Structural study and antioxidant activity determination of (2E)- N -[2-(morpholin-4-yl)ethyl]-cinnamide

Herein we report the structural study and antioxidant activity of (2 E )- N -[2-(morpholin-4-yl) ethyl)-cinnamanilide ( 1) and its salt ( 1-HCl ). Compound 1 crystallizes as orthorhombic P bca system while its salt is monoclinic P2 1 /n. The supramolecular structures are held in shape by classical (D ― H ⋅⋅⋅ A) and non classical (C ― H ⋅⋅⋅ A) interactions. The antioxidant capability of both compounds show s a structural relationship, compound 1 has moderate capability, meanwhile compound 1-HCl increases its properties owing to the presence of N-H + in the structure.


Introduction
Preservation of industrialized food containing polyunsaturated fatty acids, such as eicosapentaenoic (20:5ω-3) and docosahexaenoic (22:6ω-3) acids, has been subject of growing interest because of importance in human nutrition.ω-3-Polyunsaturated fatty acids are believed to have several health benefits, i.e. in cardiovascular disease, immune disorders, inflammation, allergies, and diabetes 1 .In foods, fatty acids oxidation is one of the most important reactions leading to a deterioration of sensorial features, shelflife and general quality.The products of oxidation cause unpleasant flavors and structural molecular changes that in turn lead to rejection of products by the consumer 2 .Several compounds have been used to retard the oxidative reaction: the butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) have been used commonly as food antioxidants.However, a growing interest exists for the use of new antioxidant compounds focused on expanding the shelf half life of foods.These food additives could eventually be used by the food industry to prevent lipid peroxidation.However, it has been reported that some of these molecules possess toxic and carcinogenic effects on health 3 .Efforts have been made to search for novel antioxidant additives leading to slow down fatty acids and lipids oxidation.Oxidative rancidity is initiated by oxygen free radical species, by the action of molecular oxygen on pre-formed free radical species from polyunsaturated fatty acids or by presence of trace amounts of metallic ions 2,4 The antioxidant activity is known to inhibit lipid peroxidation, to scavenge free radicals and active oxygen, to chelate iron and to inactivate lipoxygenase 5 .Interestingly, the same antioxidant compound may work by a number of different pathways.All of these mechanisms play a role in the antioxidant compound behaving as a stabilizing agents in foods lipids 6 .
Recently, it has been reported that cinnamic acids and other compounds behave as good free radical scavengers.8][9] It has been demostrated that caffeic acid (a cinnamic acid analogue) possesses antioxidant properties and may prevent and/or revert acute or chronic hepatic damage caused by CCl 4 administration 10 .Anti oxidant compounds can diminish the oxidative rancidity in foods, as well as prevent several related diseases, such as cancer, diabetes, Alzheimer's and Parkinson's diseases 11 .

Structure analysis
The 1 H and 13 C NMR spectra of compounds 1 and 1-HCl are in agreement with the proposed structures (Table 1).The characteristic 1 H NMR signals for compound 1 are the amide NH at 6.28 ppm and vinyl signals, the latter appear as an AX system at 6.45 and 7.65 ppm with 3 J H-H = 15.6 Hz, characteristic of a trans coupling.In 13 C NMR spectrum, amide carbonyl appears at 165.8 ppm and vinyl signals at 120.6 and 140.9 ppm, whereas the methylene HNCH 2 and NCH 2 of the pendant NHCH 2 CH 2 N fragment are at 35.6 and 56.9 ppm, respectively, and morpholine ring carbon atoms are at 66.8 and 53.2 ppm.Salt formation goes on with few changes in both 1 H and 13 C NMR spectra; in the former, a new signal at 12.4 ppm appears corresponding to the NH + proton.In 13 C NMR the most significant change occurs at the O(CH 2 ) 2 and NHCH 2 carbon atoms from morpholine and NHCH 2 CH 2 N fragments, respectively, which are shifted 3.4 and 1.9 ppm, respectively, to lower frequencies.
Compounds 1 and 1-HCl crystallized from chloroform solutions, the former as an orthorhombic system space group P bca , whereas the last crystallized as a monoclinic system space group P2 1/n , a summary of bond distances and angles are listed in Table 3, and molecular structures are shown in Figures 1(a) and 2(a), respectively.
395 meqTrolox/g measured, at similar concentration 15 .The test for protection against βcarotene-linoleic acid emulsion bleaching gives a measure of the protective capacity of compounds against oxidation.It was found that compound 1 does not provide any protection whereas compound 1-HCl reaches the same protective capacity of BHT (1 mg/mL) at 11.5 mg/mL.The bleaching kinetics of β-carotene-linoleic acid emulsion are shown in Figure 3.
Compound 1-HCl was as effective as BHT to avoid the β-carotene deterioration, thus the absorbance versus time graph remained constant, in contrast, compound 1 showed no activity.Finally, the antioxidant activity of compounds 1 and 1-HCl is not related to their chelating capability, since the test for 1 was 29% effective (35.8% for BHT 16 ) and for 1-HCl, it was 0%.Chelating capabilities are not involved in the mode of action of 1-HCl.These results are in agreement with previously reported studies in which the capacity for donating electrons seems to play a more significant role in stabilizing the antioxidant efficiency of hydroxycinnamic acids than the ability for chelating metals 17 .The above results can be explained in terms of the structure: the morpholine NH + proton in 1-HCl can be easily transferred to interrupt the chain reactions of free radicals, which are responsible for lipid oxidation.

Conclusions
The NMR analysis of (2E)-N-[2-(morpholin-4-yl)ethyl]-cinnamanilide (1) and its chlorhydrate salt (1-HCl) showed no significant differences between the two compounds.However, x-ray diffraction demonstrated that the main effect of protonation is related to the supramolecular structure in the solid state, as well as in their antioxidant capabilities.(2E)-N-[2-(morpholin-4-ARKAT USA, Inc. yl)ethyl]-cinnamide chlorhydrate (1-HCl) showed moderate antioxidant properties in comparison with BHT, whereas the free base was not active.The above results can be explained in terms of structure the morpholine NH + proton in 1-HCl can be easily transferred to interrupt the chain reactions of free radicals.

Experimental Section
General Procedures.All chemicals and solvents were of reagent grade and used as received.
Melting points were measured on a Electrothermal IA 9100 apparatus and were uncorrected.IR spectra were recorded for KBr disks using a Perkin-Elmer 16F PC IR spectrophotometer. 1  Single-crystal X-ray diffraction data for molecules 1 and 1-HCl were collected on a Bruker Smart 6000 diffractometer at 298 K with Mo Kα radiation, λ = 0.71073 Å).A semiempirical absorption correction was applied using SADABS 18 , and the program SAINT 19 was used for integration of the diffraction profiles.The structures were solved by direct methods using SHELXS 19 program of WinGX package 20 .The final refinement was performed by full-matrix least-squares methods on F 2 with SHELXL 19

Antioxidant activity
Antioxidant properties were analyzed by three different methods: 1. Measurement of free radical scavenging activity.The antiradical activity of samples was evaluated according to the procedure reported by Sumaya-Martinez et al. 2005 22 .An aliquot of sample (200 µL) was added to 1 ml of a daily-prepared solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) in ethanol (74 mg/L).The mixture was homogenized in a vortex, incubated at room temperature for 1 h at 25°C and then centrifuged at 10 000 rpm/5 min.200 µL of each supernatant were placed in a UV-VIS microplate spectrophotometer and the absorbance was determined at 520 nm.For all experiments deionized water instead of sample solution was used as blank.Trolox solution (mmol equiv/L) was used as the control.2. Antioxidant activity measured by the bleaching of β-Carotene. 23A solution of β-Carotene is prepared dissolving 2 mg of β-carotene in 10 mL of chloroform. 2 mL of this solution is pippeted into 500 ml RB flask, after chloroform was removed under vacuum (at 40°C), 40 µL of linoleic acid, 400 µL of Tween-20 and 100 mL of deionized water were added.The mixture was vigorously shaken until the formation of an emulsion.Aliquots (5 ml) of this emulsion are added to test tubes containing 200 µL of the samples solutions.BHT solution in ethyl alcohol at 0.1 mg/ml was used for comparison purposes.The tubes were homogenized in a vortex and placed in a water bath at 50°C.The course of the color disappearance of β-Carotene was followed at 0, 30, 60 and 90 min.Absorbance was measured on UV-VIS microplate spectrophotometer at 470 nm.

Table 3 .
Hydrogen bonding and contact geometry of compounds 1 and 1-HCl 13and13C NMR spectra were recorded on a Varian Mercury 300 ( 1 H, 300.08;13C, 75.46 MHz) instrument in CDCl 3 solution, measured with SiMe 4 as internal reference, following standard techniques. Elmental analyses were determined on a Perkin-Elmer Series II CHNS/O analyzer 2400 instrument.

Table 4 .
Crystal and experimental data for compounds 1 and 1-HCl