One Pot Synthesis, Quantum Chemical Stimulation and Anticancer Evaluation of Some New 8-Azacoumarin Derivatives

New anticancer agents are highly needed to overcome cancer cell resistance. Synthesis of newly pyrazole, derivatives via heterocyclic ring opening of azacoumarin promoted with grinding and ultrasonic reaction conditions. Ecient solvent less one pot synthesis can be well progressed to afford the good yield of new heterocyclic products that were characterized by IR, 1H-NMR, MS and micro-analytical data. Anticancer evaluation for the synthesized compounds exhibited good cytotoxiciy. The anti-liver cancer activity of all compounds was screened in vitro against hepatocellular carcinoma (HCC) cell lines (HepG-2) by viability assay. The synthesized compounds were evaluated for their anticancer activity and found to exhibit promising activities. All new compounds were tested for possible anti-cancer activity against HepG-2 cell lines in comparison to the reference drug doxorubicin (DOX). Compound 8 was the most active against the liver carcinoma cell line (HepG-2) giving promising half-maximal inhibitory concentration (IC50) value of 27.5 ± 1.3 μg/mL, compared with DOX with IC50 value of 0.36 ± 0.02 μg/mL. However it has weak cytotoxic effects against normal rat hepatocytes with 50% cytotoxic concentration (CC50) = 1820.5 µg/ml (= > 500 µg/ml). Compound 8 was selected to be tested in combination with ionizing gamma radiation. Gene expression levels of the cell cycle inhibitor p21 and caspase-3 was quantied. As well as, Oxidative stress was quantied by measuring the concentration of malondialdehyde (MDA), and antioxidant activity of reduced gluthatione (GSH). This study concluded that the new derivative of the azacomarin compound has an effective anti-cancer effect and it was found that using the new compound with ionizing radiation at a dose of 8 Gy improves the effectiveness of the compound on liver cancer cells.


Introduction
Many heterocyclic compounds encircling pyrazole ring are correlated with diverse pharmacological belongings.
These classes of heterocyclic compounds have synchronized the cardiovascular system, antimicrobial [ 1 , 2 , 3 ], anticancer [ 4 ], anticonvulsant [ 5 ], antiviral [ 6 , 7 , 8 ], anti-HlV [ 9 ], antifungal activity [ 10 ] and anti-leishmanial [ 11 ]. Pyrano-[2,3-b]-pyridine (8-Azacoumarin) structure is considered a key starting material for many heterocyclic compounds and screening a broad spectrum of biological activity [ 12 ]. This prompted us a speci c simple synthesis aiming to construct some new pyrazole derivatives in a single molecular framework as a unique key precursor designing new, potent, selective agent appear to be promising for anticancer evaluation. Cancer drug targeting is an effective way to treat malignancies or cancer growths. Chemotherapy can be used either individually or in conjunction with other interventions, such as surgery or radiation. Radiotherapy uses high frequency penetrating waves like x-rays or gamma rays, or particles like proton rays or neutron rays to kill or prevent the replication of cancer cells. A proliferation of naturally occurring coumarin and coumarin-related compounds and their wide therapeutic potential placed them among the most promising drugs [ 13 ].For several years, natural and arti cial coumarin derivatives have demonstrated a variety of and important therapeutic potential like antimicrobial, [ 14 ] anti-in ammatory [ 15 ], analgesic [ 16 ], anticancer [ 17 ], anticoagulant [ 18 ], antioxidant [ 19 ], antiHIV [ 20 ]. An outsized number of derivatives especially have demonstrated cytotoxic activity both in vitro and in vivo [ 21 , 22 ].
Coumarins can function on different tumor cells depending on their structure. There are various mechanisms to control the cancer cells; among this mechanisms inducing apoptosis and suppress the proliferation of cancer cells by arresting the cell cycle in phase G0 / G1, phase G2 / M [ 23 , 24 ].
Apoptosis is a crucial cytotoxicity condition caused by anticancer drugs [ 25 ]. Apoptosis or programmed cell death is a natural process that maintains a balance between cell proliferation and cell death and plays a regulatory role in regulating cell population size and homeostasis of tissues [ 26 ]. Apoptosis avoidance is known to be one of the hallmarks of cancer cells [ 27 ] Caspases, a family of proteolytic enzymes is well known to play a crucial role in the apoptotic process. Activating these proteases which are usually present inside cells as inactive zymogens result in the cleavage of several protein substrates inside the cell leading to irreversible apoptotic cell death. Caspase-3 among these is one of the most powerful downstream caspases and is called Caspase Effector [ 28 ]. Also, cyclin-dependent kinase inhibitors p21and p27 are proteins that bind and inhibit the activity of complexes of CDK2 / cycline E, CDK4 / cycline D1, and/or CDK6 / cycline D1 and thus regulate the progression of the cell cycle at phase G1 [ 29 ]. Glutathione exists in both reduced (GSH) and oxidized (GSSG) states. GSH is one of the major endogenous antioxidants produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen compounds, as well as protecting cellular protein thiol groups and maintaining exogenous antioxidants such as vitamins C and E in their reduced (active) forms [ 30 ]. Therefore, GSH is critical to ght against oxidative stress [ 31 ]. Malondialdehyde (MDA) is a lipid peroxide that can damage the plasma membrane of cells. By measuring its content, we can usually understand the degree of lipid peroxidation in the body, thereby indirectly evaluating the degree of cell damage [ 32 , 33 ].

Chemistry experimental:
Melting points are uncorrected and measured in open glass capillaries. The FT-IR Shimadzu -8400S Spectrophotometer (USA, New York) in KBr pellets used to chart IR spectra (υ max cm -1 ). 1 H-NMR and 13 C-NMR spectra were registered on 300 spectrophotometer Germany, Rheinstetten, 300 and 125MHz respectively in DMSO-d 6 solvents and TMS as internal standard. Using Shimadzu GCMS-QP-1000 EX mass spectrometer (Japan, Kyoto) to measure the mass spectra via EI technique (70 e.v.). CHN automatic analyzer used in elemental analyses and measured at Central forced armed Cairo, Egypt. Sonication (model SW 4 cleaner a Toshcon, 37 KHz, 150 W) was achieved the synthesized compounds. Check the purity of synthesized compounds with TLC. All chemical reagents and solvents were achieved from Ali-baba ne chemical company without further puri cation.   (6)

Biological evaluation:
Cell Culture: liver Cancer cell line of human origin HepG2 (human hepatocellular carcinoma) Cells were routinally cultured in DMEM media (Lonza), supplemented with 10%FBS (Lonza), 1%100u/ml penicillinand 100ug/ml streptomycin(Lonza) in a humidi ed incubator at 37 o C with an atmosphere containing 5%CO 2 . Every experiment was carried out in triplicate. At 85% con uence cells were harvested using 0.25% trypsin and were subculture into 75 cm 2 and six -well plates or 96 -well plates according to selection of experiments. Cells were allowed to attach to the surface for 24 hours prior to treatment. 8-Azacoumarin derivatives were dissolved in DMSO and diluted to appropriate concentrations.
Cytotoxicity evaluation against HepG-2 cell line: The viability of control and treated cells were evaluated using the MTT assay in triplicate. MTT assay is a laboratory test and a standard colorimetric assay (an assay which measures changes in color) for measuring cellular growth, Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) was reduced to purpleformazan in the mitochondria of living cells. A solubilization solution (dimethyl sulfoxide) was added to dissolving the insoluble purple formazan product into a colored solution. Briefly, HepG2 (hepatocellular carcinoma cell line) was seeded in 96-well plates containing 100µl of the growth medium at a density of (1x10 4 ) cells/well. Cells were permitted to adhere for 24h till con uence, washed with PBS, and then treated with different concentration of compounds in fresh maintenance medium from 500 to 15.63 µg and incubated at 37ºC for 24h. A control of untreated cells was made in the absence of the test compound. Untreated cells used as negative control. Serial two-fold dilutions of the tested compounds were added into a 96-well tissue culture plate using a multichannel pipette (Eppendorff, Germany). After treatment (24h), the culture supernatant was replaced by fresh medium. Then, the cells in each well were incubated at 37˚C with 100µl of MTT solution (5mg/ml) for 4h. After the end of incubation, the MTT solution was removed, and then 100µl of DMSO was added to each well. The absorbance was detected at 570 nm using a microplate reader (

Chemistry
It was previously reported that the thermal and microwave re ux of chalcone 1 with diversity of active methylene for instance ethyl cyanoacetate, ethyl acetoacetate and diethyl malonate with ammonium acetate produced the pyridine esters 2a-c and 2-pyridone derivatives 3a-c in good yield that outlined in Scheme 1 [43][44][45][46][47][48].
The IR spectrum of compound 4a reveals stretching absorption bands at 3434, 3329 cm -1 attributed to asymmetric and symmetric NH 2 respectively, 2223 cm -1 for CN group and 1732 cm -1 for C=O group of the coumarin ring that assigned structure to this compound. The 1 H-NMR spectrum of compound 4a shows δ 6.80 ppm corresponding to H3 in pyridine nucleus and broad singlet at δ 5.43 ppm corresponding to the NH 2 protons with absence any band at 9.8 corresponding to NH of pyridine 3 are good evidence in formation of compound 4a. So, the ultrasonic and microwave irradiation cause via the isomerization of the 2-pyridone derivative 3 (not pyridine ester 2) to the reactive lactim intermediate which reacts with another ethyl cyanoacetate affording the pyrano[2,3-b] pyridine derivatives 4a-c as a sole product (Scheme 2). Reactivity of the 3-cyano pyridine-2-one derivatives were more than the pyridine ester quantum chemical parameter [18] as outlined in supplementary le. Reaction of azacoumarin 4a with various nitrogen precursors such as hydrazine hydrate yielded interesting pyrazole 5a incorporating heterocyclic compounds that have classy antibacterial activity (Scheme 3). Moreover, reaction of azacoumarin 4b with hydrazine hydrate afforded pyrazole 5b that outlined by the mass fragmentation to con rm the structure.
Reaction of the pyrazole 5a with boiling acetic anhydride is extremely avored in the Z-con guration ⇌ Econ guration dynamic equilibrium as in the following scheme to afford the 1,3-diamino derivatives as intermediate. Bimolecular nucleophilic substitution (SN 2 ) of one of the amino groups followed by cyclization with the other amino group to afford the pyrazolo-pyrimidine derivative 6 (Scheme 4).
The appearance of broad bands at 3347-3179 attributed to OH and NH groups, 1668-1652 for carbonyl of amide groups in pyrazole moiety. The lower of hydroxy and carbonyl bands were due to intramolecular hydrogen bond which are respectable con rmation for the assigned structures of these compounds. 1 H-NMR spectrum of compound 6 exhibits signals at δ 6.80 ppm for H3 of pyridine proton, 12.22 ppm for broad singlet for acidic OH proton and signal at δ 9.80 ppm of singlet NH proton.

Anticancer Activity
The results showed that the new azacoumarin derivative was able to cause cell cycle arrest at least partly through down-regulation the expression level of the cell cycle inhibitor p21 and induced cancer cell apoptosis via caspase-3 dependent pathway. The results indicated that the irradiation plus the derivative is more effective than the derivative only. The irradiation reinforced the effect of the derivative. As the lower dose from the derivative plus irradiation is more effective than the higher dose of derivative only. Many coumarin derivatives cause caspase dependent apoptosis [ 36 ]. P21 is recognized as a potent cyclin-dependent kinase inhibitor that facilitates cell-cycle arrest by interacting with different stimuli such as p53 and DNA repair process. P21 acts both as a tumor-suppressor gene and an inhibitor of apoptosis by interacting with various molecules and transition factors [ 37 ]. In fact, prolonged treatments of cancer cells with DNA damaging agents, determine a p21 inactivation mediated by caspase-3 cleavage and reduction of p21 levels resulted in apoptosis [ 38 ]. Caspase-3 is the most frequently activated cysteine protease, which plays a vital role in both intrinsic and extrinsic apoptotic pathways. Thus, caspase-3 has been recognized as a reliable molecular biomarker for cell apoptosis [ 39 ].
While, the results of the Oxidative stress markers (GSH and MDA) showed that GSH increase in the cells treated with the azacoumarin derivative only, but decrease with irradiation. However, the cells irradiated and treated with the azacoumarin derivative showed higher GSH in compare with cells exposed to the ionizing radiation only.
GSH is critical to ght against oxidative stress [ 40 ]. On the other hand, MDA decrease in the cells treated with the azacoumarin derivative only, but increase with irradiation. However, the cells irradiated and treated with the azacoumarin derivative showed lower MDA in compare with cells exposed to the ionizing radiation only. MDA indirectly evaluating the degree of cell damage [ 41 , 42 ]. As Compound 6 was the most active against the liver carcinoma cell line (HepG-2) its cytotoxicity was evaluated against normal rat hepatocytes. it showed weak cytotoxic effects using MTT assay under the same experimental conditions for 48 hrs incubation with CC50 = 1820.5 µg/ml (= > 500 µg/ml).

DFT study
The optimization of the heterocyclic compounds is designed as minimized energetic geometrical structures of the synthesized compounds 5 and 6 (Fig 1). The electronic structures of three compounds 5 and 6 revealed entirely spread over all molecular structure and con rmed these energetic structures of synthesized compounds.

Conclusions
Synthesis and anticancer evaluation of some novel 8-azacoumarin and pyrazole derivatives can be acheived.
The most potent derivative 8 showed much better activity was produced via simple, two-step, and ecofriendly synthetic protocols. Quali ed study was concerning the result density function theory (DFT) has thru on grinding and ultrasound-assisted tools. All azacoumarin and pyrazole structures can be elucidated by elemental and spectral data. The new derivatives of the azacoumarin and pyrazole have an effective anti-cancer effect and it was found that using the new compounds with ionizing radiation at a dose of 8 Gy improves the effectiveness of the compound on liver cancer cells. The new azacoumarin and pyrazole derivatives was able to cause cell cycle arrest at least partly by down regulating the expression level of the cell cycle inhibitor p21 and up regulating caspase 3 Inducing cancer cell apoptosis via caspase-3 dependent pathway.

Declarations Author Contributions
The listed authors contributed to this work as described in the following Sameh A. Rizk and Monda M. M.
Badawy and Mohamed R. Aly gave the concepts of the work, interpreted the results and prepared the manuscript, Mohamed S. Megahed carried out the synthetic work, cooperated in the preparation of the manuscript results. All authors read and approved the nal manuscript.