Introduction

Metal-based drugs have attracted increasing attention in the biomedical field [1] as they exhibit potential applications in treating and diagnosing diseases [2,3,4]. As potential drugs, transition metal complexes have diverse biological properties such as antifungal, antibacterial, analgesic, sedative, antipyretic and anti-inflammatory agents [5,6,7]. Selecting metal junctions and ligands is crucial to designing complexes with excellent biological activity. The bioactivity of isomorphous complexes is different due to their different metal ion centers [8]. The complex at the center of the same metal ion will also cause different biological activities due to different ligands [9]. Mn-based complexes show favorable antibacterial and anticancer activities [10, 11]. Cd-based complexes also show favorable anticancer activity [12, 13].

Pyrazine carboxylic acid derivatives possess abundant O- and N-donor groups that allow metal ions to adopt multiple coordination modes, which provide the possibility of variety biological activities [14,15,16]. Vijayan synthesized a series of pyrazine carboxylic acid complexes, and the in vitro cytotoxicity of the complexes on HeLa cells indicates that complexes act as effective anticancer drugs. In addition, these complexes exhibited efficient activity against Gram-positive bacteria (Staphylococcus pneumonia, Bacillus subtilis) [17]. Gao synthesized Co(II) and Cd(II) complexes based on pyrazine carboxylic acid with good DNA binding activity. In addition, the cytotoxicity of these two complexes to human cervical cancer cells (HeLa) in vitro suggests that the complexes are effective anticancer agents [18]. Etaiw synthesized a copper pyrazine carboxylate complex, which strongly inhibited the human lung fibroblast cell line (WI-38) [19]. This indicates that the pyrazine carboxylic acid complexes have good biological activity. Thus, 5-hydroxypyrazine-2-carboxylic acid (H2pydc) was used as a ligand mainly due to its good bioactivity and strong coordination capabilities to combine with metal ions.

Analyzing how complexes interact with deoxyribonucleic acid (DNA) and bovine serum albumin (BSA) is important for the development of therapeutic drugs based on transition metal complexes [20]. Deoxyribonucleic acid (DNA), as a carrier of genetic information, dominates the process of cell growth, development and reproduction and is therefore the main target of many anticancer drugs [21]. In addition, serum albumin (SA) plays a key role in transporting metal ions and drugs to various cells and tissues in the blood. Bovine serum albumin (BSA) is a structural homolog of human serum albumin and is widely used as a model protein in research [22]. The mechanism of drug action involves the interaction of small molecule drugs with large biomolecules such as DNA/BSA. Therefore, studying the interaction between bioactive compounds and DNA/BSA is valuable for understanding the medicinal potential and mechanism of action of bioactive complexes [23].

In this work, two novel complexes, [Cd(pydc)(H2O)] and [Mn(pydc)(H2O)], were synthesized. Their structures were characterized by various means such as single-crystal X-ray diffraction, elemental analysis, thermogravimetric analysis and infrared spectroscopy. The interactions between the metal complexes and CT-DNA/BSA were thoroughly investigated using ultraviolet spectra and fluorescence spectroscopy. The quenching spectra were studied using site markers to investigate the binding sites of the complexes on the BSA structure. In addition, thermodynamic studies were performed to elucidate the type of interaction forces between the complex and DNA/BSA. The above studies showed good biological activity of the two complexes.

Experimental

Materials and methods

The chemicals and solvents were obtained from Shanghai Macklin Biochemical Co., Ltd. Infrared spectra were recorded using a BEQVZNDX 550 spectrometer in the 400–4000 cm−1 range using KBr disks. UV–Vis measurements were performed using TU-1800 beam recording spectrophotometer. Thermogravimetric analysis (TG) was performed on a Labsys Evo using hydrostatic air as the atmosphere. The fluorescence data were obtained using a Hitachi F-7000 fluorescence spectrophotometer.

Preparation of complexes 1–2

Two complexes were synthesized by similar procedures. A mixture containing CdCl2·5/2H2O (0.0500 mmol, 0.0114 g) or MnCl2·4H2O (0.0500 mmol, 0.0099 g), H2pydc (0.0500 mmol, 0.0070 g) and 5 mL of distilled water was sealed in a 15 mL Teflon-lined stainless steel vessel. The pH was adjusted to 9 by adding potassium hydroxide solution (0.5000 mol L−1) and then heated at 140 °C for 3 days. After heating, the samples were cooled to 30 °C at a rate of 5 °C h−1. The crystals that formed were collected, rinsed several times with distilled water and dried at room temperature.

[Cd(pydc)(H2O)]n: faint yellow crystals; yield: 65% (based on H2pydc); Anal. Calcd (%) for C5H4CdN2O4: C, 22.35; H, 1.49; N, 10.43. Found (%): C, 22.82; H, 1.66; N, 10.39. IR (KBr, cm−1): 3424 m, 3057 m, 1631 s, 1577 m, 1370 s, 1224 m, 1156w, 894 m, 810w, 667 m, 633 s,549w, 428w. 1H NMR (400 MHz, DMSO-d6): δ 8.05 (s, 1H), 7.98 (s, 1H) (Fig S1).

[Mn(pydc)(H2O)]n: yellow crystals; yield: 61% (based on H2pydc); Anal. Calcd (%) for C5H4MnN2O4: C, 28.43; H, 1.90; N, 13.27. Found (%): C, 28.38; H, 2.05; N, 14.35. IR (KBr, cm−1): 3427 m, 3050 m, 1631 s, 1581 s, 1363 s, 1211 m, 1152w, 892 m, 802 m, 754w, 636 s,535w, 422w. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (s, 1H), 8.08 (s, 1H).

X-ray crystallography

The crystallographic data of complexes 1 and 2 were acquired on a Bruker SMART APEX II CCD with MoKα (λ = 0.71073 Ǻ) radiation at room temperature. Structures were solved by direct methods using SHELXS-2018 and anisotropic refinement by full-matrix least squares on F2 using SHELXL-2018. All non-hydrogen atoms were refined by anisotropy. Table 1 summarizes the crystallographic parameters for complexes 1 and 2 and structural improvements. Partial bond length and angle data for complexes 1 and 2 can be found in supplemental Tables S1 and S2.

Table 1 Crystallographic and structure refinement data for complexes
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CT-DNA and BSA binding assay

The interaction experiments of the complexes with CT-DNA and BSA were carried out by UV–Vis and fluorescence spectroscopy. Binding experiments of the complexes to CT-DNA and BSA were performed in Tris–HCl buffer (10 mmol L−1 Tris–HCl, 10 mmol L−1 NaCl, pH = 7.4) and stored at 4 °C and used for no more than one week.

UV–Vis spectra

UV–Vis spectra experiments were performed at 25 °C with CT-DNA or BSA. The concentration of the complexes was kept at 20 μmol L−1 (constant), and graded concentration of CT-DNA was taken ranging from 0 to 50 μmol L−1. In the BSA UV–Vis study, the concentration of BSA was kept at 10 μmol·L−1 (constant) and graded the concentrations of complex from 0 to 50 μmol L−1.

Fluorescence experiments

Fluorescence spectroscopy was used to investigate the interaction of the complex with CT-DNA and BSA at three temperatures (CT-DNA and BSA: 25, 35 and 45 °C), and the following experiments were performed:

  1. (i)

    Ethidium bromide (EB) was used for the CT-DNA interaction study. High fluorescence is produced when EB is inserted into CT-DNA and the fluorescence intensity decreases when another complex competes with EB [24]. The CT-DNA and EB concentration was kept at of 10 μmol·L−1 (constant), and graded concentrations of the complex ranged from 0 to 50 μmol L−1. An excitation wavelength of 520 nm was used, and a scanning range of 520–700 nm was used to record the fluorescence data.

  2. (ii)

    BSA is intrinsically fluorescent. The concentration of BSA was kept 10 μmol L−1 (constant) and graded concentrations of complex ranged from 0 to 50 μmol L−1. The excitation wavelength of 280 nm was used as the excitation wavelength, and the scanning range of 200–540 nm was used to record the fluorescence curve data.

The fluorescence data were corrected according to the relationship (Eq. 1) [25, 26]:

$$F_{{{\text{cor}}}} = F_{{{\text{obs}}}} \times 10^{{(A_{{{\text{ex}}}} + A_{{{\text{em}}}} )/2}}$$
(1)

where Fcor is the fluorescence intensities corrected, Fobs the measured fluorescence value, and Aex and Aem are the absorption value of the systems at the excitation and emission wavelengths, respectively.

When the internal filter effect is not a problem or can be removed using the above methods, the observed fluorescence quenching can be explained by the Stern–Volmer Eq. (2) [27]:

$$F_{0} /F = {1} + K_{{\text{q}}} \tau_{0} \left[ {\text{Q}} \right] = {1} + K_{{{\text{sv}}}}$$
(2)

where F and F0 are the fluorescence intensities of CT-DNA + EB/BSA with and without quencher. Kq, τ0 [28, 29], Ksv and [Q] denote the bimolecular quenching constant, the BSA/EB fluorescence lifetime, the Stern–Volmer quenching constant and the quencher concentration, respectively. In addition, the number of binding sites (n) on DNA/BSA and binding constant (Ka) were calculated using Eq. (3) [30]:

$$\log (F_{0} - F)/F = \log K_{a} + n\log \left[ Q \right]$$
(3)

The thermodynamic parameters including free energy (ΔG), enthalpy change (ΔH) and entropy change (ΔS) were evaluated utilizing the van’t Hoff Eq. (4) [27] and Gibbs–Helmholtz Eq. (5) [31]:

$$\ln (K_{2} /K_{1} ) = - \Delta H/R\left( {1/T_{2} - 1/T_{1} } \right)$$
(4)
$$\Delta G = - RT\ln K = \Delta H - T\Delta S$$
(5)

where T is the temperature and R is the gas constant.

Competitive binding studies

BSA interactions were investigated using existing BSA site markers, site I (warfarin), and site II (ibuprofen). The BSA and site markers concentration was kept 10 μmol L−1 (constant), and graded concentrations of the complex ranged from 0 to 50 μmol L−1. Fluorescence spectra were recorded in a similar manner to that described above, and the Ka of the complexes 1 or 2-BSA site marker system were calculated using the modified Stern–Volmer Eq. (6) [32].

$$F_{0} /\Delta F = F_{0} /\left( {F_{0} - F} \right) = 1/\left\{ {\left( {f_{a} - K_{a} } \right)\left[ Q \right]} \right\} + 1/f_{a}$$
(6)

where F0 is the fluorescence intensity of the system in the absence of the quencher and ΔF is the difference between the fluorescence intensity of the system in the absence and presence of the quencher. fa is the fraction that contacts the fluorescent group, and Ka is the effective quenching constant that contacts the fluorescent group.

Results and discussion

Crystal structures of complexes 1 and 2

Complexes 1 and 2 are isomorphous and belong to the monoclinic P21/n space group (Table 1). Hence, the structure of complex 1 is described herein which has been reported previously as a catalyst of Knoevenagel condensation [33]. The structure of complex 2 is exhibited in Figs. S2–S4. Each Cd(II) ion is six‐coordinated by N1, N1, O1, O3 and O4 from four pyc2− together with one H2O molecules (O2), exhibiting a slightly deformed [CdN2O4] octahedron geometry (Fig. 1). Thus, in complex 1 the bond length parameters displayed the values as the Cd–O bond lengths range from 2.2171(6) to 2.3350(18) Å, while the distance of Cd–N is 2.349(2) Å and 2.303(2) Å. The O-Cd–O bond angles vary from 86.14(6)° to 170.41(6)° and the O-Cd–N bond angles vary from 71.79(6)° to 164.81(7)° and the N–Cd–N bond angle is 84.25(7)°. Furthermore, all the bond angle and length values in complexes 1 and 2 were within the range for reported octahedral manganese and cadmium complexes [34, 35].

Fig. 1
figure 1

Molecular structure of the complex 1

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Each ligand in complex 1 is completely deprotonated, and all N and O atoms are involved in coordination. As shown in Fig. 2, the adjacent Cd(II) ions form a 1D zigzag chain with two nitrogen atoms of pyrazine through a bis-monodentate coordination mode. What’s more, the oxygen atoms from hydroxyl groups and nitrogen from pyrazine rings connect the adjacent Cd(II) ions forming another 1D zigzag chain at the vertical direction. The two chains are further connected to 2D layered pore structure by Cd1-N1 and Cd1-O3. The H2pyc ligand and Cd(II) ions in the 2D layer-hole formed two adjacent 8- and 22-membered loops (Fig. 2). The 2D structure is further developed by interlayer metal–ligand interactions (Cd1···O2) and expands the structure to a 3D structure (Fig. 3).

Fig. 2
figure 2

2D structure of complex 1

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Fig. 3
figure 3

3D structure of complex 1

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The measured powder X-ray diffraction (PXRD) patterns of 1–2 are consistent with their simulated patterns from single-crystal X-ray diffraction (Fig. S5), which indicate the good phase purity of the complexes [36].

X-ray photoelectron spectroscopy (XPS)

In order to determine whether the oxidation state of Mn ions in complex 2 is + 2 or not, the X-ray photoelectron spectroscopy (XPS) has been measured. As shown in Fig. S6 in Supporting Information, the binding energy of Mn(2p3/2) contains a peak at 641.4 eV, and Mn(2p1/2) contains a peak at 656.6 eV. These values are similar to the reported Mn2+ binding energy in the literature, which suggest that the oxidation state of Mn ions in complex 2 is + 2 [37].

Thermal stability analyses

Thermogravimetric experiments were carried out to investigate the thermal stability of complexes 1 and 2, which are important parameters for bioactive materials. As depicted in Fig. 4, the thermal behavior of complexes 1 and 2 is similar. They release coordinated water molecules within the temperature range of 74.5–124.2 °C for complex 1 (found: 6.5%, calcd: 6.7%) and 99.7–147.8 °C for complex 2 (found: 8.3%, calcd: 8.5%). A continuous stabilization phase within the temperature range of 124.2–403.6 °C for complex 1 and 147.8–405.3 °C for complex 2 indicates that the structural framework remains intact in this process. Subsequently, they release the organic ligand within the temperature range of 403.6–574.3 °C for complex 1 and 405.3–602.7 °C for complex 2. The residual weights of complexes 1 and 2 after their complete conversion to CdO and MnO2 were 47.6% and 42.3%, respectively, which coincided with the calculated 47.8% and 41.7%.

Fig. 4
figure 4

Thermogravimetric diagram of complexes 1 and 2

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Infrared spectra

The possible coordination sites of the complexes concerning the metal center have been worked out by infrared spectroscopy [38]. As depicted in Fig. S7, the infrared patterns of complex 1 and complex 2 are basically consistent, which indicate that they are isomorphic. The absorption peaks observed at 3170 and 3424 cm−1 may correspond to the coordination water of the complex 1. The absorption peaks observed at 3188 and 3427 cm−1 may correspond to the coordination water of the complex 2 [31, 39]. For complexes 1 and 2, the absorption peaks of the C–N bonds of pyrazine are visible at 1577 cm−1 and 1581 cm−1, respectively. The bands defining the νasymmetric (–COO−) have been shifted to 1631 cm−1 for complexes 1 and 2, while compared to the band position at 1613 cm−1 in the free ligands (H2pydc) [40]. The above results show that the hydroxyl oxygen atom, the carboxyl oxygen atom and the N atom on the pyrazine ring all participate in the coordination. The infrared spectra are consistent with the single-crystal diffraction results.

Solubilities

The complexes 1 and 2 are insoluble in water, methanol, ethanol but soluble in dimethyl sulfoxide and tetrahydrofuran.

Solution/aqueous phase stability

UV–Vis spectra were used to investigate the solution phase stability of 1 and 2 in water and Tris–HCl buffer over a period of 48 h (Fig. S8). Demonstrating their suitability for biological studies, the absorbance curves for complexes 1 and 2 remained virtually unchanged over 48 h.

In vitro CT-DNA binding studies

UV–Vis spectra are a commonly used method to assess the mode of interaction of complexes with CT-DNA. Hypochromic and redshifts were observed in the UV spectra of the complexes interacting with CT-DNA (Fig. 5). This suggests that there is a chimeric interaction between complexes 1 and 2 and CT-DNA, which may lead to the coupling of the π orbitals in the base pairs of CT-DNA to the π* orbitals in the complexes, thus reducing the π–π* jump energy and producing bathochromism. On the other hand, the coupled orbitals are partially filled with electrons, which reduces the probability of a jump and leads to hypochromic shifts [8, 29, 41].

Fig. 5
figure 5

UV spectra of complex 1 (a) and complex 2 (b) under different CT-DNA concentrations. [complexes] = 20 μ mol L−1; From 1 to 6, the concentrations of CT-DNA were 0, 10, 20, 30, 40 and 50 μmol L−1

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To further investigate the mode of interaction of H2pydc and complexes 1 and 2 with CT-DNA, fluorescence spectra were used to evaluate the displacement process (Fig. 6).

Fig. 6
figure 6

Fluorescence quenching of CT-DNA + EB system recorded the absence and presence of in the absence and presence of complex 1 (a) and complex 2 (b). [EB] = [CT-DNA] = 10 μ mol L−1, From 1 to 6, the concentrations of the complexes were 0, 10, 20, 30, 40 and 50 μmol L−1

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EB is a cationic dye, and its fluorescence intensity significantly increases when it is precisely inserted into adjacent base pairs of CT-DNA [8]. The fluorescence intensities of the CT-DNA + EB + complex were decreased at 630 nm with increasing concentration of complexes (Fig. 6). This suggests that complexes 1 and 2 likely interact with CT-DNA via intercalation, which displaces EB from CT-DNA, thus reducing the fluorescence intensity of the system [42].

The quenching constant (KSV) was determined using the Stern–Volmer Eq. (1), and KSV and Kq were calculated and are listed in Table 2. KSV decreased with increasing temperature, and Kq was greater than the maximum dynamic quenching constant (2.0 × 1010 M−1 s−1). This indicates that the fluorescence quenching mechanism of the CT-DNA + EB complex system is static quenching [20, 43].

Table 2 Ksv and Kq of the interaction of complex 1-2 with CT-DNA + EB system at different temperatures
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As shown in Table 3, binding constant (Ka) and number of binding sites on CT-DNA (n) for complexes 1 and 2 are calculated with Eq. (2) at temperatures of 25, 35 and 45 °C (Figs. S9 and S10).

Table 3 Binding constant Ka and binding site n of complex 1–2 in CT-DNA + EB complex system at
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The number of binding sites (n) is approximately 1, indicating that there is only one independent class of metal complex binding sites on DNA. The Ka values for 1 and 2 were 1.925 × 103 and 9.853 × 103, respectively, at 25 °C. This is an indication of good binding of complexes 1 and 2 to CT-DNA [20, 44].

Thermodynamic parameters can predict the type of forces acting between the complex and the CT-DNA + EB system. Thermodynamic parameters ΔS, ΔH and ΔG were calculated by Eqs. (3) and (4). As shown in Table 4, ΔG < 0 indicates that the binding of complexes 1 and 2 to DNA is exothermic; furthermore, ΔH < 0 and ΔS < 0 indicate that the forces of CT-DNA to complexes 1 and 2 are hydrogen bonding and van der Waals forces [20].

Table 4 Thermodynamic parameters of the interaction of and complex 1–2 with CT-DNA + EB at different temperatures
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BSA binding studies

The intrinsic fluorescent properties of BSA are derived from tyrosine, tryptophan and phenylalanine residues. Still, its characteristic fluorescent behavior is mainly attributed to tryptophan residues, which emit maximum fluorescence at 345 nm [8]. As the concentration of the complexes increased, BSA showed a fluorescence quenching at 345 nm, suggesting that complexes 1 and 2 were able to bind effectively to BSA (Fig. 7). Additionally, it was noted that the fluorescent intensity of BSA reduced as the emission of wavelength increased with redshift, suggesting that the compound can quench BSA’s fluorescence and change tryptophan’s environment [29].

Fig. 7
figure 7

Fluorescence quenching of BSA by complex 1 (a) and complex 2 (b) at 25 °C [BSA] = 10 μmol L−1; From 1 to 6, the concentrations of the complexes were 0, 10, 20, 30, 40 and 50 μmol L−1

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To further elucidate the mechanism of fluorescent quencher, fluorescent quencher experiments were performed at three different temperatures (25, 35 and 45 °C) and the quantitative results were evaluated using the Stern–Volmer formula (Eq. (1)) [27]. The quencher curves of the complexes showed a good linear relationship between BSA and the quencher, consistent with a single quencher mechanism (Figs. S11 and S12).

Furthermore, the calculated quench rate constant (Kq) for complexes 1 and 2 was found to be of in the order of 1012 M−1 s−1, well above the maximum possible value for the dynamic quench constant (2.0 × 1010 M−1 s−1). This suggests that the mechanism of fluorescence quenching between the complex and BSA is static quenching [8, 45] (Figs. S11 and S12 and Table 5).

Table 5 Ksv and Kq of H2pydc and complexes 1–2 interacting with BSA at different temperatures
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The binding constants (Ka) and the number of binding sites (n) were also calculated using the Scatchard formula (2) (Figs. S13 and S14). The calculated Ka and n are given in Table 6. The number of binding sites (n) is approximately 1. This means that complexes 1 and 2 bind at a single position on the BSA molecule [8]. After the addition of the complex to the BSA solution, the absorbance at 210 nm of the UV spectrum was gradually enhanced and accompanied by a certain redshift with the increase in the concentration of the complex. This phenomenon further verified the static quenching in the mechanism of interaction between the complex and BSA, which was in agreement with the conclusion obtained from fluorescence spectroscopy (Figs. S15 and S16) [31].

Table 6 Ka and n of H2pydc and complexes 1–2 interacting with BSA at different temperatures
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Analysis of fluorescence lifetime is used for identification of the local environment of a fluorophore, and differentiation and confirmation of static and dynamic fluorescence quenching mechanisms. Lifetime studies of the intrinsic fluorophore BSA (10 μmol L−1) were performed at room temperature before and after the addition of complexes 1 and 2 separately (in 1:1 and 1:5 molar ratios with BSA). The average free BSA lifespan was found to be 5.91 ns. When each of the complexes was introduced to BSA (in 1:1 and 1:5 molar ratios), the results demonstrated a minimal change in BSA’s lifetime. These observations rule out the idea of dynamic quenching and confirm the findings of static quenching as the mechanism [46, 47] (Fig. 7).

Table 7 shows the thermodynamic parameters ΔH, ΔS and ΔG which are calculated using the van’t Hoff and Gibbs–Helmholtz equations (Eqs. (3) and (4)). ΔH < 0 and ΔG < 0 indicate that the binding of complexes 1 and 2 to BSA is exothermic and spontaneous. Furthermore, ΔH < 0 and ΔS < 0 can speculate that hydrogen bonding and van der Waals forces play key roles in the binding process of BSA to the complexes [48].

Table 7 Thermodynamic parameters related to the interaction of complexes 1–2 with BSA at different temperatures
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Warfarin (site I) and ibuprofen (site II) were chosen as site-labeling reagents for competitive substitution experiments to determine the specific binding sites of the complexes in BSA [49, 50]. The fluorescence intensity of the system gradually decreases following the addition of the site-labeling reagent to the BSA solution (Fig. 8 for complex 1, Fig. S18 for complex 2). Analysis of the binding constants in the presence of the site marker (Table 8) indicated that there was significant competition between the complex and the site marker. Comparison of the experimental results and Ksv values showed that complexes 1 and 2 compete with ibuprofen for site II.

Fig. 8
figure 8

Fluorescence spectra of the complex system of BSA and complex 1 in the presence of site markers (a: ibuprofen; b: warfarin) [BSA] = 10 μmol L−1; From 1 to 6, the concentrations of the complexes were 0, 10, 20, 30, 40 and 50 μmol·L−1

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Table 8 Binding constants of the composite system in the absence and presence of site markers at 25 °C
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Two complexes exhibit similar properties due to their isomorphism. The binding constant of DNA with the complexes was approximately 103 L mol−1 at 25 °C, whereas the binding constant of DNA with the ligand H2pydc was only 9.154 × 102 L mol−1, indicating that complexes 1 and 2 exhibit higher binding affinity with DNA than H2pydc. This is consistent with the binding force between the two complexes with BSA. It may be attributed to the chelating structure of metal ions and ligands in the complex enhancing the bonding activity between the complex and CT-DNA/BSA [51, 52]. In addition, the metal ions share a partial positive charge with the donor atoms of the ligand, leading to a decrease in the polarity of the metal ions and electron delocalization, which improves the binding affinity between the complexes and DNA/BSA [53].

Conclusion

The main focus of this study was the synthesis and characterization of complex 1 (C5H4CdN2O4) and complex 2 (C5H4MnN2O4), as well as the study of their binding to CT-DNA/BSA in vitro. The molecular structures of complexes 1 and 2 were determined by spectroscopic and analytical methods and corroborated with single-crystal X-ray diffraction results. Two complexes exhibit similar properties due to their isomorphism. A comparative study of the binding of complex 1 and complex 2 to CT-DNA in vitro showed an intercalated binding mode. Among them, hydrogen bonding and van der Waals interactions play a major role in the binding reaction. Studies have demonstrated that the complexes interact with DNA through an intercalation mechanism. The static quench mechanism was tested by performing temperature-dependent fluorescence quench experiments at three different temperatures. In addition, warfarin (site I) and ibuprofen (site II) were selected as site-labeling reagents for competitive substitution experiments and it was determined that complexes 1 and 2 preferentially bind to site II of BSA. Based on the above study, it helps to understand the mechanism of metal complexes interacting with DNA/BSA and provides reference value for the application model of drug design.