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Research Article
Synthesis and biological activity evaluation of new isatin-gallate hybrids as antioxidant and anticancer agents (in vitro) and in silico study as anticancer agents and coronavirus inhibitors
expand article infoMay Mohammed Jawad Al-Mudhafar‡, Jaafar Suhail Wadi§
‡ University of Baghdad, Baghdad, Iraq
§ Al-Rafidain University College, Baghdad, Iraq

Corresponding author: May Mohammed Jawad Al-Mudhafar ( may.mj.almudhafar@copharm.uobaghdad.edu.iq )

Academic editor: Alexander Zlatkov
© 2024 May Mohammed Jawad Al-Mudhafar, Jaafar Suhail Wadi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Al-Mudhafar MMJ, Wadi JS (2024) Synthesis and biological activity evaluation of new isatin-gallate hybrids as antioxidant and anticancer agents (in vitro) and in silico study as anticancer agents and coronavirus inhibitors. Pharmacia 71: 1-11. https://doi.org/10.3897/pharmacia.71.e124992
Open Access

Abstract

Background: The hybrid compounds hold promise for developing novel pharmaceuticals, potentially exhibiting greater activity, mainly against viruses and cancer diseases, than their components.

Objective: In this study, researchers explored the potential synergistic effects of hybrid molecules by designing and synthesizing a series of isatin-gallate hybrids, denoted as N’-(5-substituted-2-oxoindolin-3-ylidene)-3,4,5-trihydroxybenzohydrazide (3a–d).

Methods: Isatin-gallate hybrids (3a–d) were synthesized by reacting gallic hydrazide with each of the isatin analogs (2a–d). The structures of all produced compounds were described using spectrum methods such as fourier transform infrared (FTIR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR), and physicochemical attributes. The evaluation of the tested hybrids (3a–d) involved assessing their in vitro antioxidant activities using the α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method and cytotoxic activities through the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay for measuring cellular growth. Furthermore, in silico analysis was applied to the final hybrids to evaluate their effects as anticancer and anti-coronavirus agents.

Conclusion: Among the examined hybrid compounds, 3b demonstrated substantial in vitro antioxidant and cytotoxic activities. In silico analysis revealed slight variations in the anticancer activity of compounds 3a–d, with differing affinities observed across various cancer cell lines. Additionally, these compounds exhibited moderate efficacy in inhibiting coronavirus activity.

Keywords

anticancer, anti-coronavirus, antioxidant, isatin-gallolyl hybrids, in silico

Introduction

The synthesis of hybrid molecules by combining many pharmacophores in one structure may result in compounds with remarkable pharmacological properties while designing new medications. Many anticancer drugs already on the market have poor enough selectivity against cancer cells, which can have a variety of harmful side effects (Alkhzem et al. 2022). Therefore, the need to create complementary or synergistic antioxidant, anticancer, and antiviral medications with low adverse effects remains continual.

Medications derived from natural sources and their derivatives efficiently treat and prevent diseases. Polyphenol compounds like gallic acid are among the most important natural sources. Propyl gallate, a propyl ester of gallic acid, is an important semi-synthetic compound. It is prepared either biologically (enzymatically) or chemically (Arunkumar et al. 2009; Zhang et al. 2012; Antonopoulou et al. 2016). Gallic acid and propyl gallate are reported to have antioxidant, anticancer, antimicrobial, and anti-inflammatory activities (Jiang et al. 2022).

Dietary phenolic extracts were highly dependent on their structural properties, which, in turn, established their antioxidant and anticancer activities (Bakrim et al. 2022). As a result, gallic acid and its derivatives, such as benzohydrazone, have been validated in pharmaceutical chemistry (Taha et al. 2019). Different aromatic hydrazones containing hydroxyl groups were synthesized by a reaction of mono-, di-, or tri-hydroxylated aromatic hydrazides, which were prepared from different phenolic acid hydrazides with various aromatic aldehydes. The final results demonstrated that the two and three neighboring substituents of OH groups of synthesized hydrazones have the highest antioxidant activities (Moussa et al. 2020; Rahangdale and Wankhade 2023).

Gallic hydrazide Schiff bases were synthesized and evaluated for their antioxidant activities by Gwaram et al. (Gwaram et al. 2012); the DPPH assay showed that all these compounds have strong antioxidant activity.

The indole nucleus has attracted scientists to develop new compounds; they are biologically essential chemicals and possess different biological activities (Khaledi et al. 2011). Isatin is a member of the indoles’ compounds and an interesting endogenous biologically active molecule. It could undergo chemical modification to produce many organic compounds with various biological activities, such as antimicrobial, anti-inflammatory, anticancer, and antioxidant (Al-Mudhafar and Kamoon 2020; Al-Wabli et al. 2020; Ismail et al. 2022).

The combination of gallic acid and curcumin inhibited the growth of MDA-MB-231 breast cancer cells (Moghtaderi et al. 2018). Another study revealed that the paclitaxel/gallic acid combination showed the most cytotoxic effect, the largest induction of apoptosis, and a considerable elevation in P53 and Caspase 3 levels in HeLa cells (Aborehab and Osama 2019).

The scientists designed many isatin derivatives and/or hybrids with other active molecules; for example, five isatin hydrazone Schiff bases connected to the acetylenic moiety have been synthesized by Singh et al. (Singh et al. 2021). These prepared compounds showed potent antioxidant activity and remarkable activity against bacterial and fungal strains.

A series of β-Isatin aldehyde-N, N’-thiocarbohydrazone derivatives were synthesized and assayed for their in vitro antioxidant activity. The new derivatives with p-chlorophenyl substitution are the most effective antioxidants against DPPH and H2O2 scavenging activity and exhibit the highest antimicrobial activity (Kiran et al. 2013).

Furthermore, El-Serwy et al. investigated the antioxidant, anticoagulant, and fibrinolytic activity of some new isatin hydrazone-hydrazide derivatives, and most of these compounds showed potent antioxidant activity (El-Serwy et al. 2020). Other hydrazide-hydrazone derivatives of isatin with valproic acid were synthesized and assessed against two types of human tumor cell lines, including leukemia (Jurkat) and human liver (Hep-G2). It was revealed that the isatin-valproyl conjugate is active against Jurkat cells, while the 5-chloroisatin-valproyl hybrid is active against both cell lines (Jurkat and Hep-G2) (El-Faham et al. 2015).

Tumosien et al. reported on the synthesis of numerous compounds containing one or two 2-oxindole-hydrazone moieties in addition to evaluations of their antioxidant and anticancer activity after comparing the colon adenocarcinoma HT-29 cell line, which appeared to be more responsive to treatment with the hydrazone-isatin derivatives than the malignant melanoma A375 cell line. Also, it was found that bis(hydrazone-isatins) were more active than their monoforms (Tumosienė et al. 2021).

Chen et al. prepared a group of N-substituted isatin derivatives; their SARS-COV protease inhibitory effect was attributed to the isatin derivatives at C5 having electron-withdrawing groups such as NO2, Br, and I, with N1 holding the benzothiophene methyl group (Chen et al. 2005). A recent study found that rigid, bulky hydrophobic groups like naphthalen-2-ylmethyl at N1 and carboxamide groups at C5 of the isatin moiety maximize activity against the SARSCOV-2 protease (Dai et al. 2020).

Due to the synergistic effect, hybrid molecules may have greater activity than each component when designing new medications. In this research, the hybridization of gallic acid and various isatin analogs may provide new candidates with diverse biological activities, such as antioxidant and anticancer agents. Moreover, molecular docking was applied for anticancer and anti-coronavirus activities for future studies; however, experimental validation is necessary to confirm the inhibitory effect of these compounds as coronavirus inhibitors. In this study, researchers explored the potential synergistic effects of hybrid molecules by designing and synthesizing a series of isatin-gallate hybrids, denoted as N’-(5-substituted-2-oxoindolin-3-ylidene)-3,4,5-trihydroxybenzohydrazide (3a–d).

Methodology

General

An electronic melting point apparatus named the Stuart SMP30 was used to determine the melting points (uncorrected). An FTIR spectrophotometer from Shimadzu, Japan, was used to record the FT-IR spectra, with support from Specac® Quest ATR (diamond)-UK.1H‒NMR spectra were determined in deuterated dimethylsulphoxide (DMSO-d6); the chemical shifts were reported in δ (ppm). The NMR ultra-shield spectrophotometer, 500 MHz, was conducted at Tehran University (Subhi et al. 2022). The solvents and chemicals used were all obtained from commercial suppliers and were of analytical quality.

Visualization was demonstrated using the UCSF chimera tool version 1.17.3 (University of California San Francisco, San Francisco, United States) and studio discovery software version 4.5 (BIOVIA). The binding site was determined based on the already existing native ligand.

Chemical synthesis

Synthesis of 3,4,5-trihydroxybenzohydrazide (compound 1)

In a round boiling flask (0.02 mole, 3.96 g), propyl gallate (obtained from Hangzhou Hyper Chemicals Limited, LOFT49 Hangzhou, China) was added to (0.2 mole, 10 mL) 99% hydrazine hydrate (NH2-NH2) diluted in 60 mL of ethanol; this mixture was refluxed for 15 hours as shown in Fig. 1. Then, the mixture was allowed to cool to room temperature; ethanol was evaporated and poured into crushed ice. The solid product was filtered, washed with distilled water, and recrystallized with 70% ethanol. Compound 1 appeared as a white-colored powder (yield 90%). M.p. 290 °C (Rabie 2020); IR (υ cm-1): 3421 (O-H stretching vibrations of phenol), 3390 and 3298 (N-H stretching vibrations of NH2) and 3143 (N-H stretching vibrations of amide), 1601 (C=O stretching vibration of amide) (Gwaram et al. 2012).

Figure 1. 

Synthetic steps of the preparation of target hybrids (3a–d) and their intermediates.

General procedure for preparation of the target hybrids (3a–d)

An appropriate isatin analog from 2a–d (0.003 mol) was added separately to (0.003 mol) of compound 1 (Fig. 1) in the presence of 40 mL of ethanol (dried previously with anhydrous Na2SO4) and 5 drops of glacial acetic acid (catalyst). The reaction mixture was refluxed for 10–12 hours. The precipitate formed was collected by filtration, washed with ethanol, dried, and recrystallized from ethyl acetate to obtain the desired hybrids.

3,4,5-Trihydroxy-N’-(2-oxoindolin-3-ylidene)benzohydrazide (3a)

Yellow-colored powder of 85% yield. M.p. 293–295 °C; IR (υ cm-1): 3506 (O-H, phenol), 3263, 3209 (N-H, amide), 1693, 1651 (two C=O, amide groups), 1603 (C=N, imine, overlap with aromatic C=C conjugation); 1H--NMR δ ppm: 13.79 (s, NH of hydrazide, 1H), 11.34 (s, NH of amide, 1H), 9.00 (s, OH of phenol, 1H), 9.46 (s, OH of phenol, 2H), 6.91–7.60 (m, Ar-H, 6H) the HNMR result illustrated in Fig. 2a.

Figure 2. 

Docking validation for the docking process regarding EGFR (to the left) and RNA-polymerase (to the right). The red ribbons are for the native ligands in their original pose, and the orange ribbons are for the redocked native ligands.

3,4,5-trihydroxy-N’-(5-methoxy-2-oxoindolin-3-ylidene) benzohydrazide(3b)

Reddish brown-colored powder of 88% yield. M.p. 284 °C; IR (υ cm-1): 3618 (O-H, phenol), 3201, 3174 (N-H, amide), 2860 (CH3, -OCH3 group), 1693 and 1666 (two C=O, amide groups), 1605 (C=N, imine, overlap with aromatic C=C conjugation); 1H-NMR δ ppm: 13.83 (s, NH of hydrazide, 1H), 11.13 (s, NH of amide, 1H), 9.10 (s, OH of phenol, 1H), 9.43 (s, OH of phenol, 2H), 6.85–7.11 (m, Ar-H, 5H), 3.74 (s, O-CH3, 3H) the HNMR result illustrated in Fig. 2b.

N’-(5-Bromo-2-oxoindolin-3-ylidene)-3,4,5-trihydroxybenzohydrazide(3c)

Greenish-yellow-colored powder with a 71% yield. M.p. 325–327 °C; IR (υ cm-1): 3429 (O-H, phenol), 3251, 3197 (N-H, amide), 1697, and 1654 (two C=O, amide groups), 1608 (C=N, imine, overlap with aromatic C=C conjugation); 1H-NMR δ ppm: 13.72 (s, NH of hydrazide, 1H), 11.45 (s, NH of amide, 1H), 9.10 (s, OH of phenol, 1H), 9.47 (s, OH of phenol, 2H), 6.92–7.66 (m, Ar-H, 5H) the HNMR result illustrated in Fig. 2c.

N’-(5-fluoro-2-oxoindolin-3-ylidene)-3,4,5-trihydroxybenzohydrazide(3d)

Orange-colored powder with a 77% yield. M.p. 337–340 °C; IR (υ cm-1): 3556 (O-H, phenol), 3186, 3066 (N-H, amide), 1693 and 1654 (two C=O, amide groups), 1608 (C=N, imine, overlap with aromatic C=C conjugation); 1H-NMR δ ppm: 13.80 (s, NH of hydrazide, 1H), 11.35 (s, NH of amide, 1H), 9.33 (s, OH of phenol, 1H), 9.44 (s, OH of phenol, 2H), 6.89–7.42 (m, Ar-H, 5H).

Assay of biological activity

Antioxidant activity assay (in vitro)

The antioxidant activity was investigated in terms of free radical scavenging activity by the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH): The free radical scavenging activity of four different hybrids in various concentrations was measured by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Briefly, a solution of DPPH at 0.1 mM in ethanol was prepared. A total of 190 μl of DPPH solution (0.1 mL) was added to 10 μl of each derivative in various concentrations (200, 100, 50, 25, 12.5 μg/mL) in DMSO. DMSO was used as a blank. The plate was shaken vigorously and kept at room temperature for 30 min. DPPH solution has a deep violet color, with the highest absorbance at 517 nm (Tumosienė et al. 2021). The decrease in the absorbance of the reaction mixture specified the higher free radical scavenging activity and was measured at 517 nm using a spectrophotometer. Ascorbic acid was used as a positive control, and tests were done in triplicate. The concentration of each hybrid required to inhibit 50% of the DPPH free radical (IC50) was achieved using the hybrid dose-dependent inhibition curve. The DPPH radical-scavenging activity of each concentration was measured according to the following equation:

% of inhibition or DPPH scavenging effect = [A0–A1/A0] × 100

Where A0 is the absorbance of the control, A1 is the absorbance of the sample.

Cytotoxicity assay (in vitro)

Cell cultures and cell maintenance were applied in a humid environment; MCF-7, HeLa, HCT116, and WRL-68 cells were cultured using RPMI-1640 media enhanced with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotic and incubated at 37 °C in a CO2 (5%) incubator. Sub-cultured cells were loaded at a density of around 4 × 105 cells/mL. The cells were subcultured into new cell culture dishes as needed for the experiment as soon as they reached 80–90% confluency (Al-Hussaniy 2022). Depending on the cell density, cells were fed with new RPMI-1640 medium every two to three days. The cell lines were kindly provided by the Biotechnology Research Center, Al-Nahrain University/Baghdad, Iraq.

The cell growth inhibition effects of serial dilutions (25, 50, 100, 200, and 400 µg/mL) of compounds 3a, 3b, 3c, and 3d were detected by a colorimetric MTT assay. A fully confluent monolayer sheet was formed by seeding 1×105 cells per well in a 96-well microtiter plate and allowing it to proliferate for 24 h at 37 °C using a CO2 (5%) incubator. After incubation, fresh media was added, and cells were treated with compounds 3a, 3b, 3c, and 3d at desired concentrations for another 24 h. Following the treatment period, cells were washed with sterile phosphate buffered saline (PBS), and 20 µL of MTT solution (5 mg/mL in PBS) were added in each well and maintained for a further 4 h at 37 °C and 5% CO2, with regular monitoring for the formation of blue-colored formazan crystals under an inverted microscope. To solubilize the formazan crystals (MTT metabolic product), 200 µl of DMSO were added to each well after the medium in the plates was removed. At 560 nm, optical density was observed using a plate reader (Bio-Rad, Germany). The MTT assay was conducted using previously described methods (Al-Saffar et al. 2020). The IC50 values were calculated using the optical density data and the formula used to calculate the percentage of cell viability:

Cell viability (%) = Sample A560/Control A560 × 100%

In silico study

The crystal structure of epidermal growth factor receptor EGFR and RNA polymerase was retrieved from the protein data bank PDB (website: https://www.rcsb.org/). The PDB codes were 1M17 for EGFR and 7AAP for RNA polymerase. All water, ions, and ligands were removed before docking, and both proteins were energetically minimized by 500 steepest descent minimization steps. The protein is prepared for docking by adding the charges (AMBER ff14SB force field) and hydrogens. The ligands were sketched by ChemOffice and minimized by the MM2 force field. AutoDock Vina was used for docking after the validation process (Fig. 2) had taken place. The box grid dimensions for EGFR and RNA-polymerase stander dimensions and docking sizes. Visualization was demonstrated by UCSF Chimera and Studio Discovery software. The binding site was determined based on the already existing native ligand (Wadi et al. 2023).

Results

Chemistry

The synthetic route for target compounds (isatin-galloyl hybrids) is presented in Fig. 1. The structures of target hybrid compounds (3a–d) were confirmed by FT-IR and 1HNMR spectroscopy. Propyl gallate ester and hydrazine were mixed in an ethanol solvent to form the hydrazide derivative (compound 1), which was then attached to the isatin analogs (2a–d) individually via an imine link to produce different isatin-galloyl hybrids (3a–d).

Structural interpretation confirmed by FT-IR spectra of compounds 3a–d revealed the presence of -C=N- absorptions (at wavenumbers of 1603, 1605, 1608, and 1608 cm−1, respectively), confirming the formation of imines. The broad bands observed in the range (3429–3618 cm−1) were attributed to the phenolic -OH groups. The 1H-NMR spectra of the isatin-galloyl hybrids (3a–d) implied singlet signals at δ 13.79, 13.83, 13.72, and 13.80 ppm, respectively, correspond to (-CO-NH-) of hydrazide groups; other singlet signals detected at δ 11.34, 11.13, 11.45, and 11.35 ppm correspond to the -CO-NH- amide groups of the isatin part. Moreover, compound 3b displayed a singlet signal at 3.74, which was attributed to the methoxy group.

In vitro study

Antioxidant activity

A DPPH radical-scavenging experiment was used to evaluate the antioxidant activity of the produced compounds. The outcomes were contrasted with those of ascorbic acid (Table 1).

Table 1.
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The percentage of DPPH radical scavenging activity in different concentrations.

Compounds Scavenging activity% (Mean ± SD)
Concentrations
200µg/mL 100 µg/mL 50 µg/mL 25 µg/mL 12.5 µg/mL
Ascorbic acid 80.73 ± 1.87 72.73 ± 1.61 57.6 ± 2.2 39.0 ± 1.73 22.9 ± 1.83
3a 76.3 ± 3.80 63.4 ± 3.22** 54.09 ± 2.11 46.6 ± 6.19* 32.8 ± 2.93**
3b 76.77 ± 4.05 67.34 ± 0.72 55.67 ± 1.44 28.27 ± 4.60** 21.49 ± 5.32
3c 76.54 ± 3.30 62.08 ± 1.01** 52.74 ± 3.14 39.62 ± 4.13 27.7 ± 1.35
3d 44.02 ± 8.54** 34.92 ± 1.96** 22.03 ± 7.65** 17.21 ± 3.02** 14.54 ± 2.68

*: P-value < 0.05, **: P-value < 0.001, Data are mean (n = 3) ± SD.

Cytotoxic activity (MTT assay method)

The cytotoxicity of all the synthesized compounds (3a–d) was evaluated against HeLa, MCF-7, and HCT116 tumor cell lines using the MTT method. The cytotoxic potential of compounds 3a, 3b, 3c, and 3d was investigated using an MTT assay against the breast cancer cell line MCF-7 cells. According to Table 2 and Fig. 3, all compounds showed weak cytotoxic activity at lower concentrations (25 and 50 µg/mL) with no significant differences in cell inhibition patterns between the compounds. By increasing the concentration, compound 3b exhibited the most significant (p < 0.01) killing activity against MCF-7 cells compared with other compounds, with a maximum cell inhibition rate of 63.98±7.05% at 400 µg/mL. As shown in Table 2, for the MCF-7 cell line, the IC50 values vary obviously amongst different compounds.

Table 2.
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Cytotoxic activities of compounds (3a, 3b, 3c, and 3d) in human cancer (MCF-7, HeLa, and HCT116) and normal cells (WRL-68) in vitro (IC50 µg/mL) after 24 hours of incubation.

Compound Half-Maximal Inhibitory Concentration (IC50) % in µg/mL
MCF-7 HeLa HCT116 WRL-68
3a 123.5 105.0 76.27 301.8
3b 114.9 155.5 175.26 231.3
3c 174.4 163.3 147.6 200.6
3d 164.8 161.1 112.6 206.4
Figure 3. 

Mean ± SD cell viability after treatment with compounds 3a, 3b, 3c, and 3d using an MTT in vitro assay at 37 °C. A. Shows the MCF-7 cell viability %; B. Cell viability of HeLa cells; C. SD cell viability of HCT116 cells. Different letters (a, b, and c) are considered significant (p < 0.05). NS: non-significant; SD: standard deviation; n = 3.

Compounds 3a, 3b, 3c, and 3d were tested for their anticancer potentials in HeLa cells. Results in Fig. 3 demonstrate that the compounds were inactive at low concentrations (25 and 50 µg/mL), and the rate of cell reduction was insignificant between compounds. At higher concentrations, the compounds exhibited a variable degree of cell inhibition. Compounds 3c and 3d showed the most efficient capability of killing HeLa cells, with a maximum inhibition rate in cell viability of 46.6±3.31% (IC50 163.3) and 56.83±3.6% (IC50 163.3) µg/mL, respectively. Compounds 3a and 3b displayed significantly (p < 0.01) less toxicity effect against HeLa cells than compounds 3c and 3d, with an estimated IC50 value of 105.0 µg/mL for compound 3a and 155.5 µg/mL for compound 3b. The IC50 for all compounds was significantly (p < 0.01) higher in normal WRL-68 cells compared with HeLa cells, suggesting selective toxicity of these compounds against HeLa cells (Table 2).

Collectively, based on Table 2, compound 3a was considered to be the most potent among other compounds in reducing the viability of selected cancer cells, with significant selectivity toward cancer cells compared to normal WRL-68 cells. Nevertheless, compounds 3b, 3c, and 3d were positively cytotoxic to cancer cells with a higher IC50 and less selectivity for normal cells (Sagheer et al. 2022).

The anticancer efficacy of gallic acid was highly dependent on its structural properties, which, in turn, established its anticancer and antioxidant activities (Muğlu et al. 2020). Compared to the electron donor functional group (-OCH3) found on the isatin moiety, electron-withdrawing functional groups (-F and -Br) often had stronger cytotoxicity responses on cancerous cell lines under test (Kodisundaram et al. 2015). The obtained results are consistent with compounds 3c and 3d of 5-substituted at isatin moiety (-Br and -F, respectively) only against HCT116 cells, which showed the lowest IC50 value compared to 3b (-OCH3) compound that exhibited the greatest toxicity in MCF-7 and HeLa cells. Hybridized compound 3a with an unsubstituted isatin moiety may have strong cytotoxicity capabilities against HeLa and HCT116 cells but is not the most potent in MCF-7 cells (Chowdhary et al. 2022; Al-Hussaniy et al. 2023).

Molecular docking study

The results will include the binding affinity of the compounds toward the proteins, which is represented by the binding energy that was scored using box grid-based docking software (Table 3 and Figs 4–6). The docking score of the native ligands can also be seen (Sagheer et al. 2023).

Table 3.
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The binding energy of the compounds and the native ligands in kcal/mol.

EGFR
Erlotinib -6.9
Compound 3a -8.4
Compound 3b -8.1
Compound 3c -7.8
Compound 3d -8.5
SARS-CoV2 RNA-dependent RNA polymerase
RTP-favipiravir -9.2
Compound 3a -6.99
Compound 3b -6.7
Compound 3c -5.85
Compound 3d -6.42
Figure 4. 

The docked poses of all compounds in the binding site of EGFR, with labeling the most interactive residues.

Figure 5. 

The docked poses of all compounds in the binding site of RNA polymerase, with labeling of the most interactive residues. A. Compound 3a; B. Compound 3b; C. Compound 3c; and D. Compound 3d.

Figure 6. 

2D representation of the ligands’ and RNA polymerase interactions at the binding site. A. Compound 3a; B. Compound 3b; C. Compound 3c; and D. Compound 3d.

Regarding EGFR

The docking results presented in this study provide valuable insights into the binding affinities of four compounds, namely 3a, 3b, 3c, and 3d, compared to the reference compound Erlotinib (Fig. 4).

RNA polymerase

The docking results presented in this study offer valuable insights into the binding affinities of four compounds, namely 3a, 3b, 3c, and 3d, compared to the reference compound RTP-favipiravir (Figs 5, 6).

Notably, RTP-favipiravir, the reference compound, displayed the highest binding affinity with a docking score of (-9.2), underscoring its strong interaction with RNA polymerase. In contrast, hybrid compounds 3a, 3b, 3c, and 3d exhibited lower docking scores, suggesting less favorable binding affinities compared to RTP-favipiravir.

Discussion

Antioxidant effect

All investigated substances had observable DPPH radical scavenging action compared to the reference molecule (ascorbic acid), which had an IC50 (oxidative stress inhibitory concentration 50%) value of 26.35 µg/mL. Among them, compound 3b with the methoxy group at position 5 of the isatin motif showed the highest activity. Compound 3a of the unsubstituted isatin motif part was the next strength, with an IC50 value of 51.09 µg/mL. The halogenated compounds, 3c bearing (bromo group) and 3d bearing (fluoro group), showed the lowest inhibitory activity, with IC50 values of 52.29 and 187.6 µg/mL, respectively. Introducing an -OCH3 group in position 5 of the isatin motif significantly enhances the antioxidant activity. This might be explained by the methoxy group’s electron-donating characteristics, which increase the likelihood of an electron transfer from the lone pair electron on the isatin nitrogen atom by resonance. This research about the antioxidant effect has come in line with other studies published related to our compounds; research conducted on 3,4,5-trihydroxyphenylacetamide, which is very near compound 3a, shows that it was discovered that there was no direct relationship between anti-peroxidation and radical scavenging capabilities (Brandão et al. 2021; Gobinath et al. 2021).

Cytotoxic effect

Depending on IC50, MCF-7 cells showed the highest sensitivity in cell viability to compound 3b in a dose-dependent fashion, with an IC50 value of 114.9 µg/mL. The recorded IC50s of compounds 3a, 3c, and 3d in MCF-7 cells were 123.5, 174.4, and 164.8 µg/mL, respectively. On the other hand, all compounds revealed weak to moderate toxicity against the normal cells of WRL-68 with an IC50 range of 200–300 µg/mL (Table 2). In HCT116 cells, compounds 3a, 3b, 3c, and 3d were inactive at lower concentrations (25 and 50 µg/mL), and no significant variations were observed. At 100, 200, and 400 µg/mL, the compounds 3a and 3d distinctly reduced the viability of HCT116, and the maximum rate of cell inhibition was 71.59±7.52 and 65.1±4.01% at 400 µg/mL (Fig. 3). By comparing compounds 3b and 3c, compounds 3a and 3d significantly (p < 0.01) confirmed the highest toxicity against HCT116 cells with a calculated IC50 of 175.26, 147.6, 76.27, and 112.6 µg/mL. The resulting IC50 values for all compounds in HCT116 were significantly lower than the IC50 values obtained in non-cancerous WRL-68 cells (Table 2). In MCF-7, HeLa, and HCT116 cells, most of the compounds produced significant cytotoxic effects, albeit to various degrees (Table 2). The synthetic compound with three hydroxyl groups and no isatin substitution had more potent cytotoxic effects against cancer cells. However, these compounds showed decreased cytotoxic effects on ERL-68 normal cells based on the IC50 value. The results of compound 3a, in particular, seem to point to a remarkably distinct impact on cancerous cells with less toxicity toward normal cells. It was suggested that the hybrid compound 3a obtains a high level of cytotoxic action against cancer cell lines due to an isatin ring connected to gallic hydrazide. The presence of a hydroxyl group that releases electrons on a gallic acid ring that is linked to an indole skeleton exerts such toxicity (Tan et al. 2015; Tang et al. 2022).

Several studies concluded that gallic acids have good antitumor action; however, this has drawn the most attention. It has been shown to have anticancer properties in various cancer cells. It is believed that hybrid pharmaceuticals, which combine several prescriptions, may be able to stop or postpone the development of treatment resistance in cancer patients (Hanikoglu et al. 2020).

Discussion regarding EGFR

Firstly, it is evident that all four compounds exhibited superior docking scores compared to Erlotinib. Compound 3a displayed the highest binding affinity with a docking score of (-8.4), followed closely by compound 3d with a score of (-8.5). This suggests that these compounds have a strong potential for effectively binding to the EGFR active site, outperforming Erlotinib, which had a docking score of (-6.9).

The shared structural features among compounds 3a, 3b, 3c, and 3d, which include isatin, hydrazine, and trihydroxyphenyl moieties, may play a significant role in their enhanced binding affinity. These structural elements are distinct from Erlotinib’s quinazoline and anilino groups. These variations suggest that isatin, hydrazine, and trihydroxyphenyl groups in these compounds may contribute to their increased binding affinity. These structural variations could enhance the interaction with key residues in the EGFR binding pocket.

Small changes, such as the addition of a fluorine atom in 3d and a bromine atom in 3c, could, however, be responsible for the higher binding affinity. These structural changes may improve the interaction with important residues in the EGFR binding pocket (Bhatia et al. 2015).

Discussion regarding RNA polymerase

RTP-favipiravir, the reference compound, displayed the highest binding affinity with a docking score of (-9.2), underscoring its strong interaction with RNA polymerase. In contrast, hybrid compounds 3a, 3b, 3c, and 3d exhibited lower docking scores, suggesting less favorable binding affinities compared to RTP-favipiravir.

The chemical structures of these compounds significantly influence their binding affinities. RTP-favipiravir possesses a unique structure optimized for RNA polymerase inhibition, which explains its superior binding. Compounds 3a, 3b, 3c, and 3d share a common structural motif, which includes isatin, hydrazine, and trihydroxyphenyl moieties. While these structural elements differ from RTP-favipiravir, they may not be as tailored to RNA polymerase inhibition.

The observed differences in binding affinities highlight the importance of structural optimization for effectively targeting RNA polymerase. While compounds 3a, 3b, 3c, and 3d may exhibit moderate binding affinities, further structural modifications and optimization are likely necessary to enhance their potential as RNA polymerase inhibitors (Naydenova et al. 2021; Jwaid et al. 2024).

Additionally, it is important to consider that docking scores provide insights into the theoretical binding strength; however, experimental validation is essential to confirm the inhibitory activity of these compounds against RNA polymerase.

Conclusion

Throughout all these years, a wide variety of research has been carried out using isatin derivatives, which has resulted in the synthesis of a new series of isatin-gallate hybrids. The in vitro study exhibited that the hybrids showed promising activity against MCF7 cell lines. The hybrids showed less toxic effects on normal cells, which indicated that the hybrids are selective against cancer cells. Antioxidant activity screening showed that the hybrids exhibited moderate to good activity, as evidenced by the reducing power and free radical scavenging activity towards DPPH. The structure-activity relationship could be constructed, indicating that the cytotoxic activity of the hybrids may be due to the presence of the isatin and gallate moiety. Additionally, an in silico analysis assessed their anticancer and anti-coronavirus properties. The isolated derivatives were characterized using FT-IR and 1H- NMR spectral data. Results from biological activity assays indicated varied in vitro antioxidant potencies among the substituted hybrids, with the most potent being hybrid (3b) containing the -OCH3 group. For in vitro anticancer activity, hybrid (3b) also emerged as the most potent. Molecular docking studies highlighted the fluoro-substituted isatin-gallolyl hybrid (3d) as having the most active anticancer activity, with slight differences in binding energy compared to other hybrids (3a and 3b). However, regarding the anti-coronavirus inhibitory effect, compounds 3a, 3b, 3c, and 3d exhibited moderate binding affinities to RNA polymerase.

Ethics Statements

This research is an In silico study; no humans or animals were enrolled in it. However, the ethics committee at the University of Baghdad, Iraq, gave us ethical approval numbers 243-2024.

Author Contribution

The 1st author (May) contributed to the experimental design of the study, data analysis, methodology, and draft writing, providing access to research components, and the 2nd author (Jaafar) contributed to draft writing, revision, data analysis, and statistical analysis.

Data availability

All data was placed in an online repository on the Zenodo website (DOI: doi.org/10.5281/zenodo.11469230).

Contained the following data:

  • NMR for compounds 3a, 3b, 3c, and 3d is in the following files: 3a.pdf, compound-3b.pdf, 3c.pdf, and 3d.pdf, respectively.
  • IR results for compounds 1, 3a, 3b, 3c, and 3d are in the following files: IR Compound 1.jpg, IR Compound 3a.jpg, IR Compound 3c.jpg, and IR Compound 3d.jpg, respectively.

These data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

We thank the Department of Pharmaceutical Chemistry at the University of Baghdad, Iraq, for their great support.

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