Introduction

Schiff bases are a significant class of organic and inorganic chemistry, which have –CH=N– bond. Schiff bases have shown an extensive spectrum of biological activities and medicinal properties such as antibacterial [1], antimicrobial [2], antifungal [3, 4], antioxidant [5, 6], antiviral [3, 7], anti-HIV [8], antitubercular [9], anticancer [10], antiinflammatory agents [11], and anticonvulsant [12] activity.

Compounds with the structure of –NH–C(=S)NH–N= are known as thiosemicarbazones, which are important class of compounds in chemistry, biological, and pharmaceutical field. Thiosemicarbazones have showed biological and medicinal applications including antimicrobial [13], antifungal [14], antibacterial [14, 15], antioxidant [16,17,18], anti-HIV [19], anticonvulsant [13, 20], antiviral [21], antituberculosis [22], anticancer [23], enzymatic inhibition [14, 24], and cytotoxicity [13, 14] properties.

The importance of free radicals and reactive oxygen species (ROS) in the pathogenicity of diverse diseases such as inflammatory, metabolic disorders, cellular aging, reperfusion damage, and cancer has attracted considerable attention [25, 26]. Therefore, antioxidants have been reported to play an important role in protecting humans against many mortal diseases.

In view of these proofs, thiosemicarbazones including Schiff bases are significant which not only possess biological activities, but also in medicinal applications. In this paper, a new series of novel thiosemicarbazones including Schiff bases were synthesized. The chemical structure of the products was characterized by using methods FT-IR, 1H NMR, 13C NMR, and LC–MS/MS spectroscopy and elemental analysis. These compounds were performed antioxidant activity in vitro by using DPPH free radical scavenging method.

Experimental section

Instruments and reagents

All the chemical materials were bought in Sigma-Aldrich or Merck Chemical Company and were used without further purification. The solvent was spectroscopic grade. Melting points were measured using a Stuart SMP 30 melting point apparatus and were uncorrected. The elemental analysis was performed on a Eurovector EA3000-Single. Infrared spectra were recorded on a Bruker Alpha FT-IR spectrometer. 1H NMR and 13C NMR spectra (in DMSO-d6) were taken at 25 °C using a Bruker Avance DPX-400 spectrometer operating at 400 MHz and 100 MHz, respectively. The splitting patterns are indicated as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), and m (multiplied). Absorption measurements were recorded with a Shimadzu UVM-1240 UV–visible spectrophotometer. Liquid chromatography—tandem mass spectrometry (LC–MS/MS) measurements were recorded with a Shimadzu LCMS-8030 Plus spectrophotometer.

Synthesis of novel thiosemicarbazones including Schiff bases (I–XI)

Thiosemicarbazides (0.6 mmol), various aryl-substituted aldehydes (0.6 mmol), and one drop of HCl were added to aqueous EtOH (30 mL), and the mixture was refluxed at 78 °C for 3–5 h. The obtained precipitate was filtered and washed, and then, the formed colored solid was isolated and dried in air. The products were obtained good yields (63–83%) as shown in Scheme 1.

Scheme 1
scheme 1

Synthetic path for the novel thiosemicarbazones including Schiff bases (I–XI)

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Antioxidant activity measurements by using DPPH method

The DPPH method is a method showing that the ability of compounds to lose hydrogen and structurally stable radicals is associated with higher cleaning activity of the molecule.

Antioxidant activity determinations of compounds synthesized using DPPH free radical cleaning method were performed. For this, Brand-Williams et al. [27] method implementation was used with minor changes. The stock solutions of the synthesized compounds were prepared to be 250 µM in DMSO and mixed with 3 mL DPPH (250 µM) solution in different volumes (10–200 µL). Sufficient ethanol was added until the total solution volume was 5 mL. The prepared mixtures were kept in a dark room for 30 min and read against the blanks to compare with a sample without sample at 517 nm. Antioxidant activity was calculated using the equation shown below as a percentage of inhibition [28]:

$$\left( \% \right)\,{\text{inhibition}} = \left[ {\left( {A_{0} - A_{1} } \right)/A_{0} \times 100} \right]$$

Here, A0 is the absorbance of the control (empty, without compound) and A1 is the absorbance of the compound. With the help of the inhibition values obtained, IC50 (µM) values were calculated and used to evaluate antioxidant activity [29].

Results and discussion

Physical data

All the synthesized compounds are new. In Table 1, the physical data, melting points, yields, LC–MS/MS results, and elemental analysis of these compounds are presented.

Table 1 Physical data, LC–MS/MS results, and elemental analysis results of the novel thiosemicarbazones including Schiff bases
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Vibrational frequencies

In the FT-IR spectra, the signal of the aldehyde group (–CHO, two bands) of the starting material was not observed at 2780–2660 cm–1. Moreover, the symmetric and asymmetric stretching bands of the amino group (–NH2) did not show at 3520–3250 cm–1. Instead, new absorptions for the –C=N stretching bands of imine group were observed at 1599–1522 cm−1. These results indicated a successful reaction, as expected. For all compounds (I–XI), the –OH stretching vibrations were observed in the range of 3574–3376 cm–1, the amino group (= N–NH and –NH–Ar) stretching vibrations were observed in the range of 3395–3284 and 3299–3116 cm−1, aromatic and aliphatic C–H stretching vibrations were observed in the range of 3168–3017 and 2995–2962 cm−1, –C=N characteristic absorptions of imine group were observed between 1595 and 1539 cm−1, the –C=S signal of semicarbazide region was observed in the range of 1474–1352 cm−1, the –C–N group absorptions were observed at 1333–1207 cm−1, the –C–O stretching vibration was appeared in the range of 1222–1094 cm−1, and the Ar–F stretching vibration was appeared at 1172–1044 cm−1, except for compounds X and XI. Ar–Br absorption bands of compounds III and IX were observed at 629 and 591 cm−1 (see Figures S7 and S22 in supplementary material).

In compound IV, the –OH stretching vibration was observed at 3574 cm–1, while the =N–NH (amino) stretching vibration was observed at 3308 cm−1; the –NH–Ar (amino) stretching vibration was observed at 3145 cm−1. The –C=N stretching vibration was observed at 1592 cm−1; the –C=S signal of semicarbazide region was observed at 1434 cm−1; the –C–N stretching vibration was appeared at 1277 cm−1; the –C–O stretching vibration was appeared at 1220 cm−1; the –C–F stretching vibration was appeared at 1067 cm−1 as shown in Fig. 1. These values provided significant proofs for the products formation. IR values of all compounds are listed in Table 2, and spectra of them are given in supplementary material. These observations are consistent with values published formerly for similar compounds [17, 18, 30, 31].

Fig. 1
figure 1

IR spectrum of compound IV

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Table 2 FT-IR values of the compounds (cm−1)
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Interpretation of the 1H NMR spectra

The 1H NMR spectra of the synthesized compounds were detected in DMSO-d6 as solvent. For compounds I–IX, the –NH peak (= N–NH) was observed as a singlet at 11.97–11.72 ppm, and the –NH signal (–NH–Ar) was detected as a singlet at 11.40–9.82 ppm. The signal of imine (–CH=N) was observed as a singlet at 10.24–8.03 ppm. The –OH proton signal was observed as a singlet in the range of 10.36–9.06 ppm. In 1H NMR spectrum of compound IV, while the –NH peak (= N–NH) was observed as a singlet at 11.79 ppm, the –NH signal (–NH–Ar) was detected as a singlet at 10.05 ppm. The signal of imine (–CH=N) was observed as a singlet at 8.08 ppm. The –OH (H1) proton signal was observed as a singlet in the range of 9.96 ppm as shown in Fig. 2. The H2 and H5 proton coupled to the H3 and H4 proton and detected doublet peaks at 6.83–6.81 ppm, respectively. The H3 and H4 proton coupled to the H2 and H5 proton and detected doublet peaks at 7.75–7.72 ppm, respectively. The H6 proton coupled to the H7 proton and was observed as a doublet peak at 7.66–7.63 ppm. The H7 proton coupled to the H6 and H8 proton showed doublet of doublets peaks at 7.49–7.47 ppm. The H8 proton coupled to the H6, H7, and H8 proton showed doublets of triplet peaks at 7.05–6.99 ppm. The H9 proton coupled to the H7 and H8 proton showed doublet of doublets peaks at 7.42–7.36 ppm. Additionally, while the proton atoms of ethoxy (–OCH2CH3) group were detected around at 4.11–4.06 (quartet, 2H) and 1.37–1.32 (triplet, 3H) ppm for compounds I, VI, and X, the methoxy (–OCH3) signal was observed as a singlet peak around at 3.83 ppm for compounds II, VII, IX, and XI as shown in Table 3 (see also Figs. 5, 17, 20, and 29). In compounds I, VI, and X, the CH3 proton of ethoxy coupled to OCH2 protons showed as triplet peaks at 1.37–1.32 ppm, whereas CH2 proton of ethoxy coupled to CH3 protons was resonated as quartet peaks at 4.11–4.06 ppm (see Figures S2, S14, and S26 in supplementary material). The proton of CH3 coupled to NH proton was detected as doublet peaks at 3.03–3.02 and 3.01–2.99 ppm, whereas NH proton coupled to CH3 protons showed quartet peaks at 8.37–8.36 and 8.40–8.38 ppm for compounds X and XI (see Figures S26 and S29 in supplementary material). DMSO-d6 and water in DMSO (HOD, H2O) signals were shown about at 2.00, 2.50 (quintet), and 3.30 (variable, depending on the solvent and its concentration) ppm, respectively [32]. These data are in agreement with the values of previously reported for similar compounds [17, 18, 30, 31]. It is apparent that structure of the obtained compounds and electronic effects of groups/substituents in structures plays a significant role to chemical shift value. Electron-donating groups (OH, NH2, OCH3, CH3, etc.) and electron-withdrawing groups (NO2, CN, CF3, F, Cl, Br, etc.) have affected chemical shift value with mesomeric (or resonance), inductive effect [33]. Moreover, the position of these groups/substituents in the molecules plays a major role as well. Proton chemical shift values of the synthesized compounds are given in Table 3. Moreover, all tested compounds’ IR, 1H NMR, and 13C NMR spectra were given in supplementary material (Figures S1–S30).

Fig. 2
figure 2

1H NMR spectrum of compound IV

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Table 3 1H NMR (δ, ppm, in DMSO-d6) values related to synthesized compounds*
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Interpretation of the 13C NMR spectra

The 13C NMR spectra of all compounds were obtained in DMSO-d6 as solvent. The 13C NMR spectrum of the compound IV showed 14 different resonances in good agreement with the proposed structure as shown in Fig. 3. For compounds I–XI, the –C=S (C8) signals of the thiosemicarbohydrazide region were detected between 177.9 and 175.2 ppm, and the –C=N peaks were observed in the range of 144.6–138.5 ppm. In compounds I–V, the C11 carbon atoms were detected at 163.3–160.1 ppm, shifted downfield (high values, δ) due to the presence of fluorine atom (m-F) (see Figures S3, S6, S9, and S12 in supplementary material). The C10 carbon atoms were detected at 161.2–156.6 ppm, shifted downfield (high values, δ) due to the presence of fluorine atom (o-F) for compounds VI–IX. Furthermore, the C9–C14 atoms were also split into doublets due to interacting with the atomic nucleus of F except for compounds X and XI. The C1 carbon atoms were observed at 147.6–163.2 ppm, shifted downfield (high values, δ) due to the presence of hydroxyl or methoxy group (–OH or –OCH3) except for compounds III–VIII. The C2 carbon atoms were observed at 147.1–149.9 ppm, shifted downfield (high values, δ) due to the presence of hydroxyl, methoxy, or ethoxy group (–OH, –OCH3, or –OC2H5) except for compounds III–V, VIII. The C3 carbon atoms were observed at 156.4, 158.8, and 156.3 ppm, shifted downfield (high values, δ) due to the presence of hydroxyl group (–OH) for compounds III, V, and VIII, respectively (see Figures S9, S12 and S21 in supplementary material).

Fig. 3
figure 3

13C NMR spectrum of compound IV

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In 1H NMR spectrum of compound IV, the characteristic –C=N (C7) peak was observed at 144.3 ppm; the –C=S (C8) signal of the thiosemicarbohydrazide region was detected at 175.6 ppm. The carbons (C1–C6) of the aldehyde ring were observed at 163.2, 116.0, 130.0, 125.3, 130.0, and 116.0 ppm, respectively. The C1 carbon atom resonated downfield (high value, δ) due to the presence of hydroxy (–OH) group. Moreover, equivalent-symmetry carbon atoms (C2 with C6, C3 with C5) had the same chemical environment and gave the same signal. The aromatic carbons (C9–C14) of aryl-thiosemicarbohydrazone ring were observed at between 141.4 and 111.9 ppm. The aromatic carbons (C9–C14) were observed at 141.4, 141.3; 112.5, 112.3; 160.8, 160.1; 112.1, 111.9; 129.9, 129.8; 121.5; and 121.4 ppm, respectively. The C11 (160.8, 160.1 ppm) carbon atom was shifted downfield (high values, δ) due to the presence of fluorine atom. Additionally, while the carbon atoms of ethoxy (–OCH2CH3) group were detected around at 64.5 and 15.2 ppm for compounds I, VI, and X, the methoxy (–OCH3) signal was observed around at 56.3 ppm for compounds II, VII, IX, and XI. These results are suitable with the values published previously for similar compounds [17, 18, 30, 31]. The carbon chemical shift values of the synthesized compounds are given in Table 4 (see also supplementary material).

Table 4 13C NMR (δ, ppm, in DMSO-d6) values related to synthesized compounds
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Evaluation of antioxidant activity

Ascorbic acid was used as standard for antioxidant activity evaluations. Inhibition percentages of ascorbic acid and synthesized compounds calculated against concentration are given in Fig. 4.

Fig. 4
figure 4

Percentage change of inhibition calculated by DPPH method for ascorbic acid and synthesized compounds

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In this study, antioxidant inhibition percentages for the 11 different molecules we synthesized showed a regular increase in direct proportion to the increase in concentration. Among them, especially compounds I, VII, and X, although low against standard antioxidants, exhibited the highest inhibition rates among other compounds. Compound VI showed a lower percent inhibition in the concentration ranges of (0.5–2.5) µM, but showed a lower increase in concentrations of 5–10 µM and showed a stable state in percent inhibition. IC50 values were also calculated to evaluate antioxidant activities. In this study, IC50 values for ascorbic acid and compound are summarized in Table 5. Accordingly, the calculated IC50 value for ascorbic acid was 10.50 ± 0.01 µM. IC50 values for the newly synthesized compounds followed the order I > VII > X > VI > IV > IX > XI > II > V > III > VIII.

Table 5 IC50 values for the synthesized compounds
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It is possible to compare free radical scavenging activity between different molecules by knowing that antioxidant activity is inversely proportional to increase in IC50 values. According to these results, p-hydroxybenzaldehyde-derived compounds containing methoxy and ethoxy groups at different positions showed the highest antioxidant activity. However, synthesized structures (III and VIII) possessing bromine atom exhibited lower antioxidant activity. Furthermore, as Muğlu et al. [34] mentioned in their study, the presence of thio group is effective in showing antioxidant properties of compounds. In particular as mentioned in the studies of Bakır and Lawag [5], the presence of electron-donating methoxy and ethoxy structures in the compounds containing phenolic structure (I, VII, X, VI, IX, XI, II) contributed to the increase in the antioxidant activity of the compound.

Conclusions

In this paper, novel thiosemicarbazones including Schiff bases have been prepared with good yields of 63–83%. All the products were characterized by methods with FT-IR, 1H NMR, 13C NMR, and LC–MS/MS spectroscopy and elemental analyses. The in vitro antioxidant activity of the synthesized compounds was tested by the DPPH free radical scavenging method. Compound I of among the tested compounds exhibited the most satisfactory antioxidant activity against the DPPH radical. IC50 values of the thiosemicarbazones including Schiff bases ranged from 19.24 to 191.06 μM.