# One-Pot Synthesis of Novel Furochromone and Oxazocine Derivatives as Promising Antitumor Agents with Their Molecular Docking Studies

Authors: Rita M. Borik
Journal: Journal of Chemistry
 (2020)

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## Abstract

One-pot efficient synthesis of novel chromone derivatives4a–h and that of 5a–h were described in a simple method via four-component reaction between furochromone carbaldehyde, amine, isocyanate derivatives, and benzoic acid derivatives or nicotinic acid, respectively. Also, oxazocine derivatives 7a, b were prepared via reaction of visnagine carbaldehyde, ethyl acetoacetate and isocyanate derivatives 2a, b. The obtained derivatives of novel furochromone and oxazocine derivatives were evaluated as promising antitumor agents against panel of two human cell lines, hepatocellular carcinoma (HEPG2) and breast carcinoma (MCF7). The antitumor results suggested that furochromone derivatives 5a–h have activity against MCF7 in comparison with doxorubicin as the standard drug. Furthermore, the molecular docking studies of these novel derivatives of furochromone and oxazocine showed good agreement with the biological results when their binding pattern and affinity towards the active site of EGFR was investigate.


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## Body

## 1. Introduction

Cancer is uncontrolled growth of abnormal cells, one of the most widespread serious diseases and its growth and metastasis depends on angiogenesis [1–4]. So, targeting of angiogenesis is a great goal for inhibition of tumor growth, invasion, and metastasis [5, 6].Treatment of cancer using cytotoxic drugs (antineoplastics that preventing replication of cells) has many side effects [7, 8] although they are toxic to cancer cells. However, they all tend to work by interfering with some aspect of how the cells divide and multiply. For example, some work by affecting the cells’ genetic “makeup” (material which controls specific cell characteristics) and others work by blocking cells from using nutrients needed to divide and multiply. The choice of cytotoxic drugs depends on the type and stage of cancer [9]. Survey and research to get safe novel drugs with less side effects are still in continuation [10, 11].In point of this view, heterocyclic organic compounds play an important and vital role in synthesis and preparation of novel pharmacological compounds that may have a good manner in treatment of various types of tumors [12] and the human immunodeficiency virus (HIV) [13]. Our initial studies focused on the chromone and its derivatives which are important class of heterocyclic compounds. These heterocyclic compounds show a variety of pharmacological properties [14]. The tricycle-heterocyclic compounds especially dibenzooxazocine and furochromone or visnagine derivatives are used for the treatment of pain and/or inflammation [15], and also they have efficient activity against tumor cells [16–18].Imidazole derivatives are an important class of heterocyclic compounds [19, 20] that exhibiting biological and pharmacological properties [21–23]. Also, oxazocine heterocyclic compounds are active compounds against CNS disorders and are used for the treatment of pain and/or inflammation [24].So, according to this survey and in continuation of our heterocyclic synthesis of novel active compounds against some carcinoma cell lines [25], we aim to synthesize novel derivatives of furochromone and oxazocine as promising antitumor agents towards hepatic and breast cell lines (HEPG2 and MFC7) as well as the normal cell line (human normal melanocyte, HFB4) using the MTT colorimetric test [26–29] depending on their molecular docking studies via one-pot reaction of three or four components of carbaldehyde, amine, isocyanate, and benzoic acid derivatives.


## 2. Experimental

All chemicals were provided by Fluka or Aldrich companies and were used without additional purification. Elemental microanalyses were carried out at Microanalytical Unit, Central Services Laboratory, National Research Centre, Dokki, Giza, Egypt, using Vario Elementar and were found within ± 0.4% of the theoretical values. All melting points were uncorrected and were taken in open capillary tubes using electrothermal apparatus 9100. FT-IR spectra were recorded with a Perkin-Elmer Frontier. Routine NMR spectra were recorded at room temperature on a Bruker Avance TM 400 (or 300) spectrometer as solutions in dimethyl sulfoxide (DMSO-d6) or chloroform (CDCl3). All chemical shifts are quoted in δ relative to the trace resonance of protonated dimethyl sulfoxide (δ2.50 ppm), DMSO (δ39.51 ppm) or CDCl3 (δ7.28 ppm), (δ77.28 ppm), and external 85% aqueous H3PO4 (δ0.0 ppm). The mass spectra were measured with a GC Finnigan MAT SSQ-7000 mass spectrometer. The reactions were followed, the purity of the compounds was checked using TLC on silica gel-precoated aluminum sheets (Type 60, F 254, Merck, Darmstadt, Germany), and the spots were detected by exposure to UV lamp at λ254 nm. The chemical names given for the prepared compounds are according to the IUPAC system. The reported yields are based upon pure materials isolated by column chromatography. Solvents were dried/purified according to conventional procedures.


### 2.1. General Procedure for the Preparation of Compounds4a–h and 5a–h

The desired compounds were synthesized utilizing a 25 ml round bottom flask. Mixture A, aniline/or benzyl amine (1.1 mmol), was added to furochromone carbaldehyde 1 (1.1 mmol) in methanol (20.0 ml); then the mixture A was stirred for 15–30 min at room temperature. Mixture B, benzoic acid/or p-amino benzoic acid/or p-nitro benzoic acid/or nicotinic acid, respectively, (1.1 mmol) and phenyl isocyanate/or cyclohexyl isocyanate, respectively, (1.1 mmol) in methanol were stirred for 5 minutes. Then, Mixture B was poured on Mixture A. Finally, the resultant mixture was stirred at room temperature for 23–68 h, and solid K2CO3 (0.38 mmol) was added and refluxed for 76–100 h. After the reaction was completed, the crude material was concentrated and redissolved in dichloromethane. The resulting organic solution was then washed with 1 M HCl (aq). This was followed by adding a saturated aqueous solution of NaHCO3 (aq) combined with brine. The resulting organic layer was collected, dried by MgSO4, and then concentrated in vacuum at 40°C for 8 h to afford the crude material. The crude material was purified by ethanol to give the desired products 4a–h.


#### 2.1.1. N-(4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Benzamide (4a)

Product4a was separated as orange crystals, yield 59%. m.p. 210–212°C. IR (KBr, cm−1) 1674, 1656 (2C=O), 1623 (C=N). 1H NMR (500 MHz, DMSO-d6): δ = 3.99, 4.00 (s, 6H, 2OCH3); 6.22, 6.60 (dd, 2H, J = 2.01 Hz, furan ring), 7.10 (s, 1H, H7), 7.00–7.31 (m, 15H, CH arom) ppm. 13C NMR (100 MHz, DMSO-d6): δ176.2 (CO), 173.7 (CO), 164.0 (CN), 155.8, 152.9, 150.1, 147.0, 146.5, 146.1, 134.1, 133.5, 132.1, 130.0, 128.9, 128.0, 127.6, 127.1, 123.1 124,0, 122.1, 121.6 (aromatic C-H), 118.2, 111.3, 106.3, 55.0 (OCH3), 54.5 (OCH3) ppm. MS (m/z): M+ 543 (31%), 542 (17%), 541(55%). Anal. for C33H24N2O6 (544.55): Calcd. C, 72.78; H, 4.44; N, 5.14. Found C, 72.21; H, 4.02; N, 4.82.


#### 2.1.2. N-(Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl)methyl)-N-phenyl Benzamide (4b)

Product4b was separated as brown solid, yield: 52%, m.p. 182°C. IR (KBr, cm−1) 1674, 1646 (2C=O), 1621(C=N). 1H-NMR (500 MHz, DMSO-d6): δ = 1.25–1.64 (m, 10H, 5CH2 cyclohexane); 3.10 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.64, 6.56 (dd, 2H, J=2.0 Hz 1, furan ring); 7.21–7.10 (m 10H, CH armo.); 8.00 (s, 1H, H7). MS (m/z): M+ 550.6, m/e: 549 (13%), 548 (36%), 547 (64%). Anal. for C33H30N2O6 (550.21): Calcd: C, 71.99; H, 5.49; N, 5.09, Found: C, 71.42; H, 5.02; N, 4.64.


#### 2.1.3. 4-Amino-N-((4,9-dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Benzamide (4c)

Product4c was separated as yellow solid, yield: 55%. m.p. 177°C. IR (KBr, cm−1) 1622 (C=N), 1686, 1645 (2CO) and 3347 (NH2). 1H-NMR (500 MHz, DMSO-d6): δ = 3.89 (s, 6H, 2OCH3); 6.92–6.24 (m, 14H, arom.), 6.73, 6.36 (dd, 2H, J=2.01 Hz, furan ring); 7.89 (s, 1H, H7) and 8.82 (s, 2H, NH2, exchangeable D2O). 13C NMR (100 MHz, DMSO-d6): δ, 177.6 (CO), 174.1 (CO), 164.1 (CN), 156.1, 155.3, 153.8, 151,1, 147.5, 147.0, 146.3, 134.0, 133,2, 132.4, 129.0, 128.7, 127.6, 124.8, 124.3 123.1, 122.3, 121.6 (aromatic C-H), 113.0, 112,1, 56.6 (OCH3), 56.3 (OCH3). MS (m/z): M+: 558 (9%), 557 (35%), 556 (43%). Anal. for C33H25N3O6 (559.57): Calcd: C, 70.83; H, 4.50; N, 7.51, Found: C, 70.43; H, 4.24; N, 7.24.


#### 2.1.4. 4-Amino-N-(cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-N-phenylbenzamide (4d)

Product4d was separated as brown solid, yield: 56%. m.p. 196°C, IR (KBr, cm−1) 1623 (C=N), 1699, 1653 (2C=O) and 3345 (NH2). 1H-NMR (500 MHz, DMSO-d6): δ1.58–1.24 (m, 10H, 5CH2 cyclohexane); 3.02 (dd, H, CH-N cyclohexane); 4.01, 4.03 (s, 6H, 2OCH3); 6.67, 6.33 (dd, 2H, J=2.00 Hz, furan ring); 6.54–6.20 (m, 9H, arom.); 8.10 (s, 1H, H7) and 8.79 (s, 2H, NH2 exchangeable with D2O). MS (m/z): M+: 565 (11%), 564 (48%), 563 (76%). Anal. for C33H31N3O6 (565.62) Calcd: C, 70.07; H, 5.52; N, 7.43, Found: C, 69.78; H, 5.12; N, 7.01.


#### 2.1.5. N-((4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-4-nitro-N-phenylbenzamide (4e)

Product4e was separated as yellow solid, yield: 66%. m.p. 214°C. IR (KBr, cm−1) 1621 (C=N),1672, 1642 (2C=O). 1H-NMR (500 MHz, DMSO-d6): 3.76, 3.77 (ss, 6H, 2OCH3), 6.54–7.20 (m, 14H, arom); 7.28, 6.56 (dd, 2H, J=2.01 Hz, furan ring); 7.54 (s, 1H, H7). 13C NMR (100 MHz, DMSO-d6): δ, 177.3 (CO), 174.0 (CO), 164.1 (CN), 156.1, 155.0, 153.7, 151.0, 147.1, 147.0, 146.0, 134.2, 133.1, 132.3, 129.0, 128.7, 127.6, 124.4, 123.3, 122.5, 121.7 (aromatic C-H), 113.1, 112.2, 106.1, 56.1 (OCH3), 54.6 (OCH3) MS (m/z): M+: 589.15 (76.0%), 588 (32%), 587 (45). Anal. for C33H23N3O8 (589.55) Calcd C, 67.23; H, 3.93; N, 7.13, Found: C, 66.77; H, 3.41; N, 5.85.


#### 2.1.6. N-((Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-4-nitro-N-phenylbenzamide (4f)

Product4f was separated as brown solid, yield: 64%. m.p. 146°C, IR (KBr, cm−1) 1618 (C=N), 1677, 1641 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.44–1.12 (m, 10H, 5CH2 cyclohexane); 3.02 (dd, H, CH-N cyclohexane), 3.76, 3.77 (s, 6H, 2OCH3), 7.11, 6.42 (dd, 2H, J=2.01 Hz, furan ring); 7.00–6.98 (m, 9H, CH arm.); 7.56 (s, 1H, H7). MS (m/z): M+: 595.2 (14%), 593 (36%), 592 (83%), Anal. for C33H29N3O8 (595.6). Calcd C, 66.55; H, 4.91; N, 7.06, Found: C, 66.01; H, 4.33; N, 6.84.


#### 2.1.7. N-((4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Nicotinamide (4g)

Product4g was separated as brown solid, yield: 64%. m.p. 176°C, IR (KBr, cm−1) 1620 (C=N), 1664, 1634 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.99, 3.86 (s, 6H, 2OCH3), 7.54–7.00 (m, 14H, arom); 7.12, 6.33 (dd, 2H, J=2.01 Hz, furan ring); 7.74 (s, 1H, H7). 13C NMR (100 MHz, DMSO-d6): δ, 177.4 (CO), 174.1 (CO), 164.1 (CN), 156.1, 153.7, 151.0, 150.8, 149.0, 147.4, 146.2, 134.2, 133.1, 132.2, 129.0, 128.8, 127.6, 124.6, 124.3, 121.7 (aromatic C-H), 113.1, 112.3. 112.1, 106.1, 56.5 (OCH3), 56.3 (OCH3), MS (m/z): M+: 545 (9%), 544 (58%), 543 (60%). Anal. for C32H23N3O6 (545.54) Calcd C, 70.45; H, 4.25; N, 7.70, found: C, 70.00; H, 3.87; N, 7.24.


#### 2.1.8. N-(Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-N-phenylnicotinamide (4h)

Product4h was separated as brown solid, yield: 62%. m.p. 211°C, IR (KBr, cm−1) 1623 (C=N), 1672, 1642 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.41–1.22 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane), 3.96, 3.78 (ss, 6H, 2OCH3), 7.54–6.20 (m, 9H, arom.); 7.54, 6.44 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.10 (m 9H, CH arm.); 7.34 (s, 1H, H7). MS (m/z): M+: 551.21 (67.0%), 550 (35.2%), 549 (7.5%). Anal. for C32H29N3O6 (551.59) C, 69.68; H, 5.30; N, 7.62, Found: C, 69.31; H, 4.80; N, 7.02.


#### 2.1.9. 4,9-Dimethoxy-6-(1,4,5-Triphenyl-1H-imidazol-2-yl)-5H-furo [3,2-g] Chromen-5-One (5a)

Product5a was separated as brown solid, yield: 63%. m.p. 214°C. IR (KBr, cm−1) 1646 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 15H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 174.7 (CO), 158.6, 152.7, 149.8, 147.5, 146.0, 136.9, 137.1 133.1, 132.1, 129.7, 129.2, 128.6, 127.3, 124.6, 123.1, 122.5 (aromatic C-H), 118.3, 113,0, 106.1, 55.8 (OCH3), 54.8(OCH3). MS (m/z): M+: 539 (10%), 462 (38%), 401 (67%), 387 (73%), 311 (62%), 245 (66%). Anal. for C34H24N2O5 (540.56) C, 75.54; H, 4.48; N, 5.18, Found: C, 75.01; H, 4.11; N, 4.66.


#### 2.1.10. 6-(1-Cyclohexyl-4,5-diphenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5b)

Product5b was separated as brown solid, yield: 62%. m.p. 215°C, IR (KBr, cm−1) 1638 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.25–1.34 (m, 10H, 5CH2 cyclohexane); 3.00 (dd, H, CH-N cyclohexane); 3.89 (s, 6H, 2OCH3); 6.76, 6.44 (dd, 2H, J=2.01 Hz, furan ring); 7.22–7.90 (m, 10 H, CH arom.); 7.99 (s, 1H, H7). MS (m/z): M+: 546 (78%), 545 (39%), 544 (79%). Anal. for C34H30N2O5 (546.61) C, 74.71; H, 5.53; N, 5.12, Found: C, 74.22; H, 5.32; N, 4.88.


#### 2.1.11. 6-(5-(4-Aminophenyl)-1,4-diphenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g] Chromen-5-One (5c)

Product5c was separated as brown solid, yield: 64%. m.p. 186°C, IR (KBr, cm−1) 1645 (C=O) and 3346 (NH2). 1H-NMR (100 MHz, DMSO-d6): δ, 3.98 (s, 6H, 2OCH3); 6.78, 6.11 (dd, 2H, J=2.01 Hz, furan ring); 7.48 (s, 1H, H7); 7.21–7.00 (m, 14H, CH arom.) and 9.01 (s, 2H, NH2 exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6): δ, 175.0 (CO), 158.9, 153.3, 150.7, 148.0, 147.1, 145.8, 139.0, 137.1, 136.8, 132.7, 131.7, 129.2, 129.0, 128.9, 128.0, 127.1, 124.5, 123.0, 122.7, 122.1 (aromatic C-H), 118.0, 116.3, 112.7, 111.6, 105.8, 56.1 (OCH3), 56.0(OCH3). MS (m/z): M+: 555 (49%), 554 (55%), 553 (67%). Anal. for: C34H25N3O5 (555.58) C, 73.50; H, 4.54; N, 7.56, found: C, 73.00; H, 4.12; N, 7.22.


#### 2.1.12. 6-(5-(4-Aminophenyl)-1-cyclohexyl-4-phenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5d)

Product5d was separated as yellow solid, yield: 63%. m.p. 192°CIR (KBr, cm−1) 1648 (C=O) and 3346 (NH2). 1H-NMR (100 MHz, DMSO-d6): δ, 1.55–1.24 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane); 3.89 (s, 6H, 2OCH3); 6.67, 6.51 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.10 (m, 9H, CH arom.); 8.11 (s, 1H, H7) and 8.87 (s, 2H, NH2 exchangeable with D2O. MS (m/z): M+: 561 (20%), 560 (57%), 559 (51%). Anal. for C34H31N3O5 (561.63), C, 72.71; H, 5.56; N, 7.48, Found: C, 72.71; H, 5.56; N, 7.48.


#### 2.1.13. 4,9-Dimethoxy-6-(5-(4-nitrophenyl)-1,4-diphenyl-1H-imidazol-2-yl)-5H-furo[3,2-]chromen-5-one (5e)

Product5e was separated as yellow solid, yield: 61%. m.p. 222°C, IR (KBr, cm−1) 1643 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 14H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 175.5 (CO), 158.9, 153.7, 151.0, 147.6, 150.1, 145,8, 140.7, 137.8, 137.1, 132.8, 132.2, 129.8, 129.2, 129.0, 128.5, 127.7, 124.6, 123.1, 122.7, 121.5, 118.4, 113.1, 112,0, 105.2 (aromatic C-H), 56.6 (OCH3), 56.4 (OCH3). MS (m/z): M+: 585 (41%), 584 (66%), 583 (62%), Anal. for C34H23N3O7 (585.56), C, 69.74; H, 3.96; N, 7.18, Found: C, 69.21; H, 3.64; N, 6.84.


#### 2.1.14. 6-(1-Cyclohexyl-5-(4-nitrophenyl)-4-phenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5f)

Product5f was separated as brown solid, yield: 73%. m.p. 186°C, IR (KBr, cm−1) 1635 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.55–1.34 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.60, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.07 (m 9H, CH arom). 8.01 (s, 1H, H7). MS (m/z): M+: 591.2 (22%), 590 (45%), 589 (48%). Anal. for C34H29N3O7 (591.61), C, 69.03; H, 4.94; N, 7.10, found: C, 68.76; H, 4.23; N, 6.76.


#### 2.1.15. 4,9-Dimethoxy-6-(1,4-diphenyl-5-(pyridin-4-yl)-1H-imidazol-2-yl)-5H-furo[3,2-g]chromen-5-one (5g)

Product5g was separated as orange solid, yield: 64%. m.p. 196°C, IR (KBr, cm−1) 1647 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 14H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 175.2 (CO), 159.7, 153.0, 150.9, 148.2, 147.6, 145.7, 139.1, 137.3, 136.6, 132.7, 131.8, 129.6, 129.3, 129.2, 128.3, 127.2 124.2, 123.4, 122.5, 122.0 (aromatic C-H), 118.2 116.4, 112.4, 111.1, 105.7, 56.1 (OCH3), 56.0 (OCH3). MS (m/z): M+: 540 (12%), 464 (44%), 387 (74%), 311 (52%), 245 (73%). Anal. for C33H23N3O5 (541.55), C, 73.19; H, 4.28; N, 7.76, Found: C, 73.12; H, 4.24; N, 7.70.


#### 2.1.16. 6-(1-Cyclohexyl-4-phenyl-5-(pyridin-4-yl)-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5h)

Product5h was separated as brown solid, yield: 73%. m.p. 201°C, IR (KBr, cm−1) 1644 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.44–1.21 (m, 10H, 5CH2 cyclohexane); 3.00 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.64, 6.56 (dd, 2H, J=2.01 Hz, furan ring); 7.28–7.10 (m, 9H), 7.56 (s, 1H, H7). MS (m/z): M+: 547 (9%), 546 (56%), 545 (64%). Anal. for C33H29N3O5 (547.6), C, 72.38; H, 5.34; N, 7.67, Found: C, 72.01; H, 4.82; N, 7.03.


### 2.2. General Procedure for the Preparation of (7a, b)

A solution of ethyl acetoacetate (1 mmol) and 7-hydroxy-5-methoxy-4-oxo-4H-chromene-6 carbaldehyde6 (1 mmol) in dichloromethane (3 ml) was stirred, and then phenyl isocyanate/or cyclohexyl isocyanate (1 mmol) was added to the mixture. The reaction mixture was stirred for 24–36 h at room temperature. After completion of the reaction, as indicated by TLC (ethyl acetate/n-hexane, 2 : 1), the solvent was removed under vacuum, and the solid residue was washed with ether and the products 4a, b were obtained and recrystallized from ethanol.


#### 2.2.1. 5-Acetyl-7-methoxy-10-methyl-3-phenylchromeno[6,7-g][1,3]oxazocine-2,4,8 (3H)-trione (7a)

Product7a was separated as beige solid, yield: 55%. m.p. 215°C, IR (KBr, cm−1): 1775, 1770, 1658, 1587 (4C=O). 1HNMR (100 MHz, DMSO-d6): δ, 2.01 (s, 3H, CH3); 2.47 (s, 3H, COCH3), 4.06 (s, 3H, OCH3); 6.74 (s, 1H, CH12); 7.87 (d, 1H, CH=C) and 7.31–7.11 (m, 6H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 198.0 (CO), 181.2 (CO), 164.8 (CO), 164.1 (CO), 160.3, 157.5, 157.2, 152.6, 148.6, 134.3, 133.0, 129.2, 123.3, 121.6 (aromatic C-H), 114.2, 110.4, 108.1, 103.2, 55.2 (OCH3), 25.4 (COCH3), 20.1 (CH3),. MS (m/z): M+: 419.10 (66.0%), 418 (43.1%), 417 (54.2%). Anal. for C23H17NO7 (419.38), Calcd C, 65.87; H, 4.09; N, 3.34 found: C, 65.66; H,, 4.01; N, 3.31.


#### 2.2.2. 5-Acetyl-3-cyclohexyl-7-methoxy-10-methylchromeno[6,7-g][1,3]oxazocine-2,4,8 (3H)-trione (7b)

Product7b was separated as yellow solid, yield: 60%. m.p. 181°C, IR (KBr, cm−1): 1759, 1720 1658 and 1639 (4C=O). 1HNMR (100 MHz, DMSO-d6): δ, 1.01–2.26 (10H, m, 5CH2 of cyclohexyl); 2.46 (s, 3H, CH3); 2.47 (s, 3H, COCH3) 3.32 (H, s, CH–N of cyclohexyl); 4.06 (s, 3H, OCH3) 6.03 (s, 1H, CH12) and 8.23 (d, 1H, CH=C). 13C NMR (100 MHz, DMSO-d6): δ, 198.1 (CO), 182.0 (CO), 164.8 (CO), 164.1 (CO), 159.8, 157.5, 157.2, 155.2, 146.5, 133.8 (aromatic C-H), 111.1, 110.6, 108.0, 103.0, 55.9 (OCH3), 30.8 (COCH3), 28.1, 25.7, 23.0 (CH2), 21.0 (CH3). MS (m/z): M+: 425.11 (33.0%), 424 (65.1%), 423 (45.5%). Anal. for C23H23NO7 (425.43), Calcd C, 64.93; H, 5.45; N, 3.29, Found C, 64.23; H, 5.15; N, 3.21.


### 2.3. Determination of Anticancer Activities

#### 2.3.1. Cell Lines

For anticancer activity screening of the newly synthesized compounds, liver HepG2 and breast MCF-7 cell lines as well as the normal cell line (human normal melanocyte, HFB4) were obtained from National Cancer Institute, Cairo University. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) enhanced with 10% warm inactivated fetal calf serum (GIBCO), penicillin (100 U/ml), and streptomycin (100μg/ml) at 37°C in humidified air containing 5% CO2. Cells at a concentration of 0.50 × 106 were grown in a 25 cm2 flask in 5 ml of culture medium.


#### 2.3.2. Cell Viability

(1) Fast Screening. Cells were seeded in 96 wells plates. The newly synthesized compounds were applied on the two cell lines to test their anticancer activity. The compounds were tested in two distinct concentrations (0.05 μg/ml and 5 μg/ml). The two working solutions were prepared using the complete medium. Three technical replicates were carried out for each concentration. The treated cells were incubated for 48 h at 37°C and 5% CO2. Afterwards, cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The comparison was performed between the treated cells to the positive control (reference drugs) and the negative control (DMEM). The tests were performed in biological replicates.(2) IC50 Determination. Cells were seeded in 96-well plates. The synthesized compounds that showed a significant reduction in the cell viability were chosen for further analysis. Each compound was tested on the two cell lines in 4 different concentrations (5, 12.5, 25, and 50 μg/ml). The working solutions were prepared using the complete medium. Three technical replicates were performed for each concentration. The treated cells were incubated for 48 h at 37°C and 5% CO2. The viability of the cells was determined using MTT test. IC50 (50% inhibitory concentration) values were calculated with a four-parameter logistic function and presented in a mean. The test was performed in biological replicates.


#### 2.3.3. MTT Assay

The cells were washed with 50μL of PBS and then the PBS was discarded [30]. Afterwards, 50 μL of MTT working solution was applied to each well and the cells were incubated for 15–30 min at 37°C and 5% CO2. The cells were examined microscopically for formazan (black precipitate) development. The supernatant was discarded from each well and the formazan was dissolved using DMSO. The absorbance of the developed color was measured using an automated plate reader at 570 nm with a background wavelength of 670 nm. The results were presented in percentage to the values obtained from untreated cells (negative control).


### 2.4. Molecular Docking Studies

Promising biological evaluation of new furochromone and oxazocine derivatives4a–h, 5a–h, and 7a, b encourages us to study their interaction mechanism into the active site of EGFR (PDB ID: 5CAV) by molecular docking technique [31] using MOE 2008.10 program.


#### 2.4.1. Preparation of Receptor

Binding sites were generated from co-crystallized ligand within crystal protein (PDB codes: 5CAV). Water molecules were removed from the complex. Then, the crystallographic disorders and protein energy was minimized by applying CHARMM and MMFF94 force fields. By applying fixed atom constraint, the rigid binding site of protein structure was obtained. The protein essential amino acids are defined and prepared for docking process. 2D structures of tested compounds were drawn using Chem. Bio. Draw Ultra14. 3D structures were protonated, and energy was minimized by applying 0.05 RMSD kcal/mol. CHARMM force field. Then, the minimized structures were prepared for docking using prepared ligand protocol [32].


#### 2.4.2. Molecular Docking Process

Docking process was carried out using CDOCKER protocol, by employing CHARMM-based molecular dynamics (MD) scheme to dock ligands into a receptor binding site. The receptor was held rigid while the ligands were allowed to be flexible during the refinement, where each molecule was allowed to produce seven different interaction poses with the protein and then docking scores (CDOCKER interaction energy) of the best-fitted poses with the active site at (EGFR, PDB codes: 5CAV).We use all the mentioned processes to predict the proposed binding mode affinity, preferred orientation of each docking pose, and binding free energy (∆G) of the tested compounds with EGFR. The calculated interaction energies for the tested compounds were in complete agreement with experimental results which showed that our compounds are potent inhibitors against EGFR.


## 2.1. General Procedure for the Preparation of Compounds4a–h and 5a–h

The desired compounds were synthesized utilizing a 25 ml round bottom flask. Mixture A, aniline/or benzyl amine (1.1 mmol), was added to furochromone carbaldehyde 1 (1.1 mmol) in methanol (20.0 ml); then the mixture A was stirred for 15–30 min at room temperature. Mixture B, benzoic acid/or p-amino benzoic acid/or p-nitro benzoic acid/or nicotinic acid, respectively, (1.1 mmol) and phenyl isocyanate/or cyclohexyl isocyanate, respectively, (1.1 mmol) in methanol were stirred for 5 minutes. Then, Mixture B was poured on Mixture A. Finally, the resultant mixture was stirred at room temperature for 23–68 h, and solid K2CO3 (0.38 mmol) was added and refluxed for 76–100 h. After the reaction was completed, the crude material was concentrated and redissolved in dichloromethane. The resulting organic solution was then washed with 1 M HCl (aq). This was followed by adding a saturated aqueous solution of NaHCO3 (aq) combined with brine. The resulting organic layer was collected, dried by MgSO4, and then concentrated in vacuum at 40°C for 8 h to afford the crude material. The crude material was purified by ethanol to give the desired products 4a–h.


### 2.1.1. N-(4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Benzamide (4a)

Product4a was separated as orange crystals, yield 59%. m.p. 210–212°C. IR (KBr, cm−1) 1674, 1656 (2C=O), 1623 (C=N). 1H NMR (500 MHz, DMSO-d6): δ = 3.99, 4.00 (s, 6H, 2OCH3); 6.22, 6.60 (dd, 2H, J = 2.01 Hz, furan ring), 7.10 (s, 1H, H7), 7.00–7.31 (m, 15H, CH arom) ppm. 13C NMR (100 MHz, DMSO-d6): δ176.2 (CO), 173.7 (CO), 164.0 (CN), 155.8, 152.9, 150.1, 147.0, 146.5, 146.1, 134.1, 133.5, 132.1, 130.0, 128.9, 128.0, 127.6, 127.1, 123.1 124,0, 122.1, 121.6 (aromatic C-H), 118.2, 111.3, 106.3, 55.0 (OCH3), 54.5 (OCH3) ppm. MS (m/z): M+ 543 (31%), 542 (17%), 541(55%). Anal. for C33H24N2O6 (544.55): Calcd. C, 72.78; H, 4.44; N, 5.14. Found C, 72.21; H, 4.02; N, 4.82.


### 2.1.2. N-(Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl)methyl)-N-phenyl Benzamide (4b)

Product4b was separated as brown solid, yield: 52%, m.p. 182°C. IR (KBr, cm−1) 1674, 1646 (2C=O), 1621(C=N). 1H-NMR (500 MHz, DMSO-d6): δ = 1.25–1.64 (m, 10H, 5CH2 cyclohexane); 3.10 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.64, 6.56 (dd, 2H, J=2.0 Hz 1, furan ring); 7.21–7.10 (m 10H, CH armo.); 8.00 (s, 1H, H7). MS (m/z): M+ 550.6, m/e: 549 (13%), 548 (36%), 547 (64%). Anal. for C33H30N2O6 (550.21): Calcd: C, 71.99; H, 5.49; N, 5.09, Found: C, 71.42; H, 5.02; N, 4.64.


### 2.1.3. 4-Amino-N-((4,9-dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Benzamide (4c)

Product4c was separated as yellow solid, yield: 55%. m.p. 177°C. IR (KBr, cm−1) 1622 (C=N), 1686, 1645 (2CO) and 3347 (NH2). 1H-NMR (500 MHz, DMSO-d6): δ = 3.89 (s, 6H, 2OCH3); 6.92–6.24 (m, 14H, arom.), 6.73, 6.36 (dd, 2H, J=2.01 Hz, furan ring); 7.89 (s, 1H, H7) and 8.82 (s, 2H, NH2, exchangeable D2O). 13C NMR (100 MHz, DMSO-d6): δ, 177.6 (CO), 174.1 (CO), 164.1 (CN), 156.1, 155.3, 153.8, 151,1, 147.5, 147.0, 146.3, 134.0, 133,2, 132.4, 129.0, 128.7, 127.6, 124.8, 124.3 123.1, 122.3, 121.6 (aromatic C-H), 113.0, 112,1, 56.6 (OCH3), 56.3 (OCH3). MS (m/z): M+: 558 (9%), 557 (35%), 556 (43%). Anal. for C33H25N3O6 (559.57): Calcd: C, 70.83; H, 4.50; N, 7.51, Found: C, 70.43; H, 4.24; N, 7.24.


### 2.1.4. 4-Amino-N-(cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-N-phenylbenzamide (4d)

Product4d was separated as brown solid, yield: 56%. m.p. 196°C, IR (KBr, cm−1) 1623 (C=N), 1699, 1653 (2C=O) and 3345 (NH2). 1H-NMR (500 MHz, DMSO-d6): δ1.58–1.24 (m, 10H, 5CH2 cyclohexane); 3.02 (dd, H, CH-N cyclohexane); 4.01, 4.03 (s, 6H, 2OCH3); 6.67, 6.33 (dd, 2H, J=2.00 Hz, furan ring); 6.54–6.20 (m, 9H, arom.); 8.10 (s, 1H, H7) and 8.79 (s, 2H, NH2 exchangeable with D2O). MS (m/z): M+: 565 (11%), 564 (48%), 563 (76%). Anal. for C33H31N3O6 (565.62) Calcd: C, 70.07; H, 5.52; N, 7.43, Found: C, 69.78; H, 5.12; N, 7.01.


### 2.1.5. N-((4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-4-nitro-N-phenylbenzamide (4e)

Product4e was separated as yellow solid, yield: 66%. m.p. 214°C. IR (KBr, cm−1) 1621 (C=N),1672, 1642 (2C=O). 1H-NMR (500 MHz, DMSO-d6): 3.76, 3.77 (ss, 6H, 2OCH3), 6.54–7.20 (m, 14H, arom); 7.28, 6.56 (dd, 2H, J=2.01 Hz, furan ring); 7.54 (s, 1H, H7). 13C NMR (100 MHz, DMSO-d6): δ, 177.3 (CO), 174.0 (CO), 164.1 (CN), 156.1, 155.0, 153.7, 151.0, 147.1, 147.0, 146.0, 134.2, 133.1, 132.3, 129.0, 128.7, 127.6, 124.4, 123.3, 122.5, 121.7 (aromatic C-H), 113.1, 112.2, 106.1, 56.1 (OCH3), 54.6 (OCH3) MS (m/z): M+: 589.15 (76.0%), 588 (32%), 587 (45). Anal. for C33H23N3O8 (589.55) Calcd C, 67.23; H, 3.93; N, 7.13, Found: C, 66.77; H, 3.41; N, 5.85.


### 2.1.6. N-((Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-4-nitro-N-phenylbenzamide (4f)

Product4f was separated as brown solid, yield: 64%. m.p. 146°C, IR (KBr, cm−1) 1618 (C=N), 1677, 1641 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.44–1.12 (m, 10H, 5CH2 cyclohexane); 3.02 (dd, H, CH-N cyclohexane), 3.76, 3.77 (s, 6H, 2OCH3), 7.11, 6.42 (dd, 2H, J=2.01 Hz, furan ring); 7.00–6.98 (m, 9H, CH arm.); 7.56 (s, 1H, H7). MS (m/z): M+: 595.2 (14%), 593 (36%), 592 (83%), Anal. for C33H29N3O8 (595.6). Calcd C, 66.55; H, 4.91; N, 7.06, Found: C, 66.01; H, 4.33; N, 6.84.


### 2.1.7. N-((4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Nicotinamide (4g)

Product4g was separated as brown solid, yield: 64%. m.p. 176°C, IR (KBr, cm−1) 1620 (C=N), 1664, 1634 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.99, 3.86 (s, 6H, 2OCH3), 7.54–7.00 (m, 14H, arom); 7.12, 6.33 (dd, 2H, J=2.01 Hz, furan ring); 7.74 (s, 1H, H7). 13C NMR (100 MHz, DMSO-d6): δ, 177.4 (CO), 174.1 (CO), 164.1 (CN), 156.1, 153.7, 151.0, 150.8, 149.0, 147.4, 146.2, 134.2, 133.1, 132.2, 129.0, 128.8, 127.6, 124.6, 124.3, 121.7 (aromatic C-H), 113.1, 112.3. 112.1, 106.1, 56.5 (OCH3), 56.3 (OCH3), MS (m/z): M+: 545 (9%), 544 (58%), 543 (60%). Anal. for C32H23N3O6 (545.54) Calcd C, 70.45; H, 4.25; N, 7.70, found: C, 70.00; H, 3.87; N, 7.24.


### 2.1.8. N-(Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-N-phenylnicotinamide (4h)

Product4h was separated as brown solid, yield: 62%. m.p. 211°C, IR (KBr, cm−1) 1623 (C=N), 1672, 1642 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.41–1.22 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane), 3.96, 3.78 (ss, 6H, 2OCH3), 7.54–6.20 (m, 9H, arom.); 7.54, 6.44 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.10 (m 9H, CH arm.); 7.34 (s, 1H, H7). MS (m/z): M+: 551.21 (67.0%), 550 (35.2%), 549 (7.5%). Anal. for C32H29N3O6 (551.59) C, 69.68; H, 5.30; N, 7.62, Found: C, 69.31; H, 4.80; N, 7.02.


### 2.1.9. 4,9-Dimethoxy-6-(1,4,5-Triphenyl-1H-imidazol-2-yl)-5H-furo [3,2-g] Chromen-5-One (5a)

Product5a was separated as brown solid, yield: 63%. m.p. 214°C. IR (KBr, cm−1) 1646 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 15H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 174.7 (CO), 158.6, 152.7, 149.8, 147.5, 146.0, 136.9, 137.1 133.1, 132.1, 129.7, 129.2, 128.6, 127.3, 124.6, 123.1, 122.5 (aromatic C-H), 118.3, 113,0, 106.1, 55.8 (OCH3), 54.8(OCH3). MS (m/z): M+: 539 (10%), 462 (38%), 401 (67%), 387 (73%), 311 (62%), 245 (66%). Anal. for C34H24N2O5 (540.56) C, 75.54; H, 4.48; N, 5.18, Found: C, 75.01; H, 4.11; N, 4.66.


### 2.1.10. 6-(1-Cyclohexyl-4,5-diphenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5b)

Product5b was separated as brown solid, yield: 62%. m.p. 215°C, IR (KBr, cm−1) 1638 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.25–1.34 (m, 10H, 5CH2 cyclohexane); 3.00 (dd, H, CH-N cyclohexane); 3.89 (s, 6H, 2OCH3); 6.76, 6.44 (dd, 2H, J=2.01 Hz, furan ring); 7.22–7.90 (m, 10 H, CH arom.); 7.99 (s, 1H, H7). MS (m/z): M+: 546 (78%), 545 (39%), 544 (79%). Anal. for C34H30N2O5 (546.61) C, 74.71; H, 5.53; N, 5.12, Found: C, 74.22; H, 5.32; N, 4.88.


### 2.1.11. 6-(5-(4-Aminophenyl)-1,4-diphenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g] Chromen-5-One (5c)

Product5c was separated as brown solid, yield: 64%. m.p. 186°C, IR (KBr, cm−1) 1645 (C=O) and 3346 (NH2). 1H-NMR (100 MHz, DMSO-d6): δ, 3.98 (s, 6H, 2OCH3); 6.78, 6.11 (dd, 2H, J=2.01 Hz, furan ring); 7.48 (s, 1H, H7); 7.21–7.00 (m, 14H, CH arom.) and 9.01 (s, 2H, NH2 exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6): δ, 175.0 (CO), 158.9, 153.3, 150.7, 148.0, 147.1, 145.8, 139.0, 137.1, 136.8, 132.7, 131.7, 129.2, 129.0, 128.9, 128.0, 127.1, 124.5, 123.0, 122.7, 122.1 (aromatic C-H), 118.0, 116.3, 112.7, 111.6, 105.8, 56.1 (OCH3), 56.0(OCH3). MS (m/z): M+: 555 (49%), 554 (55%), 553 (67%). Anal. for: C34H25N3O5 (555.58) C, 73.50; H, 4.54; N, 7.56, found: C, 73.00; H, 4.12; N, 7.22.


### 2.1.12. 6-(5-(4-Aminophenyl)-1-cyclohexyl-4-phenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5d)

Product5d was separated as yellow solid, yield: 63%. m.p. 192°CIR (KBr, cm−1) 1648 (C=O) and 3346 (NH2). 1H-NMR (100 MHz, DMSO-d6): δ, 1.55–1.24 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane); 3.89 (s, 6H, 2OCH3); 6.67, 6.51 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.10 (m, 9H, CH arom.); 8.11 (s, 1H, H7) and 8.87 (s, 2H, NH2 exchangeable with D2O. MS (m/z): M+: 561 (20%), 560 (57%), 559 (51%). Anal. for C34H31N3O5 (561.63), C, 72.71; H, 5.56; N, 7.48, Found: C, 72.71; H, 5.56; N, 7.48.


### 2.1.13. 4,9-Dimethoxy-6-(5-(4-nitrophenyl)-1,4-diphenyl-1H-imidazol-2-yl)-5H-furo[3,2-]chromen-5-one (5e)

Product5e was separated as yellow solid, yield: 61%. m.p. 222°C, IR (KBr, cm−1) 1643 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 14H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 175.5 (CO), 158.9, 153.7, 151.0, 147.6, 150.1, 145,8, 140.7, 137.8, 137.1, 132.8, 132.2, 129.8, 129.2, 129.0, 128.5, 127.7, 124.6, 123.1, 122.7, 121.5, 118.4, 113.1, 112,0, 105.2 (aromatic C-H), 56.6 (OCH3), 56.4 (OCH3). MS (m/z): M+: 585 (41%), 584 (66%), 583 (62%), Anal. for C34H23N3O7 (585.56), C, 69.74; H, 3.96; N, 7.18, Found: C, 69.21; H, 3.64; N, 6.84.


### 2.1.14. 6-(1-Cyclohexyl-5-(4-nitrophenyl)-4-phenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5f)

Product5f was separated as brown solid, yield: 73%. m.p. 186°C, IR (KBr, cm−1) 1635 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.55–1.34 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.60, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.07 (m 9H, CH arom). 8.01 (s, 1H, H7). MS (m/z): M+: 591.2 (22%), 590 (45%), 589 (48%). Anal. for C34H29N3O7 (591.61), C, 69.03; H, 4.94; N, 7.10, found: C, 68.76; H, 4.23; N, 6.76.


### 2.1.15. 4,9-Dimethoxy-6-(1,4-diphenyl-5-(pyridin-4-yl)-1H-imidazol-2-yl)-5H-furo[3,2-g]chromen-5-one (5g)

Product5g was separated as orange solid, yield: 64%. m.p. 196°C, IR (KBr, cm−1) 1647 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 14H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 175.2 (CO), 159.7, 153.0, 150.9, 148.2, 147.6, 145.7, 139.1, 137.3, 136.6, 132.7, 131.8, 129.6, 129.3, 129.2, 128.3, 127.2 124.2, 123.4, 122.5, 122.0 (aromatic C-H), 118.2 116.4, 112.4, 111.1, 105.7, 56.1 (OCH3), 56.0 (OCH3). MS (m/z): M+: 540 (12%), 464 (44%), 387 (74%), 311 (52%), 245 (73%). Anal. for C33H23N3O5 (541.55), C, 73.19; H, 4.28; N, 7.76, Found: C, 73.12; H, 4.24; N, 7.70.


### 2.1.16. 6-(1-Cyclohexyl-4-phenyl-5-(pyridin-4-yl)-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5h)

Product5h was separated as brown solid, yield: 73%. m.p. 201°C, IR (KBr, cm−1) 1644 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.44–1.21 (m, 10H, 5CH2 cyclohexane); 3.00 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.64, 6.56 (dd, 2H, J=2.01 Hz, furan ring); 7.28–7.10 (m, 9H), 7.56 (s, 1H, H7). MS (m/z): M+: 547 (9%), 546 (56%), 545 (64%). Anal. for C33H29N3O5 (547.6), C, 72.38; H, 5.34; N, 7.67, Found: C, 72.01; H, 4.82; N, 7.03.


## 2.1.1. N-(4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Benzamide (4a)

Product4a was separated as orange crystals, yield 59%. m.p. 210–212°C. IR (KBr, cm−1) 1674, 1656 (2C=O), 1623 (C=N). 1H NMR (500 MHz, DMSO-d6): δ = 3.99, 4.00 (s, 6H, 2OCH3); 6.22, 6.60 (dd, 2H, J = 2.01 Hz, furan ring), 7.10 (s, 1H, H7), 7.00–7.31 (m, 15H, CH arom) ppm. 13C NMR (100 MHz, DMSO-d6): δ176.2 (CO), 173.7 (CO), 164.0 (CN), 155.8, 152.9, 150.1, 147.0, 146.5, 146.1, 134.1, 133.5, 132.1, 130.0, 128.9, 128.0, 127.6, 127.1, 123.1 124,0, 122.1, 121.6 (aromatic C-H), 118.2, 111.3, 106.3, 55.0 (OCH3), 54.5 (OCH3) ppm. MS (m/z): M+ 543 (31%), 542 (17%), 541(55%). Anal. for C33H24N2O6 (544.55): Calcd. C, 72.78; H, 4.44; N, 5.14. Found C, 72.21; H, 4.02; N, 4.82.


## 2.1.2. N-(Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl)methyl)-N-phenyl Benzamide (4b)

Product4b was separated as brown solid, yield: 52%, m.p. 182°C. IR (KBr, cm−1) 1674, 1646 (2C=O), 1621(C=N). 1H-NMR (500 MHz, DMSO-d6): δ = 1.25–1.64 (m, 10H, 5CH2 cyclohexane); 3.10 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.64, 6.56 (dd, 2H, J=2.0 Hz 1, furan ring); 7.21–7.10 (m 10H, CH armo.); 8.00 (s, 1H, H7). MS (m/z): M+ 550.6, m/e: 549 (13%), 548 (36%), 547 (64%). Anal. for C33H30N2O6 (550.21): Calcd: C, 71.99; H, 5.49; N, 5.09, Found: C, 71.42; H, 5.02; N, 4.64.


## 2.1.3. 4-Amino-N-((4,9-dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Benzamide (4c)

Product4c was separated as yellow solid, yield: 55%. m.p. 177°C. IR (KBr, cm−1) 1622 (C=N), 1686, 1645 (2CO) and 3347 (NH2). 1H-NMR (500 MHz, DMSO-d6): δ = 3.89 (s, 6H, 2OCH3); 6.92–6.24 (m, 14H, arom.), 6.73, 6.36 (dd, 2H, J=2.01 Hz, furan ring); 7.89 (s, 1H, H7) and 8.82 (s, 2H, NH2, exchangeable D2O). 13C NMR (100 MHz, DMSO-d6): δ, 177.6 (CO), 174.1 (CO), 164.1 (CN), 156.1, 155.3, 153.8, 151,1, 147.5, 147.0, 146.3, 134.0, 133,2, 132.4, 129.0, 128.7, 127.6, 124.8, 124.3 123.1, 122.3, 121.6 (aromatic C-H), 113.0, 112,1, 56.6 (OCH3), 56.3 (OCH3). MS (m/z): M+: 558 (9%), 557 (35%), 556 (43%). Anal. for C33H25N3O6 (559.57): Calcd: C, 70.83; H, 4.50; N, 7.51, Found: C, 70.43; H, 4.24; N, 7.24.


## 2.1.4. 4-Amino-N-(cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-N-phenylbenzamide (4d)

Product4d was separated as brown solid, yield: 56%. m.p. 196°C, IR (KBr, cm−1) 1623 (C=N), 1699, 1653 (2C=O) and 3345 (NH2). 1H-NMR (500 MHz, DMSO-d6): δ1.58–1.24 (m, 10H, 5CH2 cyclohexane); 3.02 (dd, H, CH-N cyclohexane); 4.01, 4.03 (s, 6H, 2OCH3); 6.67, 6.33 (dd, 2H, J=2.00 Hz, furan ring); 6.54–6.20 (m, 9H, arom.); 8.10 (s, 1H, H7) and 8.79 (s, 2H, NH2 exchangeable with D2O). MS (m/z): M+: 565 (11%), 564 (48%), 563 (76%). Anal. for C33H31N3O6 (565.62) Calcd: C, 70.07; H, 5.52; N, 7.43, Found: C, 69.78; H, 5.12; N, 7.01.


## 2.1.5. N-((4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-4-nitro-N-phenylbenzamide (4e)

Product4e was separated as yellow solid, yield: 66%. m.p. 214°C. IR (KBr, cm−1) 1621 (C=N),1672, 1642 (2C=O). 1H-NMR (500 MHz, DMSO-d6): 3.76, 3.77 (ss, 6H, 2OCH3), 6.54–7.20 (m, 14H, arom); 7.28, 6.56 (dd, 2H, J=2.01 Hz, furan ring); 7.54 (s, 1H, H7). 13C NMR (100 MHz, DMSO-d6): δ, 177.3 (CO), 174.0 (CO), 164.1 (CN), 156.1, 155.0, 153.7, 151.0, 147.1, 147.0, 146.0, 134.2, 133.1, 132.3, 129.0, 128.7, 127.6, 124.4, 123.3, 122.5, 121.7 (aromatic C-H), 113.1, 112.2, 106.1, 56.1 (OCH3), 54.6 (OCH3) MS (m/z): M+: 589.15 (76.0%), 588 (32%), 587 (45). Anal. for C33H23N3O8 (589.55) Calcd C, 67.23; H, 3.93; N, 7.13, Found: C, 66.77; H, 3.41; N, 5.85.


## 2.1.6. N-((Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-4-nitro-N-phenylbenzamide (4f)

Product4f was separated as brown solid, yield: 64%. m.p. 146°C, IR (KBr, cm−1) 1618 (C=N), 1677, 1641 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.44–1.12 (m, 10H, 5CH2 cyclohexane); 3.02 (dd, H, CH-N cyclohexane), 3.76, 3.77 (s, 6H, 2OCH3), 7.11, 6.42 (dd, 2H, J=2.01 Hz, furan ring); 7.00–6.98 (m, 9H, CH arm.); 7.56 (s, 1H, H7). MS (m/z): M+: 595.2 (14%), 593 (36%), 592 (83%), Anal. for C33H29N3O8 (595.6). Calcd C, 66.55; H, 4.91; N, 7.06, Found: C, 66.01; H, 4.33; N, 6.84.


## 2.1.7. N-((4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) (Phenylimino) Methyl)-N-phenyl Nicotinamide (4g)

Product4g was separated as brown solid, yield: 64%. m.p. 176°C, IR (KBr, cm−1) 1620 (C=N), 1664, 1634 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.99, 3.86 (s, 6H, 2OCH3), 7.54–7.00 (m, 14H, arom); 7.12, 6.33 (dd, 2H, J=2.01 Hz, furan ring); 7.74 (s, 1H, H7). 13C NMR (100 MHz, DMSO-d6): δ, 177.4 (CO), 174.1 (CO), 164.1 (CN), 156.1, 153.7, 151.0, 150.8, 149.0, 147.4, 146.2, 134.2, 133.1, 132.2, 129.0, 128.8, 127.6, 124.6, 124.3, 121.7 (aromatic C-H), 113.1, 112.3. 112.1, 106.1, 56.5 (OCH3), 56.3 (OCH3), MS (m/z): M+: 545 (9%), 544 (58%), 543 (60%). Anal. for C32H23N3O6 (545.54) Calcd C, 70.45; H, 4.25; N, 7.70, found: C, 70.00; H, 3.87; N, 7.24.


## 2.1.8. N-(Cyclohexylimino) (4,9-Dimethoxy-5-oxo-5H-furo [3,2-g] Chromen-6-yl) Methyl)-N-phenylnicotinamide (4h)

Product4h was separated as brown solid, yield: 62%. m.p. 211°C, IR (KBr, cm−1) 1623 (C=N), 1672, 1642 (2C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.41–1.22 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane), 3.96, 3.78 (ss, 6H, 2OCH3), 7.54–6.20 (m, 9H, arom.); 7.54, 6.44 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.10 (m 9H, CH arm.); 7.34 (s, 1H, H7). MS (m/z): M+: 551.21 (67.0%), 550 (35.2%), 549 (7.5%). Anal. for C32H29N3O6 (551.59) C, 69.68; H, 5.30; N, 7.62, Found: C, 69.31; H, 4.80; N, 7.02.


## 2.1.9. 4,9-Dimethoxy-6-(1,4,5-Triphenyl-1H-imidazol-2-yl)-5H-furo [3,2-g] Chromen-5-One (5a)

Product5a was separated as brown solid, yield: 63%. m.p. 214°C. IR (KBr, cm−1) 1646 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 15H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 174.7 (CO), 158.6, 152.7, 149.8, 147.5, 146.0, 136.9, 137.1 133.1, 132.1, 129.7, 129.2, 128.6, 127.3, 124.6, 123.1, 122.5 (aromatic C-H), 118.3, 113,0, 106.1, 55.8 (OCH3), 54.8(OCH3). MS (m/z): M+: 539 (10%), 462 (38%), 401 (67%), 387 (73%), 311 (62%), 245 (66%). Anal. for C34H24N2O5 (540.56) C, 75.54; H, 4.48; N, 5.18, Found: C, 75.01; H, 4.11; N, 4.66.


## 2.1.10. 6-(1-Cyclohexyl-4,5-diphenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5b)

Product5b was separated as brown solid, yield: 62%. m.p. 215°C, IR (KBr, cm−1) 1638 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.25–1.34 (m, 10H, 5CH2 cyclohexane); 3.00 (dd, H, CH-N cyclohexane); 3.89 (s, 6H, 2OCH3); 6.76, 6.44 (dd, 2H, J=2.01 Hz, furan ring); 7.22–7.90 (m, 10 H, CH arom.); 7.99 (s, 1H, H7). MS (m/z): M+: 546 (78%), 545 (39%), 544 (79%). Anal. for C34H30N2O5 (546.61) C, 74.71; H, 5.53; N, 5.12, Found: C, 74.22; H, 5.32; N, 4.88.


## 2.1.11. 6-(5-(4-Aminophenyl)-1,4-diphenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g] Chromen-5-One (5c)

Product5c was separated as brown solid, yield: 64%. m.p. 186°C, IR (KBr, cm−1) 1645 (C=O) and 3346 (NH2). 1H-NMR (100 MHz, DMSO-d6): δ, 3.98 (s, 6H, 2OCH3); 6.78, 6.11 (dd, 2H, J=2.01 Hz, furan ring); 7.48 (s, 1H, H7); 7.21–7.00 (m, 14H, CH arom.) and 9.01 (s, 2H, NH2 exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6): δ, 175.0 (CO), 158.9, 153.3, 150.7, 148.0, 147.1, 145.8, 139.0, 137.1, 136.8, 132.7, 131.7, 129.2, 129.0, 128.9, 128.0, 127.1, 124.5, 123.0, 122.7, 122.1 (aromatic C-H), 118.0, 116.3, 112.7, 111.6, 105.8, 56.1 (OCH3), 56.0(OCH3). MS (m/z): M+: 555 (49%), 554 (55%), 553 (67%). Anal. for: C34H25N3O5 (555.58) C, 73.50; H, 4.54; N, 7.56, found: C, 73.00; H, 4.12; N, 7.22.


## 2.1.12. 6-(5-(4-Aminophenyl)-1-cyclohexyl-4-phenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5d)

Product5d was separated as yellow solid, yield: 63%. m.p. 192°CIR (KBr, cm−1) 1648 (C=O) and 3346 (NH2). 1H-NMR (100 MHz, DMSO-d6): δ, 1.55–1.24 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane); 3.89 (s, 6H, 2OCH3); 6.67, 6.51 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.10 (m, 9H, CH arom.); 8.11 (s, 1H, H7) and 8.87 (s, 2H, NH2 exchangeable with D2O. MS (m/z): M+: 561 (20%), 560 (57%), 559 (51%). Anal. for C34H31N3O5 (561.63), C, 72.71; H, 5.56; N, 7.48, Found: C, 72.71; H, 5.56; N, 7.48.


## 2.1.13. 4,9-Dimethoxy-6-(5-(4-nitrophenyl)-1,4-diphenyl-1H-imidazol-2-yl)-5H-furo[3,2-]chromen-5-one (5e)

Product5e was separated as yellow solid, yield: 61%. m.p. 222°C, IR (KBr, cm−1) 1643 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 14H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 175.5 (CO), 158.9, 153.7, 151.0, 147.6, 150.1, 145,8, 140.7, 137.8, 137.1, 132.8, 132.2, 129.8, 129.2, 129.0, 128.5, 127.7, 124.6, 123.1, 122.7, 121.5, 118.4, 113.1, 112,0, 105.2 (aromatic C-H), 56.6 (OCH3), 56.4 (OCH3). MS (m/z): M+: 585 (41%), 584 (66%), 583 (62%), Anal. for C34H23N3O7 (585.56), C, 69.74; H, 3.96; N, 7.18, Found: C, 69.21; H, 3.64; N, 6.84.


## 2.1.14. 6-(1-Cyclohexyl-5-(4-nitrophenyl)-4-phenyl-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5f)

Product5f was separated as brown solid, yield: 73%. m.p. 186°C, IR (KBr, cm−1) 1635 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.55–1.34 (m, 10H, 5CH2 cyclohexane); 3.11 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.60, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.23–7.07 (m 9H, CH arom). 8.01 (s, 1H, H7). MS (m/z): M+: 591.2 (22%), 590 (45%), 589 (48%). Anal. for C34H29N3O7 (591.61), C, 69.03; H, 4.94; N, 7.10, found: C, 68.76; H, 4.23; N, 6.76.


## 2.1.15. 4,9-Dimethoxy-6-(1,4-diphenyl-5-(pyridin-4-yl)-1H-imidazol-2-yl)-5H-furo[3,2-g]chromen-5-one (5g)

Product5g was separated as orange solid, yield: 64%. m.p. 196°C, IR (KBr, cm−1) 1647 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 3.97 (s, 6H, 2OCH3); 6.78, 6.52 (dd, 2H, J=2.01 Hz, furan ring); 7.00 (s, 1H, H7) and 7.31–7.11 (m, 14H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 175.2 (CO), 159.7, 153.0, 150.9, 148.2, 147.6, 145.7, 139.1, 137.3, 136.6, 132.7, 131.8, 129.6, 129.3, 129.2, 128.3, 127.2 124.2, 123.4, 122.5, 122.0 (aromatic C-H), 118.2 116.4, 112.4, 111.1, 105.7, 56.1 (OCH3), 56.0 (OCH3). MS (m/z): M+: 540 (12%), 464 (44%), 387 (74%), 311 (52%), 245 (73%). Anal. for C33H23N3O5 (541.55), C, 73.19; H, 4.28; N, 7.76, Found: C, 73.12; H, 4.24; N, 7.70.


## 2.1.16. 6-(1-Cyclohexyl-4-phenyl-5-(pyridin-4-yl)-1H-imidazol-2-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one (5h)

Product5h was separated as brown solid, yield: 73%. m.p. 201°C, IR (KBr, cm−1) 1644 (C=O). 1H-NMR (100 MHz, DMSO-d6): δ, 1.44–1.21 (m, 10H, 5CH2 cyclohexane); 3.00 (dd, H, CH-N cyclohexane); 3.88 (s, 6H, 2OCH3); 6.64, 6.56 (dd, 2H, J=2.01 Hz, furan ring); 7.28–7.10 (m, 9H), 7.56 (s, 1H, H7). MS (m/z): M+: 547 (9%), 546 (56%), 545 (64%). Anal. for C33H29N3O5 (547.6), C, 72.38; H, 5.34; N, 7.67, Found: C, 72.01; H, 4.82; N, 7.03.


## 2.2. General Procedure for the Preparation of (7a, b)

A solution of ethyl acetoacetate (1 mmol) and 7-hydroxy-5-methoxy-4-oxo-4H-chromene-6 carbaldehyde6 (1 mmol) in dichloromethane (3 ml) was stirred, and then phenyl isocyanate/or cyclohexyl isocyanate (1 mmol) was added to the mixture. The reaction mixture was stirred for 24–36 h at room temperature. After completion of the reaction, as indicated by TLC (ethyl acetate/n-hexane, 2 : 1), the solvent was removed under vacuum, and the solid residue was washed with ether and the products 4a, b were obtained and recrystallized from ethanol.


### 2.2.1. 5-Acetyl-7-methoxy-10-methyl-3-phenylchromeno[6,7-g][1,3]oxazocine-2,4,8 (3H)-trione (7a)

Product7a was separated as beige solid, yield: 55%. m.p. 215°C, IR (KBr, cm−1): 1775, 1770, 1658, 1587 (4C=O). 1HNMR (100 MHz, DMSO-d6): δ, 2.01 (s, 3H, CH3); 2.47 (s, 3H, COCH3), 4.06 (s, 3H, OCH3); 6.74 (s, 1H, CH12); 7.87 (d, 1H, CH=C) and 7.31–7.11 (m, 6H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 198.0 (CO), 181.2 (CO), 164.8 (CO), 164.1 (CO), 160.3, 157.5, 157.2, 152.6, 148.6, 134.3, 133.0, 129.2, 123.3, 121.6 (aromatic C-H), 114.2, 110.4, 108.1, 103.2, 55.2 (OCH3), 25.4 (COCH3), 20.1 (CH3),. MS (m/z): M+: 419.10 (66.0%), 418 (43.1%), 417 (54.2%). Anal. for C23H17NO7 (419.38), Calcd C, 65.87; H, 4.09; N, 3.34 found: C, 65.66; H,, 4.01; N, 3.31.


### 2.2.2. 5-Acetyl-3-cyclohexyl-7-methoxy-10-methylchromeno[6,7-g][1,3]oxazocine-2,4,8 (3H)-trione (7b)

Product7b was separated as yellow solid, yield: 60%. m.p. 181°C, IR (KBr, cm−1): 1759, 1720 1658 and 1639 (4C=O). 1HNMR (100 MHz, DMSO-d6): δ, 1.01–2.26 (10H, m, 5CH2 of cyclohexyl); 2.46 (s, 3H, CH3); 2.47 (s, 3H, COCH3) 3.32 (H, s, CH–N of cyclohexyl); 4.06 (s, 3H, OCH3) 6.03 (s, 1H, CH12) and 8.23 (d, 1H, CH=C). 13C NMR (100 MHz, DMSO-d6): δ, 198.1 (CO), 182.0 (CO), 164.8 (CO), 164.1 (CO), 159.8, 157.5, 157.2, 155.2, 146.5, 133.8 (aromatic C-H), 111.1, 110.6, 108.0, 103.0, 55.9 (OCH3), 30.8 (COCH3), 28.1, 25.7, 23.0 (CH2), 21.0 (CH3). MS (m/z): M+: 425.11 (33.0%), 424 (65.1%), 423 (45.5%). Anal. for C23H23NO7 (425.43), Calcd C, 64.93; H, 5.45; N, 3.29, Found C, 64.23; H, 5.15; N, 3.21.


## 2.2.1. 5-Acetyl-7-methoxy-10-methyl-3-phenylchromeno[6,7-g][1,3]oxazocine-2,4,8 (3H)-trione (7a)

Product7a was separated as beige solid, yield: 55%. m.p. 215°C, IR (KBr, cm−1): 1775, 1770, 1658, 1587 (4C=O). 1HNMR (100 MHz, DMSO-d6): δ, 2.01 (s, 3H, CH3); 2.47 (s, 3H, COCH3), 4.06 (s, 3H, OCH3); 6.74 (s, 1H, CH12); 7.87 (d, 1H, CH=C) and 7.31–7.11 (m, 6H, CH arom.). 13C NMR (100 MHz, DMSO-d6): δ, 198.0 (CO), 181.2 (CO), 164.8 (CO), 164.1 (CO), 160.3, 157.5, 157.2, 152.6, 148.6, 134.3, 133.0, 129.2, 123.3, 121.6 (aromatic C-H), 114.2, 110.4, 108.1, 103.2, 55.2 (OCH3), 25.4 (COCH3), 20.1 (CH3),. MS (m/z): M+: 419.10 (66.0%), 418 (43.1%), 417 (54.2%). Anal. for C23H17NO7 (419.38), Calcd C, 65.87; H, 4.09; N, 3.34 found: C, 65.66; H,, 4.01; N, 3.31.


## 2.2.2. 5-Acetyl-3-cyclohexyl-7-methoxy-10-methylchromeno[6,7-g][1,3]oxazocine-2,4,8 (3H)-trione (7b)

Product7b was separated as yellow solid, yield: 60%. m.p. 181°C, IR (KBr, cm−1): 1759, 1720 1658 and 1639 (4C=O). 1HNMR (100 MHz, DMSO-d6): δ, 1.01–2.26 (10H, m, 5CH2 of cyclohexyl); 2.46 (s, 3H, CH3); 2.47 (s, 3H, COCH3) 3.32 (H, s, CH–N of cyclohexyl); 4.06 (s, 3H, OCH3) 6.03 (s, 1H, CH12) and 8.23 (d, 1H, CH=C). 13C NMR (100 MHz, DMSO-d6): δ, 198.1 (CO), 182.0 (CO), 164.8 (CO), 164.1 (CO), 159.8, 157.5, 157.2, 155.2, 146.5, 133.8 (aromatic C-H), 111.1, 110.6, 108.0, 103.0, 55.9 (OCH3), 30.8 (COCH3), 28.1, 25.7, 23.0 (CH2), 21.0 (CH3). MS (m/z): M+: 425.11 (33.0%), 424 (65.1%), 423 (45.5%). Anal. for C23H23NO7 (425.43), Calcd C, 64.93; H, 5.45; N, 3.29, Found C, 64.23; H, 5.15; N, 3.21.


## 2.3. Determination of Anticancer Activities

### 2.3.1. Cell Lines

For anticancer activity screening of the newly synthesized compounds, liver HepG2 and breast MCF-7 cell lines as well as the normal cell line (human normal melanocyte, HFB4) were obtained from National Cancer Institute, Cairo University. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) enhanced with 10% warm inactivated fetal calf serum (GIBCO), penicillin (100 U/ml), and streptomycin (100μg/ml) at 37°C in humidified air containing 5% CO2. Cells at a concentration of 0.50 × 106 were grown in a 25 cm2 flask in 5 ml of culture medium.


### 2.3.2. Cell Viability

(1) Fast Screening. Cells were seeded in 96 wells plates. The newly synthesized compounds were applied on the two cell lines to test their anticancer activity. The compounds were tested in two distinct concentrations (0.05 μg/ml and 5 μg/ml). The two working solutions were prepared using the complete medium. Three technical replicates were carried out for each concentration. The treated cells were incubated for 48 h at 37°C and 5% CO2. Afterwards, cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The comparison was performed between the treated cells to the positive control (reference drugs) and the negative control (DMEM). The tests were performed in biological replicates.(2) IC50 Determination. Cells were seeded in 96-well plates. The synthesized compounds that showed a significant reduction in the cell viability were chosen for further analysis. Each compound was tested on the two cell lines in 4 different concentrations (5, 12.5, 25, and 50 μg/ml). The working solutions were prepared using the complete medium. Three technical replicates were performed for each concentration. The treated cells were incubated for 48 h at 37°C and 5% CO2. The viability of the cells was determined using MTT test. IC50 (50% inhibitory concentration) values were calculated with a four-parameter logistic function and presented in a mean. The test was performed in biological replicates.


### 2.3.3. MTT Assay

The cells were washed with 50μL of PBS and then the PBS was discarded [30]. Afterwards, 50 μL of MTT working solution was applied to each well and the cells were incubated for 15–30 min at 37°C and 5% CO2. The cells were examined microscopically for formazan (black precipitate) development. The supernatant was discarded from each well and the formazan was dissolved using DMSO. The absorbance of the developed color was measured using an automated plate reader at 570 nm with a background wavelength of 670 nm. The results were presented in percentage to the values obtained from untreated cells (negative control).


## 2.3.1. Cell Lines

For anticancer activity screening of the newly synthesized compounds, liver HepG2 and breast MCF-7 cell lines as well as the normal cell line (human normal melanocyte, HFB4) were obtained from National Cancer Institute, Cairo University. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) enhanced with 10% warm inactivated fetal calf serum (GIBCO), penicillin (100 U/ml), and streptomycin (100μg/ml) at 37°C in humidified air containing 5% CO2. Cells at a concentration of 0.50 × 106 were grown in a 25 cm2 flask in 5 ml of culture medium.


## 2.3.2. Cell Viability

(1) Fast Screening. Cells were seeded in 96 wells plates. The newly synthesized compounds were applied on the two cell lines to test their anticancer activity. The compounds were tested in two distinct concentrations (0.05 μg/ml and 5 μg/ml). The two working solutions were prepared using the complete medium. Three technical replicates were carried out for each concentration. The treated cells were incubated for 48 h at 37°C and 5% CO2. Afterwards, cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The comparison was performed between the treated cells to the positive control (reference drugs) and the negative control (DMEM). The tests were performed in biological replicates.(2) IC50 Determination. Cells were seeded in 96-well plates. The synthesized compounds that showed a significant reduction in the cell viability were chosen for further analysis. Each compound was tested on the two cell lines in 4 different concentrations (5, 12.5, 25, and 50 μg/ml). The working solutions were prepared using the complete medium. Three technical replicates were performed for each concentration. The treated cells were incubated for 48 h at 37°C and 5% CO2. The viability of the cells was determined using MTT test. IC50 (50% inhibitory concentration) values were calculated with a four-parameter logistic function and presented in a mean. The test was performed in biological replicates.


## 2.3.3. MTT Assay

The cells were washed with 50μL of PBS and then the PBS was discarded [30]. Afterwards, 50 μL of MTT working solution was applied to each well and the cells were incubated for 15–30 min at 37°C and 5% CO2. The cells were examined microscopically for formazan (black precipitate) development. The supernatant was discarded from each well and the formazan was dissolved using DMSO. The absorbance of the developed color was measured using an automated plate reader at 570 nm with a background wavelength of 670 nm. The results were presented in percentage to the values obtained from untreated cells (negative control).


## 2.4. Molecular Docking Studies

Promising biological evaluation of new furochromone and oxazocine derivatives4a–h, 5a–h, and 7a, b encourages us to study their interaction mechanism into the active site of EGFR (PDB ID: 5CAV) by molecular docking technique [31] using MOE 2008.10 program.


### 2.4.1. Preparation of Receptor

Binding sites were generated from co-crystallized ligand within crystal protein (PDB codes: 5CAV). Water molecules were removed from the complex. Then, the crystallographic disorders and protein energy was minimized by applying CHARMM and MMFF94 force fields. By applying fixed atom constraint, the rigid binding site of protein structure was obtained. The protein essential amino acids are defined and prepared for docking process. 2D structures of tested compounds were drawn using Chem. Bio. Draw Ultra14. 3D structures were protonated, and energy was minimized by applying 0.05 RMSD kcal/mol. CHARMM force field. Then, the minimized structures were prepared for docking using prepared ligand protocol [32].


### 2.4.2. Molecular Docking Process

Docking process was carried out using CDOCKER protocol, by employing CHARMM-based molecular dynamics (MD) scheme to dock ligands into a receptor binding site. The receptor was held rigid while the ligands were allowed to be flexible during the refinement, where each molecule was allowed to produce seven different interaction poses with the protein and then docking scores (CDOCKER interaction energy) of the best-fitted poses with the active site at (EGFR, PDB codes: 5CAV).We use all the mentioned processes to predict the proposed binding mode affinity, preferred orientation of each docking pose, and binding free energy (∆G) of the tested compounds with EGFR. The calculated interaction energies for the tested compounds were in complete agreement with experimental results which showed that our compounds are potent inhibitors against EGFR.


## 2.4.1. Preparation of Receptor

Binding sites were generated from co-crystallized ligand within crystal protein (PDB codes: 5CAV). Water molecules were removed from the complex. Then, the crystallographic disorders and protein energy was minimized by applying CHARMM and MMFF94 force fields. By applying fixed atom constraint, the rigid binding site of protein structure was obtained. The protein essential amino acids are defined and prepared for docking process. 2D structures of tested compounds were drawn using Chem. Bio. Draw Ultra14. 3D structures were protonated, and energy was minimized by applying 0.05 RMSD kcal/mol. CHARMM force field. Then, the minimized structures were prepared for docking using prepared ligand protocol [32].


## 2.4.2. Molecular Docking Process

Docking process was carried out using CDOCKER protocol, by employing CHARMM-based molecular dynamics (MD) scheme to dock ligands into a receptor binding site. The receptor was held rigid while the ligands were allowed to be flexible during the refinement, where each molecule was allowed to produce seven different interaction poses with the protein and then docking scores (CDOCKER interaction energy) of the best-fitted poses with the active site at (EGFR, PDB codes: 5CAV).We use all the mentioned processes to predict the proposed binding mode affinity, preferred orientation of each docking pose, and binding free energy (∆G) of the tested compounds with EGFR. The calculated interaction energies for the tested compounds were in complete agreement with experimental results which showed that our compounds are potent inhibitors against EGFR.


## 3. Results and Discussion

### 3.1. Chemistry

Highly substituted chromone derivatives4a–h and 5a–h were synthesized via a one-pot, four-component reaction of aniline, furochromone carbaldehyde 1, acid derivatives 3a–d, namely, benzoic acid/or 4-amino-, 4-nitrobenzoic acid/or nicotinic acid, and phenyl isocyanate/or cyclohexyl isocyanate (Scheme 1). The structures of 4a–h were elucidated from their elemental and spectroscopic analyses (IR, 1H NMR, and 13C NMR) together with mass spectra which prove the structures via getting molecular ion peaks at appropriate m/z values. 1H NMR spectra of 4a as example revealed characteristic doublet peaks for furan ring protons at δ = 6.22, 6.60 ppm, while aromatic protons appeared at 7.00–7.31 ppm as multiples. 13C NMR spectrum of 4a showed 26 distinct resonances characteristic for carbon atoms 55.0 (OCH3), 54.5 (OCH3), 176.2 (CO), and 173.7 (CO) ppm. IR spectra of 4a–h showed disappearance of the aldehydic group.Scheme 1
Synthesis of compounds4a–h.The mechanism of the formation of compounds4a–h is depicted in Scheme 2. Intermediate (A) in situ is formed between acid and isocyanate. Also, nucleophilic attack of amine toward the most active site of aldehydic carbon afforded the amine intermediate (B). Intermediate (A) attacks intermediate (B) to form oxadiazine ring (D) over expulsion of carbon dioxide molecule with rearrangement to afford the desired products 4a–h (Scheme 2).Scheme 2
Proposed mechanism for formation of compounds4a–h.In the same manner, furochromone carbaldehyde1 reacted in one-pot reaction at room temperature in methanol with beneylamine together with isocyanate derivatives 2a, b with benzoic acid derivatives 3a–d to give imidazole derivatives of furochromone 5a–h in a good yield (Scheme 3). The structures of the novel compounds are elucidated via elemental and spectroscopic analysis (cf. Experimental). IR spectra showed disappearance of the aldehydic group. The 13C NMR spectrum of 5a showed 21 distinct resonances characteristic for carbon atoms.Scheme 3
Synthesis of compounds5a–h.The mechanism of formation of compounds5a–h is cited in Scheme 4, and it revealed similarity with formation mechanism of compounds 4a–h with cyclization of intermediate due to expulsion of water and carbon dioxide molecules to give the final products (Scheme 4).Scheme 4
Proposed mechanism for formation of compounds5a–h.Synthesis of new derivatives of oxazocine7a, b at one-pot three-component reaction is studied. Visnagine carbaldehyde 6, ethyl acetoacetate, and isocyanate derivatives 2a, b under mild conditions to afford derivatives 7a, b in a good yield (Scheme 5). The structures of new compounds 7a, b were confirmed based on analytical and spectral data. IR spectra showed disappearance of the characteristic band of (CHO) group and it exhibited 4C=O groups. 1HNMR (DMSO-d6) spectra of compounds (7a, b) showed a doublet around δ7.87, 8.23 ppm, respectively, for CH=C group. 13C-NMR for compound 7a as example was showed 21 signals characteristic for carbon atoms 198.0 (CO), 181.2 (CO), 164.8 (C0), 164.1 (CO) 55.2 (OCH3), 25.4 (COCH3), and 20.1 (CH3).Scheme 5
Synthesis of compounds7a, b.


### 3.2. Antitumor Activity

It has been proved that chromones have various kinds of biological activities, including antitumor, antimicrobial, antiviral, anti-inflammatory, antioxidant, and so on. Cytotoxicity refers to cell death, cell lysis, and the inhibition on cell proliferation induced by some substances.In vitro, most of chromones’s cytotoxicity against tumor cells has been tested to confirm their antitumor activity. Cell toxicity is generally evaluated using the MTT (microculture tetrazolium) or SRB (sulforhodamine B) assay. Chromones demonstrate cell toxicity against a quantity of cell lines from a great variety of tumors, including cervical epithelioid carcinoma, breast adenocarcinoma [33], hepatoma carcinoma, lung cancer, leukemia cancer, and colon cancer [34, 35].


#### 3.2.1. Cell Viability

(1) Fast Screening. Quick screening was performed to determine the newly synthesized derivatives which demonstrated a significant reduction in the cell viabilities against the two cell lines: hepatocellular carcinoma (HepG2) and breast carcinoma (MCF7). The impact of the newly derivatives on the breast carcinoma cell line (MCF7) after 48 hrs, as an example, is illustrated in (Figure 1).Figure 1
Breast carcinoma cell line (MCF7) cell viability after 48 h treatment with the synthesized derivatives.Most of the compounds exhibited a distinct reduction in the cell viability of at least one of the two cell lines. Therefore, all the compounds were further analyzed to determine their IC50 values.(2) IC50 Determination. The data calculated as the concentration of the tested samples needed to inhibit half of the cancer cells population IC50 values were calculated for each compound separately and, mean values ± SD are presented Table 1.Table 1
The antiproliferative potency of the newly prepared derivatives toward hepatic and breast cancer cell lines.
CompoundCell linesMCF-7 IC50 ± SD (μg/mL)HEPG-2 IC50 ± SD (μg/mL)Solvent (DMSO)76 ± 0.6677 ± 0.53153 ± 0.3544 ± 5.434a43 ± 0.2035 ± 0.124b37 ± 1.1743 ± 2.904c55 ± 0.5851 ± 0.374d45 ± 1.0449 ± 1.104e41 ± 11.2833 ± 2.104f50 ± 2.7964 ± 3.104g52 ± 1.5245 ± 0.474h43 ± 1.1555 ± 1.335a23 ± 1.0347 ± 2.805b20 ± 1.4433 ± 2.335c19 ± 1.2243 ± 1.575d25 ± 2.6732 ± 1.675e18 ± 1.7354 ± 1.275f24 ± 0.1045 ± 0.095g26 ± 3.2356 ± 2.595h19 ± 1.1941 ± 1.39644 ± 1.8656 ± 1.797a34 ± 1.3445 ± 1.647b33 ± 2.7939 ± 0.475 fluorouracil13 ± 0.56Doxorubicin14 ± 1.07The results stated in Table1 reveal that many compounds showed good antiproliferative activity against breast cancer cell line (MCF7) with no toxicity on normal cell line. According to National Cancer Institute guidelines, the compound with an IC50 value < 30 μg/mL is active [36]. Therefore, as almost all compounds showed inhibition of cell growth, all the compounds were further analyzed to determine their IC50. MTT test for the investigated compounds showed that most of them expressed the IC50 ranging from 18 to 64 μg/ml compared with the standard drugs in used range of concentration, and they have low toxicity on the normal cell line (Table 1). From the all tested derivatives on these two cancer cell lines, compound 5a–h had the lowest IC50 ranging from 18–26 μg/ml against breast carcinoma cell line (MCF7) (Figure 2).Figure 2
Dose-dependent response of two cell lines cells treated with the derivatives5a–h determined by MTT assay (48 h).
(a)(b)(c)(d)(e)(f)(g)(h)The abovementioned results showed that different cell lines varied greatly in their response against different synthesized compounds. This correlates well with previously reported results [37, 38], where this can be attributed to the inherent different cells in their specific membrane structure and organization.


### 3.3. Molecular Docking Studies

#### 3.3.1. Reference Ligand and Docking in EGFR Domain

The binding mode of 4ZQ exhibits an energy binding of −24.81, for RMSD (1.67). 4ZQ formed a hydrogen bond with H-bond with (Lys 745) at bond distance 3.13°A, (Met 793) at bond distance of 2.95°A and (Thr 854) at 2.79°A. Docking results of the newly synthesized compounds4a–h, 5a–h, and 7a, b which docked with EGFR protein (PDB ID: 5CAV) active site (Table 2).Table 2
Docking results of the newly synthesized compounds4a–h, 5a-h, and 7a, b docked with EGFR protein (PDB ID: 5CAV) active site.
Compd. noBinding energy score (kJ mol−1)Bonds numberAmino acid residuesLigand atomH.B.B. lengthPi4ZQ−24.8133.13,2.95,2.790Lys 745, Met 793, Thr854O of OCH3, N of pyrimidine ring, N of pyridine ring4a−26.1713.151Lys 745, Phe723O of OCH3, benzene ring4b−20.6912.941Phe723O of OCH3, benzene ring4c−24.5523.71, 2.672Phe723O of OCH3, benzene ring4d−24.7612.791Lys 745, Phe723O of OCH3, benzene ring4e−23.2223.00, 2.782Lys 745, Phe723O of OCH3, CO, benzene ring4f−19.0113.042Lys 745, Phe723O of OCH3, benzo furan moiety4g−25.0523.41,2.981Lys745,Cys797, Phe723O of OCH3, N of pyridine ring4h−25.4723.11,2.722Lys 745, Phe723O of OCH3, CO, benzene furan moiety5a−29.3112.621Lys 745, Phe723CO, imidazole ring5b−25.4722.98,2.741Lys 745, Phe723O of OCH3, CO, Benzene ring5c−30.0712.591Lys 745, Phe723CO, imidazole ring5d−26.6612.611Lys 745, Phe723CO, imidazole ring5e−26.1113.432Lys 745, Phe723O of OCH3, benzo furan moiety5f−29.1513.732Lys 745, Phe723O of OCH3, benzo furan moiety5g−27.2912.630Cys797CO5h−25.1523.01,2.691Lys745, Phe723O of OCH3, CO, benzene ring7a−18.4812.59Lys745CO7b−17.1612.63Met 793COThe results revealed that compounds5a–h showed better docking score ranging from −30.07 to −25.15 kJ/mol, compared to the co-crystallized ligand (4QZ) of −24.81 kJ/mol and root-mean-square deviation value of 1.67 (Table 2). Also, compounds 5a–h showed good binding interaction to the protein active site via formation of hydrogen bonds with the same amino acid residue (Lys 745) as the co-crystalline ligand (Table 2). The results of anticancer activity and docking studies for compounds 5c and 5e were compatible. Compounds 5c and 5e showed potent anticancer activity against MCF-7 cell line (IC50 value 19 and 18 μg/ml) compared to reference drug 5-fluorouracil (IC50 13 μg/ml), and also exhibited better docking score (−30.07 and −26.11 kJ/mol) and good binding interaction via formation of one hydrogen bond with amino acid residue Lys 745 (2.59 Å and 3.43 Å) and Pi interaction with amino acid residue Phe 723 compared to co-crystallized ligand of docking score −24.81 kJ/mol and three hydrogen bonds with Lys 745, Met 793, and Thr 854 (Figures 3–5).Figure 3
(a) The 2D depiction of the docked conformation of co-crystallized ligand (4ZQ) in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of co-crystallized ligand (4ZQ) in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)Figure 4
(a) The 2D depiction of the docked conformation of5c in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of 5c in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)Figure 5
(a) The 2D depiction of the docked conformation of5e in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of 5e in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)


## 3.1. Chemistry

Highly substituted chromone derivatives4a–h and 5a–h were synthesized via a one-pot, four-component reaction of aniline, furochromone carbaldehyde 1, acid derivatives 3a–d, namely, benzoic acid/or 4-amino-, 4-nitrobenzoic acid/or nicotinic acid, and phenyl isocyanate/or cyclohexyl isocyanate (Scheme 1). The structures of 4a–h were elucidated from their elemental and spectroscopic analyses (IR, 1H NMR, and 13C NMR) together with mass spectra which prove the structures via getting molecular ion peaks at appropriate m/z values. 1H NMR spectra of 4a as example revealed characteristic doublet peaks for furan ring protons at δ = 6.22, 6.60 ppm, while aromatic protons appeared at 7.00–7.31 ppm as multiples. 13C NMR spectrum of 4a showed 26 distinct resonances characteristic for carbon atoms 55.0 (OCH3), 54.5 (OCH3), 176.2 (CO), and 173.7 (CO) ppm. IR spectra of 4a–h showed disappearance of the aldehydic group.Scheme 1
Synthesis of compounds4a–h.The mechanism of the formation of compounds4a–h is depicted in Scheme 2. Intermediate (A) in situ is formed between acid and isocyanate. Also, nucleophilic attack of amine toward the most active site of aldehydic carbon afforded the amine intermediate (B). Intermediate (A) attacks intermediate (B) to form oxadiazine ring (D) over expulsion of carbon dioxide molecule with rearrangement to afford the desired products 4a–h (Scheme 2).Scheme 2
Proposed mechanism for formation of compounds4a–h.In the same manner, furochromone carbaldehyde1 reacted in one-pot reaction at room temperature in methanol with beneylamine together with isocyanate derivatives 2a, b with benzoic acid derivatives 3a–d to give imidazole derivatives of furochromone 5a–h in a good yield (Scheme 3). The structures of the novel compounds are elucidated via elemental and spectroscopic analysis (cf. Experimental). IR spectra showed disappearance of the aldehydic group. The 13C NMR spectrum of 5a showed 21 distinct resonances characteristic for carbon atoms.Scheme 3
Synthesis of compounds5a–h.The mechanism of formation of compounds5a–h is cited in Scheme 4, and it revealed similarity with formation mechanism of compounds 4a–h with cyclization of intermediate due to expulsion of water and carbon dioxide molecules to give the final products (Scheme 4).Scheme 4
Proposed mechanism for formation of compounds5a–h.Synthesis of new derivatives of oxazocine7a, b at one-pot three-component reaction is studied. Visnagine carbaldehyde 6, ethyl acetoacetate, and isocyanate derivatives 2a, b under mild conditions to afford derivatives 7a, b in a good yield (Scheme 5). The structures of new compounds 7a, b were confirmed based on analytical and spectral data. IR spectra showed disappearance of the characteristic band of (CHO) group and it exhibited 4C=O groups. 1HNMR (DMSO-d6) spectra of compounds (7a, b) showed a doublet around δ7.87, 8.23 ppm, respectively, for CH=C group. 13C-NMR for compound 7a as example was showed 21 signals characteristic for carbon atoms 198.0 (CO), 181.2 (CO), 164.8 (C0), 164.1 (CO) 55.2 (OCH3), 25.4 (COCH3), and 20.1 (CH3).Scheme 5
Synthesis of compounds7a, b.


## 3.2. Antitumor Activity

It has been proved that chromones have various kinds of biological activities, including antitumor, antimicrobial, antiviral, anti-inflammatory, antioxidant, and so on. Cytotoxicity refers to cell death, cell lysis, and the inhibition on cell proliferation induced by some substances.In vitro, most of chromones’s cytotoxicity against tumor cells has been tested to confirm their antitumor activity. Cell toxicity is generally evaluated using the MTT (microculture tetrazolium) or SRB (sulforhodamine B) assay. Chromones demonstrate cell toxicity against a quantity of cell lines from a great variety of tumors, including cervical epithelioid carcinoma, breast adenocarcinoma [33], hepatoma carcinoma, lung cancer, leukemia cancer, and colon cancer [34, 35].


### 3.2.1. Cell Viability

(1) Fast Screening. Quick screening was performed to determine the newly synthesized derivatives which demonstrated a significant reduction in the cell viabilities against the two cell lines: hepatocellular carcinoma (HepG2) and breast carcinoma (MCF7). The impact of the newly derivatives on the breast carcinoma cell line (MCF7) after 48 hrs, as an example, is illustrated in (Figure 1).Figure 1
Breast carcinoma cell line (MCF7) cell viability after 48 h treatment with the synthesized derivatives.Most of the compounds exhibited a distinct reduction in the cell viability of at least one of the two cell lines. Therefore, all the compounds were further analyzed to determine their IC50 values.(2) IC50 Determination. The data calculated as the concentration of the tested samples needed to inhibit half of the cancer cells population IC50 values were calculated for each compound separately and, mean values ± SD are presented Table 1.Table 1
The antiproliferative potency of the newly prepared derivatives toward hepatic and breast cancer cell lines.
CompoundCell linesMCF-7 IC50 ± SD (μg/mL)HEPG-2 IC50 ± SD (μg/mL)Solvent (DMSO)76 ± 0.6677 ± 0.53153 ± 0.3544 ± 5.434a43 ± 0.2035 ± 0.124b37 ± 1.1743 ± 2.904c55 ± 0.5851 ± 0.374d45 ± 1.0449 ± 1.104e41 ± 11.2833 ± 2.104f50 ± 2.7964 ± 3.104g52 ± 1.5245 ± 0.474h43 ± 1.1555 ± 1.335a23 ± 1.0347 ± 2.805b20 ± 1.4433 ± 2.335c19 ± 1.2243 ± 1.575d25 ± 2.6732 ± 1.675e18 ± 1.7354 ± 1.275f24 ± 0.1045 ± 0.095g26 ± 3.2356 ± 2.595h19 ± 1.1941 ± 1.39644 ± 1.8656 ± 1.797a34 ± 1.3445 ± 1.647b33 ± 2.7939 ± 0.475 fluorouracil13 ± 0.56Doxorubicin14 ± 1.07The results stated in Table1 reveal that many compounds showed good antiproliferative activity against breast cancer cell line (MCF7) with no toxicity on normal cell line. According to National Cancer Institute guidelines, the compound with an IC50 value < 30 μg/mL is active [36]. Therefore, as almost all compounds showed inhibition of cell growth, all the compounds were further analyzed to determine their IC50. MTT test for the investigated compounds showed that most of them expressed the IC50 ranging from 18 to 64 μg/ml compared with the standard drugs in used range of concentration, and they have low toxicity on the normal cell line (Table 1). From the all tested derivatives on these two cancer cell lines, compound 5a–h had the lowest IC50 ranging from 18–26 μg/ml against breast carcinoma cell line (MCF7) (Figure 2).Figure 2
Dose-dependent response of two cell lines cells treated with the derivatives5a–h determined by MTT assay (48 h).
(a)(b)(c)(d)(e)(f)(g)(h)The abovementioned results showed that different cell lines varied greatly in their response against different synthesized compounds. This correlates well with previously reported results [37, 38], where this can be attributed to the inherent different cells in their specific membrane structure and organization.


## 3.2.1. Cell Viability

(1) Fast Screening. Quick screening was performed to determine the newly synthesized derivatives which demonstrated a significant reduction in the cell viabilities against the two cell lines: hepatocellular carcinoma (HepG2) and breast carcinoma (MCF7). The impact of the newly derivatives on the breast carcinoma cell line (MCF7) after 48 hrs, as an example, is illustrated in (Figure 1).Figure 1
Breast carcinoma cell line (MCF7) cell viability after 48 h treatment with the synthesized derivatives.Most of the compounds exhibited a distinct reduction in the cell viability of at least one of the two cell lines. Therefore, all the compounds were further analyzed to determine their IC50 values.(2) IC50 Determination. The data calculated as the concentration of the tested samples needed to inhibit half of the cancer cells population IC50 values were calculated for each compound separately and, mean values ± SD are presented Table 1.Table 1
The antiproliferative potency of the newly prepared derivatives toward hepatic and breast cancer cell lines.
CompoundCell linesMCF-7 IC50 ± SD (μg/mL)HEPG-2 IC50 ± SD (μg/mL)Solvent (DMSO)76 ± 0.6677 ± 0.53153 ± 0.3544 ± 5.434a43 ± 0.2035 ± 0.124b37 ± 1.1743 ± 2.904c55 ± 0.5851 ± 0.374d45 ± 1.0449 ± 1.104e41 ± 11.2833 ± 2.104f50 ± 2.7964 ± 3.104g52 ± 1.5245 ± 0.474h43 ± 1.1555 ± 1.335a23 ± 1.0347 ± 2.805b20 ± 1.4433 ± 2.335c19 ± 1.2243 ± 1.575d25 ± 2.6732 ± 1.675e18 ± 1.7354 ± 1.275f24 ± 0.1045 ± 0.095g26 ± 3.2356 ± 2.595h19 ± 1.1941 ± 1.39644 ± 1.8656 ± 1.797a34 ± 1.3445 ± 1.647b33 ± 2.7939 ± 0.475 fluorouracil13 ± 0.56Doxorubicin14 ± 1.07The results stated in Table1 reveal that many compounds showed good antiproliferative activity against breast cancer cell line (MCF7) with no toxicity on normal cell line. According to National Cancer Institute guidelines, the compound with an IC50 value < 30 μg/mL is active [36]. Therefore, as almost all compounds showed inhibition of cell growth, all the compounds were further analyzed to determine their IC50. MTT test for the investigated compounds showed that most of them expressed the IC50 ranging from 18 to 64 μg/ml compared with the standard drugs in used range of concentration, and they have low toxicity on the normal cell line (Table 1). From the all tested derivatives on these two cancer cell lines, compound 5a–h had the lowest IC50 ranging from 18–26 μg/ml against breast carcinoma cell line (MCF7) (Figure 2).Figure 2
Dose-dependent response of two cell lines cells treated with the derivatives5a–h determined by MTT assay (48 h).
(a)(b)(c)(d)(e)(f)(g)(h)The abovementioned results showed that different cell lines varied greatly in their response against different synthesized compounds. This correlates well with previously reported results [37, 38], where this can be attributed to the inherent different cells in their specific membrane structure and organization.


## 3.3. Molecular Docking Studies

### 3.3.1. Reference Ligand and Docking in EGFR Domain

The binding mode of 4ZQ exhibits an energy binding of −24.81, for RMSD (1.67). 4ZQ formed a hydrogen bond with H-bond with (Lys 745) at bond distance 3.13°A, (Met 793) at bond distance of 2.95°A and (Thr 854) at 2.79°A. Docking results of the newly synthesized compounds4a–h, 5a–h, and 7a, b which docked with EGFR protein (PDB ID: 5CAV) active site (Table 2).Table 2
Docking results of the newly synthesized compounds4a–h, 5a-h, and 7a, b docked with EGFR protein (PDB ID: 5CAV) active site.
Compd. noBinding energy score (kJ mol−1)Bonds numberAmino acid residuesLigand atomH.B.B. lengthPi4ZQ−24.8133.13,2.95,2.790Lys 745, Met 793, Thr854O of OCH3, N of pyrimidine ring, N of pyridine ring4a−26.1713.151Lys 745, Phe723O of OCH3, benzene ring4b−20.6912.941Phe723O of OCH3, benzene ring4c−24.5523.71, 2.672Phe723O of OCH3, benzene ring4d−24.7612.791Lys 745, Phe723O of OCH3, benzene ring4e−23.2223.00, 2.782Lys 745, Phe723O of OCH3, CO, benzene ring4f−19.0113.042Lys 745, Phe723O of OCH3, benzo furan moiety4g−25.0523.41,2.981Lys745,Cys797, Phe723O of OCH3, N of pyridine ring4h−25.4723.11,2.722Lys 745, Phe723O of OCH3, CO, benzene furan moiety5a−29.3112.621Lys 745, Phe723CO, imidazole ring5b−25.4722.98,2.741Lys 745, Phe723O of OCH3, CO, Benzene ring5c−30.0712.591Lys 745, Phe723CO, imidazole ring5d−26.6612.611Lys 745, Phe723CO, imidazole ring5e−26.1113.432Lys 745, Phe723O of OCH3, benzo furan moiety5f−29.1513.732Lys 745, Phe723O of OCH3, benzo furan moiety5g−27.2912.630Cys797CO5h−25.1523.01,2.691Lys745, Phe723O of OCH3, CO, benzene ring7a−18.4812.59Lys745CO7b−17.1612.63Met 793COThe results revealed that compounds5a–h showed better docking score ranging from −30.07 to −25.15 kJ/mol, compared to the co-crystallized ligand (4QZ) of −24.81 kJ/mol and root-mean-square deviation value of 1.67 (Table 2). Also, compounds 5a–h showed good binding interaction to the protein active site via formation of hydrogen bonds with the same amino acid residue (Lys 745) as the co-crystalline ligand (Table 2). The results of anticancer activity and docking studies for compounds 5c and 5e were compatible. Compounds 5c and 5e showed potent anticancer activity against MCF-7 cell line (IC50 value 19 and 18 μg/ml) compared to reference drug 5-fluorouracil (IC50 13 μg/ml), and also exhibited better docking score (−30.07 and −26.11 kJ/mol) and good binding interaction via formation of one hydrogen bond with amino acid residue Lys 745 (2.59 Å and 3.43 Å) and Pi interaction with amino acid residue Phe 723 compared to co-crystallized ligand of docking score −24.81 kJ/mol and three hydrogen bonds with Lys 745, Met 793, and Thr 854 (Figures 3–5).Figure 3
(a) The 2D depiction of the docked conformation of co-crystallized ligand (4ZQ) in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of co-crystallized ligand (4ZQ) in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)Figure 4
(a) The 2D depiction of the docked conformation of5c in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of 5c in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)Figure 5
(a) The 2D depiction of the docked conformation of5e in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of 5e in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)


## 3.3.1. Reference Ligand and Docking in EGFR Domain

The binding mode of 4ZQ exhibits an energy binding of −24.81, for RMSD (1.67). 4ZQ formed a hydrogen bond with H-bond with (Lys 745) at bond distance 3.13°A, (Met 793) at bond distance of 2.95°A and (Thr 854) at 2.79°A. Docking results of the newly synthesized compounds4a–h, 5a–h, and 7a, b which docked with EGFR protein (PDB ID: 5CAV) active site (Table 2).Table 2
Docking results of the newly synthesized compounds4a–h, 5a-h, and 7a, b docked with EGFR protein (PDB ID: 5CAV) active site.
Compd. noBinding energy score (kJ mol−1)Bonds numberAmino acid residuesLigand atomH.B.B. lengthPi4ZQ−24.8133.13,2.95,2.790Lys 745, Met 793, Thr854O of OCH3, N of pyrimidine ring, N of pyridine ring4a−26.1713.151Lys 745, Phe723O of OCH3, benzene ring4b−20.6912.941Phe723O of OCH3, benzene ring4c−24.5523.71, 2.672Phe723O of OCH3, benzene ring4d−24.7612.791Lys 745, Phe723O of OCH3, benzene ring4e−23.2223.00, 2.782Lys 745, Phe723O of OCH3, CO, benzene ring4f−19.0113.042Lys 745, Phe723O of OCH3, benzo furan moiety4g−25.0523.41,2.981Lys745,Cys797, Phe723O of OCH3, N of pyridine ring4h−25.4723.11,2.722Lys 745, Phe723O of OCH3, CO, benzene furan moiety5a−29.3112.621Lys 745, Phe723CO, imidazole ring5b−25.4722.98,2.741Lys 745, Phe723O of OCH3, CO, Benzene ring5c−30.0712.591Lys 745, Phe723CO, imidazole ring5d−26.6612.611Lys 745, Phe723CO, imidazole ring5e−26.1113.432Lys 745, Phe723O of OCH3, benzo furan moiety5f−29.1513.732Lys 745, Phe723O of OCH3, benzo furan moiety5g−27.2912.630Cys797CO5h−25.1523.01,2.691Lys745, Phe723O of OCH3, CO, benzene ring7a−18.4812.59Lys745CO7b−17.1612.63Met 793COThe results revealed that compounds5a–h showed better docking score ranging from −30.07 to −25.15 kJ/mol, compared to the co-crystallized ligand (4QZ) of −24.81 kJ/mol and root-mean-square deviation value of 1.67 (Table 2). Also, compounds 5a–h showed good binding interaction to the protein active site via formation of hydrogen bonds with the same amino acid residue (Lys 745) as the co-crystalline ligand (Table 2). The results of anticancer activity and docking studies for compounds 5c and 5e were compatible. Compounds 5c and 5e showed potent anticancer activity against MCF-7 cell line (IC50 value 19 and 18 μg/ml) compared to reference drug 5-fluorouracil (IC50 13 μg/ml), and also exhibited better docking score (−30.07 and −26.11 kJ/mol) and good binding interaction via formation of one hydrogen bond with amino acid residue Lys 745 (2.59 Å and 3.43 Å) and Pi interaction with amino acid residue Phe 723 compared to co-crystallized ligand of docking score −24.81 kJ/mol and three hydrogen bonds with Lys 745, Met 793, and Thr 854 (Figures 3–5).Figure 3
(a) The 2D depiction of the docked conformation of co-crystallized ligand (4ZQ) in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of co-crystallized ligand (4ZQ) in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)Figure 4
(a) The 2D depiction of the docked conformation of5c in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of 5c in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)Figure 5
(a) The 2D depiction of the docked conformation of5e in the EGFR protein (PDB ID: 5CAV) binding site. (b) The 3D depiction of the docked conformation of 5e in the EGFR protein (PDB ID: 5CAV) binding site.
(a)(b)


## 4. Conclusion

The newly synthesized furochromone and oxazocine derivatives seemed to be preferred for pharmaceutical studies. The results obtained from the synthesized compounds showed reasonable medical indices especially potent activities and, besides this, their lower possible side effects due to no or weak action on normal cell lines. Compounds5a–h exhibited good antiproliferative potency against breast cancer cell line with week or no effect on normal cell lines. Their activities exceeded the tested standard drugs themselves. These findings may be used to design more effective and less harmful derivatives as potential anticancer agents.The binding mode of the newly synthesized compounds4a–h, 5a–h, and 7a, b was assessed by docking with the active site of EGFR protein (PDB ID: 5CAV). Our results exhibited that compounds 5c and 5e demonstrated better binding energy (−30.07 and −26.11 kJ/mol) and good fitting inside the active site of the protein molecular surface in comparison with the co-crystallized ligand, which agrees well with the biological results. In this way, they may be viewed as great inhibitors of EGFR protein and consequently have a high anticancer activity.



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*Source: 1474050-2020-04-29.xml*