to 2.5 times that of the remaining compounds. These compounds show strong binding ability in DNA and HSA studies. That may be an important reason for the highly cytotoxic effects of compounds on the cytotoxic activity of different cell lines. On the whole, the IC50 value of dimeric cyclopalladated compounds was less potent than monomeric cyclopalladated compounds. The lipophilicity of monomeric cyclopalladated compounds can be related to the

2.8. Experimental section Measurements Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. The cisplatin was purchased from TCI Tokyo Chemical Industry (Shanghai) Co., Ltd. (Shanghai, China). The calf thymus DNA (CTDNA), HSA and Ethidium bromide (EB) are Provided by Sigma Aldrich Chemical Company. CT-DNA, HSA and Ethidium bromide (EB) stock solutions were prepared by dissolving in Tris buffer (NaCl (50 mM), Tris–HCl (5 mM), pH being adjusted to 7.2 with NaOH (0.5 M)). ROS detection kit were purchased from Beyotime (Jiangsu, China). The CT-DNA solution was spectrophotometrically standardized using its known molar absorption coefficient at 260 -1 -1 nm (6600 M cm ). UV absorbance at 260 and 280 nm, A260/A280, is ca 1.8, indicating that DNA is pure and free of protein[39]. The stock solutions were prepared by dissolving the compounds in an aqueous solution of dimethylsulfoxide (DMSO) as co‐solvent. The

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(R)-3 (594mg, 2mmol) was added to a Methanol (30 ml) solution containing Na2PdCl4 (588mg, 2.0mmol) and NaOAc·3H2O (272mg, 2.0mmol), and stirred at room temperature for 24 h. The result reaction mixture was dried under high vacuum. The crude product was dissolved with dichloromethane and filtered. The filtrate was passed through a SiO2-column with 1:4 ethyl acetate /nhexane. Concentration of the eluted solution of two successive red bands produced compounds The first red fraction was (Rp, R, R,Rp)4 (385mg) in 44% yield. The second red fraction was (Sp, R, R, Sp)-4 (228mg) in 26% yield. (Sp, S, S, Sp)-4 (403mg) and (Rp, S, S, Rp)-4 (262mg)were respectively obtained in 46% and 30% yield starting from (S) -3 as the same way of (Rp, R, R, Rp)-4 and (Sp, R, R, Sp)-4 .

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(Rp, R, R, Rp)-4: H NMR (400 MHz, CDCl3) δ= 7.86 (s, 2H,N=CH), 3 5 4 4.78 (d, J = 13.5 Hz, 2H, H in C5H3), 4.56 – 4.17 (m, 16H, C5H5+H ,H in C5H3+OCH), 3.93 – 3.63 (m, 6H, OCH2+NCH2), 3.19 (s, 2H, NCH2), 2.10 (d, J = 22.0 Hz, 2H, OCHCH2), 1.94 (s, 4H, OCH2CH2), 1.73 – 1.59 13 (m, 2H, OCHCH2) ppm; C NMR (100 MHz, CDCl3) δ= 174.3(C=N), 1 2 5 3 102.8(C in C5H3), 86.6 (C in C5H3), 76.9(C in C5H3), 73.7(C in 4 C5H3)], 70.8(C5H5), 67.8(OCH2)67.7(OCH), 65.8(C in C5H3), 62.0 (NCH2), 28.7(OCHCH2), 25.8(OCH2CH2)ppm; MS (ES+): calcd for + C32H36Cl2Fe2N2O2Pd2 [M/2-Cl] : 401.98, Found: 402.00. Anal. Calcd for C32H36Cl2Fe2N2O2Pd2: C, 43.87; H, 4.14; N, 3.20. Found: C, 43.56; 20 o H, 4.37; N, 3.21; m.p. 184-186 C, [α] D = +389.2 (c 1.0, CHCl3). 1 (Sp, R, R, Sp)-4: H NMR (400 MHz, CDCl3) δ =7.91 (s, 2H,N=CH), 3 5 4 4.76 (s, 2H, H in C5H3), 4.47 – 4.12 (m, 16H, C5H5+H ,H in C5H3+OCH), 3.92 – 3.61 (m, 6H, OCH2+NCH2), 3.42 (s, 2H, NCH2), 2.09 (s, 2H, OCHCH2), 2.00 – 1.85 (m, 4H ,OCH2CH2), 1.78 (d, J = 9.0 13 1 Hz,2H,OCHCH2)ppm; CNMR(100MHz,CDCl3)δ=174.7(C=N),102.7(C i 2 5 3 nC5H3), 86.9(C inC5H3), 77.2(C inC5H3), 73.7(C inC5H3), 70.6 (C5H5), 4 67.9 (OCH2),67.7(OCH)66.0(C in C5H3), 62.0 (NCH2), 28.7(OCHCH2), 25.6(OCH2CH2) ppm; MS (ES+): calcd for C32H36Cl2Fe2N2O2Pd2 [M/2+ Cl] : 401.98, Found: 401.96; Anal. Calcd for C32H36Cl2Fe2N2O2Pd2: C, 43.87; H, 4.14; N, 3.20. Found: C, 43.61; H, 4.42; N, 3.28; m.p.16220 o 164 C. [α] D =-4910 (c 1.0, CHCl3). 1 (Sp, S, S, Sp)-4: H NMR(400 MHz, CDCl3) δ=7.86 (s, 2H,N=CH), 4.78 3 5 4 (d, J = 14.2 Hz, 2H, H in C5H3),4.47-4.25 (m, 16H, C5H5+H ,H in C5H3+OCH), 4.00-3.60 (m, 6H, OCH2+NCH2), 3.19 (d, J = 8.7 Hz, 2H, NCH2), 2.12 (d, J = 25.7 Hz, 2H, OCHCH2), 1.94 (d, J= 4.5 Hz, 4H, 13 OCH2CH2), 1.65 (dd, J = 13.9, 5.9 Hz, 2H, OCHCH2) ppm; C 1 2 NMR(100 MHz, CDCl3) δ=174.3 (N=CH), 102.8(C in C5H3), 86.6 (C in 5 3 C5H3), 77.0 (C in C5H3), 73.6 (C in C5H3), 70.8(C5H5), 4 67.8(OCH2),67.6(OCH)65.8 (C in C5H3), 62.6 (NCH2), 28.7 (OCHCH2), 25.8 (OCH2CH2)ppm; MS (ES+): calcd for C32H36Cl2Fe2N2O2Pd2 [M+ Cl] : 840.93, Found: 841.20; Anal. Calcd for C32H36Cl2Fe2N2O2Pd2: C, 43.87; H, 4.14; N, 3.20. Found: C, 43.69; H, 4.49; N, 3.36; m.p. 18220 o 184 C, [α] D = -384.8 (c 1.0, CHCl3). 1 (Rp, S, S, Rp)-4: H NMR (400 MHz, CDCl3) δ 7.91 (s, 2H,N=CH), 4.76 3 5 4 (s, 2H, H in C5H3), 4.46 – 4.17 (m, 16H, C5H5+H ,H in C5H3+OCH), 3.86 (dd, J = 14.9, 7.1 Hz, 2H, OCH2), 3.76 (dd, J = 14.4, 7.8 Hz, 2H, OCH2), 3.67 (d, J = 11.6 Hz, 2H, NCH2), 3.42 (s, 2H, NCH2), 2.09 (s, 2H, OCHCH2), 2.01 – 1.85 (m, 4H, OCH2CH2), 1.78 (dd, J = 19.6, 8.4 Hz, 13 2H,OCHCH2)ppm; CNMR(100MHz,CDCl3)δ=174.7(C=N),102.5(C1 in 2 5 3 C5H3), 86.9(C inC5H3), 77.2 (C inC5H3), 73.7(C inC5H3), 70.6(C5H5), 4 67.9(OCH2),67.6(OCH)66.0(C inC5H3),62.0(NCH2),28.73(OCHCH2),25. + 6(OCH2CH2)ppm;MS(ES+):calcd for C32H36Cl2Fe2N2O2Pd2 [M-Cl] : 840.93, Found: 841.02.; Anal. Calcd for C32H36Cl2Fe2N2O2Pd2: C,

2.8.1. Synthesis of Ligands (R)-3 and (S)-3.

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solution is diluted to the required concentration and stored at 5°C without using more than 4 days. The final DMSO concentration never exceeded 0.5% (v/v). Melting points were measured on a Meltemp melting point apparatus. Optical rotation was measured with Perkin Elmer model 1 341 polarimeter. H NMR spectra were recorded on Bruker AM400 NMR spectrometer. Chemical shifts were reported in parts per million (ppm) from tetramethylsilane with the solvent resonance as the internal standard (CDCl3, δ = 7.26 ppm). Spectra were reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiple), coupling constants 13 (Hz), integration and assignment. C NMR spectra were collected on commercial instruments (101 MHz) with complete proton decoupling. Chemical shifts are reported in parts per million (ppm) from the tetramethylsilane with the solvent resonance as internal 31 standard (CDCl3, δ = 77.0 ppm). 161.97 MHz ( P NMR) using standard pulse sequences at 298 K Mass spectra were recorded on UPLC-Devo TQMS system equipped with an ESI source. C, H and N elemental determination were performed on a Euro EA 3000 elemental analyser (Leeman, USA).Fluorescence emission spectra were obtained by using a Shimadzu RF-5301PC Spectrofluorophotometer at 298 K. Absorption spectra were recorded on a PerkinElmer Lambda 35 UV-Vis spectrometer using quartz cells with an absorption path length (l) of 10.0 mm. Crystallography data of compounds (Rp,R,R,Rp)-4, (Sp,R)-6 and (Rp, S)-6 were measured on a MSC/Rigaku RAXIS IIC imaging-plate diffractometer. Intensities were collected at 294 K using graphitemonochromatized Mo Kα radiation (λ = 0.71073) from arotatinganode generator operating at 50 kV and 90 mA (2θmin = 3°, 2θmax = 55°, 2-5° oscillation frames in the range of 0-180°, exposure 8 min per frame). The X-ray diffraction data were collected on Xcalibur Eos diffractometer. The structure of the molecule was elucidated using Olex2. Besides, structural refinement were achieved via the ShelXL refinement package through least-squares minimization[40].

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Ferrocenecarboxaldehyde 1 (2.14 g, 10 mmol) and (R)-2 (1.01 g, 10 mmol) were dissolved in dry toluene (100 mL). The flask containing the reaction mixture was connected to a condenser equipped with a Dean-Stark apparatus. The red solution was refluxed over an oil bath for about 6 h and then the carefully transferred into a Schleck tube, into with 5 Ǻ molecular sieve (3.0 g) were introduced. The mixture was further refluxed for 18 h and then washed with n-hexane to produce (R)-3 (446mg) yellow crystals in 75% yield. (S)-3 was obtained starting from (S)-2 as the same way of (R)-3. Since the imine is very easy to decompose, it is not further separated and purified here but it is directly injected into the next reaction. (R)-3: 1H NMR (400 MHz, CDCl3)δ8.13 (s, 1H), 4.64 (d, J = 1.7 Hz, 2H), 4.40 -4.29 (m, 2H), 4.19 (s, 5H), 4.17-4.09 (m, 1H), 3.89 (dt, J = 13.5, 6.8 Hz, 1H), 3.77 (dd, J = 14.6, 7.4 Hz, 1H), 3.56 (d, J = 5.3 Hz, 2H), 2.09-1.96 (m, 1H), 1.96-1.86 (m, 2H),1.76-1.64 (m, 1H). 2.8.2. Synthesis of the Compounds (Rp,R,R,Rp)-4, (Sp, R, R, Sp)-4, (Sp, S, S, Sp)-4 and (Rp, S, S, Rp)-4

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The compounds (Rp, R, R, Rp)-4(175mg, 0.2mmol) was added to a acetone (10 ml) solution containing PPh3 (262mg, 1mmol), stirred at room temperature for 2 h. The result reaction mixture was dried under high vacuum. The product was extracted into dichloromethane and passed through a SiO2-column with 3:1 petroleum n-hexane/ethyl acetate. Concentration of the eluted solution of one successive red band produced, and compound (Rp, R)-6 which was recrystallized from dichloromethane/n-hexane (1:5) as reddish yellow plates in a high yield [130mg (93%)]. (Sp, R)-6 (126mg), (Sp, S)-6 (127mg) and (Rp, S)-6 (122mg) were respectively obtained in 90%, 91% and 87% yield starting from(Sp, R, R, Sp)-4, (Sp, S, S, Sp)-4 and (Rp, S, S, Rp)-4 as the same way of (Rp, R)-6. 1

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(Rp, R)-6: H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.3 Hz, 1H,N=CH), 2 6 3 4 7.76 (dd,J =11.6,6.8Hz 6H;H ,H (C6H5)), 7.53-7.28 (m, 9H;H ,H , 5 5 H (C6H5)), 4.59-4.20 (m, 3H, OCH+NCH2+H in C5H3), 4.03 (s, 4 1H,H (C5H3)), 4.00 (s, 5H,C5H5), 3.88 (dd, J = 16.1Hz,8.0 Hz, 1H,OCH2), 3.77 (dd, J = 16.0Hz,7.6 Hz, 1H, OCH2), 3.32 (d, J = 1.8 Hz, 3 1H, H (C5H3)),3.28-3.08(m,1H,NCH2),2.17-2.09(m,1H,OCHCH2),2.01.83(m,2H,OCH2CH2),1.561.47(m,1H,OCHCH2).13CNMR(100MHz,CD 2 6 1 3 5 Cl3)δ=173.32(N=CH),135.0(C ,C inC6H5),132.1(C inC6H5),131.6(C ,C i 4 1 2 5 nC6H5),128.1(C inC6H5),102.9(C inC5H3),87.3(C inC5H3),77.8(C inC5H 3 4 3),76.5(C inC5H3),70.3(C5H5), 69.1(C inC5H3), 67.6(OCH2), 66.4(OCH), 61.8(NCH2), 28.7(OCHCH2), 25.6(OCH2CH2)ppm; MS (ES+): calcd for + C34H33ClFeNOPPd [M-Cl] : 664.07, Found: 664.01. Anal. Calcd for C33H34ClFeNOPd: C, 58.31; H, 4.75; N, 2.0. Found: C, 58.15; H, 4.79; 20 o N, 2.11 m.p. 176-178 C. [α] D =+1939 (c 1.0, CHCl3) 1 (Sp, R)-6: H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.2 Hz, 1H,N=CH), 2 6 3 4 7.78(dd, J=11.4,7.1Hz, 6H, H , H (C6H5)), 7.46-7.34(m, 9H, H , H , 5 5 H (C6H5)), 4.38-4.29 (m, 3H, OCH+NCH2+H in C5H3), 4.02 (s, 4 1H,H (C5H3)), 3.90-3.85 (m, 6H, C5H5 + OCH2), 3.79 (dd, J = 14.4,7.6 3 Hz, 1H, OCH2), 3.53-3.43(m, 1H,NCH2), 3.31 (s, 1H,H (C5H3)), 2.202.08 (d, J = 7.2 Hz, 1H,OCHCH2), 1.98-1.81 (d, J = 7.3 Hz, 13 2H,OCH2CH2), 1.71 – 1.55 (m, 1H,OCHCH2). C NMR (100 MHz, 2 6 1 CDCl3) δ= 173.65(N=CH), 134.9(C ,C in C6H5), 132.0 (C in 3 5 4 1 C6H5),130.4(C ,C in C6H5), 128.0(C in C6H5), 103.0(C in C5H3), 2 5 3 4 87.7(C in C5H3), 77.8(C in C5H3), 76.5(C in C5H3), 70.0(C5H5), 69.1(C in C5H3), 67.7(OCH2), 66.6(OCH),, 61.1(NCH2) 28.8(OCHCH2), 31 25.7(OCH2CH2)ppm; P NMR (162 MHz, CDCl3) δ 37.42. MS (ES+): + calcd for C34H33ClFeNOPPd [M-Cl] : 664.07, Found: 664.00. Anal. Calcd for C33H34ClFeNOPd: C, 58.31; H, 4.75; N, 2.0. Found: C, 58.34; 20 o H, 4.83; N, 1.97. m.p. 178-180 C.[α] D =-2185 (c 1.0, CHCl3). 1 (Sp, S)-6: H NMR (400 MHz, CDCl3) δ=8.14 (d, J = 8.3 Hz, 1H， 2 6 N=CH), 7.76 (dd, J = 10.8, 7.4 Hz 6H, H , H in C6H5), 7.49 – 7.32 (m, 3 4 5 5 9H, H , H , H in C6H5), 4.41 – 4.33 (m, 3H, OCH+NCH2+H in C5H3), 4 3.98 (s, 1H, H in C5H3), 3.93 (s, 5H, C5H5), 3.79 (dd, J = 15.1, 6.9 Hz, 3 1H, OCH2), 3.71 (dd, J = 15.0, 7.1 Hz, 1H, OCH2), 3.27 (s, 1H, H in C5H3), 3.19 – 3.07 (m, 1H, NCH2), 2.11-1.99 (m, 1H, OCHCH2), 1.87 – 13 1.77 (m, 2H, OCH2CH2), 1.50 -1.38 (m, 1H, OCHCH2) ppm. C NMR 2 6 (100 MHz, CDCl3) δ=173.2 (N=CH), 134.9 (C and C in C6H5), 1 3 5 4 131.98(C in C6H5), 130.4 (C and C in C6H5), 128.0 (C in C6H5),

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2.8.3. Synthesis of the Monomeric Cyclopalladated Compounds (Rp, R)-6,(Sp, R)-6,(Sp, S)-6 and (Rp, S)-6.

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103.0 (C in C5H3), 87.5 (C in C5H3), 77.8 (C in C5H3), 76.6 (C in 4 C5H3) , 70.4 (C5H5), 69.1 (C in C5H3), 67.6 (OCH2), 66.4 (OCH), 61.8 31 (NCH2), 28.6 (OCHCH2), 25.6 (OCH2CH2) ppm. P NMR (162 MHz, + CDCl3) δ=37.42. MS (ES+): calcd for C34H33ClFeNOPPd [M-Cl] : 664.07, Found: 664.19. Anal. Calcd for C33H34ClFeNOPd: C, 58.31; H, 20 o 4.75; N, 2.0. Found: C, 58.41; H, 4.64; N, 2.03. m.p. 188-190 C. [α] D =-1940 (c 1.0, CHCl3). 1 (Rp, S)-6: H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.2 Hz, 1H,N=CH), 2 6 3 4 5 7.78 (dd, 6H,H ,H (C6H5)), 7.41 (m, J = 1.6 Hz, 9H, H , H , H in 5 C6H5), 4.38-4.29 (m, J = 31.0, 6.8 Hz,3H, OCH+NCH2+H in C5H3), 4.02 4 (s, 1H, H (C5H3)), 3.91-3.85(m,6H,OCH2+C5H5), 3.79 (dd, J = 14.4,7.7Hz,1H, OCH2), 3.51-3.45 (m, 1H,NCH2), 3.31 (d, J = 2.1 Hz, 3 1H,H (C5H3)), 2.18-2.10(m, 1H, OCHCH2), 1.94-1.86 (m, 2H, 13 OCH2CH2), 1.69-1.62(m, 1H, OCHCH2). C NMR (100 MHz, CDCl3) 2 6 1 3 δ=173.6(N=CH), 134.9(C and C in C6H5), 132.0(C in C6H5), 130.4(C 5 4 1 2 and C in C6H5), 128.0(C in C6H5), 102.8 (C in C5H3), 87.6(C in C5H3), 5 3 4 77.8 (C in C5H3), 76.5(C in C5H3), 70.0(C5H5), 69.1 (C in C5H3), 67.7(OCH2),66.6(OCH),61.1(NCH2),28.8(OCHCH2)25.68(OCH2CH2)pp 31 m; P NMR (162 MHz, CDCl3) δ 37.42. MS (ES+): calcd for + C34H33ClFeNOPPd [M-Cl] : 664.07, Found: 664.01. Anal. Calcd for C33H34ClFeNOPd: C, 58.31; H, 4.75; N, 2.0. Found: C, 58.26; H, 4.84; 20 o N,2.01. m.p. 184-186 C.[α] D =+2188 (c 1.0, CHCl3)

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43.87; H, 4.14; N, 3.20. Found: C, 43.88; H, 4.15; N, 3.27; m.p. 15220 o 154 C. [α] D =+4913 (c 1.0, CHCl3).

2.8.4. DNA binding studies

A stock solution of CT-DNA was prepared in Tris-HCl buffer (5 mM Tris-HCl, 50 mM NaCl at pH 7.2). The DNA concentration is determined by the absorption spectrum (UV-Vis) using ε260 = 6600 -1 -1 M cm [39]. The DNA-binding studies were performed at room temperature by electronic absorption spectrometric experiments and luminescence measurement. A concentrated solution of metal compounds was prepared in DMSO solvent. The Tris-HCl buffer at pH 7.2 was then used to dilute the solution appropriately, containing 0.5%DMSO and 99.5% Tris-HCl buffer (5mM, pH7.2) in all the experiments. The DNA-EB solution ([DNA]/[EB] = 5) was excited at 530 nm in the absence and presence of different concentrations of compounds for EB displacement. In the absence and presence of compounds, the CD spectrum of DNA was recorded in the 320-350 nm range at 25℃, using a 1 nm bandwidth, a slit width of 0.02 mm, an average time of 0.5 s, and a 1 nm step interval. The CD spectrum of Tris-HCl buffer was subtracted from the sample spectra for data analysis. 2.8.5. Protein binding studies

The appropriate amount of HSA Tris-HCl buffer (5 mM Tris-HCl, 50 mM NaCl at pH 7.2) was dissolved in a buffer solution and stored at 4℃ further uses. A concentrated solution of metal compounds was prepared in DMSO solvent. The Tris-HCl buffer at pH 7.2 was then used to dilute the solution appropriately, containing 0.5%DMSO and 99.5% Tris-HCl buffer (5mM, pH7.2). The UV-Vis spectra of the HSA solution titrated at 280nm with various concentrations were recorded, and the changes of HSA absorption were recorded after each addition of HSA absorption. In fluorescence quenching experiments, fluorescence emission spectra of a fixed concentration of HSA solution were recorded in the wavelength range of 300-500 nm in the absence and presence of compounds (quencher). ca.345 nm (λex =280nm).

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2.8.7. Determination of ROS

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Intracellular reactive oxygen species (ROS) was detected with Reactive Oxygen Species Assay Kit according to the manufacturer’s instructions. Briefly, after treating with compounds for 24 hours, MDA-MB-231 cells ware washed twice and loaded with 10 mM 2’,7’-dichlorofluorescein (DCFH) diacetate for 20 minutes at 37℃. DCFH diacetate was deacetylated into non-fluorescent DCFH intracellularly by esterase, and DCFH was further oxidized by ROS into the fluorescent DCFH, which was determined at 488nm excitation and 525nm emission with a fluorescence microplate reader (Varioskan Flash, USA). The DCFH fluorescence intensity was representative of the intracellular ROS levels.

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Candidate cancer cells (MCF-7, HCT-116, MDA-MB-231, and HeLa) were formulated into a cell suspension with a medium containing 10% fetal bovine serum, and 200 μL per well (approximately 10000 cells) was seeded into a 96-well plate, After incubation for 24 h, a DMSO solution of the cyclopalladated compounds at a concentration of 1.506, 3.125, 6.25, 12.5, 25, 50 μM was added. The cells were incubated for 72 h at 37°C in an incubator containing 5% CO2, 95% air and 100% relative humidity. Add 20μL of MTT solution (5mg/mL in PBS buffer) to each well and incubate for 4 h to terminate the culture and carefully aspirate the culture supernatant. Add 150μL DMSO to each well and shake for 20 min to allow the crystals to be fully melted. Select the wavelength of 570nm, in the microplate reader on the determination of light absorption (OD) value. Cisplatin as a positive control, each group set up three wells. IC50 values were calculated using GraphPad Prism5.

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This work was supported by Sichuan University High Level Talent Project, Sichuan Province 1,000 Talents Plan Project.

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A series of planar chiral cyclopalladated ferrocene compounds were

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synthesized

Studied the interaction of the cyclopalladated compounds on

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Cytotoxic activity of the cyclopalladated compounds has a strong

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inhibitory effect on the viability of different cancer cell lines