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

One-pot efficient synthesis of novel chromone derivatives 4a–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.


Introduction
Cancer is uncontrolled growth of abnormal cells, one of the most widespread serious diseases and its growth and metastasis depends on angiogenesis [1][2][3][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. e 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. ese heterocyclic compounds show a variety of pharmacological properties [14]. e 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][17][18].
Imidazole derivatives are an important class of heterocyclic compounds [19,20] that exhibiting biological and pharmacological properties [21][22][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][27][28][29] depending on their molecular docking studies via one-pot reaction of three or four components of carbaldehyde, amine, isocyanate, and benzoic acid derivatives.

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-d 6 ) or chloroform (CDCl 3 ). All chemical shifts are quoted in δ relative to the trace resonance of protonated dimethyl sulfoxide (δ2.50 ppm), DMSO (δ39.51 ppm) or CDCl 3 (δ7.28 ppm), (δ77.28 ppm), and external 85% aqueous H 3 PO 4 (δ0.0 ppm). e mass spectra were measured with a GC Finnigan MAT SSQ-7000 mass spectrometer. e 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 λ 254 nm . e chemical names given for the prepared compounds are according to the IUPAC system. e reported yields are based upon pure materials isolated by column chromatography. Solvents were dried/purified according to conventional procedures.

General Procedure for the Preparation of Compounds 4a-h and 5a-h.
e 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. en, Mixture B was poured on Mixture A. Finally, the resultant mixture was stirred at room temperature for 23-68 h, and solid K 2 CO 3 (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. e resulting organic solution was then washed with 1 M HCl (aq). is was followed by adding a saturated aqueous solution of NaHCO 3 (aq) combined with brine. e resulting organic layer was collected, dried by MgSO 4 , and then concentrated in vacuum at 40°C for 8 h to afford the crude material. e crude material was purified by ethanol to give the desired products 4a-h. (4,9-dimethoxy-5- (7a, b). A solution of ethyl acetoacetate (1 mmol) and 7-hydroxy-5methoxy-4-oxo-4H-chromene-6 carbaldehyde 6 (1 mmol) in dichloromethane (3 ml) was stirred, and then phenyl isocyanate/or cyclohexyl isocyanate (1 mmol) was added to the mixture. e 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. [6,7g] [1,3] [6,7-g] [1,3] e 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 (100 μg/ml) at 37°C in humidified air containing 5% CO 2 . Cells at a concentration of 0.50 × 10 6 were grown in a 25 cm 2 flask in 5 ml of culture medium.

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

MTT Assay.
e cells were washed with 50 μL of PBS and then the PBS was discarded [36]. 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% CO 2 . e cells were examined microscopically for formazan (black precipitate) development. e supernatant was discarded from each well and the formazan was dissolved using DMSO. e absorbance of the developed color was measured using an automated plate reader at 570 nm with a background wavelength of 670 nm. e results were presented in percentage to the values obtained from untreated cells (negative control).

Molecular Docking Studies.
Promising biological evaluation of new furochromone and oxazocine derivatives 4a-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 [37] using MOE 2008.10 program.

Preparation of Receptor.
Binding sites were generated from co-crystallized ligand within crystal protein (PDB codes: 5CAV). Water molecules were removed from the complex. en, 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. e 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. en, the minimized structures were prepared for docking using prepared ligand protocol [38].

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. e 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. e calculated interaction energies for the tested compounds were in complete agreement with experimental results which showed that our compounds are potent inhibitors against EGFR.

Chemistry.
Highly substituted chromone derivatives 4a-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-, 4nitrobenzoic acid/or nicotinic acid, and phenyl isocyanate/ or cyclohexyl isocyanate (Scheme 1). e structures of 4a-h were elucidated from their elemental and spectroscopic analyses (IR, 1 H NMR, and 13 C NMR) together with mass spectra which prove the structures via getting molecular ion peaks at appropriate m/z values. 1 H 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. 13  In the same manner, furochromone carbaldehyde 1 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). e structures of the novel compounds are elucidated via elemental and spectroscopic analysis (cf. Experimental). IR spectra showed disappearance of the aldehydic group. e 13 C NMR spectrum of 5a showed 21 distinct resonances characteristic for carbon atoms. e mechanism of formation of compounds 5a-h is cited in Scheme 4, and it revealed similarity with formation mechanism of compounds 4a-h with cyclization of

Antitumor Activity.
It has been proved that chromones have various kinds of biological activities, including antitumor, antimicrobial, antiviral, antiinflammatory, 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 [30], hepatoma carcinoma, lung cancer, leukemia cancer, and colon cancer [31,32].

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). e impact of the newly derivatives on the breast carcinoma cell line (MCF7) after 48 hrs, as an example, is illustrated in (Figure 1).
Most of the compounds exhibited a distinct reduction in the cell viability of at least one of the two cell lines. erefore, all the compounds were further analyzed to determine their IC 50 values.
(2) IC 50 Determination. e data calculated as the concentration of the tested samples needed to inhibit half of the cancer cells population IC 50 values were calculated for each compound separately and, mean values ± SD are presented Table 1.
e results stated in Table 1 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 IC 50 value < 30 μg/mL is active [33]. erefore, as almost all compounds showed inhibition of cell growth, all the compounds were further analyzed to determine their IC 50 . MTT test for the investigated compounds showed that most of them expressed the IC 50 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 IC 50 ranging from 18-26 μg/ml against breast carcinoma cell line (MCF7) (Figure 2). e abovementioned results showed that different cell lines varied greatly in their response against different synthesized compounds.
is correlates well with previously reported results [34,35], where this can be attributed to the inherent different cells in their specific membrane structure and organization.          results of the newly synthesized compounds 4a-h, 5a-h, and 7a, b which docked with EGFR protein (PDB ID: 5CAV) active site (Table 2). e results revealed that compounds 5a-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). e 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 (IC 50 value 19 and 18 μg/ml) compared to reference drug 5-fluorouracil (IC 50 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 (2.59Å and 3.43Å) 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 r 854 (Figures 3-5).

Conclusion
e newly synthesized furochromone and oxazocine derivatives seemed to be preferred for pharmaceutical studies. e 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. Compounds 5a-h exhibited good antiproliferative potency against breast cancer cell line with week or no effect on normal cell lines. eir activities exceeded the tested standard drugs themselves. ese findings may be used to design more effective and less harmful derivatives as potential anticancer agents. e binding mode of the newly synthesized compounds 4a-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.
Data Availability e data supporting the findings of the study are already given within the article.