General information

Solvents and reagents were procured from Sigma Aldrich (USA) and Finar (India). Reactions were monitored using TLC with silica gel 60 F254 plates, and melting points were determined with an EI capillary instrument. PA purity was assessed by Waters Alliance HPLC (USA) using a Waters C18 column with a 20:80 water-acetonitrile mobile phase. Mass spectrometry data was procured using Agilent 6520 Q-TOF (USA), while FTIR spectra were recorded on a Shimadzu FTIR-8310 (Japan). NMR spectra used a Bruker Ascend instrument (USA) with DMSO-d6 as the deuterated solvent.

ChemistrySynthesis of intermediate methyl-8-oxo-8-(1,10-phenanthrolin-5-ylamino) octanoate

In a 10 mL tetrahydrofuran solution, 1.50 mmol of 5-amino-1,10-phenanthroline and 1.94 mmol of methyl-8-chloro-8-oxooctanoate were mixed with 6.00 mmol of triethylamine, stirred vigorously at room temperature, and heated in a microwave at 150 °C for 10 min. After cooling, the ester-containing mixture was extracted with ethyl acetate [26].

Pale yellow solid; yield: 57.08%; mp: 86 °C; retention factor (Rf): 0.93 (mobile phase: CHCl3/CH3OH; 9:3). HRMS (Q-TOF) m/z for C21H23N3O3, Calc. [M + H]+ 366.426 found. 366.185.

Synthesis of PA (N1-hydroxy-N8 -(1,10-phenanthrolin-5-yl) octanediamide)

A solution containing 53.50 mmol of hydroxylamine hydrochloride in methanol was refluxed until clear, and 53.50 mmol of potassium hydroxide in methanol was added to the above mixture and mixed at 0 °C. After cooling, a white solid formed was filtered, and the filtrate was added to 1.20 mmol of previously synthesized ester. The mixture was stirred for 24 h at room temperature. Upon completion, the pH was neutralized, and the product was extracted with ethyl acetate [26].

Beige solid; yield: 17.75%; mp: 200 °C; Rf: 0.16 (mobile phase: CHCl3/CH3OH; 4:1). Purity > 95% (HPLC). FTIR (KBr, cm−1): 3199.91 (NH); 1695.43 (C = O); 1244.09 (C-N); 3500.80 (OH).1H NMR (400 MHz, DMSO-d6, ppm): δ 10.43 (s, 1H, hydroxamic OH); 10.27 (s, 1H, amide NH); 9.12 (d, 1H, J = 3.6 Hz); 9.03 (d, 1H, J = 4.0 Hz); 8.70 (s, 1H, amide NH); 8.66 (d, 1H, J = 8.4 Hz); 8.48 (d, 1H, J = 8.0 Hz); 8.18 (s, 1H); 7.85 (dd, 1H, J = 8.4 Hz, 4.4 Hz); 7.77 (dd, 1H, J = 7.6 Hz, 3.6 Hz); 2.53 (t, 2H, J = 8.0 Hz); 2.33 (t, 2H, J = 7.2 Hz); 1.69 (m, 2H); 1.54 (m, 2H); 1.37 (m, 4H), 13C NMR (100 MHz, DMSO-d6, ppm): δ 173.86, 173.09, 150.48, 149.85, 145.83, 143.69, 136.72, 132.97,132.52, 128.62, 125.22, 124.29, 123.51, 120.34, 36.30, 33.71, 28.86, 28.74, 25.57, 24.83. HRMS (Q-TOF) m/z for C20H22N4O3, Calc. [M + H]+ 367.413 found. 367.172.

Biological evaluationCell culture

Human cancer cells (SiHa, HepG2, MCF7, Cal27) were obtained from ATCC, while foreskin fibroblast cells were generated at the Manipal School of Life Sciences. Cells were cultured in DMEM with 10% FBS, maintained in an Eppendorf CellXpert® C170 incubator at 37 °C with 5% CO2, and medium was renewed every 48–72 h.

Cell viability assay

Cancer cells (1 × 105 cells/mL) and fibroblast cells (5 × 104 cells/mL) were seeded in 96-well plates. Test compounds, dissolved in DMSO and diluted in DMEM with 10% FBS, were treated for 48 h. MTT (5 mg/mL) was added, and after 4 h, absorbance was measured at 570 and 630 nm using a VARIOSKAN™ 3001 multimode multiplate reader [27].

Cell cycle analysis

SiHA cells were synchronized through a 24 h period of serum deprivation, followed by a 48 h exposure to test compounds (IC50 and IC70) in DMEM with 10% FBS. After trypsinization, cells were fixed in 70% ethanol, treated with RNase-A, stained with propidium iodide, and analyzed using the Partec CyFlow Space with FloMax software (Germany) [28].

Apoptosis assay

SiHa cells were cultured at a density of 1 × 106 cells/mL in 10 cm plates. Upon reaching 80% confluency, cells were subjected to 48 h treatment with test compounds (IC50 and IC70). Following two PBS washes, trypsinization, and suspension in Annexin V binding buffer, cells were incubated, stained with propidium iodide, and analyzed for apoptosis using Apoptosis Detection Kit I and Partec CyFlow Space with FloMax software (Germany) [29].

Intracellular reactive oxygen species (ROS) measurement

SiHa cells were cultured at a density of 1 × 106 cells/mL in 10 cm plates. Upon reaching 80% confluency, the cells were subjected to 48 h treatment with test compounds at IC50 and IC70 concentrations. Post-treatment, cells were stained with 1 µg/mL DCFH-DA dye, incubated for 45 min at 37 ºC, and the fluorescence intensity was measured using Partec CyFlow Space and FloMax software [30].

Confocal microscopy

SiHa cells were grown on a coverslip treated with test compounds at IC50 and IC70 for 48 h, followed by 4% paraformaldehyde fixation for 10 min and treatment with 15 μg of phalloidin–tetramethylrhodamine B isothiocyanate for 30 min. After washing with PBS, cells were stained with Hoechst 33,342 (1 μg/mL), mounted on a glass slide, and imaged using a Leica SP8 confocal microscope with Leica application suite (Germany) [31].

Anchorage-dependent colony formation assay

SiHa cells (500 cells/well) were seeded in 6 cm plates. After 24 h, cells were treated with the IC50 concentration of test compounds and incubated for 21 days with media changes every three days. On day 21, colonies were stained with 0.4% crystal violet for 10 min, washed twice with PBS, and then counted [31].

In vitro cell migration assay

SiHa cells were cultured in 6-well plates using DMEM with 10% FBS until they formed a monolayer. A scratch was done on the cell monolayer by using a sterile 200 μL pipette tip. Subsequently, the cells were treated with the PA at IC50 concentration, and the extent of cell migration was assessed by analyzing the wound area at 0th, 24th, and 48th h time intervals using ImageJ software (Wayne Rasband, USA) [31].

In vitro HDAC assay

In a black 96-well plate, HDAC assay buffer, varied concentrations of test compounds, and SiHa cell nucleus extract were combined and incubated for 30 min. The enzymatic reaction commenced with the addition of the fluorescent substrate, Boc-Lys(Ac)-AMC, and lasted for 2 h at 37 °C. Reaction termination involved adding a developer, followed by an additional incubation at room temperature for 10–15 min. Fluorescence intensity was assessed using a VARIOSKAN™ 3001 multimode multiplate reader at an excitation wavelength of 354 nm and emission wavelength of 450 nm [32].

Western blot analysis

SiHa cells (2 × 105 cells/well) were cultured in 6-well plates, treated with varied concentrations of test compounds for 48 h, and lysed in RIPA buffer. After centrifugation, protein content was quantified using Bradford assay. Protein extracts were subjected to SDS-PAGE and were transferred to the nitrocellulose membrane. Following blocking, membranes were probed with CST antibodies (Acetyl-Histone H3, B-Actin) at 1:3000 dilution and secondary anti-rabbit antibodies (1:5000 dilution). iBright™ CL1500 Imaging System captured the images.

Spectrophotometric determination of complex formation

Absorption measurements were carried out using a VARIOSKAN™ 3001 multimode multiplate reader. The experiments were performed at a wavelength from 200 to 700 nm.

Inhibition of DNA synthesis

SiHa cells (5 × 106) were treated with varying test compound concentrations for 48 h. Following incubation, cells were pulsed with 5 nM 3H-thymidine for 30 min at 37 °C. After collection, washing, and total DNA extraction, radioactivity was quantified as counts per min (CPM) using a Tri-carb 2900TR liquid scintillation (Perkin Elmer, USA) [33].

dNTP pool analysis

SiHa cells were subjected to serum starvation for 24 h before treatment with PA at an IC50 concentration for 20 h. Harvested duplicate samples (1 million cells each) were washed with ice-cold PBS, and an extraction solution (6.5: 3.5 of acetonitrile and water mixture with 100 mM formic acid) was added. After incubation and centrifugation, the solution was lyophilized, reconstituted in 100 µL HPLC buffer A, and analyzed on a Waters Symmetry C18 column using HPLC. A stepwise gradient of Buffer A and B was employed, with detection at 260 and 280 nm, as mentioned by Crona et al., in 2016 [34]. Quantification was based on peak area measurements in the chromatogram, providing relative quantification of the analyzed components.

Partition coefficient studies

The partition coefficient (Kpart) of PA was determined in a 1-octanol/water system. 1-octanol was added to MOPS buffer containing PA (IC50), and OD at 276 nm was measured before and after each addition. Kpart values were calculated. [35].

In silico analysisMolecular docking

Molecular docking studies were conducted using Maestro version 11.4 (Schrodinger Inc.). The XP system in Maestro was employed for ligand–protein interaction analysis [36].

MD simulations

Molecular dynamic (MD) simulations were conducted using the Desmond module (Schrodinger Inc.). A three-step workflow included system setup, solvation with the simple point charge (SPC) solvent model, and a 100 ns simulation [36].

ADME prediction

The Swiss ADME (absorption, distribution, metabolism, and excretion) by the Swiss Institute of Bioinformatics was used for the ADME studies in PA.

Density function theory (DFT) and time-dependent density-functional theory (TD-DFT) analysis

The Fe3+ ion complexes with PA were optimized in two modes (a and b) using Becke's B3LYP functional, SVP basis set, and D3BJ damping scheme in hybrid DFT. The free molecule was optimized with a TZVP basis set and B3LYP functional. Single-point TD-DFT calculations were performed using ORCA3, and visualization utilized Avogadro software.