SPECTRA AND ANTIOXIDANT ACTIVITY OF NEW AZINE DERIVATIVE BEARING COUMARIN MOIETY

In this research, the synthesis of a new azine derivative with coumarin moiety was performed in three reaction steps, starting from 4-hydroxycoumarin. The first step in synthesis was the acetylation of 4-hydroxycoumarin to yield 3-acetyl-4-hydroxycoumarin and then the obtained 3-acetyl-4-hydroxycoumarin was reacted with hydrazine hydrate and give a corresponding hydrazone. Condensation of the hydrazone with 4-ethoxy-3methoxybenzaldehyde afforded the target compound 1-[1-(4-hydroxy-2-oxo-2H-chromen-3-yl)-ethylidene]-2-(4etoxy-3-methoxybenzylidene)-hydrazine in a good yield. The resulting azine derivative is fully spectrally characterized, including complete assignment of Hand C-NMR spectra, as well as 2D NMR (H-H COSY, NOESY, HSQC and HMBC) spectra. The antioxidant activity of corresponding hydrazone and target compound was evaluated by DPPH method where hydrazone derivative displayed a significant and target azine good antioxidant activity, with IC50 (μM) values 11.69 and 216.60, respectively.


INTRODUCTION 1
The chemistry of organic compounds undoubtedly represents an experimental field with the greatest possibilities for research that is expanding daily. Synthesis of these compounds attracts great attention to organic chemists, due to their reactivity and a wide spectrum of both biological and therapeutic activities. A similar trend is seen for azines, compounds formed by the reaction of two different or the same carbonyl compounds (mostly aldehydes or ketones) with hydrazine (Safari & Gandomi-Ravandi, 2014;Chourasiya et al., 2019). The chemical properties and biological activities of these compounds mostly are the consequence of the presence of double bonds and nitrogen atoms. It is well known that they possess antibacterial (Veena et al., 2011;Chourasiya et al., 2015), antifungal (Kurteva et al., 2011), anticonvulsant (Gul et al., 2004), antineuroinflammatory (Subedi et al., 2017), anticancer (Krezel et al., 1999;Qian et al., 2010;Liang et al., 2014) and antioxidant (Li et al., 2011) activities. Also, they behave as aldose reductase inhibitors (Meanwell et al., 1991) and MDR reversal agents (Paterna et al., 2018). Due to their ability to donate the lone electron pairs, azines have found great application as chemical sensors for many metal ions (Wei et al., 2017;Tiwari et al., 2018). In addition to the above, azines represent very good synthons in synthetic chemistry, especially for the preparation of heterocyclic compounds.
Because azines showed a similar pharmacological effect, it is expected that the synthesis of azine derivatives with 3-acetyl-4-hydroxycoumarins moiety will give a promising result. This expectation confirmed a group of compounds of this type, which showed significant CDK (cyclic-dependent kinase) inhibition and a promising anticancer effect (Abdel Latif et al., 2016). However, compounds of this type are not numerous and their literature data are very scarce, primarily in the field of NMR spectral data and biological activities with an emphasis on antioxidant activity. As part of our interest in the discovery of novel compounds with possible pharmacological potential and following our previous work (Dekić et al., 2010, Radulović et al., 2015Ristić et al., 2019), here we reported the synthesis, complete assignment of 1 H-and 13 C-NMR spectra, as well as 2D NMR spectra and antioxidant activity of new azine derivative obtained by condensation of hydrazone of 3-acetyl-4hydroxycoumarin and an aromatic aldehyde, 4-ethoxy-3methoxybenzaldehyde.

General
Chemicals and materials. All solvents and chemicals were purchased from Merck (Germany), Fluka (Germany), Acros Organics (Belgium), J. T. Baker (USA), Fisher Scientific (USA) and Sigma-Aldrich (USA). Before use all solvents (acetone, methanol, absolute ethanol, ethyl acetate, hexane and chloroform) were distilled while other chemicals were used as thus obtained. The flow of the chemical reaction was monitored by thin layer chromatography (TLC) on aluminum plates with a previously applied layer of silica gel 60 F254, layer thickness 0.2 mm, Merck (Germany). Visualization of spots on TLC plates was performed using a UV lamp (254 nm) or spraying (1:1, v/v) with an aqueous solution of sulfuric acid and then the TLC plates were heated until stains appeared. Column chromatography was used for purification and performed on silica gel 60 (particle size 40-63 μm), Fluka (Germany). Melting points were determined on MPM-HV2 melting point instrument (Germany).
UV-Vis measurements. UV spectra and absorbance were done on LLG UniSPEC2 spectrophotometer (Germany).
IR measurements. IR spectra were done on Thermo Nicolet 6700 FT-IR spectrophotometer (USA).
NMR measurements. One-dimensional (1D) and twodimensional (2D) 1 H-and 13 C-NMR spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer (Switzerland). 1 H-spectra were recorded at 400 MHz and 13 C-NMR spectra at 100.6 MHz at 25 ºC. Deuterated dimethyl sulfoxide (DMSO-d6) was used as the solvent, while tetramethylsilane (TMS) was used as the internal standard. The values of chemical shifts are given in δ (ppm) units relative to TMS (δH = 0.00 ppm) for 1 H-spectra or to the signal of residual solvent DMSO-d6 (δH = 2.50 ppm and δC = 40.45 ppm) for 13 C-and heteronuclear 2D NMR spectra. For recording 2D NMR spectra ( 1 H, 1 H-COSY, NOESY/ROESY, HSQC and HMBC) as well as for multipulse DEPT95 and DEPT135 spectra, the standard pulse sequences in the instrument software (Topspin) were used. Scalar couplings (J) are expressed in hertz (Hz).
HRMS measurements. HRMS(EI) analysis of the synthesized compounds was performed on a JOEL Mstation JMS 700 mass spectrometer (Germany). The ionization energy was 70 eV, ion trap 300 μA and the temperature of the ion source was 230 ºС.
Elemental microanalysis. Microanalysis of carbon, hydrogen, oxygen and nitrogen was performed on a Carlo Erba 1106 microanalyzer (Italy).

Antioxidant activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was used to determine the antioxidant activity of the synthesized compounds 3 and 5, with some modification (Kadhum et al., 2011). Briefly, test samples were dissolved in methanol (12.5-400 μM) and then 2 ml of sample and 1 ml of methanolic DPPH solution (0.1 mM) were placed in a test tube. The tubes were shaken and then incubated in a dark place for 40 minutes at room temperature. For ascorbic acid, which was used as a positive control, in this case, the same procedure was repeated. The percentage inhibition of DPPH radical was determined spectrophotometrically measuring the absorbance at 517 nm against a blank (methanol) by using equation (1): whereat A-absorbance of control (2 ml of methanol and 1 ml of DPPH), A1-absorbance of the test samples (2 ml of samples and 1 ml of DPPH). Based on the relationship between the percentage inhibition of DPPH radical and the concentration of the tested samples, the results are performed in triplicate and presented in IC50 values (µM).
Spectral data (HRMS(EI), IR, 1 H-and 13 C-NMR) of synthesized azine (5) were in complete agreement with the proposed structures. The IR spectra of compound (5) showed strong absorption bands at 3389 cm -1 which corresponding to the OH group, characteristic vibrations at 3092 cm -1 attributed to the Ar-H, and band at 1697 cm -1 concerning C=O (lactone carbonyl) of coumarin moiety. Characteristic vibrations at 1601 cm -1 are attributed to C=N. Intensity bands corresponding to C=C stretching vibration of the aromatic rings appeared at 1555 cm -1 . The IR spectra at 1176 cm -1 showed a characteristic C-O stretching.
High-resolution mass spectroscopy (HRMS-EI) confirmed the structure of the synthesized azine (5) with molecular formula C21H20N2O5 ((M + ) at m/z 380.1384, Δ = +1.2 mmu). In 1 H-NMR spectra of compound (5) exist seven signals with chemical shifts characteristic for aromatic methine protons which appeared as three doublets, two doublets of doublets, and two doublets of doublets of doublets. It is noticeable that in 1 H-1 H COSY and NOESY spectra these protons are separated into two groups of signals ( Figure 1). In 1 H-NMR spectra ( Table 1) three signals that appeared as doublets at 7.48, 7.41, and 7.10 ppm constitute the first group of protons, while the second group consisted of two sets of doublets of doublets at 7.95 ppm and 7.25 ppm and two doublets of doublets of doublets at 7.61 and 7.29 ppm. Based on the data from the HMBC and HSQC interactions it was possible to determine which aromatic protons are bonded to which ring (Figure 2). The second group which consists of four protons indicates that they belong to the coumarin ring. Chemical shifts at 7.95 and 7.25 ppm which appeared as doublets of doublets are assigned as protons on a position H-5 and H-8, whereby they coupling with one vicinal (H-6 and H-7) and one long-range (H-7 and H-6) protons, respectively. Analyzing of given correlations, it was concluded that proton on position H-5 in HMBC spectrum, which interacts through three chemical bonds, assigns the quaternary carbons C-4 (178.3 ppm) and C-8a (153.4 ppm). This spectrum also showed that the carbon at 133.8 ppm is C-7 by the presence of a cross peak with H-5, which then allowed the assignment of H-7 as a peak at 7.61 ppm in HSQC. Analogously, the proton at position H-8 showed interactions with C-4a (120.7 ppm) and C-6 (123.9 ppm) and thus the shift of the proton H-6 was deduced to be 7.29 ppm from HSQC. Also, a characteristic interaction was observed through two chemical bonds between the proton H-8 and the quaternary carbon C-8a, which is assigned at 153.4 ppm (Figure 2). In the HMBC spectrum, the assignment of carbons C-4a and C-8a were also confirmed by protons interactions at position H-6 and H-7, respectively. The signal at 2.96 ppm in 1 H-NMR spectrum assigned as a singlet of methyl protons attached at the 3-C=N, in HMBC spectrum showed interactions with carbon C-3 (94.1 ppm), through three bonds, and with the signal at 171.8 ppm, through two bonds, which corresponded to the carbon of imine group (MeC=N). The largest chemical shift at 16.39 ppm in the 1 H-NMR spectrum which appeared as a broad singlet, was assigned to the proton of the OH group at position C-4 on coumarin moiety. The only remaining signal in 13 C-NMR spectra at 161.3 ppm was assigned to lactone carbonyl carbon (C-2), since no interaction was observed in HSQC and HMBC spectra. Aromatic protons on the substituent side which appeared at 7.48, 7.41 and 7.10 ppm in the 1 H-NMR spectrum as a doublet, were attributed to H-2', H-6' and H-5', respectively. The signals of carbons C-2', C-6', and C-5' (Table 1) of the substituent ring, were readily connected to the aforementioned 1 H-NMR signals according to correlations in the HSQC spectrum, and further corroborated by HMBC data. The proton of OH group on coumarin moiety in NOESY spectrum showed interaction with the signal at 8.68 ppm which appeared as singlet and corresponded to the proton of azomethine group (N=CH) on the substituent side. The mentioned proton in the HMBC spectrum has two more interactions which are accomplished through three chemical bonds, whereby assigned a carbons C-2' (109.7 ppm) and C-6' (124.7 ppm), (Figure 2). Also, the position of the carbon C-1' (126.1 ppm) is confirmed by the interactions of the proton H-5' in the HMBC spectrum. In NOESY spectrum proton H-5' showed interaction with a peak that appeared as a quartet at 4.11 ppm in the 1 H-NMR spectrum and corresponded to the methylene protons of the ethoxy group. Also, methylene protons from the ethoxy group in the NOESY spectrum are in correlations with methyl protons (Figure 1) of the mentioned group which appeared as a triplet at the smallest chemical shifts of 1.37 ppm. Further, the aforementioned correlation between the methyl and methylene protons is confirmed by their interaction through two chemical bonds with the carbon atoms to which they are attached, in the HMBC spectrum. The last remaining signal (appeared as singlet) in the 1 H-NMR spectrum at 3.85 ppm belong to the protons of a methoxy group at position 3', which is confirmed by their interaction in the NOESY spectrum with a proton at position H-2'. The chemical shift of the remaining quaternary carbon C-4' (152.1 ppm) on the substituent side was determined from HMBC interactions, through three chemical bonds (H-2' and H-6').
The data provided by the NOESY spectrum were used for the determination of the spatial relationship between the coumarin core and the aryl substituent. The proton of the OH group on coumarin moiety showed a cross peak with the hydrogen of the azomethine group (N=CH, Figure 1), which makes it possible to determine the orientation of the aryl substituent ( Figure 3).  On the other hand, the proton of the azomethine group also showed simultaneous interactions with proton H-2'. All mentioned interactions indicated that the diimine bridge was created, whereby coumarin moiety and ring of the substituent, toward each other at a specific angle (Figure 3).

Antioxidant activity
Antioxidants are compounds that slow down the oxidation processes in the human body. They act as "scavengers" of free radicals, or electron donors, and thus prevent chain reactions in which other molecules in cells are damaged. In addition to natural antioxidants, synthetic antioxidants have been developed and used in practice as additives, supplements, and drugs. Due to a lack of antioxidants, many health diseases occur in humans, such as cardiovascular, carcinogenic, and inflammatory (Nojiri et al., 2004). The DPPH assay is based on monitoring the color change of a purple-colored solution of a stable DPPH radical, into a reduced yellow-colored form (DPPH-H). DPPH is a stable free radical with a delocalized free electron on nitrogen so that the molecule does not form dimers, which is often the case with most other free radicals. Delocalization allows the appearance of a purple color, with maximum absorption at 517 nm. Receiving a proton from a potential antioxidant he is reduced to hydrazine, whereby the intensity of absorption is decreased since hydrazine is not absorbed in that wavelength. A higher degree of discoloration means a higher reduction ability.
Substitution, the position of substitution, and the nature of the substituent significantly affect antioxidant activity. Thus, for example, compounds that have electron-donating groups on the phenolic ring contribute to increases in antioxidant activity, while electron-withdrawing groups usually decrease antioxidant activity (Lee et al., 2015).
As can be seen (Table 2), compound (3) showed significant antioxidant activity with IC50 value of 11.69 μM, while compound (5) showed slightly more moderate antioxidant activity (216.60 μM). In our case, ascorbic acid was used as a criterion to compare the ability to neutralize DPPH radicals with the tested compounds, i.e. as a positive control, since it is known that represented an exceptional antioxidant. As both tested compounds on the coumarin part of the molecule have an OH group that participates in the formation of a strong hydrogen bond with the nitrogen atom, indicates that the OH group probably does not have any effect on the antioxidant action. The significant antioxidant activity of hydrazone of 3acetyl-4-hydroxycoumarin (3) is a probable consequence of the electron-donating ability of the amino group and the high resonance stabilization of the formed radical. Similar to previous, although the tested compound (5) does not have an OH group on the aryl core that would certainly affect the increases in antioxidant activity, there are two electron-donating groups, methoxy and ethoxy group, which probably have influenced on the mentioned compound to exhibit a good antioxidant activity.

CONCLUSION
A new mixed azine derivative bearing coumarin moiety was synthesized and fully spectrally assigned using 1D-and 2D-NMR spectral data. The resulting azine was obtained in a good yield by condensation of hydrazone of 3-acetyl-4hydroxycoumarin and 4-ethoxy-3-methoxybenzaldehyde in a simple, rapid, and very effective way. Synthesized compounds (3) and (5) were monitored for their antioxidant activity. The antioxidant activity of the compound (3) against the stable free radical DPPH showed that this compound possessed a significant antioxidant power, while compound (5) demonstrated somewhat smaller, but still good antioxidant potential. The results obtained in this study will primarily contribute to organic synthesis and better knowledge of the structure of this group of compounds, formed by the reaction of hydrazone of coumarin and aldehydes. The obtained target azine represents a very interesting starting material for further biological and pharmacological research.