Cytotoxicity studies of coumarin analogs: design, synthesis and biological activity

Abstract

In the present study, a series of coumarin derivatives were designed, synthesized and evaluated for their antioxidant and cytotoxic properties. The title compounds, 2-(3-substituted-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid derivatives 5a–5f, were synthesized by base-catalyzed dehydrohalogenative cyclization following Hantzsch synthesis. All the newly synthesized analogues were characterized and established on the basis of mass, ¹H NMR, ¹³C NMR and IR studies. The compounds were evaluated for their in vitro antioxidant activity and found to exhibit substantial activity. The in vitro cytotoxicity was evaluated against MCF-7, MDA-231 (human breast cancer) and HT29 (human colon adenocarcinoma) cell lines by MTT assay and the results were encouraging. Compound 5b, with lower IC₅₀ values of 2.4 and 4.8 μM for MCF-7 and MDA-231, respectively, was considered to be potent among the series.

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1. Introduction

Cancer can be recognised as the second leading cause of deaths worldwide following heart diseases. The World Health Organization has estimated there will be about 12 million deaths across the world due to cancer by 2030. The pursuit of better anticancer agents endowed with cytotoxic action has been key for chemotherapy.^1,2 Epidermal growth factor receptor (EGFR) belongs to the family of protein tyrosine kinases and plays a vital role in cancer proliferation. Studies indicate that this antigen has an important role in cancer etiology and progression. EGFR is overexpressed in solid tumors,³ especially in breast, colon, and bladder cancers. Accordingly, targeting EGFR represents an ideal approach for the design of novel anti-proliferative drugs. EGFR exists on the cell surface and is activated by binding of its specific ligands, like transforming growth factor α (TGFα). On activation, EGFR undergoes a transition from an inactive monomeric form to an active homodimer. EGFR may also pair with another member of the retrovirus-associated DNA sequences (ErbB) receptor family, such as ErbB2, to produce an activated heterodimer. Activated EGFR stimulates its intrinsic intracellular protein-tyrosine kinase activity, which results in phosphorylation of several tyrosine residues in the C-terminal domain of EGFR. These downstream signaling proteins initiate several signal transduction cascades, leading to DNA synthesis and cell proliferation.^4,5 Mutations involving EGFR could lead to its constant activation which could result in uncontrolled cell division, a characteristic of cancer. EGFR is deregulated in several malignant disorders including lung, breast, colorectal, head and neck, prostate, pancreatic and other cancers.⁶ This highlights the rationale in targeting the EGF receptor for specific anticancer efficacy.

Recent studies indicate the beneficial role of cyclooxygenase-2 (COX-2) inhibition in cancer chemotherapy.⁷ PGH₂ is biosynthesized from arachidonic acid by the action of COX. COX is the rate-limiting step, which in turn limits the synthesis of other PGs. Prostaglandin H₂ (PGH₂) is consequently converted to other PGs and thromboxane A₂ by prostaglandin synthases and thromboxane synthases, respectively.

Clinical studies have indicated that prostaglandin E₂ (PGE₂), which is a product of PGH₂, exhibits a considerable role in carcinogenesis. PGE₂ elicits multiple oncogenic signals and the increased level of PGE₂ in cancer tissues indicates its considerable role in regulating a number of other secondary messengers related to the cancer phenotype.⁸ PGE₂ stimulates proliferation, leading to the expansion of tumor mass. Therefore, targeting PGE₂ by COX-2 inhibition is advantageous and holds promise for the development of newer anticancer agents.⁹ Reports on COX-2 selective inhibitors in reducing the number and also the size of intestinal adenomas in patients diagnosed with familial adenomatous polyposis¹⁰ and reduction in the occurrence of sporadic colorectal adenomas¹¹ have now shed light over COX-2 inhibition and support the use of this in cancer chemotherapy.

At the same time, increased production of reactive oxygen metabolites produces a state of oxidative stress. This oxidative stress can be considered as the main trigger of cellular damage and is recognised to exhibit a considerable role in carcinogenesis. Epidemiological investigations have provided enough evidence to support the role of reactive oxygen metabolites in the etiology of cancer. Under the action of the superoxide dismutase enzyme (SOD) there is an abnormal production of superoxide anions (O⁻˙) which results in increased production of hydrogen peroxide.¹² The hydrogen peroxide is catabolized to the highly toxic hydroxyl radical (OH˙) through Fenton and Haber–Weiss reactions. This state of increased production of reactive oxygen species in the physiological system due to environmental factors and lifestyle habits, like smoking, etc., results in oxidative stress. A chronic situation of this oxidative stress causes cross-linking in lipids, proteins and nucleic acids, leading to cellular damage and resulting in alteration of the hereditary DNA, initiating cancer development, metastasis and uncontrolled proliferation. In view of the above facts, anticancer investigations are always associated with preliminary antioxidant evaluation of the tested compounds.¹³

The chemistry of coumarin and its fused heterocyclic derivatives has received considerable attention owing to their synthetic and pharmacological importance. Literature studies highlight that coumarin derivatives have potential to be revealed as anticancer agents. The importance of the substitution on the parent coumarin moiety towards its anticancer properties is well reported.¹⁴ The pattern of substitutions influences the coumarin’s pharmacological properties and therein its therapeutic applications. Structure–activity relationship studies on the substitution indicate the introduction of an alkyloxy function at the C7 position leads to derivatives with strong potential to reduce the plasma alkaline transferase level in hepatitis and to inhibit caspase-3 activation, indicative of its potential as a cytostatic and cytotoxic agent.¹⁵ The anticancer potential of coumarin and its active metabolite, 7-hydroxycoumarin, has been established by the growth-inhibitory cytostatic activity in human cancer cell lines, such as H727 (lung), A549 (lung), MCF-7 (breast), HL-60 (leukemia) and ACHN (renal). It is also to be noted that coumarin derivatives have been reported to demonstrate activity against prostate cancer, malignant melanoma, and metastatic renal cell carcinoma in clinical trials.¹⁶ Coumarin derivatives inhibiting sulfotransferase activity, contributing significantly to their anticancer activity against breast cancer, have also been reported.¹⁷

In the present investigation, we have designed a small library of coumarin derivatives and the designed molecules were docked onto the EGFR and COX-2 enzymes. Molecular docking studies impart knowledge about the probable structural modification of the parent moiety via potential substitutions that would result in appropriate derivatives with better interactions. The designed molecules were also screened for ADMET properties. Based on the docking scores and interactions, the best-ranked molecules were selected for synthesis. The synthesized molecules were evaluated for their in vitro antioxidant activity by 2,2′-diphenyl-1-picryl hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and superoxide anion (SA) radical scavenging assay methods. The compounds were evaluated for their cytotoxic properties using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human cancer cell lines, MCF-7, MDA-231 (human breast cancer) and HT29 (human colon adenocarcinoma). The results highlight that the coumarin moiety provides a scaffold on which pharmacophores can be arranged to afford potent and selective cytotoxic agents.

2. Results & discussion

2.1. Chemistry

The target compounds 5a–5f were synthesized by a multiple step procedure as depicted in Scheme 1. 2,4-Dihydroxyacetophenone (1) was reacted with 2-bromo-2-methylpropanoic acid in the presence of potassium carbonate, establishing an ether linkage and attaining 2-(4-acetyl-3-hydroxyphenoxy)-2-methylpropanoic acid (2). The proton NMR spectrum of acid derivative 2 accounts for fourteen protons. A singlet at δ 10.3 ppm can be attributed to the hydroxy (OH) proton of the carboxylic acid group of the 2-methylpropanoic acid function. The singlet at δ 9.4 ppm can be attributed to the phenolic (OH) proton at position C3 of the phenyl moiety. The signals at δ 1.7 and 2.6 ppm values can be accounted for by the methyl protons of the dimethyl substituent of the 2-methylpropanoic acid function and the methyl protons of the acyl function, respectively. In the ¹³C NMR spectrum of acid derivative 2, the chemical shifts at δ 29.1 and 44.6 ppm can be attributed to the dimethyl substituent on C2, and the C2 of the 2-methylpropanoic acid function, respectively. The chemical shifts at δ 26.7 and 188.9 ppm can be attributed to the alpha methyl carbon and carbonyl carbon of the acyl function, respectively.

Scheme 1 The schematic representation for the synthesis of the designed compounds.

The acid derivative 2 was condensed with ethyl acetoacetate in the presence of sulphuric acid to attain the acyl derivative, 2-(3-acetyl-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (3). The ¹H NMR spectrum of the acyl derivative 3 accounts for sixteen protons, wherein the signals at δ 7.5–6.4 ppm were assigned to the aromatic protons of coumarin and a singlet at δ 2.3 ppm was attributed to the methyl protons of the acyl group (COCH₃) at C3 of the coumarin moiety. In the ¹³C NMR spectrum of acetyl derivative 3, the chemical shifts at δ 27.2 and 191.6 ppm can be attributed to the alpha methyl carbon and the carbonyl carbon of the acetyl substituent on the C3 position of the coumarin moiety, respectively. The mass spectral characterization exhibits a molecular ion peak at 305 m/z [M⁺¹] and an ion peak at 260 m/z, which can be attributed to the decarboxy fragment.

The intermediate, 2-(3-(2-bromoacetyl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (4) was prepared by bromination of the alpha methyl group of acyl derivative 3. The bromination was executed using bromine in dioxane. In the proton NMR spectrum of the bromoacyl derivative 4, a singlet at δ 3.6 ppm was assigned to the methylene proton of the bromoacyl (COCH₂Br) group. The chemical shifts at δ 32.6 and 196.4 ppm in the ¹³C NMR spectrum of bromoacyl derivative 4 were assigned to the methylene carbon and the carbonyl carbon of the bromoacyl substituent on the C3 position of the coumarin moiety, respectively.

The bromoacyl intermediate (4) was condensed with urea derivatives and 2-amino azoles as per the Hantzsch azole synthesis to undergo dehydrohalogenative cyclization in the presence of a catalytic amount of triethylamine and ferric chloride, affording the target molecules, 2-(3-substituted-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid derivatives 5a–5f. The IR spectrum of 2-(3-(2-aminooxazol-5-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5a) exhibited characteristic absorption peaks at 3445 and 1231 cm⁻¹, accounting for the amino group and C–O–C function in the oxazole moiety, respectively. In the proton NMR spectrum of compound 5a, a singlet at δ 5.4 ppm can be attributed to the amino (NH₂) proton at position C2 of the oxazole moiety. The absence of bromomethyl protons at δ 3.6 ppm and an isolated singlet at δ 7.9 ppm accounting for the CH proton at the C5 position of the oxazole moiety confirm the formation of the 2-amino oxazole moiety. The ¹³C NMR spectrum of compound 5a revealed three characteristic signals at δ 163.6, 123.7 and 156.2 ppm values, which can be attributed to the C2, C4 and C5 carbons of the oxazole moiety, respectively. The mass spectral characterization of compound 5a exhibits a molecular ion peak at 345 m/z [M⁺¹], which confirms product formation.

In the proton NMR spectrum of 2-(3-(H-imidazo[1,2-a]pyridin-2-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5d), a characteristic doublet of doublets at δ 7.2 ppm can be attributed to the protons at the C5, C6, C7 and C8 positions of the imidazo[1,2-a]pyridine moiety. Furthermore, in the ¹H NMR spectrum, an isolated singlet was observed at δ 6.9 ppm for the proton at the C3 position of the imidazo[1,2-a]pyridine moiety. The mass spectral characterization of compound 5d exhibits a molecular ion peak at 378 m/z [M⁺], a major ion peak at 333 m/z that can be assigned to the decarboxy derivative and another ion peak at 117 m/z, which can be attributed to the imidazo[1,2-a]pyridine fragment.

2.2. In silico studies

The Lipinski parameters of the molecules towards their biological efficacy were encouraging, with zero Lipinski violation. The predicted oral availability was considerable as the molecules exhibited percentage of absorption (% ABS) values ranging from 64.6 to 76.5%. The lipophilicity data suggested that the compounds were optimally lipophilic in nature, with values ranging from 1.87 to 4.68, indicating that the molecules do not breach the Lipinski requirement. The topological polar surface area (TPSA) of the designed compounds exhibited values less than 140 Å. A TPSA value greater than 140 Å indicates difficulties in the compound’s ability to permeate the cellular membrane. The molecular weight of each ligand was within the range of 344 to 434 D. The designed compounds showed positive values in the drug score calculation, with the values ranging from 0.38 to 0.79. The results indicate that the designed coumarin derivatives have potential as new drug candidates. Table 1 presents the calculated % ABS, TPSA and Lipinski parameters of the synthesized compounds.

Table 1 Predicted ADME, Lipinski parameters and molecular properties of the synthesized compounds

Sl. no.	Cpd	% ABS^a	TPSA^c	RTB^c	HBA^c	HBD^c	log P^c	MW	Violations^c	log S^b	Drug likeness^b	Drug score^b
a Data calculated as per eqn (1).b Molecular property as obtained from the OSIRIS property explorer software.c Molecular property as obtained from the Molinspiration online property calculation toolkit.
1	5a	64.6	128.8	4	8	3	1.17	344.32	0	−3.96	2.65	0.78
2	5b	69.1	115.7	4	7	3	2.54	360.39	0	−4.42	−5.02	0.38
3	5c	63.6	131.5	4	8	4	1.11	343.34	0	−4.88	−8.22	0.34
4	5d	76.5	94.1	4	7	1	2.85	378.38	0	−4.03	−0.13	0.59
5	5e	76.5	94.1	4	7	1	3.25	384.41	0	−4.03	1.11	0.71
6	5f	76.5	94.1	4	7	1	4.36	434.47	0	−3.66	2.15	0.79

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2.3. Docking studies

The docking study was performed using SYBYL-X 2.1. The designed coumarin derivatives were docked into the active site of the selected enzymes to explore, predict and understand the protein/enzyme interactions. The docking results were validated by aligning the designed ligands with the co-crystallized reference standards for the respective enzymes.

2.3.1. Molecular docking studies on EGFR (2J5F). The docking study onto the active site of EGFR (PDB: 2J5F) of the designed coumarins was encouraging. The crystal structure of the enzyme with its co-crystallized ligand N-[4-(3-bromophenylamino)quinazolin-6-yl]acrylamide (34-JAB) was downloaded from the RCSB protein data bank. The binding site of 2J5F, a typical cylindrical core between amino acid residues Lys745, Glu762, Met766, Leu788, Met793 and Thr854, concentrated the hydrophobic interaction. The reference ligand 34-JAB exhibited a docking score of 6.21 kcal M⁻¹. 34-JAB exhibited main hydrophobic interactions with the surrounding residues Thr790, Met793 and Cys797, strongly contributing to its stabilization. The hydrogen bond interactions were visualized between nitrogen N1 of the quinazoline moiety and the amino acid residue Met793 at a distance of 1.987 Å. The carbonyl oxygen of the amide group exhibited a hydrogen bonding interaction with the amino acid residue Thr854 at a distance of 1.828 Å. The docking study revealed that the coumarin derivatives 5a–5f possessed high affinity towards 2J5F, and the results were promising. These coumarin derivatives were well aligned in the hydrophobic core of the enzyme 2J5F (Fig. 1) and some of the coumarin derivatives exhibited a higher docking score in comparison to the reference ligand 34-JAB.

Fig. 1 Energy-minimized and conformationally analyzed structures aligned against common atoms interacting with 2J5F, with reference molecule 34-JAB.

Site-directed mutagenesis studies have indicated a few crucial amino acid residues of EGFR, namely Leu745, Thr790, Cys797, and Met793. These amino acids are essential for binding to EGFR and have a significant part in ligand binding.¹⁸ A few of our designed coumarin derivatives exhibit interactions with the aforementioned amino acid residues, which may provide a possible lead to active molecules (Fig. 2). Among the series, compounds 5e and 5a generated good docking scores of 7.24 and 7.14 kcal M⁻¹, respectively. In compound 5a, the amino group at position C2 of the oxazole moiety was involved in a hydrogen bond interaction with Pro794 at a distance of 2.462 Å. The carbonyl oxygen at position C2 of the coumarin moiety exhibited a hydrogen bond interaction with Met793 at a distance of 1.789 Å. The hydrogen and the hydroxyl oxygen of the carboxylic acid group of the propanoic acid fragment at position C7 of the coumarin moiety exhibited hydrogen bond interactions with the amino acid residues Glu762 and Lys748 at distances of 2.039 and 1.891 Å, respectively. Similarly, the carbonyl group of the propanoic acid fragment showed hydrogen bond interactions with Thr854 and Asp855 amino acid residues at distances of 2.089 and 2.519 Å, respectively. Compounds 5c, 5f, and 5d exhibited good docking scores of 7.08, 7.02, and 6.97 kcal M⁻¹, respectively, higher docking scores than that of the reference ligand 34-JAB. The docking studies indicated that the carboxylic acid function of the propanoic acid fragment at position C7 of the coumarin moiety of all the derivatives was involved in hydrogen bond interactions with the amino acid residues Thr854, Asp855, Lys745 and Glu762. The carbonyl oxygen at C2 of the coumarin moiety was involved in a hydrogen bond interaction with the amino acid residue Met793. However, compound 5b exhibited a docking score of 5.58 kcal M⁻¹, lower than that of the reference ligand 34-JAB.

Fig. 2 Interaction of the designed coumarins with EGFR.

All the coumarin derivatives exhibited a crash score of ≤4.4 kcal mol⁻¹. A higher crash score indicates inappropriate penetration of the ligand into the binding site of the protein, resulting in a decreased force of interaction with the amino acids. The molecular docking study indicates that all the docked coumarin derivatives have good interactions with the enzyme and have potential scope to inhibit it.

2.3.2. Molecular docking studies on the COX-2 enzyme (3LN1). The docking study onto the active site of COX-2 (PDB: 3LN1) of the designed coumarins gave significant results. In the COX-2 enzyme, 3LN1, a typical cylindrical core of 23 Å between Ser516, Ser339 and Asp510 concentrates the hydrophobic interaction. The ligand, celecoxib, exhibited a docking score of 9.69 kcal M⁻¹, with the main hydrophobic interactions being with the surrounding residues Ser339, Gly512, Leu338 and Val509, strongly contributing to the stabilization and to its inhibitory effect. The Tyr371, Trp373, and Val335 residues were involved in van der Waals’ interactions with the haloalkyl chain present in celecoxib. The hydrogen bonding interactions of the amino group protons of celecoxib were with residues Leu338 and Gln178 at distances of 2.359 and 2.213 Å, respectively. The sulfonyl oxygen of celecoxib was involved in hydrogen bonding interactions with Arg499 and Phe504 residues at distances of 2.322 and 2.751 Å, respectively.

The designed compounds, along with the reference ligand, were docked onto the target protein COX-2, 3LN1. The docking studies revealed that the designed coumarin derivatives exhibited a considerable affinity towards 3LN1. The compounds 5a–5f exhibited promising docking scores, ranging from 7.79 to 10.43 kcal M⁻¹. Compound 5c exhibited the highest docking score among the designed compounds, with a value of 10.43 kcal M⁻¹, and compound 5d had the lowest docking score (7.79 kcal M⁻¹).

The docking studies on the COX-2 protein were carried out with our designed compounds along with 34-JAB (reference standard ligand for EGFR, 2J5F) to understand the comparative interaction of this with those of our designed compounds towards anticancer applications. Compounds 5c and 5f exhibited docking scores of 10.43 and 9.71 kcal M⁻¹, respectively, higher than that of 34-JAB, indicating a better interaction to inhibit the COX-2 protein. Compound 5c was well aligned in the hydrophobic core of the 3LN1 pocket in the B-chain of the protein, which is essential for the inhibition. In compound 5c, the carbonyl oxygen of the carboxylic acid function showed hydrogen bonding with the Tyr371 residue at a distance of 2.722 Å. The 1H of the imidazole exhibited hydrogen bonding interactions with the Leu338 and Ser339 residues at distances of 2.196 and 2.402 Å, respectively. The carbonyl oxygen of the coumarin moiety at the C2 position was involved with a hydrogen bonding interaction with Phe504 at a distance of 2.558 Å (Fig. 3).

Fig. 3 Energy minimized and conformationally analyzed structures aligned against common atoms interacting with 3LN1, with reference molecule celecoxib.

The hydrogen bond interaction between the carbonyl oxygen at position C2 of the coumarin moiety with the Phe504 residue and also the hydrogen bonding interaction between the nitrogen of the azole moiety and the Arg106 residue were the major and common interactions exhibited by the designed coumarin derivatives. Table 2 presents the molecular docking results of designed compounds 5a–5f along with their crash scores and polar scores.

Table 2 Docking scores of coumarin derivatives 5a–5f

Sl. no.	Compound	Docking score towards 2J5F^a			Docking score towards 3LN1^a
		Total score	Crash score	Polar score	Total score	Crash score	Polar score
a Data expressed in kcal M^−1 as obtained from the docking utility of SYBYL 2.1 software.
1	5a	7.14	−1.33	3.06	9.19	−2.89	2.04
2	5b	5.58	−1.34	4.97	8.10	−3.67	2.32
3	5c	7.08	−0.85	4.17	10.43	−2.75	3.28
4	5d	6.97	−1.24	3.10	7.79	−4.42	2.62
5	5e	7.24	−1.37	3.63	7.81	−3.18	1.09
6	5f	7.02	−1.47	2.99	9.71	−4.19	1.31
7	34-JAB	6.21	−0.67	2.05	9.32	−4.50	0.74
8	Celecoxib	—			10.97	−0.70	2.43

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The establishment of a marked relationship between tissue prostaglandin levels in human breast tumor formation and the development of metastases and survival has been well reported.^19,20 Significant inhibition of EGFR and COX-2 by our designed coumarins indicates their potential role in acting by a dual mechanism to suppress cancer cell proliferation.

2.3.3. Docking validation. The reference standards employed were drawn and subjected to the docking protocol along with the designed series of coumarins, in order to visualize the binding affinity and strength, and to ascertain the docking process and validate it. The docking pose was compared with the co-crystallized binding pose of 34-JAB and celecoxib for 2J5F and 3LN1 protein targets, respectively (Fig. 4). The validation of the docking process is indicated by the root mean square deviation (RMSD) value and the poses for the co-crystallized and docked ligands gave values of 0.86 and 0.64 Å for 34-JAB and celecoxib, respectively. The RSMD values are affirmative that the overall docking study is corroborated, as the tolerance limit for the RMSD value is 1.5 Å. Hence, the docking results are acceptable and indicative that the docking protocol is validated.

Fig. 4 The validation of the docking protocol for the target protein. (A) Binding pose of 34-JAB onto the co-crystallized ligand 34-JAB (red) in EGFR. (B) Binding pose of celecoxib onto the co-crystallized ligand celecoxib (green) in 3LN1.

2.4. In vitro antioxidant evaluation

The newly synthesized coumarin derivatives were evaluated for their potency as antioxidants by assessing their ability to scavenge radicals. The in vitro antioxidant screening was carried out by three different methods, i.e. DPPH, SA, and ABTS radical scavenging assay methods. Ascorbic acid was employed as a reference standard. The test compounds and the standard ascorbic acid were tested at three different test concentrations of 12.5, 25 and 50 μM. The antioxidant data obtained from all the tested methods were encouraging.

The results obtained from the DPPH radical assay method indicated that the compounds exhibited significant scavenging activity ranging from 17.8 to 87.5%, in comparison to ascorbic acid (91.5% at 50 μM). The IC₅₀ values of the compounds were substantially low, ranging from 19.8 to 34.6 μM. Compound 5b exhibited higher radical scavenging potential (87.5% at 50 μM) in comparison to the other derivatives with an IC₅₀ value of 19.8 μM. The compound exhibited a substantial increase in the antioxidant efficacy with an increase in the test concentration. The statistical analysis of the antioxidant activity of compounds 5a–5f by the repeated measures Dunnett’s ANOVA method highlighted that the compounds exhibited a significance value of P < 0.001, indicating a confidence interval of 99.9% with respect to the reference standard ascorbic acid. The results of the antioxidant activity evaluated by the DPPH method highlighted that the derivatives with amino group substitution at position C2 of compounds 5a–5c exhibited significant activity (Fig. 5A).

Fig. 5 Mean increase in percentage antioxidant activity of the test compounds 5a–5f with increase in test concentrations (12.5, 25.0 and 50.0 μM): (A) DPPH method, (B) ABTS method, (C) SA method.

The results obtained from the ABTS radical assay method indicated that the compounds exhibited considerable radical scavenging properties, with values ranging from 3.1 to 81.4% in comparison to the 87.3% antioxidant activity obtained for ascorbic acid. The results highlighted that compound 5b exhibited higher radical scavenging potential (81.3% at 50 μM) with an IC₅₀ value of 30.9 μM (Fig. 5B). The statistical analysis of the antioxidant activity of compounds 5a–5f by the repeated measures Dunnett’s ANOVA method highlighted that the compounds were not significant with respect to the standard ascorbic acid.

The radical scavenging assay results from the SA method were not as similar as the DPPH and ABTS results. The results obtained from the SA radical assay method indicated that the compounds exhibited significant scavenging activity ranging from 9.7 to 83.5% in comparison to the 88.7% antioxidant activity obtained for ascorbic acid (Fig. 5C). Although the tested compounds exhibited significant antioxidant efficacy, compound 5c exhibited a better radical scavenging property (83.5% at 50 μM) than that of 5b (75.2% at 50 μM), in contrast to the DPPH and ABTS results.

Nevertheless, the antioxidant activity data from all three tested methods highlights that the derivatives with amino group substitution at position C2 of the azole series (5a–5c) exhibited significant activity. The antioxidant activities of the test compounds 5a–5f at the three different test concentrations by the DPPH, ABTS, and SA radical scavenging assay methods, expressed as the mean ± SEM, along with the IC₅₀ values obtained by regression analysis, are presented in Table 3.

Table 3 The mean radical scavenging efficacies of compounds 5a–5f at three different test strengths, along with their IC₅₀ values

Comp.	DPPH method				ABTS method				SA method
	Mean percentage free radical scavenging activity ± SEM^a			Mean IC50 value ± SEM^b	Mean percentage free radical scavenging activity ± SEM^a			Mean IC50 value ± SEM^c	Mean percentage free radical scavenging activity ± SEM^a^,^c			Mean IC50 value ± SEM^b
	12.5 μM	25.0 μM	50.0 μM		12.5 μM	25.0 μM	50.0 μM		12.5 μM	25.0 μM	50.0 μM
a Results are expressed as the mean values from three independent experiments ± SEM.b Data obtained by DPPH method was analyzed by Dunnet’s test compared with reference drug ascorbic acid. n = 3; (†) equals P < 0.1.c Data obtained by SA method was analyzed by Dunnet’s test compared with reference drug ascorbic acid. n = 3; (**) equals P < 0.01, (*) equals P < 0.05.
5a	20.87 ± 0.64	55.11 ± 0.19	79.66 ± 0.18	27.90 ± 0.22	8.44 ± 1.01	32.94 ± 1.12	69.62 ± 0.69	36.73 ± 0.31	23.33 ± 0.43	40.46 ± 0.43	73.03 ± 0.41	32.49 ± 0.10*
5b	37.19 ± 0.08	61.91 ± 0.15	87.54 ± 0.04	19.75 ± 0.08^†	13.65 ± 1.08	43.10 ± 0.92	81.38 ± 0.30	30.87 ± 0.24	26.21 ± 0.50	55.19 ± 0.09	75.15 ± 0.50	27.40 ± 0.17**
5c	19.76 ± 0.22	66.22 ± 0.04	81.26 ± 0.22	25.31 ± 0.10	6.36 ± 1.31	49.54 ± 0.56	72.01 ± 0.49	31.94 ± 0.21	16.69 ± 0.19	49.48 ± 0.34	83.47 ± 0.09	29.23 ± 0.07*
5d	21.05 ± 0.33	47.30 ± 0.44	74.05 ± 0.04	31.02 ± 0.12	3.06 ± 0.89	23.03 ± 0.66	62.09 ± 0.46	42.27 ± 0.14	13.92 ± 0.74	31.65 ± 0.99	66.34 ± 0.09	38.26 ± 0.13*
5e	17.82 ± 0.34	43.61 ± 0.26	67.88 ± 0.51	34.55 ± 0.28	4.09 ± 1.70	17.63 ± 0.29	53.09 ± 1.07	49.34 ± 0.48	9.73 ± 0.75	26.86 ± 0.57	58.35 ± 1.15	43.39 ± 0.42*
5f	25.95 ± 0.18	55.25 ± 0.49	72.79 ± 0.33	28.03 ± 0.06	8.75 ± 1.08	34.64 ± 0.96	60.25 ± 1.15	41.06 ± 0.30	14.85 ± 0.59	41.96 ± 1.09	64.71 ± 0.74	36.65 ± 0.06*
Ascorbic acid	37.31 ± 0.15	64.25 ± 0.11	91.47 ± 0.18	18.87 ± 0.09	16.90 ± 0.82	46.60 ± 0.70	87.26 ± 0.54	30.73 ± 0.22	16.86 ± 0.34	52.58 ± 0.25	88.69 ± 0.41	27.70 ± 0.10

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2.5. In vitro cytotoxicity study

The in vitro cytotoxicity evaluation was carried out against three human cancer cell lines, namely HT-29 (colon adenocarcinoma), MDA-231 and MCF-7 (breast cancer). 5-Fluorouracil and doxorubicin were employed as the reference standards and dimethyl sulfoxide (DMSO) was used as a solvent control. The results of the cytotoxicity studies highlight that the tested compounds were considerably toxic against the cancerous cell lines. Primarily, the compounds were tested on breast cancer cell line, MDA-231, and on colon adenocarcinoma cell line, HT-29. The cytotoxicity results highlighted that the compounds exhibited higher cytotoxicity towards the breast cancer cell line, MDA-231, than towards colon adenocarcinoma, HT-29. This encouraged us to test the compounds on an additional breast cancer cell line, MCF-7. The cytotoxicity results with MCF-7 were also significant. The cytotoxicity results highlight that the compounds with 2-amino substitution (5a–5c) were more cytotoxic than the other tested compounds (5d–5f), which had lower IC₅₀ values.

The cytotoxicity results towards the HT-29 cancer cell line were moderate. Compounds 5a–5c exhibited substantial cytotoxic activity with low IC₅₀ values compared with 5-fluorouracil and doxorubicin (Fig. 6A). Compounds 5a and 5b exhibited IC₅₀ values of 29.6 and 30.8 μM respectively.

Fig. 6 Mean IC₅₀ values exhibited by the test compounds 5a–5f for cytotoxicity by the MTT assay method: (A) HT-29, (B) MDA-231, (C) MCF-7.

The results for the cytotoxicity on breast cancer cell lines MDA-231 and MCF-7 were encouraging. Compounds 5a and 5b showed considerable cytotoxic activity with lower IC₅₀ values (Fig. 6B and C). Compound 5b can be considered to be a potent cytotoxic agent among the tested compounds, with IC₅₀ values of 4.84 and 2.39 μM for MDA-231 and MCF-7, respectively. Compound 5a exhibited IC₅₀ values of 7.3 and 5.1 μM for MDA-231 and MCF-7 respectively, while compound 5c, which exhibited moderate cytotoxicity for HT-29, exhibited minimal cytotoxic action on breast cancer cell lines MDA-231 and MCF-7.

Several anticancer agents possess a serious problem of safety and effectiveness with the foremost drawback being non-selectivity. These drugs are not able to distinguish between the normal healthy cells and fast growing cancerous cells. From this aspect, it is crucial to measure the cytotoxicity towards normal cell in the process of discovery of newer antitumor drugs. As a further evaluation of the selective cytotoxicity of the lead compounds, 5a and 5b were chosen for the selectivity test over normal human cell lines, using Vero (epithelial cells) and HepG2 (hepatocytes) to ascertain their safety and efficacy towards normal cells with the MTT assay. The results indicate that compounds 5a and 5b had lower cytotoxicity towards normal cell lines. The IC₅₀ values of all the tested compounds are given in Table 4.

Table 4 The mean cytotoxicity IC₅₀ values of the compounds against five different cell lines

Compound	IC50 (μM)
	HT-29^a^,^b^,^c	MDA-231^a^,^b^,^c	MCF-7^a^,^b^,^c	Vero cell line^a	HepG2^a
a Results are expressed as the mean values from three parallel experiments ± SEM.b Data was analyzed by Dunnet’s test. n = 3; (***) equals P < 0.001, (**) equals P < 0.01, (*) equals P < 0.05, with respect to reference standard 5-fluorouracil.c Data was analyzed by Dunnet’s test. n = 3; (†††) equals P < 0.001, (††) equals P < 0.01, (†) equals P < 0.05, with respect to reference standard doxorubicin. NC equals not conducted.
5a	29.57 ± 0.72**^††	7.26 ± 0.24	5.12 ± 0.09	38.65 ± 1.34	43.13 ± 2.56
5b	30.81 ± 0.15**^†††	4.84 ± 0.17	2.39 ± 0.03	57.89 ± 3.72	63.37 ± 2.84
5c	28.46 ± 1.37***^†††	56.94 ± 1.47***^†††	44.87 ± 1.91***^†††	NC	NC
5d	173.05 ± 7.16***^†††	128.19 ± 8.04***^†††	152.35 ± 8.56***^†††	NC	NC
5e	>200	181.09 ± 4.45***^†††	195.50 ± 4.62***^†††	NC	NC
5f	>200	177.15 ± 6.16***^†††	164.31 ± 9.20***^†††	NC	NC
5-Fluorouracil	2.16 ± 0.07	1.96 ± 0.087	1.86 ± 0.06	—	—
Doxorubicin	3.73 ± 0.14	3.76 ± 0.18	3.24 ± 0.15	—	—

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Our results indicated that compounds 5a and 5b induced significantly high levels of cytotoxicity as compared with the reference standards 5-fluorouracil and doxorubicin. They differ by the azole moiety; compound 5a is an oxazole derivative and 5b a thiazole derivative. The results also highlighted that the compounds with significant antioxidant potential have emerged as having considerable cytotoxic properties; this probably indicates a greater advantage with respect to the clinical aspects.

The possible differences in the cytotoxic efficacy of compounds 5a and 5b may be attributed to their partition coefficient (log10 P) values. The efficiency of a compound or drug in producing a cytotoxicity effect also depends on its accumulation in the cell. Hence, a more lipophilic nature leads to significant accumulation of the compound in the cell, resulting in considerable cytotoxicity and cell death. Compounds 5a and 5b showed clog P values of 1.17 and 2.54, respectively. The higher log P value of compound 5b indicates that it is more lipophilic and this may be co-related to its cytotoxic potential.

3. Conclusion

The in silico and molecular docking studies were encouraging. The results of antioxidant activity testing were promising as the compounds exhibited substantial antioxidant activity. The cytotoxic potential of the coumarin derivatives on human cancerous cell lines was considerable. The compounds exhibited significantly low IC₅₀ values with respect to the reference standards employed. Safety of compounds 5a and 5b on a normal cell line and selectivity towards cancerous cell lines were shown. The significant antioxidant property of compound 5b in conjunction with encouraging cytotoxicity results, especially on the human breast cancer cell lines MDA-231 and MCF-7, have shed light on these new compounds to certainly hold great promise as good active leads. Studies on the enzyme assay of the best active compounds are in progress and will be reported in the future.

4. Experimental section

4.1. Chemistry

The synthetic routes for the preparation of the target molecules were planned through a retrosynthetic approach. The most facile synthetic routes were selected based on the percentage yields of the molecules of each reaction step. The percentage yields of some reaction steps were improved by careful selection of starting materials and catalyst system. While designing the synthetic routes, priority was given to high yielding reproducible reaction steps rather than concentrating on novel synthetic routes and complex chemistry. All the reaction steps, even if reported in published literature, were selected based on our in-house laboratory facilities. Handling and disposal of all reagents and catalysts to be used in the reaction steps were studied thoroughly from the corresponding Material Safety Data Sheets (MSDS).

All the chemicals and solvents used in this study were purchased from Sigma-Aldrich Chemical Co., Spectrochem Ltd. and Sd fine chemicals of LR grade; the solvents were distilled prior to use. All commercially available reagents procured were used without further purification. TLC was used to assess the progress/completion of reactions and the purity of the synthesized compounds using aluminum backed sheets of silica gel 60 GF254 (Merck), with ethyl acetate and hexane (4 : 1) as a solvent system and iodine vapor as the visualizing agent. Melting points were determined in open glass capillaries and are uncorrected. The IR spectra were recorded using a Shimadzu FTIR-8400 instrument by the KBr disc pellet technique/liquid sampling using NaCl cells and only noteworthy absorption levels (cm⁻¹) are listed. ¹H NMR and ¹³C NMR spectra were recorded using a Bruker AC-400 MHz FT NMR spectrophotometer at 400 MHz for ¹H (100 MHz for ¹³C) with deuterated dimethyl sulfoxide (DMSO-d₆) as solvent and tetra methyl silane (TMS) as the internal standard (chemical shifts in δ, ppm). The splitting patterns were designated as follows: s: singlet; d: doublet; q: quartet; m: multiplet. The LCMS was recorded using a Shimadzu LCMS-2010A instrument by ESI. The elementary analysis was recorded using a Thermo Finnigan FLASH EA 1112 CHNS analyzer and all the compounds gave satisfactory elemental analysis. For in vitro cytotoxicity studies the cell lines were procured from the National Centre for Cell Sciences, Pune, India.

4.2. Purity analysis

All the synthesized compounds 5a–5f, were purified by silica gel column chromatography (dichloromethane : methanol, 60 : 40), followed by determination of purity of the synthesized compounds by ultra-fast liquid chromatography (UFLC, Shimadzu LC-20 A Series), using a PDA detector and XTerra C18 column (150 × 4.6 mm, 5 μm particle size). The mobile phase was composed of di-sodium hydrogen phosphate buffer (pH 3.6) and acetonitrile in the ratio 60 : 40, v/v, with a flow rate of 1.0 mL min⁻¹. 20 μL of the sample was employed as the injection volume and the detection wavelength was 254 nm. All the synthesized compounds had a purity of more than or equal to 95%.

4.2.1. Synthesis of 2-(3-acetyl-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (3). A mixture of the acid derivative 2 (23.8 g, 0.10 mol) and ethyl acetoacetate (13.0 mL, 0.10 mol) was maintained at a temperature of less than 10 °C. To this cold mixture, cold concentrated sulfuric acid (30 mL) was added in portions with constant stirring. After the completion of sulfuric acid addition, the mixture was allowed to stand overnight. The reaction mixture was poured into cold water to precipitate the acetyl derivative 3. The product was recrystallized using rectified spirit as a solvent. Yield: 87%; mp 115–117 °C; R_f: 0.72 (ethyl acetate and hexane; 4 : 1); IR: 3446 (OH), 2965 (C=C), 1754, 1718 (C=O), 1234 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 10.6 (s, 1H, OH; carboxylic acid), 7.5–6.4 (m, 3H, Ar-H), 2.3 (s, 3H, COCH₃), 2.1 (s, 6H, (CH₃)₂C), 1.6 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 191.6 (1C, COCH₃), 173.9 (1C, COOH), 167.5 (1C, CO, C2-coumarin), 157.4 (1C, C9-coumarin), 152.1 (1C, C10-coumarin), 147.9 (1C, C7-coumarin), 142.7 (1C, C3-coumarin), 128.7 (1C, C4-coumarin), 126.9 (1C, C8-coumarin), 116.2 (1C, C6-coumarin), 113.9 (1C, C5-coumarin), 46.2 (1C, (CH₃)₂C), 29.4 (2C, (CH₃)₂C), 27.2 (1C, COCH₃), 18.2 (1C, CH₃); MS: m/z (%) 305 (65) [M⁺¹], 260 (100) [C₁₆H₁₆O₄]; anal. calc. for C₁₆H₁₆O₆: C 63.15, H 5.30. Found: C 63.43, H 5.52.

4.2.2. Synthesis of 2-(3-(2-bromoacetyl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (4). A mixture of acetyl derivative 3 (15.2 g, 0.05 mol) in dioxane (30.0 mL) was cooled to a temperature of less than 10 °C. Liquid bromine (12.0 mL, 0.075 mol) diluted in 30.0 mL of dioxane was added dropwise to the above mixture while maintaining the temperature below 10 °C. The progress of the reaction was monitored by TLC. On completion of the reaction, the reaction mixture was allowed to reach room temperature. The reaction mixture was poured into cold water to obtain the brominated derivative 4. The slurry was treated with sodium metabisulphite to remove the excess bromine. The product was recrystallized using rectified spirit as solvent. Yield: 92%; mp 196–197 °C; R_f: 0.64 (ethyl acetate and hexane; 4 : 1); IR: 3483 (OH), 2931 (C=C), 1761, 1724 (C=O), 1215 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 10.4 (s, 1H, OH; carboxylic acid), 7.6–6.9 (m, 3H, Ar-H), 3.6 (s, 2H; CH₂Br), 2.0 (s, 6H, (CH₃)₂C), 1.6 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 196.4 (1C, COCH₂Br), 176.1 (1C, COOH), 169.2 (1C, CO; C2-coumarin), 162.4 (1C, C9-coumarin), 158.7 (1C, C10-coumarin), 156.2 (1C, C7-coumarin), 145.2 (1C, C3-coumarin), 129.2 (1C, C4-coumarin), 126.2 (1C, C8-coumarin), 117.9 (1C, C6-coumarin), 115.2 (1C, C5-coumarin), 46.6 (1C, (CH₃)₂C), 32.6 (1C, COCH₂Br), 29.6 (2C, (CH₃)₂C), 18.9 (1C, CH₃); MS: m/z (%) 382 (100) [M⁺]; anal. calc. for C₁₆H₁₅BrO₆: C 50.15, H 3.95. Found: C 50.35, H 4.21.

4.2.3. General procedure for the synthesis of title compounds, 2-(3-substituted-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid derivatives (5a–5f). To a mixture of bromoacyl derivative 4 (3.8 g, 0.01 mol) and urea derivatives/2-amino azole derivatives (0.02 mol) dissolved in methanol (30.0 mL), a catalytic amount of triethylamine (2.0 mL) and ferric chloride (1.0 g) were added. The mixture was heated to reflux for 4–6 h. The progress of the reaction was monitored by TLC. On completion of the reaction, the reaction mixture was concentrated and was allowed to reach room temperature. The reaction mixture was poured into cold water to obtain the title compound (5a–5f).

4.2.4. 2-(3-(2-Aminooxazol-4-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5a). Yield: 81%; mp 195–197 °C; R_f: 0.58 (ethyl acetate and hexane; 4 : 1); IR: 3445 (N–H), 3413 (OH), 3194 (C–N), 1741, 1727 (C=O), 1662 (C=N), 1231 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 10.2 (s, 1H, OH; carboxylic acid), 7.9 (s, 1H, CH; C5-oxazole), 7.6–7.2 (m, 3H, Ar-H), 5.4 (s, 2H, NH₂), 1.9 (s, 6H; (CH₃)₂C), 1.7 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 177.2 (1C, COOH), 168.5 (1C, CO, C2-coumarin), 163.6 (1C, C2-oxazole), 161.5 (1C, C9-coumarin), 159.9 (1C, C10-coumarin), 156.2 (1C, C5-oxazole), 148.5 (1C, C7-coumarin), 142.7 (1C, C3-coumarin), 141.3 (1C, C4-coumarin), 126.3 (1C, C8-coumarin), 123.7 (1C, C4-oxazole), 117.6 (1C, C6-coumarin), 113.1 (1C, C5-coumarin), 46.8 (1C, (CH₃)₂C), 29.7 (2C, (CH₃)₂C), 18.3 (1C, CH₃); MS: m/z (%) 345 (100) [M⁺¹]; anal. calc. for C₁₇H₁₆N₂O₆: C 59.30, H 4.68, N 8.14. Found: C 59.42, H 4.71, N 8.33.

4.2.5. 2-(3-(2-Aminothiazol-4-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5b). Yield: 86%; mp 235–236 °C; R_f: 0.61 (ethyl acetate and hexane; 4 : 1); IR: 3426 (N–H), 3417 (OH), 3174 (C–N), 1728, 1711 (C=O), 1643 (C=N), 1265 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 10.4 (s, 1H, OH), 7.3–6.9 (m, 3H, Ar-H), 6.3 (s, 1H, CH; C5-thiazole), 5.6 (s, 2H, NH₂), 1.8 (s, 6H; (CH₃)₂C), 1.4 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 178.2 (1C, COOH), 167.6 (1C, CO, C2-coumarin), 164.6 (1C, C2-thiazole), 158.1 (1C, C9-coumarin), 153.5 (1C, C10-coumarin), 148.4 (1C, C5-thiazole), 146.1 (1C, C7-coumarin), 141.8 (1C, C3-coumarin), 138.4 (1C, C4-coumarin), 129.2 (1C, C8-coumarin), 127.3 (1C, C4-thiazole), 118.3 (1C, C6-coumarin), 115.4 (1C, C5-coumarin), 48.2 (1C, (CH₃)₂C), 29.4 (2C, (CH₃)₂C), 18.7 (1C, CH₃); MS: m/z (%) 360 (100) [M⁺]; anal. calc. for C₁₇H₁₆N₂O₅S: C 56.66, H 4.47, N 7.77, S 8.90. Found C 56.41, H 4.34, N 7.45, S 8.76.

4.2.6. 2-(3-(2-Amino-1H-imidazol-4-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5c). Yield: 72%; mp 211–213 °C; R_f: 0.60 (ethyl acetate and hexane; 4 : 1); IR: 3431, 3426 (N–H), 3385 (OH), 3197, 3183 (C–N), 1731, 1724 (C=O), 1637 (C=N), 1246 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 11.9 (s, 1H, NH; imidazole), 10.7 (s, 1H, OH; carboxyl acid), 7.4–7.1 (m, 3H, Ar-H), 6.7 (s, 1H, CH, C5-imidazole), 6.4 (s, 2H, NH₂), 1.7 ppm (s, 6H, (CH₃)₂C), 1.4 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 176.9 (1C, COOH), 166.3 (1C, CO, C2-coumarin), 164.3 (1C, C2-imidazole), 159.1 (1C, C9-coumarin), 157.6 (1C, C10-coumarin), 151.1 (1C, C5-imidazole), 148.9 (1C, C7-coumarin), 143.6 (1C, C3-coumarin), 139.6 (1C, C4-coumarin), 134.9 (1C, C8-coumarin), 126.7 (1C, C4-imidazole), 122.3 (1C, C6-coumarin), 117.6 (1C, C5-coumarin), 47.9 (1C, (CH₃)₂C), 29.1 (2C, (CH₃)₂C), 18.6 (1C, CH₃); MS: m/z (%) 344 (100) [M⁺¹]; anal. calc. for C₁₇H₁₇N₃O₅: C 59.47, H 4.99, N 12.24. Found C 59.31, H 4.84, N 12.45.

4.2.7. 2-(3-(H-Imidazo[1,2-a]pyridin-2-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5d). Yield: 84%; mp 264–266 °C; R_f: 0.76 (ethyl acetate and hexane; 4 : 1); IR: 3451, 3433 (N–H), 3376 (OH), 3178, 3164 (C–N), 1743, 1724 (C=O), 1671 (C=N), 1253 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 10.6 (s, 1H, OH; carboxylic acid), 8.1–7.8 (m, 3H, Ar-H), 7.2 (dd, 4H, J = 9.2 Hz, ²J = 4.1 Hz, C5–C8; imidazo[1,2-α]pyridine), 6.9 (s, 1H, CH; C3-imidazo[1,2-α]pyridine), 1.9 (s, 6H, (CH₃)₂C), 1.6 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 178.4 (1C, COOH), 171.4 (1C, CO, C2-coumarin), 170.7 (1C, C9-imidazo[1,2-α]pyridine), 168.2 (1C, C2-imidazo[1,2-α]pyridine), 164.3 (1C, C9-coumarin), 162.6 (1C, C3-imidazo[1,2-α]pyridine), 159.2 (1C, C5-imidazo[1,2-α]pyridine), 154.2 (1C, C10-coumarin), 151.3 (1C, C7-coumarin), 147.4 (1C, C8-imidazo[1,2-α]pyridine), 143.1 (1C, C3-coumarin), 138.7 (1C, C4-coumarin), 134.5 (1C, C8-coumarin), 129.7 (1C, C7-imidazo[1,2-α]pyridine), 125.6 (1C, C6-imidazo[1,2-α]pyridine), 121.8 (1C, C6-coumarin), 118.5 (1C, C5-coumarin), 48.2 (1C, (CH₃)₂C), 28.8 (2C, (CH₃)₂C), 18.6 (1C, CH₃); MS: m/z (%) 378 (100) [M⁺]; anal. calc. for C₂₁H₁₈N₂O₅: C 66.66, H 4.79, N 7.40. Found C 66.47, H 4.88, N 7.54.

4.2.8. 2-(3-(Imidazo[2,1-b]thiazol-6-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5e). Yield: 56%; mp 251–252 °C; R_f: 0.68 (ethyl acetate and hexane; 4 : 1); IR: 3447, 3436 (N–H), 3413 (OH), 3165, 3143 (C–N), 1762, 1741 (C=O), 1605 (C=N), 1236 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 10.6 (s, 1H, OH; carboxylic acid), 8.2–7.9 (d, J = 8.7, 2H, C2,3-imidazo[2,1-b]thiazole), 7.8 (s, 1H, CH; C5-imidazo[2,1-b]thiazole), 7.4–7.1 (m, 3H, Ar-H), 1.9 (s, 6H; (CH₃)₂C), 1.6 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 179.2 (1C, COOH), 176.7 (1C, C6-imidazo[2,1-b]thiazole), 169.4 (1C, CO, C2-coumarin), 166.2 (1C, C8-imidazo[2,1-b]thiazole), 164.7 (1C, C5-imidazo[2,1-b]thiazole), 161.8 (1C, C9-coumarin), 159.3 (1C, C2-imidazo[2,1-b]thiazole), 157.1 (1C, C3-imidazo[2,1-b]thiazole), 153.6 (1C, C10-coumarin), 147.2 (1C, C7-coumarin), 143.1 (1C, C3-coumarin), 138.7 (1C, C4-coumarin), 134.5 (1C, C8-coumarin), 118.5 (1C, C6-coumarin), 116.7 (1C, C5-coumarin), 48.7 (1C, (CH₃)₂C), 29.5 (2C, (CH₃)₂C), 18.9 (1C, CH₃); MS: m/z (%) 384 (100) [M⁺]; anal. calc. for C₁₉H₁₆N₂O₅S: C 59.37, H 4.20, N 7.29, S 8.34. Found C 59.62, H 4.18, N 7.61, S 8.51.

4.2.9. 2-(3-(Imidazo[2,1-b]benzothiazol-2-yl)-4-methyl-2-oxo-2H-chromen-7-yloxy)-2-methylpropanoic acid (5f). Yield: 32%; mp 293–295 °C; R_f: 0.74 (ethyl acetate and hexane; 4 : 1); IR: 3436, 3422 (N–H), 3416 (OH), 3153, 3124 (C–N), 1736, 1746 (C=O), 1617 (C=N), 1229 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 10.2 (s, 1H, OH; carboxylic acid), 8.1–7.3 (m, 7H, Ar-H), 7.2 (s, 1H, CH; C3-imidazo[2,1-b]benzothiazol-2-yl), 1.8 (s, 6H, (CH₃)₂C), 1.4 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 179.2 (1C, COOH), 176.7 (1C, C12-imidazo[2,1-b]benzothiazol-2-yl), 169.4 (1C, CO, C2-coumarin), 166.2 (1C, C11-imidazo[2,1-b]benzothiazol-2-yl), 164.7 (1C, C10-imidazo[2,1-b]benzothiazol-2-yl), 162.7 (1C, C9-coumarin), 160.4 (1C, C2-imidazo[2,1-b]benzothiazol-2-yl), 157.1 (1C, C3-imidazo[2,1-b]benzothiazol-2-yl), 154.8 (1C, C10-coumarin), 149.1 (1C, C7-coumarin), 144.6 (1C, C3-coumarin), 141.9 (1C, C8-imidazo[2,1-b]benzothiazol-2-yl), 139.4 (1C, C4-coumarin), 136.2 (1C, C5-imidazo[2,1-b]benzothiazol-2-yl), 132.7 (1C, C8-coumarin), 127.6 (1C, C7-imidazo[2,1-b]benzothiazol-2-yl), 123.4 (1C, C6-coumarin), 119.6 (1C, C6-imidazo[2,1-b]benzothiazol-2-yl), 118.3 (1C, C5-coumarin), 49.1 (1C, (CH₃)₂C), 29.8 (2C, (CH₃)₂C), 18.7 (1C, CH₃); MS: m/z (%) 434 (100) [M⁺]; anal. calc. for C₂₃H₁₈N₂O₅S: C 63.58, H 4.18, N 6.45, S 7.38. Found C 63.71, H 4.22, N 6.64, S 7.52.

4.3. In silico ADME studies

In silico studies of the designed molecules were performed for Lipinski parameters, as well as TPSA, % ABS, drug-likeness and drug score in order to verify whether these designed molecules exhibit the optimal physicochemical properties. The drug-likeness and drug score were calculated using the OSIRIS property explorer software and the lipophilicity and TPSA were calculated using the Molinspiration online property calculation toolkit (http://www.molinspiration.com).²¹ The % ABS was estimated according to eqn (1).

%ABS = 109 − (0.345 ​× TPSA) (1)

4.4. Molecular docking studies

The SYBYL-X 2.1 docking program was employed in the molecular docking studies. The X-ray crystal structure of the protein enzyme was well defined and established. Using the protein preparation module the target protein was prepared and bond orders were assigned. The water and other residues were removed, hydrogen atoms were added and the protein model was charged with AMBER7 FF99. The designed coumarin derivatives and the reference standards 34-JAB (co-crystallized ligand of EGFR tyrosine kinase enzyme) and celecoxib (co-crystallized ligand of COX-2 enzyme) as ligands were drawn in ChemDraw and converted into *.sdf format. The reference standards 34-JAB and celecoxib were docked along with the coumarin molecules to validate the docking protocol for the EGFR and COX-2 enzymes, respectively. The 2D molecules were converted to 3D, and the ligands were energy minimized and charged using the Gasteiger–Marsili method. The docking of the optimized 3D-structures of the molecules was performed within a 10 Å radius to assess and to optimize the most probable binding pose of each ligand. The essential parameters in terms of hydrogen bonds and hydrophobic interactions, which govern the binding disparities for each ligand in the series, was interpreted in the form of score functions. The score of the docked molecules indicates the affinity of the ligands towards the enzyme core.

In order to visualize the importance of the possible anticancer efficacy of the molecules, the compounds were docked onto EGFR (PDB ID: 2J5F) and COX-2 (PDB ID: 3LN1). Crystallographic data of 2J5F and 3LN1 were used for docking studies since the data on 2J5F has been well established and has been used to understand the anticancer efficacy of the bound inhibitor.²² The protein structures of 2J5F and 3LN1 were determined at 3.0 and 2.4 Å resolutions, respectively. The bound conformations of co-crystallized ligands 34-JAB and celecoxib were used as controls in order to define the active site in EGFR and COX-2, respectively.

4.5. In vitro antioxidant activity

The in vitro radical scavenging of the newly synthesized compounds was carried out by three different radical scavenging methods, i.e. DPPH, SA and ABTS.^23–26 Ascorbic acid was employed as the reference for a comparative study to determine the antioxidant potential of the test compounds. All the compounds were screened at three different concentrations, 12.5, 25 and 50 μM, along with the standard ascorbic acid.

4.5.1. DPPH free radical scavenging assay. This assay is based on the ability of antioxidants to reduce the stable radical DPPH to the yellow colored diphenyl-picryl hydrazine. Methanol was employed as a solvent blank and DPPH (0.1 μM) was employed as the control. To the 2 mL solutions of test compounds, 2 mL DPPH solution (0.1 μM) was added. The solution was incubated at 37 °C for 30 min and the absorbance was measured at 517 nm. The percent free radical scavenging activity was calculated according to eqn (2).

4.5.2. ABTS radical scavenging assay. This assay is based on oxidization of ABTS to its radical cation ABTS˙⁺, which is intensely colored. Methanol was used as the solvent blank, diluted ABTS˙⁺ was employed as a positive control and ascorbic acid was employed as the reference standard. To 0.5 mL of the test compounds, 5 mL ABTS˙⁺ (7 mM) solution was added and incubated for 15 min in the dark and the absorbance was measured at 734 nm against the blank. The percent free radical scavenging activity was calculated according to eqn (2).

4.5.3. Superoxide anion radical scavenging assay. This assay is based on the generation of superoxide anion radical (O₂˙⁻) by phenazinemethosulphate (PMS)/nicotinamide adenine dinucleotide (NADH) system. To 4 mL of Tris–HCl buffer (16 mM, pH 8.0), 1 mL of nitrobluetetrazolium (300 μM) and 1 mL of NADH (936 μM) solution, 2 mL of test compounds and 2 mL of Tris–HCl solution was added for the generation of superoxide radicals. The reaction was initiated by the addition of 1 mL of PMS (120 μM) solution. The mixture was incubated for 5 min at 25 °C and the absorbance was recorded at 560 nm. The percent free radical scavenging activity was calculated according to eqn (2).

All three experiments above were done in triplicate. The percentage radical scavenging activity values of the test compounds in different concentrations were compared with the negative control and ascorbic acid using the repeated measures ANOVA with Dunnet’s test. The concentrations which produced half-maximal (IC₅₀) activity were also determined graphically from the percentage antioxidant values at various concentrations of the test compounds.

4.6. In vitro cytotoxicity activity

The synthesized compounds 5a–5f were evaluated for their in vitro anticancer potential against three different human cancer cell lines, namely MCF-7 and MDA-231 (human breast cancer) and HT29 (human colon adenocarcinoma), by the MTT assay method.²⁷ 5-Flurouracil and doxorubicin were employed as reference for the comparative determination of the cytotoxicity efficacy of the test compounds. The test compounds and the reference standards were tested at four different concentrations, 6.25, 12.5, 25 and 50 μM, in order to estimate the IC₅₀ values. The principle of the MTT assay is based on the ability of the surviving cells to reduce tetrazolium salt MTT into a blue colored product (formazan) by mitochondrial enzyme succinate dehydrogenase in the living cells. The intensity of the blue color is directly proportional to the number of cells alive.

The monolayer cell cultures were trypsinized and the cell count was adjusted to 1.0 × 10⁵ cells per mL using DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (fetal bovine serum). To each of the wells of a 96-well microtiter plate, 100 μL of each (MCF-7, MDA-231, HT29) diluted cell suspension (approximately 10 000 cells per well) was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off. The monolayer was washed once with medium and 100 μL of different test concentrations was added per well to the partial monolayer in the microtiter plates. The plates were then incubated at 37 °C for 72 h in a 5% CO₂ atmosphere. Microscopic examination was carried out and observations were recorded every 24 h for 72 h. After 72 h, the sample solutions in the wells were discarded and 20 μL of MTT (2 mg mL⁻¹) in MEM-PR (MEM without phenol red) was added to each well. The plates were gently shaken and incubated for 3 h at 37 °C in 5% CO₂ atmosphere. The supernatant was removed, 50 μL of isopropanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using eqn (3).

The concentrations which produced half-maximal (IC₅₀) activity were also determined graphically from the percentage antioxidant values at various concentrations of the test compounds. The mean IC₅₀ values of 5-fluorouracil and tested compounds were compared with the control using the repeated measures ANOVA with Dunnet’s test.

Conflict of interest

Authors declare no conflict of interest.

Acknowledgements

The authors are thankful to the Principal, JSS College of Pharmacy, Mysuru and JSS University for providing us all necessary facilities. We sincerely extend our gratitude to chairman and staff, NMR research center, Mysore University, Mysore for providing NMR and mass spectra. Authors also beholden to DBT for the financial assistance (Ref. No: BT/PR5594/MED/29/540/20l2).

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