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

Gopinath Gudipudia, Someswar R. Sagurthib, Shyam Peruguc, G. Achaiahd and G. L. David Krupadanam*a
aDepartment of Chemistry, Osmania University, Hyderabad, India 500 007. E-mail: gldkrupa@gmail.com; Tel: +91-9849128145
bDepartment of Genetics, Osmania University, Hyderabad, India 500 007
cDepartment of Biochemistry, Osmania University, Hyderabad, India 500 007
dMedicinal Chemistry Division, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, India 500 009

Received 6th September 2014 , Accepted 1st October 2014

First published on 2nd October 2014


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.


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.
image file: c4ra09945a-f1.tif
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).
image file: c4ra09945a-s1.tif
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.

image file: c4ra09945a-s2.tif
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.

image file: c4ra09945a-s3.tif
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.

image file: c4ra09945a-s4.tif
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.

image file: c4ra09945a-s5.tif
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.

image file: c4ra09945a-s6.tif
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.

image file: c4ra09945a-s7.tif
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 5th 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 linesa
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


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


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 4th position of the phenyl ring and a methoxy group on the 5th 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



image file: c4ra09945a-f2.tif
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).
image file: c4ra09945a-f3.tif
Fig. 3 H-bonding interactions between amino acid residues at the active site of the Human KRAS target and compound 16f.

image file: c4ra09945a-f4.tif
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).
image file: c4ra09945a-f5.tif
Fig. 5 H-bonding interactions between amino acid residues at active site of the Wnt target and compound 16f.

image file: c4ra09945a-f6.tif
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).
image file: c4ra09945a-f7.tif
Fig. 7 H-bonding interactions between amino acid residues at the active site of the VEGF target and compound 16f.

image file: c4ra09945a-f8.tif
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 5th position of the chromene and the 4th 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 5th 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 (6th 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 (C4a), 117.3 (CN), 121.4 (C6), 126.5 (C5), 128.4 (C7), 141.8 (C4), 157.5 (C8a).
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 (C4a), 118.9 (C5), 117.0 (CN), 125.2 (C6), 138.7 (C4), 154.9 (C8a), 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 (C4a), 113.4 (C3), 117.0 (CN), 129.6 (C5), 138.7 (C4), 155.9 (C8a), 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 (C4a), 110.3 (C3), 117.0 (CN), 133.0 (C7), 134.4 (C4), 155.1 (C5), 156.3 (C8a).

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 (C4a), 121.4 (C6), 121.5 (C5), 123.4 (C7), 132.4 (C3), 143.3 (C4), 147.5 (C8a), 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 (C4a), 121.4 (C5), 121.5 (C6), 123.4 (C3), 132.4 (C4), 143.4 (C8a), 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 (C4a), 119.8 (C5), 130.1 (C3), 132.4 (C4), 156.0 (C8a), 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 (C4a), 121.3 (C7), 127.0 (C3), 132.3 (C4), 155.1 (C5), 156.4 (C8a), 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[double bond, length as m-dash]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[double bond, length as m-dash]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[double bond, length as m-dash]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[double bond, length as m-dash]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[double bond, length as m-dash]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, H1′–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 (C2′ and C6′), 127.4 (C4), 127.7 (C3′ and C5′), 128.1 (C1), 130.2 (C2 and C6), 140.2 (C1′), 166.4 (C4), 190.3 (C[double bond, length as m-dash]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[double bond, length as m-dash]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[double bond, length as m-dash]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[double bond, length as m-dash]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[thin space (1/6-em)]:[thin space (1/6-em)]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[double bond, length as m-dash]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, H4′), 7.29 (d, J = 7.2 Hz, 2H, H2′′ & H6′′), 7.34 (s, H4), 7.37 (t, J = 8.0, 2.8 Hz, 3H, H3′′, H4′′ and H5′′), 7.69 (s, NH), 13C NMR (DMSO-d6) δ: 64.7 (C2), 114.8 (C8), 115.4 (C4a), 118.6 (C4′), 121.7 (C6), 122.4 (C5), 123.2 (C7), 124.3 (C2′′ & C6′′), 126.3 (C4′′), 127.0 (C3′′ and C5′′), 128.4 (C4), 129.1 (C1′′), 134.3 (C2′), 141.0 (C3), 143.2 (C5′), 153.2 (C8a); IR (KBr, cm−1): 3162 (N–H), 1578 (C[double bond, length as m-dash]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, H4′), 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, H2′, H3′, H5′ and H6′), 7.71 (s, NH). 13C NMR (DMSO-d6) δ: 64.7 (C2), 115.4 (C8), 115.5 (C4a), 118.9 (C4′), 121.7 (C6), 122.3 (C5), 125.9 (C7), 127.1 (C2′′ and C6′′), 128.4 (C3′′and C5′′), 129.2 (C4), 130.5 (C1′′), 133.2 (C4′′), 139.8 (C2′ and C3), 143.4 (C5′), 153.2 (C8a); IR (KBr, cm−1): 3162 (N–H), 1577 (C[double bond, length as m-dash]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, H4′), 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 H3′′), 7.2 (t, J = 7.6 Hz, H7), 7.28 (s, 2H, H4 and H5′′), 7.34 (d, J = 10.4 Hz, H2′′), 7.47 (s, NH). 13C NMR (CDCl3) δ: 64.6 (C2), 115.5 (C8), 115.84 (C4a), 118.9 (C4′), 121.75 (C6), 122.2 (C5), 125.3 (C7 and C5′′), 127.1 (C1′′), 129.2 (C4), 130.5 (C6′′ and C3′′), 133.1 (C2′′), 133.2 (C4′′), 139.8 (C3 and C2′), 143.4 (C5′), 153.2 (C8a); IR (KBr, cm−1): 3168 (N–H), 1581 (C[double bond, length as m-dash]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 H4′), 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, H2′′ and H6′′), 7.65 (d, J = 11.6 Hz, 2H, H3′′ and H5′′). 13C NMR (DMSO-d6) δ: 64.7 (C2), 115.5 (C8), 118.9 (C4a), 119.0 (C4′), 121.7 (C6), 122.3 (C5), 123.0 (C4′′), 126.3 (C7), 127.1 (C2′′ and C6′′), 129.2 (C4), 131.3 (C1′′), 133.6 (C3′′ and C5′′), 139.8 (C3 and C2′), 143.4 (C5′), 153.2 (C8a); IR (KBr, cm−1): 3017 (N–H), 1576 (C[double bond, length as m-dash]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 H4′), 7.04 (d, J = 8.0 Hz, 2H, H3′′ and H5′′), 7.23 (m, 2H, H5 & H7), 7.41 (m, 6H, H2′′′–6′′′ and H4), 7.7 (d, J = 8.4 Hz, 2H, C2′′ and C6′′), 12.2 (s, NH). 13C NMR (DMSO-d6) δ: 64.7 (C2), 69.2 (Bn–O–C), 113.6 (C3′′ and C5′′), 114.8 (C8), 115.4 (C4a), 118.3 (C4′), 121.7 (C6), 122.4 (C5), 125.6 (C7), 125.9 (C1′′), 126.9 (C2′′′ and C6′′′), 127.3 (C4′′′), 127.7 (C2′′ and C6′′), 128.4 (C3′′′ and C5′′′), 129.1 (C4), 137.1 (C3 and C2′), 140.9 (C1′′′), 142.9 (C5′), 153.2 (C8a), 157.1 (C4′′); IR (KBr, cm−1): 3061 (N–H), 1575 (C[double bond, length as m-dash]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, H2′′, H3′′, H5′′, H6′′), 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 (C4a), 115.3 (C3′′′ and C5′′′), 118.7 (C4′), 121.7 (C6), 122.3 (C5), 126.1 (C7), 126.2 (C1′′), 127.0 (C2′′ and C6′′), 129.2 (C4), 140.1 (C3 and C2′), 143.2 (C5′), 153.2 (C8a), 159.7 (C4′′); IR (KBr, cm−1): 3116 (N–H), 1577 (C[double bond, length as m-dash]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, H4′), 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, H3′′ and H5′′), 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, H2′′ and H6′′). 13C NMR (DMSO-d6) δ: 55.6 (C2), 65.5 (O–C), 113.6 (C3′′ and C5′′), 114.1 (C8), 115.8 (C4a), 120.6 (C4′), 121.6 (C6), 122.0 (C5), 126.7 (C7 and C1′′), 129.4 (C2′′ and C6′′), 130.1 (C4), 140.5 (C3 and C2′), 141.4 (C5′), 150.6 (C8a), 163.2 (C4′′); IR (KBr, cm−1): 3068 (N–H), 1573 (C[double bond, length as m-dash]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 H4′), 7.4 (t, J = 4.8 Hz, H7), 7.96 (s, H4′′), 8.15 (dt, J = 2.0 Hz, H5′′), 8.42 (d, J = 3.6 Hz, H6′′), 9.03 (s, H2′′), 12.82 (s, NH). 13C NMR (DMSO-d6) δ: 64.6 (C2), 115.5 (C8), 119.1 (C4a), 121.7 (C4′), 122.2 (C6), 122.9 (C5), 123.6 (C5′′), 127.1 (C7), 128.1 (C4), 128.8 (C3′′), 129.3 (C4′′), 131.3 (C3), 143.9 (C2′), 145.8 (C6′′ and C5′), 147.3 (C2′′), 153.2 (C8a); IR (KBr, cm−1): 3128 (N–H), 1573 (C[double bond, length as m-dash]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, H4′), 6.86 (t, J = 9.2 Hz, H8), 6.9 (td, J = 7.2, 1.2 Hz, H6), 7.04 (m, 2H, H5 and H4′′), 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, H3′′), 7.26 (s, H4), 7.27 (d, J = 3.6 Hz, H5′′). 13C NMR (DMSO-d6) δ: 64.6 (C2), 115.4 (C8), 118.9 (C4a), 121.7 (C4′ and C6), 122.2 (C5′), 122.8 (C5 and C5′′), 127.1 (C7 and C3′′), 127.6 (C4 and C4′′), 129.2 (C3 and C2′′), 14.1 (C2′), 153.2 (C8a); IR (KBr, cm−1): 3023 (N–H), 1578 (C[double bond, length as m-dash]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 H4′), 7.15 (s, H4), 7.2 (t, J = 7.2, 7.6 Hz, H6), 7.36 (t, J = 7.6, 8.0 Hz, 2H, H3′′ and H5′′), 7.44 (t, J = 7.6, 8.0 Hz, H4′′), 7.82 (d, J = 7.6 Hz, 2H, H2′′ and H6′′), 12.69 (s, NH). 13C NMR (DMSO-d6) δ: 55.6 (O–C), 64.6 (C2), 112.8 (C7), 114.8 (C4a), 118.7 (C5), 119.1 (C4′), 121.4 (C6), 122.3 (C2′′ and C6′′), 126.2 (C4′′), 128.4 (C3′′ and C5′′), 128.8 (C4), 134.3 (C1′′), 141.0 (C3 and C2′), 142.0 (C5′), 143.2 (C8a), 147.5 (C8); IR (KBr, cm−1): 3119 (N–H), 1571 (C[double bond, length as m-dash]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, H4′ & H5), 7.35 (s, 1H, H4), 7.49 (d, J = 8.4 Hz, 2H, H3′′ and H5′′), 7.6 (d, J = 8 Hz, 2H, H2′′ and H6′′). 13C NMR (DMSO-d6) δ: 55.6 (O–C), 64.5 (C2), 112.9 (C7), 115.0 (C4a), 119.1 (C5), 120.7 (C4′), 121.4 (C6), 122.9 (C4′′), 126.3 (C4), 128.7 (C2′′ and C6′′), 131.3 (C1′′), 132.4 (C3′′ and C5′′), 142.0 (C3 and C2′), 143.5 (C5′), 144.7 (C8a), 147.5 (C8); IR (KBr, cm−1): 3123 (N–H), 1576 (C[double bond, length as m-dash]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, H3′′ and H5′′), 7.16 (s, H4′), 7.4 (m, 6H, H4, Bn-5H), 7.72 (d, J = 8.4 Hz, 2H, H2′′ and H6′′), 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 (C3′′, C5′′ and C4a), 119.0 (C4′), 119.5 (C5), 121.9 (C6), 123.6 (C1′′), 126.2 (C2′′′ and C6′′′), 127.9 (C4′′′), 128.2 (C2′′ and C6′′), 128.3 (C3′′′ and C5′′′), 128.9 (C4), 137.6 (C3 and C2′), 142.5 (C5′), 143.6 (C1′′′), 148.0 (C8 and C8a), 157.7 (C4′′); IR (KBr, cm−1): 3082 (N–H), 1576 (C[double bond, length as m-dash]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, H4′), 7.08 (t, J = 8.4, 8.8 Hz, 2H, H2′′ and H6′′), 7.3 (s, H4), 7.7 (t, J = 8.8, 10.8, 2H, H3′′ and H6′′). 13C NMR (DMSO-d6) δ: 55.6 (O–C), 64.5 (C2), 112.9 (C7), 115.2 (C4a), 115.4 (C3′′ and C5′′), 118.9 (C5), 119.1 (C4′), 121.4 (C6), 122.0 (C1′′), 126.2 (C2′′ and C6′′), 126.2 (C4), 142.0 (C3 and C2′), 143.6 (C5′), 147.5 (C8a), 159.8 (C8), 162.2 (C4′′); IR (KBr, cm−1): 3081 (N–H), 1579 (C[double bond, length as m-dash]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, H4′), 6.9 (d, J = 2.8 Hz, 2H, H7 and H5), 6.96 (d, J = 8.0 Hz, 2H, H3′′ and H5′′), 7.16 (s, H4), 7.72 (d, J = 8.8 Hz, 2H, H2′′ and H6′′), 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 (C3′′ and C5′′), 118.9 (C4a), 119.5 (C4′ and C5), 121.9 (C6), 123.6 (C1′′), 126.2 (C2′′ and C6′′), 129.1 (C4), 136.5 (C3 and C2′), 142.5 (C5′), 143.6 (C8a), 148.0 (C8), 158.6 (C4′′); IR (KBr) cm−1: 3130 (N–H), 1575 (C[double bond, length as m-dash]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, H4′), 7.41 (t, J = 4.8, 6.0 Hz, H5′′), 7.93 (s, H4), 8.15 (d, J = 8.0 Hz, H6′′), 8.42 (d, J = 3.2 Hz, H4′′), 9.03 (s, H2′′), 12.82 (s, NH). 13C NMR (DMSO-d6) δ: 56.1 (O–C), 65.0 (C2), 113.5 (C7), 119.6 (C4a), 119.8 (C5), 121.9 (C4′), 123.4 (C6), 124.1 (C5′′), 128.0 (C4), 133.0 (C4′′), 136.0 (C3 and C2′), 131.8 (C3′′), 142.6 (C5), 144.4 (C6′′), 146.3 (C8a), 147.8 (C2′′), 148.0 (C8); IR (KBr, cm−1): 3085 (N–H), 1581 (C[double bond, length as m-dash]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 H4′), 7.05 (dd, J = 4.8, 3.6 Hz, H4′′), 7.14 (s, H4), 7.31 (d, J = 3.6 Hz, H3′′), 7.36 (d, J = 5.2 Hz, H5), 7.67 (d, J = 1.6 Hz, H5′′), 12.7 (s, NH). 13C NMR (DMSO-d6) δ: 56.1 (O–C), 65.0 (C2), 113.4 (C7), 114.6 (C4a), 119.6 (C5 and C4′), 121.9 (C6′), 122.0 (C5′), 123.3 (C5′′), 123.5 (C3′′), 123.8 (C4′′), 128.1 (C4), 137.0 (C2′′), 138.6 (C2′), 142.5 (C3), 143.5 (C8a), 148.0 (C8); IR (KBr, cm−1): 3086 (N–H), 1574 (C[double bond, length as m-dash]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, H4′), 6.96 (d, J = 8.0 Hz, H5), 7.27 (t, J = 7.6, 4.0 Hz, H4′′), 7.34 (s, H4), 7.39 (t, J = 7.6 Hz, 2H, H3′′ and H5′′), 7.69 (d, J = 7.2 Hz, 2H, H2′′ and H6′′), 12.12 (s, NH). 13C NMR (DMSO-d6) δ: 55.2 (O–C), 64.8 (C2), 101.5 (C8), 107.6 (C6), 115.4 (C4a), 118.7 (C4′), 124.3 (C2′′ and C6′′), 127.8 (C5), 128.5 (C4, C3′′ and C5′′), 134.2 (C1′′), 138.7 (C2′ and C3), 143.7 (C5′), 154.5 (C8a), 160.4 (C7); IR (KBr, cm−1): 3000 (N–H), 1572 (C[double bond, length as m-dash]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, H4′), 6.98 (d, J = 8.4 Hz, H5), 7.32 (s, H4), 7.5 (d, J = 8.8 Hz, 2H, H2′′ and H6′′), 7.6 (d, J = 7.6 Hz, 2H, H3′′ and H5′′). 13C NMR (DMSO-d6) δ: 55.3 (O–C), 64.7 (C2), 101.5 (C8), 107.7 (C6), 115.3 (C4a), 118.9 (C4′), 119.9 (C4′′), 126.2 (C5, C2′′ and C6′′), 127.9 (C4, C3′′ and C5′′), 131.3 (C1′′), 137.8 (C2′ and C3), 143.9 (C5′), 154.6 (C8a), 160.5 (C7); IR (KBr, cm−1): 3085 (N–H), 1570 (C[double bond, length as m-dash]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, H3′′ and H5′′), 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, H3′′′, H4′′′ and H5′′′), 7.47 (d, J = 6.8 Hz, 2H, H2′′ and H6′′), 7.72 (d, J = 8.4 Hz, 2H, H2′′ and H6′′), 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 (C3′′, C5′′ and C4a), 119.0 (C4′), 119.5 (C5), 121.9 (C6), 123.6 (C1′′), 126.2 (C2′′′ and C6′′′), 127.9 (C4′′′), 128.2 (C2′′ and C6′′), 128.3 (C3′′′ and C5′′′), 128.9 (C4), 137.6 (C3 and C2′), 142.5 (C5′), 143.6 (C1′′′), 148.0 (C7 and C8a), 157.7 (C4′′); IR (KBr, cm−1): 3007 (N–H), 1597 (C[double bond, length as m-dash]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, H4′), 7.2 (t, J = 6.8 Hz, 2H, H2′′ and H6′′), 7.73 (s, H4), 7.83 (dd, J = 8.8, 8.0 Hz, 2H, H2′′ and H6′′), 12.57 (s, NH). 13C NMR (DMSO-d6) δ: 55.3 (O–C), 64.8 (C2), 101.5 (C8), 107.6 (C6), 115.3 (C4a), 118.8 (C3′′ and C5′′), 120.0 (C4′), 126.1 (C5), 126.2 (C1′′), 127.8 (C4, C2′′ and C6′′), 139.2 (C2′ and C3), 143.7 (C5′), 154.5 (C8a), 160.0 (C4), 162.1 (C4′′); IR (KBr, cm−1): 3039 (N–H), 1572 (C[double bond, length as m-dash]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, H3′′ and H5′′), 6.6 (s, H8′), 6.9 (m, 3H, H6′, H4′), 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 (C4a), 115.3 (C3′′ and C5′′), 119.9 (C4′), 124.8 (C1′′), 125.7 (C5), 127.8 (C2′′ and C6′′), 128.5 (C4), 139.5 (C2′ and C3), 140.0 (C5′), 154.5 (C8a), 160.3 (C4′′), 160.4 (C7); IR (KBr, cm−1): 3003 (N–H), 1570 (C[double bond, length as m-dash]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, H4′), 7.4 (t, J = 6.0 Hz, H5′′′), 7.95 (s, H4), 8.15 (d, J = 8 Hz, H6′′), 8.45 (d, J = 3.2 Hz, H4′′), 9.1 (s, H2′′), 12.8 (s, NH). 13C NMR (DMSO-d6) δ: 56.1 (O–C), 65.0 (C2), 113.5 (C8), 119.6 (C4a), 119.8 (C5), 121.9 (C4′), 123.4 (C6), 124.1 (C5′′), 128.0 (C4), 133.0 (C4′′), 136.0 (C3 and C2′), 131.8 (C3′′), 142.6 (C5), 144.4 (C6′′), 146.3 (C8a), 147.8 (C2′′), 148.0 (C7); IR (KBr, cm−1): 3085 (N–H), 1581 (C[double bond, length as m-dash]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, H4′), 6.54 (dd, J = 8.4, 2.4 Hz, H8), 7.04 (t, J = 4.0, 4.4 Hz, H4′′), 7.09 (s, H4), 7.12 (d, J = 4.8 Hz, H6), 7.29 (d, J = 2.8 Hz, H3′′), 7.35 (d, J = 4.8 Hz, H5), 7.63 (d, J = 1.6 Hz, H5′′), 12.6 (s, NH). 13C NMR (DMSO-d6) δ: 55.2 (O–C), 64.7 (C2), 101.5 (C8), 107.7 (C6), 113.7 (C4a), 119.0 (C4′), 121.3 (C5′), 123.2 (C5′′), 127.5 (C5 and C3′′), 127.9 (C4′′), 136.3 (C3), 138.3 (C2′′), 143.3 (C2′), 154.5 (C8a), 160.4 (C7); IR (KBr, cm−1): 3111 (N–H), 1604 (C[double bond, length as m-dash]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, H4′), 7.26 (s, H4), 7.28 (d, J = 7.2 Hz, 2H, H2′′ and H6′′), 7.38 (dd, J = 8.0, 15.4 Hz, 3H, H3′′′, H4′′′ and H5′′′), 7.69 (s, NH). 13C NMR (DMSO-d6) δ: 56.1 (OCH3), 64.8 (C2), 104.6 (C8), 108.9 (C6), 112.1 (C4a), 114.1 (C4′), 121.6 (C2′′ and C6′′), 124.8 (C4′′), 129.7 (C1′′), 128.9 (C7, C3′′ and C5′′), 134.8 (C3 and C2′), 144.2 (C5′), 154.4 (C8a), 155.8 (C5); IR (KBr, cm−1): 3134 (N–H), 1584 (C[double bond, length as m-dash]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, H4′), 7.26 (s, H4), 7.33 (m, 4H, H2′′, H3′′, H5′′ & H6′′), 7.7 (s, NH). 13C NMR (DMSO-d6) δ: 55.7 (OCH3), 64.8 (C2), 103.6 (C8), 108.9 (C6), 111.6 (C4a), 114.9 (C4′), 126.1 (C2′′ and C6′′), 128.8 (C4, C3′′ and C5′′), 129.7 (C1′′ and C7), 132.5 (C4′′), 132.6 (C3 and C2′), 144.8 (C5′), 154.7 (C8a), 155.6 (C5); IR (KBr, cm−1): 3015 (N–H), 1582 (C[double bond, length as m-dash]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, H4′), 7.26 (s, H4), 7.29 (d, J = 2.0 Hz, H5′′), 7.32 (d, J = 2.0 Hz, H3′′), 7.44 (d, J = 2.0 Hz, H6′′), 7.71 (s, NH). 13C NMR (DMSO-d6) δ: 56.1 (OCH3), 64.7 (C2), 104.7 (C8), 108.9 (C6), 114.9 (C4′), 127.8 (C1′′ and C4), 129.8 (C7), 130.1 (C6′′), 130.7 (C3′′), 130.8 (C2′′), 131.4 (C4′′), 136.4 (C3 and C2′), 143.8 (C5′), 154.5 (C8a), 155.9 (C5); IR (KBr, cm−1): 3013 (N–H), 1583 (C[double bond, length as m-dash]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, H5′′), 8.03 (s, H4′), 8.05 (d, J = 7.2 Hz, H6′′), 8.25 (d, J = 7.6 Hz, H4′′), 8.6 (s, H2′′), 12.9 (s, NH). 13C NMR (DMSO-d6) δ: 55.6 (OCH3), 64.2 (C2), 104.2 (C8), 108.4 (C6), 111.4 (C4a), 114.3 (C4′), 118.2 (C2′′), 120.6 (C4′′), 120.8 (C5′′), 129.5 (C7 and C4), 130.0 (C6′′), 130.4 (C1′′), 136.1 (C2′), 138.6 (C3), 142.2 (C5′), 148.3 (C3′′), 153.9 (C8a), 155.9 (C5); IR (KBr, cm−1): 3092 (N–H), 1580 (C[double bond, length as m-dash]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, H2′′, H3′′, H5′′ and H6′′), 7.62 (s, NH). 13C NMR (DMSO-d6) δ: 55.7 (OCH3), 64.8 (C2), 103.6 (C8), 109.0 (C6), 111.6 (C4a), 114.9 (C4′), 120.6 (C4′′), 126.4 (C4, C2′′ and C6′′), 129.8 (C7 and C1′′), 131.8 (C3′′ and C5′′), 137.0 (C2′ and C3), 144.8 (C5′), 155.6 (C8a), 154.7 (C5); IR (KBr, cm−1): 3135 (N–H), 1583 (C[double bond, length as m-dash]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, H3′′ and H5′′), 7.1 (t, J = 8.4 Hz, H7), 7.14 (s, H4), 7.25 (s, H4), 7.34 (d, J = 7.2 Hz, 2H, H2′′′ and H6′′′), 7.39 (t, J = 6.8, 7.6 Hz, 3H, H3′′′, H4′′′ and H5′′′), 7.44 (d, J = 7.2 Hz, H2′′ & H6′′), 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 (C4a), 114.3 (C3′′ and C5′′), 115.2 (C4′), 120.7 (C1′′), 126.2 (C2′′′ and C6′′′), 127.5 (C4′′′), 128.0 (C2′′ and C6′′), 128.6 (C4, C3′′′ and C5′′′), 129.5 (C7), 136.9 (C3 and C2′), 143.7 (C5′s), 144.3 (C1′′′), 154.7 (C8a), 155.6 (C5), 158.1 (C4′′); IR (KBr, cm−1): 3072 (N–H), 1580 (C[double bond, length as m-dash]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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