Uses of 3-(2-Bromoacetyl)-2H-chromen-2-one in the Synthesis of Heterocyclic Compounds Incorporating Coumarin: Synthesis, Characterization and Cytotoxicity

In this work, 3-bromoacetylcoumarin was used as the key starting material for the synthesis of pyran, pyridine, thiophene, thiazole and pyrazole derivatives through its reaction with different reagents. The structures of the newly synthesized compounds were confirmed on the basis of their spectral data and elemental analyses. All of the synthesized compounds were screened for their in vitro anticancer activity against six human cancer cell lines, namely: human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), nasopharyngeal carcinoma (HONE1), human breast cancer (MCF) and normal fibroblast cells (WI38). The IC50 values (the sample concentration that produces 50% reduction in cell growth) in nanomolars (nM)) showed most of the compounds exhibited significant cytotoxic effect. Among these derivatives, compound 6d showed almost equipotent cytotoxic activity against NUGC (IC50 = 29 nM) compared to the standard CHS 828 (IC50 = 25 nM).


Introduction
Coumarins are a large group of naturally occurring compounds synthesized by numerous plant species as well as by some bacteria and fungi [1,2]. According to their chemical structure, they belong to the family Hybrid molecules, combining coumarins with different bioactive molecules like: pyran [22], pyridine [23], thiazole [24] and pyrazole [25] have recently been reported; these studies resulted in new compounds exhibiting significant anticancer activities.

Structure Activity Relationship
In this study, when correlating the structures of the synthesized compounds with their anticancer activity, it has been observed that most of the synthesized compounds exhibited significant cytotoxic effects with IC50 values < 900 nM. Normal fibroblast cells (WI38) were affected to a much lesser extent (IC50 > 10,000 nM).
Comparing the cytotoxicity of the thiophene derivatives 4a and 4b, one can say that the cytotoxicity of 4a was higher than that of 4b. The presence of CN group with the thiophene ring in 4a was responsible for its high potency.
Among the thiazole derivatives 5a-d, compound 5d is the most active derivative. It showed high potency against NUGC, DLDI, HA22T and HONEL with IC50 of 38, 163, 120 and 441 nM, respectively. Such high potency of 5d is due to the presence of the 4-chlorophenyl moiety with the thiazole ring. The presence of p-tolyl moiety in 5b decreases the activity relative to the unsubstituted phenyl derivative 5a. On the other hand, the introduction of 4-OCH3 group in 5c revealed better cytotoxicity against DLDI and HONEL than 5a.
Considering the bromo-4H-pyran derivatives 6a-d, compounds 6c and 6d revealed higher cytotoxic activity than 6a and 6b, both of them were active against most cancer cell lines. Compound 6d showed almost equipotent activity against NUGC (IC50 = 29 nM) compared with the standard CHS 828 (IC50 = 25 nM). At the same time, 6c exhibited the highest cytotoxicity among the four derivatives against MCF with IC50 = 89 nM. The reason for the high cytotoxicity of compounds 6c and 6d was attributed to the presence of the 4-chlorophenyl and the furan moieties, respectively.
The 5-bromo-1,4-dihydropyridine derivatives 7a-d showed optimal cytotoxic activity. Compounds 7a, 7b and 7c exhibited cytotoxic activity towards the six cancer cell lines. Compound 7c incorporating with the 4-chlorophenyl moiety showed the highest potency among the four compounds with IC50 of 32 and 43 nM against DLDI and MCF, respectively. In general, the presence of the 5-bromopyridine moiety in compounds 7a-c was responsible for their high potency.
Comparing the cytotoxicity of the pyrazole derivatives 9a and 9b, it was clear that the cytotoxicity of 9a was higher than that of 9b. It was clear that the N-phenylpyrazolyl moiety in compound 9b was responsible for its lower potency.
Among the 4H-pyran-3,5-dicarbonitrile 10a-d, compounds 10c and 10d showed higher cytotoxicity than 10a and 10b. Such high potency was attributed to the presence of 4-chlorophenyl group in the case of compound 10c, and the furan moiety in case of compound 10d, together with the pyran ring.

Chemistry
All melting points were determined on a Stuart apparatus and the values given are uncorrected. IR spectra (KBr, cm −1 ) were determined on a Shimadzu IR 435 spectrophotometer (Faculty of Pharmacy, Cairo University, Egypt). 1 H-NMR and 13 C-NMR spectra were recorded on Bruker Ascend 400 MHz spectrophotometers (Microanalytical Unit, Faculty of Pharmacy, Cairo University, Egypt) using TMS as the internal standard. Chemical shift values were recorded in ppm on δ scale. The electron impact (EI) mass spectra were recorded on a Hewlett Packard 5988 spectrometer (Microanalysis Center, Cairo University, Egypt). Elemental analyses were carried out at the Microanalysis Center, Cairo University, Egypt; found values were within ±0.35% of the theoretical ones. The progress of the reactions was monitored using thin layer chromatography (TLC) sheets precoated with UV fluorescent silica gel Merck 60F 254 and were visualized using UV lamp. The 3-(2-bromoacetyl)-2H-chromen-2-one (1) [30,31] was obtained using the reported procedure by the reaction of 3-acetylcoumarin in chloroform solution with bromine together with continuous stirring.   or furfural (0.96 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL) was heated under reflux for 2 h, left to cool to room temperature, poured onto ice/water, and neutralized by hydrochloric acid. The precipitated solid was collected by filtration, washed with water and crystallized from ethanol.    or furfural (0.96 g, 0.01 mol) in absolute ethanol (40 mL) containing ammonium acetate (0.5 g) was heated under reflux for 3 h, left to cool to room temperature, poured onto ice/water, and neutralized with hydrochloric acid. The precipitated solid was collected by filtration, washed with water and crystallized from ethanol.  A solution of compound 8 (2.13 g, 0.01 mol) and either hydrazine hydrate (0.5 g, 0.01 mol) or phenylhydrazine (1.08 g, 0.01 mol) in absolute ethanol (40 mL) was heated under reflux for 2 h, left to cool to room temperature, poured onto ice/water containing few drops hydrochloric acid. The resulting product was collected by filtration, washed with water and crystallized from ethanol.