Introduction

Thiocarbohydrazide is a promising unit to synthesize new polyfunctional thiocarbohydrazones (TCHs) after condensation with an aldehyde or ketone [1]. Biological activities of TCHs are the functions of their parent aldehyde or ketone moiety, and therefore it is very important to foresee the optimal structural fragments giving the improved biological activity [2]. Also, TCHs contain an azomethine (–C=N–) linkage that may connect two or more aromatic/heterocyclic ring to form various molecular hybrids with interesting biological properties [3]. In addition, TCHs and structurally similar thiosemicarbazones are well-studied anticancer agents [4,5,6,7], but also exert other biological activities [2, 8, 9].

TCHs are structurally versatile molecules with several possible solution structures including configurational (E or Z) isomers around imine bond and thione/thiol tautomers [10,11,12]. The state of equilibria depends on energy of appropriate form, solvent/solute interactions and intra/intermolecular hydrogen bonding [13]. Hydrogen bonding interactions determine the geometry and interaction/functionality of biologically active compounds. Therefore, knowledge of the mechanism of hydrogen bond formation and subsequent influences on molecular properties is very important for the design of molecules with the improved biological profile. A photochromism of 2-hydroxyphenyl and 2-pyridyl(carbohydrazones) results in E/Z isomerization driven by the intramolecular hydrogen bond in E-2-hydroxyphenyl and Z-2-pyridyl derivatives [14]. The bis(hydrazones) with 2-pyridyl moiety represent an interesting class of tunable molecular switches where the isomerism from stable E,E isomer to metastable E,Z and Z,Z forms can be controlled photochemically or electrochemically [15].

2-Acetylpyridine and 2-hydroxybenzaldehyde are aromatic carbonyl compounds and constitute the compounds with promising biological activities [16, 17], probably due to their chelating ability toward vital metal ions [18, 19]. TCHs are able to interact with many metalloenzymes essential for microbes [12], while sulfur atom increases lipophilicity which is generally positively correlated with potency [20]. The Schiff bases are also known for their antioxidant activities. Two aromatic rings of diarylhydrazones linked with (carbo)hydrazide moiety represent an interesting pharmacophore for antioxidant activity [21]. The extended conjugation is the characteristics of natural antioxidants such as lycopene and β-carotene, and can be achieved through the rapid equilibrium between the tautomeric forms. The tautomeric equilibrium between thione and thiol tautomers of TCHs extends the conjugation and might provide a structural basis for the excellent radical scavenging ability and antioxidant activity.

The assessment of exact aqueous structure of isomerizable, biologically important molecules is a prerequisite for further drug development. Mass spectrometry studies showed that bis-TCHs might exist as a mixture of two cyclic (2-amino-l,3,4-thiadiazolidine and 1,2,4-triazolidine-3-thione) and one linear form in a gas phase, with the fragmentation peaks corresponding to thiol form. On the other hand, the structure of TCHs in biologically relevant media is not studied in more details. The 1H NMR spectra of the same compounds indicated that only linear, thione form is present in DMSO-d6 solution [22]. The addition of water into DMSO solution of salicylaldehyde benzoylhydrazone induces the conformational and configurational changes [23].

In this study, we focus on the structural effects responsible for the increased antioxidant potency of TCHs observed upon the addition of water into DMSO solution of compounds. The combination of various spectroscopic techniques and quantum chemical calculations provided the new findings on the isomerism of TCHs in a solution and its impact on antioxidant activity.

Experimental

Materials

Salicylaldehyde, methyl 2-pyridyl ketone, and thiocarbohydrazide (98%) were obtained from Acros Organics (BVBA, Geel, Belgium). All solvents (reagent grade) were obtained from commercial suppliers and used without further purification.

Syntheses of compounds

The compounds 1 [18], 2 [24], 3 [19], and 4 [25] (Fig. 1) have been published earlier. All these compounds are synthesized by literary procedures and their purity was verified through NMR characterization. For the compounds 1 and 3, there is no complete literary characterization so it is presented in main text, while the characterization of compounds 2 and 4 is given in Electronic Supplementary Material (ESM).

Fig. 1
figure 1

Structures of investigated mono- and bis-TCHs along with the atom numeration

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[(2-hydroxyphenyl)methylene]-carbonothioic dihydrazide (1)

Yellow solid; Yield 76%; M.p. 186–187 °C; IR (KBr, cm−1) vmax: 3452s (OH), 3196s (NH2), 3069m (NH), 2999w (CHaryl), 1483s (amide II), 1282vs (C=S); Anal. Calc. For C8H10N4OS (210.06 g mol−1): C, 45.70; H, 4.79; N, 26.65; S, 15.25%, Found: C, 45.66; H, 4.77; N, 26.45; S, 15.18%. The solvent for recrystallization: methanol.

E:Z Isomers ratio (81:19). Isomer E: 1H NMR (500 MHz, DMSO-d6, δ ppm): 4.85 (s, 2H, H2–N4); 6.77–6.92 (m, 2H, H–C4, H–C6); 7.20 (t, 1H, H–C5, 3J5,6 = 6.3 Hz); 7.98 (d, 1H, H–C3, 3J3,4 = 6.5 Hz); 8.33 (s, 1H, H–C7); 9.73 (s, 1H, H-C1(OH)); 9.85 (s, 1H, H–N3,); 11.35 (s, 1H, H–N2). 13C NMR (126 MHz, DMSO-d6, δ ppm, TMS): 116.06 (C6); 119.28 (C4); 120.36 (C2); 127.20 (C3); 130.96 (C5); 139.81 (C7); 156.29 (C1); 175.91 (C8); 15N NMR (DMSO-d6 at 298 K): 96.22 (N4); 133.27 (N3); 166.38 (N2); 310.74 (N1).

Isomer Z: 1H NMR (500 MHz, DMSO-d6, δ ppm): 4.85 (s, 2H, H2–N4); 6.77-6.92 (m, 2H, H–C4, H–C6); 7.26–7.35 (m, 1H, H–C3, H-C5); 8.62 (s, 1H, H–C7); 9.28–9.61 ((br.m.ovlp. 2H, H–N3, H-C1(OH)); 11.78 (s, 1H, H–N2). 13C NMR (126 MHz, DMSO-d6, δ ppm, TMS): 116.06 (C6); 119.28 (C4); 129.25 (C2); 130.30 (C5); 127.61(C3); 147.57 (C7); 155.14 (C1); 171.68 (C8) (ESM, Fig. S1).

E-[1-(2-pyridinyl)ethylidene]-carbonothioic dihydrazide (3)

Yellow solid; Yield 88%; M.p. 179–181 °C; IR (KBr, cm−1) vmax: 3265 s (NH2), 3166 m (NH), 3000w (CHaryl), 1500vs (amide II), 1235s (C=S); Anal.Calc. For C8H11N5S (209.27 g mol−1): C, 45.91; H, 5.30; N, 33.47; S, 15.32%, Found: C, 45.82; H, 5.21; N, 33.32; S, 15.41%. The solvent for recrystallization: ethanol.

Thione/Thiol form ratio (97:3) Thione form: 1H NMR (500 MHz, DMSO-d6, δ ppm): 2.38 (s, 3H, CH3); 4.98 (s, 2H, H–N4); 7.36 (dd, 1H, H–C5); 7.78 (td, 1H, H–C4, 3J4,3= 8.1 Hz ); 8.50–8.59 (m, 2H, H–C3, H–C6); 9.95 (s, 1H, H-N3); 10.30 (s, 1H, H–N2); 13C NMR (126 MHz, DMSO-d6, δ ppm, TMS): 12.46 (CH3); 121.55 (C3); 124.28 (C5); 136.71 (C4); 148.72 (C7); 148.81 (C6); 155.20 (C2); 176.68 (C8); 15N NMR (DMSO-d6, δ ppm): 72.47 (N4); 136.01 (N3); 162.31 (N2); 310.70 (NAr); 313.11 (N1).

Thiol form: 1H NMR (500 MHz, DMSO-d6, δ ppm): 2.37 (3H, CH3); 4.98 (2H, H–N4); 7.58 (1H, H–C5); 8.08 (1H, H–C4, 3J4,3= 8.1 Hz); 8.77 (2H, H–C3, H–C6); 9.54 (s, 1H, H–N3), 14.14 (s, 1H, H–S); 13C NMR (126 MHz, DMSO-d6, δ ppm, TMS): 23.74 (CH3); 121.74 (C3); 123.35 (C5); 137.28 (C4); 148.27 (C7); 149.33 (C6); 159.93 (C2); 171.97 (C8) (ESM, Fig. S2).

Free radical scavenging antioxidant assay

The proton donating ability of TCHs was assayed using a protocol for the determination of radical scavenging activity [26]. Compounds were dissolved in DMSO and diluted into ten different concentrations. Commercially available DPPH· (2,2-diphenyl-1-picrylhydrazyl radical) was dissolved in methanol at a concentration of 6.50 × 10–5 mol L−1. Into a 96-well microplate, 140 μL of DPPH· solution was loaded and 10 μL DMSO solution of the tested compounds was added, or pure DMSO (10 μL) as the control. The microplate was incubated for 30 min at 298 K in the dark and the absorbance was measured at 517 nm using a Thermo Scientific Appliskan. All the measurements were carried out in triplicate. The scavenging activity of the compounds was calculated using Eq. (1):

$$ \mathrm{Scavenging}\ \mathrm{activity}\ \left(\%\right)=\frac{\left({A}_{\mathrm{control}}-{A}_{\mathrm{sample}}\right)}{A_{\mathrm{control}}}\times 100 $$
(1)

where Asample and Acontrol refer to the absorbances at 517 nm of DPPH· in the sample and control solutions, respectively.

IC50 values were calculated from the plotted graph of scavenging activity against the concentrations of the samples. IC50 is defined as the total antioxidant concentration necessary to decrease the amount of the initial DPPH· by 50%. IC50 was calculated for all compounds based on the percentage of DPPH· scavenged. Ascorbic acid was used as the reference compound (positive control) with concentrations from 50 to 500 mg L–1.

Samples preparation for UV measurements

Stock solutions of all compounds were prepared by dissolution of the weighed samples in DMSO. The working solutions was prepared in a 10.0 mL volumetric flask by adding appropriate volume of stock solution and water, and diluted to 10.0 mL with DMSO. Working sample, used for the measurement of UV–Vis spectra, was prepared in a such a way to obtain V(DMSO)/V(H2O) mixed solvents ratio of 4/1 [27] (Fig. 2). Concentration of compound in the obtained solution was 5 × 10−5 mol L−1.

Fig. 2
figure 2

The UV/Vis spectra changes induced by the addition of water in the DMSO solution of a) 1; b) 2; c) 3; d) 4

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Physical measurements

Elemental analyses (C, H, N) were performed by the standard micromethods using the ELEMENTAR Vario ELIII C.H.N.S=O analyzer.

Fourier-transform infrared (FTIR) spectra were obtained using FTIR BOMEM MB 100 in the form of KBr pellets. FTIR spectra were recorded in the transmission mode between 400 and 4000 cm–1 with a resolution of 4 cm–1. Abbreviations used for IR spectra are as follows: vs, very strong; s, strong; m, medium; w, weak.

NMR spectral assignments and structural parameters were obtained by combined use of 1D (1H and 13C, Figs. S3–S10) and 2D (COSY, NOESY, 1H-13C HSQC, and 1H-13C HMBC) spectra are given in ESM, Figs. S11–S24. The NMR spectra were recorded on Bruker Avance 500 equipped with broad-band direct probe. Chemical shifts are given on δ scale relative to tetramethylsilane (TMS) as an internal standard for 1H and 13C. Abbreviations used for NMR spectra are as follows: s, singlet; dd, doublet of doublets; ddd, double double doublet.

Computational chemistry studies

The initial geometry of each isomeric form was obtained using MMFF94 force field [28]. Afterward, conformational seach was performed using AMMP program [29] and Vega ZZ 3.1.0 GUI [30]. Boltzman jump search parameters were used, while all flexible torsions were rotated by 15°. Dielectric constant was set to 78.4 (water) and other settings were retained at default values. The lowest energy conformer of each form was additionally optimized using DFT with B3LYP functional and 6-311++G(d,p) basis set. Non-specific solvent effects were simulated using the water IEF-PCM model. Frequency calculations were performed in order to obtain zero-point corrected energies and other thermodynamic parameters. No imaginary vibrational frequencies were present in the optimized geometries, confirming that the geometry reported represent the minimum on the potential energy surface. The absorption spectra are simulated using time-dependent DFT method with CAM-B3LYP functional [31] and the same basis set as for geometry optimization, while solvent effects were simulated using IEF-PCM model of DMSO. Gauge independent atomic orbital (GIAO) method [32] was used for predicting the NMR chemical shifts of diastereoisomers in DMSO. The rotational barrier was calculated by stepwise rotation around C2–C7 bond by 20° and geometry optimization of each rotamer at DFT/ωB97XD/6-311g(d,p) level to account for dispersion interactions [33]. All calculations were performed in Gaussian09 [34].

Results and discussion

The antioxidant activity of TCHs and the water-induced structural changes

It is known that thione antioxidants, such as ergothioneine [35] and thiosemicarbazones [36, 37], show good free-radical scavenging activities. In this study, antioxidant activity of four TCHs (Fig. 1) was analyzed using a DPPH method [26].

Mono- and bis-TCHs with 2-hydroxyphenyl group on azomethine carbon (comps. 1 and 2) were better DPPH· scavengers than compounds bearing 2-acetylpyridine moiety (3 and 4) (Table 1). Generally, the presence of hydroxyl groups increases the antioxidant activity of TCHs [7].

Table 1 Antioxidant activities of mono- and bis-TCHs, determined in the DPPH assay and expressed as IC50 values (in mM)
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Interestingly, it was observed that the addition of 20% water in DMSO solution of DPPH· increase the antioxidant activity of the investigated compounds (Table 1). To explain this phenomenon, we performed structural investigation of studied molecules to get the fundamental information necessary for establishing the structure-property relationships. Two possible structural changes are hypothesized: E/Z isomerism around imine bond and thione/thiol tautomerism.

E/Z isomers around imine bond(s) can contribute to diverse chelating properties as well as pharmacodynamics profiles [38]. Thione-thiol tautomerism is also important for the biological activity of compounds as different tautomers have different ADMET properties as well as different hydrogen bonding preferences in the interaction with the biological targets [39]. To further characterize the solution structure of studied compounds, E/Z isomerization, as well as thione-thiol tautomerism were examined in DMSO/water mixtures and discussed with the aid of quantum chemical calculations.

A variety of spectroscopic methods could be used for analysis of the state of tautomeric equilibria, i.e., study of the mechanism of tautomer/isomer transformation. Absorption spectra of isomers/tautomers are generally different and could be useful for detection/quantification of solvent induced isomerization/tautomerization [40]. For structurally related aroylhydrazones, it has been reported that one type of isomer prevails in non-aqueous media, while water induces isomerization into another configurational isomer [41]. It was shown that E/Z photoisomerization of pyridine-substituted semicarbazide Schiff bases is followed by a red shift of the absorption maximum [42, 43]. Also, the addition of water could support the tautomeric conversion of the existing geometric isomers, as reported for structurally similar thionamide/thiolimine isomerization [44].

In order to analyze water-induced structural changes, the electronic absorption spectra of four TCHs (Fig. 1) were recorded in DMSO and DMSO/water mixture. The obtained spectra (Fig. 2) show the changes in the position and intensity of absorption peaks, i.e., solvent induced E/Z isomerization or thione-thiol tautomeric equilibria shift, with the addition of water in DMSO solution of TCHs. Water-induced formation of thiolimine tautomer is usually accompanied by the appearance of a new band at ~ 400 nm [44], while E/Z-photoisomerization also causes the red-shift of the absorption maximum [42, 43]. Therefore, based only on UV–Vis spectra of the investigated compounds, it is very difficult to elucidate which of these two phenomena occurs.

1H NMR experiments were useful for resolving this uncertainty. The spectra of 1 and 2 in DMSO-d6/H2O mixture didn’t show a characteristic downfielded signal for iminothiolic SH group at ~ 15 ppm [10], excluding the possibility for thione/thiol (thioamide-iminothiol) tautomerism (ESM, Figs. S3 and S4). Based on these experiments, it was concluded that 1 and 2 undergo E/Z isomerization upon addition of water in the DMSO solution.

The possible mechanism on water induced E-to-Z isomerization is hypothesized. The addition of water in DMSO solution might destabilize the ground state of 1 by disturbing the intramolecular hydrogen bond, thereby reducing the energy barrier for E-to-Z isomerization (ESM, Fig. S1). The 1H NMR spectroscopy supports this hypothesis, as the peak at 11.73 ppm, which is assigned to Z-isomer, increases while simultaneously the peak of E-isomer at 11.37 ppm decreases (ESM, Fig. S3). After the addition of water, the E/Z ratio is changed from 81:19 to 21:79. Literature describes that 1H NMR chemical shifts of N2–H proton of structurally similar thiosemicarbazones move downfield by 0.22 ppm upon UV irradiation, as a consequence of E/Z photoisomerization [42]. The preference of 1 toward E-configuration was assessed by 2D NOESY spectrum, showing through-space correlations between H-atoms at C7/C3 and OH/N3-H (ESM, Fig. S14).

Although being symmetrical, 2 shows two distinct 1H NMR signals for both hydrazide and imine nitrogen protons (ESM, Fig. S4a). The presence of two distinct signals for NH protons was already reported for bis(hydrazones) [25]. The addition of water into DMSO solution of 2 induces smaller changes in UV–Vis spectrum (Fig. 2b). Two 1H NMR signals originating from N2–H and N2’–H at 12.10 and 11.92 ppm (ESM, Fig. S4a) merged upon the addition of H2O, and the intensity of new peak corresponds to two protons (ESM, Fig. S4b).

In order to explain this effect, we predicted the 1H NMR shifts for (E,E)-, (E,Z)- and (Z,Z)-forms of 2 using GIAO method. The difference in chemical shifts of N2–H and N2’–H (Δδ) (Fig. 1) decreases from 0.97 to 0.17 ppm upon (E,E)- to (E,Z)-isomerization (ESM, Table S1), while complete transition to (Z,Z)-isomer yields a symmetrical structure with negligible Δδ value of 0.01 ppm. Therefore, the observed overlapping of two N–H peaks in 1H NMR spectrum (ESM, Figs. S4a and S4b) might originate from E-to-Z isomerization.

According to 1H NMR spectra, the compound 2 is present in a solution as 2-(E,Z) form in DMSO, and upon addition of water the equilibrium shifts to 2-(Z,Z) form. The small percentage of water, present in a hygroscopic DMSO-d6, might be enough to initialize the 2-(E,E) to 2-(E,Z) isomerization to some extent. 13C-NMR spectrum of compound 2 showed two signals of olefinic carbons, suggesting the existence of two diastereomeric carbazone moieties (ESM, Fig. S8) and further confirming the presence of 2-(E,Z) isomer in a solution. In addition, correlation between H-atoms at N2 and N2’ in the NOESY spectrum of 2 (ESM, Fig. S17), which may be present only in the case of the E/Z and the Z/Z isomers (ESM, Table S1), additionally confirms this claim.

On the contrary, compound 4 showed symmetric structure and no diastereomeric moieties were recognized in its 13C-NMR spectrum (ESM, Figs. S10 and S22). 1H NMR data suggest a symmetric structure in which the C=S bond is located on the symmetry line of the molecule (ESM, Fig. S6). It could be supposed that the methyl groups limit the flexibility of a molecule by increasing the energy barrier for isomerization, so it preferentially adopts a symmetric conformation [25].

Although 1 and 2 exhibits E/Z isomerization, compounds 3 and 4 undergo thione/thiol tautomerism upon the addition of water. This is confirmed by the appearance of highly downfield signal characteristic for –SH proton in 1H NMR spectrum (14.14 and 15.41 ppm, ESM, Figs. S5a and S6a) [10, 45]. The E- and (E/E)-configuration of hydrazone moiety in compounds 3 and 4 can be evidenced by NOESY cross-correlations of H-atoms from -CH3 and N2-H (ESM, Figs. S21 and S24). Furthermore, the absence of through-space correlation between H-atoms at -CH3 and C3-H which is expected for Z-isomer (ESM, Fig. S2; 3-b-Z form) further confirms the claim.

The E-to-Z isomerization around C=N double bond can proceed via two mechanisms: torsional route around C=N bond and in-plane inversion, where the transition state has a linear geometry with N atom being sp hybridized [46]. As torsional pathway cannot be omitted from the considerations on possible isomerization mechanisms [47], it is likely that the torsional route is a preferential mechanism for E/Z isomerization of TCHs, similar to azobenzene [46]. In sterically congested systems, E/Z isomerization via torsional route is hindered. This might explain the observed E/Z isomerization in 1 and 2, and the lack of isomerization in sterically more demanding 3 and 4.

After the addition of water to DMSO solution of 3 and 4, the proportion of thiol form increases. The intensity of corresponding 1H NMR signals increased from 0.03 to 0.18 for 3 (ESM, Fig. S5), and from 0.17 to 0.35 in case of 4 (ESM, Fig. S6), which means that the ratio of thione/thiol forms is changed from 97:3 to 85:15 for compound 3, and from 85:15 to 66:35 for compound 4.

Additionally, a new absorption maximum appears in the UV–Vis spectra at 400 nm for 3 (Fig. 2c) and 425 nm for 4 (Fig. 2d). In the simulated UV–Vis spectra of 4, the absorption maximum of thiol form is red-shifted by ~ 20 nm compared to thione, and additional peak appears at ~ 270 nm (ESM, Fig. S25).

The water-induced thione/thiol tautomerization is more pronounced in 4 (NMR signal ratio of E-thione:thiol is 1:0.35) compared to 3 (E-thione:thiol 1:0.18). This result enforces the significance of hydrogen bonding established by intermolecular solvent/solute interactions, which prevail over intramolecular H-bond (IHB) in a cyclic structure of 3-Z form (ESM, Fig. S2; 3-b-Z form). Although the strong IHB observed in Z-isomers of bis(2-pyridil)hydrazones [15] yields a highly downfield 1H NMR signal at ~ 15 ppm, the signal observed in the NMR spectra of 3 and 4 does not originate from intramolecularly bonded N2–H but from thiolic –SH atom.

To evaluate the relative strength of IHB in compound 3 (2-acetylpyridyl TCH) and sterically less hindered 2-formylpyridyl derivative, the rotational barriers for the rotation of pyridine ring around thiocarbohydrazide moiety are calculated at DFT/ωB97XD/6-311g(d,p) level. To quantify the sterical contribution to the overall rotational barrier of 3, a hypothetical derivative with pyridine N atom at position 4 is also considered.

The relative energies of comp. 3 and two structural analogs, i.e., mono-TCHs based on 4-acetylpyridine (4-ap; (Z)-[1-(4-pyridinyl)ethylidene]carbonothioic dihydrazide) and 2-formylpyridine (2-fp; ((Z)-(2-pyridinylmethylene)carbonothioic dihydrazide), were comparatively analyzed with respect to stepwise change of rotation around C2–C7, i.e., torsional angle change. Obtained results are presented in Fig. 3.

Fig. 3
figure 3

Graphical presentation of relative energy of comp. 3 and two structural analogs versus stepwise rotation around C2–C7 bond, in respect to the most stable rotamer

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The higher stability of planar structure was obtained for 2-fp and 2-ap TCH, where IHB was formed. Oppositely, steric repulsion in TCH based on 4-ap means higher energy and rotation for 60° bring to structure with minimum energy. Also, obtained results indicate weaker IHB in comp. 3 than in TCH based on 2-fp, with local maxima at 90° rotation corresponding to energy necessary for hydrogen bond breakage. Somewhat higher energy is necessary for hydrogen bond breakage in TCH based on 2-fp due to stronger IHB, where larger steric interference in comp. 3 cause decrease of relative energy. Overall relative energy for both TCHs based on 2-fp and 2-ap are similar at 180° rotation as steric component is more pronounced for 2-ap compared to 2-fp derivatives. In general, weaker IHB was obtained for comps. 3 than for sterically less hindered TCH based on 2-fp. This claim is in line with hydrogen bond formation of (Z)-(2-pyridinyl)-based carbonic dihydrazide, where the presence of the methyl group on azomethine carbon, instead of hydrogen atom, reduces the IHB ability [14].

The effect of configurational isomerism and thione/thiol tautomerism on the increase of antioxidant activity of TCHs

Aiming to explain how water-induced structural changes improve the antioxidant activity of TCHs, we performed detailed electronic structure and bond dissociation enthalpy (BDE) calculations. The radical scavenging ability of a molecule (M–H) is associated with the ability to transfer H atom to any free radical (for example DPPH·), as shown by the reaction:

$$ \mathrm{M}\hbox{--} \mathrm{H}+{\mathrm{DPPH}}^{\cdotp}\to {\mathrm{M}}^{\cdotp }+{\mathrm{DPPH}}_2 $$

Among many potential mechanisms of this process (HAT, hydrogen atom transfer; SET, single-electron transfer; SPLET, sequential proton loss electron transfer), the HAT is the simplest and most widely studied one. The HAT potency of a compound is determined by the BDE of molecule, where H atom of the most labile X–H bond (where X can be any heteroatom) will be abstracted first. The BDE value can be calculated by the equation:

$$ \mathrm{BDE}\ \left(\mathrm{M}\hbox{--} \mathrm{H}\right)={\Delta}_f\mathrm{H}\left({\mathrm{M}}^{\cdotp}\right)+{\Delta}_f\mathrm{H}\left(\mathrm{H}\right)\hbox{--} {\Delta}_f\mathrm{H}\left(\mathrm{M}\mathrm{H}\right), $$
(2)

where ΔfH(M·) is the enthalpy of formation (EOF) of the radical formed by hydrogen atom abstraction, ΔfH(H) is the EOF of hydrogen atom, and ΔfH(MH) is the EOF of a molecule. The stabilization of radical species by the resonance, conjugation, and intramolecular hydrogen bonding leads to the lower values for BDE, and is usually followed by the increased in vitro antioxidant activity [48].

The relative stability of E- and Z-isomers of both thione and thiol tautomers of compounds 1 and 3 was studied by quantum chemical calculations at DFT/B3LYP/6-311++G(d,p) level using IEF-PCM model of water. The results obtained (ESM, Figs. S27 and S28) may serve only for qualitative purpose as PCM model do not account for explicit hydrogen bonding between solvent and solute. Such explicit interactions may play a significant role in lowering/increasing the energy barrier for isomerization or tautomerization.

The most stable isomer of 1 appeared to be the 1-E-thione form (ESM, Fig. S27). E-to-Z isomerism breaks the pseudo 6-membered ring formed by intramolecular hydrogen bonding between the –OH group and N1 atom. The E-thione form was also the most stable form for compound 3, although the intramolecular H-bond between the N2–H and NPy apparently decreases the energy difference between two geometric isomers (ESM, Fig. S28).

As the addition of water induces E/Z isomerization of 1 and almost halve the IC50 values toward DPPH· (Table 1), we performed BDE calculations to see if the appearance of Z-isomer facilitates the HAT. Both 1-E and 1-Z isomers were modeled in thione tautomeric form. As can be seen from Table 2, Z-isomers have 3-5 kcal/mol lower BDEs of –N–H atoms attached to C=S (–N3–H and –N2–H), while isomerization does not influence the BDE of terminal –NH2 group (–N4–H). The most striking difference is observed for –OH group, where E/Z isomerization decreased the BDE by 11 kcal/mol. Upon isomerization to Z-isomer, the phenolic –OH group is released from intramolecular hydrogen-bonded 6-membered ring. This effect makes the –OH group more prone to interaction with DPPH·, while ground state destabilization decreases the BDE (Eq. (2)) and facilitates HAT.

Table 2 The zero-point corrected energy and enthalpy of 1–4 in different configurational/tautomeric forms. The bond dissociation enthalpies for corresponding X–H bonds are shown. The percentage of spin delocalization is reported, calculated as 1–spin located at heteroatom which remained after geometry optimization of a radical
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The isomerization of 2 from E,E to Z-Z decreases the BDE of O and N3 radicals by ~ 7 and 5 kcal/mol, respectively (Table 2).

Spin density distribution map helps visualizing the extent of spin delocalization, which influences the overall stability of a radical formed by HAT. The spin density distribution of O (Fig. S29) and N-radicals (Fig. 4) of 1-Z and 2-(Z,Z) forms is more delocalized compared to E-isomers. The additional spin delocalization corroborates with the increased radical scavenging potency upon isomerization.

Fig. 4
figure 4

Spin density distribution for radicals formed by hydrogen atom abstraction from the lowest energy N–H bond in 1 and 2

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In order to study the influence of thione/thiol tautomerism on antioxidant activity of 3, the BDEs of several bonds were calculated for thione and thiol tautomers, starting from E-geometry. Thione/thiol tautomerism decreases the lowest BDE by 7 kcal/mol (Table 2), supporting the trends in experimentally obtained antioxidant activities.

Although one may expect –SH group as the most probable site for interaction with DPPH·, we found that –N3-H bond of 3 is more labile, with ~ 10 kcal/mol lower BDE (Table 2). To see if such a trend is maintained for bis-derivative, we performed BDE calculations for compound 4. The BDE of –N2–H bond of (E,E)-thione form was 87.06 kcal/mol. Isomerization to (E,E)- thiol form decreased the BDE of –N2–H to 80.32 kcal/mol, while –SH had a similar BDE value (81.77 kcal/mol). The presence of two almost equally reactive sites should lead to the increase of the antioxidant potency of 4, and this effect explains the 5-fold drop in the IC50 values upon the addition of water (from 0.67 to 0.13 mM, Table 1).

As can be seen from Fig. 5, thiol form of 3 and 4 has more delocalized spin which stabilizes the radical species, decreases the corresponding BDE, and finally increases the antioxidant activity.

Fig. 5
figure 5

Spin density distribution of radicals formed by HAT from N3–H bond of 3-E and N2–H bonds of 4-(E,E) isomers, for both thione and thiol tautomers

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Conclusion

In conclusion, the presence of hydroxyl groups increases the antioxidant activity of TCHs as 1 and 2 were more potent radical scavengers compared to 2-acetylpyridine analogs 3 and 4. The addition of water affects the intramolecular hydrogen bond strength in 1-E form facilitating E-to-Z isomerization, and thus increases the antioxidant activity due to formation of “free” –OH group in 1-Z isomer with 11 kcal/mol lower BDE. Electron spin delocalization is more pronounced in Z-isomers of 1 and 2.

The addition of water into DMSO solution of 3 and 4 causes thione-to-thiol tautomerization, as seen from the appearance of red-shifted UV–Vis absorption maximum and a deshielded 1H NMR signal of –SH proton. The presence of thiol form in a solution is correlated with the increase in antioxidant activity. Two low-energy bonds of 4 (–N–H and –S–H) along with the delocalized spin density of thiol radical rationalize the observed increase in the radical scavenging activity.

The results of this study provide new insights on the solution structure of TCHs as biologically active compounds, and also highlight the importance of detailed structural characterization in studying structure-antioxidant relationships of TCHs or other isomerizable compounds.