Rational design and synthesis of novel 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazole derivatives as an anti-angiogenesis and anti-cancer agent

Abstract

Based on earlier proven pharmacophore analogues of cancer a novel 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazoles (13–16) were rationally designed and synthesized by the reaction of chromene-3-carboxylic acids (10a–d) with substituted acyl bromides in the presence of TEA followed by refluxing with NH₄OAc in toluene. Compounds 13–16 were screened in vitro for the inhibition of KRAS/Wnt and their anti-angiogenesis properties. Compound 16f has been identified as a potent anti-angiogenesis molecule, which can be considered as a new lead structure. The molecular docking analysis displayed the higher binding affinity of 16f with KRAS, Wnt and VEGF.

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1. Introduction

Several researchers are working to discover new drugs based on natural products to treat cancer, but the success rate is very less because of toxicity issues, as well as the inability of compounds to selectively act on cancer cells. Epidemiological studies have reported that diets rich in isoflavones, particularly soybeans and soya products reduce the incidence of various cancers. In vitro and in vivo animal studies showed that the isoflavones genistein (1) and daidzein (2)^1–8 are promising agents for cancer chemoprevention and inhibition of tumor progression (Fig. 1). Isoflavones inhibit cell growth at concentrations greater than 20 μM. The inhibition of cell proliferation by isoflavones may involve the interference with signaling via the epidermal growth factor receptor kinase, effects on the cell cycle, capsase or the transforming growth factor β signaling.

Fig. 1 Structures of isoflavone based anti-angiogenesis molecules, genistein (1) and daidzein (2) and phenoxodiol (3).

Tumor angiogenesis is a complex dynamic process necessary for the growth of all tumors.^9–12 A tumor cannot grow through a defined volume if it is not vascularized. Among the angiogenic factors secreted by the tumor cells, the Vascular Endothelial Growth Factor (VEGF) is one of the important. Most human cancer cells express elevated levels of VEGF and VEGF receptors on their surface. An anti-angiogenic drug impairs the VEGF pathway and tumor vasculature by targeting VEGF (for e.g.: bevacizumab¹³ a monoclonal antibody) or their receptors. In the last two decades several small molecules were evaluated as anti-angiogenic agents.¹⁴ A few of the successfully derived natural product anticancer drugs include vincristine (acute lymphocytic leukemia),^15,16 taxol (ovarian cancer)¹⁷ and etoposide (lung cancer).¹⁸

The synthetic analogs of the isoflavones, daidzein (2) and phenoxodiol (3)^l9–23 showed strong apoptotic and anti-angiogenic activities in ovarian carcinoma in vitro and reduced the tumor volume of ovarian xenografts in vivo. Phenoxodiol (3) is a dihydroxylated-3-arylchromene. Synthetic heterocyclic compounds containing nitrogen and oxygen are commonly explored as anticancer agents with anti-angiogenesis as the mode of action.⁴ These compounds are chromenes with an aryl imidazole substitution at the 3-position of the chromene. The imidazole moiety is present in a wide range of naturally occurring compounds. It is a common scaffold in many significant biomolecules, including biotin, histamine, the essential amino acid histidine, and the pilocarpine alkaloids.²⁴ The use of imidazole and its derivatives in chemical processes, especially in pharmaceuticals have increased because of their ability to form hydrogen bonds with the active sites of several enzymes.²⁵ The incorporation of the imidazole nucleus is an important synthetic strategy in rational drug discovery.

We rationally designed and synthesized novel 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazoles (13a–j; 14a, e–j; 15a, e–j; 16a–f) that are structurally similar to the natural isoflavones and phenoxodiol and are considered potential anti-angiogenic agents. Molecular models enable us to build the three dimensional shape of the small molecules and its target. It allows us to check whether the shape of a potential lead is complementary to the shape of its target. It also allows us to calculate the binding energy liberated when a molecule binds to an active site. Molecular modelling has reduced the need to synthesize every analogue of a lead compound. The synthesized compounds were characterized by IR, NMR, and mass spectrometry. Of the synthesized compounds, fourteen compounds were screened in the Eli Lilly's Open Innovation Drug Discovery programme (OIDD). Therefore, these leads used competitive biological activity against cancer cell lines. Experimental procedures for the angiogenesis assay, cellular imaging and endothelial and nuclear staining were also carried out.

2. Results and discussion

2.1 Chemistry

1,3-Dimethoxybenzene (5) on regioselective lithiation with n-BuLi at 0 °C in THF–TMEDA yielded 2-lithiated dimethoxybenzene, which on treatment with DMF yielded 2,6-dimethoxybenzaldehyde (6),²⁶ Selective mono demethylation with AlCl₃ afforded 2-hydroxy-6-methoxybenzaldehyde (8a).^26,27 2,4-Dihydroxybenzaldehyde (7) was prepared from resorcinol by Vilsmeier–Haack Formylation.^28,29 Compound-7 on methylation with CH₃I yielded 8b (Scheme 1). 3-Methoxysalicylaldehyde (vanillin) and salicylaldehydes are commercially available. The treatment of the substituted salicylaldehydes (8a–d) with acrylonitrile and 1,4-diazabicyclo[2.2.2]octane (DABCO) as a catalyst in the Baylis–Hillman reaction³⁰ afforded chromene-3-nitriles (9a–d), which in turn were hydrolyzed to chromene-3-carboxylic acids³¹ (10a–d) (Scheme 2). The chromene-3-carboxylic acids (10a–d) were reacted with substituted acyl bromides (12a–j) (Scheme 3) in the presence of TEA followed by refluxing with NH₄OAc in toluene to obtain 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazoles³¹ (13–16) (Schemes 4–7).

Scheme 1 Synthesis of the substituted salicylaldehydes. Reagents and conditions: (a) MeI, K₂CO₃, acetone, rt, 12 h; (b) n-BuLi, TEMDA, dry DMF, dry THF, 0 to 5 °C; (c) AlCl₃, DCM, −15 °C, rt, 6 h; (d) DMF, POCl₃, acetonitrile.

Scheme 2 Synthesis of the substituted chromene-3-carboxylic acids (10a–d). Reagents and conditions: (a) acrylonitrile, DABCO, reflux, 10 h; (b) NaOH, H₂O, reflux, 6 h.

Scheme 3 (a) Synthesis of the substituted phenacyl bromides (12a–h). (b) Synthesis of 2-bromo-1-(pyridin-3-yl)ethanone (12i). (c) Synthesis of 2-bromo-1-(thiophen-2-yl)ethanone (12j). Reagents and conditions: (a) bromine, diethyl ether, DCM, 0 °C to rt, 2 h; (b) bromine, 33% HBr in acetic acid, 0 to 60 °C, 2 h; (c) bromine, DCM, rt, 2 h.

Scheme 4 Synthesis of the 2-(2H-chromen-3-yl)-5-aryl-1H-imidazoles (13a–c, e–j): Reagents and conditions: (a) TEA, DCM, rt, 12 h; (b) NH₄OAc, toluene, reflux, 12 h.

Scheme 5 Synthesis of the 2-(8-methoxy-2H-chromen-3-yl)-5-aryl-1H-imidazoles (14a, e–j). Reagents and conditions: (a) TEA, DCM, rt, 12 h; (b) NH₄OAc, toluene, reflux, 12 h.

Scheme 6 Synthesis of the 2-(7-methoxy-2H-chromen-3-yl)-5-aryl-1H-imidazoles (15a, e–j). Reagents and conditions: (a) TEA, DCM, rt, 12 h; (b) NH₄OAc, toluene, reflux, 12 h.

Scheme 7 Synthesis of the 2-(5-methoxy-2H-chromen-3-yl)-5-aryl-1H-imidazoles (16a–f). Reagents and conditions: (a) TEA, DCM, rt, 12 h; (b) NH₄OAc, toluene, reflux, 12 h.

2.2 Biological activity

2.2.1 Anti-cancer activity. Of the 29 chromenyl imidazoles synthesized in this study, 14 compounds (16a–f, 15h, 14f, h, j, 13f, g, i, j) were selected for screening from the OIDD chemo informatics filter. Compounds (16a–f, 15h, 14f, 14h, j, 13f, g, i, j) were subjected to in vitro primary screening in the KRAS-Wnt SL (Synthetic Lethal) in the basal viability of colon cancer cell lines (SW480, DLD-1, HCT116, GSK 3b in pretreated viability HCT116, HT-29, RKO, SW837, Colo320, SNU-C1) and the % inhibition (at 0.2, 2.0 and 20 μM) was calculated by the IC₅₀ values (summarized in Table 1). According to the IC₅₀ values from their concentration–response curves, compounds 13g, 16a, 16e and 16f were found to be the potent inhibitors. Especially, compound 16f, which showed IC₅₀ values in the range of 0.37–8.3 μM on the different cancer cell lines tested. The colorectal cancer cell line inhibitions depend on the nature of the substituents on the phenyl ring of the imidazole nucleus and the methoxy group of the chromene nucleus. IC₅₀ values were also determined for three other compounds in the basal TCF/TK Luciferase DLD-1 assay wherein 16f showed IC₅₀ = 4.731 μM (Table 2). It was observed that compounds possessing a methoxy group on the 5^th position of chromene nucleus demonstrated augmented cancer inhibitory activity; benzyloxy substitution was found more favorable towards cancer inhibition over the other substitution.

Table 1 Synthetic lethal primary SP assay: IC₅₀ of novel 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazoles (13–16) on K-RAS/WNT expression and the basal viability of cancer cells in different colon cancer cell lines^a

Primary CRC
S. no.	Basal_viability ATP SW480	Basal_viability ATP DLD-1	Basal_viability ATP HCT116	GSK 3b inh pretreated_viability ATP HCT116	Basal_viability ATP HT-29	Basal_viability ATP RKO	Basal_viability ATP SW837	Basal_viability ATP Colo320	Basal_viability ATP SNU-C1
	IC50 (μM)	IC50 (μM)	IC50 (μM)	IC50 (μM)	IC50 (μM)	IC50 (μM)	IC50 (μM)	IC50 (μM)	IC50 (μM)
a IC50 (μM) of 14 compounds based on the inhibition of GSK-3b and the basal viability in CRC cell lines.
13f	8.45	10.25	>50.0	>50.0	>25.0	9.42	8.26	6.82	>25.0
13g	4.833	20.0	5.072	8.23	6.716	3.551	4.18	1.118	2.782
13i	11.45	>50.0	16.17	>50.0	8.98	9.16	16.32	2.22	>50.0
13j	15.54	13.56	14.23	10.33	>50.0	16.55	29.15	>50.0	>25.0
14f	8.98	22.18	5.48	2.77	12.43	14.03	22.00	10.33	>50.0
14h	6.77	21.07	7.13	3.98	11.11	14.98	21.07	14.39	>50.0
14j	6.98	33.17	9.33	3.43	7.09	14.32	33.13	14.76	>50.0
15h	12.76	22.67	14.18	6.72	6.03	17.16	22.62	>25.0	12.15
16a	5.974	12.701	4.046	5.541	6.25	6.175	16.59	9.408	13.29
16b	8.65	21.15	19.54	8.53	12.19	9.32	18.32	>50.0	>50.0
16c	9.15	10.43	8.98	7.21	14.06	8.95	32.15	11.25	>50.0
16d	6.18	12.87	7.09	9.23	16.32	6.74	30.98	>50.0	>50.0
16e	2.5	3.917	1.1	0.959	0.8	1.111	25.0	1.017	1.051
16f	0.831	1.279	0.138	0.179	0.962	1.095	8.303	0.388	3.725

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Table 2 Wnt reporter assay: IC₅₀ based on the luciferase reporter activity of 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazoles (13g, 16a, e, f)^a

S. no.	Basal_TCF/TK-luciferase DLD-1	Basal_CMV-luciferase DLD-1
	IC50 (μM)	IC50 (μM)
a All basal viability in IC50 (μM).
13g	>50.0	>50.0
16a	>50.0	>50.0
16e	10.804	>50.0
16f	4.731	>50.0

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2.2.2 Anti-angiogenic activity. The three compounds (16a, 16e, 16f) were screened for anti-angiogenesis activity and they exhibited greater than 50% inhibition of the tube area at 10 μM in the primary single point assay. These three were further screened at different concentrations to obtain CRC (concentration response curves) and IC₅₀ values (Table 3). Only compound 16f with benzyloxy substitution at the 4^th position of the phenyl ring and a methoxy group on the 5^th position of the chromene nucleus showed significant activity with an IC₅₀ of 0.89 μM (Fig. 2), and the remaining two compounds showed IC₅₀ values greater than 10 μM. The compounds lacking the benzyloxy substituent failed to exhibit significant antiangiogenic activity; thus, indicating the importance of the benzyloxy group for binding to the receptor and the subsequent inhibitory activity. Thus, our studies resulted in a new lead molecule with potent anti-angiogenic activity, which was better than the earlier synthesized compounds (isoflavone metabolite 6-methoxyequol; IC₅₀ value of 3).³² The novel compound 16f could be studied further to discover a new clinical and therapeutic agents, which are useful in cancer chemotherapy.

Table 3 Anti-angiogenesis assay: anti-angiogenic, cell cycle effects influenced by 5-(4-(benzyloxy)phenyl)-2-(5-methoxy-2H-chromen-3-yl)-1H-imidazole (16f) on CRC cell lines and ADSC/ECFC co-culture cell lines

	Primary CRC		Secondary
	VEGF_ADSC/ECFC Angiotube area	VEGF_ADSC/ECFC Angio Nuc area	Hela CellCyc 4N	Hela CellCyc MI	Hela CellCyc 2N	Hela CellCyc cell number
	IC50 (μM)	EC50 (μM)	EC50 (μM)	IC50 (μM)	IC50 (μM)	EC50 (μM)
16f	0.89	>10.0	1.445	1.357	>10.0	1.029

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Fig. 2 Anti-angiogenesis activity of compound 16f.

2.3 Molecular docking studies

2.3.1 Binding of 16f to Human KRAS receptor. The docking of compound 16f in the binding site of the Human KRAS receptor illustrated that the chromene and imidazole ring play a decisive role in inhibiting angiogenesis activity. The docking results showed hydrogen bonding interactions between the oxygen of the chromene ring and (i) the amine group of Asn 26 with a bond distance of 1.1 Å, (ii) ND2 of Asn 26, d = 2.5 Å and (iii) His 27, d = 3.2 Å. The methoxy oxygen in the chromene ring also showed hydrogen bonding with His 27, d = 2.8 Å. Further hydrogen bonding interactions were also observed involving the two nitrogen atoms of the imidazole ring with the backbone carbonyl of Gln 25 (d = 2.6 Å and d = 2.3 Å) and Ile 24 (d = 3.4 Å). The chromene aromatic ring of 16f was surrounded by the Asn 26, His 27, Gln 25 and Ile 24 amino acid residues (Fig. 3 and 4).

Fig. 3 H-bonding interactions between amino acid residues at the active site of the Human KRAS target and compound 16f.

Fig. 4 Surface representation of the KRAS receptor and ligand complex 16f.

2.3.2 Binding of 16f to Wnt receptor. Docking of compound 16f in the active site of the Human Wnt receptor showed hydrogen bonding between the nitrogen atom of the imidazole ring with the backbone carbonyl of Gln 25 CB (d = 1.66 Å) and His 27 (d = 3.03 Å). The oxygen atom of the chromene ring showed interactions with the amine group of Tyr 100 (d = 1.905 Å) (Fig. 5 and 6).

Fig. 5 H-bonding interactions between amino acid residues at active site of the Wnt target and compound 16f.

Fig. 6 Surface representation of the Wnt receptor and ligand complex 16f.

2.3.3 Binding of 16f to VEGF receptor. 16f in the active site of the VEGF receptor showed two hydrogen bonding interactions between the nitrogen atom of the imidazole ring with Arg 82 (d = 2.37 Å and d = 2.05 Å). Further hydrogen bonding interactions from the oxygen atom of the chromene ring and amine group of Arg 105 (d = 1.93 Å) was observed (Fig. 7 and 8).

Fig. 7 H-bonding interactions between amino acid residues at the active site of the VEGF target and compound 16f.

Fig. 8 Surface representation of the VEGF receptor and ligand complex 16f.

For the other chromenyl imidazoles synthesized their docking studies did not show closer interactions with the KRAS, Wnt and VEGF receptors.

3. Conclusion

Several new 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazoles (13–16) were rationally designed, synthesized and subjected to in vitro primary anticancer screening against several cancer cell lines (SW480, DLD-1, HCT116, GSK 3b in pretreated viability HCT116, HT-29, RKO, SW837, Colo320 and SNU-C1). Among them four compounds (13g, 16a, 16e and 16f) were subjected to the basal TCF/TK Luciferase DLD-1 assay. The results of the in vitro cancer assay indicated that all the compounds showed considerable cancer inhibition. However, the substitution of the methoxy group at the 5^th position of the chromene and the 4^th position of the benzyloxy group on the phenyl ring of the imidazole was found to be more favorable for cancer inhibition. Whereas, the compounds with no methoxy group at the 5^th position on the chromene showed diminished cancer activity. Among all the screened compounds, 16f was found to be the most potent inhibitor of cancer with the highest selectivity. Furthermore, in the anti-angiogenesis assay compound 16f showed potent activity in inhibiting the VGF_ADSC/CFC Angiotube area. The molecular docking analysis also exhibited the higher binding affinity of 16f with the KRAS, Wnt and VEGF ligands. Hence, 16f could be considered as a lead structure in the development of a new series of anti-angiogenesis/anticancer agents.

4. Experimental section

4.1 General experimental methods

Unless otherwise specified, all the solvents and reagents were obtained from commercial suppliers. Solvents were purified as per the procedures given in the “Text book of practical organic chemistry” by Vogel (6^th Edition). All the reactions were performed under a nitrogen atmosphere unless otherwise noted. Column chromatography was performed using Merck silica gel, 60–120 mesh. ¹H NMR spectra were recorded on a Bruker spectrometer at 400 MHz, ¹³C NMR spectra were obtained on 100.6 MHz with tetramethylsilane as the internal standard, and the chemical shifts (δ) are reported in ppm as (δ) shift (multiplicity, coupling constant, proton count). The mass spectral analysis was accomplished using electro spray ionization (ESI) techniques.

4.2 Preparation of 1,3-dimethoxybenzene (5)

To a stirred solution of resorcinol 4 (20 g, 0.18 mol) and acetone (200 ml) in an ice both was added K₂CO₃ (62.72 g, 0.45 mol) and methyl iodide (25.8 ml, 0.399 mol) drop wise over a period of 10 min. After complete addition, the solution was stirred overnight at rt. After the completion of the reaction, acetone was evaporated under reduced pressure. The crude was diluted with water and extracted two times with ethyl acetate. The combined ethyl acetate extracts were dried (Na₂SO₄) and evaporated. The residue was purified by chromatography, by elution with ethyl acetate–pet-ether to obtain the title compound 5 (22 g, 87.69%). ¹H NMR (CDCl₃) δ: 3.78 (s, 6H, 2 × OCH₃), 6.4 (t, J = 2.0 Hz, H₅), 6.5 (dd, J = 2.0 Hz, H₄), 6.52 (dd, J = 1.6 Hz, H₆), 7.2 (t, J = 2.0, H₂). ¹³C NMR (CDCl₃) δ: 56.5 (2 × O–C), 100.4 (C₂), 107.5 (C₄ and C₆), 129.8 (C₅), 162.7 (C₁ and C₃).

4.3 Preparation of 2,6-dimethoxybenzaldehyde (6)

A suitable RB flask was charged with 1,3-dimethoxybenzene (5) (21.5 g, 0.155 mol), N,N,N′,N′-tetramethylethylenediamine (TMEDA, 27.6 ml, 0.176 mol) and dry THF (200 ml). At 0 °C, 1.6 M n-butyl lithium (116.77 ml, 0.186 mol) was added. The solution was stirred at 5 °C for 30 min and dry DMF (18.3 ml, 0.233 mol) was slowly added and the temperature was maintained less than 10 °C (38 °C adiabatic temperature rise from the addition). The solution was stirred for 2 h, and then quenched with 4 M HCl under cold water and the stirring continued for 30 min at 5 to 15 °C. The reaction mixture was filtered and washed with water subsequently followed by pet-ether to obtain compound 6 as an off white solid (18.4 g, 71.15%). ¹H NMR (CDCl₃) δ: 3.89 (s, 6H, 2 × OCH₃), 6.4 (t, J = 2.0 Hz, H₅), 6.58 (d, J = 2.0 Hz, 2H, H₃ and H₅), 7.46 (t, J = 8.8 Hz, H₄), 10.51 (s, CHO). ¹³C NMR (CDCl₃) δ: 56.06 (O–C), 103.8 (C₃ and C₅), 114.3 (C₁), 135.9 (C₄), 162.2 (C₂ and C₈), 189.4 (CHO).

4.4 Preparation of 2,4–dihydroxybenzaldehyde (7)

To a well cooled (0–5 °C) solution of resorcinol (4) (20 g, 0.18 mol) in acetonitrile (200 ml), dry DMF (21.24 ml, 0.272 mol) and freshly distilled dry POCl₃ (25.34 ml, 0.272 mol) were added with constant stirring at 0–5 °C. The salt that separated was filtered and washed with cold acetonitrile. To this salt, water was added and heated at 50 °C for 0.5 h, and then cooled. The solid that separated was filtered, washed with cold water, and dried to obtain 2,4-dihydroxybenzaldehyde (7) (18 g, 72%). ¹H NMR (CDCl₃) δ: 6.38 (d, J = 1.2 Hz, H₃), 6.5 (dd, J = 10.4, 2.0 Hz, H₅), 7.35 (d, J = 8.8 Hz, H₆), 9.64 (s, 2-OH), 10.1 (s, 4-OH), 11.42 (s, CHO). ¹³C NMR (CDCl₃) δ: 102.8 (C₃), 109.2 (C₅), 114.5 (C₁), 135.8 (C₆), 164.3 (C₂), 165.8 (C₄), 194.0 (CHO).

4.5 Preparation of 2-hydroxy-6-methoxybenzaldehyde (8d)

To a RB flask with AlCl₃ (22.02 g, 0.165 mol) and dichloromethane (120 ml), which was cooled to −15 °C, a solution of 2,6-dimethoxybenzaldehyde (6) (18 g, 0.108 mol) in dichloromethane (60 ml) was slowly added while maintaining the temperature below 0 °C. The solution was slowly warmed to ambient temperature and allowed to stir for 6 h. A solution of conc. HCl in water was slowly added while maintaining the temperature below 25 °C, and then dichloromethane was evaporated under vacuum distillation. The product was collected by filtration and washed with a solution of conc. HCl and water. After drying, compound 8d was obtained (13 g, 78.9%). ¹H NMR (CDCl₃) δ: 3.89 (s, OCH₃), 6.37 (d, J = 8.4 Hz, H₃), 6.53 (d, J = 8.4 Hz, H₅), 7.42 (t, J = 8.4 Hz, H₄), 11.97 (s, CHO). ¹³C NMR (CDCl₃) δ: 55.8 (O–C), 100.8 (C₅), 109.9 (C₃), 110.8 (C₁), 138.4 (C₄), 162.4 (C₆), 163.6 (C₂), 194.3 (CHO).

4.6 Preparation of 2-hydroxy-4-methoxybenzaldehyde (8c)

To a stirred solution of 7 (18 g, 0.13 mol) and acetone (150 ml) in an ice, both K₂CO₃ (35.88 g, 0.26 mol) and methyl iodide (12.18 ml, 0.195 mol) was added drop wise over a period of 10 min. After the complete addition, the solution was stirred overnight at rt. After the completion of the reaction, the acetone was evaporated. The crude was diluted with water and extracted two times with ethyl acetate. The combined ethyl acetate extracts were dried (Na₂SO₄) and evaporated. The residue was purified by chromatography, eluting with ethyl acetate/pet-ether to obtain the title compound 8c (13 g, 68.4%). ¹H NMR (CDCl₃) δ: 3.85 (s, OCH₃), 6.42 (d, J = 2.0 Hz, H₃), 6.54 (dd, J = 8.4, 2.4 Hz, H₅), 7.42 (d, J = 8.4 Hz, H₆), 9.71 (s, OH), 11.49 (s, CHO). ¹³C NMR (CDCl₃) δ: 56.0 (O–C), 103.0 (C₃), 108.0 (C₅), 114.0 (C₁), 135.9 (C₆), 162.0 (C₂), 164.2 (C₄), 194.0 (CHO).

4.7 General procedure for the synthesis of substituted-2H-chromene-3-carbonitriles (9a–d)

Substituted salicylaldehydes (1.0 mol) (8a–d) with an excess of acrylonitrile in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO, 0.25 mol) as a catalyst was stirred at 80 °C for 10 h, to undergo the Baylis–Hillman reaction. The excess acrylonitrile was evaporated under reduced pressure, and then the reaction mixture was dissolved in ethyl acetate, washed with 1 N NaOH followed by 1 N HCl. The organic phase was dried over anhydrous Na₂SO₄, filtered and the solvent was evaporated under vacuum and the residue was subjected to column chromatography using silica gel (60–120) to obtain the product substituted-2H-chromene-3-carbonitriles 9a–d, in quantitative yields.

4.7.1 2H-Chromene-3-carbonitrile (9a). Yellow solid, yield 75%. ¹H NMR (CDCl₃) δ: 4.8 (d, J = 1.2 Hz, O–CH₂), 6.86 (d, J = 8.0, H₈), 6.96 (td, J = 7.6, 0.8 Hz, H₆), 7.09 (dd, J = 7.6, 1.2 Hz, H₅), 7.153 (s, H₄), 7.25 (td, J = 8.0, 0.8 Hz, H₇). ¹³C NMR (CDCl₃) δ: 71.4 (C₂), 109.0 (C₃), 114.2 (C₈), 115.7 (C_4a), 117.3 (CN), 121.4 (C₆), 126.5 (C₅), 128.4 (C₇), 141.8 (C₄), 157.5 (C_8a).

4.7.2 8-Methoxy-2H-chromene-3-carbonitrile (9b). Off white solid, yield 70%. ¹H NMR (CDCl₃) δ: 3.88 (s, OCH₃), 4.86 (d, J = 1.2 Hz, OCH₂), 6.74 (m, H₆), 6.93 (m, 2H, H₄ & H₇), 7.17 (m, H₅). ¹³C NMR (CDCl₃) δ: 55.5 (OCH₃), 64.4 (C₂), 105.4 (C₃), 113.5 (C₇), 115.9 (C_4a), 118.9 (C₅), 117.0 (CN), 125.2 (C₆), 138.7 (C₄), 154.9 (C_8a), 162.8 (C₈).

4.7.3 7-Methoxy-2H-chromene-3-carbonitrile (9c). Off white solid, yield 72%. ¹H NMR (CDCl₃) δ: 3.8 (s, OCH₃), 4.7 (s, OCH₂), 6.42 (d, J = 2.0 Hz, H₈), 6.51 (dd, J = 8.4, 2.4 Hz, H₆), 7.02 (d, J = 8.8 Hz, H₅), 7.13 (s, H₄). ¹³C NMR (CDCl₃) δ: 55.5 (OCH₃), 64.4 (C₂), 99.3 (C₈), 101.9 (C₆), 108.9 (C_4a), 113.4 (C₃), 117.0 (CN), 129.6 (C₅), 138.7 (C₄), 155.9 (C_8a), 163.4 (C₇).

4.7.4 5-Methoxy-2H-chromene-3-carbonitrile (9d). Off white solid, yield 70%. ¹H NMR (CDCl₃) δ: 3.85 (s, OCH₃), 4.74 (s, OCH₂), 6.48 (t, J = 8.0 Hz, 2H, H₆ and H₈), 7.21 (t, J = 8.4 Hz, H₇), 7.53 (s, H₄). ¹³C NMR (CDCl₃) δ: 55.8 (O–C), 63.9 (C₂), 100.4 (C₆), 104.1 (C₈), 109.0 (C_4a), 110.3 (C₃), 117.0 (CN), 133.0 (C₇), 134.4 (C₄), 155.1 (C₅), 156.3 (C_8a).

4.8 General procedure for the synthesis of substituted-2H-chromene-3-carboxylic acid (10a–d)

A mixture of 5-methoxy-2H-chromene-3-carbonitrile and 10% sodium hydroxide was refluxed for 6 h. The reaction mixture was acidified with conc. HCl and the separated product was washed with water, dried, and recrystallized with ethanol to afford the desired compounds.

4.8.1 2H-Chromene-3-carboxylicacid (10a). Yellow solid, yield 90%. ¹H NMR (DMSO-d₆) δ: 4.9 (d, J = 1.2 Hz, OCH₂), 6.8 (d, J = 8.0 Hz, H₈), 6.95 (td, J = 7.2, 7.6, 0.8, 1.2 Hz, H₆), 7.26 (td, J = 8.0, 7.6, 1.6, 1.2 Hz, H₇), 7.32 (dd, J = 7.6, 7.2, 1.6, 1.2 Hz, H₅), 7.45 (s, H₄), 12.86 (s, COOH). ¹³C NMR (DMSO-d₆) δ: 63.5 (C₂), 115.0 (C₈), 120.7 (C_4a), 121.4 (C₆), 121.5 (C₅), 123.4 (C₇), 132.4 (C₃), 143.3 (C₄), 147.5 (C_8a), 165.4 (COOH).

4.8.2 8-Methoxy-2H-chromene-3-carboxylicacid (10b). Off white solid, yield 85%. ¹H NMR (DMSO-d₆) δ: 3.75 (s, OCH₃), 4.88 (s, OCH₂), 6.9 (m, 2H, H₆ & H₇), 7.0 (dd, J = 7.2, 2.8 Hz, H₅), 7.42 (s, H₄), 12.8 (s, COOH). ¹³C NMR (DMSO-d₆) δ: 55.6 (O–C), 63.9 (C₂), 115.0 (C₇), 120.7 (C_4a), 121.4 (C₅), 121.5 (C₆), 123.4 (C₃), 132.4 (C₄), 143.4 (C_8a), 147.5 (C₈), 165.5 (COOH).

4.8.3 7-Methoxy-2H-chromene-3-carboxylicacid (10c). Off white solid, yield 80%. ¹H NMR (DMSO-d₆) δ: 3.7 (s, OCH₃), 4.89 (s, OCH₂), 6.45 (s, H₈), 6.55 (d, J = 8.8 Hz, H₆), 7.22 (d, J = 8.8 Hz, H₅), 7.4 (s, H₄), 12.69 (s, COOH). ¹³C NMR (DMSO-d₆) δ: 55.3 (O–C), 64.2 (C₂), 101.2 (C₈), 107.9 (C₆), 113.9 (C_4a), 119.8 (C₅), 130.1 (C₃), 132.4 (C₄), 156.0 (C_8a), 162.3 (C₇), 165.6 (COOH).

4.8.4 5-Methoxy-2H-chromene-3-carboxylicacid (10d). Off white solid, yield 85%. ¹H NMR (DMSO-d₆) δ: 3.82 (s, OCH₃), 4.83 (s, OCH₂), 6.47 (d, J = 8.4 Hz, H₆), 6.61 (d, J = 8.4 Hz, H₈), 7.23 (t, J = 8.4 Hz, H₇), 7.55 (s, H₄), 12.73 (s, COOH). ¹³C NMR (DMSO-d₆) δ: 55.8 (O–C), 63.6 (C₂), 104.2 (C₆), 108.4 (C₈), 110.0 (C_4a), 121.3 (C₇), 127.0 (C₃), 132.3 (C₄), 155.1 (C₅), 156.4 (C_8a), 165.4 (COOH).

4.9 General procedure for the synthesis of 2-bromo-1-(substituted-phenyl)ethanone derivatives (12a–h)

Following the literature method,³³ to a stirred solution of acetophenone (5 g, 0.041 mol) in diethyl ether (30 ml) in an ice bath a solution of bromine (2.08 ml, 0.041 mol) in dichloromethane (30 ml) was added drop wise over 10–15 min. After the addition was complete, the solution was stirred for 2 h at rt. The solvent was evaporated under reduced pressure and triturated with pet-ether and placed in a freezer for few h, the separated solid was filtered to obtain the desired compounds 12a–h.

4.9.1 2-Bromo-1-phenylethanone (12a). Off white solid, yield 85%. ¹H NMR (CDCl₃) δ: 4.46 (s, CH₂), 7.5 (t, J = 8.0, 7.6 Hz, 2H, H₃ and H₅), 7.61 (t, J = 7.2 Hz, H₄), 7.99 (d, J = 8.4 Hz, 2H, H₂ and H₆). ¹³C NMR (CDCl₃) δ: 30.9 (C–Br), 128.8 (C₂ and C₆), 128.9 (C₃ and C₅), 131.7 (C₄), 133.9 (C₁), 191.2 (C=O).

4.9.2 2-Bromo-1-(4-chlorophenyl)ethanone (12b). Brown solid, yield 70%. ¹H NMR (CDCl₃) δ: 4.41 (s, CH₂), 7.34 (d, J = 8.8, 2H, H₃ & H₅), 7.84 (d, J = 8.4, 2H, H₂ and H₆). ¹³C NMR (CDCl₃) δ: 30.5 (C–Br), 129.3 (C₃ and C₅), 130.4 (C₂ and C₆), 132.2 (C₁), 132.6 (C₄), 190.4 (C=O).

4.9.3 2-Bromo-1-(2,4-chlorophenyl)ethanone (12c). Brown oil, yield 60%. ¹H NMR (CDCl₃) δ: 4.25 (s, CH₂), 7.5 (s, H₃), 7.65 (m, 2H, H₅ and H₆). ¹³C NMR (CDCl₃) δ: 30.5 (C–Br), 126.9 (C₅), 130.4 (C₃), 132.0 (C₆), 132.8 (C₂), 133.2 (C₁), 142.1 (C₄), 190.4 (C=O).

4.9.4 2-Bromo-1-(3-nitrophenyl)ethanone (12d). Off white solid, yield 70%. ¹H NMR (CDCl₃) δ: 4.48 (s, CH₂), 7.74 (t, J = 8.0 Hz, H₅), 8.33 (d, J = 8 Hz, H₆), 8.48 (d, J = 8.4 Hz, H₄), 8.81 (s, H₂). ¹³C NMR (CDCl₃) δ: 30.8 (C–Br), 122.7 (C₂), 123.5 (C₄), 127.6 (C₅), 133.6 (C₆), 137.2 (C₁), 145.3 (C₃), 190.9 (C=O).

4.9.5 2-Bromo-1-(4-bromophenyl)ethanone (12e). Brown solid, yield 75%. ¹H NMR (CDCl₃) δ: 4.41 (s, CH₂), 7.65 (d, J = 8.8 Hz, 2H, H₃ and H₅), 7.85 (d, J = 8.4 Hz, 2H, H₂ and H₆). ¹³C NMR (CDCl₃) δ: 30.5 (C–Br), 129.3 (C₄), 130.2 (C₂ & C₆), 132.2 (C₃ & C₅), 132.3 (C₁), 190.2 (C=O).

4.9.6 1-(4-(Benzyloxy)phenyl)-2-bromoethanone (12f). Off white solid, yield 65%. ¹H NMR (CDCl₃) δ: 4.39 (s, CH₂–Br), 5.14 (s, Bn–CH₂), 7.03 (dd, J = 6.8, 2.0 Hz, 2H, H₃ and H₅), 7.4 (m, 5H, H_1′–5′), 7.97 (dd, J = 6.8, 2.0 Hz, 2H, H₂ and H₆). ¹³C NMR (CDCl₃) δ: 30.5 (C–Br), 69.7 (Bn–CH₂), 114.2 (C₃ and C₅), 127.1 (C_2′ and C_6′), 127.4 (C₄), 127.7 (C_3′ and C_5′), 128.1 (C₁), 130.2 (C₂ and C₆), 140.2 (C_1′), 166.4 (C₄), 190.3 (C=O).

4.9.7 2-Bromo-1-(4-fluorophenyl)ethanone (12g). Off white solid, yield 85%. ¹H NMR (CDCl₃) δ: 4.42 (s, CH₂), 7.17 (t, J = 8.8, 8.4 Hz, 2H, H₃ and H₅), 8.03 (dd, J = 9.2, 5.6 Hz, 2H, H₂ and H₆). ¹³C NMR (CDCl₃) δ: 30.5 (C–Br), 112.4 (C₃ and C₅), 129.3 (C₂ and C₆), 130.4 (C₁), 160.3 (C₄), 190.2 (C=O).

4.9.8 2-Bromo-1-(4-methoxyphenyl)ethanone (12h). Brown solid, yield 70%. ¹H NMR (CDCl₃) δ: 3.38 (s, O–CH₃), 4.4 (s, CH₂), 6.96 (d, J = 8.8 Hz, 2H, H₃ and H₅), 7.97 (d, J = 8.8 Hz, 2H, H₂ and H₆). ¹³C NMR (CDCl₃) δ: 30.8 (C–Br), 55.5 (O–C), 114.3 (C₃ and C₅), 128.2 (C₁), 131.3 (C₂ and C₆), 164.1 (C₄), 189.9 (C=O).

4.9.9 2-Bromo-1-(pyridin-3-yl)ethanone (12i). Following the literature method,³⁴ 3-acetyl pyridine (11i) (5 g, 0.04 mol) was added to 33% HBr–AcOH (50 ml) with stirring. Bromine (2.04 ml, 0.04 mol) was then added and the reaction mixture was heated to 60 °C for 2 h. The solution was cooled to rt and diethyl ether (50 ml) was added. The separated solid was filtered, washed with diethyl ether and dried to obtain 2-bromo-1-(pyridin-3-yl) ethanone (12i). White solid 5.6 g, 70%. ¹H NMR (CDCl₃) δ: 4.39 (s, CH₂), 7.9 (m, 3H, H₄, H₅ and H₆), 9.13 (s, H₂). ¹³C NMR (CDCl₃) δ: 31.5 (C–Br), 121.3 (C₅), 123.3 (C₁), 131.6 (C₆), 145.4 (C₄), 147.5 (C₂), 190.2 (C=O).

4.9.10 2-Bromo-1-(thiophen-2-yl)ethanone (12j). Following the literature method,³⁵ a solution of bromine (1.98 ml, 0.039 mol) in dichloromethane (20 ml) was added drop wise to 2-acetylthiophene (11j) (5 g, 0.039 mol) in dichloromethane (30 ml) over 20 min. The reaction mixture was stirred at 25 °C for 2 h and neutralized with a saturated solution of NaHCO₃. The organic layer was washed with water followed by brine and dried over Na₂SO₄, and filtered. The removal of the solvent afforded a residue, which was purified by column chromatography (silica gel, ethyl acetate–pet-ether 5 : 95 as the eluent). Yellow oil; yield 6 g, 73.6%. ¹H NMR (CDCl₃) δ: 4.37 (s, CH₂), 7.17 (dd, J = 4.8, 4.0 Hz, H₄), 7.73 (dd, J = 3.6, 1.2 Hz, H₃), 8.81 (dd, J = 4.8, 1.2 Hz, H₅). ¹³C NMR (CDCl₃) δ: 31.1 (C–Br), 128.6 (C₄), 133.7 (C₃), 135.4 (C₅), 140.7 (C₂), 184.4 (C=O).

4.10 General procedure for the preparation of 2-(substituted-2H-chromen-3-yl)-5-aryl-1H-imidazoles (13–16)

To a mixture of substituted-2H-chromene-3-carboxylic acid (10a–d) (1.0 mol) and triethylamine (3.0 mol) in dichloromethane in an ice, acyl bromides (12a–f) (1.2 mol), was added and the solution was stirred overnight at rt. The reaction mixture was diluted with chloroform and successively washed with water and a brine solution, and dried over Na₂SO₄, after the removal of the solvent under reduced pressure; the residue was washed with pet-ether and dried under vacuum. The residue was dissolved in toluene and treated with ammonium acetate (5.0 mol) under a nitrogen atmosphere. The reaction mixture was refluxed for 12 h and was monitored by TLC. The reaction mixture was quenched with saturated NaHCO₃. The product was extracted with ethyl acetate (2 times), and successively washed with water followed by a brine solution, and dried over Na₂SO₄. After the removal of the solvent under reduced pressure, the crude product was purified by column chromatography using petroleum ether–ethyl acetate.

4.10.1 (2H-Chromen-3-yl)-5-phenyl-1H-imidazole (13a). Yellow solid; yield 50%; mp. 165–170 °C; ¹H NMR (CDCl₃) δ: 5.29 (s, O–CH₂), 6.85 (t, J = 8.4, 9.2 Hz, H₆), 6.9 (d, J = 7.2 Hz, H₈), 7.03 (d, J = 7.2 Hz, H₅), 7.14 (t, J = 8.0, 7.6 Hz, H₇), 7.25 (s, H_4′), 7.29 (d, J = 7.2 Hz, 2H, H_2′′ & H_6′′), 7.34 (s, H₄), 7.37 (t, J = 8.0, 2.8 Hz, 3H, H_3′′, H_4′′ and H_5′′), 7.69 (s, NH), ¹³C NMR (DMSO-d₆) δ: 64.7 (C₂), 114.8 (C₈), 115.4 (C_4a), 118.6 (C_4′), 121.7 (C₆), 122.4 (C₅), 123.2 (C₇), 124.3 (C_2′′ & C_6′′), 126.3 (C_4′′), 127.0 (C_3′′ and C_5′′), 128.4 (C₄), 129.1 (C_1′′), 134.3 (C_2′), 141.0 (C₃), 143.2 (C_5′), 153.2 (C_8a); IR (KBr, cm⁻¹): 3162 (N–H), 1578 (C=N); MS (ESI, m/z): 275.07.

4.10.2 5-(4-Chlorophenyl)-2-(2H-chromen-3-yl)-1H-imidazole (13b). Brown solid; yield 45%; mp. 180–185 °C; ¹H NMR (CDCl₃) δ: 5.33 (s, O–CH₂), 6.86 (s, H_4′), 6.91 (d, J = 8.0 Hz, H₈), 6.94 (td, J = 7.2, 7.6, 0.8, 1.2 Hz, H₆), 7.1 (dd, J = 7.6, 1.2 Hz, H₅), 7.19 (td, J = 8.0, 7.6, 1.6, 1.2 Hz, H₇), 7.28 (s, H₄), 7.38 (d, J = 8.0 Hz, 4H, H_2′, H_3′, H_5′ and H_6′), 7.71 (s, NH). ¹³C NMR (DMSO-d₆) δ: 64.7 (C₂), 115.4 (C₈), 115.5 (C_4a), 118.9 (C_4′), 121.7 (C₆), 122.3 (C₅), 125.9 (C₇), 127.1 (C_2′′ and C_6′′), 128.4 (C_3′′and C_5′′), 129.2 (C₄), 130.5 (C_1′′), 133.2 (C_4′′), 139.8 (C_2′ and C₃), 143.4 (C_5′), 153.2 (C_8a); IR (KBr, cm⁻¹): 3162 (N–H), 1577 (C=N); MS (ESI, m/z): 308.97, 310.98.

4.10.3 5-(2,4-Dichlorophenyl)-2-(2H-chromen-3-yl)-1H-imidazole (13c). Yellow solid; yield 45%; mp. 190–195 °C; ¹H NMR (CDCl₃) δ: 5.34 (s, OCH₂), 6.88 (s, H_4′), 6.91 (d, J = 9.2 Hz, H₈), 6.95 (t, J = 8.4 Hz, H₆), 7.12 (d, J = 8.8 Hz, 2H, H₅ and H_3′′), 7.2 (t, J = 7.6 Hz, H₇), 7.28 (s, 2H, H₄ and H_5′′), 7.34 (d, J = 10.4 Hz, H_2′′), 7.47 (s, NH). ¹³C NMR (CDCl₃) δ: 64.6 (C₂), 115.5 (C₈), 115.84 (C_4a), 118.9 (C_4′), 121.75 (C₆), 122.2 (C₅), 125.3 (C₇ and C_5′′), 127.1 (C_1′′), 129.2 (C₄), 130.5 (C_6′′ and C_3′′), 133.1 (C_2′′), 133.2 (C_4′′), 139.8 (C₃ and C_2′), 143.4 (C_5′), 153.2 (C_8a); IR (KBr, cm⁻¹): 3168 (N–H), 1581 (C=N); MS (ESI, m/z): 342.93, 344.95.

4.10.4 5-(4-Bromophenyl)-2-(2H-chromen-3-yl)-1H-imidazole (13e). Pale yellow solid; yield 52%; mp. 165–170 °C. ¹H NMR (CDCl₃) δ: 5.34 (s, OCH₂), 6.88 (m, 3H, H₈, H₆ and H_4′), 7.11 (d, J = 7.6 Hz, H₅), 7.19 (t, J = 6.4 Hz, H₇), 7.39 (s, H₄), 7.54 (d, J = 15.6 Hz, 2H, H_2′′ and H_6′′), 7.65 (d, J = 11.6 Hz, 2H, H_3′′ and H_5′′). ¹³C NMR (DMSO-d₆) δ: 64.7 (C₂), 115.5 (C₈), 118.9 (C_4a), 119.0 (C_4′), 121.7 (C₆), 122.3 (C₅), 123.0 (C_4′′), 126.3 (C₇), 127.1 (C_2′′ and C_6′′), 129.2 (C₄), 131.3 (C_1′′), 133.6 (C_3′′ and C_5′′), 139.8 (C₃ and C_2′), 143.4 (C_5′), 153.2 (C_8a); IR (KBr, cm⁻¹): 3017 (N–H), 1576 (C=N); MS (ESI, m/z): 354.94, 355.94.

4.10.5 5-(4-(Benzyloxy) phenyl)-2-(2H-chromen-3-yl)-1H-imidazole (13f). Pale yellow solid; yield 45%; mp. 170–175 °C. ¹H NMR (DMSO-d₆) δ: 5.12 (s, OCH₂), 5.48 (s, Bn–OCH₂), 6.9 (m, 3H, H₈, H₆ and H_4′), 7.04 (d, J = 8.0 Hz, 2H, H_3′′ and H_5′′), 7.23 (m, 2H, H₅ & H₇), 7.41 (m, 6H, H_{2′′′–6′′′} and H₄), 7.7 (d, J = 8.4 Hz, 2H, C_2′′ and C_6′′), 12.2 (s, NH). ¹³C NMR (DMSO-d₆) δ: 64.7 (C₂), 69.2 (Bn–O–C), 113.6 (C_3′′ and C_5′′), 114.8 (C₈), 115.4 (C_4a), 118.3 (C_4′), 121.7 (C₆), 122.4 (C₅), 125.6 (C₇), 125.9 (C_1′′), 126.9 (C_2′′′ and C_6′′′), 127.3 (C_4′′′), 127.7 (C_2′′ and C_6′′), 128.4 (C_3′′′ and C_5′′′), 129.1 (C₄), 137.1 (C₃ and C_2′), 140.9 (C_1′′′), 142.9 (C_5′), 153.2 (C_8a), 157.1 (C_4′′); IR (KBr, cm⁻¹): 3061 (N–H), 1575 (C=N); MS (ESI, m/z): 381.06.

4.10.6 2-(2H-Chromen-3-yl)-5-(4-fluorophenyl)-1H-imidazole (13g). Pale yellow solid; yield 40%; mp. 160–165 °C. ¹H NMR (CDCl₃) δ: 5.31 (d, J = 1.6 Hz, 2H, OCH₂), 6.84 (s, H₄), 6.88 (d, J = 8 Hz, H₈), 6.92 (td, J = 8.0, 1.6, 7.2, 1.2 Hz, H₆), 7.09 (m, 5H, H₅, H_2′′, H_3′′, H_5′′, H_6′′), 7.17 (td, J = 7.6, 1.2, 8.0, 1.6 Hz, 1H, H₇), 7.31 (s,H₄), 7.7 (s, NH). ¹³C NMR (DMSO-d₆) δ: 64.7 (C₂), 114.6 (C₈), 115.1 (C_4a), 115.3 (C_3′′′ and C_5′′′), 118.7 (C_4′), 121.7 (C₆), 122.3 (C₅), 126.1 (C₇), 126.2 (C_1′′), 127.0 (C_2′′ and C_6′′), 129.2 (C₄), 140.1 (C₃ and C_2′), 143.2 (C_5′), 153.2 (C_8a), 159.7 (C_4′′); IR (KBr, cm⁻¹): 3116 (N–H), 1577 (C=N); MS (ESI, m/z): 293.07.

4.10.7 2-(2H-Chromen-3-yl)-5-(4-methoxyphenyl)-1H-imidazole (13h). Light green solid; yield 42%; mp. 150–155 °C. ¹H NMR (CDCl₃) δ: 3.88 (s, OCH₃), 5.44 (s, OCH₂), 6.84 (s, H_4′), 6.86 (d, J = 8 Hz, H₈), 6.93 (td, J = 7.2, 0.8, 7.6, 1.2 Hz, H₆), 6.97 (d, J = 8.8 Hz, 2H, H_3′′ and H_5′′), 7.16 (dd, J = 7.6, 2.0, 7.2, 1.6 Hz, H₅), 7.25 (td, J = 8.0, 1.6, 7.6, 1.2 Hz, H₇), 7.59 (s, H₄), 7.71 (s, NH), 7.92 (d, J = 8.8 Hz, H_2′′ and H_6′′). ¹³C NMR (DMSO-d₆) δ: 55.6 (C₂), 65.5 (O–C), 113.6 (C_3′′ and C_5′′), 114.1 (C₈), 115.8 (C_4a), 120.6 (C_4′), 121.6 (C₆), 122.0 (C₅), 126.7 (C₇ and C_1′′), 129.4 (C_2′′ and C_6′′), 130.1 (C₄), 140.5 (C₃ and C_2′), 141.4 (C_5′), 150.6 (C_8a), 163.2 (C_4′′); IR (KBr, cm⁻¹): 3068 (N–H), 1573 (C=N); MS (ESI, m/z): 305.

4.10.8 3-(2-(2H-Chromen-3-yl)-1H-imidazol-5-yl) pyridine (13i). Brown solid; yield 45%; mp. 265–270 °C. ¹H NMR (DMSO-d₆) δ: 5.23 (s, OCH₃), 6.76 (d, J = 8.0 Hz, H₈), 6.96 (t, J = 7.2 Hz, H₆), 7.17 (m, 3H, H₅, H₄ and H_4′), 7.4 (t, J = 4.8 Hz, H₇), 7.96 (s, H_4′′), 8.15 (dt, J = 2.0 Hz, H_5′′), 8.42 (d, J = 3.6 Hz, H_6′′), 9.03 (s, H_2′′), 12.82 (s, NH). ¹³C NMR (DMSO-d₆) δ: 64.6 (C₂), 115.5 (C₈), 119.1 (C_4a), 121.7 (C_4′), 122.2 (C₆), 122.9 (C₅), 123.6 (C_5′′), 127.1 (C₇), 128.1 (C₄), 128.8 (C_3′′), 129.3 (C_4′′), 131.3 (C₃), 143.9 (C_2′), 145.8 (C_6′′ and C_5′), 147.3 (C_2′′), 153.2 (C_8a); IR (KBr, cm⁻¹): 3128 (N–H), 1573 (C=N); MS (ESI, m/z): 276.02 [M + 1]⁺.

4.10.9 2-(2H-Chromen-3-yl)-5-(thiophen-2-yl)-1H-imidazole (13j). Brown solid; yield 47%; mp. 170–175 °C. ¹H NMR (DMSO-d₆) δ: 5.27 (d, J = 1.6 Hz, OCH₂), 6.84 (s, H_4′), 6.86 (t, J = 9.2 Hz, H₈), 6.9 (td, J = 7.2, 1.2 Hz, H₆), 7.04 (m, 2H, H₅ and H_4′′), 7.15 (td, J = 8.0, 1.6, 7.6, 1.2 Hz, H₇), 7.23 (dd, J = 4.8, 0.8, 5.2, 1.2 Hz, H_3′′), 7.26 (s, H₄), 7.27 (d, J = 3.6 Hz, H_5′′). ¹³C NMR (DMSO-d₆) δ: 64.6 (C₂), 115.4 (C₈), 118.9 (C_4a), 121.7 (C_4′ and C₆), 122.2 (C_5′), 122.8 (C₅ and C_5′′), 127.1 (C₇ and C_3′′), 127.6 (C₄ and C_4′′), 129.2 (C₃ and C_2′′), 14.1 (C_2′), 153.2 (C_8a); IR (KBr, cm⁻¹): 3023 (N–H), 1578 (C=N); MS (ESI, m/z): 281.12 [M + 1]⁺, 282.16 [M + 2]⁺, 283.05 [M + 3]⁺.

4.10.10 2-(8-Methoxy-2H-chromen-3-yl)-5-phenyl-1H-imidazole (14a). White solid; yield 55%; mp. 145–150 °C. ¹H NMR (DMSO-d₆) δ: 3.78 (s, OCH₃), 5.19 (s, OCH₂), 6.78 (dd, J = 6.8, 2.0, 7.2, 2.4 Hz, H₇), 6.9 (m, 2H, H₅ and H_4′), 7.15 (s, H₄), 7.2 (t, J = 7.2, 7.6 Hz, H₆), 7.36 (t, J = 7.6, 8.0 Hz, 2H, H_3′′ and H_5′′), 7.44 (t, J = 7.6, 8.0 Hz, H_4′′), 7.82 (d, J = 7.6 Hz, 2H, H_2′′ and H_6′′), 12.69 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.6 (O–C), 64.6 (C₂), 112.8 (C₇), 114.8 (C_4a), 118.7 (C₅), 119.1 (C_4′), 121.4 (C₆), 122.3 (C_2′′ and C_6′′), 126.2 (C_4′′), 128.4 (C_3′′ and C_5′′), 128.8 (C₄), 134.3 (C_1′′), 141.0 (C₃ and C_2′), 142.0 (C_5′), 143.2 (C_8a), 147.5 (C₈); IR (KBr, cm⁻¹): 3119 (N–H), 1571 (C=N); MS (ESI, m/z): 305.06 [M + H]⁺.

4.10.11 5-(4-Bromophenyl)-2-(8-methoxy-2H-chromen-3-yl)-1H-imidazole (14e). Yellow solid; yield 55%; mp. 155–160 °C. ¹H NMR (CDCl₃-d₆): δ 3.89 (s, 3H, OCH₃), 5.34 (s, 2H, OCH₂), 6.71 (d, J = 6.8 Hz, 1H, H₇), 6.83 (m, 3H, H₆, H_4′ & H₅), 7.35 (s, 1H, H₄), 7.49 (d, J = 8.4 Hz, 2H, H_3′′ and H_5′′), 7.6 (d, J = 8 Hz, 2H, H_2′′ and H_6′′). ¹³C NMR (DMSO-d₆) δ: 55.6 (O–C), 64.5 (C₂), 112.9 (C₇), 115.0 (C_4a), 119.1 (C₅), 120.7 (C_4′), 121.4 (C₆), 122.9 (C_4′′), 126.3 (C₄), 128.7 (C_2′′ and C_6′′), 131.3 (C_1′′), 132.4 (C_3′′ and C_5′′), 142.0 (C₃ and C_2′), 143.5 (C_5′), 144.7 (C_8a), 147.5 (C₈); IR (KBr, cm⁻¹): 3123 (N–H), 1576 (C=N); MS (ESI, m/z): 386.24 [M + 2]⁺.

4.10.12 5-(4-(Benzyloxy)phenyl)-2-(8-methoxy-2H-chromen-3-yl)-1H-imdazole (14f). Pale green solid; yield 40%; mp. 150–160 °C. ¹H NMR (DMSO-d₆) δ: 3.77 (s, OCH₃), 5.12 (s, OCH₂), 5.16 (s, OCH₂), 6.82 (dd, J = 6.4, 2.0, 6.8, 2.4 Hz, H₇), 6.9 (m, 2H, H₆ and H₅), 7.04 (d, J = 6.8 Hz, 2H, H_3′′ and H_5′′), 7.16 (s, H_4′), 7.4 (m, 6H, H₄, Bn-5H), 7.72 (d, J = 8.4 Hz, 2H, H_2′′ and H_6′′), 12.5 (s, NH). ¹³C NMR (DMSO-d₆) δ: 56.1 (O–C), 65.1 (C₂), 69.7 (Bn–O–C), 113.3 (C₇), 115.4 (C_3′′, C_5′′ and C_4a), 119.0 (C_4′), 119.5 (C₅), 121.9 (C₆), 123.6 (C_1′′), 126.2 (C_2′′′ and C_6′′′), 127.9 (C_4′′′), 128.2 (C_2′′ and C_6′′), 128.3 (C_3′′′ and C_5′′′), 128.9 (C₄), 137.6 (C₃ and C_2′), 142.5 (C_5′), 143.6 (C_1′′′), 148.0 (C₈ and C_8a), 157.7 (C_4′′); IR (KBr, cm⁻¹): 3082 (N–H), 1576 (C=N); MS (ESI, m/z): 411.04 [M + 1]⁺.

4.10.13 5-(4-Fluorophenyl)-2-(8-methoxy-2H-chromen-3-yl)-1H-imidazole (14g). Pale green solid; yield 45%; mp. 135–140 °C. ¹H NMR (CDCl₃) δ: 3.9 (s, OCH₃), 5.36 (s, OCH₂), 6.72 (d, J = 7.2 Hz, H₇), 6.82 (d, J = 7.2 Hz, H₅), 6.88 (t, J = 7.2 Hz, H₆), 6.88 (s, H_4′), 7.08 (t, J = 8.4, 8.8 Hz, 2H, H_2′′ and H_6′′), 7.3 (s, H₄), 7.7 (t, J = 8.8, 10.8, 2H, H_3′′ and H_6′′). ¹³C NMR (DMSO-d₆) δ: 55.6 (O–C), 64.5 (C₂), 112.9 (C₇), 115.2 (C_4a), 115.4 (C_3′′ and C_5′′), 118.9 (C₅), 119.1 (C_4′), 121.4 (C₆), 122.0 (C_1′′), 126.2 (C_2′′ and C_6′′), 126.2 (C₄), 142.0 (C₃ and C_2′), 143.6 (C_5′), 147.5 (C_8a), 159.8 (C₈), 162.2 (C_4′′); IR (KBr, cm⁻¹): 3081 (N–H), 1579 (C=N); MS (ESI, m/z): 323.09 [M + 1]⁺.

4.10.14 2-(8-Methoxy-2H-chromen-3-yl)-5-(4-methoxyphenyl)-1H-imidazole (14h). Brown solid; yield 40%; mp. 150–155 °C. ¹H NMR (DMSO-d₆) δ: 3.77 (s, 2 × OCH₃), 5.17 (s, OCH₂), 6.78 (dd, J = 6.0, 2.8 Hz, H₆), 6.89 (s, H_4′), 6.9 (d, J = 2.8 Hz, 2H, H₇ and H₅), 6.96 (d, J = 8.0 Hz, 2H, H_3′′ and H_5′′), 7.16 (s, H₄), 7.72 (d, J = 8.8 Hz, 2H, H_2′′ and H_6′′), 12.61 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.5 (O–C), 56.1 (O–C), 65.1 (C₂), 113.2 (C₇), 114.5 (C_3′′ and C_5′′), 118.9 (C_4a), 119.5 (C_4′ and C₅), 121.9 (C₆), 123.6 (C_1′′), 126.2 (C_2′′ and C_6′′), 129.1 (C₄), 136.5 (C₃ and C_2′), 142.5 (C_5′), 143.6 (C_8a), 148.0 (C₈), 158.6 (C_4′′); IR (KBr) cm⁻¹: 3130 (N–H), 1575 (C=N); MS (ESI, m/z): 335.12 [M + 1]⁺.

4.10.15 3-(2-(8-Methoxy-2H-chromen-3-yl)-1H-imidazole-5-yl)pyridine (14i). Brown solid; yield 40%; mp. 225–230 °C. ¹H NMR (DMSO-d₆) δ: 3.78 (s, OCH₃), 5.19 (s, OCH₂), 6.8 (d, J = 6 Hz, H₇), 6.9 (m, 2H, H₅ and H₆), 7.19 (s, H_4′), 7.41 (t, J = 4.8, 6.0 Hz, H_5′′), 7.93 (s, H₄), 8.15 (d, J = 8.0 Hz, H_6′′), 8.42 (d, J = 3.2 Hz, H_4′′), 9.03 (s, H_2′′), 12.82 (s, NH). ¹³C NMR (DMSO-d₆) δ: 56.1 (O–C), 65.0 (C₂), 113.5 (C₇), 119.6 (C_4a), 119.8 (C₅), 121.9 (C_4′), 123.4 (C₆), 124.1 (C_5′′), 128.0 (C₄), 133.0 (C_4′′), 136.0 (C₃ and C_2′), 131.8 (C_3′′), 142.6 (C₅), 144.4 (C_6′′), 146.3 (C_8a), 147.8 (C_2′′), 148.0 (C₈); IR (KBr, cm⁻¹): 3085 (N–H), 1581 (C=N); MS (ESI, m/z): 306.12 [M + 1]⁺.

4.10.16 5-2-(8-Methoxy-2H-chromen-3-yl)-5-(thiophen-2-yl)-1H-imidazole (14j). Pale green solid; yield 40%; mp. 155–160 °C. ¹H NMR (DMSO-d₆) δ: 3.77 (s, OCH₃), 5.14 (s, OCH₂), 6.78 (dd, J = 6.4, 1.6, 6.8, 2.0 Hz, H₆), 6.91 (m, 2H, H₇ and H_4′), 7.05 (dd, J = 4.8, 3.6 Hz, H_4′′), 7.14 (s, H₄), 7.31 (d, J = 3.6 Hz, H_3′′), 7.36 (d, J = 5.2 Hz, H₅), 7.67 (d, J = 1.6 Hz, H_5′′), 12.7 (s, NH). ¹³C NMR (DMSO-d₆) δ: 56.1 (O–C), 65.0 (C₂), 113.4 (C₇), 114.6 (C_4a), 119.6 (C₅ and C_4′), 121.9 (C_6′), 122.0 (C_5′), 123.3 (C_5′′), 123.5 (C_3′′), 123.8 (C_4′′), 128.1 (C₄), 137.0 (C_2′′), 138.6 (C_2′), 142.5 (C₃), 143.5 (C_8a), 148.0 (C₈); IR (KBr, cm⁻¹): 3086 (N–H), 1574 (C=N); MS (ESI, m/z): 311.01 [M + 1]⁺, 312.16 [M + 2]⁺, 313.06 [M + 3]⁺.

4.10.17 2-(7-Methoxy-2H-chromen-3-yl)-5-phenyl-1H-imidazole (15a). Yellow solid; yield 50%; mp. 155–160 °C. ¹H NMR (CDCl₃ + DMSO-d₆) δ: 3.78 (s, OCH₃), 5.28 (s, OCH₂), 6.46 (s,H₈), 6.48 (d, J = 2.4 Hz, H₆), 6.81 (s, H_4′), 6.96 (d, J = 8.0 Hz, H₅), 7.27 (t, J = 7.6, 4.0 Hz, H_4′′), 7.34 (s, H₄), 7.39 (t, J = 7.6 Hz, 2H, H_3′′ and H_5′′), 7.69 (d, J = 7.2 Hz, 2H, H_2′′ and H_6′′), 12.12 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.2 (O–C), 64.8 (C₂), 101.5 (C₈), 107.6 (C₆), 115.4 (C_4a), 118.7 (C_4′), 124.3 (C_2′′ and C_6′′), 127.8 (C₅), 128.5 (C₄, C_3′′ and C_5′′), 134.2 (C_1′′), 138.7 (C_2′ and C₃), 143.7 (C_5′), 154.5 (C_8a), 160.4 (C₇); IR (KBr, cm⁻¹): 3000 (N–H), 1572 (C=N); MS (ESI, m/z): 305.06 [M + 1]⁺.

4.10.18 5-(4-Bromophenyl)-2-(7-methoxy-2H-chromen-3-yl)-1H-imidazole (15e). Yellow solid; yield 45%; mp. 175–180 °C. ¹H NMR (CDCl₃) δ: 3.79 (s, OCH₃), 5.27 (s, OCH₂), 6.47 (s, H₈), 6.5 (d, J = 2.0 Hz, H₆), 6.8 (s, H_4′), 6.98 (d, J = 8.4 Hz, H₅), 7.32 (s, H₄), 7.5 (d, J = 8.8 Hz, 2H, H_2′′ and H_6′′), 7.6 (d, J = 7.6 Hz, 2H, H_3′′ and H_5′′). ¹³C NMR (DMSO-d₆) δ: 55.3 (O–C), 64.7 (C₂), 101.5 (C₈), 107.7 (C₆), 115.3 (C_4a), 118.9 (C_4′), 119.9 (C_4′′), 126.2 (C₅, C_2′′ and C_6′′), 127.9 (C₄, C_3′′ and C_5′′), 131.3 (C_1′′), 137.8 (C_2′ and C₃), 143.9 (C_5′), 154.6 (C_8a), 160.5 (C₇); IR (KBr, cm⁻¹): 3085 (N–H), 1570 (C=N); MS (ESI, m/z): 386[M + 2]⁺.

4.10.19 5-(4-(Benzyloxy)phenyl)-2-(7-methoxy-2H-chromen-3-yl)-1H-imidazole (15f). Yellow solid; yield 45%; mp. 320–330 °C. ¹H NMR (DMSO-d₆) δ: 3.75 (s, OCH₃), 4.88 (s, OCH₂), 5.12 (s, OCH₂), 6.55 (d, J = 8.4, 2H, H_3′′ and H_5′′), 7.11 (d, J = 8.4 Hz, H₆), 7.25 (d, J = 8.0 Hz, H₅), 7.41 (s, H₄), 7.44 (t, J = 7.6 Hz, 3H, H_3′′′, H_4′′′ and H_5′′′), 7.47 (d, J = 6.8 Hz, 2H, H_2′′ and H_6′′), 7.72 (d, J = 8.4 Hz, 2H, H_2′′ and H_6′′), 12.6 (s, NH). ¹³C NMR (DMSO-d₆) δ: 56.1 (O–C), 65.1 (C₂), 69.7 (Bn–O–C), 113.3 (C₈), 115.4 (C_3′′, C_5′′ and C_4a), 119.0 (C_4′), 119.5 (C₅), 121.9 (C₆), 123.6 (C_1′′), 126.2 (C_2′′′ and C_6′′′), 127.9 (C_4′′′), 128.2 (C_2′′ and C_6′′), 128.3 (C_3′′′ and C_5′′′), 128.9 (C₄), 137.6 (C₃ and C_2′), 142.5 (C_5′), 143.6 (C_1′′′), 148.0 (C₇ and C_8a), 157.7 (C_4′′); IR (KBr, cm⁻¹): 3007 (N–H), 1597 (C=N); MS (ESI, m/z): 411.04 [M + 1]⁺.

4.10.20 5-(4-Fluorophenyl)-2-(7-methoxy-2H-chromen-3-yl)-1H-imidazole (15g). Yellow solid; yield 50%; mp. 165–170 °C. ¹H NMR (DMSO-d₆) δ: 3.75 (s, OCH₃), 5.17 (s, OCH₂), 6.49 (d, J = 2.4 Hz, H₈), 6.55 (dd, J = 8.0, 2.4, 8.4, 2.8 Hz, H₆), 7.1 (d, J = 8.4 Hz, H₅), 7.14 (s, H_4′), 7.2 (t, J = 6.8 Hz, 2H, H_2′′ and H_6′′), 7.73 (s, H₄), 7.83 (dd, J = 8.8, 8.0 Hz, 2H, H_2′′ and H_6′′), 12.57 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.3 (O–C), 64.8 (C₂), 101.5 (C₈), 107.6 (C₆), 115.3 (C_4a), 118.8 (C_3′′ and C_5′′), 120.0 (C_4′), 126.1 (C₅), 126.2 (C_1′′), 127.8 (C₄, C_2′′ and C_6′′), 139.2 (C_2′ and C₃), 143.7 (C_5′), 154.5 (C_8a), 160.0 (C₄), 162.1 (C_4′′); IR (KBr, cm⁻¹): 3039 (N–H), 1572 (C=N); MS (ESI, m/z): 323 [M + 1]⁺.

4.10.21 2-(7-Methoxy-2H-chromen-3-yl)-5-(4-methoxyphenyl)-1H-imidazole (15h). Brown solid; yield 45%; mp. 135–140 °C. ¹H NMR (DMSO-d₆) δ: 3.75 (s, OCH₃), 3.85 (s, OCH₃), 5.5 (s, OCH₂), 6.45 (m, 2H, H_3′′ and H_5′′), 6.6 (s, H_8′), 6.9 (m, 3H, H_6′, H_4′), 7.45 (m, 1H), 7.6 (m, H₅), 7.8 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.0 (O–C), 58.3 (O–C), 64.8 (C₂), 101.5 (C₈), 107.6 (C₆), 114.0 (C_4a), 115.3 (C_3′′ and C_5′′), 119.9 (C_4′), 124.8 (C_1′′), 125.7 (C₅), 127.8 (C_2′′ and C_6′′), 128.5 (C₄), 139.5 (C_2′ and C₃), 140.0 (C_5′), 154.5 (C_8a), 160.3 (C_4′′), 160.4 (C₇); IR (KBr, cm⁻¹): 3003 (N–H), 1570 (C=N); MS (ESI, m/z): 334.96 [M + 1]⁺.

4.10.22 3-(2-(7-Methoxy-2H-chromen-3-yl)-1H-imidazol-5-yl)pyridine (15i). Brown solid; yield 50%; mp. 240–245 °C. ¹H NMR (DMSO-d₆) δ: 3.78 (s, OCH₃), 5.2 (s, OCH₂), 6.8 (d, J = 6.0 Hz, H₈), 6.85 (m, 2H, H₅ and H₆), 7.19 (s, H_4′), 7.4 (t, J = 6.0 Hz, H_5′′′), 7.95 (s, H₄), 8.15 (d, J = 8 Hz, H_6′′), 8.45 (d, J = 3.2 Hz, H_4′′), 9.1 (s, H_2′′), 12.8 (s, NH). ¹³C NMR (DMSO-d₆) δ: 56.1 (O–C), 65.0 (C₂), 113.5 (C₈), 119.6 (C_4a), 119.8 (C₅), 121.9 (C_4′), 123.4 (C₆), 124.1 (C_5′′), 128.0 (C₄), 133.0 (C_4′′), 136.0 (C₃ and C_2′), 131.8 (C_3′′), 142.6 (C₅), 144.4 (C_6′′), 146.3 (C_8a), 147.8 (C_2′′), 148.0 (C₇); IR (KBr, cm⁻¹): 3085 (N–H), 1581 (C=N); MS (ESI, m/z): 306 [M + 1]⁺.

4.10.23 2-(7-Methoxy-2H-chromen-3-yl)-5-(thiophen-2-yl)-1H-imidazole (15j). Brown solid; yield 40%; mp. 120–124 °C. ¹H NMR (DMSO-d₆) δ: 3.75 (s, OCH₃), 5.14 (s, OCH₂), 6.49 (s, H_4′), 6.54 (dd, J = 8.4, 2.4 Hz, H₈), 7.04 (t, J = 4.0, 4.4 Hz, H_4′′), 7.09 (s, H₄), 7.12 (d, J = 4.8 Hz, H₆), 7.29 (d, J = 2.8 Hz, H_3′′), 7.35 (d, J = 4.8 Hz, H₅), 7.63 (d, J = 1.6 Hz, H_5′′), 12.6 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.2 (O–C), 64.7 (C₂), 101.5 (C₈), 107.7 (C₆), 113.7 (C_4a), 119.0 (C_4′), 121.3 (C_5′), 123.2 (C_5′′), 127.5 (C₅ and C_3′′), 127.9 (C_4′′), 136.3 (C₃), 138.3 (C_2′′), 143.3 (C_2′), 154.5 (C_8a), 160.4 (C₇); IR (KBr, cm⁻¹): 3111 (N–H), 1604 (C=N); MS (ESI, m/z): 311.01 [M + 1]⁺, 312.16 [M + 2]⁺, 313.06 [M + 3]⁺.

4.10.24 2-(5-Methoxy-2H-chromen-3-yl)-5-phenyl-1H-imidazole (16a). White solid; yield 60%; mp. 165–170 °C. ¹H NMR (CDCl₃) δ: 3.86 (s, OCH₃), 5.28 (s, OCH₂), 6.47 (d, J = 8.4 Hz, H₈), 6.54 (d, J = 8.4 Hz, H₆), 7.1 (t, J = 8.4 Hz, H₇), 7.17 (s, H_4′), 7.26 (s, H₄), 7.28 (d, J = 7.2 Hz, 2H, H_2′′ and H_6′′), 7.38 (dd, J = 8.0, 15.4 Hz, 3H, H_3′′′, H_4′′′ and H_5′′′), 7.69 (s, NH). ¹³C NMR (DMSO-d₆) δ: 56.1 (OCH₃), 64.8 (C₂), 104.6 (C₈), 108.9 (C₆), 112.1 (C_4a), 114.1 (C_4′), 121.6 (C_2′′ and C_6′′), 124.8 (C_4′′), 129.7 (C_1′′), 128.9 (C₇, C_3′′ and C_5′′), 134.8 (C₃ and C_2′), 144.2 (C_5′), 154.4 (C_8a), 155.8 (C₅); IR (KBr, cm⁻¹): 3134 (N–H), 1584 (C=N); MS (ESI, m/z): 305.13 [M + 1]⁺.

4.10.25 5-(4-Chlorophenyl)-2-(5-methoxy-2H-chromen-3-yl)-1H-imidazole (16b). Yellow solid; yield 60%; mp. 170–174 °C. ¹H NMR (CDCl₃) δ: 3.85 (s, OCH₃), 5.27 (s, OCH₂), 6.47 (d, J = 8.4 Hz, H₈), 6.54 (d, J = 8.0 Hz, H₆), 7.11 (t, J = 8.4, 8.0 Hz, H₇), 7.16 (s, H_4′), 7.26 (s, H₄), 7.33 (m, 4H, H_2′′, H_3′′, H_5′′ & H_6′′), 7.7 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.7 (OCH₃), 64.8 (C₂), 103.6 (C₈), 108.9 (C₆), 111.6 (C_4a), 114.9 (C_4′), 126.1 (C_2′′ and C_6′′), 128.8 (C₄, C_3′′ and C_5′′), 129.7 (C_1′′ and C₇), 132.5 (C_4′′), 132.6 (C₃ and C_2′), 144.8 (C_5′), 154.7 (C_8a), 155.6 (C₅); IR (KBr, cm⁻¹): 3015 (N–H), 1582 (C=N); MS (ESI, m/z): 339.08 [M + 1]⁺, 341.11 [M + 2]⁺.

4.10.26 5-(2,4-Dichlorophenyl)-2-(5-methoxy-2H-chromen-3-yl)-1H-imidazole (16c). Off white solid; yield 55%; mp. 190–195 °C. ¹H NMR (CDCl₃) δ: 3.87 (s, OCH₃), 5.27 (s, OCH₂), 6.48 (d, J = 7.6 Hz, H₈), 6.55 (d, J = 8.0 Hz, H₆), 7.12 (t, J = 8.4, 8.0 Hz, H₇), 7.18 (s, H_4′), 7.26 (s, H₄), 7.29 (d, J = 2.0 Hz, H_5′′), 7.32 (d, J = 2.0 Hz, H_3′′), 7.44 (d, J = 2.0 Hz, H_6′′), 7.71 (s, NH). ¹³C NMR (DMSO-d₆) δ: 56.1 (OCH₃), 64.7 (C₂), 104.7 (C₈), 108.9 (C₆), 114.9 (C_4′), 127.8 (C_1′′ and C₄), 129.8 (C₇), 130.1 (C_6′′), 130.7 (C_3′′), 130.8 (C_2′′), 131.4 (C_4′′), 136.4 (C₃ and C_2′), 143.8 (C_5′), 154.5 (C_8a), 155.9 (C₅); IR (KBr, cm⁻¹): 3013 (N–H), 1583 (C=N); MS (ESI, m/z): 373.06 [M + 1]⁺, 375.02 [M + 2]⁺.

4.10.27 2-(5-Methoxy-2H-chromen-3-yl)-5-(3-nitrophenyl)-1H-imidazole (16d). Orange solid; yield 55%; mp. 245–250 °C. ¹H NMR (DMSO-d₆) δ: 3.86 (s, OCH₃), 5.18 (s, OCH₂), 6.52 (d, J = 8.0 Hz, H₈), 6.64 (d, J = 8.0 Hz, H₆), 7.14 (t, J = 8.4, 8.0 Hz, H₇), 7.44 (s, H₄), 7.66 (t, J = 8.0 Hz, H_5′′), 8.03 (s, H_4′), 8.05 (d, J = 7.2 Hz, H_6′′), 8.25 (d, J = 7.6 Hz, H_4′′), 8.6 (s, H_2′′), 12.9 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.6 (OCH₃), 64.2 (C₂), 104.2 (C₈), 108.4 (C₆), 111.4 (C_4a), 114.3 (C_4′), 118.2 (C_2′′), 120.6 (C_4′′), 120.8 (C_5′′), 129.5 (C₇ and C₄), 130.0 (C_6′′), 130.4 (C_1′′), 136.1 (C_2′), 138.6 (C₃), 142.2 (C_5′), 148.3 (C_3′′), 153.9 (C_8a), 155.9 (C₅); IR (KBr, cm⁻¹): 3092 (N–H), 1580 (C=N); MS (ESI, m/z): 350.1 [M + 1]⁺.

4.10.28 5-(4-Bromophenyl)-2-(5-methoxy-2H-chromen-3-yl)-1H-imidazole (16e). Off white solid; yield 60%; mp. 195–200 °C. ¹H NMR (CDCl₃) δ: 3.85 (s, OCH₃), 5.26 (s, OCH₂), 6.47 (d, J = 8.4 Hz, H₈), 6.54 (d, J = 8.0 Hz, H₆), 7.11 (t, J = 8.4, 8.0 Hz, H₇), 7.17 (s, H4′), 7.33 (s, H₄), 7.49 (d, J = 8.0 Hz, 4H, H_2′′, H_3′′, H_5′′ and H_6′′), 7.62 (s, NH). ¹³C NMR (DMSO-d₆) δ: 55.7 (OCH₃), 64.8 (C₂), 103.6 (C₈), 109.0 (C₆), 111.6 (C_4a), 114.9 (C_4′), 120.6 (C_4′′), 126.4 (C₄, C_2′′ and C_6′′), 129.8 (C₇ and C_1′′), 131.8 (C_3′′ and C_5′′), 137.0 (C_2′ and C₃), 144.8 (C_5′), 155.6 (C_8a), 154.7 (C₅); IR (KBr, cm⁻¹): 3135 (N–H), 1583 (C=N); MS (ESI, m/z): 385.02 [M + 1]⁺, 386.02 [M + 2]⁺.

4.10.29 5-(4-(Benzyloxy)phenyl)-2-(5-methoxy-2H-chromen-3-yl)-1H-imidazole (16f). Pale green solid; yield 54%; mp. 175–180 °C. ¹H NMR (CDCl₃) δ: 3.86 (s, OCH₃), 5.09 (s, OCH₂), 5.27 (d, J = 0.8 Hz, Bn–OCH₂), 6.47 (d, J = 8.4 Hz, H₈), 6.54 (d, J = 8.0 Hz, H₆), 7.02 (d, J = 8.8 Hz, 2H, H_3′′ and H_5′′), 7.1 (t, J = 8.4 Hz, H₇), 7.14 (s, H₄), 7.25 (s, H₄), 7.34 (d, J = 7.2 Hz, 2H, H_2′′′ and H_6′′′), 7.39 (t, J = 6.8, 7.6 Hz, 3H, H_3′′′, H_4′′′ and H_5′′′), 7.44 (d, J = 7.2 Hz, H_2′′ & H_6′′), 7.6 (s, NH). ¹³C NMR (CDCl₃); δ 55.6 (OCH3), 64.9 (C₂), 70.1 (Bn–O–C), 103.6 (C₈), 108.9 (C₆), 111.7 (C_4a), 114.3 (C_3′′ and C_5′′), 115.2 (C_4′), 120.7 (C_1′′), 126.2 (C_2′′′ and C_6′′′), 127.5 (C_4′′′), 128.0 (C_2′′ and C_6′′), 128.6 (C₄, C_3′′′ and C_5′′′), 129.5 (C₇), 136.9 (C₃ and C_2′), 143.7 (C_5′s), 144.3 (C_1′′′), 154.7 (C_8a), 155.6 (C₅), 158.1 (C_4′′); IR (KBr, cm⁻¹): 3072 (N–H), 1580 (C=N); MS (ESI, m/z): 411.14 [M + 1]⁺.

5. Biological assay³⁶

5.1 Angiogenesis assay

Human clonal endothelial colony forming cells (ECFC, Engenator) were cultured in an EGM-2MV media containing 10% FBS (EGM-2MV + FBS). The ECFCs were expanded to passage 7, rapidly frozen and stored in liquid N₂ until further use. Human adipose derived stem cells (ADSC, Zen Bio) were derived from pooled donor samples, cultured in EGM-2MV + FBS, rapidly frozen and stored in liquid N₂ until further use. On day 1, the ECFCs were quickly thawed, diluted into a pre-warmed EGM-2 media containing 10% FBS (EGM-2 + FBS), and incubated for 24 h. The ECFCs were washed with PBS, trypsinized and replated with EGM-2 + FBS for 48 h. On the third day, the ADSCs were quickly thawed and diluted into pre-warmed EGM-2MV + FBS. The ADSCs were collected by centrifugation and suspended in the Optimized Media (TCS Cell works). The ADSCs (5000 per well) were added to a 384 well Cell Bind plate (Corning), incubated at room temperature for 5 min, and then placed overnight in a CO₂ incubator. On the fourth day, the ECFC cultures from day 2 were washed with PBS, trypsinized and resuspended with EGM-2 + FBS. The cells were collected by centrifugation, suspended in the Optimized Media and passed through a sterile 23 gauge needle. The ECFCs (500 per well) were overlaid onto the ADSC feeder layer from day 3 and returned to the CO₂ incubator. After 2 h, rhVEGF (10 ng ml⁻¹; R&D Systems) and the compounds at the indicated concentrations were added (0.5% (v/v) DMSO final) and incubated for 96 h. The maximum and minimum responses correspond to rhVEGF + 0.5% (v/v) DMSO orrhVEGF + 500 nM sutent (Sunitinib), respectively.

5.2 Endothelial and nuclear staining

Co-cultures were fixed with 3.7% formaldehyde–PBS for 20 min, treated with 0.1% TX-100–PBS for 20 min and washed twice with PBS, and incubated overnight with 63 ng ml⁻¹ mouse Anti-Human CD31 antibody (BD Pharmigen, 550389) in PBS containing 1% BSA at 4 °C. Following the PBS wash, the samples were then incubated with 3 μg ml⁻¹ Alexa 488 goat secondary antibody in PBS for 1 h, and then washed twice with PBS before staining the cellular DNA with Hoechst stain (2 μg ml⁻¹).

5.3 Cellular imaging

The fixed and stained ECFC–ADSC co-cultures were analyzed with an Array Scan VTI (Thermo Scientific) using a 5× objective and collecting 4, non-adjacent frames per well; the nuclei and CD31 were visualized using the XF93-Hoechst and XF93-Alexa-488 dichroic mirror emission filter pairs, respectively. The endothelial tube features were determined with the Cellomics Tube Formation V3 application by the analysis of the CD31 channel. Relative cell death in the ECFC–ADSC co-culture was measured by the loss of valid nuclear objects as determined by the Target Activation V3 applications.

6. Molecular docking studies

With a view to find a correlation between the anti-angiogenesis activity of the 5-(substituted aryl)-2-(5-methoxy-2H-chromen-3-yl)-1H-imidazoles (16a–f, 15h, 14f, h, j, 13f, g, i, j) and their binding with the target proteins viz.; KRAS, Wnt and VEGF, the docking studies were performed employing the AutoDocksoftware.^37,38 The receptor–ligand interactions and the corresponding interaction energies were identified after the docking studies.

Since the Human KRAS protein is a target in anti-angiogenesis,^39–43 we selected the crystal structure of the Human KRAS receptor protein with PDB ID: (4EPX·pdb) from the protein data bank.⁴⁴ The docking studies were performed with fourteen molecules (16a–f, 15h, 14f, 14h, 14j, 13f, 13g, i, j) to investigate the receptor interactions. All of the fourteen molecules were constructed using the tools of the SYBYL software 2 and the structures were minimized using 500 steps of the steepest descent followed by 500 steps of the conjugate gradient methods and the structure was stabilized at 1100 steps. Later the binding/catalytic sites of protein 4EPX·pdb were identified using the biopolymer module of the SYBYL-X (Tripos) software.

Further, the molecules were loaded into the AutoDock software,⁴⁵ and converted into pdb structures by applying the charge assigning method of Kolman and Gasteiger Huckle, and then saved into a pdbqt form. Later, polar atoms were added to the protein and the protein structure was maintained in the grid N-dimension (Grid sizes for receptor X = 11.393, Y = 1.498, Z = 9.595; ligand X = 40, Y = 36, Z = 38) for fourteen molecules. The virtual screening studies were performed using the AutoDock 4.0 software to predict the interactions based on the Genetic and Lamarckian algorithms.⁴⁶ Docking was performed and 140 results were generated on the basis of their energy values. The results were arranged in chronological order and analyzed.

Similarly the above procedure was also applied to perform the docking studies for the Wnt receptor PDB ID: (3FOA.pdb)⁴⁷ and PDB ID: Vegf (2VPF.pdb).⁴⁸

Acknowledgements

The Biology assays were carried out by Eli Lilly Open Innovation Drug Discovery (OIDD) (https://openinnovation.lilly.com). We thank Dr Vittal Venkatasatya Kurisetty for helpful discussions in the analysis of biological assay data.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra09945a

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