Design, Synthesis and Anticancer Activity of New Polycyclic: Imidazole, Thiazine, Oxathiine, Pyrrolo-Quinoxaline and Thienotriazolopyrimidine Derivatives

In this article, we showed the synthesis of new polycyclic aromatic compounds, such as thienotriazolopyrimidinones, N-(thienotriazolopyrimidine) acetamide, 2-mercapto-thienotriazolo-pyrimidinones, 2-(((thieno-triazolopyrimidine) methyl) thio) thieno-triazolopyrimidines, thieno-pyrimidotriazolo-thiazines, pyrrolo-triazolo-thienopyrimidines, thienopyrimido-triazolopyrrolo-quinoxalines, thienopyrimido-triazolo-pyrrolo-oxathiino-quinoxalinones, 1,4-oxathiino-pyrrolo- triazolothienopyrimidinones, imidazopyrrolotriazolothienopyrimidines and 1,2,4-triazoloimidazo- pyrrolotriazolothienopyrimidindiones, based on the starting material 2,3-diamino-6-benzoyl-5- methylthieno[2,3-d]pyrimidin-4(3H)-one (3). The chemical structures were confirmed using many spectroscopic ways (IR, 1H, 13C, −NMR and MS) and elemental analyses. A series of thiazine, imidazole, pyrrole, thienotriazolopyrimidine derivatives were synthesized and evaluated for their antiproliferative activity against four human cancer cell lines, i.e., CNE2 (nasopharyngeal), KB (oral), MCF-7 (breast) and MGC-803 (gastric) carcinoma cells. The compounds 20, 19, 17, 16 and 11 showed significant cytotoxicity against types of human cancer cell lines.


Introduction
Cancer is one of the most recent serious diseases that afflict humans and ultimately leads to their death. From here, researchers began to develop and discover much effective anticancer therapeutics, such as treatment of cancer using chemotherapy [1] and heterocyclic compounds, such as thienopyrimidine, triazolopyrimidine, quinoxaline and imidazole derivatives containing anticancer drugs [2], as follows: Azathioprine, Pimonidazole, Dacarbazine, Misonidazole, Fadrozole, Tipifarnib, Bendamustine, Indimitecan and Nilotinib. Therefore, the chemical studies of each of thiophene, pyrimidine, triazole, thiazine, pyrrole, quinoxaline and imidazole nucleus play a significant role in the synthesis of a diversity of fused heterocyclic compounds having a wide range of biological and pharmacological activities. Accordingly, previous scientific studies confirmed that thienopyrimidine derivatives have various biological and pharmacological activities, such as antibiotics [3], antimicrobial [4,5], anticonvulsants [6], antiviral [7], antioxidant and antitumor agents [8], anticancer [9] and mitotic arrest of breast cancer [10], anti-inflammatory and analgesic activities [11,12], antiglaucoma agents [13], platelet aggregation inhibitors [14], anti-hyperlipidemia [15], antidepressant, anti-inflammatory and antimicrobial activities [16]. In addition, the thiazolopyrimidine, 1,2,4-triazolopy-rimidine and thienotriazolopyrimidinone derivatives possess such biological activities as [14], anti-hyperlipidemia [15], antidepressant, anti-inflammatory and antimicrobial activities [16]. In addition, the thiazolopyrimidine, 1, 2, 4-triazolopy-rimidine and thienotriazolopyrimidinone derivatives possess such biological activities as anti-inflammatory and analgesic activity [17,18], antimicrobial [19] and anticancer activity through a potential of the enzyme (PARP-1) inhibition [20]. Lately, in connection to continuing work in the synthesis and biological evaluation of new polycyclic fused thienopyrimidine, purine and 1,2,4-triazole systems, purine derivatives are of great importance and wide applications in many biological activities, such as antitumor [21] and the potential xanthine oxidase (XO)-inhibitory activities, as well as many biological activities when fused with 1,2,4-triazole ring, shown in Figure 1, as the following: 7β-D-ribofuranosyl-1,2,4-triazolopurines (A) [22], 1,2,4-triazolopurines (B) [23], thienotriazolopyrimidinones (C) and 2-sub-thienotriazolopyrimidinones (D) as a new class of the XO inhibitors [24]. Additionally, Azathioprine (E) is an anticancer drug that possesses considerable potential, because of its ability to interfere with DNA prepared and then stop growth and division cells and it is used for the treatment of metastatic malignant melanoma and cell carcinoma of the pancreas [25]. The nitrogen atoms containing heterocycles, especially, display a different range of biological activities, due to their similarities with numerous synthetic and natural molecules with recognized biological activities [26]. The benzimidazole and imidazole rings have been generally used as the substantial basic structure for the development of therapeutic molecules of biological and pharmaceutical activities. An example of five-membered heterocycles is imidazole, which is spread between the significant biological building blocks. Thus, many drugs contain imidazole, such as the following: Sertaconazole is used as an antifungal agent [27]. Omeprazole is antiulcer and controls the acid secretion in the stomach and it is considered clinically superior to H2-receptor antagonists [28,29]. Mizolastine is an antihistaminic and potent antagonist at H1 receptor sites for the treatment of allergic rhinoconjunctivitis and urticarial [30]. Candesartan is used as a receptor antagonist, because it contains a bulky lipophilic group, carboxylic acid, the biphenyl group that is more efficient than the tetrazole analogue [31]. Azanidazole is an antiparasitic drug and used as an antibacterial and antiprotozoal drug [32]. Maribavir is an antiviral drug, which is used in the treatment and prevention of human cytomegalovirus (HCMV) disease in hematopoietic stem cell/bone marrow transplant patients [33], as shown in Figure 2. The nitrogen atoms containing heterocycles, especially, display a different range of biological activities, due to their similarities with numerous synthetic and natural molecules with recognized biological activities [26]. The benzimidazole and imidazole rings have been generally used as the substantial basic structure for the development of therapeutic molecules of biological and pharmaceutical activities. An example of five-membered heterocycles is imidazole, which is spread between the significant biological building blocks. Thus, many drugs contain imidazole, such as the following: Sertaconazole is used as an antifungal agent [27]. Omeprazole is antiulcer and controls the acid secretion in the stomach and it is considered clinically superior to H 2 -receptor antagonists [28,29]. Mizolastine is an antihistaminic and potent antagonist at H 1 receptor sites for the treatment of allergic rhinoconjunctivitis and urticarial [30]. Candesartan is used as a receptor antagonist, because it contains a bulky lipophilic group, carboxylic acid, the biphenyl group that is more efficient than the tetrazole analogue [31]. Azanidazole is an antiparasitic drug and used as an antibacterial and antiprotozoal drug [32]. Maribavir is an antiviral drug, which is used in the treatment and prevention of human cytomegalovirus (HCMV) disease in hematopoietic stem cell/bone marrow transplant patients [33], as shown in Figure 2.

General Information
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Tokyo, Japan). Elemental analyses were performed on Vario EL (Elementar, Langenselbold, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University and National Research Centre. The IR spectra (KBr disc) were recorded using a Perkin-Elmer 1650 spectrometer (Waltham, MA, USA). NMR spectra were determined using JEOL 270 MHz and JEOL JMS-AX 500 MHz (JEOL, Tokyo, Japan) spectrometers with Me4Si as an internal standard. Mass spectra were recorded on an EI Ms-QP 1000 EX instrument (Shimadzu, Tokyo, Japan) at 70 eV. Biological evaluations were done by the anticancer unit, Mansoura University, Faculty of Pharmacy (Department of Pharmacognosy), 35516, Egypt. All starting materials and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).  19.2, 121.3, 128.6, 129.3, 131.5, 132.3, 135.3, 142.2, 146.4, 149.5, 158.1, 168

General Information
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Tokyo, Japan). Elemental analyses were performed on Vario EL (Elementar, Langenselbold, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University and National Research Centre. The IR spectra (KBr disc) were recorded using a Perkin-Elmer 1650 spectrometer (Waltham, MA, USA). NMR spectra were determined using JEOL 270 MHz and JEOL JMS-AX 500 MHz (JEOL, Tokyo, Japan) spectrometers with Me4Si as an internal standard. Mass spectra were recorded on an EI Ms-QP 1000 EX instrument (Shimadzu, Tokyo, Japan) at 70 eV. Biological evaluations were done by the anticancer unit, Mansoura University, Faculty of Pharmacy (Department of Pharmacognosy), 35516, Egypt. All starting materials and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).  19.2, 121.3, 128.6, 129.3, 131.5, 132.3, 135.3, 142.2, 146.4, 149

General Information
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Tokyo, Japan). Elemental analyses were performed on Vario EL (Elementar, Langenselbold, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University and National Research Centre. The IR spectra (KBr disc) were recorded using a Perkin-Elmer 1650 spectrometer (Waltham, MA, USA). NMR spectra were determined using JEOL 270 MHz and JEOL JMS-AX 500 MHz (JEOL, Tokyo, Japan) spectrometers with Me4Si as an internal standard. Mass spectra were recorded on an EI Ms-QP 1000 EX instrument (Shimadzu, Tokyo, Japan) at 70 eV. Biological evaluations were done by the anticancer unit, Mansoura University, Faculty of Pharmacy (Department of Pharmacognosy), 35516, Egypt. All starting materials and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

General Information
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Tokyo, Japan). Elemental analyses were performed on Vario EL (Elementar, Langenselbold, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University and National Research Centre. The IR spectra (KBr disc) were recorded using a Perkin-Elmer 1650 spectrometer (Waltham, MA, USA). NMR spectra were determined using JEOL 270 MHz and JEOL JMS-AX 500 MHz (JEOL, Tokyo, Japan) spectrometers with Me4Si as an internal standard. Mass spectra were recorded on an EI Ms-QP 1000 EX instrument (Shimadzu, Tokyo, Japan) at 70 eV. Biological evaluations were done by the anticancer unit, Mansoura University, Faculty of Pharmacy (Department of Pharmacognosy), 35516, Egypt. All starting materials and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

Synthesis of N-(4-(3-Acetyl-6-Benzoyl-7-Methyl-8-oxo-3,8-Dihydrothieno[2,3-d][1,2,4] Triazolo[1,5-a]Pyrimidin-2-yl)Phenyl) Acetamide (5)
Method A: To a warm (60-80 • C) solution of sodium ethoxide (prepared by dissolving 0.23 g of sodium metal in 35 mL ethanol), compound 4 (4.01 g, 10 mmol) was added, and heating (60-80 • C) was continued for 40 min. The mixture was allowed to cool to room temperature, and acetic anhydride (10 mmol) was added. The mixture was stirred under reflux for 8-10 h, allowed to cool to room temperature and finally poured into 100 mL cold water. The solid product precipitate was filtered off and washed with 100 mL water. The solid obtained was filtered off, dried and crystallized from the proper solvent to produce compound 5. Method B: A mix of compound 4 (4.01 g, 0.01 mol) and acetic anhydride (30 mL) was refluxed with heat for one hour, checked via (TLC). After cooling, the solution was concentrated and poured into crushed ice. The separated solid was filtered off, dried and recrystallized from benzene as yellowish crystals in 74% yield, m. p. > 350 • C. IR  A mixture of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g, 0.01 mol), the appropriate aromatic aldehyde (0.01 mol) and the catalytic amount of potassium iodide (0.3 g, 0.002 mol) in dimethyl-formamide (40 mL) was heated and refluxed for 20-22 h at 180-200 • C; the progress of reaction was monitored by TLC, and, at the end of reaction, the solvent was allowed to cool to room temperature and poured into water (100 mL). Then the obtained solid products were filtered off, dried and crystallized from the appropriate solvent to give compounds 6a-c in good yields, respectively.  121.2, 128.1, 128.6, 128.7, 128.9, 129.5, 130.4, 132.1, 138.7, 142.4, 143.6, 148 To a suspension of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g, 0.01 mol) and red mercury (II) oxide (2.16 g, 0.01 mol) in 40 mL of dimethylformamide was added 1.83 g, 0.01 mol, of the corresponding methylphenylcarbamodithioate in 5 mL of DMF at room temperature. The mixture was refluxed for 15-18 h under control (TLC). After cooling, the mixture was filtrated, and to the filtrate were added 50 mL of water. The precipitate thus obtained was filtrated off, washed with water, dried and recrystallized from dioxane in 76% yield as yellow crystals, m. p.  To a solution of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g, 0.01 mol) and anhydrous potassium carbonate (1.4 g, 0.01 mol) in dry acetone (50 mL), ethyl-chloroformate (1 mL, 0.01 mol) was added to the solution dropwise; after the completion of the addition, the mixture was refluxed for 14-17 h, allowed to cool at room temperature, and then poured into the cool water. The final product was obtained by being filtrated off, washed with ethanol and recrystallized from methanol in 84% yield as yellowish crystals, m. p. > 350  (9) KOH (0.56g, 0.01mol) was dissolved through stirring in 20 mL of anhydrous methanol in a 250 mL flask. Carbon disulfide (0.89 g, 0.01 mol) was dissolved in anhydrous methanol (5 mL) and added dropwise to the stirring solution, followed by reflux. The solution of 2,3-diamino-benzoyl-methyl-thienopyrimidinone (3) (3 g, 0.01 mol) in methanol (30 mL) was added to the above reaction mixture and stirring under reflux (75-80 • C) for 8-10 h. Initially, the solution was yellow, which then slowly turned to brown, and, as the reaction progressed, the evolution of hydrogen sulfide was observed. The reaction mixture was monitored via TLC (Ethyl-acetate: petroleum ether, 1:3). After completion of the reaction, the mixture was poured into a beaker containing ice water and acidified with 4N, HCl to maintain pH 4-5. The final precipitate was separated, then filtered and recrystallized from the benzene in 70% yield as yellow crystals, m. p. Chloroacetic acid (0.95 g, 0.01 mol) was dissolved in 35 mL of 4N, HCl and stirred into the mixture for nearly 30 min. 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g, 0.01 mol) was added to the above solution with constant stirring; we continued stirring with reflux (100 • C) for nearly 7-9 h. The solid product was established by TLC (Ethyl acetate: petroleum ether, 1:1). The above hot solution was poured into ice cold water with stirring, followed by dropwise addition of con NH 3 ; then the yellowish precipitate was filtered. The precipitate was then recrystallized from the dioxane in 79% yield as yellowish crystals, m. p.      and then allowed to cool and poured into ice water. The formed solid product was collected by filtration and crystallized from the proper solvent. Method B: A solution of compound 19 (4.34 g, 0.01 mol) and benzohydrazide (1.36 g, 0.01 mol) in ethanol (40 mL) containing sodium ethoxide [prepared by dissolving sodium metal (0.23 g, 0.01 mol) in ethanol] was heated under reflux for 11-15 h, the reaction mixture was cooled and the deposited precipitate was filtered off, washed with ice water/ethanol and acidified with 10% HCl. The formed precipitate was filtered, dried and crystallized from dioxane in 73% yield as yellowish crystals, m. p. > 350

Human and Animal Rights
No humans or animals were used in the study. The research was conducted according to ethical standards in vitro.

Chemicals and Drugs
Types of human carcinoma cancer cell line (CNE2, KB, MCF-7 and MGC-803) are derived from the National Cancer institute, Cairo University, Cairo, Egypt, and 5-Fluorouracil and DMSO were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

Materials and Methods (In Vitro Cytotoxicity)
The in vitro cytotoxicity of the synthesized compounds against different cancer cell lines was performed with the MTT assay, according to the method found in [34,38,40,48]. The MTT assay is based on the reduction of the soluble 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazoliumbromide (MTT) into a blue-purple formazan product, mainly by mitochondrial reductase activity inside living cells. The cells used in the cytotoxicity assay were cultured in the suitable cell culture medium (RPMI 1640) medium supplemented with 10% fetal calf serum. Cells suspended in the medium (2Y' 104/mL) were plated in 96-well culture plates and incubated at 37 • C in a 5% CO 2 incubator. After 12 h, the test sample (2 mL) was added to the cells (2Y' 104) in 96-well plates and cultured at 37 • C for 3 days. The cultured cells were mixed with 20 mL of MTT solution and incubated for 4 h at 37 • C. The supernatant was carefully removed from each well, and 100 mL of DMSO was added to each well to dissolve the formazan crystals, which were formed by the cellular reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well was measured by a microplate reader using a test wavelength of 570 nm. The results were expressed as the IC 50 , which is the concentration of the drugs inducing a 50% inhibition of cell growth of treated cells, when compared to the growth of control cells. Each experiment was performed at least 3 times. There was a good reproducibility between replicate wells with standard errors below 10%.