Synthesis and screening of anticancer potentials of some new terephthaldehyde-derived nitrone compounds

Purpose: To synthesize and screen some new nitrone compounds derived from terephthaldehyde for their anticancer potential. Methods: Six new compounds (H, p-Me,p-Br, p-Cl, o-Cl and m-Me) were synthesized via a condensation reaction between terephthaldehyde and a variety of aryl hydroxylamine compounds derived from nitrobenzene and its derivatives. The chemical structures of these compounds were identified using elemental CHN analysis and were elucidated using Fourier Transform infra-red (FT-IR), H-nuclear magnetic resonance (H NMR), mass spectrometry (MS), and elemental analysis. The anticancer effects of the compounds were screened in vitro with respect to their cytotoxicity on MCF7 human cancer cells line. The IC50 values were obtained by MTT assay and their effects on apoptosis of MCF-7 cells were assessed using Acridine orange-ethidium bromide AO/EtBr staining method under a fluorescence microscope. Results: Only four compounds (2b, 2d, 2e, and 2f) inhibited more than 50 % of the growth of MCF-7 cells. The strongest anti-proliferation effect against MCF-7 cells was exhibited by 2f (m-Me), producing more apoptosis which increased membrane disruption and consistency of lysosome vacuoles; it also exhibited higher cytotoxic effects on human cancer cell lines (IC50 < 7.5) than the other synthesized compounds. Conclusion: The new nitrone compounds (2b, 2d, 2e, and 2f) synthesized from terephthaldehyde exhibit some anticancer properties, and so are potential anticancer agents.


INTRODUCTION
Nitrones are 1,3-dipolar compounds which contain the azomethine group (-C=N + -O -) [1] and exert biological and chemical effects. In addition, the azomethine group possesses an oxygen atom bonded to the nitrogen atom (-CH=NO-) by co-ordinate bond [2]. These compounds possess numerous beneficial and pharmacological effects such as anticancer [3], antifungal [4], neuroprotective [5], antibacterial [6] and antioxidant [4] properties. The most effective method used in the synthesis of nitrones involves a condensation reaction between hydroxylamines and carbonyl compounds. This reaction smoothly proceeds with high yield [7,8].
Cancer is an ailment where numerous anomalous cells grow wildly, paying little mind to the customary standards of cell division [9]. Cancer ranks second after heart disease among diseases that result in the high death rate of patients, Cancer represents about 23 % of all demise cases in the united kingdom with breast cancer MCF-7 being the most wide-spread [10]. The exact cause of cancer has not yet been determined, in spite of the fact that the hereditary viewpoint is engaged with 5 to 10 % of cancer [11]. Different causes incorporate diseases, unhealthiness, weight, absence of physical movement, contamination and tobacco use. These variables influence the movement of biogenes legitimately or by implication that can prompt cancer [11]. The target of the present examination was to investigate the potential anticancer impacts of some new nitrones derived from terephthaldehyde against the MCF-7 cell line.

Instruments
The melting points of the synthesized nitrone compounds were determined on the Gallenkimp apparatus, while CHN analysis was carried out on UoroVector model/EA3000A. The FT-IR 8400S SHIMADZU apparatus was used to record the IR spectra using KBr disks. Bruker model ultra-shield 500 MHz (Switzerland) was used to obtain 1 H-NMR spectra at Tehran University, Iran. Deuterated dimethyl sulfoxide used as a solvent, with TMS (tetra-methyl silane) as the internal standard.

Synthesis of N-substitutedphenylhydroxylamine (1a, 1b, and 1f)
In a 500-mL conical flask, a mixture of the corresponding nitrobenzene (0.205 moles), NH 4 Cl (0.235 moles) and water (400 ml) was stirred vigorously. Zinc dust powder (31 g) was added portion-wise for 20 min. The stirring continued for 20 min. The mixture was filtered off then washed with hot water. Thereafter, to saturate the filtrate, NaCl was added. Then by using an ice-bath for cooling the reaction mixture. The resultant hydroxylamine was filtered and recrystallized from toluene and petroleum ether [12].

Synthesis of N-substitutedphenylhydroxylamine (1c and 1d)
The above method was modified using 70 % ethanol. In 200 ml of 70 % ethanol, NH 4 Cl (0.115 moles) and the appropriate halonitrobenzene (0.1 mole) was dissolved. The mixture was stirred with heating at 60 o C for 45 min. Thereafter, the mixture was saturated with NaCl then purified while hot. Desired hydroxylamine was filtered and recrystallized from toluene and petroleum ether [12].

Synthesis of nitrones (2a-2d and 2f)
A mixture of terephthaldehyde (0.02 moles) and arylhydroxylamine i.e. 1a-1d and 1f (0.081 moles) in 20 ml of absolute EtOH was stirred overnight. The desired nitrone was filtered and purified from ethanol [13]. The procedures used for the synthesis of nitrones are summarized in Table 1.

Synthesis of nitrone (2e)
To avoid decomposition of N-(o-chlorophenyl) hydroxylamine, the previous procedure was modified as follows: A mixture of ochloronitrobenzene (0.036 moles) and ammonium chloride (0.046 moles) in 25 ml of 60 % ethanol was stirred at 10 o C. While stirring at 10 o C, 0.065 mol of zinc dust was added portionwise over a period of 1h. The precipitate formed was filtered and washed three times with 8 ml of boiling ethanol, and 0.018 moles of terephthaldehyde were added to the alcoholic filtrate. At room temperature, the mixture was stirred for 20 hour, next the precipitated nitrone was filtered and recrystallized from absolute ethanol.
Acridine orange (AO), fetal bovine serum, trypsin-EDTA, ethidium bromide (EB) and dimethyl sulfoxide (DMSO) were given from Sigma-Aldrich (St Louis, MO, USA) . Kapa Biosystems, Inc. BioSource International (Belgium) provided a penicillin-streptomycin solution. All chemicals applied were of the diagnostic evaluation in this examination. All solutions and buffers were set up as substantive previously [14].

Cell viability
The effects of the six chemical compounds (H, p-Me, p-Br, p-Cl, o-Cl, and m-Me) on the viability of MCF-7 cell line were evaluated using SUNRICE Tecan absorbance reader (Schooler) for measuring the absorbance at 620 nm. The growth medium was decanted and the cell culture was washed once with 2 ml trypsinversene solution.
Approximately 2-3 ml of trypsin-Bersin was added to the cell culture, then culture flask was rocked gently, then the culture was incubated at 37 °C until the cells separated from the flask. The cells were dispensed in a 5ml growth medium and redistributed at the required density into the culture. The cells were brooded in a humidified climate containing 5% CO 2 at 37 °C [14].

MTT assay
This examine was finished by a prior qualified technique [15]. The cells were seeded in 96-well microtiter plates with RPMI medium at a concentration of 1×10 5 cells/ml, then were incubated overnight for attachment. At that point, (100 μg/mL) from each compound were added in triplicate to the wells at various concentrations (25, 50, 100, 200, and 300 μg/mL), followed by incubation for 72 hr. Thereafter, MTT was added to the cells at a concentration of 2 μg/mL, followed by further incubation for 3 hr. Next the incubation time, the MTT was aspirated from the wells then supplanted with DMSO to break up the formazan crystals formed. The cells were additionally incubated for 15 min before deciding the absorbance of each well at 620 nm. The percentage of cytotoxicity was determined using Eq 1. (1) where A and B are the absorbance of the control, and test samples, respectively.

Acridine orange-ethidium bromide (AO/EtBr) double recolouring
The capability of the compounds to stimulate the death of the MCF-7 cells was tried utilizing the AO/EtBr recolouring method [16]. The cells were seeded into 96-well microtiter plates in RPMI medium at a concentration of1×10 5 cells/ml and were incubated overnight for attachment. Then, the cells were incubated by (100 μg/mL) with each of (p-Me, p-Cl, o-Cl and m-Me) for 24 h, followed by washing using PBS. Thereafter, 100 μL of dual fluorescent dye was added to the wells prior to the visualization of the cells under a fluorescence microscope.

Determination of IC 50
The concentration of compounds which produced 50 % inhibition of cell development (IC 50 ) was resolved from cell survival (%) versus drug concentration (μg) curves. yielding an equation used to compute the concentration of substance required to produce a half decrease in cell viability (IC 50 ) as past outlined [17,18].

Statistical analysis
Both unpaired t-tests with Graph Pad Prism 6 were used statistically analyzed the data obtained. The values are introduced presented as mean ± standard deviation (SD) of triplicate estimations. Both Bonferroni's post-test and One-way ANOVA were utilized for factual investigation [19]. Analysis of the non-linear regression curve for IC 50 determination was done with Graph Pad PRISM version 6 (Graph Pad Software, Inc., La Jolla, CA, USA)

RESULTS
The results of the mass spectral characterization of the synthesized compounds are listed in Table  1, along with other physical data.  Table 2 shows the elemental (CHN) data for the synthesized nitrones (2a-2d). The outcome supports the framework of the nitrone compounds.

FT-IR spectra
The medium absorption band observed at (1673 -1593 cm -1 ) for all nitrone compounds (Table3) was due to the stretching vibration of the C=N group. The FT-IR spectra of nitrones showing a very strong band at regions 1180-1215 cm -1 and 1068 -1095 cm -1 can are due to the stretching frequencies of C-N and N-O bonds, respectively.
Stretching frequencies of the aromatic C-H occurred at 3047 -3128 cm -1 . Out-of-plane bending (o.o.p bend) and in-plane bending (in. pl. bend) of aromatic C-H appeared at regions 837 -860 cm -1 and 1393 -1420 cm -1 , respectively. All infrared spectra of nitrones showed forceful bands at 1454 -1481 cm -1 and at 1500 -1558 cm -1 due to the stretching frequencies of the aromatic C=C group. Nitrones 2b and 2f showed weak bands for aliphatic C-H bond at 2920 and 2924 cm -1 , respectively [20,21].  Table 4. The signals of a the 1 H-NMR spectrum of compound 2f are summed up in Table 4.

Effect of 2a-2f on cell viability of (MCF-7) cell line
At Conversely, neither compound 2a nor compound 2c was able to induce inhibition of cell growth in MCF-7 cell line after 72 h at the concentration of 100 μg/ml used in the assay (Figure 1). Consequently, these compounds were not used in subsequent studies.   The results in Figure 5, Table 5 were revealed that the ability of these compounds to inhibit MCF-7 proliferation was more than 50 % at all concentrations tested. Bar chart E shows that the compound 2f had the highest inhibition (59.27 %) at (300 µg/ml) concentration when compared to the other compounds and MCF-7 untreated cells (p < 0.0001).

Morphological changes in MCF-7 cell nucleus
AO/EtBr recolouring technique was utilized to study the changes in MCF-7 nuclear morphology after treatment with the different compounds (2b, 2d, 2e and 2f). This method is applied to recolour explicit pieces of the cell and to decide the particular apoptotic markers of atomic alterations.  As shown in Figures (6), all of the nitrones compounds, caused cell death but the 2f (m-Me) compound produces the most apoptosis when exposure of MCF-7 cells to the compounds led to increased membrane disruption and the formation of lysosome vacuoles, as As shown in (Figure 4) when compared to untreated control cells (A, B, C, and D).

DISCUSSION
Generally, the outcome exhibit differences in anti-proliferative, apoptosis and cytotoxic effects amongst the tested compounds. The present study confirmed through the results that these nitrone compounds synthesized from terephthaldehyde (2b, 2d, 2e, and 2f) are effective and have the capability for inhibiting the growth of breast cancer MCF-7 cell by 50 %, but there was variance in the degree of inhibition.
More interesting, the study confirmed that nitrone compounds synthesized are new of their kind and there are no previous studies to indicate and support our results. It was found that the potency of cell growth inhibition correlated with the position of the electron-withdrawing nitro group on the tested compounds.
Figures 2 shows that all of the nitrone compounds caused cell death. These results proved that the compounds 2b, 2d, 2e and 2f are potential sources of efficacious and cytotoxic materials. In any case, the 2f nitrone compounds were discovered the best exacerbates that delivered more apoptosis when contrasted with the other. This compound showed high cytotoxicity through restraint of MCF-7 multiplication, relative to different mixes and untreated MCF-7 cells. Furthermore, the study assumed the effects of 2f and 2b might be due to the existence of the methyl group at 4-and 3positions, which may increase their lipophilicity. This is most likely because of the presence of the azomethine group (-CH=N-) which possesses biological and chemical properties. Moreover, the azomethine group possesses an oxygen atom bonded. to the nitrogen atom (-CH=NO-) by coordinate bond [2].
These chemical groups are important due to their anticancer effects. Through the results, it turns out that the most significant inhibition at 300 µg/ml produced by 2f compound, showing stain in certain parts of the cell indicating specials apoptotic signs of nuclear alterations. The study assumed the mechanism action of this compound when the introduction of compounds to the cells prompted expanded layer disturbance and the development of lysosome vacuoles when contrasted with untreated control cells ( Figure 5). This might be because of their capacity to enter the cell layer and influence capability to penetrate the cell membrane and affect the RNA levels of p53, Bax, bcl-2, caspase-3, and caspase-9 [23,24].

CONCLUSION
The new nitrones compounds (2b, 2d, 2e, and 2f) synthesized from terephthaldehyde exhibit some anticancer properties, and so are potential anticancer agents. The strongest antiproliferation effect against MCF-7 cells was exhibited by 2f (m-Me) which produced more apoptosis than the other compounds. Thus, 2f possesses the potential for further development into an effective anticancer and antioxidant agent.