Discovery of new thiazolo[4,5-b] pyridine-based 1,2,3-triazoles as potent antioxidant agents: in vitro and in silico investigation

2.1.1 Preparation of 7-substituted-aryl-thiazolo-pyridine-6-carbonitrile (5a & 5b) (Pagadala et al., 2015)

A 100 mL flask holding equimolar quantities of substituted benzaldehyde, thiazolidine-2,4-dione, malononitrile, ammonium acetate (NH₄OAc), and triethylamine was incorporated with 5 mL of ethanol. The mixture was stirred at room temperature for 10 min, and the reaction was monitored by thin-layer chromatography (TLC) with a 70:30 hexane/ethyl acetate eluent. After the reaction finished, the mixture was poured into a beaker, and the solid product was collected by filtration. Purification was achieved by recrystallization from ethanol, yielding the target compounds (5a & b) in high purity and good yields.

2.1.2 Synthetic approach for the 5-amino-7-(substituted-aryl)-oxo-(propynyl)-dihydrothiazolopyridine-6-carbonitrile (7a & 7b) (Mareddy et al., 2013):

Propargyl bromide (2.2 mmol) and compound (5) (1 mmol) in ethanol (6 mL) were introduced to a reaction vessel containing potassium carbonate (2.5 mmol). At room temperature, the solvent lasted for 2 h. After the reaction came to an end, the reaction mix was progressively emptied onto crushed ice. The final product was the solid, which was then filtered, dried, and washed with ether to purify it.

2.1.3 Synthetic procedure for thiazolopyridine-carbonitrile-triazole derivatives (9a-j):

A mixture comprising compound (7) in a combination solvent system [THF:H₂O (1:1)] was supplemented with various substituted azides (8a-e) (1 mmol). The following steps involved adding CuSO₄ (0.05 mmol) and sodium ascorbate (0.15 mmol) at RT. Later, TLC helped continuously monitor the reaction’s progress until the starting reactants disappeared. Then, it was extracted using CHCl₃ (30 mL), and the solvent was dried with anhydrous Na₂SO₄. A crude was concentrated by removing the solvent at a reduced pressure and was subjected to additional refinement. Following purification, the target products were separated using a solvent solution of 60:40 hexane to ethyl acetate and silica gel chromatography (Scheme 1). The supplementary information contains the instrumentation details and spectral characterization data for all the compounds (SI-I).

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Scheme 1.

Synthetic route for Thiazolo[4,5-b]pyridine-6-carbonitrile linked substituted aryl-1,2,3-triazole analogues.

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2.2.1 Nuclear magnetic resonance (NMR)

Compound 9a: ¹H NMR (400 MHz, DMSO-d₆) δ 9.10 (s, 1H, CH), 8.89 (d, J = 8.5 Hz, 2H, Ar-H), 8.19 (d, J = 9.0 Hz, 2H, Ar-H), 7.49 – 7.31 (m, 2H, Ar-H), 7.14 – 7.03 (m, 3H, Ar-H), 6.45 (s, 2H, NH₂), 4.90 (s, 2H, CH₂), 3.82 (s, 3H, OCH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.38, 157.50, 153.43, 146.50, 143.43, 136.22, 133.50, 131.58, 131.02, 128.99, 125.90, 122.28, 121.27, 120.34, 115.59, 114.44, 56.12, 51.01; HRMS of [C₂₃H₁₇N₇O₂S + H]⁺ (m/z) 456.1912; Calcd: 456.1895; FTIR (ATR, cm^-1): 3037, 2842, 2300, 1594, 1276.

3.2.1 Antioxidant assay

Three complementary tests (β-carotene-linoleic acid, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS^•+) scavenging, and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•) scavenging) were used to assess the antioxidant properties of thiazolopyridine-linked triazoles (9a-j) using in vitro techniques. Table 1 illustrates the significant antioxidant potential of all the produced compounds compared to the standards. When benchmarked against control antioxidants (butylated hydroxyanisole (BHA) and α-tocopherol (α-TOC)), derivatives 9i (IC₅₀ = 13.06 ± 0.50 µM) and 9e (IC₅₀ = 17.30 ± 0.58 µM) demonstrated superior radical scavenging capacity within the tested series. Their significantly lower IC₅₀ values indicate enhanced potency relative to the reference compounds. The phenyl ring connected to the triazole-thiazolopyridine scaffold of these compounds has electron-donating methoxy groups, which are responsible for their improved scavenging capabilities.

Further, the methoxy substituent on the phenyl ring is what makes compounds 9i and 9e the most effective in the ABTS^•+ radical scavenging action, with IC₅₀ values of 15.18 ± 0.25 µM and 18.52 ± 0.18 µM, respectively. The majority of these compounds exhibited adequate scavenging activity of ABTS^•+. Additionally, 9i and 9e were shown to be the most active in the lipid peroxidation inhibition assay, with respective values of 09.10 ± 0.46 µM and 16.08 ± 0.18 µM, which is again due to the electron-releasing methoxy group. The results varied from moderate to weak for most of the series; however, all investigated substances showed some degree of lipid peroxidation inhibitory efficacy.

3.3.1 Binding affinities and interaction profile analysis of 9a-j with NRF2-KEAP1 and catalase

NRF2, a key transcription factor regulating antioxidant enzymes like catalase, SOD, and GPx, is normally suppressed by KEAP1, which facilitates its breakdown. During oxidative conditions, NRF2 detaches from KEAP1, enters the nuclei, and stimulates expression of genes that hold antioxidant response elements, thereby strengthening the cell’s protective mechanisms against oxidative damage. Small-molecule modulators targeting the NRF2/KEAP1 pathway can promote NRF2 activation and catalase function, mitigating OS-related damage. Docking studies help elucidate how these compounds interact with NRF2-KEAP1 and catalase, providing insights into their potential to strengthen cellular antioxidant mechanisms (Dallakyan, and Olson, 2015)

Docking simulations on NRF2-KEAP1 and Catalase proteins (Fig. 2) revealed the molecular interactions of compounds 9e and 9i, featuring a central 2-oxo-7-phenyl thiazolo-pyrimidine core with varying substituents. The key distinction between them is the presence of a chloro group on the 7-phenyl ring in 9i. As close structural analogs, 9e and 9i exhibited binding energies (in kcal/mol) of -9.3 and -9.4, within the NRF2-KEAP1 protein-protein interaction site (Heightman et al., 2019). These low molecular weight compounds function as KEAP1 blockers, critically influencing redox homeostasis by promoting NRF2 signaling pathways, which amplify the endogenous antioxidant capacity of cells. The reference inhibitor J6Q displayed a slightly stronger binding energy of -10.2 kcal/mol (Table S1). Overall, 9e and 9i demonstrated binding affinities and interaction profiles comparable to J6Q, highlighting their potential as NRF2-KEAP1 inhibitors.

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Fig. 2.

Binding energy plot of compounds 9e and 9i with NRF2-KEAP1 and catalase.

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The compounds 9e and 9i exhibited a similar interaction profile due to their close structural similarity. Three hydrogen bonds stabilized interactions with residues of ARG483, SER555, and SER508. The backbone scaffold, containing the 2-oxo and sulfur atoms of the thiazole ring, engaged in hydrogen bonding with SER508 and ARG483, while the 5-amino group established hydrogen bonds with SER555. The binding interface revealed multiple stabilizing forces; ARG415 and ARG483 engaged in π-cation contacts with both the core structure and pyrimidine-attached phenyl group. TYR525 established π-π stacking with the central framework, whereas ALA556 participated in π-alkyl contacts involving methoxy-substituted phenyl and triazole rings. TYR334 mediated alkyl interactions with a methyl group. Carbon-hydrogen bonds formed with GLY462, GLY509, and SER363, complemented by van der Waals forces from neighboring residues, collectively reinforced complex stability.

Compounds J6Q, 9e, and 9i shared conserved interactions, hydrogen bonds with SER508/SER555, and hydrophobic contacts with TYR525/ALA556, implying analogous binding modes. This molecular recognition pattern indicates their capacity to interfere with KEAP1-NRF2 association, potentially upregulating cellular antioxidant pathways. The hinge region, involving the methylene group between the scaffold and the methoxy-substituted phenyl ring on the triazole, showed similar orientations, closely overlapping with the binding pose of J6Q. This consistent binding orientation is illustrated, highlighting the superimposition of pharmacophore features, further supporting the compounds’ similar interaction profiles.

The compounds 9e and 9i also displayed superior binding energies within the HEM binding site of the catalase (Table S2). Compounds 9e and 9i exhibited binding affinities (in kcal/mol) of -10.8 and -11.0, respectively, higher than their binding affinities with the NRF2-KEAP1 complex.

Compound 9e predominantly exhibited hydrophobic interactions and H-bonding with ARG112 through the OMe. ALA357 showed π-σ interactions, whereas PHE161 and HIS75 participated in π-π stacking interactions with the aromatic scaffolds of the backbone structure and phenyl substituent. Several residues, including PRO162, PRO158, ARG354, and ALA133, were involved in hydrophobic contacts through π-alkyl and alkyl interactions. TYR358 established hydrogen bonds via π-donor interactions, while SER114 formed carbon-hydrogen bonds.

Compound 9i exhibited dual hydrogen bonding with ARG112 through its methoxy group, mirroring the interaction pattern observed for 9e and an additional H-bond interaction with TYR358 through the amino group, but not present in 9e, and the remaining hydrophobic interaction profile had close similarities with the 9e compound. HEM is a prosthetic group in catalase that contains a central iron atom coordinated to a porphyrin ring. Similarities with the HEM interaction profile, along with strong binding energies of 9e and 9i, hold potential for positive outcomes for the antioxidant activity.

The binding interactions have been visually represented in Figs. 3 and 4, showing the 2D interaction profiles, highlighting the diverse interactions exhibited by compounds 9e and 9i with NRF2-KEAP1 and catalase. Fig. 5 illustrates the interaction profile of J6Q and HEM, providing a comparative view of the similarities in interactions with compounds 9e and 9i. Fig. S1 illustrates the compound binding orientations, including reference molecule J6Q, within their respective protein complexes and their respective pocket binding sites (Putnam et al., 2000). Common hydrogen bond interactions among these compounds have been highlighted in Fig. S2, while Fig. S3 displays the superimposed binding conformations, highlighting critical pharmacophoric elements. The conserved residue interactions align with J6Q’s binding mode, indicating analogous mechanistic behavior that may contribute to antioxidant effects. These results justify additional exploration to optimize these lead compounds as promising antioxidant candidates.

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Fig. 3.

(a-b) 2D molecular representation of interactions of 9e and 9i with the active site residues of the NRF2-KEAP1 protein. Interactions were displayed as color-coded dashed lines; green lines indicate the H-bonds.

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Fig. 4.

(a-b) 2D molecular representation of interactions of 9c and 9i with the active site residues of the catalyze protein HEM binding domain. Interactions were displayed as color-coded dashed lines; green lines indicated the H-bonds.

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Fig. 5.

Interaction profiles of J6Q in (a) NRF2-KEAP1 and (b) HEM in catalase, highlighting critical hydrogen bond and hydrophobic interactions. These profiles serve as a reference for comparing the interaction patterns of compounds 9e and 9i.

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