Synthesis and cytotoxic evaluation of novel chromenes and chromene(2,3-d)pyrimidines

The synthesis of novel compounds starting from 2-amino-8-(2-chlorobenzylidene)-4-(2-chlorophenyl)-5,6,7,8tetrahydro-4H-chromene-3-carbonitrile 2 has been studied. Diarylidene cyclohexanone reacts with malononitrile to afford compound 2. Compound 2 reacts with benzoyl chloride to afford compound 3. N-(8-(2-chlorobenzylidene)4-(2-chlorophenyl)-3-cyano-5,6,7,8-tetrahydro-4H-chromen-2-yl)benzamide 3 reacts with acetic anhydride to afford compound 4. Compound 2 reacts with acetic anhydride to afford 9-(2-chlorobenzylidene)-5-(2-chlorophenyl)-2methyl-3,5,6,7,8,9-hexahydro-4H-chromeno[2,3-d]pyrimidin-4-one 5. Chromene derivative 2 reacts with formic acid to give compound 6. Compounds 4–6 react with phosphorus oxychloride to give compounds 7a–c. Chromeno[2,3-d] pyrimidine derivatives 7a–c react with hydrazine hydrate to afford compounds 8a–c. Chromeno[2,3-d]pyrimidine derivatives 8a,b react with xylose and glucose to give compounds 9a–d. Chromeno[2,3-d]pyrimidine derivatives 9a–d react with acetic anhydride to give compounds 10a–d. Screening of most of the synthesized compounds against A-549, CaCo-2, and HT-29 cell lines were done. 2-Amino-8-(2-chlorobenzylidene)-4-(2-chlorophenyl)-5,6,7,8tetrahydro-4H-chromene-3-carbonitrile 2 gives high cytotoxic activity against A-549 and HT-29 cancer cell lines as compared to doxorubicin as the reference drug.

In addition, 4H-chromene derivatives have shown spasmolytic, diuretic, anticoagulant, and antianaphylactic activities (Ghorbani-Vaghei et al., 2011). 4H-Chromene derivatives bind to the Bcl-2 protein and initiate apoptosis in cancer cells. The Bcl-2 protein improves neoplastic cell proliferation by preventing normal cell turnover. Increasing Bcl-2 gene expressions are present in many types of human cancers and can result in cancer cell resistance to chemotherapy and radiotherapy. Therefore, Bcl-2 protein-binding compounds are promising compounds as anticancer agents (Ghorbani-Vaghei et al., 2011). Aminochromene derivatives have also shown antihypertensive and anti-ischemic behavior (Ghorbani-Vaghei et al., 2011).

Experimental section
The apparatus used was as in a previously reported study . Compound 1 (diarylidene cyclohexanone) was prepared according to previously known literature (Kumar et al., 2011).

General procedure for the preparation of compounds 7a-c
A mixture of compounds 4-6 (0.01 mol), 30-ml phosphorus oxychloride, and 2 g phosphorous pentachloride was refluxed for 6 hours. Then, the reaction mixture was cooled and filtered. The precipitate was filtered and crystalized from ethanol to give compound 7a-c.

General procedure for the preparation of compounds 8a-c
A mixture of compounds 7a-c (0.01 mol), 1-ml hydrazine hydrate in 30-ml dioxane was refluxed for 4 hours. Then, the reaction mixture evaporated under reduced pressure. The residue was crystallized from ethanol to give compounds 8a-c.

General procedure for the preparation of compounds 9a-d
A mixture of compounds 8a,b (0.01 mol), 40-ml ethanol, 5-ml distilled water, 1-ml acetic acid, and glucose or xylose (0.01 mol) was refluxed for 6 hours. The reaction mixture evaporated under reduced pressure. The residue crystallized from ethanol to give compounds 9a-d.

General procedure for the preparation of compounds 10a-d
A mixture of compounds 9a-d (0.01 mol) and 10-ml acetic anhydride was refluxed for 20 hours. Then, the reaction mixture was poured into water and the solid formed filtered, dried, and crystallized from ethanol to give compounds 10a-d.

Cytotoxic activity
The cytotoxic activity was carried out based on a previously reported procedure (Yousif et al., 2019c).

RESULTS AND DISCUSSION
Diarylidene cyclohexanone 1 reacts with malononitrile in triethylamine to produce 2-Amino-8-(2-chlorobenzylidene)-4-(2-chlorophenyl)-5,6,7,8-tetrahydro-4H-chromene-3carbonitrile 2. Compound 2 has been previously reported (Wang et al., 2004a;Jin et al., 2005;Wang et al., 2004b;Kumar et al., 2011). The method of preparation of compound 2 was a modified method, by using triethylamine as a weak base instead of sodium methoxide in a solvent-free reaction. The proposed structure is in agreement with spectral data. The IR of compound 2 shows the absorption band for CN group and NH 2 group and shows the disappearance of carbonyl group absorption band. Mass spectroscopy for compound 2 shows a molecular ion peak at m/z 409.

Cytotoxic activity
The cytotoxic activity of the new synthesized compounds was carried out against three different cancer cell lines, namely adenocarcinomic human alveolar basal epithelial cells A-549, human epithelial colorectal adenocarcinoma cells CaCo-2, and human colorectal adenocarcinoma cell line using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Yousif et al., 2019c). The results are presented in Table 1 as cytotoxic activity of the synthesized compounds at 100 μM on the three cell lines. The results show that compounds 9b,d and 10b,d have moderate cytotoxic activity toward A-549 cell lines when compared to doxorubicin as the reference drug. Compounds 3-6, 7b, and 8a-b have a weak cytotoxic activity toward A-549 cell lines. Compound 2 has high cytotoxic activity toward CaCo-2 cell lines when compared to doxorubicin as the reference drug. Compounds 5, 6, 9b, and 10b have a weak cytotoxic activity toward CaCo-2 cell lines. Compound 2 shows high cytotoxic activity toward HT-29 cell lines. Compounds  3, 5, 6, 7a-b, 8a-b, 9b,d, and 10b,d show a weak cytotoxic activity toward HT-29 cell lines.
From the aforementioned biological activity, we can deduce the structural activity relationship. The presence of the amino group at position 2 and the cyano group at position 3 in compound 2 increases the cytotoxic activity toward CaCo-2 and HT-29 cell lines. The presence of the hydrazine group linked to glucose in compounds 9b,d makes the cytotoxic activity moderate toward A-540 cell lines. The presence of the triazolo ring linked to acetylated glucose in compound 10b,d makes the cytotoxic activity moderate toward A-549 cell lines. The disappearance of the amino group in compound 3 and the presence of the pyrimidine ring linked to chromene afford a weak cytotoxic activity toward A-549 cell lines. The presence of the pyrimidine ring linked to chromene and chlorine atom at position 4 in compound 7b makes cytotoxic activity weak toward A-549 cell lines. Also, the presence of the pyrimidine ring linked to chromene and hydrazine function group at position 4 in compound 8a,b makes the cytotoxic activity weak toward A-549 cell lines. The presence of the pyrimidine ring linked to chromene in compounds 5,6 makes the cytotoxic activity toward CaCo-2 cell lines weak. Also, the presence of the pyrimidine ring linked to the chromene and hyrazino function group and linked to glucose in compound 9b makes cytotoxic activity weak towards CaCo-2 cell lines. In addition, the presence of the pyrimidine ring and triazolo ring linked to chromene and acetylated glucose in compound 10b makes the cytotoxic activity weak toward CaCo-2 cell lines.

CONCLUSION
Novel compounds derived from chromene have been synthesized and structurally elucidated using mass spectroscopy, infrared, and nuclear magnetic resonance spectroscopy. Screening of most of the synthesized compounds against A-549, CaCo-2, and HT-29 cell lines has been made.