Green synthesis and characterization of new carbothioamide complexes; cyclic voltammetry and DNA/methyl green assay supported by silico ways versus DNA-polymerase

2.1 Reagents

To synthesize thiosemicarbazide derivative as 2-(3,4-dihydronaphthalen-1(2H)-ylidene)-N-ethyl hydrazine-1-carbothioamide (HL), N-ethyl hydrazinecarbothioamide and 3,4-dihydronaphthalen-1(2H)-one were obtained from Sigma & Aldrich. Additionally, on the other side the three complexes were prepared from [Ni(OAc)₂].4H₂O, [Zn(OAc)₂].2H₂O and [UO₂(OAc)₂].2H₂O compounds that purchased also from Sigma & Aldrich. Glacial acetic, ethanol (EtOH) and dimethyl sulfoxide (DMSO) were used as they obtained due to their extra-pure nature. The ATCC's agency provided the microbial strains as a holding for biological products and vaccinations. Tris-buffer, MgSO₄, and methyl green/DNA have also been purchased from Institute Fermentation of Osaka (IFO).

2.2 Synthesis of the ligand (HL)

The HL ligand was synthesized by mixing N-ethyl hydrazine carbothioamide (0.01 mol, 1.19 g) and 3,4-dihydronaphthalen-1(2H)-one (0.01 mol,1.46 g) in EtOH (50 mL) with addition of glacial acetic acid (three drops) (Alkhamis et al., 2022). The reaction was kept under reflux for 3hr, then the reddish brown precipitate was isolated by filtration and washed by EtOH several times. The dried solid (under CaCl₂) was analyzed to estimate its elemental percentages in addition to other analytical data (Table 1). Notice: HL is the abbreviation of the ligand which suggested based on the labile hydrogen existed in thioamide group.

2.3 The strategy of green synthesis for the complexes

The weights of reactants applied for green synthesis of complexes were reported in Scheme 1. The molar ratio utilized was 1:1 either from the ligand or the metal ion that mixed together in ball milling technique (Scheme S1) without solvent (Alkhamis et al., 2022). Just a little amount of distilled water was added to facilitate grinding of reactants that continued up to 20 min. The TLC test confirmed that the produced solid complexes were free from reactants after a deliberate washing by distilled water which followed by EtOH. These complexes were technically analyzed to investigate their chemical and structural formulae that appeared by 1metal to 2 ligand.

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Scheme 1

The synthesis strategy y of the ligand (HL) and its metal complexes.

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2.4 Equipments of analysis

For simplicity, the majority of the analysis tools were illustrated in the supplementary file as photos, and each tool's specifications were typed beneath its photo (Schemes S2 & S3). The discussion section will also include a separate report on each technique's measuring circumstances. The two ligand' NMR spectra were recorded using Bruker WP (500 MHz) in DMSO‑d₆ solvent and TMS as an internal reference. Using JENWAY type 4070 Conductance Bridge, the electrical conductivity of the complexes was assessed for 1 mmol in DMSO solvent. Additionally, through using Johnson Matthey Magnetic Susceptibility Balance, the magnetic moments were calculated by determining the magnetic susceptibility at room temperature. By utilizing a typical procedure for complexometric titration, the Ni(II) and Zn(II) ratios were determined (Vogel, 1989). While the uranium percentage was measured gravimetrically upon complete combustion for weighted sample till reach to U₃O₈ formula which cooled and then weight.

2.5 Geometry optimization

The Materials Studio software (Modeling and Simulation Solutions for Chemicals and Materials Research and Studio, 2009), the DFT method, and the DNP basis set (Frisch et al., 2009) were all used in conjunction with the DMOL3 tool to improve the structures of most compounds and compute critical parameters. Based on GGA and PBEPBE functions, the best exchange–correlation function was identified (Hehre et al., 1986). Additionally, by using B3LYP/6-31G (d) level on the DFT theory, the MEP map was established. Unfortunately, we couldn't optimize the UO₂(II) complex due to selecting of suitable conditions is not easy to reach.

2.6 Cyclic voltammetry

Nickel acetate solution was subjected to this electro-analytical method either in absence or in existence of the coordinating ligand. The cell used is a small beaker which contains 30 mL of 0.L M KCl as a supporting electrolyte. The glassy carbon working electrode (GCWE), Ag/AgCl/KCl (saturated) is the reference electrode, and the platinum wire is an auxiliary electrode used. These electrodes were linked with a potentiostat after being dipped in KCl solution.

2.7 Biological activity

1. Antimicrobial activity screening (MIC)

In accordance with procedure shown in Part A of supporting materials, the disc diffusion method was used to calculate the minimum inhibitory concentration (MIC) for the investigated drugs against various microorganisms (Al-Hazmi et al., 2020). Gram positive (S. aureus & B. subtilis) and Gram negative (E. coli & P. aeuroginosa) bacteria as well as fungi were used to assess the ligand and its associated complexes (C. albicans & A. flavus). As is widely recognized, the term MIC was employed to identify the limiting concentration that was successful in preventing the development of both bacteria and fungus throughout the experiment. The MIC test is the first stage usually employed prior clinical studies in the field of medicines. It involves examining the activity of candidates against target fungi or bacteria.

• B)

  DNA/methyl green binding test

Methyl green reacts reversibly with polymerized DNA; the molecule is stable at neutral pH, whereas the free methyl green decreased. According to the process outlined in Scheme S4 (Burres et al., 1992), the absorption of methyl green had nearly totally vanished after 24 hr in the solution of substitution reaction. Colorimetric technique was used to assess how compounds that can bind to DNA displace methyl green from the molecule. By calculating the IC₅₀ value, which shows the concentration of the compound that causes the absorption to be reduced to half value, the genotoxicity of the currently used compound can be evaluated (Burres et al., 1992).

2.8 Quantum-based silico tests

This part of study aims to confirm the binding efficiency of HL ligand that appeared perfectly invitro assay. Various quantum-based programs were implemented for this virtual study to evaluate and to compare the biological efficiency of HL ligand. The ligand structure was optimized and saved in a suitable format to be used in docking programs (Pharmit link, MOE module & Swiss ADME). The technical details of each methodology were typed in the supporting file (Parts B and C).

3.1 Features of molecular masses

[Ni(L)₂(H₂O)₂], [Zn(HL)₂₍OAc)₂] and [UO₂(L)₂].2H₂O were the formulae of the three complexes derived from thiosemicarbazide derivative (HL)(Table 1). The chemical formulae were suggested based on elemental analysis basically which supported firstly by their incomplete solubility in DMSO or DMF. Such sparingly soluble complexes must have a non-electrolytic nature. The metal (1 mol) to ligand (2 mol) ratio, 1:2, was suggested for all complexes. These bis- ligands were coordinated to complete the stable formulae beside the other secondary ligand either the acetate group or water molecules. A weighted sample (0.2 g) of each complex was kept in oven (at 70 °C) for 1 h and then cooled in dry desiccators under CaCl₂. The samples weight is kept constant except the UO₂(II) complex (0.191 g). This reduction in the complex weight was calculated and assigned to 2H₂O molecules that hydrated the coordination sphere of UO₂(II) complex. These samples were returned again to the oven (at 120 °C) for extra 1 h and after cooling they weighted. The Ni(II) complex weight (0.188 g) was reduced in accordance with two coordinating water molecules that decomposed during heating. These observations suggested the presence of crystal water molecules with UO₂(II) complex whereas these molecules are covalently attached with nickel atom in the coordination sphere of its complex.

Further, the molecular mass of [Zn(HL)₂₍OAc)₂] complex (i. e.) was confirmed from its mass spectrum (Fig. 1) that performed over m/z range of 50–800, potential difference of 70 eV and heating rate of 40 °C/min. The molecular ion peak was recorded at m/z = 678.07 (calcd. 678.21) which assigns to M⁺. The successive fragmentation peaks may be assigned as follow in Scheme 2. The isotopes of zinc were recorded at m/z = 66 and 67.

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Fig. 1

The mass spectrum of [Zn(HL)₂₍OAc)₂] complex.

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Scheme 2

Suggested fragmentation for the mass spectrum of Zn(II) complex.

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3.2 Analysis of IR and NMR spectra

Four absorption bands at 3242, 3309, 1049, and 1624 cm⁻¹ in the IR spectrum of HL were assigned to υ(NH)₁, υ(NH)₂, ʋ(C⚌S), υ(C⚌N) vibrations, respectively (Abdel-Rhman et al., 2019; Abu-Dief et al., 2021; El-Asmy et al., 2015, Hosny et al., 2019). (Table 2, Figure S1). The profile of nuclear magnetic resonance (¹H NMR, DMSO‑d₆) (Figure S2), showed two signals at 9.80 and 10.91 ppm that were attributed to the protons of (NH)₁ and (NH)₂. Additionally, there are two signals associated to protons in the ethyl group at 2.52 ppm and 3.47 ppm. Furthermore, several signals in the range of 7.13 to 8.34 ppm signify the aromatic protons. Finally, the spectrum's ¹³C NMR (Figure S3) revealed two signals at 180.06 and 168.35 ppm, respectively, that are indicative to (C⚌S) and (C⚌N). Moreover, aromatic carbons are responsible for the signals from 124.1 to 140.88 ppm range.

The IR spectra of [Ni(L)₂(H₂O)₂], and [UO₂(L)₂].2H₂O complexes (Figures S1) revealed a red shift of υ(C⚌N) vibration and disappearance of υ(C⚌S) and υ(NH)₁with the instantaneous appearance of υ(C–S) bands at 610–620 and 1615–1625 cm⁻¹ regions (Nakamoto, 1970). This feature suggests a mononegative bidentate mode for each coordinating ligand via the donors of azomethine (C⚌N), and deprotonated thiol (⚌C—S^-). The vibrations of new M−L bonds that recorded (Table 2) were assigned to υ(M—N) and υ(M—S) bands (Nakamoto, 1970). Also, ρ_r(H₂O) and ρ_w(H₂O) bands in the Ni(II) complex were appeared at 824 and 529 cm⁻¹, respectively. While, the broadness centered at ≈ 3300 cm⁻¹in the spectrum of UO₂(II) complex supports the presence of hydrated water molecules which already suggested (Qurban et al., 2022).

Regarding the UO₂(II) complex, the dioxouranium ion exhibited two vibrations, υ₃ & υ₁, respectively at 906 and 868 cm⁻¹. The force constant (F) of U⚌O bond could being estimated based on McGlynn equation (McGlynn et al., 1961) as follow; (υ₃)² = (1307)² (F_U−O)/14.103. The estimated F_U−O value (6.776 mdaynÅ⁻¹) was used to calculate the U-O bond length (R_U−O = 1.740 Å) from this equation; R_U−O = 1.08 (F_U−O)^−1/3 + 1.17. The calculated values are within the normal range of uranyl complexes (Qurban et al., 2022; McGlynn et al., 1961).

The IR spectrum of [Zn(HL)₂(OAc)₂] complex (Figures S1) revealed a red shift of υ(C⚌N), and υ(C⚌S) vibrations which detects the neutral bidentate mode of bonding for each ligand. Azomethine nitrogen and thione sulfur were the coordinating atoms towards the zinc atom. This proposition was confirmed by the appearance of υ(Zn–N) and υ(Zn–S) bands at 485 and 551 cm⁻¹, respectively. Additionally, the vibrations of acetate group were appeared by a difference (Δν > 180 cm⁻¹) refers to its mono-dentate binding nature (Nakamoto, 1970).

On the other hand the NMR spectra of Zn(II) complex were performed in DMSO‑d₆ (Figures S2 & S3) to elucidate the suggested structure of the complex. The ¹H NMR spectrum showed two signals at δ 9.89, &10.96 ppm which assignable to (NH)₁, and (NH)₂ protons, respectively. The chemical shifts are almost the same as those of the free ligand, confirming that these groups are not involved in coordination with zinc ion. Moreover, the ¹³C NMR spectrum of the complex indicated the participation of both (C⚌S), and (C⚌N) in the coordination because they were shifted to down field.

3.3 UV–Vis spectral analysis

Electronic transitions within metal ion complexes are a good indicator for several characteristics based on the field of coordinating ligand. The transitions of intra-ligand and charge transfer are significantly affected after complexation. While, the ligand field transitions within the splitting of d-orbitals are the major indicator for the geometry of paramagnetic complexes (Lever, 1968). So, the UV–Vis spectrum of green Ni(II) complex was done for its solution in DMSO solvent along 200–1000 nm range (Al-Qahtani et al., 2021). The bands recorded at 440 and 735 nm may be assigned to ³A_2g→³T_1g(P,υ₃) and ³A_2g→³T_1g(F,υ₂) transitions, respectively. These transitions are particular to the octahedral geometry of d⁸ systems. The ligand field parameters as; crystal field splitting energy (Dq = 903), Racah parameter (B = 813) and nephelauetic ratio (β = 0.78) were calculated and found close to the known values of octahedral geometry (Lever, 1968; Al-Qahtani et al., 2021). The nephelauetic ratio reflects the high polarity of new covalent bonds formed between the metal and the ligand. Moreover, the magnetic moment value (μ_eff = 3.33 BM) is closer to that reported for octahedral form (Cotton et al., 2003).

3.4 Analysis of scan electron microscope (SEM) and EDX patterns

The use of accelerated beam of electrons that focused on solid samples enables to investigate the surface topography of the sample. The SEM images were obtained and displayed for investigation (Figure S4). The image of Zn(II) complex showed a uniform surface of well-spherical particulates that appeared with tiny size in micrometer range. While, the images of Ni(II) and UO₂(II) complexes exhibited a non-uniform ice-rocky shape that aggregates various spherical particles (Alkhamis et al., 2021).

The complex's Ni(II), Zn(II), or U(VI) element singlet-set of signals (Figure S5) from the EDX analysis indicate the coordination mechanism properly. For the inner layers to be penetrated in this study, accelerating x-ray photons are necessary (K- or L-shell). These photons stimulate the electrons, which subsequently produce positive gaps that readily draw other electrons from higher levels (L- or M−shell). This relaxation is coupled with radiation emitted that draw peaks of K-series (Ka1, Ka2, or Kb) or L-series. Depending on each element's distinct electronic configuration, these peaks distinguish them; the L-series being the most essential (Alkhamis et al., 2021). The wt/At% was also calculated in relation to the indicator element (heavier element as the metal).

3.5 Geometry optimizing

The geometry of HL ligand and its complexes (Figure S6) except the UO₂(II) one was optimized under DFT method. Whereas, 3D-maps (Fig. 2& S7) were demonstrated by using B3LYP/6-31G (d) basis set. These maps as well as the estimated computational parameters (Table S1) offer a well information about the coordination process fully.

1. General features

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Fig. 2

HOMO & LUMO levels of (A)HL ligand, (B)Ni(II) and (C)Zn(II) complexes.

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The ligand geometry of minimum energy content (Figure S6) exhibited a suitable position of C(10) = N(11) and C(13) = S(14) groups that able to coordinate without bond strain. The charges of N¹¹ (-0.197790) and S¹⁴ (-0.067486) donors describe their sufficient nucleophilicity for coordination with the metal ions. The bond lengths of C¹⁰ = N¹¹ (1.28182 Å) and C¹³ = S¹⁴ (1.58265 Å) groups are significantly lengthen after the coordination of their donors (N & S) towards the metal ions (Alaysuy et al., 2022, Hosny et al., 2023). Further, the angles of S(14)-C(13)-N(12), N(12)-N(11)-C(10) and C(17)-N(15)-C(13) were 122.26°, 121.92° and 122.52°, respectively. These angles reflect the regularity of sp² hybridized-carbons in HL structure. While such angles were slightly shifted in the two complexes due to some normal changes happened with coordination (Figure S6).

• B)

  3D- models of frontier orbitals and MEP

The HOMO and LUMO patterns were demonstrated (Fig. 2) to evaluate the distribution of electron-cloud on definite functional groups. As seen, the HOMO level in HL ligand was concentrated on the functional groups that contained the donor atoms mainly, while the LUMO level was dispersed over the whole molecule (Alatawi et al., 2022). The feature of the two orbitals did not change strongly in the two complexes while they are seeming focused around the nickel center more than the zinc one. The energies of the two orbitals were computed (Table S1) and the gap between them (ΔE = E_LUMO- E_HOMO) are significantly lower in the two complexes as expected.

The maps of electrostatic potential (Figure S7) were demonstrated to differentiate the nucleophilic, electrophilic, and zero-potential zones. Such zones were chromatically characterized by red, blue and green colors. As noticed in HL map, the nucleophilic zone (red) seems focused on N(11) and S(14) donors which supporting their priority in coordination among the other donors. This nucleophilicity was reduced in the two complexes depending on the coordination of these donor atoms. Further, the zero-potential zone is normally prevailing on the whole carbon skeleton.

• C)

  Computational parameters

The energy of kinetic, binding, spin electrostatic, polarization and exchange–correlation beside the summation of atomic energy as well as the dipole moments were estimated and tabulated (Table S1). The values of total energy, summation of atomic energy and binding energy of the two complexes are lower than the free ligand indicating the high stability of such complexes. The exchange–correlation energy and electrostatic energy values are also lowered in the complexes to confirm their priority in stability of bonds. The dipole moment of HL ligand and its corresponding complexes are relatively comparable to each other's and their values detect the reduced polarity over the whole molecules. This feature is preferable in biological application due to the ability of such compounds to miscible with cell-lipids and then may contact directly with active sites in biological systems (Abumelha et al., 2020).

3.6 Cyclic voltammetry

This study offers essential parameters that reflect a light on the changes of electrochemical behavior of nickel ion (i. e.) in presence of the ligand. This may help to understand the coordination process within the complexes in solid state as the main aim in this research. Notice: the equations implemented for this study were typed in the supporting material file (part D) to reduce the similarity in the manuscript as possible.

1. Electrochemical behavior of Ni²⁺ in absence and presence of HL at 288.55 K

The redox behavior of Ni²⁺in 0.1 M KCl (supporting electrolyte) was studied electrochemically at 288.55 K under glassy carbon electrode (working one), potential window of 1 to −1 V and a scan rate 0.1 V/s (Fig. 3). In absence of HL ligand, the electroactivity of Ni²⁺ solution was suggested due to appearance of one cathodic and anodic peaks on the voltammogram which attributing to (Ni²⁺/Ni⁰) and (Ni⁰/ Ni²⁺), respectively. The catiodic potential peak, Epc, arises to −0.4525 V in the forward scan, while the anodic potential peak, Epa, reaches to 0.0425 V in the reverse scan. The increase in all peak currents designates and supports the presence of diffusion process (Almehmadi et al., 2022). Adding HL ligand by various concentrations leads to lower shift of the redox peak due to the covalency between the Ni²⁺ ions and the ligand (Fig. 3).

• B)

  Stability constant and Gibbs free energies for the Ni²⁺-HL complex

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Fig. 3

Influence of different concentrations of Ni(II) in absence of HL (A) and in presence of HL (B) using glassy carbon electrode in 0.1 M KCl at 288.55 K and scan rate 0.1 V.S^-1.

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Adding HL ligand to Ni²⁺ ions, the elevated stability constants (β_ML) and Gibbs free energies (ΔG) suggest the successive building to stable complex in solution as shown in Table 3. To calculate β_ML and ΔG, we must apply these Eqs. (1)–(3):

Where (E˚M) is the valid peak potential of the Ni⁺² ion at zero ligand concentration, (E˚Ml) is the valid peak potential of the complex after each addition for the ligand amount, (j = [L]/[M]) is the molar ratio, and (CL) is the molar concentration of the ligand. Finally, increasing the ligand concentration the βML and ΔG values are increased.

3.7 Invitro assays

1. Antimicrobial screening

Gram positive (S. aureus & B. subtilis), Gram negative (E. coli & P. aeuroginosa), and fungi (C. albicans & A. flavus) were chosen for this test concerning the HL ligand and its corresponding complexes. The MIC method was applied, and the results were aggregated in Table 4 and graphed in Fig. 4 (Raman et al., 2003). Two references were screened for comparative evaluation, the first is Ampicillin as the antibacterial reference, and the second is Colitrimazole as antifungal reference. The results point to the superiority of the ligand towards all bacteria which appeared more than the reference. Also, the antifungal screening results are also excellent. The antimicrobial activity of Ni(II) complex comes right after the ligand activity. Unfortunately, the behaviour of rest complexes is ineffective. As reported in various researches (Shah et al., 2016), the existence of functional groups containing nitrogen or sulphur atom enables the chemical compound to interact with electron-deficient centres in biological systems. This is noticed by a distinguish way with the free HL ligand, while this feature was reduced with the complexes. This may refer to the coordination of some functional groups with the metal ions which may cause incapability of such groups for other contributions. Fortunately, the activity of Ni(II) complex is highly significant which is due to the toxic role of nickel atom inside living cells.

• B)

  DNA/methyl green test

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Fig. 4

Antimicrobial activity in terms of MIC (µg/mL).

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Although the bioactivity of newly chemicals are investigated from many scientists, understanding the real mechanism happened inside the living cells is still an area of disagreement. But the best way for choosing the pharmaceutical compound is not area of disagreement between them, they concentrated on the binding activity of such compound with the DNA. The affinity of methyl green to stain the DNA via binding at pH = 7 was clearly recorded in a reversible reaction in between (Nagar, 1990). The magnitude of decay in the green color staining the DNA in presence of a tested compound measures the reactivity of such compound in replacing methyl green. This decay was monitored at λ = 630 nm and the concentration of the tested compound causes the absorption reduction to its half value (IC₅₀) was determined for each compound (Table 5). The ligand and [Ni(L)₂(H₂O)₂] complex exhibited significant genotoxicity towards the DNA, particularly the ligand (Ramesh and Maheswaran, 2003). Consequently, controlling the cell-DNA via its combination with the compound is the main target to inhibit cell division, resulting induced apoptosis. When the damage of DNA is becoming serious and cannot easily corrected so the cell will death. This damage based on the deterioration of DNA helix due to intercalation of HL ligand with the grooves or through H-bonding. We know the role of aromatic ring in intercalation with DNA, which considered the first part from the ligand that really interacted (Abdel-Rahman et al., 2021). Prospective research is required to determine whether genetic information can improve therapeutic efficacy and safety by assisting with drug selection and dose. Despite the fact that there still many obstacles to overcome, the integration of pharmacogenetics into clinical practice is anticipated to have a substantial impact on health in the upcoming years.

3.8 Quantum-based in-silico tests

1. Pharmit server and MOE module

Here we aimed to examine the extent of effectiveness for binding between the ligand and DNA-polymerase to evaluate the degree of conformity with in-vitro results. Three proteins were chosen from PDB as 1bpy, 5szt and 1zqa (Scheme S5) for this docking purpose using either the pharmit server (Daina et al., 2017, Fenton et al., 2002; Maiorov and Crippen, 1994) or MOE module (Ver. 2018). Pharmit server, showed a best interaction for the ligand with the pockets of 1bpy and 5szt proteins (Fig. 5). This simulation was ended by searching on analogous drug in MolPort database, a drug with code number MolPort-002–822-252 (Scheme 3) has extent of similarity with HL ligand in its binding with DNA. The Root Mean Square Deviation value (RMSD = 0.266) of the drug is very small which points to the high similarity of such drug with the tested ligand in their biological effectiveness (Maiorov and Crippen, 1994).

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Fig. 5

Pharmit interaction for the HL ligand towards DNA-polymerase proteins.

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Scheme 3

The structure of MolPort-002–822-252.

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Such distinguished behavior of the ligand towards DNA-polymerase was confirmed by MOE-docking (Fig. 6). The ligand interacted perfectly to DG 6(T) residue in 1bpy protein via H-donor bond (length 3.3 Å) and the scoring energy value of the docking pose was excellent (S = −6.1512 kcal/mol). While, the allosteric binding with 5szt protein was conducted through various aminio acid residues to produce a pose also with excellent scoring energy (−5.9887 kcal/mol) (Hosny et al., 2020; Katouah et al., 2020).

• B)

  ADME parameters

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Fig. 6

Effective Interaction of HL with two DNA-polymerase proteins.

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The absorption, distribution, metabolism and excretion (ADME) are the parameters targeted to investigate the ligand (smaller size) by using Swiss-link. The bioavailability radar (Fig. 7) which exhibited XLOGP3 (lipophilicity), polarization, saturation limit (sp³ ≥ 0.25), size, flexibility and solubility indexes on six axis reflects the drug-likeness and physicochemical properties of the ligand. The pink region detects the limits of the optimum values of such indices (Alkhamis et al., 2021). The values of XLOGP3 (2.61), saturation (0.38), size (247.3), polarity (TPSA = 68.51), and flexibility (4) are lower enough to judge on distinguish HL drug-like and physicochemical properties. The lipophilic feature of the ligand was also supported by LogP_{o/w (}2.74) and LogS (-3.02) values that indicate the lower solubility of HL in polar solvent as water (Alkhamis et al., 2021).

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Fig. 7

The bioavailability radar (A) and BOILED-Egg (B) for the ligand via Swiss-ADME study.

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Additionally, the permeability of skin (Log K_p), P-glycoprotein (P-gp) substrate, gastrointestinal tract absorption (GI), brain barrier absorption (BBB), and cytochromes P450 (CYP) were the indices of ligand pharmacokinetics. The behavior of HL ligand towards P-glycoprotein (P-gp) and cytochrome P450 (CYP) enzymes was investigated based on the importance of such protein (P-gp) or these enzymes. The P-gp could protect the nerve cells from xenobiotics although its excretion in tumor cells will resist the anticancer drug action. On the other side, a group of cytochromes which is called iso-enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4) is essential for de-accumulation process (via metabolism) of remaining drug inside the cell (Abu-Dief et al., 2021). The HL ligand doesn't play as a substrate of P-gp protein, which is considered favorable for protecting tissues and organisms. Further, the ligand did not inhibit three CYP-enzymes just inhibit CYP1A2 & CYP2C19. So, the drug metabolism process inside the cell did not affected seriously. The log Kp (-5.96 cm/s) that reflects the relation between lipophilicity and size of the compound, appeared with smaller negative value, indicates the high extent of skin permeability for dug.

Furthermore, the BOILED-Egg image (Fig. 7) which point to a relation between WLOGP and TPSA, indicates the ability of the ligand (red point) to absorb in brain barrier as appeared in yellow region (yolk) (Abu-Dief et al., 2021). While, there is no ability for such compound to absorb in gastrointestinal tract (white zone). Also, the red point reflects the non-substrate function of the ligand towards P-gp (PGP-) protein. The interaction pathway of the ligand inside the living cells (Figure S8) was oriented towards cathepsin L protease; leukocyte elastase protease by 26.7 %, P2X purinoceptor 7 (by homology) ligand-gated ion channel by 20.0 %, and isocitrate dehydrogenase [NADP] cytoplasmic enzyme by 13.3 %. Finally, the interaction of the HL ligand with DNA-polymerase cannot be ignored either from in vitro or in silico evaluations. This study may be the out of point for other extensive studies aimed at deepening the biological efficacy of the carbothioamide derivative and enriching the rich history of this family of compounds with biological excellence.