Synthesis and Identification of Some Imino Chalcone Derivatives with Evaluating their Anti-oxidant Activity

This work involves the preparation of high yield iminochalcon compounds (B1B15) through two parts. The first part involves the preparation of 2,4-dihydroxy Chalcone (A1-A15) by the condensation of 2,4-dihydroxy acetophenone with aryl aldehyde in the presence of sodium hydroxide (40%) as a catalyst. The second part includes the preparation of iminochalcon from the condensation of p-hydroxy aniline with 2,4-dihydroxy chalcone derivatives ) A1-A15) in the presence of some drops of conc. H2SO4. Thin-layer chromatography ((TLC) was used to control the chemical reaction . These new derivatives were characterized by using FT-IR and 1H-NMR spectroscopy. These synthesized compounds were also assessed by the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method, through which the compounds (B1-B15) were evaluated for their antioxidant activity. The compound B3 was found to have the strongest antioxidant activity (IC50= 23.91 μg/mL) as compared to that of the common standard of ascorbic acid (IC50=31.95 μg/mL).


Introduction
Schiff bases are prepared through the condensation between equimolars of primary amine (aromatic , aliphatic, or related derivatives) and carbonyl compounds (aldehydes or ketones, aromatic ,

Lihumis and Aowda
Iraqi Journal of Science, 2021, Special Issue2, pp: 1-16 2 aliphatic, or related derivatives) using basic or acidic media in alcoholic solvent [1]. They are characterized by the -N = CH-bond, which is essential in the process of transamination and racemization in biologic structures [2].They are used in various biochemical and biological activities, such as antitumor [3], anticancer and anti-tubercular [4], antimicrobial [5]antibacterial [6]and antibiofilm formation in methicillin-resistant staphylococcus aureus [7]. Chalcones have a C^^(A)-CO-CH = CH-C^ (B) structure, where two aromatic rings (A and B) are bound by an aliphatic three carbon chain [8]. Chalcone condenses in ethanol with substituted aniline in the presence of 2,3 drops of H 2 SO 4 to provide chalcone imine. Chalcones and their derivatives hold a special significance among pharmaceutical and synthetic compounds [9]. The chalcone nuclei are important components of several drugs [10]. The synthesis of imines from the condensation of carbonyl compounds (aldehydes or ketones) with amines as a nucleophile is typically used to prepare chalcones and Schiff bases [11]. Chalcone (1,3-diaryl-2-prapen-1-one) and Schiff bases (substituted benzylidene aniline) belong to the commonly used compounds in companies of natural intermediates. They have broad spectrums of biological activities, such as those of antioxidants [12], anti leshmanial [13], antifungal [14]and antimicrobial [15].α,β-Unsaturated ketimines, which are obtained from chalcone and amine condensation, possess different pharmacological properties [16]. Both chalcones and Schiff bases are essential for the synthesis of different active organic compounds, such as flavones [17], indazol-3-one and thioxo pyrimidines [18],pyrazoloins [19],β-lactams [20], sulfonamide derivatives [21], and metal complexes [22]. They are also used to minimize photosensitivity of photographic emulsions in color photography [23]and mesomorphic properties of dimer containing chalcone [24].

Materials and Methods
Chemicals used in this work are supplied from Sigma Fluka, MERCK, BDH and CDH and are used without further purification.

2.1Instruments
The melting points of the compounds prepared were determined using the SMP30 melting point instrument. The uncorrected FT-IR spectra were recorded on SHMADZU FT-IR 8400Series Japan)" using the KBr disk method . TLC was performed for silica gel G and spots were visualized by I 2 vapors. The H1-NMR spectra were obtained using DMSO as a solvent and TMS as an internal standard with NMR spectrometer (Bruker, Ultra Shield 400 MHZ, Switzerland).

Synthesis of Chalcone Derivatives [25] (A1-A15)
A total of 40 % NaOH(10 mL) and 0.01mol of 2,4-dihydroxy acetophenone (1.52gm ) were added to 15 ml absolute EtOH in 100ml round bottom flask with 30 minutes of stirring. The substituted aldehyde (0.01 mol) heated at 40-45 0 C on water bath for 4-5 hours. The precipitate formed was left for overnight . The reaction mixture was completed by TLC using petroleum ether: ethylacetate (4:1), then washed with water and ethanol. Crushed ice was acidified with 45% (50ml) HCl and added . The precipitate was filtered and washed with 1% NaHCO 3 solution and water. The precipitate (yelloworange product) was crystallized from EtOH . Its physical properties are shown in Table-1.

Results and Discussion
The formation of Schiff bases from an aldehyde (or) ketone is a reversible reaction and usually occurs under acid (or) base catalysis or heating. The formation is usually powered by the separation of

Lihumis and Aowda
Iraqi Journal of Science, 2021, Special Issue2, pp: 1-16 6 The mechanism of the preparation of α, β-unsaturated ketimines from aldehydes (or) ketones was developed in an acid-catalyzed process which begins with the addition of the primary amine to the carbonyl group by nucleophile [27], as shown in scheme 1.3.

Scheme 1.3-Mechanism of Schiff's base synthesis ( acid-catalyzed )
The FT-IR spectra of the prepared compounds (B1-B15), which are listed in Table-3 , did not show the band assigned to the υ (C=O) of chalcone derivative at (1674-1647) cm-1. The absorption bands of C=N at (1590-1630) cm-1 were observed. The stretching vibration of C=N was moved to low due to the conjugation of the C=N bond with the aromatic ring [28]. A wide band of the hydrogen-bonded phenolic hydroxyl group (bonding O-H) was observed at (3307-3417 cm-1). The C = C, aromatic two peaks at (1489-1586) cm-1 were also shown. Two bands of absorption appeared at (3050-2959) cm-1 belonging to (C-H, stretching) of the aromatic and aliphatic groups, respectively, and a (C-N) appeared at a stretching frequency of (1230-1020)cm-1, as shown in Figures-(1-6).

Antioxidant Activities of the Prepared Compounds[29]
DPPH (1.3mg / ml) was prepared as a normal solution in methanol 100μl DPPH was added in 3ml of methanol and absorbance at 517 nm was noted. Various compound concentrations (25,50,75, and 100 μg / ml) were prepared. Sample (1 ml) was diluted to 3 ml and 100 μl of DPPH was applied. Test tubes were placed in light for 30 minutes to complete the reaction., Absorbance of each test tube was measured after 30min at 517 nm on UV-VIS spectrophotometer against methanol as a blank, as in Figures-(11, 12, 13).
The results of antioxidant activity are shown in Table-5 and indicate that the majority of the synthesized compounds displayed moderate to strong antioxidant activity in comparison to normal (ascorbic acid) activity (IC50=31.95 μg / mL). The highest activity was attributed to the p-OH group on the ring B in compounds B3 and B2 (IC50= 23.91 μg / ml and 28.82 μg / ml, respectively) . Conversely, compound B8 showed a low activity of m-NO 2 (IC50=123.87 μg / mL). For this reason, the (OH) ring in groups A and B confers high antioxidant activity. The p-OH group in B2 and B3 displayed greater free radical scavenging behavior than that of the m-NO 2 group of B8 compound. The standard reference made of ascorbic acid showed IC50 value of 31.95 μM. Compared to the reference, the strength of antioxidant functions of the tested compounds are in the following order: ascorbic acid >B3>B2>B6>B1>B11>B4>B10>B7>B9>B13>B12> B15> B14>B5>B8.

Conclusions
In the present study, substituted 2,4-dihydroxy-N-hydroxyl phenyl chalcone imines were synthesized with a percentage yield range of 54-90%. The structures of all these synthesized compounds were established on the basis of spectral data (FT-IR and 1 H-NMR). It is also interesting to note that the synthesized chalcone imines had strong antioxidant activities.