Introduction

Branched-chain amino acids (BCAAs)—isoleucine, leucine, and valine—are essential constituents of proteins and play a fundamental role in plant growth, metabolism, and stress adaptation. Their biosynthesis in higher plants proceeds through a conserved metabolic pathway regulated by acetolactate synthase (ALS; also known as acetohydroxyacid synthase, AHAS), which catalyzes the first common step in their formation1. Since ALS is absent in animals, it represents an ideal biochemical target for selective herbicidal action. Consequently, a wide range of herbicides targeting this enzyme has been developed and commercialized, owing to their high potency, low application rates, and minimal mammalian toxicity2.

ALS-inhibiting herbicides are classified into five major chemical families: sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinylthio-benzoates (PTBs), and sulfonylaminocarbonyltriazolinones (SCTs)3. Among these, triazolopyrimidines have emerged as an important class due to their broad-spectrum weed control, excellent crop selectivity, and favorable environmental fate4. Florasulam, a representative member of this class, is widely employed in wheat and barley cultivation for post-emergence control of broadleaf and certain grass weeds. Compared with other ALS inhibitors such as sulfonylureas and imidazolinones, Florasulam exhibits improved crop tolerance, faster degradation in soil, and a distinct ALS binding profile that reduces the likelihood of cross-resistance5,6. These features make Florasulam not only an effective herbicide but also an ideal molecular model for studying the mechanisms of ALS inhibition.

Structurally, Florasulam [N-(2,6-difluorophenyl)−8-fluoro-5-methoxy-[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide] possesses a fused heteroaromatic triazolopyrimidine core substituted with both electron-withdrawing (fluoro and sulfonyl) and electron-donating (methoxy) groups. These substituents collectively modulate the electronic distribution, dipole moment, and lipophilicity, thereby governing hydrogen-bonding potential, molecular polarity, and target-enzyme binding affinity. Recent QSAR and 3D-QSAR studies have demonstrated that for structurally diverse ALS inhibitors—including sulfonylureas and triazolopyrimidine-2-sulfonamides—steric and electrostatic fields, hydrophobicity, and local electronic parameters (HOMO-LUMO gap, MEP, partial charges) are key determinants of herbicidal potency7. Yang et al. systematically established these correlations, revealing that small electronic perturbations in the triazolopyrimidine ring markedly influence ALS inhibition efficiency7. Chen et al. further reported that the position of fluorine and methoxy substituents in N-(2,6-difluorophenyl)−5-methoxy triazolopyrimidines critically controls ALS-binding orientation and selectivity8. Moreover, environmental and mechanistic reviews of triazolopyrimidinesulfonamides emphasize that physicochemical features such as polarity, acidity, and hydrogen-bonding capacity influence not only bioactivity but also degradation and persistence profiles9. Hence, comprehensive QSSR (quantitative structure–selectivity relationship) and QSAR analyses are essential to link Florasulam’s molecular structure to its herbicidal function and environmental behavior.

In the present study, an integrated experimental–computational methodology was employed—combining single-crystal X-ray diffraction (SCXRD), Hirshfeld surface and energy-framework analyses, DFT/TD-DFT-based electronic structure calculations, and molecular docking supported by molecular-dynamics simulations—to quantitatively elucidate the structure–activity and structure–selectivity relationships (QSAR/QSSR) of Florasulam. This combined approach enables correlation of experimental solid-state features with computed electronic descriptors, providing predictive insight for the rational design of next-generation triazolopyrimidine herbicides.

Experimental and computational details

General remarks

The Florasulam (FL) compound procured from Sigma Aldrich. The infrared spectrum of the FL compound was recorded in the range of 4000–450 cm− 1 with a resolution of 1.0 cm− 1 using the KBr disc method on the Perkin Elmer Spectrum1 FT-IR instrument (Spectrum One: FT-IR Spectrometer) employing Fourier transform technique.

Single crystal XRD analysis

Single crystals of FL were obtained using a slow solvent-evaporation method. Approximately 50 mg of FL was dissolved in 20 mL of methanol, and the solution was allowed to evaporate slowly at room temperature. The beaker was covered with perforated aluminum foil to regulate the evaporation rate. After approximately one week, colorless, needle-shaped single crystals suitable for crystallographic analysis were obtained. Single crystals withdimensions of 0.33 × 0.29 × 0.24 mm3 was selected and mounted on a Bruker APEX-II CCD diffractometer with monochromated MoKα radiation (λ = 0.71073 Ǻ) at 296(2) K. The data was processed with SAINT and corrected for absorption using SADABS10. The structure was solved by the direct method using the program SHELXT and refined by using the program SHELXL11 by full-matrix least squares technique on F2 using anisotropic displacement parameters for all non-hydrogen atoms. The carbon bound hydrogen atoms were positioned with idealized geometry using a riding model with Carm–H = 0.93 Å/Cmethyl–H = 0.96 Å. H atoms were refined with isotropic displacement parameters (set to 1.2–1.5 times of the Ueq of the parent atom). The hydrogen atoms of -NH was located from the difference Fourier map and were refined freely. The ORTEP diagram of the compound is given in Fig. 1 and the packing of molecules in the crystal lattice is shown in Fig. 2. The crystallographic data and refinement parameters are summarized in Table 1. Geometric parameters for hydrogen bonds (Å, °) operating in the crystal structure are listed in Table 2. Selected bond lengths, bond angles and torsional angles are listed in Table 3.

Hirshfeld surface analysis

Hirshfeld surface12 analyses were carried out and finger print plots13 were plotted using the software CrystalExplorer 17.514. The dnorm plots were mapped with colour scale in between − 0.18 au (blue) and 1.4 au (red). The 2D fingerprint plots were displayed by using the expanded 0.6–2.8 Å view with the de and di distance scales displayed on the graph axes. When the cif files of all the compounds were uploaded into the CrystalExplorer software, all bond lengths to hydrogen were automatically modified to typical standard neutron values i.e., C–H = 1.083 Å and N–H = 1.009 Å. Electrostatic potential surface was calculated using the Tonto program available in the CrystalExplorer software using B3LYP method and 6-311G(d, p) basis set.

Energy framework analyses and interaction energy calculations

CrystalExplorer17.5 was used to analyze and visualize the pair wise and overall interaction energies. The tube size (scale factor) used in all the energy frameworks was100, and, the lower cut-off energy value was set to 5 kJ/mol. The energy components calculated within this.

Table 1  Crystal data and structure refinement for FL
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Table 2 Geometric parameters for hydrogen bonds and other intermolecular contacts (Å, º) operating in the crystal structure of FL
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Table 3 Geometrical parameters of the title molecule (FL)
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method are electrostatic, polarization, dispersion and exchange-repulsion and finally the total interaction energy. The energy calculations were done at the B3LYP/6-31G(d, p) level of theory and using experimental crystal structure cif file.

Fig. 1
figure 1

ORTEP view of FL Thermal ellipsoids are drawn at 50% level of probabilities

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Fig. 2
figure 2

(a) A partial view of the packing in the crystal of FL demonstrating the formation of 1D ribbon (N-H…N and C-H…O interactions are shown as thin blue lines; (b) Formation of 2D-sheet in the crystal of FL where in the 1D ribbons are interlinked by bifurcated S = O…π interactions

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Computational details

Gaussian 09 software15 was used to conduct DFT and TD-DFT calculations at the B3LYP/6–311 + + G(d, p) theory level. The first structure generated from the asymmetric unit of FL was used to determine the ground-state geometry. An overlay of the structure obtained from the XRD data and optimized DFT structure showed that the two structures exactly overlap with one another with RMSD of 0.000 Å. The DFT-B3LYP approach with dispersion correction (D3)16 was used to optimize the structure and obtain infrared spectral data. The integral equation formalism variant of the polarizable continuum model (IEFPCM)17 was used solvent computations. Non-covalent interactions were comprehensively examined using ALIE, ELF, LOL, RDG, and TDM analyses with the Multiwfn program18. Iso-surfaces were demonstrated using the VMD program19. The UV-Vis spectrum and electronic properties, including HOMO-LUMO, were computed using the TD-DFT method. AIM 2000 program20 has been used to determine intramolecular interactions through quantum topological atoms in molecules (QTAIM). Sites of protein-ligand interactions have been discovered using Autodock Tools version 4.2.1 program21 and docking parameters have been examined using Discovery studio visualization software22. Macromolecule flexibility and impact of mobility on drug-protein interactions have been effectively studied using the computational analysis defined as molecular dynamics simulation23. Using the GROMACS 5.1 programme, the ligand-enzyme complex simulation has been executed24. Ligand parameters have been examined using Swiss Param online server25 in the framework of CHARMM27 all-atom force field.

Result and discussion

Molecular and crystal structure

The molecular structure of FL is shown in Fig. 1 (ORTEP view of FL). In the molecule, the triazolo pyrimidine ring with the attached fluorine & sulphur atoms and the attached methoxy group (excluding all hydrogen atoms) are nearly planar with rmsd 0.050(1) Å. Also, the aniline ring with the two substituted fluorine atoms (excluding all hydrogen atoms) is nearly planar with rmsd 0.037(1) Å. The molecule has ‘V’ shape with the two planar fragments tapering towards S1 atom and making dihedral angle of 31.2(5)o. The conformation of the N5–C7 bond in the central –C6–S1(O2)–N5(H5)–C7 segment is gauche with respect to the S1 = O1 and S2 = O2 bonds with the central torsion having a value of −68.8(2)o. The V shaped confirmation is stabilized by an intramolecular interaction C12–F2···π involving the π electrons of triazolo ring (Table 2).

The crystal structure is very interesting as the packing of molecules is not dictated by the usual sulfonamide N–H···O = S hydrogen bonds which generally features crystal structures of most of the sulfonamides. The crystal packing is driven by a pair of N5–H5···N4 hydrogen bonds (Table 2; Fig. 2a) between two molecules related by inversion centre forming a ring R22(10) motif. These dimers are further expanded into a one-dimensional ribbon by formation of another dimeric pair via C9–H9···O3 interactions between inversion centre related molecules of the adjacent R22(10) dimers forming a R22(26) ring motif. This ribbon propagates parallel to (1\(\:\stackrel{-}{1}\)0) plane (Fig. 2a). In addition to the structure directing N–H···N and C–H···O interactions, the structure also features relatively weaker but significant attractive S = O···π interactions involving π electrons of the electron rich π-systems and the lone pair of S atom or S = O double bond. The inversion related molecules of the adjacent ribbons are interconnected via structure directing-bifurcated S1 = O1···π interactions (involving the ring electrons of both the aromatic rings of triazolo pyrimidine) bifurcated at O1 atom forming a two-dimensional sheet in the (1\(\:\stackrel{-}{1}\)0) plane (Fig. 2b). It is apparent that the sulfonyl groups are utilized in expanding the supramolecular architecture built at the primary level rather than being themselves involved in building the architecture in the primary level. Finally, the two-dimensional sheet is stabilized by π···π interactions involving the π electrons of the phenyl rings.

Fig. 3
figure 3

(a-h). Various surface properties plotted over the Hirshfeld surface of FL: (a) dnorm plot displaying N-H···N hydrogen bonds; (b) dnorm plot displaying C-H···O interactions; (c) and (d) dnorm and shapeindex displaying O···π interactions; (e) and (f) Electrostatic potential N-H···N and O···π interactions respectively; (g) and (h) Shapeindex and curvedness plots displaying π ···π interactions

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Hirshfeld surface analysis

Dark-red spots are observed in the vicinities of nitrogen N4 atom as well as N5 bound H5 atom (Fig. 3a) suggesting the presence of strong N–H···N hydrogen bond dimeric pairs. Also, a pair of dark red spots in the vicinities of O3 atom and C9 bound H atom of FL (Fig. 3b) suggests the presence of a pair of C–H···O interactions. The dark reds spots on the dnorm surface are the characteristics of atoms participating in strong hydrogen bonds, thereby, confirming the presence of dimeric pairs of N5–H5···N4 and C9–H9···O3 hydrogen bonds in the crystal. This is further confirmed by the electrostatic potential surfaces (Figs. 3e). Dark blue patches in the vicinities of H5 and H9 atoms and, and, dark red patches in the vicinities.

Fig. 4
figure 4

FPs-full contacts as well delineated to individual atom···atom contacts of FL and percentage contribution of each contact to the Hirshfeld surface

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of N4 and O3 atoms demonstrates that these are respectively low and high electron densities regions and are therefore involved in strong non-covalent interactions. The dnorm surface also display two faint red spots near the two lone pairs of electrons of O1 atom and centroids of triazolo and pyrimidine rings which indicates the presence of relatively weaker O1···π interactions involving π electrons of triazolo pyrimidine ring and bifurcated at O1. This is also supported by shape index and electrostatic potential curves plotted over Hirshfeld surface (Fig. 3d and f respectively). A dark blue patch near O1 atom & a dark red dip in the surface near the triazolo and pyrimidine rings, and, dark red region near O1 atom and a dark blue patch on the electrostatic potential surface above triazolo and pyrimidine rings are because of S1 = O1···π interactions. Shape index and curvedness curves help in identifying π···π stacking interactions. The π···π stacking interactions in the shape index appear as alternating red and blue triangles above the rings participating in the stacking, while they are characterized by a flat surface in the curvedness plot above the participating rings. Both these characteristics are observed in the shape index and curvedness plots (Fig. 3g and h) of the molecule and therefore gives evidence for π···π stacking interactions in the crystal.

The analysis of FP plots not only gives a quantitative picture of contributions of different atom···atom contacts to the crystal structure but also estimates the nearest atom…atom contact distances which can be correlated to the donor···acceptor distances in an intermolecular interaction. The FPs (of overall contacts and individual atom…atom contacts) of FL are shown in Fig. 4. All the FPs are symmetric about the di = de line which is due to the homomeric nature of the interactions in the crystal. The greatest contribution to the Hirshfeld surface is from H···F/F···H dispersions contributing 23.4%. O···H/H···O and N···H/H···N contacts contribute respectively 15.8% and 13.5% which can be attributed to the presence of C9–H9···O3 and N5–H5···N4 interactions. The contributions of 4.4% and 3.2% by C···C and O···C contacts are because of π···π and S = O···π interactions operating in the crystal. The appearance of a pair of sharp spikes in the FPs of O···H/H···O and N···H/H···N contacts are because of C9–H9···O3 and N5–H5···N4 interactions each existing as dimeric pairs. The closest di + de distance for the N···H/H···N contacts occur at 2.1 Å which is very close to the H5···N4 contact distance of 2.19 Å observed in N5–H5···N4 interaction (Table 2). Also, the closest di + de distance for the O···H/H···O contacts occur at 2.4 Å which is very close to the H9···O3 contact distance of 2.5 Å observed in C9–H9···O3 interaction (Table 2). The centroid-to-centroid perpendicular distance of 3.6397 Å observed for the π···π interactions (Table 2) correlates with the closest di + de distance of 3.6 Å observed in the FP of C···C contact. The closest di + de distance of 3.1 Å observed in the FP of O···C contact is very close to the O···π distances of 3.0466(4) Å and 3.2343(4) Å observed for S = O···π contacts (Fig. 4). Thus, the Hirshfeld surface and FP analysis gives confirmation -qualitative as well as quantitative-to the types and nature of intermolecular interactions in FL.

Energy framework analysis

Energy frameworks offer quantitative as well as qualitative analysis of the energetics of each intermolecular interactions as well as different supramolecular frameworks existing in the structure26,27. Also, it reveals the nature of interactions as whether it is attractive or repulsive, electrostatic or dispersive etc. The energy framework of the 1D-ribbon comprising of alternating pairs of N–H···N and C–H···O interactions is displayed in Fig. 5, whereas, that of the overall 2D-sheet is displayed in Fig. 6. when viewed along a, b and c axes respectively.

Figures S1 and S2 (please refer supplementary material) display the energy topology of the overall structure when viewed along a, b and c axes respectively.

The formation of 1D-ribbon as the primary mode of crystal packing is supported by the energy framework analysis. The alternating zig-zag tubes (whose size is proportional to interaction energy) propagating parallel to (1\(\:\stackrel{-}{1}\)0) plane (Fig. 3) is due to the alternating dimeric pairs of N5–H5···N4 and C9–H9···O3 interactions. Both interactions are attractive in nature with dimeric pairs constituted by N5–H5···N4 interactions having energy of −63.4 kJ/mol and that formed by C9–H9···O3 interactions having − 67.5 kJ/mol. Both the interactions are having electrostatic as well as dispersive nature, with N–H···N interaction having greater contribution from electrostatic than dispersive, while C–H···O having nearly equal contribution from the two.

The energy framework analysis supports the formation of 2D sheet in the crystal structure. The energy framework of the adjacent ribbons is interconnected by energy frameworks of the S = O···π interactions constituting a grid like framework (Fig. 3a–c). These interactions have both electrostatic and dispersive contributions are attractive in nature and contributes an energy of −9.9 kJ/mol. Analysis of the dispersive contribution to the energy framework of the crystal (Fig. 3b) shows that the adjacent ribbon frameworks are also interlinked via energy framework of the π···π interactions. This interaction is absent in the electrostatic contribution (Fig. 3a) suggesting that the π···π stacking is purely dispersive in nature. Also, these.

Fig. 5
figure 5

Energy framework of 1D-ribbon of FL: (a) Coulombic contribution (b) dispersive contribution (c) overall energy and (d) energy annotations

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Fig. 6
figure 6

Energy framework of 2D-sheet of FL: (a) Coulombic contribution (b) dispersive contribution (c) overall energy and (d) energy annotations

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interactions stabilizes the supramolecular framework as the energy involved with these interactions is −24.4 kJ/mol.

Optimized geometry

Florasulam is a heterocyclic organic molecule with a fused ring structure and several functional groups that give rise to its herbicidal properties. It has a triazolopyrimidinecore a fusion of a 1,2,4-triazole ring and a pyrimidine ring which is required for biological activity. A methoxy group (-OCH₃) at the 5th position of the pyrimidine ring28. A fluorine atom at the 8th location of the fused ring increases lipophilicity and improves binding to the ALS enzyme. A sulfonamide group (-SO₂NH-) is added to the 2-position, connecting to a substituted aromatic ring. The sulfonamide is linked to a 2,6-difluorophenyl ring, which aids in both electronic stability and enzyme interactions29. The molecule has planarity across its aromatic and heterocyclic rings, which allows for adequate contact with enzyme active sites. Electron-withdrawing groups (fluorine and sulfonamide) increase binding affinity and activity in biology. C–H fall within the expected range of 1.08–1.09 Å, typical for aromatic and sp² hybridized carbons. C–C bond lengths in the range of ~ 1.37–1.39 Å, consistent with conjugated π-systems. C–F bonds typically range between 1.35 and 1.38 Å, observed here as:

C12–F2 = 1.35 Å, C8–F3 = 1.37 Å. S = O double bonds observed at ~ 1.44–1.45 Å (O2–S1 = 1.44 Å), matching standard sulfonyl bond lengths. The C–N bond lengths are in the range 1.32–1.41 Å, indicating a mix of aromatic, imine, or amine linkages. C = O bond (carbonyl group) is at 1.196 Å (approx.), typical of double bonded carbonyls. The bond lengths in aromatic rings exhibit an alternating pattern of short and long, which corroborates the presence of aromatic conjugation. The geometry of the sulfonyl (–SO₂–) group is accurately depicted by the short S = O bonds and the longer S–O bonds, signifying a pronounced double bond nature. Fluorinated aromatic domains exhibit marginally elongated C–F bond lengths, frequently augmenting molecule stability and lipophilicity. Shortened C–N bonds in heterocycles signify resonance stability throughout the fused ring system (Figure S3).

This optimized structure indicates effective electronic delocalization, structural stability, and potential bioactive interactions, aligning with its pharmacological molecular design.

Vibration analysis

A crucial method in chemistry for determining the different functional groups present in organic molecules is vibrational spectroscopy. Florasulam made composed of 32 atoms, has 90 vibrational modes and belongs to the C₁ group30. Implementing the 6–311 + + G(d, p) basis set, conceptual vibrational investigations were performed utilizing the B3LYP functional. Table 4 contains the scaled and unscaled theoretical vibrational frequencies, actual FT-IR measurements, and the mode allocations (which appear in PED%) for each. Figure S4 displays an analysis of Florasulam’s predicted & experimental FT-IR spectra. Because of constraints in the basis set and the disregard for electron correlation effects, computed vibrational frequencies are generally more elevated than their experimental equivalents. In order to rectify this, the DFT-calculated frequencies were scaled by 0.961431. The estimated frequencies and the experimental results accord well after scaling.

SO2 vibrations

The SO₂ group’s symmetric stretching modes are frequently found between 1135 and 1165 cm− 1, whilst its asymmetric stretching vibrations are commonly found in the spectral range of 1310–1360 cm− 1Superscript>32. The IR spectra of the current investigation showed a significant band at 1293 cm− 1 that corresponded to the asymmetric SO₂ stretching vibration. The vibration was anticipated to occur at 1299 cm− 1 by theoretical calculations using the B3LYP technique, with a 70% PED contribution. Conversely, the symmetric SO₂ stretching vibrations were calculated at 1097 cm− 1 with weak strength and were experimentally found as medium-intensity bands in the 1140–1146 cm− 1 range. The computed outcomes displayed minor variations from the experimental data because of the impact of hydrogen bonding contacts.

NH, SN and CS vibrations

ν(N–H) stretching vibrations are generally detected in the 3300–3500 cm− 1 range, according to published literature. The ν(N5–H5) vibrations were calculated at 3416 cm−¹ using the B3LYP functional and the 6–311 + + G(d, p) basis set. However, the FT-IR spectra did not clearly show these vibrations. Typically, the S–N stretching mode manifests at approximately 905 ± 30 cm− 133. The Florasulam molecule has a weakly observed band at 839 cm− 1, which corresponds to the theoretical v(S1–N5) stretching mode estimated at 847 cm− 1 via DFT. ν (C–S) are frequently tricky to identify in FT-IR spectra because the strength of absorption varies greatly depending on the molecule makeup and structure. The spectrum range of these vibrations is broad, ranging from 1035 to 245 cm− 1. Florasulam has a prominent FT-IR band at 769 cm− 1, which is due to the ν(C6–S1). The estimated value is 756 cm− 1.

C–N vibrations

The experimental FT-IR spectra shows prominent bands at 1352 cm− 1 (very strong) and 1342 cm− 1 (s), indicating C–N stretching vibrations. The theoretically estimated values at 1381 cm− 1 and 1342 cm− 1, which indicate PED contributions of 32% and 39%, respectively, are in good agreement with these results34. Identifying C–N deformation modes is difficult because they intersect with other vibratory modes, which commonly occur within 1266 and 1382 cm− 1. The observed spectrum assigns the in-plane bending vibration β(C–N–C) at 1077 cm− 1 (vs.), which corresponds to the theoretical value of 1047 cm− 1. Furthermore, the C–C–N bending mode is present at 549 cm− 1(s) and nearly matches the frequency of 547 cm− 1 that was computed. These allocations are supported by the PED assessment, which shows that the corresponding vibrational modes contribute significantly.

C–H and CH3 vibrations

Aromatic C–H stretching vibrations produce distinctive absorption bands in the 3100–3000 cm− 1 area35. Experimental FT-IR peaks were detected in the Florasulam molecule at 3108 cm− 1 (vw) and 3069 cm− 1 (extremely strong), which correspond to C–H ring stretching modes. The computed spectrum projected modes at 3089 and 3066 cm− 1, with PED contributions of 99% and 100%, respectively. Between 1300 and 1000 cm− 1, there are sharp bands of variable intensity that are ascribed to β(C–H) vibrations36. The C–H bending modes were identified in the experimental FT-IR spectrum as being represented by the bands at 1448 cm− 1 (s), 1418 cm− 1 (vs.), and 1169 cm− 1 (vs.). These bands are in good accordance with the estimated values at 1438, 1445, and 1154 cm− 1, respectively.

Asymmetric & symmetric stretching vibrations of the –CH₃ group (νASCH3 and νSSCH3) are typically detected in the 2962–2872 cm− 1 range37. These –CH₃ stretching modes are responsible for the faint bands at 2952 and 2901 cm− 1 and the significant absorption at 3009 cm− 1 (vs.) in the FT-IR spectra. The hyperconjugation among the aromatic ring system and the methyl group is one of the electrical factors that affects variances in CH₃ stretching frequency.

Table 4 Vibrational assignment of FL
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C–C vibrations

In rings, ν(C–C) vibrations usually occur between 1400 and 1650 cm− 138 and can involve both C = C and C–C stretching modes. A significant C = C stretching vibration is visible in the Florasulam molecule’s FT-IR spectra at 1601 cm− 1, which is in good agreement with the scaled theoretical wavenumber of 1606 cm− 1. ν(C–C) vibrations were recorded at 1583 cm− 1, 1528 cm− 1, and 1476 cm− 1 in the simulated spectra, with very strong intensities and PED contributions of 53%, 52%, and 38%, respectively. C–C stretching vibrations were found at 1566, 1549, and 1445 cm− 1 in the predicted spectra. These correlates to very strong intensities at 1583 cm− 1, 1528 cm− 1, and 1476 cm− 1, indicating potentially significant PED contributions of 53%, 52%, and 38%, respectively.

Fig. 7
figure 7

HOMO and LUMO plot of FL

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HOMO-LUMO analysis

According to Frontier Molecular Orbital (FMO) hypothesis39, the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) play an important role in assessing a compound’s biologic activity. Understanding their energy levels is crucial for comprehending molecular operations since the HOMO donates electrons while the LUMO accepts them. Ionization potential is correlated with HOMO energy; a higher HOMO energy generally corresponds to a higher ionization potential. Similarly, LUMO energy level effects a molecule’s electron affinity. The computed energy parameters in the gas phase and in solvents (DCM, methanol, and ethyl acetate) reveal that solvation stabilizes both HOMO and LUMO orbitals, increasing the energy gap (ΔE) and enhancing molecular stability. Among the solvents, ethyl acetate shows the largest ΔE, indicating maximal stability and minimal reactivity, whereas the gas phase exhibits the smallest ΔE, consistent with higher reactivity. These results underscore the influence of solvent polarity on the electronic structure and reactivity of the molecule.

To assess the reactivity and biological significance of the chemicals in this investigation, global reactivity descriptors derived from Koopmans’ theorem were utilized40. Table 5 summarizes these descriptors, which were computed using quantum chemistry techniques. Figure 7 show HOMO and LUMO plot. Florasulam’s HOMO-LUMO energy gap was measured to be 3.455 eV. By shedding light on both structural and electrical properties, quantum descriptors improve our comprehension of chemical phenomena. A molecule’s capacity to withstand variations in its electronic framework is measured by its chemical hardness (η); a bigger energy gap often denotes better stability. The inverse relationship between hardness and chemical softness (σ), on the other hand, suggests a molecule’s reactivity, especially when the energy gap is tiny41. While a compound’s ability to receive electrons is measured by its electrophilicity index (ω), its chemical potential (µ) indicates its propensity to form bonds with other species42. Florasulam’s robust electrophilic nature and good reactivity profile were indicated by its elevated electrophilicity index (ω = 18.228 eV), softness (σ = 0.289 eV), and chemical potential (µ = −7.936 eV).

Table 5 Calculated energy values of title compound by B3LYP/6–311 + + G(d, p) for gas and solvents
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UV analysis

TD-DFT and the B3LYP basis set, in addition to the IEFPCM, utilized a variety of solvents, including DCM, methanol, and ethyl acetate in both experimental and theoretical43. Comparative investigation demonstrated that TD-DFT is a reliable method for forecasting electronic transitions, hence validating the correlation of experimental UV-Vis spectral bands to particular electronic excitations44. This combination of theory and experimentation helps us understand the electronic structure-property correlations of the compounds we analyzed, which is important for creating possible drugs with the right electronic properties. Table S1 provides information regarding of the maximum absorption wavelength, the excitation energy, and the oscillatory strength. In Figure S5A and B, the computed and observed spectra that are caused by the various solvents (DCM, methanol, and ethyl acetate) are displayed. Different electron transitions are represented by peaks in the ultraviolet absorption spectra that are located at DCM: 262 nm, methanol: 259 nm and ethyl acetate: 260 nm, (in experimental) and in theoretical the absorbance values are (DCM: 257 nm, methanol: 258 nm and ethyl acetate: 261 nm) respectively. Generally, theoretical λcalc values matched practical λmax, proving the trustworthiness of the computation method. Basis set approximations, solvent effects, and vibrational coupling not fully addressed in the computational model cause minor differences between theoretical and actual values.

MEP analysis

Molecular electrostatic potential (MEP) reveals the charge distribution of molecules based on electron density and chemical reactivity sites. MEP is connected to molecular electro-negativity, partial charge, dipole moment, and chemical reactivity45. Figure S6 displays hues (red-orange-yellow-green-blue) that correspond to the electrical density distribution. Positive patches (blue, green) indicate nucleophilic reactivity, while red and yellow indicate maximal negative potential. Green represents zero potential46. MEP map revealed negative regions (red) around oxygen and sulphur atoms, and positive regions (blue green) around the hydrogen atoms of ring groups (Fig. S6). Red regions hold the greatest potential for electrophilic assault. In contrast, the blue areas showed the highest nucleophilic attack reactivity47. Sulfonyl (SO₂) oxygens → Electrophilic attack sites are in the most negative area. Nucleophilic attack sites are in the most positive area, where hydrogen is linked to nitrogen. The MEP surface shows that the molecule can strongly bind to biological proteins through hydrogen bonding and electrostatic interactions. This makes it even more important for finding new drugs.

Topological analysis

ELF & LOL analysis

In order to identify specific functional groups or molecules that are most likely to take part in chemical interactions, especially those related to the effects of drugs, topological approaches are used to study the structures of molecules48. ELF is a measurement of the likelihood of discovering an electron with the same spin that is close to a reference electron that is positioned at a specific location. For moderate ELF values (0.5), the series drops from brown and yellow to green, whereas high ELF values (about 0.8–1.0) are colored white. Blue represents the bottom end of the scale. Since both LOL and ELF rely on the kinetic-energy density, their chemical compositions are comparable49.

However, LOL only acknowledges that localized orbital gradients are maximal when localized orbitals coincide, in contrast to ELF, which is based on assessing the pair of electrons density. While lesser values (< 0.5) depict locations where electrons are likely to be delocalized, larger values in the interval 0.5 and 1.0 suggest regions with bonding and nonbonding localized electrons50. In areas where electron localization dominates the electron density, the LOL achieves high values (> 0.5). A high value in that area indicates a high localization of electrons because of the existence of a nuclear shell or a covalent bond (a single pair of electrons) in that area51. The existence of extremely localized bonding and nonbonding electrons is indicated by the extremely large ELF zones surrounding oxygen atoms in Figure S7(A). Green represents the covalent relationship that exists between the hydrogen and carbon atoms. The delocalized electron cloud surrounding hydrogen atoms is visible in the blue areas surrounding them. The yellow hue and almost high LOL value show the covalent areas between the nitrogen and oxygen and some of carbonatoms52. The blue circles surrounding the hydrogen and carbon nuclei represent the electron depletion zones between the inner and valence shells (Figure S7(B).

RDG and NCI analysis

The Reduced Density Gradient (RDG) analysis provides a visual representation of the regions within molecules where intra- and intermolecular non-covalent interactions occur, using the electron density and its associated derivatives53,54. The plotting of RDG against sign (λ2) ρ creates an RDG scatter graph, where sign (λ2) ρ represent the second eigen-value of the Hessian matrix that measures electron density. The sort of interaction is determined by the sign (λ2) ρ value: an attractive interaction is indicated by a sign (λ2) ρ < 0, a repulsive interaction is shown by a sign (λ2) ρ > 0, and a weak interaction, lsuch as a van der Waals interaction, is indicated by a sign (λ2) ρ close to zero55. Figure S8(B) shows the RDG plot of monomer (i) and dimer (ii).

Strong repulsive interactions, mainly observed in the centers of aromatic rings of the 2,6-Difluorophenyl ring and pyrimidine ring are indicated by the red region, while the blue region indicates strong attractive interactions, corresponding to the strong N–H···N intermolecular hydrogen bonding. Additionally, the C–F··· π intra molecular interaction visualized as a green disc56. Figure S8(A) shows the NCI plot of monomer (i) and dimer (ii).

Table 6 Electron density (ρ), laplacian of electron density (∇2ρ), bond ellipticity (ε) and intramolecular interaction energy (EHB) of non-covalent interactions
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QTAIM analysis

R. F. W. Bader’s Quantum Theory of Atoms in Molecules (QTAIM)57 provides a robust framework for understanding chemical bonding through the topology of the electron density (ρ). In this approach, the presence of a bond path between two atoms indicates an interaction, and the corresponding bond critical point (BCP) represents the point of minimum electron density along that path58.

QTAIM analysis was carried out on the optimized Florasulam monomer and dimer structures using the AIM2000 program to identify the key intra- and intermolecular noncovalent interactions responsible for their stability.

The monomer displays an intramolecular C12–F2···π interaction with a Laplacian (∇²ρ) of 0.0223 a.u., an electron density (ρ) of 0.0057 a.u., and an interaction energy of −1.27 kcal/mol, consistent with a weak-to-medium electrostatic interaction. In contrast, the dimer features a significantly stronger intermolecular N5–H5···N4 hydrogen bond, characterized by a Laplacian (∇²ρ) of 0.1473 a.u., an electron density (ρ) of 0.05028 a.u., and an interaction energy of −11.17 kcal/mol. Additional weak halogen bonds further contribute to the stabilization of the dimer. Taken together, these findings indicate that Florasulam’s stability results from a synergistic combination of noncovalent interactions: moderate intramolecular halogen–π interactions in the monomer and a network of weak halogen bonds reinforced by a strong N–H···N hydrogen bond in the dimer. This interplay of interactions enhances molecular rigidity and may influence the physicochemical properties and potential biological activity of Florasulam (Figure S9 and Table 6).

Fig. 8
figure 8

BDEs of hydrogen atoms and all single acyclic bonds of FL

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Sensitivity towards autoxidation

Computational molecular modelling approaches, particularly bond dissociation energy (BDE) analysis, are widely employed to pinpoint structural sites that may initiate molecular breakdown. Hydrogen-abstraction BDEs (H-BDEs) are especially informative because they reflect a compound’s propensity to undergo autoxidation, while BDEs of other single acyclic σ-bonds help identify alternative positions where degradation could begin. As many agrochemical and pharmaceutical residues ultimately reach aquatic environments, understanding their stability in water and their susceptibility to oxidative or non-oxidative pathways is essential59. Given the relevance of oxidation reactions in the environmental transformation of such compounds, this study also examined the potential of Florasulam (FL) to undergo autoxidation.

The BDE calculations revealed no hydrogen-abstraction sites within the FL structure with energies below 90 kcal·mol⁻¹ (Fig. 8). Because values under this threshold are typically associated with high autoxidation reactivity, the consistently high H-BDEs indicate that FL is inherently resistant to radical-driven oxidative attack. All computed abstraction energies exceeded 90 kcal·mol⁻¹, supporting the conclusion that FL is unlikely to participate in autoxidation under typical environmental or atmospheric conditions60,61.

To explore other possible degradation routes, BDEs were evaluated for all single, non-cyclic bonds. The weakest bonds were identified as N–S (bond 10, 52.55 kcal·mol⁻¹), S–C (bond 11, 53.56 kcal·mol⁻¹), and O–C (bond 7, 57.71 kcal·mol⁻¹). These relatively low dissociation energies highlight these sites as the most probable points of initial bond scission. Breakage at these positions would lead either to the loss of the 2,6-dichlorobenzene substituent or [1,2,4]triazolo[1,5-c]pyrimidine ring, outlining feasible non-oxidative degradation pathways.

Biological traits

Molecular Docking

In plants and a variety of microorganisms, acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS), is an essential enzyme that produces the branched-chain amino acids valine, leucine, and isoleucine. Animals do not have this enzyme, hence AHAS is a great target for selective herbicide development. This work used molecular docking techniques to investigate the connections between recognized AHAS-inhibiting herbicides and the enzyme’s active site. Florasulam’s herbicidal capability was assessed by analyzing its binding affinity with four AHAS protein structures (PDB IDs: 1YHY, 1YI0, 1Z8N, and 3EA4). Favourable binding energies and significant molecular interactions were found by docking research, underscoring Florasulam’s prospects as an intriguing lead chemical for herbicide development. Protein-ligand interactions depend heavily on hydrogen bonding, which enhances the stability and selectivity of the complex. Hydrogen atoms bound to significantly electronegative atoms like oxygen, nitrogen, or fluorine (contributors) and adjacent electronegative atoms containing lone pairs (acceptors) are usually the ones involved in these relationships. in spite of their electronegative properties and capacity to engage in halogen bonding, halogens are also purposefully included in medication design to improve the binding performance of ligands62,63. Figure 9 shows docking plots.

The B.E. for Florasulam with proteins 1YHY, 1YI0, 1Z8N, and 3EA4 were determined to be −5.37, −6.06, −5.70, and − 5.76 kcal/mol, respectively, as shown in Table 7. The associated inhibition constants were 36.15 µM, 66.7 µM, 59.56 µM, and 116.04 µM. Florasulam had an extremely high binding affinity for protein 1YI0, implying a stronger and more stable association and consequently enhanced herbicidal ability. Upon closer examination of docking positions, it was discovered that Florasulam’s oxygen atoms O1 and O2 were actively forming hydrogen bonds with amino acid residues like VAL415, SER186, ARG246, PRO247, GLY309, ASP414, and ILE394, with bond lengths varying between 1.87 and 3.19 Å. In the protein 2P1P, halogen bonding was also detected with ASP185, GLY245, and GLY371 at distances of 2.55, 3.42, and 3.12 Å. The 3EA4 protein was subjected to halogen bonding to LEU349 at a distance of 2.59 Å and hydrogen bonding to HIS352, GLY350, SER507, and LEU332. Substantial hydrogen and halogen bonds among Florasulam and amino acid residues in the chosen AHAS proteins’ active areas lend credence to the compound’s potential as an aggressive herbicidal agent.

Fig. 9
figure 9

2D and 3D docking plot of FI with the proteins (1YHY, 1YI0, 1Z8N, and 3EA4)

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Table 7 Molecular Docking result of thiamine molecule with different antifungalproteins
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Fig. 10
figure 10

Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and Radius of Gyration (Rg) plots for (A) ALS protein (1YI0), (B) Florasulam ligand, and (C) the Florasulam–ALS (1YI0) complex

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MD simulation

Molecular dynamics (MD) models use sophisticated computational algorithms to faithfully reproduce the motions that take place in both macro and micro-molecules under circumstances that closely mimic those found in life. In this investigation, a 10-nanosecond MD simulation was run on the protein-ligand complex of 1YI0 and Florasulam, which was found to have the highest binding affinity. This simulation aimed to investigate intermolecular relationships structural changes, and molecular oscillations during the span of the simulation64. The computations were performed using the GROMACS 5.1 software tool65, and the SwissParam service provided the ligand parameters25. The RMSD, which displays variations in atomic positions over time, is one of the main instruments utilized in trajectory analysis. Understanding structural stability and structural alterations can be gained by examining the RMSD with special attention to the ligand and the protein backbone. Protein 1YI0’s RMSD stabilized about 0.26 nm, indicating structural consistency and bolstering the idea that greater structural diversity equates to improved stability. Reliable structural behavior is shown by the complex and its reference’s RMSD values closely aligning.

To evaluate flexibility at the residue level, RMSF was also computed. Greater RMSF values indicate that the protein–ligand complex is more flexible. The 1YI0 protein shows variations up to about 0.38 nm, as seen in Fig. 10, suggesting that ligand binding has decreased structural stiffness. This rise in residue-level motion is indicative of a dynamic complex that may stabilize via interactions. A measurement of the protein’s tertiary structure’s uniformity was also made using the Rg. Rg indicates how closely a protein is pleated; more compact structures have lesser Rg values. The 1YI0–Florasulam complex exhibits perpetually low Rg values in Fig. 10 suggesting a protein-ligand organization that is structurally compact and densely packed.

Conclusion

This work thoroughly examined the structural, vibrational, electrical, and biological aspects of Florasulam using an integrated experimental and computational methodology. Single-crystal X-ray diffraction demonstrated that Florasulam exhibits a V-shaped conformation with a dihedral angle of 31.2°, maintained by intramolecular C–F···π interactions. The one-dimensional ribbon structure extended into two-dimensional sheets via N–H···N, C–H···O, and S = O···π interactions, was validated using Hirshfeld surface analysis, with H···F (23.4%), O···H (15.8%), and N···H (13.5%) as predominant contributors. Vibrational analysis conducted at the B3LYP/6–311 + + G(d, p) level closely aligned with the experimental FT-IR data following the use of a 0.9614 scaling factor. The asymmetric SO₂ stretching at 1293 cm⁻¹ (experimental) and 1299 cm⁻¹ (theoretical) exhibited remarkable concordance. The HOMO-LUMO study indicated an energy gap of 3.455 eV, signifying substantial chemical stability. However, the MEP analysis identified negative electrostatic areas surrounding oxygen and sulphur atoms, conducive to electrophilic assault. Florasulam exhibits strong resistance to autoxidation, as indicated by consistently high H-BDE values, but remains susceptible to degradation through cleavage of specific low-energy heteroatom-containing σ-bonds. These findings highlight non-oxidative bond-scission pathways as the most plausible routes governing its environmental transformation. Molecular docking validated Florasulam’s potential herbicidal efficacy, with binding energies between − 5.37 and − 6.06 kcal/mol, especially with the ALS enzyme (PDB ID: 1YI0), establishing critical hydrogen and halogen bonds. Furthermore, MD simulation confirmed the structural stability of the Florasulam-ALS complex, with an average RMSD of 0.26 nm. The findings unequivocally affirm Florasulam’s chemical stability, reactivity, and biological efficacy, substantiating its role as a selective herbicide and offering substantial evidence for its prospective use in sustainable weed management.