Synthesis, chemo - informatics, and anticancer evaluation of ﬂ uorophenyl - isoxazole derivatives

: The current study aimed to design and synthe size a novel series of ﬂ uorophenyl - isoxazole - carboxamide derivatives and evaluate their antiproliferative activities. Anticancer activities of the novel compounds were evalu ated by MTS assay against four cancer cell lines, including liver ( Hep3B, HepG2 ) , cervical ( HeLa ) , and breast ( MCF - 7 ) , and α - fetoprotein tumor marker, cell cycle analysis, and annexin V tests. Chemo - informatics analysis showed that all synthesized derivatives 2a – 2f obeyed Lipinski ’ s rule. Compound 2f was the most potent compound against Hep3B and Hep - G2 cancer cell lines with IC 50 values of 5.76 and 34.64 µg/mL, respectively. Moreover, compounds 2a – 2c and 2e showed potent inhibitory activity against Hep3B with an IC 50 value range of 7.66 – 11.60 µg/mL. Hep3B secretions of α - fetoprotein ( α - FP ) results showed that compound 2f reduced the secretion of Hep3B to 168.33 ng/mL and compound 2d reduced the secretion to value approximately 598.33 ng/mL, in comparison with untreated cells ’ value of 1116.67 ng/mL. Furthermore, cell cycle analysis showed that the 2f compound induced arrest in the G2 - M phase in 6.73% of the total cells and that was lower than the activity of the positive control doxorubicin ( 7.4% ) . Moreover, 2b and 2f compounds reduced the necrosis of Hep3B to 4 - folds and shifted the cells to apoptosis.


Introduction
Cancer is a disease that has spread in recent decades, and it is one of the main reasons for mortality and death worldwide [1][2][3]. In 2016, about 17.2 million new cases of cancer and 8.9 million deaths were registered around the world. From 2006 to 2016, there was an increase in cancer cases of about 28% [4]. However, in 2018, the number of deaths was estimated at 9.6 million, and according to the World Health Organization (WHO), the distribution of the most common cancers was as follows: breast (2.09 million), lung (2.09 million), colorectal (1.80 million), prostate (1.28 million), and skin cancer (1.04 million) [3].
Hepatocellular carcinoma (HCC), the fourth most common type of cancer, was one of the leading causes of cancer-related deaths in 2020. Approximately 80% of cases found in Asia and the African regions arise due to hepatitis B, C, and chronic alcohol use. The early stage of HCC is asymptomatic and the patients are diagnosed in the advanced stages. Surgical therapy was associated with postoperative complications and a high risk of recurrence. Radio-frequency ablation, microwave ablation, radioembolization, molecular targeted therapies, and chemotherapy remain the alternative ways of treatment [5,6].
Cancer occurs due to the abnormal and uncontrolled proliferation of living cells and damage of the genes that regulate the cell cycle [7]. The cancer therapy protocols include surgery, radiation, immunotherapy, gene therapy, and chemotherapy. Nowadays, chemotherapy is an effective way to stop and eliminate cancer growth. New methods of treatment depend on mechanisms involved in cancer progression [3,8].
Chemotherapy has been widely used, particularly against various cancer types [9]. However, many agents were extracts from plants with various pharmacological activities [10,11] and some of them with cytotoxic activity [12]. One of the most extracted compounds, the Combretastatin, was isolated from the African Combretum caffrum plant and it was successfully modified to find new analogs with potent anticancer activity. In recent years, fosbretabulin (combretastatin A-4 phosphate) ( Figure 1) was approved by the FDA and utilized for the treatment of thyroid cancer [13,14].
Many types of research attempt to combine or hybridize various chemical moieties to develop new anticancer agents [15,16]. Many compounds that have halogen-aryl with heterocyclic-carboxamide as a linker with methoxyphenyl were synthesized and evaluated as anticancer agents (st.1, Figure 2), and many other compounds were synthesized, which combined the heterocyclic-carboxamide with the methoxyphenyl moiety (st.2, and st.3; Figure 2) [17]. However, heterocyclic compounds that contain nitrogen with oxygen atoms have diverse medical and biological activities. One of these molecules is the isoxazole ring, which has various biological activities such as anticancer, antituberculosis, insecticidal, antibacterial, and antifungal [5]. Many researchers have focused on this heterocycle (isoxazole) and have synthesized compounds with anticancer activity (st.4, and st.5; Figure 2) [17][18][19][20].
Regarding the mentioned data, the current study aims to synthesize novel fluorophenyl-isoxazole-carboxamide analogs with different substituents and evaluate their anticancer activity on various cancer cell lines including, HeLa, MCF-7, HepG2, and HepB3, utilizing different anticancer tests (α-fetoprotein, cell cycle analysis, and apoptosis/necrosis).

Chemical reagents and instruments
All the used chemical reagents were purchased from Alfa Aesar (Massachusetts, United States) and Sigma-Aldrich (Schnelldorf, Germany). SMP-II digital melting point apparatus was used without correction to determine the melting points of the synthesized compounds. Proton and carbon nuclear magnetic resonance (NMR) spectra were recorded in DMSO-d6 and were performed on a Bruker 300 MHz-Avance III High-Performance Digital FT-NMR spectrometer at the Doping and Narcotics Analysis Laboratory in the Faculty of Pharmacy, Anadolu University, Turkey. Tetramethylsilane (TMS) was used as an internal standard. All   chemical shifts were recorded as δ (ppm). High-resolution mass spectrometer data (HRMS) were collected using a Waters LCT Premier XE Mass Spectrometer (high sensitivity orthogonal acceleration time-of-flight instrument) using ESI (positive proton) method; the instrument is coupled to an ACQUITY Ultra Performance Liquid Chromatography system (Waters Corporation, Milford, MA, USA) at Pharmacy Faculty, of Gazi University, Ankara-Turkey.

Synthesis
This product was purified by column chromatography n-hexane:ethyl acetate solvent system (3:2

Chemo-informatics properties of synthesized compounds
Regarding the basis of Lipinski's rule of five (RO5) and chemo-informatics properties, the synthesized compounds were evaluated and multiple online servers were employed, such as Molsoft (http://www.molsoft.com/) and Molinspiration (http://www.molinspiration.com/), to predict the bioactivity score and molecular properties of newly designed compounds.

Biological methods
Cell culture, MTS assay, apoptosis, α-fetoprotein, cell cycle, and flow cytometry. HCC (Hep3B and HepG2), cervical adenocarcinoma (HeLa), and breast carcinoma (MCF7) were used as cancer cell lines which were cultured in RPMI-1640 media and accomplished with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin antibiotics. Then, the cells were matured in a moist atmosphere with 5% CO 2 at 37°C. In a 96-well plate, the cells were seeded at 2.6 × 104 cells/well. After 72 h, the cells were confluent and media was changed and then the cells were incubated with various concentrations (500, 100, 50, 10, and 1 µg/mL) of the evaluated compounds (2a-2f) for 24 h. The viability of cells was assessed by Cell Tilter 96® Aqueous One Solution Cell Proliferation (MTS) Assay regarding the manufacturer's procedures (Promega Corporation, Madison, WI). However, at the end of the treatment, about 20 μL/100 μL of MTS solution/media was added to each well, and for 2 h, they were incubated at 37°C. Finally, the absorbance was measured at 490 nm [22].
DMEM was applied to culture Hep3B, using a serum of 10% fetal bovine and we detect tumor activity by using α-FP (α-fetoprotein) as a marker. We indicate Hep3B cell by smearing HBsAg on its surface (Water et al., 1998). Hep3B was incubated with each compound in 10 µl/mL for 24 h. After that, a commercially obtainable ELISA kit from R & D Systems, Inc., USA, was used to assess the level of α-FP in the medium, then harvested, and trypsinized the Hep3B cells (0.05% trypsin/0.53 Mm EDTA); after that, was washed and finally analyzed for cell cycle and apoptosis by the mechanism of flow cytometry.
After Hep3B cells were harvested, they were adjusted to 10 6 /mL buffer (in saline containing 1% albumin: the biological industries, Israel) for 10 min to found out their purity, which has staining properties, considering they were fixed with 4% paraformaldehyde. After that, for 20 min, cells were permeabilized with 0.1% saponin in PBS, then, for 30 min, stained using antihuman HBsAg monoclonal antibody (R&D system, USA), all at room temperature. After that, according to manufacture direction, we made apoptosis and viability measurements, by staining fragmented DNA using propidium-iodide PI, while annexin V-conjugated to FITC to stain phosphatidylserine (R&D systems, Minneapolis, Mn) [23].
Annexin-V (+) and propidium-iodide (−) define apoptosis, while annexin-V (−) and propidium-iodide (−) indicate the presence of viable cell. In each experimental test, unstained controls were used, such as FMO and IgG isotypes. Propidium-iodide was utilized to analyze the cell cycle by quantitation of the contents of DNA. Seventy percent of cold ethanol at 40 C was added to fixed Hep3B for 30 min at least, then the cell was washed 2 times using PBS. Finally, discard the supernatant after Rotate at 2,000 rpm. To make sure that DNA was exclusively stained, we treated the cells with ribonuclease (50 mcg of 100 mcg/mL RNase), and then 5 mcL of 50 mcg propidium-iodide/100 mL was applied to stain them; after that flow cytometer was used as an analyzer (Becton-Dickinson LSR 11, Immunofluorometry systems, Mountain View, CA).
According to the manufacturer's procedures, the apoptosis and viability analyses were done, and the used staining was the phosphatidylserine staining by annexin V-conjugated to FITC and propidium-iodide staining of DNA fragments (R&D Systems, Minneapolis, MN). Viable cells were specified as negative for both annexin-V and propidiumiodide (−), while the apoptosis was specified as negative for propidium-iodide (−) and as positive for annexin-V (+). In each experimental tuning, unstained controls were used, such as those mentioned before (FMO and IgG isotype) [24].
Cell cycle test was processed by using propidiumiodide to quantitate the DNA contents. The Hep3B cells were settled in cold 70% of ethanol for at least 30 min at 4°C and then the cells were washed twice in PBS. Spin at 2,000 rpm and the supernatant was discarded. To be sure that just DNA is stained, the treatment with ribonuclease (50 μL of 100 μg/mL RNase) was performed on the cells, and then stained with 5 μL of 50 μg of propidium-iodide/ 100 mL, and by the flow cytometer (Becton-Dickinson LSR II, Immunofluorometry systems, Mountain View, CA), was measured [24].
Hep3B, HeLa, and MCF7 cancer cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium and supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37°C. The cells were seeded at 2.6 × 10 4 cells/well in a 96-well plate. After 72 h, the cells were confluent; the medium was changed and cells were incubated with various concentrations (500, 100, 50, 10, and 1 µg/mL) of the synthesized compounds for 24 h. Cell viability was assessed with the Cell Tilter 96® Aqueous One Solution Cell Proliferation (MTS) assay according to the manufacturer's instructions (Promega Corporation, Madison, WI). Briefly, at the end of the treatment, 20 μL of MTS solution per 100 μL of the medium was added to each well and incubated at 37°C for 2 h. The absorbance was measured at 490 nm [25].
Ethical approval: The conducted research is not related to either human or animal use.

Chemistry
The synthesis of novel fluorophenyl-isoxazole-carboxamide derivatives (2a-2f) was outlined in Scheme 1. The coupling reaction to form the fluorophenyl-isoxazole-carboxamide compounds 2a-2f was afforded by using EDCI and DMAP as activating agent and covalent nucleophilic catalysts, respectively. After the coupling step, they were reacted with the aniline derivatives [26], EDCI (1-ethyl-3-(30-dimethylamino)carbodiimide, and are usually used for peptide or amide coupling, and there is no need for additional amine. The carbodiimide reacts with the COOH (carboxylic acid) to form the anhydride mixed with Oacylisourea; the produced intermediate directly reacts with the aniline or amine derivatives to produce the desired final amide product as well as urea as side-product [27]. The synthesis of these derivatives was confirmed HRMS, the ppm values for all compounds were less than 2.8, the difference between the calculated mass and the founded mass for all compounds was less than 0.0011 g/mol, for example, compound 2a's calculated mass was 387.1356 g/mol in comparison with founded mass 387.1265 g/mol. Further, they were purified by column chromatography using different solvent systems (n-hexane: ethyl acetate, and dichloromethane: ethyl acetate). The 1 H-NMR peaks confirmed the synthesis of these products. A singlet peak of one proton for N-H amide in the range of 9.30−10.47 ppm was observed in each compound. Multiple signals in the aromatic region were observed, and singlet peaks integrated for 3 protons were observed around 2.60 ppm which refers to the methyl group on the isoxazole ring. The 13 C-NMR spectrum showed a C signal of carbonyl around 170.5 ppm, and at 12.5 ppm, the signal of aliphatic carbon methyl.

LO5 and chemo-informatics properties of synthesized compounds (2a-2f)
The predicted chemo-informatic characteristics were processed by computational tools. Results showed that  Table 1) [28]. Our predicted data showed that all synthesized compounds were within the standard range, which showed their good oral bioavailability character. However, drug score parameter was used to evaluate the synthesized compounds according to their hydrogen bonding properties, molecule size, hydrophobicity, flexibility, and electronic distribution and the results showed that all the synthesized structures showed excellent drug score values (0.45-0.95), which represents its better drug-likeness behavior and may be assumed as drug candidate agents against their targets. The overall predicted values of all synthesized compounds are listed in Table 1.

Biological evaluations 3.3.1 Cytotoxic evaluation of the compounds 2a-2f
The MTS test was utilized on MCF-7, HeLa, Hep3B, and HepG2 cancer cell lines to assess the cytotoxicity of the produced new compounds. As shown in Table 2, five concentrations were used (500, 100, 50, 10, and 1 µg/mL). Based on the results shown in Table 2, the compound 2f showed the most potent activity on Hep3B and HepG2 cancer cell lines, with IC 50 values of 5.76 and 34.64 µg/mL, respectively, while compounds 2a−2c and 2e showed IC 50 values below 11.6 µg/mL against the Hep3B cancer cell line. However,   [21]. However, in this current series, the same compound with fluorophenyl showed better activities against Hep3B with IC 50 7.66 µg/mL, as well as almost all of these new series compounds were more potent than the previous work.

Alpha-fetoprotein results
According to the results of the MTS test and because the synthesized compounds showed potent activities against Hep3B, the inhibitory effects on cell proliferation were used and medium levels of α-FP were evaluated. Actually, all synthesized compounds, except the 2d compound, showed a reduction of secretion with values below 598 ng/mL. The compound 2f was the most active compound in the MTS assay and decreased the Hep3B secretions of α-FP to 168.33 ng/mL in comparison with untreated cells with a value of 1116.67 ng/mL. The results showed that the 2f compound has anticancer activity on the Hep3B cancer cell line (Figure 3).

Cell cycle analysis of Hep3B cells
The flow cytometry analysis was used to verify the ability of 2f and other active compounds (2b, 2c, and 2e) in changing the behaviors for each phase in the cell cycle of Hep3B cell lines. These changes of the normal disturbances in the cell cycle phases were investigated using the boiled solution of the active derivatives to induce cell cycle progression, and they were compared with positive control anticancer drug doxorubicin (Dox). The data in Figure 4 show a similar proportion of cells in the G1 phase following treatment with 2f compound as compared with DOX. The 2f compound showed also similar behaviors as the DOX in reducing proportions of cell percentages in the S, as well as the G2-M phase (P < 0.05). These data showed significant changes in cell cycle parameters in different phases, especially in S and the G2/M phase (mitosis state); these results indicate a significant delay in the mitotic phase and can confirm the potent anticancer activity of the 2f compound.

Apoptosis versus necrosis test
The next step of the current study was to determine whether the synthesized compounds induced programmed  cell death (apoptosis) and necrosis. Apoptosis was defined as AnnexivV+/PI−, while late apoptotic or necrotic cells were defined as AnnexivV+/PI+. Figure 5 illustrates that Hep3B untreated cells possess a baseline apoptotic cell population of 43.0%, while 2b, 2e, and 2f compounds reduced apoptosis to 8.3, 14.53, and 10.6%, respectively. An annexin-V+/PI+ fraction from 2b, 2e, and 2f compounds significantly increased apoptosis/necrosis cells to 69.67, 67.66, and 66.33%, respectively, as compared with 28.83% in untreated cells. These findings support the 2f compound's potent anticancer properties by increasing the necrotic activity of annexin-V+/PI+ in the Hep3B cell line, thereby hastening their death.

Conclusion
The synthesized fluoro-isoxazole-carboxamide derivatives showed anticancer activity on the hepatocellular cancer cell lines. Five compounds showed potent activity against the Hep3B cancer cell line, with IC 50 values close to the positive control Dox. The most potent compound 2f demonstrated potent anticancer activity in different in vitro tests. The MTS assay showed potent activity against Hep3B, with IC 50 around 5 µg/mL, moderate activity against HepG2, with IC 50 values around 35 µg/mL, and weak activity against HeLa, with IC 50 values around 90 µg/mL. The α-FP tumor marker analysis of 2f derivative decreased the Hep3B secretions to 168 ng/mL compared with 1,116 ng/mL in the negative control (untreated cells), indicating less proliferation and tumorigenicity of Hep3B. For further evaluation of the molecular effects of the novel synthesized compounds, the 2f compound induces cell cycle arrest in the mitotic phase (G2/M) and is similar to Dox positive control values. In future projects, further analogs and derivatives based on the lead compound 2f will be designed and synthesized as promising anticancer agents. More in vivo research is needed to confirm the efficacy and create appropriate pharmaceutical dosage forms for the most active substances.