Synthesis, structural characterization, DNA/HSA binding, molecular docking and anticancer studies of some D-Luciferin complexes

2.1 Materials

D-luciferin (LN) was supplied from NANJING YANST BOI-TECH CO.LTD. Metal chloride salts (CuCl₂·2H₂O, MnCl₂·4H₂O, NiCl₂·6H₂O, ZnCl₂, CoCl₂), calf thymus DNA (CT-DNA) type 1A36 and human serum albumin (HSA) were purchased from Sigma-Aldrich (Hamburg, Germany). Doubly distilled water (pH 7.10) and DMSO were used as solvents. All chemicals and solvents were of analytical grade and used without further purification.

2.2 Instrumentation

The elements’ content of C%, H%, S% and N% was determined using CE-440 elemental analyzer. IR spectra of ligand and its synthesized metal complexes were recorded on Bruker Alpha in the range of 250–4000 cm⁻¹. The melting point was measured using Stuart SMP10 automated melting point system. ¹H NMR spectrum was examined by a Bruker ultrashield 600 MHz spectrometer using DMSO‑d₆ as a solvent and TMS as an internal reference. Magnetic measurements were performed at room temperature using a Sherwood Scientific Magnetic Susceptibility Balance (MSB). EPR spectrum of Cu⁺² complex in solid was recorded using the continuous wave Bruker EMX PLUS spectrometer (Bruker Biospin, Rheinstetten, Germany) and collected with Bruker Xenon Software. Conductivity measurements were measured by the OHAUS-STARTER 3100C conductivity meter at 25 ℃ using 1 × 10^-3 M solutions in DMSO. Thermogravimetric analysis was performed using PerkinElmer TGA apparatus under a nitrogen atmosphere at a heating rate of 10 ℃/min for the temperature range 50–800 ℃. The UV–Vis absorption spectra of ligand and its complexes were recorded in DMSO using (1 × 10^-4M) solution in the range of 200–800 nm on MutiSpec-1501 spectrophotometer fitted with a quartz cell 1.0 cm path length. The emission of compounds was measured using a HITACHI F-7000 fluorescence spectrophotometer.

2.3 Molar ratio method

The stoichiometry of the complexes was studied using the mole ratio method (Hussien and Salama, 2016; Basaleh et al., 2022a). The metal concentration was kept constant at (0.72 × 10^-4M), while the ligand concentration was varied (0–2.52 × 10^-4 M), as presented in Table S1. The absorbance of sample solutions was measured in DMSO in the range of 200–800 nm at room temperature. The relation between the maximum absorbance against [M]/[L] + [M] was plotted. The inflection of the obtained line shows the molar ratio of the complex.

2.4 Synthesis of D-luciferin metal complexes

All complexes with the general formula [M(LN)₂Cl₂]H₂O, which M = Mn⁺², Co⁺², Ni⁺², Cu⁺² and Zn⁺², were synthesized employing a 1:2 metal to ligand ratio(M:L). After conforming the ligand salt to the acidic form, pH of the mixture was adjusted to 8.00 by adding drops of NaOH (0.5 M)(deprotonated carboxylate). By adding the metal chloride salts (1 mmol) dissolved in distilled H₂O to the ligand solution, the pH of the mixture was decreased, and the ligand returned to origin form. The resulted precipitate after refluxing for two hours was filtered and left in the air to evaporate. The metal complex was washed several times with ethanol, followed by diethyl ether, and allowed to dry overnight.

[Mn(LN)₂Cl₂]H₂O: Yield: 80.30 %, M.wt: 704.47 g/mol, color: dark brown, M.p: >300 ℃, elemental analysis of (C₂₂H₁₈MnCl₂N₄O₇S₄) found%(calc%): C: 38.40 (38.49), H: 2.34 (2.35), N: 7.96 (8.16) and S: 18.55(18.68), IR(cm⁻¹): 1488 (C = C), 1577 (C = N), 815 (C-S), 1555 (COO^–)as, 1380 (COO^–)s, 1244 (C-O) and 355 (M−S), Λ_m(10^-3 M DMSO): 31.25 Ω⁻¹ cm² mole^-1, µeff: 5.81B.M.

[Co(LN)₂Cl₂]H₂O: Yield: 52.17 %, M.wt: 708.47 g/mol, color: dark brown, M.p: >300 ℃, elemental analysis of (C₂₂H₁₈CoCl₂N₄O₇S₄) found%(calc%): C: 38.14 (38.27), H: 2.38 (2.34), N: 8.04 (8.11) and S: 18.49 (18.57), IR(cm⁻¹): 1481 (C = C), 1582 (C = N), 809 (C-S), 1551 (COO^–)as, 1380 (COO^–)s, 1243 (C-O) and 368 (M−S), Λ_m(10^-3 M DMSO): 15.62 Ω⁻¹ cm² mole^-1, µeff: 4.43B.M.

[Ni(LN)₂Cl₂]H₂O: Yield: 68.57 %, M.wt: 708.23 g/mol, color: dark brown, M.p: >300 ℃, elemental analysis of (C₂₂H₁₈NiCl₂N₄O₇S₄) found%(calc%): C: 38.18 (38.28), H: 2.25 (2.34), N: 8.20 (8.12) and S: 18.40 (18.58), IR(cm⁻¹): 1483 (C = C), 1582 (C = N), 816 (C-S), 1558 (COO^–)as, 1380 (COO^–)s, 1237 (C-O) and 348 (M−S), Λ_m(10^-3M DMSO): 18.75 Ω^-1cm²mole^-1, µeff: 3.32B.M.

[Cu(LN)₂Cl₂]H₂O: Yield: 69.57 %, M.wt: 713.08 g/mol, color: dark brown, M.p: >300 ℃, elemental analysis of (C₂₂H₁₈CuCl₂N₄O₇S₄) found %(calc%): C: 38.10 (38.02), H: 2.40 (2.32), N: 8.20 (8.06) and S: 18.35 (18.45), IR(cm⁻¹): 1488 (C = C), 1582 (C = N), 816 (C-S), 1555 (COO^–)_as, 1382 (COO^–)_s, 1240 (C-O) and 363 (M−S), Λ_m(10^-3M DMSO): 3.12 Ω^-1cm²mole^-1, µeff: 1.73B.M.

[Zn(LN)₂Cl₂]H₂O: Yield: 51.39 %, M.wt: 714.95 g/mol, color: reddish brown, M.p: >300 ℃, elemental analysis of (C₂₂H₁₈ZnCl₂N₄O₇S₄) found%(calc%): C: 37.83 (37.92), H: 2.25 (2.31), N: 8.10 (8.04) and S: 18.31 (18.40), IR(cm⁻¹): 1487 (C = C), 1583 (C = N), 811 (C-S), 1555 (COO^–)as, 1382 (COO^–)s, 1243 (C-O) and 367 (M−S), Λ_m(10^-3M DMSO): 28.12 Ω^-1cm²mole^-1.

2.5 Theoretical calculations

The input files were built, and Gaussian 09 software was used to calculate the atomic and molecular properties of the free ligand and its metal complexes.

The free ligand and its synthesized metal complexes were optimized by GAUSSIAN 09 (Frisch et al., 2009) software package using hybrid DFT (B3LYP) (Becke, 1993) ) in the gas phase. The LanL2DZ (Hay and Wadt, 1985) and 6-311G(d,p) (McGrath and Radom, 1991) were used as mixed basis sets where the 6-311g(d,p) basis set was implemented for all atoms except for the metal atom and LanL2DZ basis set for the metal center . The vibrational frequencies were calculated to ensure that the optimized geometry corresponds to the minimum of the potential energy surface (absence of any imaginary frequency) and were scaled by a factor of 0.966 (Standards and Technology, 2018) . Gauss View (Dennington et al., 2016) software was used to visualize the output files and determine the HOMO-LUMO energies. The following equations were used to compute the reactivity descriptors: energy gap (ΔE), absolute electronegativities (χ), chemical potential (Pi), global hardness (ɳ), chemical softness (S), and electrophilicity (ω) (Alomari et al., 2022; Frisch et al., 1988; Hay and Wadt, 1985; Dennington et al., 2016, Sharfalddin et al., 2022): $$\mathrm{\Delta }\text{E}={\text{E}}_{\text{LUMO}}-{\text{E}}_{\text{HOMO}}$$ $$\chi =-({\text{E}}_{\text{LUMO}}+{\text{E}}_{\text{HOMO}}/2)$$ $$\text{Pi}=-\chi$$ $$\eta =\left({\text{E}}_{\text{LUMO}}-{\text{E}}_{\text{HOMO}}/2\right)$$ $$\text{S}=1/2\eta$$ $$\omega =Pi2/2\eta$$

2.6 Biological applications

3.1 . Molar ratio method

The molar ratio method was employed to examine the complexes’ stoichiometry (Tirmizi et al., 2012). The absorbance values of each addition of metal ion to LN ligand were plotted against the ratio [M]/([L] + [M]) and presented in (Fig. 1) while the rest of complexes are shown in (Fig. S1). According to the results, a ratio of 1:2 metal to ligand was observed in all complexes at an infliction point of 0.33.

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Fig. 1

The molar ratio spectrum of CuLN.

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3.2 . Molar conductance and magnetic susceptibility

The molar conductivity of the ligand and its metal complexes was investigated in DMSO at (1 × 10^-3M), and it was in the range (3–31) Ω^-1cm²mol⁻¹. The low conductance values of compounds confirm their non-electrolytic nature (Ali et al., 2013). The magnetic measurements of complexes were done at room temperature. The magnetic moment of [Mn(LN)₂Cl₂]H₂O is 5.81B.M, which undoubtedly suggests high spin octahedral geometry with five unpaired electrons. The [Co(LN)₂Cl₂]H₂O complex has a magnetic moment of 4.43B.M, corresponds to three unpaired electrons which indicates that this complex has high spin octahedral geometry. The magnetic moment value of 3.32B.M for [Ni(LN)₂Cl₂]H₂O, due to the presence of two unpaired electrons, possesses an octahedral geometry. Similarly, the octahedral geometry of [Cu(LN)₂Cl₂]H₂O complex was suggested by its magnetic moment 1.73B.M, which is equal to the spin only value anticipated for one unpaired electron(Chandra and Kumar, 2005; Alamri et al., 2021).

3.3 Fourier transform infrared spectroscopy (FTIR)

The IR spectra of the complexes were compared with the free ligand to identify the coordination sites that may be involved in complexation. A peak’s location or intensity is expected to change after complexation. The IR spectra of the ligand and its synthesized complexes were recorded in the range of 250–4000 cm⁻¹ and presented in (Fig. 2) and (Table 2). All complexes showed almost no change in the (C = N) vibration band at 1580 cm⁻¹ in the ligand’s spectrum, indicating that the nitrogen atom was not involved in coordination (Althagafi et al., 2019b). The distinctive band of carbonyl (C = O) was replaced by antisymmetric (COO^–)_as and symmetric (COO^–)_s vibrational bands, which were observed at 1558 and 1380 cm⁻¹, respectively in the spectrum of ligand, remained almost unchanged for all complexes, confirming the non-involvement of carboxylate ion in coordination (Ferraro, 1961). The thiazole sulfur appears around 748 cm⁻¹ in the spectrum of ligand which has been shifted to higher wavenumbers in the range between 809 and 816 cm⁻¹ in the complexes. The shift is due to the coordination of sulfur atoms of thiazole rings to metal ions(Sathyanarayanmoorthi et al., 2013). The presence of a broad band in the region of 3200–3600 cm⁻¹, proved the existence of lattice water molecule in all complexes. The formation of complexes was further confirmed by the appearance of new bands in the regions of 348–368 cm⁻¹ in the spectra of complexes which associated with the stretching frequency of (M−S) (Chandra and Kumar, 2005; Tyagi et al., 2017).

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Fig. 2

A comparison of the IR spectra of ligand and its synthesized metal complexes.

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3.4 Nuclear magnetic resonance spectroscopy (¹H NMR)

The ¹H NMR spectral data of the ligand and its diamagnetic Zn(II) complex were performed in DMSO‑d₆ with respect to TMS as an internal reference to confirm the binding mode of ZnLN complex. In the spectrum of LN, the multiplet signals between 8 and 6.5 ppm were due to aromatic protons in the benzene ring (Alanazi et al., 2021). The signals at 3.5 and 4.4 are assigned to CH₁ and CH₂, respectively. The observed signal of the phenolic proton appears upfield than the predicted value which supposed to be at 9 ppm. There is inconsistence with the experimental value that appears at 3.5 ppm. This variation between the calculated and observed peaks implies interactions between LN and the solvent molecules. An intermolecular hydrogen-bonding is formed between hydrogen in OH and oxygen in DMSO‑d₆ that is responsible for the upfield chemical shift (Odai et al., 2009). Similar chemical shift patterns can be found in the spectrum of ZnLN as in LN’s spectrum. The chemical shifts of aromatic and thiazolyl proton-based signals are almost unaffected. However, the signal of CH₂ is slightly shift downfield to 4.97 due to the complexation through sulfur atoms. The broadness of the signals in the complex can be attributed to the presence of the positive charge on metal ion (Alanazi et al., 2021). The strong peak in the spectra of ligand and its Zn(II) complex at 2.5 is caused by solvent residues of DMSO (Gottlieb et al., 1997). A comparison between the ¹H NMR spectra of LN and ZnLN complex is illustrated in (Fig. 3) and (Table 3).

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Fig. 3

The ¹H NMR spectral changes of ZnLN complex (a) and the free ligand (b).

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3.5 Electron paramagnetic resonance spectroscopy (EPR)

The EPR spectrum of the solid [Cu(LN)₂Cl₂]H₂O complex was recorded at room temperature in (Fig. 4). The complex possesses an axial symmetry with the following order gz > gx = gy in which g|| is equivalent to (gz) and g⊥ is equivalent to (gx = gy). In the axial symmetry, the g||(2.0080) > g⊥(2.0047) > g_e(2.0023), this arrangement is common for an elongated octahedron which indicates that the unpaired electron is localized in the dx²-y² orbital of Cu(II) as the ground state(Alamri et al., 2021). The g average (g_ave) value was computed using the following equation g_ave= (g||+2g⊥)/3 and it equals to 2.0058 which is lower than 2.3, implying that the Cu(II) metal ion is surrounded by a covalent environment (Ibrahim et al., 2015; Sharfalddin et al., 2021b). Moreover, the equation G = (g||-g_e)/(g⊥-g_e) has been used to calculate the value of the exchange interaction between the copper (II) centers in the polycrystalline solid. If G > 4, it implies a minimal exchange interaction, while G < 4 it denotes a significant exchange interaction. The calculated G was found to be 2.375 which revealed a considerable exchange interaction between Cu(II) centers (Ibrahim et al., 2015).

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Fig. 4

EPR spectrum of CuLN complex.

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3.6 Thermal gravimetric analysis (TGA)

Thermogravimetric analysis of the synthesized metal complexes was performed under nitrogen atmosphere within a temperature range of 50–800 ℃ and presented in (Fig. 5) and (Table 4). The analysis was carried out to verify the molecular structure of the synthesized metal complexes and examine their thermal stability. As shown in (Fig. 5), the TG curves of the metal complexes are almost identical to each other and undergo the same thermal degradation processes. All complexes decomposed in two steps except CuLN which decomposed in three steps. A residue of metal oxides and carbon atoms left at a high temperature after the final step. A good correlation was found between the results of thermal analysis, IR and the suggested formula from the elemental analysis of metal complexes.

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Fig. 5

TGA curves of synthesized metal complexes.

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The thermogravimetric curve of [Mn(LN)₂Cl₂]H₂O complex exhibits two decomposition steps. MnLN started to degrade thermally at a temperature between 52 and 75 ℃ due to the dehydration of one water molecule with a weight loss of 2.5% (calc. 2.56%). When the loss of a water molecule occurs at this temperature range, the water molecule is of lattice type (Aziz et al., 2020). This is followed by decomposition of organic moiety including 5C₂H₂ + 4H₂S + 4NO + CO + Cl₂ in the temperature range 75–780 ℃ representing a weight loss of 70.5% (calc. 70.6%). The final products contain MnO and ten carbon atoms as residues with a mass loss of 27% (calc. 27.11%). The thermogravimetric curve of [Co(LN)₂Cl₂]H₂O demonstrated that the decomposition occurred in two step. The dehydration of one water molecule in the first stage at 57–75 ℃ accounted for a mass loss of 2.5% (calc. 2.56%). The second step accompanied with a mass loss of 70% (calc. 70.2%) in the range between 75 and 800 ℃ which attributed to the decomposition of 5C₂H₂ + 4H₂S + 4NO + CO + Cl₂. The final products involved CoO and ten carbon atoms as residues with a weight loss of 27.50% (27.52%). The thermogravimetric curve of [Ni(LN)₂Cl₂]H₂O displayed two degradation steps. The first step occurred in the range of 53–125 ℃, which corresponds to the dehydration of one water molecule with a mass loss of 2.5% (calc. 2.54%). The second step took place between 125 and 785 ℃ representing a weight loss of 77.5% (calc. 77.78%), which attributed to the decomposition of the organic moiety 7C₂H₂ + 4H₂S + 4NO + CO + Cl₂. The residues that remained after the final step include six carbon atoms and NiO as metal oxide accounted for mass loss of 20% (calc. 20.7%). The thermogravimetric curve of [Cu(LN)₂Cl₂]H₂O complex has three degradation stages. The initial step corresponded to the dehydration of one water molecule between 50 and 80 ℃ with a mass loss of 2.5% (calc. 2.52%). This was followed by a decomposition of C₂H₂ + H₂S + NO in the second step that occurred within the temperature range of 80–310 ℃ accompanied for a mass loss of 12% (calc. 12.94%). The final step accounted for the loss of 3C₂H₂ + 3H₂S + 3NO + Cl₂ with a mass loss of 55.5% (calc. 56.33%) at 310–750 ℃. The residue consists of 12 carbon atoms and CuO as metal oxide, the weight loss was 30% (calc. 31.35%). The thermogravimetric curve of [Zn(LN)₂Cl₂]H₂O complex undergo two degradation steps starting with the dehydration of one water molecule in the range of 50–100 ℃ with a mass loss of 2.5% (calc. 2.52%). The second weight loss of 73.5% (calc. 73.5%) occurred between 100 and 790 ℃ accounted for the decomposition of the organic moiety 6C₂H₂ + 4H₂S + 4NO + CO + Cl₂, leaving ZnO and eight carbon atoms as final products with a mass loss of 24% (calc. 24.8%).

3.7 Kinetic studies of thermal analysis

The Coats-Redfern integral (CR) (Coats and Redfern, 1963) and the approximation of the Horowitz-Metzger equation (HM) (Horowitz and Metzger, 1963) were applied to calculate the thermodynamic parameters, including the activation energy of decomposition Ea (kJ mol⁻¹), enthalpy ΔH (kJ mol⁻¹), entropy ΔS (J mol^-1K⁻¹), Gibbs free energy ΔG (kJ mol⁻¹) and Arrhenius factor A (S^-1), as represented in (Table S2). According to the results, the high Ea values of all complexes implied that they are extremely thermally stable. The complexes were endothermic and endergonic, as shown by the positive values of ΔH (ΔH > 0) and ΔG (ΔG > 0), respectively. For all complexes, ΔS was negative, suggesting a slow rate of spontaneous decomposition. The correlation coefficients obtained from CR and HM plots for the thermal decomposition, which were determined to be within the range of (0.90–0.98), showed a reasonable fitting with the linear function (Alamri et al., 2021). The plots of Coats-Redfern and Horowitz-Metzger are shown in (Table 5) and (Table S3).

Table 5 Coats-Redfern (CR) and Horwitz-Metzger (HM) plots of NiLN complex.

Complex	Step	Coats-Redfern (CR)	Horowitz-Metzger (HM)
NiLN	1st
	2nd

3.8 Theoretical investigation

3.8.1

3.8.1 Geometry optimization

The optimized geometry for the two D-luciferin isomers namely, cis and trans, were studied using B3LYP/6-311G** for the free ligand. The optimized structures of the ligand are presented in Fig. 6. The trans isomer is more stable than the cis isomer by 6.51 kcal/mol. The small energy difference between the two isomers revealed the possibility of cis–trans isomerization specially during the complex formation. The transition state for the isomerization is slightly higher than the cis isomer by a 2.1 kcal/mol.

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Fig. 6

Optimized structures of trans and cis isomers of D-luciferin.

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The average Natural Bond Orbital (NBO) charges on heteroatoms of the trans isomer are −0.64, −0.45, 0.32 for oxygen, nitrogen and sulfur respectively. On the other hand, the most negative carbon atom on the ligand is located on the CH₂ group next to the sulfur atom with NBO charge of −0.50. Even though the negativity charge of oxygen and nitrogen atoms were higher than of sulfur, the collected data from IR, NMR, EPR and TGA of the metal complexes suggested that LN binds to the metal ions as a bidentate ligand via two sulfur atoms (Sudhaharan and Reddy, 2000).

Optimized geometries and numbering systems of ligand and its metal complexes are shown in (Fig. 7) while the other metal complexes structures are shown (Fig.S3). The selected optimized geometrical parameters, such as bond lengths and bond angles are illustrated in (Table 6).

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Fig. 7

The optimized geometry with numbering system for the free ligand and Mn(II) complex.

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Table 6 Selected geometric bond lengths and bond angles of the optimized ligand and its metal complexes.

Compound	LN	MnLN	CoLN	NiLN	CuLN	ZnLN
Bond length (Å)
C12-S11	1.83	1.89	1.90	1.90	1.89	1.89
C10-S11	1.81	1.91	1.89	1.90	1.88	1.89
C4-S7	1.74	1.80	1.81	1.81	1.81	1.80
C8-S7	1.79	1.86	1.88	1.88	1.88	1.86
C10-C8	1.47	1.46	1.47	1.47	1.46	1.46
M−S7	–	2.43	2.86	2.79	2.79	2.39
M−S11	–	2.57	2.77	2.64	2.56	2.59
M−S17	–	2.52	2.88	2.79	2.53	2.99
M−S24	–	2.52	2.76	2.65	2.29	2.53
Bond angle (°)
C10-S11-C12	88	87.87	86.41	86.63	88.53	88.47
C8-S7-C4	88.27	86.68	86.12	86.32	88.23	86.13
S7-C8-C10	120.28	119.88	116.14	116.71	120.90	119.45
S11-C10-C8	118.73	118.64	116.40	115.24	119.33	118.08
S7-M−S11	–	64.53	84.11	87.79	70.84	76.69
S17-M−S24	–	63.47	85.76	87.93	43.30	75.85
S7-M−S17	–	114.80	94.66	92.42	151.15	96.61
S24-M−S11	–	116.20	94.32	92.83	117.43	119.42
Cl-M−Cl	–	179.30	179.25	179.32	176.04	170.29

The alteration in the free ligand after complexing with the metal ion is a practical sign for the coordination reaction. The bond length in the free ligand has maintained after complexing with metal ions except for the length of C10-S11which showed elongation with 1–4 Å. The M−S bond's lengths were between 2.0 and 2.50 Å which presented a covalent bond character (Sharfalddin et al., 2021b). Among the selected angles around the coordination sites, S11-C10-C8 bond angle showed negligible change in the metal complexes except MnLN compound which was shorter by 5°. The new angles around the respective central metal ion suggested a distorted octahedral geometry for the synthesized complexes.

3.8.2

3.8.2 Global reactivity descriptors

The chemical reactivity of compounds was estimated by calculating quantum chemical parameters with the density functional theory (DFT) approach. These parameters describe the stability, reactivity and suggest the most bioactive compound. Therefore, the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) were extracted from the chk files and presented in (Fig. 8 and Fig.S4) with the difference between their energies (ΔE). For the ligand, the HOMO and LUMO orbitals are distributed around the whole molecule with energy gap of 4.20 eV. The interaction of the metal ions with the ligand has lowered the energy gap and showed the ability of these compounds to electron transition, good reactivity and stability (Alomari et al., 2022).

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Fig. 8

HOMO and LUMO charge density maps and the energy gap of the ligand and Mn(II) complex.

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The synthesized complexes were organized based on their reactivities as following LN > CoLN > CuLN > MnLN > NiLN > ZnLN. Small ΔE values indicate low stability and high chemical reactivity of the compound. Therefore, ZnLN has the smallest energy gap which means it is the most bioactive compound and has the highest reactivity compared with other synthesized complexes. The HOMO orbitals are located in one LN molecule in most of the metal complexes while the LUMO orbitals are centered around the metal center in CuLN, CoLN and NiLN complexes. In the case of MnLN and ZnLN, these orbitals were distributed over one of the ligand molecules. This observation allowed the compound to react as electrophile or nucleophile in the biological environment (Alshehri et al., 2022).

The softness of the molecules is an expression of easily reacting of the compound with other molecules while hardness describes the property of being rigid and resistant to react with other molecules. The quantum chemical descriptors were calculated and listed in (Table 7). Among the metal complexes, ZnLN and CuLN complexes have the highest values of absolute hardness (ɳ) and absolute softness (σ), respectively. The positive value of electrophilicity indicator (χ) and the negative value of the electronic chemical potential (Pi) revealed the capability of the new compounds to act as double sword to interact with cations and anions. The maximum charge that an electrophile may accept from the environment is presented as ΔNmax. The high value of ΔNmax for ZnLN complex showed its high ability to accept electrons from the surrounding biological media and attract shared electrons when forming a chemical bond(Basaleh et al., 2022b).

Table 7 HOMO and LUMO energy (eV) and quantum parameters calculated based on energy gaps for the free ligand and its metal complexes.

Sample code	HUMO	LUMO	Δ E	χ	ɳ	σ	Pi	S	ω	ΔNmax
LN	−6.28	−2.08	4.20	4.18	2.10	0.48	−4.18	1.05	4.16	1.99
MnLN	−5.17	−5.13	0.04	5.15	0.02	50.00	−5.15	0.01	2.58	257.50
CoLN	−4.99	−4.48	0.51	4.74	0.26	3.92	−4.74	0.13	2.37	18.57
NiLN	−4.34	−4.31	0.03	4.33	0.02	66.67	−4.33	0.01	2.16	288.33
CuLN	−3.70	−3.63	0.07	3.67	0.04	28.57	−3.67	0.02	1.83	104.71
ZnLN	−4.89	−4.87	0.02	4.88	0.01	100.00	−4.88	0.00	2.44	488.00

4.1 Ct-DNA binding studies

In these studies, calf thymus DNA (CT-DNA) is used due to its affordability, ease of availability, and structural resemblance to human DNA (Wani et al., 2020). Four different techniques were employed to study CT-DNA interaction with the ligand and its metal complexes: ultraviolet–visible spectroscopy (UV–Vis), fluorescence emission spectroscopy, viscosity measurements and molecular docking.

4.1.1

4.1.1 UV–Vis absorption spectroscopy

DNA exhibits a distinctive band at 260 nm corresponds to π-π* intraligand transitions of the chromophoric groups in purine (adenine and guanine) and pyrimidine (cytosine and thymine) moieties (Sirajuddin et al., 2013; Veeralakshmi et al., 2017). The absorbance spectra of ligand and complexes were recorded in the absence and presence of increasing amounts of CT-DNA. As shown in (Fig. 9), all the compounds showed an increase in the absorbance with the continuous rise of CT-DNA concentration. A significant hyperchromic effect with no shift in the maximum absorbance implies the presence of groove binding between LN and its metal complexes with CT-DNA (Arjmand et al., 2013). The binding constants (K_b) were calculated from Wolfe-Shimmer equation and presented in (Table 8). The values of binding constants of the ligand and its metal complexes are lower compared to the famous intercalator ethidium bromide (EB) that equals to 1 × 10⁷ M⁻¹ (Kosiha et al., 2018). According to the results, the complexes’ strong binding affinity toward CT-DNA is shown by the high K_b constants; LN has the highest K_b value compared to the metal complexes.

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Fig. 9

The absorption spectra of LN and its metal complexes in the absence and presence of increasing concentration of CT-DNA.

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Table 8 Binding constants of the ligand and its metal complexes with CT-DNA.

Sample code	Kb (M−1)
LN	2.00 × 106
MnLN	1.00 × 105
CoLN	1.00 × 106
NiLN	2.00 × 105
CuLN	1.00 × 105
ZnLN	1.00 × 106

4.1.2

4.1.2 Fluorescence emission spectroscopy

Emission experiments were performed to further study the interaction between the complexes and CT-DNA. LN and its synthesized metal complexes exhibited a high blue emission in DMSO, as shown in (Fig. S2)Fig. S4. With an excitation wavelength of 360 nm, the emission spectra of the compounds were recorded in the range 400–700 nm after incubation for 5 min at room temperature. The investigated compounds showed a broad band with a maximum at 530 nm due to the strong charge transfer from benzothiazole ring to thiazoline ring (Hiyama et al., 2012; Vieira et al., 2012). As shown in (Fig. 10), the intensity of the fluorescence emission enhanced after adding amounts of CT-DNA to a fixed amount of complexes, showing a strong interaction between complexes and DNA (Arjmand et al., 2013). The change in the environment surrounding the metal center and the degree of complex insertion into the hydrophobic environment inside the DNA helix are the causes of the rise in emission intensity. The hydrophobic environment inside the DNA helix makes it harder for solvent molecules to reach the binding site, preventing the quenching effect. It is apparent that the DNA helix effectively shielded complexes, which resulted in a decrease in vibrational modes of relaxation and an increase in emission intensity. The rise in emission intensities showed that the complexes bind to DNA into its hydrophobic pocket along the major and minor grooves (Tabassum et al., 2012). The binding constants K_b were calculated from Scatchard equation by plotting the log [(I₀-I)/I] against log [DNA] as presented in Fig. 11 and the value are listed in (Table 9).

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Fig. 10

The fluorescence spectra of ligand and metal complexes in the absence and presence of increasing concentrations of CT-DNA.

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Fig. 11

The binding constants Kb of LN and its metal complexes using Scatchard equation.

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Table 9 The binding parameters of LN and its complexes.

Sample code	Kb (M−1)
LN	5.06 × 105
MnLN	3.33 × 105
CoLN	4.54 × 105
NiLN	2.53 × 105
CuLN	3.04 × 105
ZnLN	3.88 × 105

4.2 HSA binding studies

Human serum albumin (HSA) was chosen due to its potential utility as a transporter for the delivery of specific anticancer drugs and its high concentration in human serum, accounting for 55% of the protein in blood plasma (Babgi et al., 2021). The binding of HSA with the LN and its synthesized metal complexes was studied by UV–Vis absorption spectroscopy, fluorescence emission spectroscopy and molecular docking.

4.2.1

4.2.1 UV–Vis absorption spectroscopy

The absorption spectrum of HSA shows a distinctive band at 278 nm which is caused by phenyl rings in aromatic amino acids such as tryptophan, phenylalanine and tyrosine (Chen et al., 2020). The absorption titration experiments were performed by keeping a constant concentration of HSA and varying the concentrations of ligand and complexes. As seen from the spectra in (Fig. 12) (Fig.13), the incremental addition of the studied compounds to HSA results in an increase of the absorbance due to the destruction of the tertiary structure of HSA and the extension of additional aromatic acid residues into the aqueous environment (Chen et al., 2020). The intrinsic binding constants (K_b) were calculated using Benesi-Hildebrand equation and listed in (Table 10). The high values of K_b constants (10⁴M⁻¹) revealed a strong binding affinity with HSA (Alanazi et al., 2021).

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Fig. 12

Effect of ligand and its metal complexes on the viscosity of CT-DNA.

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Table 10 Binding constants of LN and its metal complexes with HSA.

Sample code	Kb (M−1)
LN	1.40 × 104
MnLN	1.14 × 104
CoLN	1.26 × 104
NiLN	1.08 × 104
CuLN	1.49 × 104
ZnLN	1.52 × 104

4.2.2

4.2.2 Fluorescence emission spectroscopy

Fluorescence quenching spectroscopy has been shown to be an effective technique for investigating the structural changes of HSA and its interaction with the investigated compounds. By comparing the intrinsic fluorescence intensity of HSA before and after quenching, it was possible to identify the changes in its conformation (Mahaki et al., 2019). By exciting the protein at 280 nm, the emission spectra of LN and its complexes with HSA solutions were observed within the wavelength range of 300–600 nm, as shown in (Figs. 13 and 14). The fluorescence spectra of HSA were recorded in the absence and presence of increasing concentrations of LN and its metal complexes. The spectra revealed that there is a strong emission band at 339 nm, which is due to the presence of tryptophan residues, more specifically Trp-214 in subdomain IIA (Lazou et al., 2020). This band gradually diminishes as the concentration of the investigated compounds increases, indicating that LN and its metal complexes are thought to be inhibitors of the protein’s emission. The maximum emission wavelength of HSA with LN has a slight blue shift during the interaction, suggesting that LN’s binding to HSA changed the microenvironment of the tryptophan residues and caused the HSA residues to be in a more hydrophobic environment (Chen et al., 2015). Additionally, a peak at around 530 nm simultaneously arises with the increase in compounds concentration, which attributed to the fluorescence emission of studied compounds as mentioned earlier in the fluorescence with CT-DNA. The new peak at 560 nm corresponds to change of the solvent medium by adding DMSO to the buffer solution of HAS HSA and resulting an excited-state of anionic species of D-luciferin and its metal complexes (Kuchlyan et al., 2014; Mahaki et al., 2019). The K_SV values of the compounds were calculated using Stern-Volmer equation from the plot of I₀/I versus [ligand or metal complex]. The fluorescence quenching rate constant K_q was derived using the formula (K_SV/τ₀) and listed in (Table 11). The value of K_q for all compounds are in the range of 10¹¹M^-1s⁻¹, which is greater than the limiting diffusion quenching rate constant K_diff (2 × 10¹⁰ M⁻¹s⁻¹) of biomolecules indicating that the type of quenching is static (Chen et al., 2015; Lazou et al., 2020).

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Fig. 13

The absorption spectra of HSA in the absence and presence of increasing amounts of LN and its metal complexes.

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Fig. 14

The fluorescence quenching spectra of HSA in the absence and presence of increasing concentration of ligand and metal complexes.

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Table 11 HSA binding parameters for ligand and complexes.

Sample code	KSV (M−1)	Kq (M−1s−1)
LN	1.69 × 104	1.6910×11
MnLN	6.30 × 104	6.3010×11
CoLN	4.01 × 104	4.0110×11
NiLN	1.33 × 104	1.3310×11
CuLN	5.63 × 104	1.0010×11
ZnLN	3.33 × 104	3.3310×11

4.3 Molecular docking

Molecular docking is a valuable technique to mimic the interactions between small molecules and biomacromolecules. The studies on molecular docking have further verified the experimental results. Docking scores with higher negative values were displayed along with the two-dimensional and three-dimensional models. Based on the best-ranked binding free energy, intermolecular energy, and electrostatic energy, the docking results are shown in (Table 12). According to Tables (12–14) and (S4-S15), the following results were obtained:

1. LN and its metal complexes bind in the minor groove of DNA from A = T rich region through hydrogen bonds and van der Waal interactions. LN binds more strongly to DNA than complexes since it has a more negative binding energy (S = -7.94 kcal mol⁻¹). The same result was obtained from the experimental studies of electronic absorption spectroscopy.

2. Docking of LN and its metal complexes with 1h9z, 3eqm and 4 fm9 proteins revealed that complexes have higher negative free binding energy scores than the free ligand. This is because the metal ion overlapped with the ligand orbitals upon chelation and decreased the metal polarity, which increases the complexes' lipophilicity and ability to cross the lipid plasma membrane (Sharfalddin et al., 2021b; El-Boraey et al., 2022).

3. Based on docking scores, ZnLN complex binds to 1h9z, 3eqm and 4 fm9 proteins with −9.81, −10.39 and −8.98 kcal mol⁻¹ through hydrogen bonds, indicating it has the highest binding affinity compared with other complexes. This is consistent with the experimental cytotoxicity results.

Table 13 2D and 3D representation of LN and NiLN with 1bna of DNA.

Sample code	3D model	2D model
LN
NiLN

Table 14 2D and 3D representation of the metal complexes with 1h9z, 3eqm and 4 fm9.

Protein	Sample code	3D model	Site view	2D model
1h9z	LN
	CuLN
3eqm	LN
	ZnLN
4 fm9	LN
	CoLN

4.4 Cytotoxicity assay

The anticancer activity of the ligand and its synthesized metal complexes has been investigated against the human breast cancer cell line (MCF-7) and the human liver carcinoma cell line (HepG-2). MCF-7 was selected because breast cancer is the most relevant cancer in women globally and because this cell line responds to estrogen by proliferating, while HepG-2 was chosen because liver cancer is the third frequent cause of fatalities worldwide (Sung et al., 2021; Al-rashdi et al., 2022). Cisplatin was used as a standard control for comparison reasons. As listed in (Table 15), the inhibitory effect was assessed by the half maximal concentration IC₅₀. The results revealed that ZnLN complex showed the highest activity against both cell lines compared to cisplatin and other investigated compounds, as shown in (Fig. 14) (Fig. 15) and (Fig. 16). The IC₅₀ values of the investigated compounds against MCF-7 cell line follow the order: ZnLN > cisplatin > NiLN > CoLN > MnLN > CuLN > LN, while Hep-G cell line follow the order: ZnLN > CoLN > MnLN > NiLN > cisplatin > CuLN > LN. The cytotoxicity results are consistent with the docking values. The effect of ZnLN on both cell lines at different concentrations were shown in (Fig. 16) (Fig. 17) and the rest of complexes are shown in (Fig S3). A significant change in cell morphology was observed in both cell lines as well as a reduction in cell density.

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Fig. 15

Inhibition and IC₅₀ of LN complexes against MCF-7 cell line.

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Fig. 16

Inhibition and IC₅₀ of LN complexes against HepG-2 cell line.

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Fig. 17

The effect of ZnLN against MCF-7 and HepG2 cell lines at different concentrations.

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