The research team, led by Maimoonah M. Khalaf, Safa M. Shawkat, and colleagues at Tikrit University, focused on nitrogen-containing heterocycles, a class of ring-shaped molecules that has underpinned drug discovery for more than a century. Tetrazoles, five-membered rings built from one carbon atom and four nitrogen atoms, have been studied since their first synthesis in 1855 and appear in numerous pharmaceutical agents, largely because their high nitrogen content allows them to form multiple hydrogen bonds with biological targets and to mimic the carboxylic acid groups found in many natural metabolites. Imidazole rings, another privileged structural motif, are embedded in a wide range of approved medicines and exhibit broad biological activity thanks to their electron-rich character and ability to interact with enzymes and receptors.

The synthetic route began with 1,1′-biphenyl-4,4′-dicarbaldehyde, a rigid biphenyl scaffold bearing two aldehyde groups, which was condensed with 4-cyanoaniline in absolute methanol using catalytic glacial acetic acid. After seven hours of stirring, the resulting Schiff base, compound S1, was isolated in an excellent 90 percent yield as a light yellow solid melting at 239 to 241 degrees Celsius. Spectroscopic analysis confirmed the transformation: the aldehyde carbonyl band vanished from the infrared spectrum while a sharp azomethine stretch appeared at 1631 reciprocal centimeters, accompanied by the characteristic nitrile band at 2222 reciprocal centimeters inherited from the cyanoaniline starting material. Proton NMR spectroscopy showed the diagnostic imine singlet at 8.72 parts per million alongside sixteen aromatic protons, and elemental analysis of carbon, hydrogen, and nitrogen matched theoretical values closely.

From this versatile Schiff base intermediate, the team branched into two distinct heterocyclic families. Refluxing S1 with sodium azide in tetrahydrofuran at 64 to 66 degrees Celsius for eleven hours triggered a cycloaddition across the imine double bonds, producing the bis-tetrazoline derivative S2 in 83 percent yield. Infrared spectroscopy documented the complete disappearance of the azomethine band and the emergence of new features: a secondary amine stretch at 3390 reciprocal centimeters, an endocyclic N=N vibration at 1454 reciprocal centimeters, and ring C-N stretches near 1194 and 1165 reciprocal centimeters. In the proton NMR spectrum, the imine signal was replaced by a singlet at 5.16 parts per million for the saturated C-5 proton of the newly formed 2,5-dihydro-1H-tetrazole ring and a singlet at 4.67 parts per million for the ring N-H proton, a strongly shielded position consistent with a non-aromatic, partially saturated tetrazoline core rather than a fully aromatic tetrazole.

The second branch exploited Schiff base chemistry with amino acids. Condensing S1 with glycine in refluxing ethanol delivered the bis(5-oxoimidazolidine) compound S3 in 77 percent yield, while the parallel reaction with L-alanine produced the methyl-substituted analogue S4 in 70 percent yield. Both orange to red crystalline products showed the hallmark signatures of successful ring closure: disappearance of the azomethine and primary amine bands, appearance of secondary amine stretches near 3365 and 3340 reciprocal centimeters, and a strong imidazolidinone carbonyl at 1678 reciprocal centimeters. The NMR data sealed the structural assignments, with ring N-H signals between 8.23 and 8.39 parts per million, saturated ring methine protons near 6.1 parts per million, a methylene singlet at 4.36 parts per million for S3, and, for S4, the telltale quartet-doublet pattern of a methyl-bearing stereocenter at 4.86 and 1.82 parts per million. The authors note that equipment limitations prevented acquisition of carbon-13 NMR, high-resolution mass spectrometry, and HPLC purity data, but the convergent infrared, proton NMR, and elemental analysis evidence firmly supports the proposed structures.

Biological testing followed standard agar well diffusion protocols against two clinically relevant pathogens: Gram-positive Staphylococcus aureus isolated from tonsillitis patients and Gram-negative Escherichia coli from urinary tract infections, both obtained from the Advanced Biological Sciences Laboratory at Tikrit University. Bacterial lawns standardized to the 0.5 McFarland turbidity equivalent were grown on Mueller-Hinton agar, and each compound was tested at 0.01, 0.001, and 0.0001 milligrams per milliliter with DMSO as the negative control and ampicillin as the reference drug. The results were unambiguous. The Schiff base S1, lacking any heterocyclic ring, was inactive against both organisms at every concentration. The tetrazoline S2, by contrast, dominated the screen: it produced a 46 millimeter zone of inhibition against E. coli at the highest dose, substantially exceeding ampicillin’s 30 millimeters under the same conditions, along with 40 and 34 millimeter zones at the two lower doses, while against S. aureus it yielded an 18 millimeter zone.

The imidazolidinone derivatives S3 and S4 showed moderate but genuine activity against both organisms, with inhibition zones in the range of 6 to 12 millimeters depending on concentration. Intriguingly, the methyl group installed in S4 did not help; activity slightly decreased at moderate and low concentrations, a pattern the authors attribute to steric hindrance blocking access to cellular targets. The overall hierarchy makes chemical sense: the tetrazoline ring carries a higher density of nitrogen atoms and therefore greater electron density and hydrogen-bonding capacity than the oxoimidazolidine system, and its electronic character appears to facilitate penetration through the lipid-rich outer membrane that makes Gram-negative bacteria such formidable drug targets. The study thus provides a clean structure-activity lesson, showing that antibacterial potency rises with nitrogen content and electron density while bulky substituents can quietly sabotage otherwise promising scaffolds.

To probe mechanism, the researchers docked the active compounds into two essential bacterial enzymes: biotin carboxylase from E. coli, which catalyzes the ATP-dependent carboxylation of biotin during fatty acid synthesis, and biotin protein ligase from S. aureus, which covalently attaches biotin to metabolic carboxylases. Docking parameters were validated by redocking the native ligands, with heavy-atom root-mean-square deviations of 0.85 and 1.12 angstroms, comfortably below the accepted 2.0 angstrom threshold. Within the E. coli enzyme active site, S3 recorded the best binding energy at minus 8.3 kilocalories per mole, closely followed by S2 and S4 at minus 8.2, with hydrogen bonds to tyrosine 380 and aspartate 382 stabilizing the tetrazoline complex. In the S. aureus ligase pocket, S2 achieved the strongest overall affinity of minus 9.1 kilocalories per mole, anchored by a hydrogen bond to asparagine 124, while S3 and S4 each scored minus 7.9. The nitrogen-rich tetrazole core emerged as a strong bioisostere that drives polar contacts within these druggable pockets.

Complementing the docking work, density functional theory calculations at the B3LYP/6-31G(d,p) level yielded frontier molecular orbital energies, energy gaps, dipole moments, chemical hardness, chemical potential, and electrophilicity indices for all four compounds. Chemical hardness rose progressively from S2 at 0.078265 electron volts through S3 at 0.088975 to S4 at 0.089505, indicating the tetrazoline occupies a reactive sweet spot balancing stability and softness, while the inactive Schiff base S1, despite having the smallest hardness value and narrowest energy gap, remained biologically inert, a reminder that electronic softness alone cannot deliver antibacterial efficacy without the right pharmacophoric and steric features. S2 also displayed a moderate chemical potential that positions it ideally for interaction with enzyme active sites. Taken together, the synthesis, screening, docking, and quantum chemical results converge on a single message: nitrogen-dense 2,5-dihydro-1H-tetrazole scaffolds grafted onto rigid biphenyl backbones deserve serious attention in the ongoing search for antibacterial agents capable of countering resistant Gram-negative bacteria, and the integrated experimental-computational pipeline demonstrated here offers a replicable template for designing the next generation of target-specific antimicrobials.

Subject of Research: Synthesis, antibacterial evaluation, molecular docking, and DFT analysis of biphenyl-based bis(tetrazoline) and bis(imidazolidin-4-one) derivatives

Article Title: Synthesis, characterization, antibacterial evaluation, molecular docking, and DFT analysis of biphenyl-based Bis(tetrazoline) and Bis(imidazolidin-4-one) derivatives

Article References: Khalaf, M. M., Shawkat, S. M., Saleh, M. Q., Salih, M. M., Saleh, M. J., Saleh, J. N., & Abdulmajeed, A. Z. (2026). Synthesis, characterization, antibacterial evaluation, molecular docking, and DFT analysis of biphenyl-based Bis(tetrazoline) and Bis(imidazolidin-4-one) derivatives. Results in Chemistry, 30, Article 103867. https://doi.org/10.1016/j.rechem.2026.103867

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103867

Keywords: tetrazole derivatives, Schiff base, imidazolidin-4-one, antibacterial activity, molecular docking, DFT calculations, Escherichia coli, Staphylococcus aureus, heterocyclic compounds, biphenyl scaffold, antibiotic resistance, biotin carboxylase