Synthesis, Antioxidant activity and Structure-Activity Relationship of gallic hydrazones Analogues

A series of gallic acid hydrazones were designed and synthesized as new potential anti-oxidant agents. Most of these compounds are potent antioxidants. The strongest compounds are 11 and 15 (EC 50 : 6.42 μg·mL −1 , 6.86 μg·mL −1 , DPPH) and (EC 50 : 12.85μg·mL −1 , 12.49μg·mL −1 ABTS), more potent than the positive control Trolox. Furthermore, the promising compounds 11 and 15 exhibited very low cytotoxic activity against HEK293 cell (IC 50 >56.4 µM). The SAR study revealed that the pattern of hydroxyl, methoxy and methyl substituents on the gallic hydrazones framework can increase the antioxidant properties of the prototype compounds. Moreover, the results also showed that the activity increased with the number of the groups and increased following hydroxyl > methoxy > methyl. Overall, the present study suggests that the designed compounds may serve as lead molecules for developing novel anti-oxidative agents in food industry.


Introduction
Free radicals and ROS play an important role in the pathogenesis of many diseases, the identi cation and synthesis of novel antioxidant to combat the harmful effects of free radicals and ROS attracting much attention for their versatile properties in pharmacology [1][2][3][4]. In particular, natural antioxidants, such as vitamin C, gallic acid, and polyphenols, are widely used to scavenge free radicals and to combat the harmful effects of ROS [5][6][7][8][9][10]. It is well known that donating group such as phenolic hydroxyl hydrazino and amino groups showed positive in uence for the antioxidant activities, many efforts have been focussed on designing antioxidants containing phenolic hydroxyl groups [2,11,12]. Gallic acid which has three phenolic hydroxyl groups is a well-known natural antioxidant exhibits extensively biological activities, such as anti-oxidative, anti-tyrosinase, anti-in ammatory, anti-fungal, anti-cancer, and so on, which has versatile applications in medicine, food and pharmaceutical industries because of its unique physiochemical characteristics, non-toxicity, biodegradability, abundant availability, and low cost [13][14][15][16][17].
Recently, hydrazones were reported as potent antioxidants due to azomethine group increasing free radical scavenging ability have been extensively investigated [5,7,18]. Meanwhile, the N-H proton of phenylhydrazones is necessary for the radical scavenging activity of this compound in Fig 1 [19].
Based on the ndings and in continuation of our works on the eld [20], the present work report here the synthesis and antioxidant properties of gallic acid hydrazones and their structure-activity relationships based on the presence, position and number of different substituents (hydroxyl, methoxy and methyl) on the phenyl ring.

Synthesis
The gallic acid esters were prepared from gallic acid in re uxing methanol and the presence of H 2 SO 4 as catalyst. The reaction of esters with hydrazine hydrate in ethanol afforded hydrazides. Finally, condensation of hydrazide with different aromatic aldehydes in methanol produced the desired gallic acid hydrazones Analogues 1-14 in 68−85% yield.

Antioxidant activity
The antioxidant activities of the newly prepared compounds were measured against DPPH and ABTS radicals assay respectively, according to the literatures [21,22]. Trolox and gallic acid were also determined for comparison.
The results indicated that most the synthesized compounds possessed potent DPPH radical scavenging activity compared to the positive control Trolox. As shown in Table 1, most the synthesized compounds possessed potent DPPH radical scavenging activity compared to the positive control Trolox and gallic acid. The antioxidant activity of gallic acid hydrazones were enhanced in comparison with gallic acid, it could be concluded that NH group was important contributors to their DPPH radical scavenging activity.
The derivatives 11 and 15, both containing six hydroxyl groups with EC 50 value of: 6.42 μg·mL −1 and 6.86 μg·mL −1 were the most e cient highlighting the importance of hydroxyl group. Compounds 8, 9 and 12 although less active than compounds 11 and 15, also exhibit prominent DPPH radical scavenging activity, the results are in agreement with previous study that catechol group is the main contributor to the antioxidant capacity of phenolic compounds [23].
Additionally, introduction of methyl groups on the phenyl ring in the 2-and 3-positions increases the effects but the higher activity is obtained with 2-hydroxyl or 3-hydroxyl. The effect of hydroxy′ s position is also studied, compounds 2, 4 and 7 differing in the position of the hydroxyl group, compounds 7 (4position) displayed better activities than compounds 2 (2-position) and 4 (3-position), the relative DPPH radical scavenging activity decreases in the following sequence: 4-position > 3-position > 2-position.
The same tendency was observed in the ABTS radical scavenging activity studies. As shown in Table 1. Most of synthesized compounds showed signi cant ABTS radical cation scavenging activity, the order of ABTS radical cation scavenging activity of the antioxidants was mostly in accordance with DPPH radical scavenging activity. The compounds (11 and 15) with EC 50 value of 12.85 μg·mL −1 and 12.49 μg·mL −1 were the most potent compounds too.
In view of the results, the number of active groups was highly related to the antioxidant activity of the compound, and generally more active groups resulted in higher activity. Additionally, electron donating substituent like methoxy can increase their antioxidant activity too and increased following hydroxyl > methoxy > methyl. Such phenomenon was consistent with previous study [24]. In summary, the hydrogendonating ability of phenoxyl radical in uence the antioxidant activity of the prototype compounds.

Cytotoxicity of promising compounds 11 and 15
In order to check the safety pro le of promising antioxidant, it was selected to test its cytotoxicity against human normal cell line (HEK293). The result showed that compounds 11 and 15 displayed much lower cytotoxic activity for HEK293 (IC 50 >56.4μg·mL −1 ). The result revealed that the compounds 11 and 15 can display signi cant antioxidant activity at low concentrations without inducing cytotoxicity.

Conclusions
In conclusion, we have designed and synthesized a number of gallic acid hydrazones as new potential antioxidant. The in vitro antioxidant properties of the compounds in terms of reducing ability and radical scavenging activity were assessed by using ABTS and DPPH tests, respectively. Compound 11 and 15 which both have six hydroxyl groups were the most potent radical scavenger in the tsets. This nding might be attributed to the fact that the antioxidant activity was strongly dependent on the number of hydroxyl groups in the grafted phenolic moieties. The SAR study revealed that the pattern of hydroxyl, methoxy and methyl substituents on the gallic acid hydrazones framework can increase the antioxidant properties of the prototype compounds. Furthermore, the results also showed that the activity increased with the number of the groups and increased following hydroxyl > methoxy > methyl.
Therefore, it could be concluded that this class of compounds had a good safety pro le for their potential application in the food industry and proved that compound 11 and 15 might be potentially used as an antioxidant agents with a high potency and low toxicity in pharmaceutical, and food industries. Further studies on the relevant action mechanisms and structural modi cation of identi ed hits are on-going.

Chemistry
All reactions were performed with commercially available reagents and solvents without further puri cation. All reactions were monitored by thin-layer chromatography (TLC). 1 H and 13 C NMR spectra were recorded on Bruker AVANCE-III HD 400MHz or NEO 500MHz (Bruker Daltonics Inc., Germany) spectrometers using TMS as a reference. Mass spectra were recorded on a Bruker APEXII49e spectrometer (Bruker Daltonics Inc., Germany) with ESI source as ionization.
General procedure for the synthesis of gallic hydrazones 1-17.
The reaction route is outlined in Scheme 1. The intermediate compounds gallic hydrazide was prepared according to the reported methods [20]. Brie y, the gallic acid esters were prepared from gallic acid in re uxing methanol and the presence of H 2 SO 4 as catalyst. The reaction of esters with hydrazine hydrate in ethanol afforded hydrazides. Finally, condensation of hydrazide with different aromatic aldehydes in methanol produced the desired gallic hydrazones analogues 1-17 in 68−85% yield.   163.38, 148.61, 147.64, 146.00, 138.11,   137.27, 135.37, 133.56, 125.73, 124.08, 107.   The DPPH radical-scavenging activities of test compounds were determined according to the reported method [14]. Brie y, the test compounds at various concentrations were added to 3mL of DPPH solution (0.1 mM in DMSO) and the reaction mixture was shaken vigorously. After incubation at room temperature for 10 min, the absorbance of this solution was determined at 517 nm after 10, 30, and 60 min, using a spectrophotometer. All of the assays were performed three times. The concentration of a certain compound necessary to decrease the initial DPPH concentration by 50% (EC 50 μg/mL) was determined by linear regression analysis of data obtained by plotting the scavenging rate % against the concentrations of that compound.

ABTS radical-scavenging assay
The ABTS radical-scavenging activities of test compounds were determined according to the reported method [22,23]. ABTS radical cation was produced by reacting 7 mM aqueous ABTS solution with 2.45 mM potassium persulphate and the mixture was allowed to stand in the dark at room temperature for 12-16 h before use. An aliquot (0.1 mL) of DMSO solution of different antioxidant concentrations was added to 3.9 mL of the ABTS solution. Absorbance at 734 nm was recorded at different time intervals on a UV-vis spectrophotometer.

Cytotoxicity assay
Considering that compounds 11 and 15 were the most potent antioxidant agents, they were selected to investigate the cytotoxicity against human normal cell line (HEK293) according to the reported method [24,25]. Cells were seeded in 96-well plates and then treated with different concentrations of compounds 11 and 15, they were dissolved in DMSO as a 100µM stock solution and then diluted to the different concentrations. Two independent experiments in triplicate were done for determination of cell viability inhibition for the compound. The IC 50 values were calculated from concentration-response curves and expressed as means ± SD.

Conclusion
We have designed and synthesized a number of gallic acid hydrazones as new potential antioxidant. The in vitro antioxidant properties of the compounds in terms of reducing ability and radical scavenging activity were assessed by using ABTS and DPPH tests, respectively. Compound 11 and 15 which both have six hydroxyl groups were the most potent radical scavenger in the tsets.
This nding might be attributed to the fact that the antioxidant activity was strongly dependent on the number of hydroxyl groups in the grafted phenolic moieties. Furthermore, cytotoxicity assay showed that they had lower cytotoxic activity for HEK293.
Therefore, it could be concluded that this class of compounds had a good safety pro le for their potential application in the food industry and proved that compound 11 and 15 might be potentially used as novel antioxidant agents with a high potency and low toxicity.
The SAR study revealed that the pattern of hydroxyl, methoxy and methyl substituents on the gallic acid hydrazones framework can increase the antioxidant properties of the prototype compounds. Furthermore, the results also showed that the activity increased with the number of the groups and increased following hydroxyl > methoxy > methyl. Overall, the present study suggests that the designed compounds may serve as lead molecules for developing novel anti-oxidative agents in pharmaceutical, cosmetic, and food industries.