The target at the heart of the review is dihydrofolate reductase, or DHFR, an enzyme that has long been validated as essential to the parasite’s survival. DHFR catalyses the reduction of dihydrofolate to tetrahydrofolate using NADPH as an electron donor, a conversion that underpins the synthesis of purines, thymidylate and certain amino acids required for DNA replication and cell proliferation. When inhibitors occupy the enzyme’s active site, tetrahydrofolate levels collapse, nucleotide production grinds to a halt and the parasite can no longer divide. Established antifolate drugs such as pyrimethamine and cycloguanil exploit exactly this vulnerability, competitively binding the parasite enzyme and blocking the DHF-to-THF conversion. The problem is that resistance to these drugs has emerged through point mutations in the DHFR gene that alter the active site and reduce inhibitor binding affinity, which is why the search for new scaffolds capable of tolerating or evading these mutations has become so urgent.

The structural biology of the parasite enzyme explains both the promise and the difficulty of this endeavour. Plasmodium falciparum DHFR exists as a bifunctional enzyme coupled with thymidylate synthase, forming a homodimer with extensive interactions between the TS domains. Like all DHFR enzymes, it features a polypeptide fold built around an eight-stranded mixed beta-sheet flanked by alpha-helices. Crystallographic studies of the enzyme complexed with cofactors and inhibitors have revealed the intricate network of interactions at the active site, with residues such as Asp54 and Phe58 engaged in hydrogen bonding and pi-pi interactions that stabilise inhibitor binding. The enzyme is remarkably similar to its Plasmodium vivax counterpart, with roughly 66 percent sequence identity and the capacity to be superimposed with a root mean square deviation of about 0.87 angstroms, raising the possibility that inhibitors designed against one species could serve against both.

Resistance to classical antifolates has been mapped at atomic resolution, and the story is a textbook example of molecular evolution. X-ray crystallography shows that pyrimethamine and cycloguanil form key hydrogen bonds with conserved active-site residues including Asp54 and Ile164, along with a critical water-mediated hydrogen bond to the Ser108 side chain. The S108N mutation disrupts this precise interaction network: the asparagine substitution sterically occludes the inhibitor’s binding mode and eliminates the favourable hydrogen bond with the drug, while the natural substrate continues to be accommodated through alternative conformations. In the quadruple-mutant form of the enzyme, the steric hindrance imposed by Asn-108 is severe enough to defeat rigid inhibitors entirely. This structural insight has driven the design of next-generation antifolates with built-in flexibility, and the review devotes considerable attention to these efforts.

Among the next-generation inhibitors, WR99210, a 4,6-diamino-1,2-dihydro-1,3,5-triazine derivative, stands out. Its flexible (2,4,5-trichlorophenoxy)propoxy side chain allows the molecule to overcome the steric barrier imposed by Asn-108 in the quadruple-mutant enzyme, maintaining potency against both wild-type and pyrimethamine-resistant forms of PfDHFR. A related compound, PS-15, is metabolically converted in the body to WR99210 and is thought to offer improved absorption or reduced toxicity while preserving the active drug in vivo. The clinical candidate P218 takes the strategy further, binding tightly to both wild-type and mutant enzymes with high selectivity for the parasite protein and forming an additional hydrogen bond with the main chain of Cys15, an interaction that is largely insensitive to the resistance mutations. These molecules illustrate the design principle the review advocates: atomic-level precision in maintaining critical interactions within a rapidly evolving active site.

So where does benzothiazole fit in? The authors argue that this scaffold offers a distinct mechanism of action independent of the pathways compromised in artemisinin resistance, which involve mutations in PfKelch13 and enhanced efflux via PfCRT. Benzothiazole derivatives can bind free heme and inhibit heme polymerisation, disrupting the parasite’s haemoglobin detoxification pathway and leading to toxic heme accumulation, while also showing DHFR inhibition during erythrocytic infection. This dual capability means benzothiazole-based compounds could offer synergy potential in novel combinations and retain activity against multidrug-resistant strains. The review systematically surveys the in vitro and in vivo antiplasmodial activity of these compounds across multiple Plasmodium species, along with the synthetic chemistry and structure-activity relationships that govern their potency.

The synthetic accessibility of benzothiazole is one of its practical strengths. The classic route, first reported by Hugershoff in 1900, condenses 2-aminothiophenol with aldehydes or carboxylic acids, proceeding through imine formation, cyclodehydration to a benzothiazoline and air oxidation to the aromatic product, with yields typically between 70 and 90 percent. Oxidative cyclisation approaches using iodine, bromine, DDQ or copper catalysis offer milder conditions, while visible-light photocatalysis with ruthenium tris-bipyridine and oxygen as the terminal oxidant exemplifies modern efficiency. Green chemistry strategies push yields to 85 to 98 percent using microwave irradiation over zinc chloride or silica-supported reagents, solvent-free grinding with montmorillonite clay, or glycerol as a bio-based solvent. Post-synthetic functionalisation at the C-2 and C-6 positions, including palladium-catalysed direct arylation and nucleophilic substitution on 2-chlorobenzothiazoles, enables the construction of diverse compound libraries for biological screening.

The biological data compiled in the review show genuine promise. In work by Oyeneyin and colleagues published in 2024, quinoline-urea-benzothiazole hybrids were evaluated against P. falciparum, with compound 13 showing the greatest beta-haematin inhibition activity at an IC50 of 0.201 micromolar, far outperforming chloroquine at 18.26 micromolar and comparing favourably with artemether-lumefantrine and pyrimethamine. In a series of substituted 2-aminobenzothiazoles studied by Thakkar and colleagues, a meta-nitro derivative emerged as the most potent DHFR inhibitor with an IC50 of 0.0312 millimolar, while para-chloro and para-bromo analogues also showed appreciable activity, suggesting that halogen substitution at the para position contributes favourably to interactions with the DHFR active site. Earlier studies by Hout and colleagues found that certain benzothiazoles act on multiple parasite life-cycle stages, including mature and young schizonts, with some compounds disrupting mitochondrial membrane potential, while Sarkar and colleagues demonstrated that benzothiazole-hydrazone conjugates show superior antimalarial and iron-chelating action in murine models, with a 3,4-dihydroxy benzothiazole showing the highest efficacy among hydroxy-substituted variants.

The structure-activity relationships distilled from these studies point to clear design rules. The amino group at the C-2 position is essential for hydrogen bonding with DHFR active-site residues, particularly Asp54, while hydrophobic residues such as Ile14 and Leu164 stabilise the ligand through van der Waals contacts. Introducing aryl or heteroaryl groups at C-2 increases hydrophobicity and enables pi-pi stacking with Phe58, and electron-withdrawing substituents such as halogens or nitro groups on the aryl ring, especially at the para position, enhance binding affinity through improved electronic interactions. Hybridisation of benzothiazole with pharmacophores such as pyrimidine, triazine and triazole via acetamide or urea linkers achieves multiple simultaneous binding contacts, and bioisosteric replacements in the linker region can improve selectivity. Substitutions at positions C-4 through C-7 modulate the molecule’s electronic distribution and shape, facilitating a better fit into the enzyme’s hydrophobic pocket. Docking and structural studies suggest the benzothiazole core forms hydrogen bonds with Asp58 while hydrophobic interactions with Phe58, Ile14 and Leu164 create a favourable environment that enhances both selectivity and potency.

The authors are candid about the challenges that remain. The DHFR enzyme is prone to mutations at its binding site, and if benzothiazole-based antifolates target the same structural elements as older drugs, their efficacy could be similarly compromised. In vivo validation of promising derivatives lags behind encouraging in vitro results, and pharmacokinetic properties such as lipophilicity must be optimised to balance membrane penetration with solubility. Toxicity is a real concern: the antitumour benzothiazole 5F203 formed adducts in the lungs and liver that persisted in mouse lungs, and in a clinical trial involving 50 patients, lung and liver toxicity were observed without a maximum tolerated dose being defined. The path forward, the review concludes, lies in rational scaffold optimisation at C-2, robust target validation, hybrid designs that simultaneously disrupt folate metabolism, heme detoxification and other validated targets such as PfATP6 and PfPI4K, and combination strategies that raise the barrier to resistance. If those hurdles can be cleared, benzothiazole-based hybrids may well represent a leading frontier in the next generation of antimalarial drug discovery.

Subject of Research: Benzothiazole derivatives as inhibitors of Plasmodium falciparum dihydrofolate reductase for antimalarial drug discovery

Article Title: Review on benzothiazole as a privileged nucleus for Plasmodium DHFR inhibition with structure guided comprehensive analysis

Article References: Sarma, K., Ahmed, N., Banu, Z. W., Patowary, L., Gogoi, D., Devi, N. B., & Kalita, J. M. (2026). Review on benzothiazole as a privileged nucleus for Plasmodium DHFR inhibition with structure guided comprehensive analysis. Discover Chemistry, 3(1), Article 446. https://doi.org/10.1007/s44371-026-00905-0

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00905-0

Keywords: malaria, benzothiazole, dihydrofolate reductase, antimalarial drugs, drug resistance, structure-activity relationship, Plasmodium falciparum, molecular docking, heme detoxification, antifolates, medicinal chemistry, drug discovery

The motivation behind the study lies in one of the central dilemmas of modern renewable energy. Silicon photovoltaics dominate the market, but their production is expensive, energy-intensive and dependent on complex industrial processes. Organic photovoltaic cells, first developed in the 1990s, offer an attractive alternative: they absorb light efficiently, are cheap to manufacture, and can be made into flexible, lightweight panels. Their Achilles heel has always been efficiency, which still trails crystalline silicon by a considerable margin. Closing that gap requires better molecular materials, and triazine-based compounds, with their highly conjugated electronic systems and dual ability to trap and transport electrons, have emerged as promising building blocks for the next generation of organic semiconductors.

The synthetic route devised by the team is elegantly simple. A diazonium ion was generated from 3-amino-5,6-dimethyl-1,2,4-triazine using sodium nitrite and concentrated sulfuric acid at temperatures between minus five and zero degrees Celsius in dimethyl sulfoxide. This reactive intermediate was then coupled with three 2-aminobenzothiazole derivatives bearing different substituents, an ethoxy group, a nitro group and a methoxy group, yielding three azo compounds designated 4a, 4b and 4c. In a further reaction, the diazonium ion was coupled with the parent triazine itself to produce a symmetrical bis-triazine derivative, compound 5, in an impressive 95.7 percent yield. Establishing the exact structures of these products was no trivial matter, because azo couplings can proceed through several different pathways. The researchers resolved the ambiguity using a combination of infrared spectroscopy, one- and two-dimensional nuclear magnetic resonance, high-resolution mass spectrometry and elemental analysis, showing for example that compound 4a forms by electrophilic substitution on the benzothiazole ring while compound 4b arises from addition at the ring nitrogen.

With the molecules in hand, the team turned to quantum chemistry. All four compounds were modeled using the B3LYP hybrid functional with the 6-311+G(d,p) basis set, a combination chosen for its proven reliability on conjugated organic systems. Calculations were performed in the gas phase and in two polar solvents, methanol and dimethyl sulfoxide, using the polarizable continuum model to mimic the real environments in which such materials would be used. The authors are candid about the limitations of their approach: global hybrid functionals like B3LYP tend to underestimate the excitation energies of charge-transfer transitions and to overestimate hyperpolarizabilities compared with long-range-corrected alternatives, but they retained it for internal consistency across the full set of properties and because it correctly reproduces the qualitative trends observed experimentally for this series.

The electronic structure calculations revealed a striking split within the family. Compounds 4a and 4c, which carry electron-donating alkoxy groups, behave almost identically despite differing only by a methyl group in their side chains. Their HOMO-LUMO energy gaps shrink from roughly 3.3 electron volts in the gas phase to about 2.95 electron volts in polar solvents, a solvent-induced narrowing that favors visible-light absorption. Orbital analysis showed that in these two molecules the HOMO sits predominantly on the donor benzothiazole-alkoxy fragment while the LUMO resides on the acceptor triazine unit, a textbook donor-acceptor architecture that drives intramolecular charge transfer across the conjugated azo bridge. Compound 4b, bearing the strongly electron-withdrawing nitro group, proved far less sensitive to solvation and emerged as the most electronically stable member of the series, with the highest electrophilicity index and ionization potential, marking it out as a natural electron acceptor.

Charge transport, the lifeblood of any semiconductor, was assessed through reorganization energies calculated within the framework of Marcus theory. These values measure how much a molecule must geometrically distort when it gains or loses an electron, and low values translate into faster, more efficient charge hopping. Here again the alkoxy compounds shone: 4a and 4c posted hole and electron reorganization energies of just 0.61 to 0.67 electron volts, with electron values only slightly exceeding hole values, suggesting a nearly balanced ambipolar character that is highly prized in organic electronics. Compound 5 showed moderate values, while the nitro-substituted 4b was a dramatic outlier, with a hole reorganization energy of 4.09 electron volts, roughly 6.7 times that of 4c, indicating that its geometry changes too profoundly upon oxidation to serve as an efficient charge carrier. The authors benchmarked their results against pentacene, the reference p-type organic semiconductor, whose hole reorganization energy of 0.08 to 0.12 electron volts remains far lower; their dyes are competitive but not yet optimal.

Perhaps the most eye-catching results concern nonlinear optics, the branch of photonics concerned with materials whose optical response changes with light intensity. Such materials enable frequency doubling, optical switching and signal processing. The first-order hyperpolarizability of compounds 4a and 4c surged from around 45 to 46 times ten to the minus thirty esu in the gas phase to roughly 171 times ten to the minus thirty esu in polar solvents, a solvent-driven amplification of more than a factor of three. Set against para-nitroaniline, the classical benchmark donor-pi-acceptor chromophore, whose hyperpolarizability reaches only about 20 to 25 times ten to the minus thirty esu even in water, the two alkoxy dyes outperform the reference by a factor of seven to eight. Intriguingly, compound 4b, despite its strong acceptor credentials, showed a hyperpolarizability less than half that of its alkoxy siblings, demonstrating that a large dipole moment alone does not guarantee a strong nonlinear response and that the spatial delocalization of charge separation matters more.

The optoelectronic and optical calculations rounded out the picture. Compounds 4a and 4c exhibited high dielectric constants and refractive indices above 2.4 in methanol, values typical of high-performance pi-conjugated organic materials and attractive for light confinement in integrated photonic waveguides. Time-dependent DFT placed the lowest-energy absorption of 4a and 4c in the blue-green region of the visible spectrum, near 480 and 490 nanometers respectively, with mixed multi-orbital transitions characteristic of intramolecular charge transfer. The high-energy pi to pi-star bands were reproduced with excellent accuracy against the experimental ultraviolet-visible spectra, with errors below three percent, while the lowest charge-transfer band showed the larger deviations expected from the known weaknesses of B3LYP. Thermodynamic calculations confirmed that all four compounds are stabilized in polar media, with the nitro compound 4b showing the lowest Gibbs free energy and hence the greatest thermodynamic stability.

The authors are careful to frame their conclusions as computationally motivated hypotheses rather than proven device performance. No solar cell or light-emitting device was fabricated in this study, and experimental validation at the material and device level will be needed before any of these dyes reaches a photovoltaic panel or a photonic modulator. Nevertheless, the work illustrates a powerful and increasingly standard paradigm in materials discovery: synthesize a chemically diverse family of candidate chromophores, pin down their structures rigorously, and use solvent-explicit quantum chemical screening to decide which members deserve the expense of device fabrication. On that basis, compounds 4a and 4c, with their narrow gaps, balanced charge transport and record-beating nonlinear optical response, have earned their place at the front of the queue, while the stable but transport-limited 4b may find its calling as an electron acceptor in multilayer organic architectures.

Subject of Research: Synthesis and DFT characterization of triazine-benzothiazole hybrid azo dyes as pi-conjugated materials for organic photovoltaic and nonlinear optical applications

Article Title: Synthesis photophysical characterization and DFT Study of triazine benzothiazole hybrid azo dyes as π conjugated materials for organic photovoltaic and nonlinear optical applications

Article References: Tsemeugne, J., Ebode, R. D. P. N., Fomekong, L. T., Eckhardt, P., Kamsi, R. A. Y., Mvot, C. A., Ottou, M. T. A., Ngoupo, A. T., Sielinou, V. T., Ejuh, G. W., Mkounga, P., Opatz, T., Ndjaka, J.-M. B., Sopbué, E. F., & Nkengfack, A. E. (2026). Synthesis photophysical characterization and DFT Study of triazine benzothiazole hybrid azo dyes as π conjugated materials for organic photovoltaic and nonlinear optical applications. Discover Chemistry, 3(1), Article 554. https://doi.org/10.1007/s44371-026-00999-6

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00999-6

Keywords: azo dyes, triazine, benzothiazole, DFT, organic photovoltaics, nonlinear optics, pi-conjugated materials, charge transport, reorganization energy, hyperpolarizability, TD-DFT, organic semiconductors