In silico molecular docking, ADME study and synthesis of new 1,3-diazetidin-2-one derivatives with high anti-proliferative activity

Synthesis of methyl 2-((2,3dimethylphenyl)amino) benzoate [M1]

Mefenamic acid was dissolved in 15 ml of dry methanol at a ratio of 4.3 mmoles, and the mixture was chilled to a temperature between 0 and -5°C. To guarantee full solubilization, a small amount of tetrahydrofuran solvent was added. Thionyl chloride was gradually added to the solution dropwise until 0.32 ml (4.5 mmol) had been added. The mixture was heated in a water bath at 40°C for three hours, followed by eight hours of reflux stirring at 65°C. It was evaporated and then treated with 25 ml of ice water after it had warmed to room temperature. The product is recrystallized from 100% ethanol to produce a pure product, using a silica gel-coated TLC plate to monitor the reaction.¹⁶ The purity of the synthesized compounds was assessed through recrystallization and confirmed by determining a sharp melting point. Physical properties, R_f values, FT-IR, and NMR spectra interpretations of the synthesized compounds are illustrated in Table 1.

Synthesis of 2-((2,3-dimethylphenyl)amino) benzohydrazide [M2]

Compound [M2] was synthesized by dissolving 1 mmol of compound [M1] in 15 mL of absolute ethanol. Add an excess of 0.25 mL (5 mmol) of hydrazine hydrate, 99.99%, to the solution. The mixture was refluxed for 15 hours. After cooling, ice distilled water were added to precipitate the solution. Recrystallization from absolute ethanol gave compound [M2] a light yellow powder. Monitor the reaction with silica gel-coated TLC plates.¹⁷

General procedure for the synthesis of Schiff base compounds [M3a-e]

Each aromatic aldehyde (1 mmol) was combined with 10 mL of 100% ethanol before adding 2-3 drops of glacial acetic acid to begin the synthesis of the compounds [M3a-e]. The mixture was then swirled for 30 minutes at room temperature. Then mix 1 mmol of compound [M2] with 15 ml of 100% ethanol for 30 minutes at room temperature, reflux the mixture for 3 to 6 hours at 75 °C. After filtering, washing with cold water, recrystallization from pure ethanol. Follow the development of the reaction on a TLC plate with silica coating.¹⁸

General procedure for the synthesis of 1,3-diazetidin-2-one derivatives [M4a-e]

Anhydrous 1,4-dioxane (25 ml) was first mixed with 1 mmol of (M3a-e), and then 1.5 mmol of phenyl isocyanate was slowly added dropwise while maintaining a temperature between 0 and -5°C. The resultant mixture was refluxed for (5-7) hours after being agitated for 3 hours at room temperature. The resultant solid was washed with a solution of ethyl acetate: petroleum ether (1:1), filtered, and dried after the solvent had evaporated at room temperature. The process of recrystallizing was done from pure ethanol.¹⁴

The synthesis method of the compound M3a-e and M4a-e is shown in Figure 1.

Figure 1. Scheme (1): Synthesis of (M3a-e and M4a-e).

Computational methods

GOLD Suite (v. 2021.2.0), a licensed CCDC tool, was utilized for the docking technique to examine the possible binding of certain 1,3-diazetidin-2-one derivatives to EGFR. A nonproprietary alternative that can be suggested are AutoDock and AutoDock Vina. Additionally, using the CCDC Hermes Visualizer program (version 2021.2.0), bond lengths, brief contacts, hydrogen bond interactions, and interactions between proteins and ligands were estimated. Nonproprietary alternative that can be suggested in this instance are PyMOL, jmol and ChimeraX. The ligands from Chem Sketch were transformed into SMILE names using the Swiss ADME program, and the physicochemical characteristics and pharmacokinetic statistics of molecular atoms were predicted. Additionally, using BOILED EGG,¹⁹ the lipophilicity and polarity of molecules are estimated.²⁰ The component amino acids of the EGFR protein crystal structure now exist in ionized and tautomeric forms because hydrogen atoms have taken the role of water molecules in the structure. With default settings for all parameters, CheBio3D (version 16.0), MM2 force field, and CHEMPLP fitness algorithm were used to assess each proposed solution. Nonproprietary alternatives to CheBio3D are Avogadro, MarvinSketch, and BKChem. Additionally, the hydrogen atoms' dependence on distance and angle was taken into account when calculating the steric complementarity between the protein and its ligand. The candidate solutions are then evaluated, and the binding mode, docking position, and binding energy for each candidate solution are determined by a piecewise linear potential function. These findings assess how our created compounds interact with the EGFR's amino acid residues.²¹

In vitro cytotoxicity

Cell culture

A human lung cancer cell line (A549) from ATCC has been purchased by Al Mustansiriyah University Cell Bank Tissue Culture Research Center and is currently housed at the Faculty of Pharmacy.

Storage and resuscitation of cell line

For 24 hours, cells were kept frozen in liquid nitrogen (-80°C). Add 10 ml of fresh media after thawing at 37 °C, and the cells were separated via a centrifuge. The cells are then transfered to a 75 cm² culture flask after resuspending them in 25 ml of fresh media.²²

Cell maintenance

The cells are incubated at 37°C in 95% humidified air and 5% CO₂, A549 cells were cultured in RPMI 1640 medium. The medium was supplemented with antibiotics such as penicillin-streptomycin-amphotericin B100X and 1% L-glutamine. An addition of 10% of fetal bovine serum (FBS) was made to enhance the medium. In sterile 75 cm² flasks, the cells were grown and when 90% confluency was reached, they were subcultured by washing with 5 ml of phosphate-buffered saline (PBS) and detached from the bottom of the flask using trypsin solution that was incubated for 2 min at 37°C. Using a hemocytometer, check the cell count after transferring a cell suspension to a 50 ml conical tube and centrifuging at 1200 rpm for 3 minutes. After decanting the supernatant, resuspend cell pellets in supplemented growth medium. Complete growth medium should also be added in equal volume.²³

Cell viability by MTT colorimetric assay

In an effort to uncover new information, we conducted an experiment to evaluate how synthetic chemicals [M4a-e] would impact the survival of lung cancer cell lines by administering the MTT colorimetric assay. Our approach involved dispensing 100 μl of a cell suspension, with a concentration of (5×10³) cells/well, into each individual well of a 96-well flat-bottom tissue culture plate and leaving them undisturbed for a total of 24, 48, or 72 hours. After the first 24 hours, we introduced each chemical, with a 5 μm concentration, to the cells. Incubate the culture in a medium containing 10 μl of MTT solution (5 mg/ml MTT powder in PBS) at 37°C for two hours after the recovery period (24 h, 48 h, and 72 h). Cast aside the medium after 2 hours, and attribute 100 μl DMSO to each well, and put it in a dark room at room temperature for around 15-20 minutes. Using a Multiscan Reader, the optical density of each plate (well) was measured through a transmitted wavelength range of 560-600 nm. To calculate the cell growth inhibition (percent cytotoxicity), the formula [Percent inhibition = (A-B/A)*100] was implemented, with [A and B] representing the tested optical density substance and the standard (erlotinib), respectively. Performances were completed in duplicate.²⁴

Determination of the half maximal inhibitory concentration (IC50)

Using the dose response curve, the IC50 of the test compound can be calculated. For in vitro MTT assays, IC50 is the concentration of the test chemical [M4a-e] required to reduce cell viability by 50%. Using the results of the in vitro MTT assay, IC50 values were calculated for the study drug 72 hours after exposure to the cells. The concentration ranges of the substances [N4a-c] used for the calculation of IC50 values were (500, 250, 125, 12.5, 62.5, 31.25, 15.62, 7.81, 3.90 and 1, 95 μm).

Interpretation of synthesis results

The identification of the synthesized compounds was made through the examination of their melting point and R_f values. The FT-IR analysis revealed that the hydroxyl group of mefenamic acid had disappeared, as seen from the absence of its broad peak at 2500-3200 cm^-1. Instead, a signal was detected at 1732 cm^-1 for compound (M1), indicating the presence of a carbonyl group of ester (-C=O) in place of the carboxylic acid (C=O) found in mefenamic acid. The stretching of the ester was observed at 1224 cm^-1 for compound (M1), while the singlets of (-OCH₃) of ester were detected at 3.87 (δ) in the ¹H-NMR results. The broad peak of the hydroxyl group at 12.99 (δ) for mefenamic acid had disappeared. In the ¹³C-NMR results, a peak at 168.71 (δ) was observed for the carbonyl of the ester in (M1), while the methyl group showed a peak at 52.19 (δ).

Infrared analysis (FT-IR) of compound (M2) revealed the disappearance of a broad band at (1732 cm^-1) associated with stretching of the carbonyl (C=O) of the (M1) ester and the appearance of a New bands are assigned to stretches of the amide carbonyl (C=O) and to the (-NHNH₂) group of (M2) at (3351, 3329, 3188 cm^-1). The ¹H-NMR spectrum showed a broad singlet at 4.31 (δ) for the NH₂ proton of the hydrazide and a singlet at 9.52 (δ) for the NH proton of the hydrazide (M2). ¹³C-NMR showed that the methyl ester peak disappeared and the amide carbonyl at (172.73) (δ) became visible.

The FT-IR characteristic absorption bands of (M3a-e) showed υNH stretching of amide at (3344-3325 cm^-1) and combination band of υC=O stretching of amide and υC=N stretching at (1664-1622 cm^-1). ¹H-NMR spectra of compounds (M3a-e) showed singlet for N=CH-Ar (imine proton) at (8.34-8.88) (δ), and disappearance of the broad singlet for NH₂ protons of hydrazide. ¹³C-NMR showed the peak of (-CH) imine at (~143-150) (δ).

The characteristic FT-IR absorption band of N-H stretching of secondary amides appears at (3325, 3286 cm^-1) as two peaks due to hydrogen bonding; in the presence of hydrogen bonding, the N-H stretching splits into two peaks: one relatively low frequency peak and a higher frequency peak. Lower frequency peaks correspond to N-H stretching in hydrogen-bonded NH groups, while higher frequency peaks correspond to N-H stretching in non-hydrogen-bonding NH groups. In addition, υC=O at (1745, 1730 cm^-1) is a diazetidine ring, υC=O at (1650, 1641 cm^-1) is an amide ring, at (3136, 3102 cm^-1), the -CH segment of the diazetidine ring. ¹H-NMR results show that the characteristic single peak of the imine proton has disappeared, while the (-CH) proton of the diazetidine ring is at (6.96-6.99) (δ) was increased. ¹³C-NMR shows that the characteristic single peak of imine carbon disappears, the (-CH) peak of diazetidinine is at (61.86-66.82) (δ), and the carbonyl peak of diazetidinine is at (152 –161) (δ).

Interpretation of docking results

A genetic algorithm called GOLD was used to dock flexible ligands to protein binding sites, predicting the optimal molecular interaction between the expected compound (M4a-e) and the active binding site of the EGFR protein.²⁴ The PLP fitness scores rank inhibitory activities, and docking studies suggest that all predicted compounds have excellent binding energies with active receptor pockets, potentially showing promising activity with EGFR proteins. The preliminary molecular docking perfectly correlated with subsequent cytotoxicity (in-vitro) studies for compounds (M4a-e) against lung cancers. Amino acids of the epidermal growth factor receptor (PDB: 1M17) interacted with the novel compounds through hydrogen and short contact bonds, including VAL 702, LYS 721, ASP831, THR830, LEU820, MET769, LEU768, GLY772, THR766, LEU764, CYS773, CYS721, and CYS 773. All anticipated final compounds exhibited higher binding energies than the standard drug erlotinib, ranging from (84.70) to (92.77), with a PLP fitness value of (76.20) as shown in Table 2.

Interpretation of ADME results

The use of in silico tools to predict the pharmacokinetic properties of potential drug candidates during the lead generation and optimization stages has been shown to increase the chances of surviving the high turnover rates of drug discovery. To expedite the drug discovery process, efforts have been made to integrate pharmacokinetic and developmental considerations early in the research process, with a focus on identifying compounds with greater potential to optimize binding.²⁵ All predicted compounds had a bioavailability of 0.55 and a TPSA of less than 140 A⁰, except compounds M4c and M4e, which had a bioavailability of 0.17, indicating that they entered the systemic circulation. These compounds do not cross the blood-brain barrier (BBB). In addition, none of the compounds had an affinity for P-gp, the protein responsible for preventing chemotherapy drugs from being taken up by cells. Since these compounds are P-gp non-substrates, they are not excreted from the cell by efflux transporters.²⁶ In addition, all predicted compounds complied with the Lipinski rule of five, with the exception of M4c and M4e, which had two violations, as shown in Table 3.

Interpretation of cytotoxicity results

Based on the anti-proliferative evaluation²⁷ on the A549 cell line, we found that the compounds M4c and M4e had outstanding and promising anticancer activity against this cell line for the treatment of lung cancer with IC50 values of 1.75 and 2.05 μm, respectively, after 72 hours of administration, they are more active than the reference product erlotinib with IC50 (11.5) μm after 72 hours. The results of the cytotoxicity studies of the terminal compounds (M4c and M4e) were consistent with the predictions of the molecular docking studies. All synthesized compounds showed excellent anti-proliferative activity except compound (M4d) with IC50 (11.33) μm comparable to erlotinib (11.5) μm and compound (M4b) with slightly lower IC50 (14, 01) μm, higher than that of erlotinib as shown in Figure 2, and A549 cells were also observed by microscopy after 48 hours after exposure to 62.5 μm synthetic compounds (M4a-e). Morphological changes, compared with A549 cells (control cells) in Figure 3.

Figure 2. IC50 dose-response curves of synthesized compounds after 72 hours.

(a)=M4a, (b)=M4b, (c)=M4c, (d)=M4d, (e)=M4e.

Figure 3. The morphological changes of the A549 cell line treated with synthetic compounds for 48 hours under the microscope.

a: control, b: M4a, c: M4b, d: M4c, e: M4d, f: M4e.