Design, synthesis and in vitro biological activities of coumarin linked 1,3,4-oxadiazole hybrids as potential multi-target directed anti-Alzheimer agents

2.1.1 Synthesis of ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate (2)

A mixture of 7-hydroxy-4-methyl coumarin (1) (5.6 mM) and ethylchloroacetate (8.5 mM) in dry dimethylformamide (DMF) (25 mL) was refluxed at 80 °C on a water bath for 10 h in the presence of potassium carbonate. After the completion of the reaction, potassium carbonate was filtered, and the mixture was poured on to the ice-cold water to obtain the precipitates of the product. The crude product was filtered and recrystallized using ethanol to obtain the TLC pure compound ethyl 2-[(4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate. m.p. 90–91 °C (Literature; 88–90 °C- (Khan and Akhtar 2003)); yield:78%.

2.1.2 Synthesis of 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy) acetohydrazide (3)

It was prepared as per the reported method. Briefly, hydrazine hydrate (15 mM) was mixed with compound 2 (10 mM) in 25 mL of absolute ethanol and the resulting mixture was gently refluxed for 4–6 h. The reaction mixture was concentrated and then cooled to obtain a solid product. Crude solid product was recrystallized in ethanol to get TLC pure compound. m.p. 201–203 °C (Literature; 198–200 °C- (Khan and Akhtar 2003)); yield: 89.9 %.

2.1.3 General procedure for the synthesis of 7-(5-substituted aryl)-1,3,4- oxadiazol-2-yl) methoxy)-4-methyl-2H-chromen-2-one (4a-n)

To a mixture of compound 3 (10 mM) and various substituted phenolic acids (10 mM), 3 mL of cyclizing agent phosphorus oxychloride (POCl₃) was added drop wise and then the mixture was refluxed for approximately 6 h. The progress of the chemical reaction was monitored by TLC and after confirmation of the completion of reaction, the content was cooled, poured on to crushed ice and stirred continuously to neutralize the acidic content present. Furthermore, the mixture was neutralized by adding sufficient quantity of 10% sodium bicarbonate solution until neutral pH was obtained. The resulting solution was filtered and purified by recrystallization using ethanol: DMF (1:1) to obtain pure crystalline coumarin − 1,3,4-oxadiazole hybrid derivative in good yield.

2.1.4 Synthesis of ethyl 2-oxo-2H-chromene-3-carboxylate (5)

It was synthesized by reacting a mixture of salicylaldehyde (10 mM) and diethylmalonate (10 mM) in the presence of L-proline (30 mg) as a catalyst following Knoevenagel Condensation reaction. The reaction -mixture was heated for 30–40 min in solvent free condition which upon cooling produced yellow solid. The obtained product was recrystallized using ethanol to get pure yellow crystalline compound.. m.p. 94–95 °C (Literature; 92 °C (Karade et al., 2007)); yield: 75.2%.

2.1.5 Synthesis of 2-oxo-2H-chromene-3-carbohydrazide (6)

It was prepared as per the reported method. Briefly, hydrazine hydrate (15 mM) was mixed with a solution of compound 5 (10 mM) in 25 mL of absolute ethanol and the resulting solution was refluxed for 4–6 h. The reaction mixture was concentrated and then cooled to obtain a solid product. It was recrystallized in ethanol to obtain TLC pure compound. m.p. 136–137 °C (Literature; 136–137 °C (Khan and Akhtar 2003); yield: 47.6 %.

2.1.6 General procedure for the preparation of 3-(5-substituted aryl)-1,3,4-oxadiazol-2-yl))–2H-chromen-2-one (7a-m)

To a mixture of compound 6 (10 mM) and various substituted phenolic acids (10 mM), 3 mL of cyclizing agent phosphorus oxychloride (POCl₃) was added drop wise and then the mixture was refluxed for approximately 6 h. The reaction mixture on usual work up yielded the crude solid product which was filtered and purified by recrystallization using ethanol: DMF (1:1) to obtain pure crystalline coumarin − 1,3,4-oxadiazole hybrid derivative in moderate to good yield.

2.2.1 DPPH radical scavenging (antioxidant activity) assay of the designed hybrid ligands

In vitro antioxidant activity of the synthesized trihybridized coumarin linked 1,3,4-oxadiazole derivatives 4(a-n) and 7(a-m) was investigated using 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical assay. Gallic acid was used as the standard antioxidant. Various concentrations (100, 50, 40, 30, 20 and 10 μM) of the synthesized derivatives (4a-n and 7a-m) and positive control were used for the assay. To evaluate the antioxidant activity 25 μL of the test sample/standard was added to 96 well plate and was mixed thoroughly with 175 μL of DPPH in methanol solution (0.004% w/v). A control well was also maintained which did not contain sample to cancel out the inherent activity of solvent in the experiment. The plate was then kept aside in dark at room temperature for 30–45 min. The optical density of the test and standard solution was measured at 517 nm using 96 microplate bioanalyzer reader (Epoch, Biotek) (Lolak et al., 2020). The radical scavenging activity was calculated using the standard formula.

IC₅₀ of the designed ligands was calculated using non-linear regression analysis of the % inhibition and concentration (μM). The antioxidant property of the designed ligands was compared with the antioxidant capability of gallic acid. All the tests were carried out in triplicate and the results are reported as mean ± standard deviation (SD).

2.2.2 Evaluation of AChE inhibitory activity

A colorimetric assay was employed to evaluate AChE inhibitory activity of the designed ligands. β-naphthyl acetate (NA) was employed as the substrate for the enzyme AChE while fast blue-B salt (FB) was used as the diazonium dye forming reagent. β-NA is hydrolyzed by the enzyme AChE into naphthol and acetate. The formed naphthol then reacts with the fast blue B to form diazonium dye that gives a stable purple color. For the quantitative estimation of the dye formed, absorbance of the color intensity was measured after the reaction using a bio analyzer. For the spectrometric analysis each compound prepared in different concentrations (100, 50, 40, 30, 20, 10 μM) and 50 μL of it was added in triplicate in different wells. 50 μL of NA (0.25 mg/mL in methanol) and 50 μL of AChE were added to each well and mixed thoroughly. The microplate was then incubated at room temperature for 40 min. After 40 min, 10 μL of FB salt (0.25 mg/mL) solution in water was added to the incubated mixture and mixed well with the pipette. The absorbance of the mixture was determined at 600 nm. Blank wells containing no samples were also used to nullify the effect of solvents in the mixture. Standard drug galantamine, an AChE inhibitor, was used to compare the results (Khokar et al., 2021).

2.2.3 Evaluation of BuChE inhibitory activity

BuChE inhibitory Kit (Catalog#K516-100) was purchased from BioVision. The inhibitory assay was performed according to the method described in the product information leaflet. Briefly, 20 μL of desired concentrations of the samples were added to each well in triplicate. The enzyme BuChE was diluted 50 folds. 8 μL of the enzyme was added to each well and mixed thoroughly. The volume was adjusted to 95 μL with the assay buffer. Control well was also prepared in the same way but without adding a sample. 5 μL of DTNB solution prepared was also added to the reaction mixture. The total volume in each well was made up to 100 μL.100 μL of 120-fold diluted BuChE substrate was then added and mixed well. The plate was then kept for incubation in the dark for 20–30 min. The absorbance was measured at 412 nm and % BuChE inhibitory activity was calculated (Lolak et al., 2020).

2.2.4 Evaluation of COX inhibitory activity of the synthesized hybrids

COX activity assay kit (item no.760151) was purchased from Cayman Chemicals. The assay was performed according to the procedure outlined in the product information leaflet. Briefly, 110 µL of assay buffer, 10 µL of hemin, 10 µL of COX enzyme and 40 µL of sample were added to the sample wells in triplicate. After Incubation for 5 min, the contents were mixed uniformly in each well followed by addition of 20 µL of colorimetric substrate to each well. To initiate the reaction, 20 µL of arachidonic acid solution was added to the wells. The plates were shaken carefully and the incubated for 5 min at 25 °C.

The control well was prepared in the same manner by omitting the sample compounds. The absorbance of the mixture was recorded at 590 nm using a microplate reader and % COX inhibition was calculated (Dey et al., 2003).

2.4.1 Visualization of docking poses and docking analysis

Molecular docking studies were performed with mcule.com webserver which generated top five hits. The poses were analyzed for interaction. The best minimal binding energy pose for ligand–protein interaction was saved as a PDB file to further study docking conformation and types of bonding interaction. The downloaded poses were visualized for the ligand-target interaction using Discovery studio Visualizer. The 2D image was used to study the amino acids residues of the active site of the receptor interacting with the designed ligands.

3.5.1 Molecular docking studies

Molecular docking studies were carried out to study the AChE/BuChE- ligand interactions and to validate the results of in vitro experimental studies. Binding energy scores of the best pose of synthesized ligands with both the target receptors (2CMF and 4BDS) are presented in the Supplementary Table 1. Series 1a compounds showed better binding energy scores (-7.3 to 12.0 Kcal/Mol) than series 1b compounds (-1.3 to −9.5 Kcal/Mol) on to the AChE enzyme. Compounds 4g and 4e which showed the best in vitro AChE inhibitory activity showed binding energy scores of −9.7 and −10.1 Kcal/Mol. Compound 4e contains 3,4-dihydroxy phenyl ring structural fragment which comes from the 3,4 dihydroxy benzoic acid while 4g was prepared using gallic acid and therefore bears 3,4,5 trihydroxyphenyl attached at the 5th position of oxadiazole ring in the hybrid molecule. Both the molecules are from series 1a and in contrast to series 1b compounds consists of a 4-methyl group on the coumarin ring and an additional –O-CH₂- as a connecting linker between the coumarin and oxadiazole pharmacophores. Docking results of 4e shows that the oxygen atom of the oxadiazole moiety interacts with the TYR121 present in the active site of the AChE receptor (2CMF). Apart from hydrogen bond interaction, π-π stacking (TRP84, PHE330, PHE331), van der Waals interaction and π-alkyl (TYR334) interactions were also observed (Fig. 4). It exhibited a docking score of −10.1 kcal/mol. The compound could inhibit 50% of the AChE enzyme at 29.56 µM ± 3.95. Compound 7e which is a corresponding compound in the series 1b with the identical 3,4-dihdroxy phenyl substituent showed a much lower docking score of −7.1 kcal/mol even though it can form two hydrogen bonds with the enzyme amino acid residues. The hydroxy group on the meta position on the phenyl ring and nitrogen atom in the 1,3,4-oxadiazole moiety formed hydrogen bond with the SER200 amino acid residue on the active site of the receptor. However, compound 7e upon in vitro AChE inhibitory assay showed an IC₅₀ value of 49.65 µM ± 2.98 which is much higher than its corresponding compound in series 1b. Also, this compound is less selective for AChE (SI = 0.76) over BuChE in comparison to 4e (SI = 1.55). Furthermore, 4e upon docking on to the BuChE formed three hydrogen bonds, two through carbonyl oxygen (SER198 & ALA199) and one through hydroxyl group (TYR128) and showed a binding energy score of −9.6 kcal/mol. It also showed other interactions with amino acid residues including π-π (PHE329), π-alkyl (VAL288, LEU286), π-sigma (TRP231) and van der Walls interactions (Fig. 4). Thus, based on these results it can be proposed that a linker connecting the two pharmacophores (oxadiazole with coumarin) and presence of a methyl group in the coumarin moiety might help in increasing the selectivity and potency of the trihybridized molecule towards AChE. In compound 4g, oxygen atom of the 1,3,4-oxadiazole moiety forms one hydrogen bond with the TYR121 and another hydrogen bond between m-OH group and PHE288 amino acid residue in the active site of the AChE receptor (Fig. 4). It showed an IC₅₀ value of 28.68 ± 2.91 µM for AChE inhibition and was found to be 1.65 times more selective towards AChE over BuChE with the binding energy score of −9.7 kcal/mol. The corresponding compound (7g) with similar structural fragment in series 1b exhibited an IC₅₀ of 43.29 ± 3.44 µM against AChE and a lower docking binding score of −6.5 kcal/mol. Interactions of 7g with the BuChE enzyme shows that it forms three hydrogen bonds with the receptor amino acid residues and has a docking score of −8.1 kcal/mol. The p- hydroxyl group on the phenyl ring forms a hydrogen bond with LEU286 and m-hydroxy group forms a hydrogen bond with TRP231. Increase in the number of hydrogen bond did not increase the interaction with the receptor which might be because hydrogen bond should be placed in favorable distance and favorable orientation for the formation of strong interaction with the receptor. On the other hand, the most promising 4g also forms three hydrogen bonds (HIS438, SER198, ALA199) along with π-π (PHE329, TRP82), π-alkyl (VAL288, LEU286), π-sigma (TRP231) and van der Walls interactions with amino acid residues on the BuChE receptor and showed much improved binding energy score of −10.0 kcal/mol in comparison to 7g (Fig. 4).

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Fig. 4

2D image of active site amino acid residue interacting with compound (A) 4g with AChE receptor; (B) 4g with BuChE receptor; (C) 4e with AChE receptor and (D) 4e with BuChE receptor.

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3.5.2 Prediction of molecular properties, toxicity and pharmacokinetic profile

Molecular properties predicted with the help of molinspiration indicated that all the synthesized hybrid compounds obey’s Lipinski’s rule of five and are expected to be orally bioavailable. Bioactivities predicted with the help of PASS cheminformatic online software revealed that almost all the compounds possess MAO inhibitory, antioxidant and anti-inflammatory activities. However, compounds 7j, 7m and 7l were noted to be devoid of anti-inflammatory activity. OSIRIS online software predicted series 1b compounds to be non-toxic (non-mutagenic, non-tumorigenic, non-irritant on skin with no reproductive toxic effect). However, series 1a compounds could produce reproductive toxicity. Admet-SAR predicted high gastrointestinal absorption and moderate to good BBB permeability. All the compounds are predicted to be safer in rat acute toxicity, AMES toxicity, fish toxicity, honeybee toxicity and acute oral toxicity. Molecular properties, biological activity by PASS, OSIRIS toxicity and pharmacokinetic properties data of synthesized compounds are presented in the supplementary information.