Microwave assisted synthesis, photoisomerization study and antioxidant activity of a series of N-acylhydrazones

2.1 Synthesis

2.2 Photoisomerization

Stock solutions (4.10^-6 M) of compounds 4(a-d) were prepared using DMSO as solvent for a first set of experiments and methanol in another set. Samples were exposed to UV radiations at 365 nm with a low-pressure lamp (40 W; model Vilber VL340.BL, light intensity 413 mW/cm²). The reactor was placed 12 cm below the lamp. Samples' spectra were registered as function of time.

2.3 Antioxidant effect

To evaluate the antioxidant capacity of our molecules, we prepared solutions with a concentration of 100 µg/ml in DMSO. Increasing volumes were sampled, supplemented with methanol up to 200 µl, and then mixed with a DPPH solution in methanol (2.4 µg/ml). The prepared samples were stirred and left at ambient temperature in the dark for 30 min. After this incubation period, the absorbance (A) was measured at 517 nm, using methanol as the blank control and ascorbic acid as control. Tests were performed three times, and the IC50 values, which are the required concentration for each compound to inhibit 50 % of the DPPH free radicals, were calculated and presented as the mean value ± standard deviation. IC50 were determined following equation (1).

3.1 Synthesis

The synthetized acylhydrazones were prepared from salicylic acid following the three-step route illustrated in Fig. 1. 2-hydroxybenzohydrazide (3) was obtained in good yield from ethyl 2-hydroxybenzoate (2) and hydrazine hydrate in ethanol. Finally, N-acylhydrazones (4) were prepared by a coupling reaction between 2-hydroxybenzohydrazide and different natural and substituted aldehydes to afford the final compounds in interesting yields (Fig. 1). For this last step, we used a microwave-assisted synthesis method and results were compared to those obtained by a conventional synthetic method.

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Fig. 1

Synthetic route to N-acylhydrazone derivatives from salicylic acid.

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With the conventional method, reactions were conducted at reflux in ethanol for four hours and compounds 4(a-f) were obtained in 69–80 % yields. Conversely, for the chemistry method, reaction conditions such as solvent, temperature and reaction time were optimized.

Reactions were carried out in ethanol and in dichloromethane at ambient temperature, 30 °C and 60 °C, the irradiation power was set at 80, 100 and 150 W, while the irradiation duration was varied from 2 to 10 min. Results allowed us to conclude that the optimal conditions were to run reactions under an irradiation power of 150 W and a temperature of 60 °C. Results summarized in Table 1 show that compounds 4(a-f) were afforded in excellent yields (95–98 %) with both solvent. Reactions conducted in DCM were achieved in 4 min while those in ethanol required 10 min to be completed.

Interestingly, microwave assisted synthesis was proved to be a simple, effective and prompt method for the preparation of compounds 4(a-f) and DCM was demonstrated better solvent for the reaction. In a previous work (Andrade and Barros, 2010) compound 4a was prepared with a comparable method using microwave assisted synthesis by reacting hydrazide with benzaldehyde in absence of solvent and catalysts but the reaction conditions were set to 200 W under 4.1 bar and a temperature of 201 °C. Authors obtained the resulting product 4a in 7 min with 89 % yield. Comparing to those results, we can conclude that we developed an effective method using a lower power (150 W) and a lower temperature (60 °C). This synthetic pathway permitted us to prepare the target compounds in higher yields (98 %) in less time.

Structures of the synthetized products were established using various spectral analyses, including FTIR, ¹H NMR, ¹³C NMR, and mass spectrometry. Relevant spectral data are provided as supplementary material (Fig. S1-S18).

In FTIR spectra of compounds (4a-f), the imine function in the N-acylhydrazone fragment revealed a characteristic band at 1600–1611 cm⁻¹ range, and the carbonyl group revealed a signal at 1680 cm⁻¹. In compounds 4(e-f), the azo unit gave characteristic absorption bands in the region of 1530–1490 cm⁻¹.

In ¹H NMR, HC = N protons appear as singlets in the 8.78 to 8.33 ppm region, the amide protons produce a broad singlets signal in the 11.89 to 11.68 ppm range and aromatic protons resonate in general as doublets or multiplets in the 7.86–6.3 ppm area. Methoxy group resonates as sharp singlet at 3.78 ppm. The ¹³C NMR spectra also reveal signals corresponding to the imine (C = N) and amide (–NHCO) moieties at 159.65–166.05 ppm and 159.83–145.07 ppm respectively. The –OCH3 group resonates at 56.37 ppm.

3.2 UV–visible absorption and photoisomerization study

The UV–visible spectra of 4(a-d) were monitored at room temperature in the region 200–800 nm in two solvents: methanol (Fig. 2) and DMSO (Fig. 3). All the N-acylhydrazone compounds 4(a-d) exhibit three main absorption bands, the first one appeared in the visible region with a weak intensity and was attributed to n-π* transition characteristic of the N-acylhydrazone fragment. This band appeared at 392–438 nm in methanol and 384–456 nm in DMSO. The spectra of 4(a-d) display two other bands assigned to π-π* transitions at 288–324 and 298–344 nm in MeOH, and at 270–300 and 312–328 nm in DMSO. We noticed also that the presence of the methoxy group in the structure of compound 4d affects the absorption bands of both n-π* and π-π* transitions in either MeOH or DMSO solutions, resulting in a significant redshift.

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Fig. 2

Absorption spectra of N-acylhydrazones 4(a-d) (4.10^-6 M) in MeOH.

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Fig. 3

Absorption spectra of N-acylhydrazones 4(a-d) (4.10^-6 M) in DMSO.

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The solvent nature has a notable effect on the absorbance of each molecule (Karmakar et al., 2021). In effect, changing methanol with DMSO induced a decrease in the absorbance of compounds 4b and 4c, which have a hydroxyl group on the phenyl ring while the absence of this group in 4a led to an increase in absorbance around 320 nm. At the same time, we noticed the decrease of the band at 328 nm for compound 4d and the enhancement of absorbance of the band at 456 nm. These spectra changes are probably due to hydrogen bond formation between the solvent and the hydroxyl or NH groups.

Photoisomerisation is an important property of a molecule that attract great interest due to its diverse applications. This property was studied for NAHs 4(a-d) by evaluating the differences in the UV–Vis spectra of these compounds before and upon UV irradiation as function of time. The disparities in the spectra of compounds 4(a-d) were recorded in two solvents: methanol and DMSO. The resulting UV–visible spectra are displayed in Fig. 4 and Fig. 5. Results analysis allowed us to conclude that all the studied compounds demonstrated effective photochromic behavior.

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Fig. 4

UV–visible spectra of 4(c) in MeOH (4.10^-6 M) recorded as function of time before and upon irradiation at 365 nm and after exposure to visible light for 60 min.

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Fig. 5

UV–visible spectra of 4(c) in DMSO (4.10^-6 M) recorded as function of time before and upon irradiation at 365 nm and after exposure to visible light for 60 min.

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In both MeOH and DMSO solvents, compound 4c, upon 10 min of irradiation at 365 nm, exhibited a drastic decrease in the intensity of the λmax in the π–π* band. Simultaneously, the lower energy band of the characteristic n-π* transition of the acylhydrazone group, underwent a hyperchromic shift and, at the same time, a slight red shift (10 nm for DMSO and 6 nm for MeOH). These changes indicated clearly the light-induced trans to cis isomerization of the imine bond in N-acylhydrazones 4(a-d).

E/Z isomerization process strongly depends on the polarity of the solvent and its ability to stabilize the photoinduced transition state since the interactions between the solvent and the solute influence the distribution of isomers during this process (Cordier et al., 2004) (Galić et al., 2012). When a solution of acylhydrazones 4(a-d) in methanol are exposed to UV light, a complete trans to cis isomerization was detected after 30 min. Methanol is a polar protic solvent that can form intermolecular interactions with hydroxyl groups and the hydrazide group of solute compounds through hydrogen bonds. These interactions stabilize the formed isomers and lead to a faster isomerization. However, DMSO, a polar aprotic solvent, can also interact through hydrogen bonds with specific functional groups of the solute inducing stabilization effects distinct from methanol. These interactions might modify the dynamics of the compounds involved in the photoisomerisation process, thereby influencing the rate of isomerization. Consequently, the trans to cis isomerization was achieved in 45 min in DMSO. In addition, the presence of different substituents, including hydroxyl groups (OH) and methoxy (OCH₃), affects the intermolecular interactions, thus also affected the isomerization rate.

Immediately after switching off the UV light, as the visible light-return time progressed, we observed a decrease in the intensity of π–π* transition and an increase in the intensity of the n–π* transition. As reported in the spectra presented in Fig. 3 and Fig. 4, the trans form was completely recovered after 60 min in MeOH and 90 min in DMSO. These finding proved the reversible isomerization from cis- to trans-form of 4c compound. The formation of isopiestics points, indicating the coexistence of cis and trans-forms, further confirms the isomerization. Similar results were obtained for compounds 4a, 4b and 4d.

3.3 Antioxidant activity.

The anti-radical activity is demonstrated by the aptitude of a substance to neutralize free radicals, which are unstable molecules produced naturally in the body or introduced by external factors. This characteristic contributes to protecting cells against oxidative damage associated with health benefits, including the prevention of chronic diseases. Several methods are employed to assess anti-radical activity in in vitro tests (Abd Alkareem and Waheed, 2022) (Bale, 2022). In our study, antioxidant assays were conducted following the 2,2 Dipheny-1-picrylhydrazyl (DPPH) radical scavenging test reported in literature [Kedare et al., 2011), (Hani et al., 2023) and using ascorbic acid as reference. Half-maximal inhibitory concentrations (IC50) are summarized in Table 2. Data show that compounds 4e and 4f exhibited a significant capacity for scavenging DPPH free radicals although it is less than the ascorbic acid. However, molecules 4a-d revealed a very high IC50 values, making it challenging to determine whether their activity is very low or null compared to 4e and 4f.

Molecules possessing OH or NH mobile protons groups in their structures are probable scavengers of reactive oxygen species (ROS) and several studies have demonstrated the relation between radical scavenging property and the facility of an H-atom transfer, mostly for the hydroxyl group, to free radicals (Santos-Sanchez et al., 2019).

For our compounds, this promising activity is not only due to the presence of the OH group that is generally known to be the active group for DPPH scavenging but can be attributed to the presence of the azobenzene group. In effect, this later induces a substantial conjugation in the structure that offers to the system various resonance structures and consequently high stability of these molecules. We noticed also that the presence a donating group such as –OCH₃ on the central core of the molecule was favorable for this activity.