Coordination behavior, structural, statistical and theoretical investigation of biologically active metal-based isatin compounds

Abstract

Isatin-core-containing ligands were synthesized by reacting an equimolar ratio of isatin with ethanolamine and anthranilic acid, respectively. The ligands were characterized by using FT‐IR, UV–Vis, ¹H-NMR, ESIMS and elemental analysis. The synthesized ligands were further complexed with transition metal ions in a 1:2 molar ratio. Metal complexes were also characterized by physical, analytical and spectral methods in addition to molar conductance and magnetic moment measurements. The DFT calculations at B3LYP/6–311 +  + G(d,p) level were accomplished for comparative study of isatin-based compounds. A good agreement among experimental UV–Vis, FT-IR and corresponding DFT results was found, which confirmed the purity of the studied compounds. The antimicrobial profile of all the compounds was evaluated by testing their antibacterial activity against two species of Gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes) and two species of Gram-negative bacteria (Pseudomonas syringae, Escherichia coli) and antifungal activity against three species of fungi (Aspergillus niger, Rhizopus stolonifer and Trichoderma spirale) using the disk diffusion method. Moreover, the compounds were also investigated for antioxidant activity using phosphomolybdate and DPPH assays. The ligands revealed moderate activity against targets, which increased further upon complexation with different metal ions due to the chelation process. In addition, ANOVA and Tukey test for the experimental results of biological activities for all the prepared compounds supported the significant difference in all preclinical treatments’ performance. The results showed that the isatin metal-based compounds are potent for being active as antimicrobial and antioxidant prototypes.

Graphical abstract

Introduction

Synthetic chemistry is playing an innovative role in preparing bioactive compounds (Yakan et al. 2020). Isatin (1H-indol-2,3-dione) is a privileged scaffold and is one of the most important series of polyfunctional heterocyclic derivatives that exhibit versatile activity profiles and tolerated well in humans. Due to its wide range of biological activities, it has gained many promising applications in pharmaceutical chemistry (Kakkar, 2019). It is also considered as a versatile substrate for the synthesis of different isatin-based compounds, such as indoles and quinolines, and also as a starting material for drug synthesis (Havrylyuk et al. 2012). These compounds have been reported to show numerous biological activities including antibacterial (Guo 2019), antifungal (Dar et al. 2019), antiviral (De Moraes et al. 2019), anti-HIV (Li et al. 2018), anti-inflammatory (Brandão et al. 2020), anticancer (Ibrahim et al. 2016), anti-amyloid (González et al. 2009), antiulcer, antioxidant and anti-leishmanial activities (Sonmez et al. 2019). Isatin analogous was reported to display neuropharmacological and neurophysiological effects like antimycobacterial activity (Penthala et al. 2010).

Among isatin derivatives, isatin imines (Schiff bases) were found to exhibit a wide spectrum of biomedical activities. Isatin scaffold-bearing Schiff bases have also been successfully employed as ligands, thus forming complexes with numerous transition metal cations. So, over the years, the research on the metal complexes of isatin-derived Schiff-base ligands has gained much interest (Bashiri et al. 2021). The metal complexes of isatin-derived Schiff bases have exhibited multifarious progress in the pharmacological industry because of their therapeutic and diagnostic properties (Shakir et al. 2016). Out of these complexes, Cu(II) complexes of isatin imines have attracted much more consideration, mainly owing to their effective anticancer activities. Likewise, many isatin-derived imines and their respective Cu(II) complexes have also been reported in the literature to demonstrate promising antimicrobial and cytotoxic action against various cells (Pervez et al. 2016). Isatin-derived metal complexes have also shown moderate to significant antibacterial action against various bacterial strains. Considering the antibacterial properties, mono-isatin-derived metal complexes displayed weaker activity than the bis-isatin-derived metal complexes (Guo, 2019).

Bacterial resistance is one of the most important worldwide health issues due to the multidrug resistance of pathogenic species (Kaur et al. 2010). Currently, the design, study and synthesis of biologically active metal-based chelates from Schiff base ligands with nitrogen and oxygen donor atoms are one of the embryonic areas of research owing to their competent pharmacological actions (Sumrra et al. 2021a, b, c; Ejidike and Ajibade 2015). Predominantly, the Schiff base ligands possessing nucleophilic substituents such as –OH, –SH and –NH₂ at ortho position to the imine linkage were found to be more appropriate for complexation with transition metal cations, thus producing more stable metal chelates (Sumrra et al. 2021a, b, c). These types of nitrogen- and oxygen-coordinated metal complexes are widely studied due to their excellent biological and physicochemical properties (Sakthikumar et al. 2019). They are predominant among the type of compounds used as pharmaceuticals (Dharsini et al. 2020), antioxidants, sensitizers and dyestuff (Ejidike and Ajibade 2016). They are significant topics of structural and synthetic research due to their structural versatility (Pedreño et al. 2005), easy synthetic approach and more significant biological activity than the non-coordinated ligands (Nagesh and Mruthyunjayaswamy 2015).

The reactivity of transition metal chelates is influenced by the type of coordinating ligands, the oxidation state of the central metal ion and the coordination geometry of the metal complexes (Sumrra et al. 2020). Existing antibiotic resistance is one of the leading sources of motivation to develop and explore new effective agents for inhibiting bacterial growth (Mitra et al. 2013). The metal ions complexed with bioactive agents are more effectual, for the reason that both the components (ligands and metal ions) have the potential to affect various stages of pathogenic life cycles. In most cases, metal complexes are capable of catalyzing life-sustaining metabolic reactions. The metallic part of the complex is typically responsible for effectively binding with DNA, thus damaging the vital genetic material of microbes (Zafar et al. 2021).

Recently, the quantum chemical approaches established on density functional theory (DFT) have been credibly used to accelerate the high throughput drug screening (Akman et al. 2020). These computational methodologies have been acknowledged for their accuracy and efficiency when it comes to predict and understand the electronic structure of newly synthesized compounds to correlate their experimentally observed structural features (Sagaama et al. 2020). The chemical reactivity of molecules was predicted by employing the frontier molecular orbitals (FMOs) accompanied by eminent descriptors (Jomaa et al. 2020). Moreover, various electronic properties including FMO energy gaps and absorption wavelengths together with oscillator strengths have been calculated through TD-DFT method (Issa et al. 2020).

In continuance of our previous work concerning metal-based isatin compounds (Khalid et al. 2020), in the work reported here we aimed to synthesize some more divalent and tetravalent complexes with our target isatin-based ligands. Spectral characterization and molecular modeling were employed for the structural elucidation of synthesized compounds. Theoretic studies using DFT and time-dependent DFT computations have been executed and combined with experimental results throughout this work for structural, electronic and vibratory characterization. The biological activity of synthesized compounds with moderate to significant biological interactions was explored by their FMO energies gaps. In vitro biological activities were performed to study the antimicrobial and antioxidant activity profile of all the compounds. According to the results, all the studied compounds were found to have therapeutic potential and exhibited exciting properties from the pharmacological aspect which will ultimately prove the capability of these active components in new medicinal products.

Experimental

All the required chemicals were ordered from Merck, Sigma–Aldrich (A.R), and consumed as received without additional refinement. Stuart apparatus was utilized to measure the melting points of all compounds. Infrared spectra were collected using a Nicolet, iS5 FT-IR spectrophotometer in the working range of 4000–400 cm⁻¹. UV–Vis spectra were recorded on a Hitachi UV-3200 spectrophotometer using DMF (dimethylformamide)-based solutions. The C, H and N analysis was performed on a high tech, 2400 CHN elemental analyzer. ¹H-NMR spectra were documented on a 300 MHz Bruker instrument. For the electron impact mass spectrometric analysis, the isatin compounds (including the ligands and their complexes) were dissolved in methanol and inserted directly by the use of a direct insertion pump into the LTQ XL linear ion trap mass spectrometer (Thermo Scientific, USA) fitted out with an electrospray ionization (ESI) probe. Capillary temperature was fixed at 350 °C, while the flow of sheath gas (N₂) at 30 arbitrary units. All the samples were analyzed in both modes: positive- and negative-ion mode with a 10-µL/min flow rate. Then, the data were collected at both types of full-scan modes with a mass scan m/z ranging from 50 to 700 using Xcalibur software.

By using the full-scan ESI–mass spectra, the synthesized compounds were characterized. ESI–MS gave detailed m/z peaks by making [M + Na]⁺, [M-H]⁻ and [M + H]⁺ ions. The ESI–MS/MS fragmentation pattern of the known peaks was utilized to further fragment the daughter ions to confirm the molecular identity of each ligand and complex. Molar conductivity of synthesized complexes was determined by the use of Inolab Cond 720 Conductivity Bridge at room temperature with 0.001 molar DMF-based solutions. The magnetic susceptibility of the synthesized complexes was executed with the magnetic susceptibility balance (MSB) (Mk1 design) using MnCl₂ as standard.

Synthesis of ligands (F1H) & (F2H)

The isatin-based ligands, namely 3-[(2-hydroxyethyl)imino]-1,3-dihydro-2H-indol-2-one (F1H) and 2-{[2-oxo-1,2-dihydro-3H-indol-3-ylidene]amino}benzoicacid (F2H), were prepared by implementing a previously described protocol (Arunachalam et al. 2010) as shown in Scheme 1. For the synthesis of ligand (F1H), to the refluxed ethanolic solution of isatin (1.47 g, 10 mmol) in a double-neck flask, the solution of ethanolamine (0.60 mL, 10 mmol) was added with constant stirring after 1 h of refluxing. The reaction progression was continuously noted through TLC (thin-layer chromatography). The precipitates obtained after 21 h were cooled at room temperature, followed with filtration, washed with boiling ethanol and then recrystallized in an equimolar mixture of ether and ethanol to get pure product. Similarly, the other ligand (F2H) was synthesized with the same protocol by using anthranilic acid instead of ethanolamine.

Scheme 1

Synthesis of isatin-derived Schiff bases (F1H) and (F2H)

3-[(2-Hydroxyethyl)imino]-1,3-dihydro-2H-indol-2-one (F1H)

Yield (%): 65; m.p (°C): 126; color: dark brown; IR (cm⁻¹): 3420 (OH), 3241 (NH), 1715 (C = O), 1644 (C = N); ¹H-NMR (DMSO-d₆) δ ppm: 3.34–3.99 (t, aliphatic protons), 7.62 (s, 1H, NH), 6.75–7.64 (m, aromatic protons), 4.02 (s, 1H, OH); ESIMS m/z (%): 163.92 (100), 147.92 (58), 174.00 (56), 191.92 (38), 180.42 (8), 131.00 (6), 118.92 (2); Anal. Calcd. (%) for C₁₀H₁₀N₂O₂; 190.1: calculated; C 63.15, H 5.30, N 14.73: found; C 63.07, H 5.27, N 14.69.

2-{[2-Oxo-1,2-dihydro-3H-indol-3-ylidene]amino}benzoicacid (F2H)

Yield (%): 77; m.p (°C): 185; color: dark orange; IR (cm⁻¹): 3389 (OH), 3220 (NH), 1710 (C = O), 1632 (C = N); ¹H-NMR (DMSO-d₆) δ ppm: 11.04 (s, 1H, OH), 7.51 (s, 1H, NH), 6.89–7.62 (m, aromatic protons); ESIMS m/z (%): 266.25 (100), 205.17 (28), 179.17 (20), 146.08 (10), 247.25 (5), 136.08 (4), 118.00 (2); Anal. Calcd. (%) for C₁₅H₁₀N₂O₃; 266.3: calculated; C 67.67, H 3.79, N 10.52: found; C 67.51, H 3.68, N 10.41.

Synthesis of metal complexes

The ethanolic solution of respective metal salt [hydrated iron(II) sulfate, cobalt(II) chloride, nickel(II) chloride, copper(II) chloride, zinc(II) chloride and vanadyl(IV) sulfate] (0.5 mmol) was added to the magnetically refluxing ethanolic solution of isatin-based ligand (1 mmol) in two neck flask, after 1 h of refluxing (Scheme 2). The reaction progress was continuously checked through TLC. The recovered precipitates of the product were cooled at room temperature, followed with filtration, washed with hot ethanol and afterward recrystallized in the equimolar mixture of ether and ethanol.

Scheme 2

Synthesis of metal-based isatin-derived Schiff base compounds (1)–(12)

Antimicrobial activity

Antibacterial activity

All the synthesized ligands and their derived 3d-metal complexes have been tested for antibacterial activity using two species of Gram-positive bacteria (S. aureus, S. pyogenes) and two species of Gram-negative bacteria (P. syringae, E. coli) by disk diffusion test (Sumrra et al. 2016). The nutrient broth, petri plates and filter paper strips were autoclaved at 121 °C for half an hour. Partially liquid agar media were distributed to petri plates and allowed to solidify; then, the bacterial inoculum was dispersed on the media using a glass spreader. Round strips of filter paper were arranged on the media, and 20 µL sample (25 mg/mL concentration in DMSO solvent) was injected on disks using a micropipette. Amoxicillin was used as a standard in this procedure. Then, the petri plates were stacked in an incubator at 37 °C and the inhibition zone was measured in millimeter after 48 h.

Antifungal activity

Newly prepared isatin-based ligands and their complexes were checked for in vitro antifungal activity against three fungal strains (A. niger, R. stolonifer and T. spirale) by disk diffusion test (Sumrra et al. 2016). The media (prepared by adding 2 g glucose, 2 g agar, 0.02 g potassium dihydrogen phosphate, 0.005 g magnesium sulfate, 0.005 g calcium chloride and 0.02 g ammonium sulfate), petri plates and filter paper strips were autoclaved at 121 °C for half an hour. Semi-liquid media were distributed to petri plates and left for setting; then, the fungal inoculum was dispersed on the media using a glass spreader. Round strips of filter paper were arranged on the media, and 20 µL sample (25 mg/mL concentration in DMSO solvent) was injected onto disks using a micropipette. Fungom was used as a standard in this procedure. Then, the petri plates were stacked in an incubator at 37 °C and the inhibition zone was measured in millimeter after 48 h.

Antioxidant activity

Antioxidant activity of prepared compounds was determined using two methods, i.e., DPPH and phosphomolybdate assays.

DPPH method

Antioxidant activity of prepared compounds was checked through the recommended procedure using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical (Bakır and Lawag 2020). Five different concentration levels (50, 100, 150, 200 and 250 µg/mL) of the compounds and ascorbic acid (standard) were prepared, which were then poured in the test tubes and mixed with freshly prepared DPPH (2 mL, 2 mM) solution and then kept in the dark for half an hour. After that, the absorbance of compounds and standard was noted at 517 nm wavelength, and the percentage inhibition was quantified by using the following formula (Eq. 1).

$$Percentage \, \left( \% \right) \, inhibition = \frac{{\left( {Blank - Sample} \right)}}{Blank} \times \, 100.$$

(1)

Phosphomolybdate method

The total antioxidant capacity of isatin-derived Schiff bases (F1H) and (F2H) as well as their metal chelates was determined through the recommended procedure (Sumrra et al. 2018). Respective compound solution (0.2 mL) was poured in reagent solution (2 mL) that was prepared through intermingling sodium phosphate (28 mM), ammonium molybdate (NH₄)₂MoO₄ (4 mM) and 0.1 M H₂SO₄ (5 mL). This mixture was warmed up to 95 °C temperature for 35 min, and the absorbance reading was then noted at 695 nm wavelength using UV–Vis spectrophotometer.

Statistical analysis

The statistical analysis was accomplished by the use of Minitab software (Keeling and Pavur, 2007) to interpret the experimental outcomes of biological studies, including antibacterial, antifungal and antioxidant activities.

Computational details

Computational calculations of the investigated isatin-derived ligands (F1H) and (F2H) as well as their selected metal complexes were accomplished by DFT using B3LYP (Becke, 3 parameters, Lee–Yang–Parr) level of theory in combination with basis set 6–311 +  + G(d,p) through Gaussian 09 D.0.1 package (Frisch et al. 2009). The B3LYP functional is the modified version of the Becke hybrid functional and is most commonly used hybrid functional. All the input data files of the studied compounds were obtained by using GaussView software (Dennington et al. 2009).

In DFT calculations, at the start, the geometries of all the studied compounds were completely optimized by employing DFT/B3LYP in combination with 6–311 +  + G(d,p) basis set. After that, using the frequency calculations at the same basis set, the stability profile was confirmed for all the optimized geometrical structures. The lack of negative eigenvalues out of all the computed frequencies signified proper minimal level of their potential energy surfaces.

Neutral structures of all the investigated compounds were obtained after optimization of their geometries. The UV–visible analysis was accomplished using TD-DFT with B3LYP/6–311 +  + G(d,p) (Brémond et al. 2010). The IR, FMO, NBO, MEP and MAC calculations were done on these optimized geometries by the use of same settings. Finally, the output data were interpreted by means of Chemcraft, GaussView, Avogadro and GaussSum programs.

Results and discussion

Physical characterization

The isatin Schiff base ligands (F1H) and (F2H) have been prepared by the condensation reaction of isatin moiety with ethanolamine and anthranilic acid, respectively, in an equimolar ratio using ethanol as solvent. The prepared isatin-based ligands were complexed with metallic cations of vanadium, iron, cobalt, nickel, copper and zinc. All the compounds were stable in air and moisture. All the compounds were synthesized with a good yield ranging from 62 to 84%. Melting points of ligands and decomposition points of complexes provided a strong indication about the formation of both ligands and their resultant metal complexes.

All the 3d-metal chelates were obtained in intense colors except zinc complexes. They were completely soluble in ethanol (EtOH), methanol (MeOH), dimethyl sulfoxide (DMSO) and dimethylformamide (DMF), but moderately soluble in other organic solvents. The proposed CHN data showed high agreement with the practically observed data. The data of elemental analysis together with some physical properties for all the compounds are depicted in Table 1.

Table 1 Physical, elemental and analytical characteristics of synthesized compounds

Spectral characterization

The ligands were spectrally characterized through ¹H-NMR, FT-IR, UV–Vis and mass spectrometry. The experimental IR and UV–Vis results of the both isatin ligands and their selected metal complexes have also been compared with theoretical findings obtained by DFT calculations. But the complexes were also characterized using conductance and magnetic moment values.

FT-IR spectral characterization

The formation of these two Schiff bases (F1H) and (F2H) was proven from the absence of (NH₂) and (C = O) peaks in their spectra. The experimental IR spectra of isatin-based ligands showed characteristic bands of (C = N) at 1632–1644 cm⁻¹ that gave a strong intimation about the condensation of reactants (Ismail et al. 2021). Both ligands also exhibited bands at 1710–1715, 3110–3191, 3220–3241 and 3389–3420 cm⁻¹ due to v(C = O), v(C–H), v(NH) and v(OH) groups, respectively. With the purpose of determining the involvement of coordinating sites in chelate formation, the IR spectral data of complexes were studied in comparison with free ligands. The IR data for all the synthesized metal complexes showed the appearance of some new peaks along with the disappearance and shifting of some already existing peaks that gave a clue about metal and ligand coordination, as depicted in Table S1 and Figs. S1–S3.

The disappearance of (OH) peaks at 3389–3420 cm⁻¹ of ligands and downward shifting of v(C–O) peak from 1389–1390 to 1362–1387 cm⁻¹ signified the coordination of isatin-based ligands with metal cations through deprotonated oxygen atom of the hydroxyl group (Ouari et al. 2015). For complexes, a new peak was observed in the range of 448–491 cm⁻¹ owing to v(M–O) that confirmed the metal–ligand bond through oxygen (Al‐Hazmi et al. 2020). The bonding action of azomethine–nitrogen with metal cations was supported by the downward shift of frequency from 1632–1644 cm⁻¹ to 1610–1631 cm⁻¹ in all metal chelates. Another new peak was also observed ranging from 517 to 532 cm⁻¹ because of v(M–N) that showed the formation of metal–ligand bond through nitrogen (Chohan et al. 2010). For the vanadium complexes of both ligands, a new peak was observed at 840 and 869 cm⁻¹, respectively, indicating v(V = O) vibration (Grivani et al. 2012). All these signals supported the coordinating action of metal ions with the isatin-derived ligands.

UV–Vis spectral characterization

The electronic spectra of isatin-based compounds have been taken in DMF solutions (10^–1 M) in the scan range of 200–800 nm. The UV–Vis spectra of ligands (F1H) and (F2H) demonstrated two and three absorption bands 289 and 400 nm and 250, 400 and 490 nm because of π–π* and n–π* electronic transitions, correspondingly. In the metal complexes, new bands supported the formation of strong (M–N) and (M–O) bonds. The vanadyl(IV) complexes have exhibited their representative bands at 378–385, 542–554 and 749–768 nm which ascribed to the B₂ → E_π, B₂ → B₁ and B₂ → A₁ electronic transitions, respectively. The high-intensity bands as a result of charge transfer from metal to ligand were observed at 327–334 nm. All these transitions signified the square pyramidal environment around VO(IV) ions. The Fe(II) complexes have shown their two representative electronic transitions comprising ⁵E_g and ⁵T_2g at 376–383 and 512–532 nm, approved their octahedral configuration.

The low-energy electronic bands of Co(II) complexes at 512–546 and 639–662 nm due to ⁴T_1g(F) → ⁴T_1g(P) and ⁴T_1g(F) → ⁴T_2g(F) electronic transitions together with a band of high energy at 344–352 nm attributable to metal to ligand charge transfer (MLCT), signified their high spin octahedral geometry (Kargar et al. 2021). Similarly, for Ni(II) complexes, three absorption bands appeared at 387–419, 604–627 and 765–782 nm as a result of ³T_2g(F) → ³T_1g(P), ³A_2g(F) → ³T_1g(F) and ³A_2g(F) → ³T_2g(F) electronic transitions, while the most intense band of MLCT was found at 329–336 nm, thus evidencing their octahedral geometry. The octahedral geometry for Cu(II) complexes was confirmed owing to the occurrence of two electronic transitions reflecting ²B_1g → ²E_g and ²B_1g → ²A_1g excitations at 517–529 and 631–648 nm, correspondingly, while the third band due to MLCT having high intensity was obtained at 337–342. For Zn(II) complexes, only a strong band was obtained for MLCT at 324–339 nm owing to their diamagnetic nature (Hassan and Sumrra 2021).

¹H-NMR spectral characterization

The ¹H-NMR spectra of both isatin-based ligands (F1H) and (F2H) were documented using DMSO-d₆ as a solvent. The examination of spectra showed the characteristic peak of N–H proton at 7.51–7.62 ppm as a singlet. The aromatic protons of both ligands were observed as a multiplet at 6.75–7.64 ppm. The O–H proton of ligands was obtained at 4.02 and 11.04 ppm, respectively. For ligand (F1H), two signals were also observed as a triplet at 3.34–3.99 ppm for aliphatic protons of ethanolamine. All the observed signals supported the formation of both ligands and their proposed structures, as shown in Figs. S4 and S5.

Mass spectral characterization

Mass spectra of ligands (F1H) and (F2H), as well as vanadium and zinc complexes of ligand (F2H), showed various peaks at different intensities indicating molecular ion peaks, base peaks and other fragments (Table S2). For ligands (F1H) and (F2H), the molecular ion peaks appeared at m/z = 191.92 and 266.25, correspondingly that confirmed the formation of ligands (Figs. S6 and S7). The fragmentation design of isatin-based ligands showed the C–C, C = C and C = N bond breaking. The base peak and molecular ion peak of ligand (F2H) were found at the same fragment. But for ligand (F1H), the highly intense base peak was found at m/z = 163.92. All the peaks confirmed the formation of both ligands. The proposed structures of metal complexes were also confirmed through the mass spectra as depicted in Figs. S8 and S9. The complex VO(F2)₂ exhibited a molecular ion peak at m/z = 598.00, while the base peak at m/z = 388.17. Similarly, the molecular ion peak for Zn(F2)₂ complex was observed at m/z = 627.17, whereas the most intense and stable peak was determined at m/z = 266.25.

Molar conductance and magnetic moments

Measurement of molar conductance for all the metal chelates was performed in DMF solvent. The results of molar conductance (11.8–19.3 Ω⁻¹cm²mol⁻¹) displayed the non-conductor identity of the metal complexes.

The magnetic moments of all synthesized chelates were determined by magnetic susceptibility balance (Table S1). The copper and vanadium complexes showed magnetic moment values ranging from 1.72 to 1.93 BM, indicating the occurrence of one unpaired electron (Maurya et al. 2015). The measured magnetic moments of cobalt complexes were found to be 4.21–4.33 BM, which showed the existence of three unpaired electrons. The nickel complexes displayed a magnetic moment at 3.01–3.15 BM, revealing the occurrence of two unpaired electrons (Hanif and Chohan 2013). The magnetic moments of the iron complexes were found to be 5.03–5.07 BM, showing the presence of four unpaired electrons in the d-orbital. The zinc complexes showed zero magnetic moment value that indicated the diamagnetic nature of these complexes due to the d¹⁰ system (Chandrasekar et al. 2021). The data of molar conductance and magnetic moments of the metal complexes supported the suggested structures of the metal complexes.

Geometry optimization

The optimized geometrical structures of the both isatin-derived ligands and their selective metal chelates were visualized through Chemcraft software. Figure 1 illustrates the molecular geometries for all the compounds together with their atom numbering.

Fig. 1

Optimized structures of isatin-based compounds

Frontier molecular orbital (FMO) analysis

Frontier molecular orbitals are used to explore and describe the optical parameters, stability, chemical reactivity and electrical parameters. HOMO and LUMO act as donor and acceptor, and electron excitation occurs from HOMO to LUMO. In this study, the FMOs energies, i.e., E_HOMO, E_LUMO, and their energy gaps ΔE_HOMO–LUMO∆H–L were quantified by the use of B3LYP/6–311 +  + G(d,p) for both isatin-derived ligands, their selective metal complexes as well as reference compounds (ascorbic acid, indomethacin and gallic acid) (Hassan et al. 2021) as depicted in Table 2 and displayed in Fig. 2 and Fig. 3. The antimicrobial action and antioxidant aptitude of compounds are strongly associated with the spatial distribution of occupied molecular orbitals signifying most appropriate sites in investigated compounds that are susceptible for attack by reactive agents besides free radicals. The overlapped FMOs indicated extremely responsive behavior of the studied compounds concerning the antioxidant and antimicrobial action.

Table 2 Energies of frontier molecular orbitals and computed global reactivity descriptors for isatin-based compounds and reference compounds

Fig. 2

Frontier molecular orbitals of ligands (F1H) and (F2H) [color codes for orbitals; yellow (positive) and blue (negative) in HOMO, while pink (positive) and green (negative) in LUMO]

Fig. 3

Frontier molecular orbitals of standard compounds [color codes for orbitals; red and orange colors illustrate the positive, while both blue colors illustrate the negative part of orbitals]

The DFT data revealed that ligands (F1H) and (F2H) contain 366 and 497 molecular orbitals, out of which orbital numbers 50, 69, and 51, 70 acts as HOMO and LUMO, correspondingly. For ligands (F1H) and (F2H), the energies of HOMO and LUMO were found to be − 6.540, − 6.331 and − 2.548, − 2.819 eV, respectively. The energy gaps of the above said orbitals were obtained to be 3.992 and 3.512 eV, respectively, for both ligands. The descending order of E_LUMO for all the studied compounds was found as: Ni(F2)₂ (− 3.255) > VO(F2)₂ (− 3.057) > (F2H) (− 2.819) > (F1H) (− 2.548) > Cu(F1)₂ (− 2.507) > Fe(F1)₂ (− 2.338) > indomethacin (− 2.160) > gallic acid (− 1.630) > ascorbic acid (− 1.160). The opposite trend for E_HOMO was observed as; ascorbic acid (− 6.710) > (F1H) (− 6.540) > gallic acid (− 6.440) > (F2H) (− 6.331) > indomethacin (− 6.000) > VO(F2)₂ (− 5.214) > Ni(F2)₂ (− 5.170) > Cu(F1)₂ (− 4.341) > Fe(F1)₂ (− 4.300). The susceptibility in the ΔE_HOMO–LUMO was noted as: ascorbic acid (5.550) > gallic acid (4.840) > (F1H) (3.992) > indomethacin (3.840) > (F2H) (3.512) > VO(F2)₂ (2.157) > Fe(F1)₂ (1.962) > Ni(F2)₂ (1.951) > Cu(F1)₂ (1.834).

In the HOMO and LUMO of ligand (F1H), the electronic charge was accumulated over the whole structure of ligand, 3-[(2-hydroxyethyl)imino]-1,3-dihydro-2H-indol-2-one with the exception of methylene (CH₂) and hydroxyl (OH) groups, while in the HOMO of ligand (F2H), the electronic charge density was partially distributed on the entire structure of ligand, 2-{[2-oxo-1,2-dihydro-3H-indol-3-ylidene]amino}benzoicacid. But in the LUMO of ligand (F2H), the charge density was only focused on the azomethine linkage and anthranilic acid part of the ligand. Likewise, in HOMO of Fe(F1)₂ and VO(F2)₂, the charge density was concentrated on the respective metal centers and the coordinating groups, while in the HOMO of Cu(F1)₂ and Ni(F2)₂, the charge density was spared over the whole structure but mostly on the respective metal centers and the coordinating groups. Moreover, in LUMO of metal complexes, electronic charge density was scattered on the entire structures.

Global reactivity descriptors

The density functional theory (DFT) is effective in quantifying numerous quantum chemical reactivity descriptors, which are utilized to evaluate the chemical reactivity besides the reactive site peculiarity of chemical systems. The FMO band energies were used to calculate global reactivity descriptors (as shown in equations S1–S6), which gave information about the behavior of the studied compounds such as stability, reactivity and softness–hardness as shown in Table 2 (Sumrra et al. 2021a, b, c). The decreasing order of IP was found as ascorbic acid (6.710) > (F1H) (6.540) > gallic acid (6.440) > (F2H) (6.331) > indomethacin (6.000) > VO(F2)₂ (5.214) > Ni(F2)₂ (5.170) > Cu(F1)₂ (4.341) > Fe(F1)₂ (4.300). While the EA order was: Ni(F2)₂ (3.255) > VO(F2)₂ (3.057) > (F2H) (2.819) > (F1H) (2.548) > Cu(F1)₂ (2.507) > indomethacin (2.160) > gallic acid (1.630) > ascorbic acid (1.160).

Among the studied ligands, (F1H) contained the greatest IP value as 6.540 eV, whereas (F2H) exhibited the lowest value as 6.331 eV. Similarly, (F2H) possessed maximum EA value, 2.819 eV; however, (F1H) exhibited minimum EA value, 2.548 eV. Therefore, the electron-donating and accepting behaviors of our ligands (F1H) and (F2H) were measured by their IP and EA amplitudes, which are actually linked to HOMO–LUMO energy gap [∆E(eV)]. As a result, the values of IP were found to be greater in magnitude than the EA, signifying that ligands (F1H) and (F2H) contained good electron-losing potential.

The decreasing order for chemical potential was: (F2H) (− 4.575) > (F1H) (− 4.544) > Ni(F2)₂ (− 4.213) > ascorbic acid (− 4.170) > VO(F2)₂ (− 4.136) > indomethacin (− 4.080) > gallic acid (− 4.030) > Cu(F1)₂ (− 3.424) > Fe(F1)₂ (− 3.319). Any molecule having smaller value of chemical potential was preferred as a less stable and more reactive molecule and vice versa. The chemical potential for examined ligands (F1H) and (F2H) was observed to be greater than the other studied compounds. The electronegativity order follows the same trend: (F2H) (4.575) > (F1H) (4.544) > Ni(F2)₂ (4.213) ascorbic acid (4.170) > VO(F2)₂ (4.136) > indomethacin (4.080) > gallic acid (4.030) > Cu(F1)₂ (3.424) > Fe(F1)₂ (3.319). Among the studied ligands, (F2H) has higher electronegativity value (4.575) (Table 2). The obtained data have shown that both the ligands (F1H) and (F2H) could be considered as chemically hard molecules with strong electron-donating ability besides maximal kinetic stability.

The subsequent descending order of hardness was obtained as: ascorbic acid (2.540) > gallic acid (2.400) > (F1H) (1.996) > indomethacin (1.920) > (F2H) (1.756) > VO(F2)₂ (1.079) > Fe(F1)₂ (0.981) > Ni(F2)₂ (0.958) > Cu(F1)₂ (0.917), whereas the softness decreased in the following order: indomethacin (1.560) > gallic acid (1.340) > ascorbic acid (1.130) >  > Cu(F1)₂ (0.545) > Ni(F2)₂ (0.522) > Fe(F1)₂ (0.510) > VO(F2)₂ (0.464) > (F2H) (0.284) > (F1H) (0.250). Any chemical system with small ∆E value was considered as reactive, soft and unstable. For the investigated compounds, calculated values of global softness (σ) were smaller in contrast to the global hardness (η); thus, the synthesized isatin-derived compounds were relatively stable and unreactive.

The order of hardness and FMO energy gap was found notably similar and can be summarized as (F1H) > (F2H) > VO(F2)₂ > Fe(F1)₂ > Ni(F2)₂ > Cu(F1)₂. The trend for electrophilicity index was: Ni(F2)₂ (9.266) > VO(F2)₂ (7.929) > Cu(F1)₂ (6.392) > (F2H) (5.959) > Fe(F1)₂ (5.615) > (F1H) (5.170) > indomethacin (4.330) > ascorbic acid (3.420) > gallic acid (3.390). The compounds with minimum values of E_HOMO exhibited reduced electron-contributing aptitude, demonstrating that the studied metal complexes might possess superior electron-donating capability in comparison with ligands and some standard compounds. These outcomes came to end with the conclusion that the metal complexes would be better biological active and antioxidant contenders.

Molecular electrostatic potential (MEP) analysis

A molecular electrostatic potential (MEP) plot is utilized to conceptualize different charge distributions in compounds. MEP is basically the interpretation of the three-dimensional electron charge density through graphical interpretation. And by using this 3D MEP plot, the physical and chemical characteristic features of a molecule can be discussed. MEP plot consists of various standard colors: orange, red, blue, yellow and green, indicating the level of electrostatic potential. The descending order for the level of electrostatic potential was: blue > green > yellow > orange > red. MEP analysis helps to recognize negative, positive and neutral zones of the investigated molecules by different shadings. On the MEP map, red color shows a highest negative potential which is actually electron-rich area and considered as a fitting spot for attack by electrophilic species, yellow color also specifies a slightly electron-rich area, and blue indicates a highly positive potential which is basically an electron-deficient zone and could be a better fitting spot for attack by a nucleophilic species. The light blue represents a slightly electron-deficient zone, whereas the green color signifies a neutral zone (Mermer et al. 2020).

In this study, the MEP surfaces of investigated compounds were figured at the B3LYP functional with 6–311 +  + G(d,p) level of theory and the pictographic display is presented in Fig. 4, which gives the valuable evidence about the possible electrophilic and nucleophilic sites within the compounds. It could be seen from the MEP surfaces that the red color displaying the electron-rich zone was displayed on the oxygen atoms. Therefore, the possibility of electrophilic attack is maximum at these sites. Blue and green zones were spread over the hydrogen and some carbon atoms specifying the electron-deficient zone areas and are implied as the best sites for the encounter of nucleophilic attack. From red, blue and green colors, it is evident that sites for various reactions were present in all the investigated compounds.

Fig. 4

MEP maps for isatin-derived compounds

Mulliken atomic charges (MAC) analysis

Mulliken population analysis has a very important part in the quantum chemical operation to molecular framework because atomic charges largely affect the electronic structure, polarizability and properties of molecules (Al-Amiery et al. 2012). Mulliken atomic charges for isatin-derived ligands and their selected metal complexes were computed through B3LYP/6–311 +  + G(d,p) and are given in Tables S3 and S4. Mulliken atomic charges were also represented as histograms in Fig. 5. The optimized structures of both ligands with atoms involved in these histograms are shown in Fig. 1. The Mulliken atomic charges showed the existence of greater electronegative atoms: N15 = − 0.191, O22 = − 0.295 and O24 = − 0.269 for ligand (F1H), while O14 = − 0.246, N15 = − 0.197, O28 = − 0.235 and O30 = − 0.839 for ligand (F2H), which has positive charge distribution on carbon atoms, while all hydrogen atoms carry positive charge (as shown in Fig. S10).

Fig. 5

Histogram of Mulliken atomic charges for isatin-based compounds

The MAC data of metal complexes have also shown unequal redistribution of the electronic charge density over the benzene rings due to the presence of atoms with high electronegativity in the structure such as nitrogen and oxygen. On the other hand, metals of the studied complexes Fe(F1)₂, Cu(F1)₂, VO(F2)₂ and Ni(F2)₂ were found to have greater electropositive values of Fe29 = 1.078, Cu29 = 0.692, V41 = 1.036 and Ni41 = 0.984, respectively.

Natural bond orbital (NBO) analysis

NBO analysis is a very excellent tool to analyze the interactions in a molecular framework. The stabilization energy obtained through this analysis directly affects the hyper-conjugative associations between donors and acceptors. NBO analysis was performed to compare and analyze the coordinating capability of ligands. NBO analysis provides an explanation of the molecular structure by a set of two center localized antibonding and bonding orbitals along with valence lone pair and one center core pair orbital. The analysis is established on the second-order perturbation theory concerned with the Fock matrix. The findings of NBO analysis were used to study and explore the second-order interactions between lone pair (LP) orbitals and empty anti-bonding orbitals (σ* or π*) as a measure for probable coordination ability and localization of lone pair (Weinhold et al. 2016).

The NBO calculations for isatin-based ligands and their selective metal complexes were measured at DFT/B3LYP/6–311 +  + G(d,p) method to obtain the perturbation theory. Very large data were obtained through analysis, but here only promising transitions which have a greater value of stabilization energies are shown in Table S5. The optimized structures of studied compounds with numbering are shown in Fig. 1. For ligand (F1H), the transfer of charge from π(C₂‒C₃) → π*(C₁‒C₆), π(C₈‒O₂₄) → π*(C₇‒N₁₄) leads to the stabilization energies as 251.32 and 62.29 kJmol⁻¹_, respectively, that showed strong delocalization within the system. For ligand (F2H), the charge transfer from π(C₁–C₆) → π*(C₂–C₃), π(C₁₇–C₁₉) → π*(C₁₈–C₂₀) and π(C₁₇–C₁₉) → π*(C₂₂–C₂₃) leads to the stabilization energies as 355.00, 288.62 and 208.60 kJmol⁻¹_, respectively, that showed strong delocalization within the system.

The interactions of lone pairs (LPs) of nitrogen and oxygen with different antibonding orbitals were considered in both ligands. All the LPs mainly have p character, and they are practically filled with two electrons. The interaction of LPs with antibonding orbitals results in the donation of electrons from LPs to antibonding orbitals. The stabilization energy E⁽²⁾ related to these interactions could be utilized as a measure of the involvement of the lone pairs in the intramolecular delocalization.

The greater stabilization energy value results in highly intensive interaction between electron-donating LPs and electron-accepting antibonding orbitals (Srivastava and Misra 2014). The LP of N₁₄ and N₁₅, interacted with antibonding orbitals, lone pair (1) N₁₄ → σ*(C₇–C₈), N₁₅ → π*(C₂–C₃) and N₁₅ → π*(C₈–O₂₄) resulted in significant stabilization by 15.94, 36.78 and 54.40 kJmol⁻¹, respectively, in ligand (F1H), while the LP of N₁₅ and N₁₆, interacted with antibonding orbitals, lone pair (1) N₁₆ → σ*(C₇–C₈), N₁₆ → π*(C₁₇–C₁₉), N₁₅ → π*(C₁–C₆) and N₁₅ → π*(C₇–O₁₄) gave rise to enormous stabilization by 19.10, 21.26, 43.12 and 65.05 kJmol⁻¹, respectively, in ligand (F2H).

In comparison, the interactions of the second lone pair of oxygen contributed less amount of stabilization (less than half of that of nitrogen). The LP of O₂₄ interacted with antibonding orbitals, lone pair (2) O₂₄ → σ*(C₇–C₈) and O₂₄ → σ*(C₈–N₁₅) caused substantial stabilization by 21.50 and 26.76 kJmol⁻¹, respectively, signifying the strong conjugative interaction of O to C–C, C–N and C–O in ligand (F1H). The LP of O₁₄, O₂₈ and O₃₀ interacted with antibonding orbitals, lone pair (2) O₃₀ → σ*(C₁₉–C₂₇), O₁₄ → σ*(C₇–C₈), O₁₄ → σ*(C₇–N₁₅), O₃₀ → σ*(C₂₇–C₂₈) and O₂₈ → π*(C₂₇–C₃₀) resulted in significant stabilization by 20.20, 26.47, 32.52, 38.54 and 51.16 kJmol⁻¹, respectively, specifying the strong conjugative interaction of O to C–C, C–N and C–O in ligand (F2H). In both ligands (F1H) and (F2H), the main p character of N(1) lone pair orbitals along with little participation in the intramolecular hyperconjugation exhibits behavior relevant to pure LP orbitals.

Therefore, on the basis of the results of NBO analysis, it can be established that nitrogen atoms of azomethine [N₁₄ in (F1H) and N₁₆ in (F2H)] are more reactive and have the maximum ability to coordinate with metal ions than oxygen atoms. Thus, the reduced energy involved in the hyper-conjugative interactions of lone pair of nitrogen suggested that it has a comparatively greater coordination tendency. Similarly, the π → π* interactions such as π(C₁₈‒C₁₉) → π*(C₁₇‒N₂₅), π(C₄‒C₅) → π*(C₃‒N₁₁), π(C₃₅‒C₃₆) → π*(N₃₀‒C₃₄) and π(C₃₄‒C₃₅) → π*(N₃₀‒Ni₄₁) yielded 27.15, 11.65, 12.52 and 43.39 kJmol⁻¹ stabilization energies for complexes Fe(F1)₂, Cu(F1)₂, VO(F2)₂ and Ni(F2)₂, respectively. Moreover, the LP → π* interactions were seen as N₁ → π*(C₈–C₉), N₁ → π*(C₂–C₁₀), N₁₁ → π*(C₁₂–C₁₃) and N₁₁ → π*(N₃₀–Ni₄₁) afforded stabilization energies of 37.63, 29.88, 29.15 and 62.47 for complexes Fe(F1)₂, Cu(F1)₂, VO(F2)₂ and Ni(F2)₂, correspondingly. This NBO analysis showed strong interactions, stabilization energies, coordination tendency and stability of studied compounds.

Computational IR analysis

The experimental IR spectra of both ligands and their respective metal complexes were also compared with theoretical computed data, and results showed a close agreement between both values. The experimental and computed data of ligands are given in Tables S6 and S7, along with their intensities and vibrational assignments. The vibrational frequencies were calculated at DFT/B3LYP with 6–311 +  + G(d,p) basis set, as illustrated in Fig. 6 for ligands and in Fig. S11 for complexes. Generally, the computed vibrational frequencies by DFT are obtained slightly higher than the experimental ones, which have been improved by using a scale factor of 0.9724 (Altürk et al. 2017). Infrared (IR) spectroscopy has been acknowledged as an effective approach in the hydrogen-bond research (Issaoui et al. 2009; Rekik et al. 2007a). Vibrational properties and frequencies are strongly affected by hydrogen-bond interactions (Rekik et al. 2007b). A redshift in the stretching vibrations, band broadening and increase in intensity of C–O and OH vibrations are considered as the most common consequences of hydrogen bonding (Issaoui et al. 2010).

Fig. 6

Experimental and computed vibrational spectra of ligands (F1H) and (F2H) with the broadening parameter of 10 cm⁻¹

C–H vibrations: The C–H stretching vibrations were easily predicted through DFT frequencies and are generally fall in the 3000–3100 cm⁻¹ range of IR spectra (Prabavathi et al. 2014). The experimental C–H stretching frequencies of both ligands appeared at 3110–3191 cm⁻¹ range, which agreed well with the theoretical frequencies obtained at 3022–3198 cm⁻¹. The vibrations of C–H (bending) are generally observed in the 1000–1300 cm⁻¹ range (Arivazhagan et al. 2015). The computed values (1039–1303 cm⁻) well support experimental values found at 1052–1325 cm⁻¹. For ligand (F1H), the C–H aliphatic bands were observed experimentally at 2917 and 2989 cm⁻¹ which are well agreed with the theoretical values at 2991 and 3022 cm⁻¹. The small variation in the experimental and computed frequencies is may be due to solid-state interactions of ligands for spectral studies, but no interactions for gaseous sample utilized for computational studies.

Ring vibrations: The stretching ring vibrations are normally observed at 1400–1650 cm⁻¹. The theoretical frequencies observed at 1513 and 1598 cm⁻¹ showed a close agreement to the experimental values at 1563 and 1625 cm⁻¹ as weak bands indicating carbon–carbon single and double bonds.

N–H vibrations: These vibrations for ligands (F1H) and (F2H) were observed at 3241 and 3220 cm⁻¹, respectively, while the computed frequencies were found at 3634 and 3635 cm^−1. This difference in computed and experimental values may be attributed to the presence of solid-state intermolecular interactions in the compounds during the spectral study, while such interactions are ignored for the sample by considering it as a single molecule in the gas phase used for computational studies.

C = O vibrations: The experimental C = O stretching vibrations were observed at 1709–1715 cm⁻¹ for both ligands, which are consistent with the computed frequencies at 1769–1795 cm⁻¹.

Computational UV–Vis analysis

The TD spectra for isatin Schiff base ligands and their selected metal complexes were carried out through TD-DFT in the gas phase. For ligand (F1H), the computed spectral analysis showed bands π → π* and n → π* electronic transitions at 402, 375, 289, 277, 262 and 235 nm, which agreed well with the experimentally observed peaks at 400 and 289 nm, as given in Fig. 7. Similarly, for ligands (F2H), the computed spectra showed π → π* and n → π* electronic transitions at 490, 389, 331, 310, 297 and 289 nm, which were found to be in good agreement with the experimentally observed peak values at 490, 400 and 250 nm. These intra-ligand transitions are commonly attributable to the presence of (C = C), (C = O) and (C = N) groups in the structures of both ligands.

Fig. 7

Experimental and calculated electronic spectra of ligands (F1H) and (F2H) with the broadening parameter of 2000 cm⁻¹ for (F1H) and 3000 cm⁻¹ for (F2H)

The theoretical and experimental wavelengths, excitation energies, oscillator strengths and atomic orbital contributions of ligands and their selected metal complexes are given in Tables S8 and S9, whereas the theoretical spectra of complexes are shown in Fig. S12. The computed data also showed some other transitions which can be considered as forbidden because their oscillator strengths are almost zero. Therefore, the DFT-based vibrational modes computed for both isatin-derived ligands and their corresponding complexes were found to be in good rational agreement with the experimental findings.

Antimicrobial activity

Antibacterial activity

Isatin-based ligands and their corresponding transition metal chelates were tested for antibacterial activity against two Gram-positive (S. pyogenes, S. aureus) and two Gram-negative (P. syringae, E. coli) bacterial strains. The results of antibacterial activity are displayed in Table 3 and Fig. 8 as inhibition zones in millimeters and compared with standard drug amoxicillin. The results revealed that ligand (F2H) was inactive against D and showed moderate activities for other strains. Similarly, metal complexes also showed good results for antibacterial activity. The complex Ni(F2)₂ showed maximum activity for A and moderate activity for B, C and D, while Cu(F2)₂ showed maximum activity, i.e., 32 mm and 25 mm for B and D, and significant results for A and C as 24, 26 mm, respectively. All other complexes showed good results, and the increase in the activity of complexes in contrast to ligands was due to the chelation process.

Table 3 Antibacterial and antifungal activity of ligands and their metal chelates

Fig. 8

Antibacterial and antifungal activity of synthesized isatin-based compounds

The antibacterial profile of these compounds has been compared with the similar type of isatin-derived Schiff bases, and their resulting metal complexes have already been published in the literature. All the values of inhibition zones were in good agreement with the reported results (Table S10). But in most cases, the compounds have exhibited more antibacterial properties than the published results.

Antifungal activity

The antifungal activity of metals based isatin ligands was checked using three fungal strains: A. niger (E), R. stolonifer (F) and T. spirale (G). The results were recorded as inhibition zones in millimeters as shown in Table 3 and Fig. 8. The results were also compared with the reference antifungal drug, Fungom. The results revealed that the ligand (F1H) was inactive against F, but exhibited reduced activity for E. And the ligand (F2H) was inactive against two fungal strains F and G. The complexes Zn(F1)₂ and Co(F2)₂ were inactive against E, and VO(F1)₂ was inactive against F strain. All the complexes of ligand (F2H) were inactive against G except complex Zn(F2)₂ that showed significant activity of 30 mm and VO(F2)₂ that showed the least activity of 7 mm. Overall complexes Ni(F1)₂, Cu(F1)₂ and Zn(F2)₂ exhibited significant activity, while the other complexes showed good to moderate activity against all fungal strains. The observed increase in the activity of complexes in contrast to the ligands was due to the chelation process.

The antifungal potential of these compounds has been compared with the similar type of isatin-derived Schiff bases, and their resulting metal complexes have already been published in the literature. All the values of inhibition zones were in good agreement with the reported results (Table S10). But in most cases, the compounds have exhibited more fungicidal properties than the published results.

Antioxidant activity

Antioxidant activity of metals based isatin ligand was checked by DPPH radical scavenging assay and phosphomolybdate assay.

DPPH assay

The diphenylpicrylhydrazide (DPPH) is stable and free radical, which was utilized to determine the radical scavenging activity of isatin-based ligands and their derived metal complexes. Different concentrations (i.e., 50, 100, 150, 200 and 250 µg/mL) of as-synthesized compounds and ascorbic acid (standard) were used for this activity, and absorbance was measured at 517 nm. After that, the inhibition results of synthesized compounds were compared with ascorbic acid. The results also showed that percentage inhibition was increased by increasing the concentration of compounds, as shown in Fig. 9 and Table S11. The results revealed weak to moderate activity for synthesized ligands and complexes. Ascorbic acid has shown maximum DPPH scavenging activity. Among the synthesized compounds, highest activity was shown by Fe(F2)₂ complex, i.e., 93%. Cobalt complex Co(F2)₂ also showed well activity, i.e., 53%.

Fig. 9

Antioxidant activity of synthesized isatin-based compounds

Phosphomolybdate assay

The total antioxidant capacity of compounds was evaluated using a phosphomolybdate assay. In this method, a reagent solution (obtained by mixing sodium phosphate (0.028 M), ammonium heptamolybdate (0.04 M) and sulfuric acid (5 mL) was used and measured absorbance at 695 nm. The results revealed that VO(F2)₂ complex showed the highest absorbance, i.e., 0.8376. The results also revealed that most of metal complexes showed higher absorbance than their parent ligand, as shown in Table S12 and Fig. 10.

Fig. 10

Total antioxidant capacity of synthesized isatin-based compounds

Statistical analysis

One-way analysis of variance (ANOVA) is a statistical and advanced research method that is concerned with comparing means of different samples. It was used to interpret the experimental outcomes of biological activities for all the prepared compounds and to see the significant difference in the performance of all treatments. Furthermore, multiple comparison test, particularly Tukey’s test, was used to determine the most significant treatment effect among all compounds. For antibacterial activity, the results obtained through ANOVA and Tukey test showed that performance of B has a highly significant effect as compared to other strains A, C and D as shown in Table S13.

Similarly, for antifungal activity the results showed that no treatment has significant effect because for all E, F and G strains the p-value is greater than 0.05 as shown in Table S14. For antioxidant activity, the results showed that performance of 50 µg/mL and 250 µg/mL has significant effect than all other concentrations as shown in the table of Tukey pairwise comparison the p-value for this is 0.02 which is less than 0.05. The graphs of interval plots and Tukey test are shown in Figs. 11 and S13–S17.

Fig. 11

Tukey plot for 50, 100, 150, 200 and 250 µg/mL

Drug-likeness analysis

In silico drug-likeness assessment involves the detailed examination of the structural and physiochemical features of the compounds to order to determine their potential to develop as an oral drug-like candidate. To determine whether the lipophilicity and biocompatibility of the compounds are associated with synthetic compounds, computational engine called Molinspiration (version 2018.10) has been used to evaluate and present physiochemical properties as depicted in Table 4 (Noreen and Sumrra 2021). The octanol–water partition coefficient (miLogP) for both ligands (F1H) and (F2H) was less than five (0.91–3.28), signifying a stronger permeability, higher mobility and a more soluble character all over the cell membrane (Fig. 12). All the metal complexes have negative value of miLogP ranging from − 2.27 to − 6.04, that signified their high affinity having strong interactive process with the targeted proteins. The lower values of miLogP have displayed that these compounds have ability to bind with targeted proteins. The TPSA (total polar surface area) potentials for these synthetic compounds indicate that when they are used as drugs, they have the ability to dissolve well with the purpose of attaching to a specific purpose. For the studied compounds, TPSA ranges from 5.45 to 171.10. The ligand (F1H) exhibited the lowest value of TPSA, while the complexes have shown the highest value of TPSA.

Table 4 Drug-likeness properties of synthetic compounds by Lipinski’s rule

Fig. 12

Drug-like properties of the synthesized ligands (F1H) and (F2H) and their respective metal complexes determined by the Lipinski law

The molecular weight of both the synthesized ligands (F1H) and (F2H) was less than 500 g/mol, demonstrating their superior ability to move, digest and disperse. All metal complexes, on the other hand, have a higher molecular mass ranging from 434.22 to 597.43 g/mol, which may reduce their readable drug-likeness aptitude (move, digest or disperse) to some extent. In agreement with Lipinski law, hydrogen-bond acceptors (oxygen and nitrogen) along with donor atoms were determined in the accepted range in all hydrogen-bond tests (OH and NH). All the metal complexes were found to have the maximum rotative relations of 3 with regard to Lipinski law. No rotary hydrogen was found for the ligands. The ligands (F1H) and (F2H) have exhibited less volume as 169.47 and 226.26, respectively, while their metal complexes had greater volume ranging from 360.25 to 490.73.

Conclusion

Assessment of the intrinsic structural modifications inflicted by the coordination of transition metals in the molecular geometries of isatin Schiff base ligands is of primary importance for detailed characterization and thoughtful structure–activity relationships. We made an effort to scrutinize the coordinating aspects of isatin ligands as efficacious metal-binding entities involving a combined synthetic and computational approach. Two new isatin-based ligands, namely 3-[(2-hydroxyethyl)imino]-1,3-dihydro-2H-indol-2-one (F1H) and 2-{[2-oxo-1,2-dihydro-3H-indol-3-ylidene]amino}benzoic acid (F2H), were synthesized and complexed with divalent and tetravalent 3d-metal ions. The synthesis was carried out efficiently using the condensation method, and spectroscopic studies supported the anticipated structures of the isatin compounds. The vibrational frequencies and electronic transitions of the studied compounds have been DFT-computed and then compared with experimental findings. Overall, the DFT findings revealed excellent concurrence with the experimentally observed data, which confirmed the purity of synthesized compounds.

For ligands (F1H) and (F2H), the FMO energy gap (HOMO–LUMO) was 3.992 eV and 3.512 eV, respectively. The low FMO energy gap revealed the stability of the ligand (F1H). The minimum E_HOMO values exhibited reduced electron-contributing aptitude, demonstrating that both ligands (F1H) and (F2H) might have better electron-donating capability in comparison with other compounds. NBO analysis concluded that nitrogen atoms of azomethine [N₁₄ in (F1H) and N₁₆ in (F2H)] are more reactive and able to coordinate with metal ions than oxygen atoms. The synthesized isatin compounds have shown varying degrees of inhibition effects on the growth of tested fungal and bacterial species. Besides the antimicrobial activity, all the compounds have also exhibited a moderate to significant antioxidant profile. Statistical analysis also validated the experimental outcomes of biological activities. In conclusion, these synthesized and studied isatin metal-derived compounds stand out as potential candidates in developing effective drugs against microbial infection.