The synthetic route itself is elegantly simple. Substituted aromatic aldehydes, methyl cyanoacetate and barbituric acid are combined in a 1:1 mixture of ethanol and water at 85 degrees Celsius with just 0.03 mol percent of Bi(OTf)3. The catalyst orchestrates a domino Knoevenagel-Michael sequence: it coordinates to the ester oxygen of methyl cyanoacetate, sharpening the electrophilicity of the carbonyl carbon, while the aldehyde-derived enolate attacks to form a carbon-carbon bond. The resulting alpha,beta-unsaturated intermediate is then struck by the weakly nucleophilic nitrogen of barbituric acid, and intramolecular cyclization closes the fused pyridopyrimidine ring. Eight derivatives, bearing nitro, bromo, hydroxyl, methyl, methoxy or unsubstituted phenyl groups, were isolated in yields ranging from 76 to 91 percent within two to five hours, with minimal side products and no need to purify intermediates.

What makes the protocol genuinely green is the solvent system and the catalyst. Bismuth(III) salts have gained a reputation as environmentally benign alternatives to conventional Lewis acids because they are air- and moisture-stable, functionally tolerant, commercially available and far less corrosive than many metal halides. Compared with earlier methods, the advantages are clear. A DMAP-catalyzed protocol requires ultrasonic irradiation in toxic DMF, ZrO2 nanoparticles demand nanomaterial synthesis and raise agglomeration concerns, and the sulfonated SBA-15 catalyst involves a cumbersome multistep preparation. The bismuth route runs in a drinkable solvent mixture under ordinary reflux, and the catalyst can be recovered, although reusability tests showed a gradual decline: the model reaction yielded 79 percent in the first cycle, 74 percent in the second, 65 percent in the third and only 33 percent in a fourth run that stretched to nine hours. The authors suggest that immobilization strategies could extend catalyst lifetime in future work.

On the computational side, the team optimized all eight molecules at the B3LYP/6-31G(d,p) level of density functional theory, confirming that each geometry corresponds to a true energy minimum. Frontier molecular orbital analysis revealed that the nitro-substituted compounds 4a and 4b possess the smallest HOMO-LUMO gaps, at roughly 0.135 electron volts, making them the most electronically soft and reactive members of the series, primed for donor-acceptor interactions with biological macromolecules. In contrast, the bromo, methyl and unsubstituted derivatives 4c, 4f and 4g showed the largest gaps and the greatest hardness, indicating higher kinetic stability. Mulliken charge analysis and molecular electrostatic potential maps pinpointed the carbonyl oxygens and ring nitrogens as the electron-rich hotspots most likely to engage in hydrogen bonding with protein targets, while the N-H hydrogens carried the complementary positive charge.

The biological evaluation used the broth dilution method to determine minimum inhibitory concentrations against four bacterial strains, the Gram-negative Escherichia coli and Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus and Streptococcus pyogenes, benchmarked against ampicillin, and three fungal strains, Candida albicans, Aspergillus niger and Aspergillus clavatus, benchmarked against griseofulvin. The structure-activity trends were striking. The unsubstituted compound 4g and the methoxy-bearing 4h were the standout antibacterial agents, with MIC values as low as 65 and 62 micrograms per milliliter respectively against E. coli and S. aureus, figures that actually beat ampicillin on those strains. The methyl derivative 4f was the most potent antifungal, matching griseofulvin’s 100 micrograms per milliliter against A. niger. Electron-donating groups such as methyl and methoxy generally enhanced activity, likely by improving lipophilicity and membrane penetration, while nitro groups tended to raise MIC values.

To explain these observations mechanistically, the researchers docked all eight compounds into two clinically significant enzymes: bacterial beta-lactamase, the molecular engine of antibiotic resistance, and sterol 14-alpha demethylase CYP51, the fungal enzyme that builds ergosterol membranes. Using AutoDock 4.2 with a Lamarckian genetic algorithm, they found that compounds 4b and 4h bound beta-lactamase most favorably, with estimated free energies of binding of minus 9.30 and minus 9.43 kilocalories per mole, stabilized by networks of hydrogen bonds, pi-alkyl contacts and pi-cation interactions with residues such as ARG 661, PRO 298 and TYR 611. Against CYP51, compound 4b again led with minus 7.79 kilocalories per mole, and the methyl-substituted 4f formed six hydrogen bonds within the active site, consistent with its antifungal potency. Torsional free energies were nearly uniform across the series, indicating that differences in affinity stem from noncovalent interaction networks rather than ligand flexibility.

Molecular dynamics simulations then stress-tested the most promising complexes over 100-nanosecond production runs using the Maestro-Desmond package with the OPLS3e force field. The 4f-beta-lactamase complex held a backbone root mean square deviation averaging 1.60 angstroms, comfortably below the 2.0 angstrom threshold that validates docking reliability, and stabilized after an initial 30-nanosecond equilibration. The 4d-CYP51 complex averaged 1.40 angstroms, closely tracking the reference antifungal ligand VT1 at 1.35 angstroms. Root mean square fluctuation analysis showed that flexibility was confined to loop and terminal regions, leaving the binding pockets rigid. Contact histograms revealed that water-bridged hydrogen bonds, involving residues such as Ser64, Gln120, Thr122 and Tyr132, together with hydrophobic contacts, were the main glue holding the complexes together throughout the simulations.

The pharmacoinformatic layer of the study added yet another dimension. POM analysis, which classifies bioactivity based on dipolar interactions between electron-rich and electron-deficient centers, identified three distinct antitumor pharmacophore regions built from NH donor to carbonyl oxygen acceptor pairs, plus a single antibacterial pharmacophore involving an NH-CO motif. OSIRIS toxicity screening found no major mutagenic, tumorigenic, irritant or reproductive risks across the series, with compounds 4d and 4g earning the highest drug scores at 0.70 and 0.87. Molinspiration calculations confirmed that all eight molecules respect Lipinski’s criteria almost entirely: molecular weights below 500 daltons, five or fewer rotatable bonds, moderate polarity and acceptable lipophilicity, with only the heavily nitrated pair showing a single violation.

Taken together, the work demonstrates a rare degree of coherence between experiment and computation. The compounds with the best MIC values, notably 4f, 4g and 4h, also posted strong docking energies and stable dynamic behavior, while the electrostatic potential maps independently flagged the same carbonyl and ring nitrogen atoms that docking identified as interaction points. The authors conclude that pyrido[2,3-d]pyrimidines represent a promising scaffold for further antimicrobial development, and that targeted substitution of the aromatic ring is the key lever for tuning activity. With antimicrobial resistance declared one of the top global public health threats, a synthetic method that is cheap, green and high-yielding, paired with a validated computational pipeline for prioritizing candidates, offers exactly the kind of integrated approach the field has been calling for. The next step will be translating these laboratory and in silico hits into lead compounds capable of surviving the far harsher tests of animal models and, eventually, the clinic.

Subject of Research: Green synthesis and antimicrobial evaluation of pyrido[2,3-d]pyrimidine derivatives catalyzed by bismuth(III) triflate

Article Title: Bi(OTf)₃-catalyzed one-pot synthesis and antimicrobial evaluation of bioactive pyridopyrimidines supported by DFT, molecular dynamics, POM and pharmacophore analyses

Article References: Mehta, A. W., Abdel-Megid, M., Patil, R. C., Ahmed, S., Salem, M. E., Abu-Rayyan, A., Shtaiwi, A., Hajam, Y. A., Bhat, A. R., Eissa, M. E., Agisho, H. A., Mujahid, M. H., Yamari, I., Raza, N., & Zbancioc, A. M. (2026). Bi(OTf)₃-catalyzed one-pot synthesis and antimicrobial evaluation of bioactive pyridopyrimidines supported by DFT, molecular dynamics, POM and pharmacophore analyses. Results in Chemistry, 30, Article 103849. https://doi.org/10.1016/j.rechem.2026.103849

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103849

Keywords: pyridopyrimidines, bismuth(III) triflate, multicomponent reaction, green chemistry, antimicrobial activity, molecular docking, molecular dynamics, DFT, beta-lactamase, CYP51, drug discovery, pharmacophore

The clinical backdrop is grim. Candida albicans lives harmlessly on the skin and mucous membranes of most healthy people, but in patients whose immune defenses are compromised it turns opportunistic, causing infections that range from thrush and invasive candidiasis to deadly bloodstream sepsis. Treatment options have narrowed rather than widened over the decades: the azole antifungals, which include fluconazole, have been used so widely that resistant strains now circulate in hospitals worldwide, and the newer echinocandins, though valuable, are expensive and have their own limitations. Resistance in Candida typically emerges through mutations in the drug’s protein target, overexpression of that target, or efflux pumps that simply expel the drug from the cell before it can act. A compound that must be defeated twice, at two biologically distinct targets, presents a far taller order for the pathogen’s evolutionary toolkit.

The first of the two targets examined in the study is CYP51, also known as lanosterol 14-alpha-demethylase, a cytochrome P450 enzyme that sits at the heart of ergosterol biosynthesis. Ergosterol is the fungal equivalent of cholesterol: it lodges in the cell membrane and governs the membrane’s fluidity, integrity and the function of embedded proteins. Without it, the fungal cell membrane becomes leaky and disorganized, and the cell cannot survive. The azole class works by blocking CYP51, which is precisely why resistance to azoles usually maps onto mutations in this enzyme or its gene. A molecule that inhibits CYP51 by a different binding mode, or that is structurally unrelated to azoles, could in principle evade the resistance mutations that have eroded fluconazole’s effectiveness.

Using molecular docking, Shukla and Dubey found that both compounds lodge themselves into the CYP51 active site with convincing affinity. The computed binding energy was minus 7.4 kilocalories per mole for 1PB1 and minus 7.6 kilocalories per mole for 45R, scores that fall into the range typically considered indicative of stable ligand-protein complexes. But docking alone is only a hypothesis. The researchers then treated Candida albicans cultures with each compound and profiled the cells’ sterol content by gas chromatography-mass spectrometry. The telltale signature of CYP51 inhibition appeared: lanosterol, the enzyme’s substrate, accumulated in the treated cells because the blocked enzyme could no longer convert it downstream. The consequence was visible in the ergosterol assays, which showed that ergosterol production fell by roughly 69.92 percent, give or take 2.3 percent, in cells exposed to 1PB1 and by 55.48 percent, give or take 1.79 percent, in cells exposed to 45R. In plain terms, both compounds starved the fungus of the lipid that its membranes depend on.

The second target is less conventional and, in some ways, more intriguing. Exo-beta-1,3-glucanase is a cell wall enzyme that clips beta-glucan, the sugar polymer that gives the fungal wall much of its structural strength. Unlike beta-1,3-glucan synthase, the target of the echinocandin class, exo-glucanase participates in the trimming and remodeling of wall glucan, processes the fungus relies on during growth, cell division and the transition between its yeast and hyphal forms, which is central to its virulence. Because it is a wall-associated enzyme rather than a membrane-bound one, and because it has not been the focus of a major drug class, exo-beta-1,3-glucanase represents a comparatively fresh angle of attack against the pathogen.

Docking against this enzyme produced even stronger scores than for CYP51: minus 8.4 kilocalories per mole for 1PB1 and minus 8.1 kilocalories per mole for 45R. To verify that these predicted interactions translate into real enzymatic inhibition, the researchers ran in vitro enzyme assays. The results were striking. 1PB1 suppressed exo-beta-1,3-glucanase activity by a maximum of 94.6 percent, with an uncertainty of 4.7 percent, at a concentration of 52.7 micrograms per milliliter, while 45R achieved 91.77 percent inhibition, with an uncertainty of 1.7 percent, at 313.2 micrograms per milliliter. Nearly complete shutdown of a wall-remodeling enzyme, in a simple biochemical assay, is a potent result, and it places both compounds among the stronger exo-glucanase inhibitors described for this organism.

Static docking pictures can be misleading, however, because proteins and their bound ligands are constantly in motion. To address this, the team turned to molecular dynamics simulations, which track the atom-by-atom jostling of the protein-ligand complexes over simulated time. The simulations showed that the complexes formed by both compounds with both targets remained conformationally stable over the course of the trajectories, holding their bound poses far more securely than the corresponding unliganded, or apo, forms of the enzymes held their own structures. Stable dynamics are the computational equivalent of a firm handshake: they suggest that the binding observed in docking is not a fleeting artifact but a durable physical interaction capable of blocking the enzyme’s function in a living cell.

What makes the study noteworthy is not any single number but the convergence. A compound that inhibits only CYP51 can be defeated by the resistance mutations already widespread in clinical Candida populations. A compound that inhibits only a wall-remodeling enzyme might be dodged by compensatory changes in wall architecture. But a compound that must simultaneously be neutralized at CYP51 and at exo-beta-1,3-glucanase faces a double evolutionary barrier, and the combination of membrane destabilization and wall weakening may act synergistically on the fungal cell, which depends on both barriers staying intact. The dual-target profile also hints at the possibility of lower effective doses, since each partial hit reinforces the other. The work builds on the same group’s earlier characterization of ADP4 metabolites, which have previously shown anti-Candida and antibiofilm activity, and on a growing literature that mines Streptomyces species, the same bacterial genus that gave the world streptomycin and tetracycline, for molecules with antifungal potential.

Considerable distance remains between these results and a medicine on a pharmacy shelf. Docking scores are estimates, enzyme assays are conducted outside the cell, and the leap from a biochemical inhibition curve to a safe, absorbable, non-toxic drug in a human patient is long and statistically unforgiving. The compounds’ pharmacokinetics, their behavior in animal models of candidiasis, their toxicity toward human cells, and their activity against resistant clinical isolates all remain open questions. Nevertheless, the study adds two chemically distinctive scaffolds, a trimethyl cyclohexyl ketone and an amino decalin, to the short list of natural products with experimentally supported, dual mechanisms against Candida albicans. At a moment when fungal infections cause an estimated millions of severe cases each year and the antifungal pipeline runs thin, a soil bacterium offering molecules that hit the pathogen at two vulnerable points at once is exactly the kind of lead the field has been waiting for. The next challenge, as always in natural product drug discovery, will be turning a promising hit into a therapy.

Subject of Research: Dual-target antifungal compounds from Streptomyces chrestomyceticus ADP4 against Candida albicans

Article Title: New anti-fungal compounds 1PB1 and 45R from Streptomyces chrestomyceticus ADP4 target exo-β-1,3-glucanase and ergosterol biosynthesis in Candida albicans

Article References: Shukla, J., & Dubey, A. K. (2026). New anti-fungal compounds 1PB1 and 45R from Streptomyces chrestomyceticus ADP4 target exo-β-1,3-glucanase and ergosterol biosynthesis in Candida albicans. The Journal of Antibiotics. https://doi.org/10.1038/s41429-026-00958-4

Image Credits: AI Generated

DOI: 10.1038/s41429-026-00958-4

Keywords: antifungal compounds, Candida albicans, Streptomyces chrestomyceticus, CYP51, ergosterol biosynthesis, exo-beta-1,3-glucanase, molecular docking, molecular dynamics, natural products, drug resistance, The Journal of Antibiotics, dual mode of action