The chemicals required for synthesis of indazolylchromones were obtained from manufacturers. All the chemicals were utilized as received and not purified unless stated otherwise. The TLC plates (20 × 20 cm) coated with silica gel (having F254 fluorescence indicator) were used for reaction monitoring. 100–200 mesh size silica gel was used for column chromatography. Melting points of synthesised compounds were measured by Buchi M-560, reported values were not adjusted/corrected. NMR spectra of (¹H and ¹³C) of synthesised compounds were recorded by JEOL JNM-ECZ400/SI. The mass spectrometry was carried out by ABSCIEX triple TOFTM 5600+ having Turbo Ion Spray. ED₅₀ values were estimated with the SPSS statistical package. Molecular docking was carried out using molsoft software.

2-hydroxyacetophenone derivatives 2a-2n were synthesised by reacting 2- hydroxyacetophenone 1 and bromoalkanes or iodoalkanes of different chain lengths in a molar ratio of 1:1.2 by continuous stirring for 6 h at 60 °C in the presence of K₂CO₃ and acetone. These 2-hydroxyacetophenone derivatives 2a-2t (1 mmol) on reaction with N,N-dimethylformamidedimethylacetal (DMFDMA) (2 mmol, 2 molequiv) overnight at 90 °C resulted in formation of enaminones 3a-3t. The chromones 4a-4t were synthesized by reacting enaminones 3a-3t, (1 mmol) and iodine (2 mmol, 2 molequiv), in 30 mL CHCl₃ at room temperature for 8 h with continuous stirring. The pure chromones were obtained in 67%–89% yield by column chromatography of synthesised crude compounds. Finally, conjugated addition of 3-iodochromones 4a-4t with indazole 5 gave 2-Indazolyl-chromones 6a-6t in the yield range of 76%–94%. (Scheme 1, Table 1). All synthesized 2-Indazol-1-yl-chromen-4-one derivatives were characterized using IR, ¹H-NMR, and ¹³C-NMR analysis. The spectral analysis of all synthesised compounds has been provided in supporting document.

Molecular docking was carried out to predict the interaction of most active compounds, 6t and 6f with Cytochrome P450 14alpha -sterol demethylase (CYP51) enzyme using molsoft software and the respective docking scores were calculated to compare the interaction of compounds with the lanosterol 14α-demethylase (LDM) enzyme.

The aquatic toxicity potentials of the synthesized compounds were assessed using Ecological Structure Activity Relationship (ECOSAR) model. It is a free and easy to use computer programme developed by US EPA. It predicts the median lethal concentration (LC₅₀), median effective concentration (EC₅₀), and chronic value (ChV) for various species.

In the present work, total twenty indazolylchromones (6a- 6t) were synthesized, out of which 19 compounds (6b- 6t) were found novel. The indazolylchromones were synthesized by 4 steps synthetic scheme (scheme- I). In the last step, conjugated addition of 3-iodochromones 4a-4t with Indazole 5 is the Michael type addition reaction where indazole act as Michael donor (nucleophile) and chromone as Michael acceptor. In the first step indazole attacks chromone ring at 2-position and gives an intermediate A, which on removal of H-1 results in the formation of 2-(1-Indazolyl) chromones (Sugita et al., 1996).

The above synthetic method resulted in the synthesis of 2-(1-Indazolyl) chromones in 76%–94% yield. All the synthesised compounds were characterised by IR, ¹H NMR and ¹³C NMR. In all synthesised compounds 6a-6t, typical peak at δ 6.83–6.93 (1H, s, H-3) appeared due to elimination of iodine from 3- Iodochromones. The peaks at δ 8.21–8.35 (2H, s, H- 3′), δ 7.74–8.19 (1H, d, J = 8, H-4′), δ 7.31–7.48 (1H, t, J = 8, H-5′), δ 7.55–7.67 (1H, t, J = 7.2, H- 6′) and δ 7.66–7.85 (1H, d, J = 8, H-7′) were assigned to protons of indazolyl ring in ¹H-NMR spectra of all the compounds and validates the synthesis of indazolylchromones. In ¹³C-NMR, the peaks at δ 96.10–96.42 (C-3), 123.76–125–85 (C-5′), 121.76–123.99 (C-4′), 155.20–157.96 (C-2), 129.06–135.57 (C- 6′), 114.31–119.07 (C-7’) and at δ 176.44–177.23 for carbonyl carbon (C=O) were prominent for all the synthesised compounds. In IR spectra stretching of 1,675–1,677 (C=O), 1,365–1,368 (C-N) and 1,532–1,537 (pyrone ring C=C stretch) supported the NMR data. The spectroscopic data revealed that in the above-synthesized compounds, the N atom at the one-position of the indazole was linked to chromone ring at C-2 position.

All the synthesised indazolylchromones (6a- 6t) exhibited antifungal activity against S. rolfsii (Table 2), but compound 6t (Figure 1) exhibited the highest activity (ED₅₀ = 10.10 mg L^-1) and performed at par with that of Hexaconazole 5% SC (ED₅₀ = 8.57 mg L^-1), a commercial fungicide. Among the alkoxy derivatives of indazolylchromones (6a- 6n), butoxy derivative 6f, 2-Indazol-1-yl-7-butoxy chromen-4-one, was found most active with ED₅₀ = 16.18 mg L^-1. It was observed that alkoxy derivatives having even number of carbon chain length were more active as compared to odd number of carbon chain length. This trend was observed up to carbon chain length of C- 6. After C-6 there was no regular trend was observed (Figure 2).

In case of F. oxysporum, all the indazolylchromones (6a- 6t) showed antifungal activity (Table 3), compound 6f with ED₅₀ = 27.82 mg L^-1was found most active (Figure 3), but that was less than commercial fungicide carbendazim 50% WP (ED₅₀ = 9.01 mg L^-1). Antifungal activity of synthesised compounds (6a- 6t) against F. oxysporum was less than their antifungal activity against S. rolfsii.

The docking study of most potent compounds, 6f and 6t revealed that both compounds have different orientations, which in turn results in different modes of binding in the active site of CYP51.

In Figure 4, it has been observed that in compound 6f, the indazole ring is having π-π with TRY 76 and hydrophobic interactions with MET 79, PHE 255, PHE 78, VAL 434, LEU 321 residues. The chromone ring was found to have hydrophobic interactions with ALA 256 and HEM 460 residues. The butoxy chain of chromone ring was observed to be engaged in hydrophobic interactions with LYS 97, PHE 83, LEU 100 and ARG 96 residues.

In case of compound 6t (Figure 5), it has been observed that the indazole ring is having π-π with TRY 76 and hydrophobic interactions with PHE 78, LEU 321and HEM 460 residues. The chromone ring was found to have hydrophobic interactions with GLN 72, ALA 73, MET 79 THR 80 and PHE 83 residues.

In commercial fungicide hexaconazole (Figure 6), triazole ring showed hydrophobic interaction with HEM 460, HIS 259, THR 260 and ALA 256 residues. The benzyl ring was found to be engaged with PHE 255, HIE 101 PHE 83 and LEU 100 residues.

The docking studies revealed that compound 6t with highest negative binding energy value (−17.77 kcal mol⁻¹) showed better binding with lanosterol enzyme as compared to compound 6f (−17.65 kcal mol⁻¹). These docking studies supports the in vitro bioassay result where compound 6t (ED ₅₀ = 10.10 mg L^-1) exhibited higher fungicidal activity than compound 6f (ED ₅₀ = 16.18 mg L^-1).

The acute toxicity predictions for fish (LC₅₀,96 Hr), daphnid (LC₅₀, 48 Hr) and green algae (EC₅₀, 96Hr) and the chronic toxicity predictions (ChV) were compared and tabulated in table 5. Fish LC₅₀ values varied from 1.64 × 10⁻⁶ to 0.685 mg/L whereas the chronic toxicity values varied from 1.86 × 10⁻⁴ to 0.042 mg/L among the different compounds, the highest toxicity was shown by the alkoxy substituted compounds. Among the alkoxy substituted compounds, the toxicity was found to be increasing with increase in the alkyl chain. Lowest toxicities were exhibited by compounds with halo substitutions. Among the halogenated compounds, dichlorinated compound exhibited the highest toxicity but less toxic as compared to alkoxy derivatives (Table 4). As per ECOSAR prediction, all the chemicals are inside AD and not missing predictions. It was observed that the acute as well as chronic toxicity values have negative linear correlations with the log K_OW values (Figures 7, 8).