Synthesis of 1-[(Aryl)(3-amino-5-oxopyrazolidin-4-ylidene) methyl]-2-oxo-1,2-dihydroquinoline-3-carboxylic Acid Derivatives and Their Breast Anticancer Activity

This research aimed to produce new 1-[(aryl)(3-amino-5-oxopyrazolidin-4-ylidene) methyl]2-oxo-1,2-dihydroquinoline-3-carboxylic acid derivatives and check their anticancer effect against the breast cancer MCF-7 cell line. The 2-oxo-1,2-dihydroquinoline-3-carboxylic acid (4) compound was obtained by hydrolyzing ethyl 2-oxo-1,2-dihydroquinoline-3-carboxylate (2) with thiourea and anhydrous potassium carbonate ethanol, which was then treated with ethyl 3-substituted 2-cyanoacrylates (6) in the presence of triethylamine in diethyl formamide to give 1-[2-(ethoxy)carbonyl-2-cyano-1arylvinyl]-2-oxo-1,2-dihydroquinoline-3-carboxylic (7a,d). Cyclization of compound 7 with hydrazine hydrate ethanol inferred the association of 1-[(aryl)(3 amino-5-oxopyrazolidin-4-ylidene)methyl-2oxo-1,2-dihydroquinol-3-carboxylates (8a,d). Spectroscopic and micro-analytical techniques such as IR, NMR, and elemental analysis were used to validate the structure of the synthesized organic compounds. The anticancer effects of the synthesized compounds 7a–d and 8a–d were tested by using the MTT assay on the MCF-7 cell line. When compared to the reference compound Dox, the compounds 7b,c and 8a–c demonstrated strong anticancer activity against the MCF-7 cell line. The anticancer effects of the synthesized compounds 7a–d and 8a–d were tested against the MCF-7 cell line, using MTT assay. The compounds 7b,c and 8a–c showed significant anticancer activity compared to the reference compound Dox against the MCF-7 cell line.


Introduction
Quinoline, a heterocyclic nitrogen compound, has been list by the Food and Drug Administration as a chemotherapy compound [1]. Quinolines are a common pharmacological scaffold that can be found in a wide range of synthetic and natural bioactive compounds [2]. The chemistry of quinolines has been extensively studied over the last century, with various and fascinating biological activities, such as antibacterial, antifungal, anti-inflammatory, anti-malaria, and anticancer properties [3][4][5][6][7][8]. Quinoline derivatives are effective against cancer cells in the breast, prostate, gastrointestinal, colon, and liver [9][10][11][12]. Similar compounds, such as camptothecin and its analogs (irinotecan and topotecan) [13,14], as well as bosutinib [15][16][17], have been used in clinical trials.
The anticancer hybrid drug approach is a cutting-edge synthetic strategy that involves either combining or blending the hepatotoxic moieties of several drugs into a novel molecular structure [18,19]. Better anticancer agents resulted from combining these active pharmacophores in a new molecular architecture [18][19][20][21][22]. Pyrazoloquinolines correspondingly are used for many applications, including antiviral activity against the herpes simplex virus [23], caspase activators, anticancer activity [24,25], and apoptosis inducers [25]. Several quinoline-pyrazole hybrids with potent anti-proliferative activity against human liver, breast, and colon cancer cell lines were developed by Pirol et al. [26]. As a result, finding new medications to treat diseases and infections without causing serious adverse effects in patients is critical.
Consequently, quinoline derivatives are among the most effective molecules, with several studies stating that they have a broad variety of biological activities while being safe for patients [27,28]. Therefore, using a taster of multiple functional groups on the quinoline scaffold to create a new anticancer drug is a good idea. As a result of these findings, it was deemed valuable to synthesize some new 2-oxo-1,2-dihydroquinoline-3-carboxylic acids with pyrazolinone moieties at the N-position to obtain a safe and effective new anticancer drug compound.

Chemistry
Melting points (m.p.) of the synthesized compounds were measured in an electrothermal digital melting-point device. IR and NMR spectra were verified on a Shimadzu 470 c as KBr pellets and Bruker 400 DRX-Avance spectrometer in DMSO-d 6 as a solvent, respectively. The elemental analysis was achieved on a Perkin-Elmer 2400 series II CHN elemental analyzer. Chemicals that were used in synthesis were purchased from Aldrich, Merck, or Fluka Chemical Companies.

Synthesis of the Starting Materials (2)
Ethyl 2-oxo-1,2-dihydroquinoline-3-carboxylate (2) was obtained by fusion of the mixture 2-aminobenzaldehyde (1) (0.01 mol) and diethyl malonate (0.01 mol) in the presence of piperidine (2 mL) on a hot plate, for 2-3 min; ethanol (30 mL) was added to the reaction mixture and heated under reflux for 2 h. The reaction mixture was cooled, poured into water, and neutralized with dilute hydrochloric acid (2%). The resulting product was filtered off, washed with water, dried, and, finally, the product was crystallized from ethanol to give 2 as colorless crystals, yield 76%, m.

General Procedures for the Preparation of Ethyl 3-aryl-2-cyanoccrylamates (6a-d)
A mixture of ethyl cyanoacetates (0.01 mol), appropriate aromatic aldehydes (namely, benzaldehyde, 4-N,N-(dimethyl)amino benzaldehyde, 0.01 mol), and triethyl amine (0.03 mol) in ethanol (50 mL) was refluxed for 2 h. After cooling, the solution was poured into water and neutralized with dilute acetic acid (2%). The solid formed was washed with water, dried, and recrystallized from a suitable solvent to give the compounds 6a-d. A mixture of compound 7 (0.01 mol) and hydrazine hydrate (0.02 mol) in ethanol (50 mL) was heated under reflux for 2 h, then cooled, poured into ice-water, and neutralized with a few drops of acetic acid. The precipitate formed was filtered, washed with water, and dried. Finally, the product was recrystallized from a suitable solvent to give 8.

In vitro Anticancer Effect of the Compounds 7a-d and 8a-d Against MCF-7 Cell Line
A cytotoxicity test was conducted, using the MTT technique, to investigate the effect of the synthesized compounds 7a-d and 8a-d as anticancer medications [29]. Cells were started at the concentration 10 4 cells/well and distributed in a 96-plate and allowed to bind to the plate for 24 h before adding the synthesized compounds. Six wells were set for each dose. The 96-plates were incubated at 37 • C with 5% CO 2 . MTT was added to each well at a final concentration of 0.5 mg/mL after two days, and the plates were incubated at 37 • C for an additional four hours in the presence of 5% CO 2 . An ELISA reader was used to calculate color intensity. To create a survival curve for the MCF-7 cancer cell line after the particular concentration of the synthesized compounds, a link between the survivor curve and drug consumption was drawn. Data are calculated as the mean ± of three different experiments.

Cell-Cycle Analysis
To investigate the effect of the synthesized 7c compound on the cell cycle of MCF-7, cells at the concentration of 2 × 10 5 /well were incubated for two days with compound No. 7 at its IC 50 value (1.73 µM). After being exposed to this procedure, cells were washed twice with ice-cold phosphate saline (PBS) buffer, centrifuged, and fixed in ice-cold 70% ethanol at 4 • C for 30 min. Next, cells were washed in 1xPBS solution for 30 min at 37 • C and then collected by centrifugation at low speed (2000 rpm) for 5 min. Finally, cells were stained with a propidium iodide solution at a final concentration of 1 µg/mL. The samples were gently mixed and left at room temperature for 20 min in the dark. A BD FACSCanto flow cytometer was used to look at the DNA material (BD Biosciences Systems, San Jose, CA, USA). FACSDiva software (BD Biosciences Systems, San Jose, CA, USA) was used to interpret the data (BD Biosciences Systems).

Statistical Analysis
Statistical comparisons were accomplished by a one-way ANOVA with the Duncan test, using IBM SPSS version 26 (IBM, Armonk, NY, USA). A probability level of 0.05 or lower was considered statistically significant; ** p < 0.01.
The structure of the compounds obtained was determined (7a-d) by spectral data. In the infrared spectra, large expansion bonds belonging to OH, C=O for ester, amide, and acid groups, C=C, and CN were observed. 1 H-NMR spectra of 7a-d compounds showed two sharp signals at δ = 1.27-1.34 ppm as triple signal and δ = 4.25-4.35 ppm as quadruple signal due to protons of the ethoxy groups (OCH 2 CH 3 ) (Supplementary Materials Figures S1-S8).
Regarding compounds 7a-d, two protons appeared as singlet signal at δ 8.88-8.89 ppm refer to the H-4 of quinolinone ring and hydroxyl function (OH) for the carboxylic acid groups. The entire aromatic proton peaks in the 1 H-NMR spectra were also recorded at estimated regions (Supplementary Materials Figures S1-S8). The 13 C-NMR spectra of compounds 7a-d displayed three-carbon signals at δ 164.81-162.99 ppm, 162.99-162.28 ppm, and δ 162.51-160.81 ppm due to carbons of three carbonyl function (C=O) of ester, amide, and acid (Supplementary Materials Figures S1-S8). In addition, the carbons of cyano groups (CN) for the compounds 7a-d in the 13 Figures S1-S8).

In Vitro Cytotoxic Activity Against MCF-7 Cell Line
To examine the anticancer activity of the prepared quinolone derivatives, the effect of the synthesized compounds on the viability of the MCF-7 cell line was measured by using colorimetric MTT assay after 48 h of incubation. The percentage of cell viability of quinolone derivatives and reference compound are presented in Table 1. The reference compound in our assay is Doxorubicin (Dox). Interestingly, among the quinolone derivatives compound, 1-[2-(ethoxy)carbonyl-2-cyano-1-(4-hydroxy-3-methoxy)phenylvinyl]-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (compound 7c) was found to be superior to the other quinolone derivatives in terms of anticancer activity and showed significant antiproliferative activity compared to the reference compound Dox with IC 50 value of 1.73 ± 0.27 µg/mL.

Cell-Cycle Analysis
Due to the significance of the cell cycle in the process of tumor cell proliferation, MCF-7 cell growth inhibition, as a result of cell-cycle arrest, was evaluated by using DNA flow cytometric assay. In this assay, MCF-7 cells were dealt with compound 7c at a concentration of 1.73 µM value for 48 h. It is evident from Figure 1 that compound 7c induced cell arrest on 36.04% of cells at the G2/M phase, in comparison with the untreated cells, which had 9.88%. Moreover, compound 7c revealed G0 phase arrest marked by the appearance of a peak at the G0 phase of the cell-cycle distribution profile, which indicates MCF-7 cell apoptosis ( Figure 1).
These data suggest that compound 7c causes perturbations during the cell-cycle progression, especially at the G2/M stage. Cell cycle and apoptosis play remarkable roles in the regulatory mechanisms of the development and growth of the cell. The impacts of numerous substances that are utilized as anticancer drugs were discovered to be through the capturing of the cell cycle during the stages G0/G1, S, and G2/M, which exhilarated and initiated apoptosis [30][31][32][33]. Consequently, we presume that the impact of compound 7c on the MCF-7 cell line was through the instabilities influences of the compound 7c in cell-cycle progression particularly at the G2/M stage.

Conclusions
In conclusion, we have developed a rapid and efficient synthetic route for the synthesis of 1-[(aryl)(3-amino-5-oxopyrazolidin-4-ylidene) methyl]-2-oxo-1,2-dihydroquinoline-3carboxylic acid derivatives. The present synthetic pathway has the advantages of operational simplicity, moderate reaction conditions, and good to high yield of bioactive products. Our method is simple, as no extraordinary apparatus, reagents, or chemicals for workup are required, and the compound formed is filtered and purified just by simple crystallization. This synthesis is also beneficial in terms of economy, as well as avoiding any hazardous chemicals. The effect of the synthesized compounds on the viability of MCF-7 cell line was measured by using colorimetric MTT assay after 48 h of incubation. The compounds 7b, 7c, 8b, and 8c showed significant anticancer activity compared to the reference compound Dox against the MCF-7 cell line.