Antituberculosis, antimicrobial, antioxidant, cytotoxicity and anti-inflammatory activity of Schiff base derived from 2,3-diaminophenazine moiety and its metal(II) complexes: structural elucidation, computational aspects, and biological evaluation

2.1 Chemicals

To conduct the current research, high-purity analytical reagents were used in the experiments including the following chemicals: 4-diethylaminosalicylaldehyde (98 %, Aldrich), 2,3-diaminophenazine (90 %, Aldrich), cobalt nitrate hexahydrate (98 %, Fluka), nickel nitrate hexahydrate (98.5 %, Fluka), copper nitrate trihydrate (99 %, Aldrich), cadmium nitrate tetrahydrate (98 %, Aldrich), dimethyl sulfoxide (99 %, Ficher), N,N-dimethyl formamide (99.9 %, Merck), methanol anhydrous (99.8 %, Aldrich), and ethanol (99.8 %, Fluka).

2.2 Apparatus

Molar conductivity measurements were obtained for solid compounds in dimethylformaldehyde (DMF) solution at room temperature by Jenway 4510 conductivity meter. Magnetic susceptibility calculations were performed using the Faraday method (Faraday equilibrium) at room temperature. The Perkin-Elmer 2400 CHN was utilized for elemental analysis. Also, the metallic content was determined using a Thermo Scientific iCE 3300 spectrometer atomic absorption spectrometer with an auto-sampler model. The FT-IR spectra were recorded using a Thermo Scientific 6700 and a KBr disc. The Beckman Coulter DU 800 spectrometer was used to obtain electronic spectra and dimethylsulfoxide (DMSO) as solvent. The mass spectra of compounds were performed by a direct input unit (DI-50) with the Shimadzu QP-5050 GC-MS. The ¹H and ¹³C NMR spectra were recorded using a Bruker high-performance Avance III NMR spectrometer at 400 MHz, with DMSO‑d₆ as the solvent. The ESR spectra of the powder complexes were recorded at room temperature using a Jeol JES-FE 2XG spectrometer. Most of the analyses were carried out by the microanalysis team located at Cairo University in Giza, Egypt. To determine the antimicrobial and antioxidant activity, biological tests were conducted at the Department of Microbiology at Omar Al-Mukhtar University. Cytotoxicity and anti-tuberculosis activity were evaluated at the Faculty of Veterinary Medicine in collaboration with the Animal Health Center in the Laboratory of Preventive Medicine and Public Health.

2.3 Synthesis of ligand

The Schiff base ligand was prepared by 4-diethylaminosalicyaldehyde (1.93 g, 10 mmol) was added to 25 mL of hot methanol, followed by the addition of 2,3-diaminophenazine (1.05 g, 5 mmol) dissolved in 25 mL of methanol (Figure 1). ^{13 , 15} The mixture was heated and refluxed at 90 °C for 3 h. After condensation, the mixture was left to cool down to room temperature, resulting in the formation of orange precipitated crystals. The crystals were then isolated by filtration.

Figure 1:

Synthesis of 2,3-diaminophenazine Schiff base (H ₂ L).

Characterization of 6,6′-((1E-1′E)-(phenazine-2,3-diylbis(azanylylidene))bis-(metha-nylylidene))bis(3-(diethylamino)phenol) (H ₂ L): color: orange; yield: 70 %; m.p.: 132 °C; elemental analysis for organic ligand, m.wt: 560.702 g mol⁻¹ (C₃₄H₃₆N₆O₂) calculated: C, 72.83 %; H, 6.47 %; N, 14.99 %; found: C, 72.80 %; H, 6.40 %; N, 5.05 %. FT-IR (KBr, cm⁻¹): 3,047 ν(C–H)_arm; 3,359 ν(O–H); 1,244 ν(C–N)_phe; 1,610 ν(C=N)_azo; 1,025 ν(C–O). ¹H NMR (300 MHz, DMSO‑d₆) δ 13.46 (m, 1H, OH), δ 13.36 (m, 1H, OH), δ 8.62 (m, 2H, CH=N), δ 8.52 (dd, J = 6.2, 3.4 Hz, 2H, Ar–H), δ 8.34 (s, 4H, Ar–H), δ 7.73 (dd, J = 6.2, 3.3 Hz, 2H, Ar–H), δ 7.38 (d, J = 2.1 Hz, 1H, Ar–H), δ 7.30 (d, J = 8.9 Hz, 1H, Ar–H), δ 7.09 (dd, J = 8.7, 2.2 Hz, 2H, Ar–H), δ 6.89 (J = 8.7, 2.5 Hz, 2H, Ar–H), δ 4.79 (q, J = 6.9 Hz, 4H, CH₂), δ 2.49 (t, J = 7.1 Hz, 6H, CH₃). ¹³C NMR (125 MHz, CDCl₃) δ 187.16, 183.09, 175.56, 169.66, 168.65, 168.01, 157.51, 156.52, 150.17, 149.16, 148.98, 146.03, 144.00, 71.80, 39.52.

2.4 Synthesis of complexes: a general produce

The complexes were prepared by mixing: Co(NO₃)₂·6H₂O (1.456 g, 5 mmol); Ni(NO₃)₂·6H₂O (1.454 g, 5 mmol); Cu(NO₃)₂·2H₂O (1.208 g, 5 mmol) and Cd(NO₃)₂·4H₂O (1.542 g, 5 mmol) with the (2.803 g, 5 mmol) of organic ligand H ₂ L (molar ratio 1:1) in solvent mixture DMF/EtOH/H₂O (volume ratio 10:3:3 cm³). ¹⁵ The mixture was returned at 90 °C for 24 h, the prepared complexes were left to cool at room temperature, then the crystals were filtered. Different analytical methods were used characterize the ligand and its complexes with metal ions. The results obtained for the metal complexes were listed and summarized as follows:

2.5 Antimicrobial activity

In order to evaluate the antimicrobial activity of the ligand and its complexes, the agar-well diffusion method was used to test compounds and compare the efficacy with tetracycline, ciprofloxacin, and amphotericin B as standard drugs. ^{12 , 13} For the objective of comparison, the standard drugs and complexes were subject to identical concentrations and conditions. The antimicrobial activity of the complexes was subsequently assessed in vitro against two kinds of fungal: A. flavus and C. albicans strains as well as four bacterial strains: B. subtilis, S. aureus (+Gram), E. coli, and P. aeruginosa (−Gram).

2.6 Antioxidant activity

The assay measured the radical scavenging effect of stabilized 2,2-diphenyl-1-picrylhydrazyl (DPPH). ¹³ The 1 μg of ligand and its complexes were dissolved in 1 mL of DMSO, and their antioxidant activities were compared to ascorbic acid. 250 μL of each solution was added to 1 mL of DMSO/DPPH solution, which was prepared by dissolving 6 mg DPPH in 50 mL DMSO. The total volume was then adjusted to 3 mL by adding DMSO. The mixtures were incubated for 30 min at room temperature. The absorbance was measured at 517 nm wavelength, and the blank sample containing 3 mL of DMSO and DPPH was measured as well. The measurements were done in triplicate, and the value of radical scavenging activity was calculated using the following equation:

Where: A_control: DPPH absorbance of radical + DMSO after 30 min.

A_sample: DPPH absorbance + sample after 30 min.

Where: AE: antiradical efficiency.

IC₅₀: concentration that had 50 % inhibition.

2.7 Cytotoxicity assay

The cytotoxicity assay was conducted using brine shrimp larvae, according to the protocol developed by Mayer et al. ²⁵ This method has been found to correlate well with cytotoxic activity, while it is also cost-effective. Shrimp eggs were incubated in a rectangular dish filled with seawater with the help of the Department of Preventive Medicine and Public Health Laboratory at Omar Al-Mukhtar University. To create an uneven section in the plate, a perforated tool was used. About 50 mg of eggs were sprayed into a large dark chamber, while the smaller chamber was exposed to normal light. After two days, the larvae were collected in a pipette with a lighted side. To perform the test, stock solutions of each test compound were prepared by dissolving 10 mg of the compound in 10 mL of DMSO. Different concentrations of compounds were added to separate vials and the final volume was adjusted to 10 mL with seawater after two days. Once the shrimp larvae were ready, we added 10 larvae to each vial and incubated them for 24 h. The vials were then observed using a magnifying glass and the number of surviving individuals in each vial was counted. The test was performed in duplicate and Finney computer software was used to analyses the data and determine the LD₅₀. The results were compared with a control group positive for the anticancer drug bleomycin.

2.8 Anti-inflammatory activity

The anti-inflammatory activity of the organic ligand and its complexes was evaluated using a BSA (bovine serum albumin, 98 %, Aldrich),) assay. ²⁶ The test was conducted in triplicate with aspirin and sodium diclofenac as standard drugs. Compounds were prepared in DMSO with dilute phosphate buffer (0.2 M, pH 7.4) at concentrations of 15, 25, 50, 100, and 200 µg L⁻¹. 1 mL of BSA (1 mM) in phosphate buffer was dissolved in 4 mL containing various concentrations of the compounds. The BSA was denatured in a water bath at 70 °C for 15 min after incubating the solutions at 37 °C for 20 min. After allowing the solutions to cool at ambient temperature, the test solutions exhibited turbidity. Finally, the absorbance was measured at 660 nm using a UV–Vis spectrometer, and the % denaturation was determined as a control in the absence of any drug and the inhibition of BSA was assessed using the following formula:

Where: X: absorbance of the control.

Y: absorbance of the compounds.

2.9 Anti-tritubercular activity

All compounds were evaluated in vitro for anti-TB using the proportion method. ²⁷ To perform this method, a Lowenstein–Jensen (LJ) medium was prepared by mixing fresh chicken eggs with mineral salt and adding 2 % malachite green. A stock solution of the test compound was created in DMF and then filtered through a 1 µg porous membrane. It was then diluted to 1, 2 and 3 μg/mL. Each solution was transferred to a sterile universal container and put in isolation at 85 °C for 45 min. Control experiments were also conducted without the presence of the test compound. Mel (LJ) was inoculated with 10⁻⁴ CFU mL⁻¹ of standard M.TB H _37RV and the clinical isolate. They were successively incubated at 37 °C for 28 days. The tested compounds were also incubated for 14 days and compared with pyrazinamide (PZA) as a standard.

2.10 Computational studies

The computational studies mentioned below were performed on the organic ligand and its metal complexes against active anti-tuberculosis compounds to support in vitro results.

3.1 Chemistry

Metal ion complexes of Co(II), Ni(II), Cu(II) and Cd(II) were prepared in the solid state. They are soluble in some organic solvents, such as DMF and DMSO, and not soluble in water. The low molar conductivity of the complexes was measured in 10⁻³ M in DMF solution at ambient temperature, indicating that they are non-electrolyte in nature. ³² The thermal stability of the complexes was determined up to 253 °C using TGA. The square planar geometry of the N and O atoms of the Schiff base ligand in the complexes was determined using various spectral analysis methods.

3.2 Spectral and physical methods

The current study, investigated the nature of chemical bonding between atoms, using thermal decomposition and various spectral and physical techniques for analysis, as shown below.

3.3 Elemental analysis

The elemental components and the purity of the prepared complexes were determined by (C %, H %, N % and M %) elemental analysis and are mentioned in (Table 1).

3.4 Molar conductivity

The molar conductivity of the prepared complexes was measured using a 10⁻³ M dimethylformamide (DMF) solution. The obtained results showed that the C1, C2, C3, and C4 complexes have low molar conductivity values (17.9, 17.4, 16.8, and 18.5 Ω⁻¹ cm² mol⁻¹). These values confirmed that the complexes are non-electrolytic in nature. ³²

3.5 Mass spectra

The peaks of the complexes’ mass spectra were recorded by correlating the peaks of the molecular ions (m/z) with the molecular mass of the complexes. The bonds of the complexes show molecular ionic peaks at m/z 617.2, 616.2, 621.2, and 672.1 for C1, C2, C3, and C4, respectively, which is consistent with the proposed molecular mass values for complexes (Figures S1–S4).

3.6 FT-IR spectra

Table 2 presents the FT-IR data for the ligand and their corresponding metal complexes. In the FT-IR spectra, the free Schiff base ligand H ₂ L displays a sharp band at 1,630 cm⁻¹, which is attributed to the ν(C=N) azomethine group. ¹⁴ Additionally, the broadband at 3,359 cm⁻¹ indicates the presence of the ν(OH) phenolic group in the ligand. ^{23 , 33} Furthermore, the range of 1,237 cm⁻¹ shows the occurrence of ν(C–O) phenolic. The broadband is observed in regions 1,547 and 1,258 cm⁻¹ due to the (C=C) and (C–N), respectively. ³⁴ Upon chelating, the bands are shifted higher or lower indicating the participation of the phenolic oxygen atom in coordination with the metal ion. Based on the non-electrolytic nature of metal complexes, it can be concluded that metal ions coordinate with phenolic oxygen due to their bivalency satisfaction. ³⁵ The absence of broadband attributed to ν(OH) phenolic in the FT-IR spectra of metal complexes indicates the coordination of the central metal bonds of C1, C2, C3, and C4 by the phenolic oxygen atom. ³⁶ This shift is attributed to the decrease in the property of the double bond between ν(C=N) azomethine, which occurred due to the entry of nitrogen into coordination with the metal ion. ³⁷ The ν(M−O) and ν(M–N) complexes have frequency patterns of 525, 526, 523, and 541 cm⁻¹; and 443, 441, 443 and 487 cm⁻¹, respectively. ^{38 , 39}

3.7 Electronic absorption spectra and magnetic moments

The stereochemistry of the organic ligand H ₂ L and its complexes is well examined by UV–Vis spectra, which were evaluated in DMSO at ambient temperature (Figure S6). The absorption band recorded at (273 nm, 36,630 cm⁻¹) was assigned to π→π* transitions that correspond to the phenolic ring of the ligand. ⁴⁰ The absorption band at (406 nm, 24,630 cm⁻¹) is attributed to n→π transitions of azomethine. ⁴¹ compared with the organic ligand, the Co(II) and Ni(II) complexes showed three absorption bands, while the Cu(II) complex contained two absorption bands, while the Cd(II) complex showed one absorption band due to the d¹⁰ electronic configuration and charge transfer from the ligand to the Cd(II) ion and are mentioned in (Table 3). ⁴² Magnetic moment values for C1, C2, and C3 complexes were found in the range of 2.1, 1.6, and 1.5 BM, respectively, and the C4 complex did not show a value for the magnetic moment due to the electrical configuration of d¹⁰. ⁴³

3.8 ¹H NMR and ¹³C NMR spectra

¹H and ¹³C NMR analysis provides the positions of proton and carbon signals for the Schiff base ligand H ₂ L and its C4 (Table 4, Figures S7 and S8). Two phenol δ OH signals were observed at 13.43 and 13.46 ppm, while the δ CH=N signal of the azomethine proton appeared at 8.62 ppm in the ¹H NMR spectra of the ligand. ²³ Fourteen aromatic δ C–H protons were recorded in the range of 4.79 ppm, and there were four aliphatic δ CH₂ protons and six δ CH₃ protons in the ligand. ⁴⁴ In the C4, the phenol proton at δ OH 13.43 and 13.46 ppm in the ligand disappears due to its entry into the complex. ¹³ Moreover, the signal shift of the azomethine group of the ligand indicates the formation of the complex via the nitrogen atom of azomethine. In the ¹³C NMR spectra, a signal appears at 187.16 ppm attributed to two azomethine carbons in the ligand. Signals also appear at 175.56–144.00 ppm are attributed to the carbons in the aromatic ring of quinoxaline, C27, C28, C29, C30, C31, C32, C33, C35, C37, C38, C39, C40 and C41. The signal for the aliphatic CH₃ group appears at 39.52 ppm, C11, C12, C25 and C26, while the aliphatic CH₂ signal appears at 71.80 ppm, C10, C11, C23 and C24. ⁴²

3.9 ESR spectra

ESR spectra of a metal complex provide information about the metal and its environment within the complex. ⁴⁵ This includes details about the geometric structure and location arrangement of the Schiff base and the metal. Figure 2 illustrates the ESR spectra of the copper complex at room temperature. The data suggests that the delocalized electron is in the dx²–y² orbital and its ground state is ²B_1g with square planar geometry. The metal–ligand bonds have a covalent nature with a g_‖ value of less than 2.0023. Hathaway’s statement suggests that if G < 4, there are some exchange interactions, while if G > 4, there is little interaction between the copper ions. The properties of the square planar geometry can be determined by the symmetry axis parameter G, which has a value of 1.609, indicating significant exchange interactions between the copper centers. ⁴⁶ Furthermore, the correlation parameters α²(in-plane σ bonding), β²(in-plane π bonding), and γ²(out-plane π bonding), were predicted based on the values of the basic parameters derived (g_‖, g_┴ and A_‖) as shown in Table 5. The resulting values indicate that K_2‖ > K_2┴, suggesting the presence of a large external bond in the copper complex, confirming its strong covalent nature.

Figure 2:

The ESR spectra of H ₂ L and C1, C2, C3 and C4 at room temperature.

3.10 Thermal analysis

In the current work, we conducted thermal analysis to investigate the thermal stability of Schiff base complexes, synthesized during the research. The aim was to determine the presence or absence of solvent molecules, and whether they were located inside or outside the inner coordination sphere of the complex. ^{47 , 48} The ligand and its complexes underwent thermogravimetric analysis from 0 to 600 °C with a heating rate of 10 °C min⁻¹. All thermal decomposition data for the ligand and its complexes shown in (Figure 3) are listed in (Table 6). The thermal curve pattern for the decomposition of C1, C2, and C3 complexes converges in three steps, with a slight difference in C4, which has four steps. The complexes begin to decompose in the first step at a temperature range at 218–243 °C with the loss of the two azomethine groups and part of the organic ligand. In the second step, the remaining organic ligand decomposes at 235–520 °C, leaving the metal oxide as a final product behind.

Figure 3:

TGA curve of H ₂ L and C1, C2, C3 and C4.

3.11 Geometrical structures

The geometries of ligand and the metal complexes were calculated after optimizing the angle and bond lengths using the Gaussian 09 program package. ⁴⁹ Density functional theory (DFT) method has been used, the B3LYP functional along with the 6-31G(d) basis set for ligands and LANL2DZ for metals in the solvent phase. ⁵⁰ Structure data for the selected bond lengths are listed in (Table 7 and 8). The optimized conformations of all complexes are listed in (Figure 4). Structure data for the selected bond lengths are listed in (Table S2). The bond lengths of all complexes of the optimized conformations in (Figure 11) are listed in (Tables S3–S4). The theoretical data showed several points:

1. The lengths of bonds belonging to the coordinated atoms suffer elongation from the free ligand, due to the entry of MO and MN into coordination.

2. The MN bond is longer than the MO bond in all complexes.

3. There was no significant change in the other bond lengths of the atoms.

Figure 4:

The optimized conformations of H ₂ L and C1, C2, C3 and C4.

The two basic orbitals that play a crucial role in chemical stability are the highest molecular orbital, HOMO, and the unoccupied molecular orbital, LUMO. The HOMO refers to the ability to donate an electron, while LUMO refers to the ability to gain an electron as an electron acceptor. The (Table 9 and Figure 5) list the HOMO and LUMO energies obtained from calculations of complexes with C1, C2, and C3. Moreover, additional parameters such as HOMO–LUMO energy gap E_g, absolute electronegative χ, chemical potentials Pi, absolute hardness η, absolute softness σ, global electrophilicity ω, global softness S and additional electronic charge ΔN_max were calculated and are listed in (Table 9). ^{51 , 52} The energy gap E_g. between HOMO and LUMO is an important metric for predicting the stability of compounds. A lower value of the energy gap E_g. indicates a higher reactivity of the compound. ⁵² Compared to the other complexes, the C3 complex was found to be the most reactive. The energy gap values of the complexes C1, C2, C3, and C4 are 0.76, 0.73, 0.43, and 2.57 eV, respectively.

Figure 5:

Molecular orbital distribution plots of HOMO, LUMO state, and MESP of the compounds.

Understanding the polarizability, acid-base behavior, structural properties of a molecule, and electrophilic attack sites through electrostatic potentials is crucial and has various applications in quantum chemical calculations. Figure 5 demonstrates the significance of the MESP (Molecular Potential Surface) chart, which was introduced by Gauss View 6.0.1 in the chart, the red, green, and blue colors indicate the negative, neutral, and positive ESP (Electrostatic Potential) regions, respectively. Figure 6 depicts the most positive element of the Cd(II) atom. Generally, the highest negative ESP region is observed in the phenolic oxygen atoms, the two nitrogen atoms of the Schiff base, and the two nitrogen atoms of the phenazine moiety.

Figure 6:

Molecular electrostatic potential map of compounds.

3.12 Biological activity

3.12.1 Antimicrobial activity

The antibacterial and antifungal activities of a ligand and its complexes were tested against six different microorganisms, including B. subtilis, S. aureus, E. coli, P. aeruginosa, A. flavus, and C. albicans. The tests were conducted using a DMSO solvent with a concentration of 1 mg mL⁻¹, and the well-diffusion method was employed to determine the results. The standard drugs Ciprofloxacin, Tetracycline, and Amphotericin B were also screened at the same concentration. The data presented in (Table 10 and Figure 7) demonstrate that the complexes exhibit moderate to good antibacterial activity as compared to standard drugs. The following points provide a summary of the obtained results:

1. The ligand and its complexes were found to have only slight activity against C. albicans, except the C3 which showed more significant activity.

2. The ligand and its complexes exhibited higher activity against bacterial strains and, in some cases, higher than the standard drugs tested. iii. In some cases, the data showed that activity increased after complexation compared to the free ligand. ⁵³ This can be attributed to the chelation theory, which proposes that complexation reduces the polarity of the bond between the central atom and the ligand, making it easier for the complexes to penetrate the lipid layer of the cell membrane. ⁵⁴

Table 9:

The selected bond length (Å) of complexes.

H2L	Start atom	End atom	C1	Start atom	End atom	C2	Start atom	End atom	C3	Start atom	End atom	C4	Start atom	End atom
CNC 122.38	C21	N1	N–Co–N 86.25	N1	N2	N–Ni–N 86.84	N1	N2	N–Cu–N 87.52	N1	N2	N–Cd–N 86.99	N1	N2
CNH	C23	H35	N–Co–O 94.41	N1	O1	N–Ni–N 94.40	N1	O1	N–Cu–O 94.24	N1	O1	N–Cd–O 93.57	N1	O1
CNH	C23	H35	N–Co–O 179.41	N1	O2	N–Ni–O 178.74	N1	O2	N–Cu–O 176.26	N1	O2	N–Cd–O 178.83	N1	O2
CNC	C22	N2	N–Co–O 179.34	N2	O2	N–Ni–O 178.74	N2	O1	N–Cu–O 176.26	N2	O1	N–Cd–O 178.83	N2	O1
COH 123.83	C1	H37	O–Co–O 85.00	O1	O2	N–Ni–O 94.41	N2	O2	O–Cu–O 83.81	N2	O2	O–Cd–O 93.57	N2	O2
COH 123.83	C7	H38	Co–NC 125.22	Co	C21	Ni–NC 126.19	Ni	C21	Cu–NC 125.57	Cu	C21	Cd–NC 124.86	Cd	C21
			Co–NC 125.29	Co	C23	Ni–NC 121.71	Ni	C23	Cu–NC 109.59	Cu	C23	Cd–NC 126.86	Cd	C23
			Co–OC 130.59	Co	C1	Ni–OC 133.91	Ni	C1	Cu–OC 132.04	Cu	C1	Cd–OC 131.15	Cd	C1
			Co–OC 130.67	Co	C7	Ni–OC 133.91	Ni	C7	Cu–OC 132.03	Cu	C7	Cd–OC 131.15	Cd	C7

Table 10:

Antimicrobial activity of the investigated complexes.

Compound	Antimicrobial activity (inhibition zone diameter in mm)
	B. subtilis (+G)	S. aureus (+G)	E. coli (−G)	P. aeruginosa (−G)	A. flavus (fungus)	C. albicans (fungus)
DMSO, control	NA	NA	NA	NA	–	–
Ciprofloxacin	7	5	7	7	–	–
Tetracycline	27	28	32	23	–	–
Amphotericin B	–	–	–	–	21	20
HL	25	30	18	14	7	5
C1	36	33	24	22	5	5
C2	28	30	24	26	8	8
C3	28	32	25	24	14	14
C4	38	37	28	16	7	27

Figure 7:

Antimicrobial activity of the compounds and standard drugs.

3.12.2 Antioxidant activity

The antioxidant activity of the ligand and its complexes was evaluated by comparing them with ascorbic acid, which was used as a standard. The DPPH assay results for the ligand and its complexes as shown in (Figure 8) indicated that they had higher antioxidant activity than the standard. The IC₅₀ values revealed that the ligand and its complexes exhibited antioxidant activity in the following order: C3 > C2 > C1 > C4 > H ₂ L.

Figure 8:

Antioxidant activity of compounds and standard (ascorbic acid).

3.12.4 Anti-inflammatory activity

Inflammation can be caused by various factors such as temperature, external stress, and organic solvents, which can disrupt protein structure and lead to pathogenic diseases. Inflammation can be beneficial as a defense mechanism against tissue damage, but it can also contribute to serious illnesses such as asthma, arthritis, etc.(Table12 and Figure9) ³⁰

1. The study showed that all compounds were highly effective in inhibiting inflammation, and the percentage of inhibition rate increased with the concentration, as shown in (Table 12). The anti-inflammatory activity of the ligand can be observed, which is comparable to its complexes with enhanced efficacy.

2. It has been observed that the complexes exhibit a trend of C4 > C3 > C2 > H ₂ L > C1 in terms of activity. This is due to the property of protein denaturation present in the complexes, which helps prevent the formation of adema and water retention and indicates a decrease in inflammation. ⁵⁵ Other factors such as hydrophilicity, chelation, and metallic effects may also impact biological activity. ⁵³

3. From the data listed in (Table 12 and Figure 9), the results indicate that C4 complex is significantly active as an anti-inflammatory with 34.74 % at 200 μg mL⁻¹, which can be compared with standard drugs.

Table 12:

Anti-inflammatory activity of ligand and their complexes.

Symbol	Inhibition % ± SD
	15	25	50	100	200
H2L	17.93 ± 0.02	20.68 ± 0.05	24.97 ± 0.08	29.03 ± 0.06	34.51 ± 0.05
C1	16.59 ± 0.04	20.84 ± 0.04	25.03 ± 0.02	29.77 ± 0.05	34.86 ± 0.02
C2	18.57 ± 0.08	21.07 ± 0.06	25.18 ± 0.10	30.26 ± 0.05	35.18 ± 0.02
C3	19.23 ± 0.06	21.84 ± 0.09	26.15 ± 0.10	31.16 ± 0.07	35.93 ± 0.02
C4	21.07 ± 0.06	23.54 ± 0.03	27.94 ± 0.08	34.75 ± 0.06	40.08 ± 0.09
Diclofenac sodium	23.86 ± 0.02	29.57 ± 0.05	33.16 ± 0.08	38.75 ± 0.02	41.63 ± 0.02

Figure 9:

Anti-inflammatory activity of compounds and standard drug as % inhibition.

3.12.5 Anti-tritubercular activity

In vitro, the activity of the compounds and standard drug against M. tuberculosis was evaluated using the proportion method, ²⁷ PZA was used as a positive control, and the results obtained were quite encouraging. The findings are listed in Table 13. Among the compounds examined, C4 was found to be the most effective, followed by C2, which showed higher activity compared to PZA against M. tuberculosis (Figure 10). The rest compounds showed activity similar to that of the standard drug against tuberculosis.

Table 13:

Anti-tuberculosis activity data for compounds.

Compound	MIC (μg mL−1)
H2L	2.45 ± 0.018
C1	3.16 ± 0.07
C2	2.68 ± 0.10
C3	2.74 ± 0.04
C4	4.05 ± 0.05
PZA	3.2 ± 0.05

Figure 10:

The antituberculosis activity of compounds and (PZA) standard drug.

3.13 Molecular docking

A molecular docking study was carried out using AutodockVina to simulate docking with the target amino acids of the protein (PDB ID:1DQZ) and verify the binding properties of the compounds designed as tuberculosis drugs (Figure 11). ³¹ Table 14 summarizes the binding affinity and type of binding between compounds with active sites in the amino acids of the protein. The major residues of the diethylamine site and the phenazine moiety’s aromatic ring form hydrophobic interactions via Pi-alkyl bonding, for Instance, VAL-B163 and PRO-B237. Pi-anion binding was only observed in C2, which interacts electrostatically through the aromatic ring of the phenazine moiety with ASP-B234. Based on the binding affinity values of the compounds prepared, we found that the highest value was obtained for compound C4.

Figure 11:

Predicted position of (a) H ₂ L; (b) C1; (c) C2; (d) C3; (e) C4 from docking analysis and binding interactions with amino acids.

The C4 exhibited a higher binding capacity with the protein of M. tuberculosis compared to other compounds, as shown by docking studies, which is consistent with the laboratory results.

3.14 ADMET analysis

Many compounds cannot be used as medicine due to various reasons such as insufficient absorption, distribution, high levels of toxicity, or excretion. These effects are collectively known as ADMET. ³⁰ To determine whether an organic ligand and its synthesized metal complexes have a toxic effect after ingestion or have a good pharmacokinetic profile, they need to be tested thoroughly. To calculate ADMET parameters, the AdmetSAR web server was used. The server calculates pharmacokinetic, pharmacodynamic parameters and human intestinal absorption, etc. ³¹ The organic Schiff ligand and its metal(II) complexes exhibit promising pharmacological properties for human intestinal absorption (Table 15). Negative values indicate a low P-glycoprotein inhibitor and CYP2D9, while the ligand and its metal complexes also show similar values for oral toxicity. Additionally, it is considered safer than some drugs that disrupt liver function as it exhibits negative results for hepatotoxicity. Although positive nephrotoxicity values are moderate but pose a still risk as a drug design.