of Kufa for Chemical Sciences

New formazan (FOZ) derivative was synthesized and evaluated as an anti-breast cancer and antioxidant agent. Chemical structure of FOZ was proved by CHN, FT-IR, 1 H-NMR and Mass techniques. The anti-proliferative activity of FOZ was estimated toward breast cancer cells. The synthesized compound displayed significant anti-breast cancer activity towered MCF-7 cells. The MTT examination demonstrated that 16 µg/ml of FOZ and doxorubicin ( Dox ) as a standard drug suppressed cell growth by 83.33% and 92.66%, respectively after 48hrs. The formazan antioxidant activity was estimated in vitro at various concentrations (25-400ug/ml) and the outcomes showed that 400 μg/ml of FOZ was more efficient compared to ascorbic acid.


Introduction
Cancer is a category of diseases characterized by aberrant cell growth and the capacity to invade or spread to other body regions (1). Breast cancer (BC), which accounts for (12%) of all new instances of cancer each year worldwide, became the most widespread cancer in the world (2). Eight million female alive by the end of 2020 have been diagnosed with BC in the past five years (3). One of the best methods for extending a patient's life is chemotherapy. However, the challenge of drug resistance has put many chemotherapeutic medications in a situation where their therapeutic impact has been decreased (4). Additionally, the harmful effects of chemotherapeutic agents on healthy cells result in unpleasant side effects for the patients. These factors led to increased interest in the development of new kinds of anticancer medicines that exhibit effective and focused toxicity on tumor cells (5).
Antioxidants inhibit oxidation by reacting with reactive and unstable free radicals to prevent free radical-related diseases like carcinogenesis, cardiovascular and aging (6). A number of endogenous systems pathological states or exposure to a variety of physiochemical conditions, our bodies produce free radicals (reactive oxygen-nitrogen species) (7). There must be equilibrium between antioxidants & free radicals. Oxidative stress is caused by free radicals overpowering the body's capacity to control them. Therefore, free radicals negatively impact DNA, lipids and proteins and cause various disorders in humans. In order to manage this oxidative stress, antioxidants from an external source might be applied (8).
In this study, we present synthesis and identification of new formazan compound (FOZ). Likewise, cancer cells were utilized in an anti-cancer activity investigation. The research displayed that the FOZ has cytotoxic effect against MCF-7. The examination of the antioxidant properties yielded data that supported the FOZ's strong anti-DPPH radical properties.

Experimental 2.1. Materials and Instruments
All chemical supplies were bought from Merck and Sigma-Aldrich. FT-IR spectra were collected using a Shimadzu FTIR spectrophotometer. The 1 HNMR spectrum was obtained utilizing a Bruker (360 MHz) NMR spectrophotometer. On a FisonsTrio1000 spectrometer, GC-mass spectra were captured. EM-017 analyzer carried out the CHN test.

Synthesis of N,N-Dimethyl-4-((2-phenylhydrazono)methyl)aniline
The Schiffbase was prepared by the reaction of 0.01 mole of 4-(Dimethylamino)benzaldehyde dissolved in 35ml of ethyl alcohol with 0.01 mole of phenylhydrazine with a 2 drops of anhydrous acetic acid. The mixture was refluxed for (4 hrs). Utilizing methanol, the solid product was filtered & recrystallized (19

Antioxidant Activity
The scavenging activity of FOZ and vitamin C (standard) were tested utilizing DPPH • methods. Sample solutions were prepared in different concentrations (25,50,75,100,200, 300 and 400) μg/ml and added to same volume of DPPH • (0.1 mM) dissolved in methyl alcohol. The mixture was incubated for 50 min at 25 0 C. The mixture's absorbance at (517 nm) was tested to determine its antioxidant activity. (21,22). The percentage of inhibition was calculated utilizing following equation: % scavenging activity = (A control − A test sample / A control ) × 100

Anticancer Activity
To measure viability of MCF-7 cells, they were seeded in 96-well plates and treated as needed after 24 hours of seeding. Optical density was assessed at (500 nm), and % cell viability was calculated in comparison to un-treated cells (23,24).

Chemistry
Formazan compound (FOZ) was synthesized in two phases. According to Scheme 1, the initial step involved producing the Schiff base through reaction of p-Dimethylaminobenzaldehyde with phenyl hydrazine. The imine group was coupled with the diazonium salt of 4,4'-diaminodiphenylmethane in the second step to synthesis the FOZ.

Structure characterization
The structure of FOZ was determined basis on the of FT-IR, 1

Fig2: FT-IR diagram of FOZ
The FOZ formation has been proved by the 1 H-NMR. The protons of (N-H) group were recorded in 9.67ppm. The proton of methylene group (Ph-CH 2 -Ph) of appeared at δ 3.92ppm. 1 H-NMR diagram showed a long and sharp peak at 2.5ppm and 3.3ppm due to DMSO-d 6 solvent.

Fig3: 1 H-NMR spectrum of FOZ
The structure of formazan was further confirmed by the GC-mass spectra. The GCmass of FOZ showed the correct molecular ion that appeared at m/z: 698 (M+, R%1).

Antioxidant Activity
It is common to quantify the capacity of substances to act as a scavenger for free radicals or to assess antioxidant activity by utilizing a DPPH• radicals that has an absorbance at (517 nm) (25). By donating an electron or H atom, the antioxidant chemicals in the media change the DPPH• into a more stable DPPH molecular product. The antioxidant effect can be assessed spectrophotometrically because the reduced form of DPPH changes from purple to a pale yellow color (26). The results of antioxidant activity of samples were compared to the ascorbic acid (standard) (Fig5). At concentration 25-400 μg/ml, FOZ show 20% to 89.33% and ascorbic acid show 26-98% (Table 1). The pi electrons of the aromatic systems in the FOZ molecule can compensate for the electron deficiency and prevent oxidation (27).   (Fig.1). The findings revealed that 16 g/ml of doxorubicin and FOZ reduced cell growth by 92.66% and 83.33%, respectively. Due to their intercalating or covalent binding capabilities with DNA and interactions with the cell membrane, aromatic compound FOZ have shown anticancer effects (28,29).

Conclusions
Successful synthesis of the novel formazan (FOZ), which inhibits the growth of breast cancer (MCF-7) cells and has antioxidant properties. Using CHN, FT-IR, 1HNMR, and GC-Mass, the structure of FOZ was characterized. Compared to doxorubicin, a common drug, MTT findings for FOZ showed good cytotoxicity. Studies on the antioxidant potential have shown that FOZ has a strong anti-DPPH radical effect.