Design and synthesis of newer 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine analogues as anticancer agents

ABSTRACT

Aim

Cancer is the second leading cause of death and chemotherapy is widely used and well–known for treating cancer, yet it has lots of adverse side effects, making the search for novel compounds imperative. We reported here design, synthesis, DFT analysis, anticancer evaluation and in–silico studies of new 1,3,4–oxadiazoles (4a–e).

Material and methods

IMC-038525 and IMC-094332 tubulin inhibitors’ oxadiazole-linked aryl cores inspired the innovative compounds, and synthesis was accomplished in two steps followed by their characterization by spectral data. The HOMO and LUMO energy gap (ΔE) was determined to investigate compounds’ (4a–e) stability followed by their anticancer activity at 10 μM and in-silico studies.

Results and conclusion

5-(4-Nitrophenyl)-N-(naphthalene-2-yl)-1,3,4-oxadiazol-2-amine (4b) demonstrated substantial anticancer activity against a few cell lines like SR, MDA-MB-435, MOLT-4, K-562, and HL-60(TB). 5-(3,4,5-Trimethoxyphenyl)-N-(naphthalene-2-yl)-1,3,4-oxadiazol-2-amine (4e) demonstrated promising anticancer activity against cell lines, UO–31, NCI–H226, CAKI–1, PC-3, and MCF7. The molecular docking against tubulin’s colchicine binding site (PDB ID: 1AS0), displayed a docking score of –7.295 Kcal/mol and a H–bond interaction with Ala317 residue for the ligand 4e. The ligand 4e was found to interacted 24 amino acids of the tubulin protein in MD simulation investigation with moderate local conformational changes with ligand 4e (< 1 Å).

1. Introduction

The development of novel biological molecules based on molecular recognition is an important goal in medicinal chemistry that has gained a lot of attention [1]. These days, due to the broad range of pharmacological value, researchers concentrate on nitrogen-containing heterocyclic compounds [2]. The structure of compounds is the primary dynamic source for functioning as a novel, biologically active species. Oxadiazole holds a unique place in the domains of pharmaceutical and medicinal chemistry because of the range of tasks it may accomplish [3]. One oxygen and two nitrogen atoms compose up the five-member heterocyclic ring of the oxadiazole, which has the chemical formula C₂H₂N₂O. Oxadiazoles are available in four distinct isomeric forms, including 1,3,4-oxadiazole, 1,2,5-oxadiazole, 1,2,4-oxadiazole, and 1,2,3-oxadiazole [4]. The 1,3,4-oxadiazole ring is generally advantageous in terms of water solubility, lipophilicity profiles, and stability under thermal and metabolic conditions [5]. The substitution reaction of 1,3,4-oxadiazoles can take place by a variety of methods, including photochemical, thermal, electrophilic, and nucleophilic reactions [6]. The heterocyclic nucleus 1,3,4-oxadiazole is found in a wide-variety of therapeutic agents, such as raltegravir [7], furamizole [8], nesapidil [9], tiodazosin [10], zibotentan [11], AZD1979 [12], etc. The chemical and biological attributes of 1,3,4-oxadiazole-containing compounds have been studied extensively [3,13]. The 1,3,4-oxadiazole analogues garnered a great attention due to their biological and pharmacological activities against cancer [14,15], tuberculosis [16], HIV [17], bacteria [18], inflammation [19], seizure [20], malaria [15], and many other biological activities [21] as reported in the recent literature.

Cancer is the second leading cause of death, with nearly 10 million deaths and 19.3 million new cases globally in 2020 [22,23]. The numeral figure of new cancer cases globally is anticipated to surpass 28.4 million by 2040 [23]. At least one-third of the 10 million new cases of cancer that occur each year can be avoided by adopting healthy dietary habits, abstaining from alcohol and tobacco use, immunizing against the viral hepatitis B virus, and controlling one’s weight [24]. Breast (11.6%), lung (11.6%), colorectal (10.2%), and prostate (7.1%) cancers are the most common cancer types reported in 2018 [25]. Lung cancer is the most common cause of cancer-related death in developed nations, accounting for around 30% of all cancer-related deaths [24,25]. Chemotherapy is widely used and well known for treating cancer, yet it has a lot of adverse side effects [26]. Consequently, the pursuit of new compounds is always admired. In continuance, we explored here some of the 1,3,4-oxadiazoles inspired by their miraculous biological activities and study their antiproliferative effect against 59 National Cancer Institute (NCI) cell lines at single dose assay at 10⁻⁵ M. The oxadiazole linked aryl core (blue colored) of IMC-038525 [27,28], IMC-094332 [27,28], and FABT [29] was taken as design approach for the newer oxadiazoles as anticancer agents as shown in Figure 1. The compound sanguinarine, which includes a naphthyl ring and several 2-naphthyl combretastatin analogues, has been extensively documented as an anticancer agent and a tubulin inhibitor [30]. The bicyclic benzdioxane ring replaced of IMC-038525 and IMC-094332 was replaced with 2-naphthyl ring (bicyclic ring). Furthermore, the 1,3,4-thiadiazole ring of another anticancer compound, FTAB, has been replaced isosterically with 1,3,4-oxadiazole ring in the title compounds (4a–e). Several clinically approved anticancer agents, including paclitaxel and the vinca alkaloids, vincristine and vinblastine, have tubulin as their molecular target [31]. The tubulin inhibitors disrupt the dynamics of tubulin-microtubules, which prevents mitosis during cell division and kills cancerous cells [32]. Cell division and growth depend on microtubules, which are dynamic structures made up of α- and β-tubulin heterodimers [33]. Cancer has also been associated with the expression of particular tubulin isotypes, but the precise mechanism is still unknown [34]. These compounds (4a–e) share a basic pharmacophore with tubulin inhibitors (IMC-038525 and IMC-094332) and many of the 2-naphthyl combretastatin analogues as well as naphthyl containing sanguinarine were reported as tubulin inhibitors. Therefore, a hypothesized mechanism of anticancer effects was examined through molecular docking and a dynamic strategy targeting tubulin, a crucial target for many anticancer agents.

Figure 1.

Design of new 2,5-disubstituted-1,3,4-oxadiazoles.

2. Material and methods

2.1. General

All the chemicals were procured from SD fine, CDH and Sigma Aldrich and were used without any additional purification. The melting point (in °C) was recorded by open tube capillary method. Bruker Alpha-II, Bruker Avance 400/AvIII HD-300 (FT NMR), and Waters Alliance 2695/HPLC-TQD mass spectrometers recorded Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), and mass spectral data, respectively. The purity was analyzed using Perkin-Elmer 2400 elemental analyzer.

2.2. General method for the synthesis of N-(naphthalen-2-yl)hydrazine carboxamide (3)

2-Naphthyl isocyanate (1) (25 mmol; ~4.23 g) solution in acetonitrile was added drop-wise to an excess of hydrazine hydrate (2) in acetonitrile. The reaction was carried out in an ice bath with continuous stirring for half an hour to obtain a cream colored precipitate. The precipitate was filtered, washed with water and finally air-dried to obtain N-(naphthalen-2-yl)hydrazine carboxamide (3). Yield: 88%; Mp 258°C.

2.3. General method for the synthesis of 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine analogues (4a-e)

An equimolar quantity of N-(naphthalen-2-yl)hydrazine carboxamide (3) (1 mmol, 201 mg) and appropriate aromatic aldehydes (Ar−CHO) (1 mmol) was dissolved in ethanol (5 mL) with slight warming followed by an addition of water (8 mL) and sodium bisulfite (2 mL; 10% mol). The reaction mixture was refluxed for 8 h on water-bath with vigorous stirring [35]. The reaction’s progress was monitored by thin layer chromatography (TLC; acetone-n-hexane, 8:2 and chloroform-methanol, 9:1). Upon completion the reaction mixture was concentrated to one-third of initial volume, cooled, poured into the crushed ice. The resultant precipitate was filtered and the solid thus obtained was washed with plenty of water, dried and crystallized with absolute ethanol to obtain 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine analogues (4a–e).

2.3.1. 5-(4-chlorophenyl)-N-(naphthalene-2-yl)-1,3,4-oxadiazol-2-amine (4a)

Yield: 88%; Mp 102–104°C; IR: 3202 (NH), 1597 (N=C), 1142 (−O−), 698 (C−Cl) cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 7.44 (d, 2 h, J = 2.4 hz, ArH), 7.85 (d, 2 h, J = 2.4 hz, ArH), 7.57–7.93 (m, 7 h, ArH), 8.72 (s, 1 h, ArNH); ¹³C NMR (75 MHz, DMSO-d₆): δ 164.16, 160.78, 142.19, 135.09, 134.03, 130.04, 128.67, 128.03, 127.19, 126.09, 125.18, 124.52, 123.97, 121.26, 119.16, 108.42; Anal. Calc. for C₁₈H₁₂ClN₃O: C, 67.19; H, 3.76; N, 13.06 found: C, 67.12; H, 3.72; N, 13.10%. LC-MS m/z = 321.1 (M)⁺, 323.1 (M + 2)⁺.

2.3.2. 5-(4-nitrophenyl)-N-(naphthalene-2-yl)-1,3,4-oxadiazol-2-amine (4b)

Yield: 82% Mp 118–120°C; IR: 3261 (NH), 1597 (N=C), 1543 (NO_{2, unsym str.}), 1343 (NO_{2 sym str.}), 1162 (−O−) cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 7.44–7.93 (m, 7 h, ArH), 8.21–8.20 (d, 2 h, J = 2.4 hz, ArNO₂), 8.34–8.31 (d, 2 h, J = 9.9 hz, ArH), 8.84 (s, 1 h, ArNH); ¹³C NMR (75 MHz, DMSO-d₆): δ 164.16, 159.81, 147.13, 142.15, 134.72, 134.32, 130.91, 128.68, 128.05, 127.11, 126.01, 125.20, 124.76, 121.07, 119.19, 109.47; Anal. Calc. for C₁₈H₁₂N₄O₃: C, 65.06; H, 3.64; N, 16.86 found: C, 65.01; H, 3.66; N, 16.90%. LC-MS m/z = 332.0 (M)⁺, 333.0 (M + 1)⁺.

2.3.3. 5-(4-methoxyphenyl)-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine (4c)

Yield: 89%; Mp 130–132°C; IR: 3242 (NH), 1592 (N=C), 1228 (−O−CH₃), 1159 (−O−) cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 3.83 (s, 3 h, OCH₃), 7.02 (d, 2 h, J = 6.0 hz, ArH), 7.98 (d, 2 h, J = 6.1 hz, ArH), 8.01–8.22 (m, 7 h, ArH), 8.76 (s, 1 h, ArNH); ¹³C NMR (75 MHz, DMSO-d₆): δ 164.21, 161.66, 160.78, 142.90, 134.94, 128.68, 127.27, 126.84, 126.06, 125.17, 124.20, 121.10, 119.12, 115.85, 114.23, 109.27, 56.21; Anal. Calc. for C₁₈H₁₃N₃O₂: C, 71.28; H, 4.32; N, 13.85 found: C, 71.20; H, 4.35; N, 13.89%. LC-MS m/z = 317.1 (M)⁺.

2.3.4. 2-(5-(naphthalen-2-ylamino)-1,3,4-oxadiazol-2-yl)phenol (4d)

Yield: 90%; Mp 126–128°C; IR: 3287 (OH), 3214 (NH), 1526 (C=N), 1194 (−O−) cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 7.46–8.26 (m, 11 h, ArH), 8.90 (s, 1 h, ArNH), 11.52 (s, 1 h, ArOH); ¹³C NMR (75 MHz, DMSO-d₆): δ 164.31, 159.34, 155.31, 133.74, 133.68, 128.01, 127.82, 127.65, 127.34, 125.96, 125.76, 125.57, 124.86, 122.64, 120.94, 118.71, 110.91, 108.01; Anal. Calc. for C₁₈H₁₃N₃O₂: C, 71.28; H, 4.32; N, 13.85 found: C, 71.21; H, 4.35; N, 13.88%. LC-MS m/z = 303.1 (M)⁺, 304.1 (M + 1)⁺.

2.3.5. 5-(3,4,5-trimethoxyphenyl)-N-(naphthalene-2-yl)-1,3,4-oxadiazol-2-amine (4e)

Yield: 91%; Mp 108–110°C; IR 3290 (NH), 1578 (C=N), 1231 (−O−CH₃), 1183 (−O−) cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 3.76 (s, 3 h, OCH₃), 3.86 (s, 6 h, OCH₃), 7.13 (s, 2 h, ArH), 7.16–7.88 (m, 7 h, ArH), 8.89 (s, 1 h, NH); ¹³C NMR (75 MHz, DMSO-d₆): δ 164.31, 159.84, 153.34, 139.08, 133.18, 128.01, 127.88, 127.68, 127.34, 125.97, 125.57, 124.87, 122.64, 120.94, 118.71, 108.01, 60.78, 56.70; Anal. Calc. for C₂₁H₁₉N₃O₄: C, 66.83; H, 5.07; N, 11.13 found: C, 66.79; H, 5.10; N, 11.18%. LC-MS m/z = 377.1 (M)+, 378.1 (M + 1)⁺.

2.4. Density functional theory (DFT) computations

DFT calculation on the compounds (4a–e) was performed using the Schrödinger Jaguar Module. The DFT calculations used Becke’s three-parameter exchange potential and Lee-Yang-Parr correlation functional (B3LYP) theory with split-valence double-zeta basis set 6-31 G* [36]. Quantum chemical properties such as frontier molecular orbitals (FMOs) and electrostatic potential were calculated. The global reactivity parameter was then computed using the Parr and Pearson interpretation as well as Koopmans theorem [37,38].

2.5. Anticancer activity

Compounds (4a–e) were tested for antiproliferative activity against over 59 cell lines at 10⁻⁵ M. The National Cancer Institute (NCI US) protocol was followed for antiproliferative activity [39–42]. The antiproliferative activity was measured as growth percent (GP) and percent growth inhibition (PGI) at 10⁻⁵ M. The GP and PGI are correlated as: $\mathit{PGI}=100-\mathit{GP}$.

2.6. Molecular docking studies

The protein data bank provided a 3D X-ray crystallographic structure of tubulin complexed with colchicine with a resolution of 3.58 Å with r value 0.233 (obs.) [43]. Chain B was selected for grid preparation and the colchicine binding site was used for molecular docking investigation [44].

2.7. Molecular dynamic (MD) simulation

MD simulation was used to study the stability of one of the promising compounds (4e) binding to tubulin protein. It facilitates the assessment of time-dependent protein–ligand interaction, complex system conformational dynamics, and protein–ligand interaction patterns. Using Desmond version of 6.5 by D.E. Shawn Research MD simulation program, system builder used SPC solvent model to solvate protein–ligand complex, then 70 Na⁺ and 34 Cl⁻ counter ions were added to make it electrically neutral [45,46]. The protein–ligand combination was energy minimized and relaxed under NPT ensemble using Desmond’s default method with an addition of 0.15 M NaCl to simulate the physiological environment [47]. The MD simulation ran for 100 ns with the NPT ensemble at 300 K and 1 bar of pressure, maintained by a Nose-Hoover thermostat and a Martyna-Tobias-Klein barostat during the simulation [48,49]. At every 100 ps snapshot was recorded, which yielded 1000 fames, which were analyzed by the simulation interaction diagram tool.

2.8. ADMET prediction

SwissADME and ProTox-II predicted the ADMET profile of compounds (4a–e) [50,51].

3. Results and discussions

3.1. Chemistry

The oxadiazoles were prepared in two steps starting from 1-naphthyl isocyanate (1) as shown in Figure 2. In the first step an excess of hydrazine hydrate (2) in acetonitrile (MeCN) was stirred at cold condition (0–5°C) using ice bath and subsequently 2-naphthyl isocyanate (1) (25 mmol; ~4.23 g) solution in acetonitrile was added drop-wise for 30 min to obtain a cream colored precipitate [52]. The precipitate was filtered, washed with ample water and finally air-dried at room temperature to obtain N-(naphthalen-2-yl)hydrazinecarboxamide (3). The reaction offers a benefit of obtaining N-(naphthalen-2-yl)hydrazinecarboxamide in a single step from 2-naphthylisocyanate (1), hence minimizing effort of two steps synthesis from anilines [53]. In the subsequent step, an equal mixture of intermediate compound 3 (1 mmol; 201 mg) and aromatic aldehyde (1 mmol) was refluxed in water-ethanol (2:1) solvent system for 8 h with an adding 10% mol NaHSO₃ [35]. Surplus amount of solvent was removed once reaction completed, and pouring the concentrate into the crushed ice, filtering, washing with water, drying, and recrystallizing with absolute ethanol to yield 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine analogues (4a–e). TLC was used to monitor the reaction’s progress throughout, with eluants, acetone-n-hexane (8:2) and chloroform-methanol (9:1). The yields of the compounds (4a–f) were found to be ranging between 82% and 91%. The structure of compounds was confirmed using spectral analysis. The IR spectra showed the phenolic function (Ar−OH) present in compound 4d at 3287 cm⁻¹, and the unsymmetric as well as symmetric stretching of NO₂ function present in compound 4b at 1543 and 1343 cm⁻¹ respectively, while the C−Cl function present in compound 4a at 698 cm⁻¹. The methoxy function (O−CH₃) present in compounds 4c and 4e was observed at 1228 and 1231 cm⁻¹ respectively. The peak of oxadiazole (−O−) present in the compounds (4a–e) were detected at 1142–1194 cm⁻¹, while C=N peaks were detected at 1526–1597 cm⁻¹. The secondary amines (NH) present in the compounds (4a–e) were detected at 3202–3297 cm⁻¹. The nuclear magnetic resonance (¹H NMR and ¹³C NMR) of the compounds (4a–e) was recorded in the solvent DMSO-d₆. The methoxy (−OCH₃) group of the compounds 4c and 4e was observed as singlet at δ 3.76–3.86 ppm, while phenolic function (Ar−OH) of compound 4d was identified as singlet at δ 11.52 ppm. The aromatic protons of the compounds were observed as multiplets, doublet, and singlet depending upon the nature of protons present and ranging between δ 7.02–8.39 ppm, while NH peaks were as singlet and were observed at δ 8.72–8.90 ppm. Similarly, the ¹³C NMR data were in full harmony with the number of carbon atoms present in the compounds (4a–e). The ¹³C NMR of the most active compound (4e) among the series showed 18 distinct types of carbon atoms present in the compounds observed at δ 164.31, 159.84, 153.34, 139.08, 133.18, 128.01, 127.88, 127.68, 127.34, 125.97, 125.57, 124.87, 122.64, 120.94, 118.71, 108.01, 60.78, and 56.70 ppm.

Figure 2.

Synthetic protocol of newer 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine (4a-e): reagents and conditions: (i) MeCN, 0–5 °C, 30 min, 89% yield; (ii) ArCHO, H₂O-EtOH (2:1 v/v), NaHSO₃ (10% mol), 95–100 °C, 8 h, 82–91%, yield.

3.2. Density functional theory (DFT) computations

3.2.1. Molecular reactivity analyses

The stability and reactivity of a molecule depend on its frontier molecular orbitals (FMOs), which consists of highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) [54]. HOMOs can donate, while LUMOs can accept electrons. Functionally, ligands with high electron transfer capacity may bind to the protein better because they interact better with the target protein structure. Also, the energy gap (ΔE) helps to identify the most active molecules. The motif’s narrower energy gap facilitates molecular charge transport. A smaller HOMO-LUMO gap leads to increased excitation of π-electrons from low to high energy regions [55,56]. This ease of electron mobility increases energy distribution in molecules, which stabilizes and usually improves activity. A molecule with a larger HOMO-LUMO energy gap ought to be harder, more stable, and less reactive [54,56]. The B3LYP/6-31 G* basis set was used to compute the graphical representations of FMOs of the compounds (4a–e) as shown in Figure S1 (supplementary materials). Red and blue represent positive and negative phases, respectively. Replacing the substituent clearly has minimal influence on the HOMO and LUMO energy. In all compounds except 4c, the HOMO level located primarily on the amino-oxadiazole ring, while the LUMO level was primarily delocalized over the conjugated π-system. The charge density of compounds 4a and 4b differed from compound 4c, hence their HOMO orbitals were different. In compounds 4a and 4b, LUMO covers the halogen-substituted aromatic ring attached to the 2-amino oxadiazole ring, whereas LUMO extends the naphthalene moiety in compound 4c, which was replaced with a methoxy group. The nitro substituent significantly altered the 4b compound’s numerical HOMO and LUMO values. All compounds had a HOMO-LUMO energy gap of 0.149 to 0.154 eV except compound 4b, which had 0.114 eV.

The HOMO/LUMO values were utilized to calculate global reactivity descriptors such as chemical potential (µ), softness (S), hardness (η), electronegativity (X), and electrophilicity index (ω) as shown in Table 1. A compound’s structure, stability, and global chemical reactivity are explained by global chemical reactivity descriptors [57]. The title compounds were highly polarizable due to their greater softness value (≈6.6 eV). All compounds have higher ionization potentials (IPs) than EAs, indicating a greater electron-releasing tendency. The compound 4b had the highest softness (8.754 eV), smallest HOMO – LUMO gap (0.114 eV), and hardness (0.057 eV) among the series of compounds, it may be more reactive. The IPs of all produced compounds are higher than the electrophilicity index (ω), indicating a stronger nucleophilic nature.

Table 1.

The results of DFT analyses of the compounds 4a-e..

Parameters	4a	4b	4c	4d	4e
HOMO (eV)	−0.205	−0.214	−0.195	−0.196	−0.197
LUMO (eV)	−0.056	−0.100	−0.044	−0.045	−0.043
ΔE (eV)	0.149	0.114	0.151	0.151	0.154
Ionization Potential (IP) (eV)	0.205	0.214	0.195	0.196	0.197
Electron Affinity (EA) (eV)	0.056	0.100	0.044	0.045	0.043
Electronegativity (Χ)	0.131	0.157	0.120	0.121	0.120
Chemical Hardness (η)	0.075	0.057	0.076	0.076	0.077
Chemical Softness (S)	6.697	8.754	6.611	6.602	6.506
Chemical Potential (µ)	−0.131	−0.157	−0.120	−0.121	−0.120
Electrophilic Index (ω)	0.114	0.216	0.095	0.096	0.093
ESPmin (kcal/mol)	−37.72	−33.51	−41.82	−52.76	−38.79
ESPmax (kcal/mol)	48.06	51.55	44.48	53.33	51.24
mean ESP (kcal/mol)	2.11	3.42	0.64	0.49	−0.63

3.2.2. Molecular electrostatic potential (ESP) analysis

Molecular electrostatic potential (ESP) is used to calculate electrostatic distribution potential and visualize the reactive site on compounds with electrophilic and nucleophilic reactions [55]. The ESP surface map is a valuable tool for comprehending the distribution of reactive sites. The electrostatic potential (ESP) surface was computed using the B3LYP/6-31 G* method for all synthesized compounds. This was done to investigate potential sites for nucleophilic and electrophilic interactions. The resulting ESP surface is depicted in Figure S2 (supplementary materials). The maximum negative area in the ESP is the favored location for electrophilic attack, as illustrated by the red color, while most positive regions are in blue, and zero potential regions are visualized in green. The change in the distribution of the electrostatic potential surrounding the drug may be primarily responsible for the variance in its binding affinity with the active site receptor [56]. The compounds undergo changes in molecule size, shape, and the orientation of negative, positive, and neutral electrostatic potentials based on the type of atoms, their electrical nature, and the molecular properties. The minimum, maximum, and mean electronic potential are summarized in Table 1. The oxadiazole ring had the lowest potential due to oxygen atom. The molecular docking showed that the highly electropositive nitrogen atom of the secondary amine function formed a hydrogen bond with tubulin protein’s amino acid residues Ala317 and Ala352.

3.3. Anticancer activity

All the compounds were screened for antiproliferative activity against nine panels of nearly five dozen cancer cell lines. The NCI-US protocol was followed for antiproliferative activity at 10 µM drug concentrations and their results were recorded as growth percent (GP) and percent growth inhibitions (PGI). A comprehensive explanation of anticancer screening was given in our previous work [58]. The compound 4a and 4c displayed anticancer activity with mean GP of 102.23 and 100.85 respectively. The breast cancer cell line MDA-MB-468 expressed good sensitivity with PGI of 71.40 percent, breast cancer cell line MCF7 expressed moderate sensitivity with PGI of 43.16 percent, the cell lines, LOX IMVI, CAKI-1, and KM12 expressed less sensitivity with PGI of 14.46, 12.86, and 10.25 percent respectively, while rest of the cell lines expressed very less sensitivity with PGI less than 10 percent against the compound 4a. The breast cancer cell lines, MCF7 displayed moderate sensitivity with PGI of 41.43 percent, the cell lines, MDA-MB-468, CAKI-1, NCI-H226, and M14 expressed less sensitivity with PGI 23.79, 18.84, 16.61, and 15.02 respectively, while rest of the cell lines expressed very less sensitivity with PGI less than 15 percent against the compound 4c. The compounds 4d and 4e expressed anticancer activity with mean GP of 89.16 and 89.45 respectively. The cell lines MDA-MB-435, SR, K-562, and MDA-MB-468 expressed moderate sensitivity against the compound 4d with PGI of 60.31, 57.28, 52.35, and 51.44, while cell lines MCF7, and KM12 expressed significant sensitivity with PGI 47.83, and 36.85 percent respectively. Some of the cell lines SNB-19, SNB-539, RXF-393, CAKI-1, HCC-2998, SF-295, and HS578T expressed less sensitivity with PGI of 26.33, 18.7, 17.89, 17.3, 17.17, 16.2, and 15.55 respectively, while rest of the cell lines expressed very less sensitivity with PGI less than 15 percent against the compound 4d. The cell lines UO-31, NCI-H226, CAKI-1, PC-3, MCF7, and RXF 393 expressed moderate sensitivity against the compound 4e with PGI of 46.86, 45.16, 34.12, 32.34, 32.00, and 30.80 respectively, and the cell lines, UACC-62, MDA-MB-231/ATCC, LOX IMVI, T-47D, KM12, RPMI, NCI-H23, SF-295, EKVX, M14, HS 578T, HCT-15, SK-MEL-5, and SF-268 expressed less sensitivity with PGI of 28.13, 26.31, 24.40, 24.02, 23.41, 21.90, 20.56, 20.48, 20.38, 20.11, 18.93, 15.62, 15.34, and 15.26 percent respectively and rest of the cell lines showed very less sensitivity with PGI less than 15 percent against the compound 4e. The cell lines SR, MDA-MB-435, MOLT-4, K-562, HL-60(TB), MCF7, MDA-MB-468, U251, HCT-15, and HT29 expressed good sensitivity with PGI of 98.83, 97.31, 96.69, 95.65, 91.53, 89.01, 81.10, 80.57, 80.27, and 80.23 respectively, while cell lines RXF 393, LOX IMVI, M14, CCRF-CEM, SW-620, NCI/ADR-RES, COLO 205, RPMI-8226, SNB-19, NCI-H522, and KM12 moderate sensitivity against the compound 4b with PGI of 66.67, 60.32, 56.87, 56.43, 56.22, 55.36, 55.00, 53.83, 53.51, 53.24, and 52.86 respectively. The cell lines HCT-116, NCI-H23, T-47D, SNB-75, IGROV1, HCC-2998, SK-MEL-5, SF-539, MALME-3 M, HOP-92, UACC-62, SK-MEL-2, CAKI-1, A549/ATCC, NCI-H226, and MDA-MB-231 displayed significant sensitivity against the compound 4b with PGI of 44.18, 43.33, 41.74, 41.39, 41.28, 40.90, 39.80, 37.23, 35.58, 35.55, 35.38, 35.23, 34.30, 33.84, 32.09, and 30.83 respectively, while cell lines SF-295, HOP-62, PC-3, NCI-H460, SK-MEL-28, SF-268, OVCAR-8, DU-195, EKVX, and 786-O, displayed less sensitivity against the compound 4b with PGI of 29.64, 28.74, 27.55, 24.98, 23.02, 22.61, 21.17, 20.84, 18.59, and 17.09 respectively, and rest of the cell lines displayed very less than with PGI less than 15 percent. The anticancer activity of compounds 4a–e against 59 cancer cell lines at 10 µM are given in Table S1 (supplementary materials) and five most sensitive cell lines against the compound 4a–e are given in Table 2. The comparative anticancer activity of compound (4a–e) on the PGI scale are shown in Table 3. The overall anticancer activity in terms of mean GP followed as 4-NO₂ >3,4,5-(OCH₃)₃ >2-OH > 4-OCH₃ >4-Cl. In order to compare the anticancer activity of each compound with that of imatinib and 5-fluorouracil (5-FU), the average GP was calculated for the cancer panel and the results are shown in Table S2. The most active compound 4b, demonstrated superior anticancer activity to imatinib against all cancer cell lines and superior anticancer activity to the standard drug, 5-FU, against leukemia, melanoma, and ovarian cancer. The compound 4e demonstrated superior anticancer activity to imatinib against all the cell lines except non-small lung cancer cell lines. The anticancer activity data of the 5-FU and imatinib were retrieved from NCI website [39].

Table 2.

In vitro anticancer activity of 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine (4a-e.)

S. No.	Compound	Most sensitive cell lines	GP	PGI
1	4a	MDA-MB-468 (Breast cancer)	28.60	71.40
		MCF7 (Breast cancer)	56.84	43.16
		LOX IMVI (Melanoma)	85.54	16.46
		CAKI-1 (Renal cancer)	87.14	12.86
		KM12 (Colon cancer)	89.75	10.25
2	4b	SR (Leukemia)	1.17	98.83
		MDA-MB-435 (Melanoma)	2.69	97.31
		MOLT-4 (Leukemia)	3.31	96.69
		K-562 (Leukemia)	4.35	96.65
		HL-60(TB) (Leukemia)	8.47	91.53
3	4c	MCF7 (Breast cancer)	58.57	41.43
		MDA-MB-468 (Breast cancer)	76.21	23.79
		CAKI-1 (Renal cancer)	83.39	16.61
		NCI-H226 (Non-small cell lung cancer)	81.16	18.84
		M14 (Melanoma)	84.98	15.02
4	4d	MDA-MB-435 (Melanoma)	39.69	60.31
		SR (Leukemia)	42.72	57.28
		K-562 (Leukemia)	47.65	52.35
		MDA-MB-468 (Breast cancer)	48.56	51.44
		MCF7 (Breast cancer)	52.17	47.83
5	4e	UO-31 (Renal cancer)	53.14	46.86
		NCI-H226 (Non-small cell lung cancer)	54.84	45.14
		CAKI-1 (Renal cancer)	65.88	34.12
		PC-3 (Prostate cancer)	67.66	32.34
		MCF7 (Breast cancer)	68.00	32.00

Table 3.

Comparative anticancer activity of 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amine (4a-e) on the PGI scale.

S. No.	Compounds	Cell line (percent growth inhibition; PGI)
		>68	67.99 to 50.00	49.99 to 30.00
1	4a	MDA-MB-468 (71.40)	–	MCF7 (43.16)
2	4b	SR (98.83) MDA-MB-435 (97.31) MOLT-4 (96.69) K-562 (95.65) HL-60(TB) (91.53) MCF7 (89.01) MDA-MB-468 (81.10) U251 (80.57) HCT-15 (80.27) HT29 (73.45)	RXF 393 (66.67) LOX IMVI (60.32) M14 (56.87) CCRF-CEM (56.43) SW-620 (56.22) NCI/ADR-RES (55.36) COLO 205 (55.00) RPMI-8226 (53.83) SNB-19 (53.51) NCI-H522 (53.24) KM12 (52.86)	HCT-116 (44.18) NCI-H23 (43.33) T-47D (41.74) SNB-75 (41.39) IGROV1 (41.28) HCC-2998 (40.90) SK-MEL-5 (39.80) SF-539 (37.23) MALME-3 M (35.58) HOP-92 (35.55) UACC-62 (35.38) SK-MEL-2 (35.23) CAKI-1 (34.30) A549/ATCC (33.84) NCI-H226 (32.09) MDA-MB-231 (30.83)
3	4c	–	–	MCF7 (41.43)
4	4d	–	MDA-MB-435 (60.31) SR (57.28) K-562 (52.35) MDA-MB-468 (51.44)	MCF7 (47.83) KM12 (36.85)
5	4e	–	–	UO-31 (46.86) NCI-H226 (45.16) CAKI-1 (34.12) PC-3 (32.34) MCF7 (32.00)

(-) None of the cell lines displayed PGI of this percent.

The structure–activity relationship (SAR) established with the anticancer data showed the 4-nitro substitution (4b) in the oxadiazole linked phenyl ring demonstrated a more promising anticancer activity than 3,4,5-trimethoxy substitution (4e) followed by 2-hydroxy (4d) and 4-methoxy (4c) substitutions. The 4-chloro substitution displayed less significant anticancer activity. The role of other substituents like fluoro, bromo, methyl and others at ortho, meta, and para position in the phenyl ring are required for further exploration of the SAR. The present study might be beneficial and present an exciting opportunity for further research.

3.4. Molecular docking studies

The molecular docking simulation against the colchicine binding site of tubulin (PDB ID: 1SA0) was carried out for the ligands (4a–e) and their docking score and interactions are shown in Table 4. Prior to these simulations, the docking protocol was validated by re-docking the co-crystallized ligand (colchicine) into the colchicine binding site and comparing the resulting docked pose to the original crystal structure. The root mean square deviation (RMSD) value obtained was 1.065 Å, which confirms the reliability of the docking protocol, as an RMSD value below 2 Å is generally accepted as valid (Figure S3) (supplementary materials) [59]. Efficient binding was observed for the ligands 4a–e with docking score of −6.679 to −7.912 kcal/mol. The ligands 4b and 4c showed similar type of interactions displayed π-cationic interaction with naphthyl ring with the residue Lys352, while ligand 4d interacted with the residues Ala317 (with NH) and Thr179 (with OH) through H-bond. The ligand 4e displayed H-bond interaction with the residue Ala317 (with NH), while ligand 4a displayed H-bond interaction with the residue Ala317 (with NH) and π-cationic interaction with phenyl ring with the residue Lys352. The superimposed 3D image of ligands 4a-e are shown in Figure S4 (supplementary materials). The ligand 4e that showed H-bond interaction with the residue Ala317 is shown in Figure 3. The ligand 4e (docking score = −7.295 kcal/mol) displayed promising anticancer activity displayed strong interaction within the active site of tubulin exhibited promising anticancer as well as antioxidant activities. The superimposed 3D interaction of ligand 4b and colchicine at the colchicine binding site in molecular docking is shown in Figure S5 (supplementary materials). The 2D interaction revealed π-cationic with the residue Lys352 for the ligand 4b while colchicine demonstrated two H-bonds with the residues Val315 and Ser178. The interaction of rest of the compounds are given in Figure S6 (supplementary materials).

Table 4.

The molecular docking studies of 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amines (4a-e) against the active site of tubulin (PDB ID: 1SA0).

S. No.	Compound	Docking score	Glide emodel	Interaction(s)
1	4a	−7.242	−59.702	H-bond (Ala317; 2.43 Å); π-Cation (Lys352; 6.27 Å)
2	4b	−6.697	−59.795	π- Cation (Lys352; 6.33 Å)
3	4c	−6.679	−58.908	π- Cation (Lys352; 6.40 Å)
4	4d	−7.912	−66.927	H-Bond (Ala317; 2.53 Å and Thr179; 1.79 Å)
5	4e	−7.295	−68.116	H-Bond (Ala317; 2.54 Å)
6	IMC-038525	−8.069	−77.353	−
7	IMC-094332	−7.487	−77.268	−

(−) = No electrostatic interactions.

Figure 3.

3D and 2D image of ligand 4e against the active site of tubulin (PDB ID: 1SA0).

3.5. Molecular dynamics (MD) simulation

The protein does not stay constant to fit the incoming ligand to its receptor inside the bodily functions. There are continual random structural rearrangements that necessitate flexible docking assessment of the protein and ligand. MD simulations enable flexible docking by simulating motions at an atomic scale using Newtonian physics. These can be well understood by molecular dynamics studies, which recreate the identical biological conditions [60]. Since the compound 4e demonstrated significant anticancer activity, therefor, MD simulation was performed for the ligand indicating the significant anticancer activity and docking score (compound 4e), in complex with tubulin protein to analyze the dynamic stability and conformational behavior. Several parameters were displayed and assessed, including the ligand and protein RMSD, protein root mean square fluctuation (RMSF), and the histogram reflecting the interaction between the ligand and amino acids.

The RMSD is the mean deviation in movement of atoms in the protein – ligand complex in contrast to the starting (reference) frame, for a given time period. The RMSD values of protein backbone (Cα atoms) atoms bonded to the compound represent how the protein backbone stays inside and changes structurally with time in reference to the starting point [61]. The highest RMSD value of the tubulin protein’s Cα backbone is 3.2 Å, indicating that the tubulin-4e complex stayed stable during the simulation period. In ligand RMSD with respect to protein, an initial drift was observed till 5 ns, after which a gradual equilibrium was shown till reaching 80 ns, fluctuating within the range of 3.0 Å, and then returned again to an equilibrium state within a means of fluctuation of 2.2 Å (Figure 4(a)). In contrast, for the colchicine complex, the RMSD remained stable between 1.2 Å and 2.8 Å from 0 ns to 50 ns. Subsequently, a minor drift was observed; the RMSD decreased and stabilized at an average of 2.4 Å for the remainder of the simulation (Figure S7A) (supplementary materials).

Figure 4.

MD simulation analysis of compound 4e in complex with tubulin protein (PDB ID: 1SA0) (a) time dependent RMSD of tubulin-4e complex (protein Cα-atoms RMSD is shown in gray while RMSD of compound 4e with respect to protein are shown in maroon) (b) protein amino acids RMSF (c) ligand 2D interaction diagram and (d) protein – ligand contact histogram obtained from MD trajectory.

Fluctuations of specific amino acids influences the stability of the protein-ligand complex. The residue-wise fluctuations or local changes in protein backbone atoms during simulation are denoted by RMSF. Protein RMSF plot analysis can provide information about the protein’s flexible regions [62]. Figure 4(b) shows the RMSF plot of the tubulin-4e complex. Both the active ligand 4e and colchicine (Figure S7B) (supplementary materials) was found to interact different amino acids of the tubulin protein, including Asn 101 (0.882 Å), Ser 178 (1.085 Å), Thr 179 (0.931 Å), Ala 180 (0.816 Å), Glu 183 (0.713 Å), Tyr 224 (1.168 Å), Cys 241 (1.137 Å), Leu 242 (1.193 Å), Gln 247 (1.391 Å), Leu 248 (1.037 Å), Asn 249 (1.023 Å), Ala 250 (1.2 Å), Leu 252 (0.651 Å), Lys 254 (0.765 Å), Leu 255 (0.831 Å), Val 257 (0.78 Å), Asn 258 (0.87 Å), Met 259 (0.769 Å), Ala 316 (0.894 Å), Val 318 (0.852 Å), Lys 352 (0.955 Å), Thr 353 (0.989 Å), Ala 354 (1.085 Å), and Ile 378 (0.662 Å). It was obvious that the binding site amino acids showed the minor local conformational changes with compound 4e (˂ 1 Å). This finding confirmed the conformational stability of the binding pocket during the simulation. In this plot both tails (N-terminal regions) have greater RMSF values, which corresponds to the predicted high flexibility of their loop structure (white bar), in contrast to other α-helices (blue bar) and β-sheets (pink bar), which are typically stiffer.

The 2D binding interaction of ligand 4e during the simulation is depicted in Figure 4(c) (supplementary materials), where Thr353 interacted through hydrogen bonding with secondary amine for 71% of the simulation time. Additionally, the oxadiazole ring and the trimethoxy phenyl ring interacted with Lys352 for 65% and 57% of the simulation time, respectively. In the tubulin-colchicine complex, the carbonyl group interacted with Thr179 for 54% of the simulation time, while the amino group engaged with the positively charged residue Lys352 for 83% of the simulation duration (Figure S7C) (supplementary materials). To assess the interactions of the ligands with the protein, each contact with the amino acid was evaluated via the protein – ligand contact histogram. This method quantifies the frequency and nature of the interactions throughout the simulation period. It was noted that over the normalized course of the trajectory, in the tubulin-4e, only two amino acids, i.e., Thr353, and Asn258, were involved in hydrogen bonding and these hydrogen bonds are critical for stabilizing the ligand within the binding pocket, facilitating direct interactions that influence the ligand’s orientation and affinity for the protein, while major hydrophobic interaction was observed with nonpolar amino acid residues, Ala180, Tyr224, Leu248, Leu255, Ala316, Ala316, Lys352, Ala354, and Ile378. These hydrophobic contacts are essential in driving the binding process, as they minimize the exposure of hydrophobic regions of the ligand and protein to the aqueous environment. Such interactions can lead to a more favorable free energy change, promoting ligand binding. Moreover, hydrogen bonding mediated by amino acids was found to be essential for stability, as demonstrated in this complex with residues Asn101, Ser178, Ala180, Glu183, Gln247, Asn249, Ala250, and Thr353 and these hydrogen bonding interactions highlighted a cooperative network of contacts that fortified the ligand in place. These residues not only provide anchoring points but also help maintain the overall conformation of the binding site, ensuring optimal positioning of the ligand for effective interaction (Figure 4(d)). In the case of colchicine, this ligand forms hydrogen bonds with three specific amino acids: Thr179, Cys241, and Lys352. Each of these residues contributes uniquely to stabilizing the complex. The hydrogen bond formed with Thr179 may assist in positioning colchicine correctly for optimal interaction with the binding site, while the interaction with Cys241 could enhance stabilization through sulfur-containing side-chain interactions. In addition to hydrogen bonding, colchicine engages in robust hydrophobic interactions with residues, such as Leu248, Leu255, Ala316, Val318, and Ala354. The significance of these interactions lies in their contribution to the ligand’s binding affinity. By favorably interacting with the hydrophobic regions of the protein, colchicine effectively buries its hydrophobic surface, which was critical for the formation and stability of the complex. Furthermore, similar to the tubulin-4e complex, colchicine benefited from water-mediated hydrogen bonds. These transient interactions can serve as additional stabilizing forces, allowing for dynamic adaptability in the binding interface, which may enhance the overall stability of the ligand-protein complex (Figure S7D) (supplementary materials).

3.6. ADMET prediction

The title compounds were predicted for their ADMET studies by SwissADME software and ProTox II and their results are shown in Table S3 (supplementary materials). The number of hydrogen bond acceptor and donor were found ranging from 3 to 6 (<10) and 1 − 2 (<5) respectively, while log p values were predicted between 2.67 and 3.63 (≤5). The total polar surface area (TPSA) was predicted from 50.95 to 96.77 and percentage of absorption (%Abs) were calculated ranging from 91.42 to 75.61% from the equation %Abs = 109 − 0.345 × TPSA [63]. The number of rotatable bonds were predicted as 3 to 6 (<10) and molecular weight ranging from 303 to 378 (<500). Most of compounds were predicted to have sufficient lipophilicity to cross the blood-brain barriers and to be absorbed from the GIT and none of the compounds violated Lipinski rule of five [64]. The compounds (4a-f) were also predicted to be toxicity-free, except hepatotoxicity (HEP). The compounds 4b and 4e were also predicted to have slightly mutagenicity (MUTAGEN) and immunotoxicity (IMMUNO) respectively.

4. Conclusion

We discussed the synthesis, anticancer activity, and molecular docking studies of newer 5-aryl-N-(naphthalen-2-yl)-1,3,4-oxadiazol-2-amines (4a–e) in this study. The compounds were prepared efficiently in excellent yield. The DFT studies were carried out to analyze HOMO-LUMO energy gap of compounds (4a–e). Among the title compounds, 4b has the highest softness (8.754 eV), as well as the smallest HOMO – LUMO gap (0.114 eV) and hardness (0.057 eV), indicating that it could be more reactive than the others, and the compound 4b demonstrated good anticancer activity against a few cell lines like SR, MDA-MB-435, MOLT-4, K-562, HL-60(TB), MCF7, MDA-MB-468, U251, HCT-15 and HT29 expressed good sensitivity against the compound 4b with PGI of 98.83, 97.31, 96.69, 95.65, 91.53, 89.01, 81.10, 80.57, 80.27, and 73.45, respectively, at 10⁻⁵ M. The cell line MDA-MB-468 displayed good sensitivity against the compound 4a with PGI of 71.40 percent. The compound 4e exhibited the promising anticancer activity and docking score. A few cell lines like UO-31, NCIH226, CAKI-1, PC-3, MCF7, and RXF 393 expressed sensitivity against the compound 4e with PGI of 46.86, 45.16, 34.12, 32.34, 32.00, and 30.80, respectively, at 10⁻⁵ M. The compounds were studied for molecular docking against the active site of tubulin (PDB ID: 1AS0), where the ligand 4e (with a docking score of −7.295 kcal/mol) showed H-bond interaction with the residue Ala317. The MD simulation were also performed in complex with tubulin protein and ligand 4e to examine dynamic stability and conformational behavior. All of the compounds passed the ADME prediction tests, indicating that they could be absorbed more effectively when taken orally.

Funding Statement

This research was funded by Taif University, Saudi Arabia, Project number (TU-DSPP-2024-77).

Article Highlights

• The designed 1,3,4-oxadiazole analogues were synthesized efficiently in two steps.

• DFT computations were accomplished and HOMO/LUMO analyzed followed by their anticancer evaluations.

• The compounds 4b demonstrated a significant anticancer activity against cancer cell lines, SR, MDA-MB-435, MOLT-4, K-562, and HL-60(TB) at 10 µM.

• The compounds 4e showed a promising anticancer activity against cancer cell lines, UO-31, NCI-H226, CAKI-1, PC-3, MCF7, and RXF 393 at 10 µM.

• The compound 4e displayed a H-bond interaction with Ala317 residue in docking studies with a docking score of −7.295 Kcal/mol and moderate local conformational deviations (˂ 1 Å) in the MD simulation studies.

Data availability statement

All data generated or analyzed during this study are included in this published article.

Conflicts of interest

The author confirms that this article content has no conflicts of interest.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2504335