Introduction

The biological activity of the compounds is a function of their physical and chemical parameters which depend on their molecular structure [1]. Thus, it is important to accurately determine the structure of a chemical compound as it will reflect in its molecular properties [2, 3]. An elongation of the alkyl chain may result in a better fit to the active site, thereby increasing the potency of the drug. It could also result in an increase of lipophilicity, which is related to the ease of drug penetration into a molecular target. For example, thioconazole, which is non-polar and poorly soluble in blood, is only used in antifungal skin infections. However, after the introduction of a hydroxyl group and more polar heterocyclic rings, fluconazole was obtained with better solubility and efficiency in systemic fungal infections [4]. On the other hand, the molecular structure, apart from pharmacokinetic properties, also influences pharmacodynamics. For example, the key to local anesthesia is the benzoyl group in the cocaine structure [5]. Thus, it is so important to investigate and precisely determine the structure of the molecule before any biological testing.

One of the important parameters for biological activity is the phenomenon of tautomerism. Antonov calls tautomers chemical chameleons because of their ability to change structure quickly depending on conditions [6]. Prototropic tautomerism is a proton displacement between two polar atoms of a given compound [7]. Related to the low-energy barrier between the tautomers, many factors affect the tautomerization process [8]. It is determined by the ratio of the tautomers, which depends on the molecular structure and the type of chemical compound, solvent, temperature, pressure, concentration, and pH [7]. The ratio of tautomers determines the most preferred tautomeric form in a particular environment. Tautomers differ in their molecule shapes, proton, and acceptor–donor properties, and therefore, depending on the tautomeric form, they can be involved in different molecular interactions with the molecular targets. In the light of above, determination of the most stable tautomer plays a key role in drug design and discovery.

One of the most common of tautomerism is the keto-enol tautomerism, occurring in the compounds which possess a carbonyl group. In nature, the most common example of keto-enol tautomerism is nitrogen bases, which form hydrogen bonds only by shifting the tautomeric equilibrium into the keto form [9]. Tautomeric equilibrium studies are of great importance because of the key role of tautomerism in the optimization of the biological activity of medicinal compounds [9,10,11,12,13,14,15,16]. The tautomeric form may determine the stability of the ligand, which was, e.g., shown by Senthilkumar and Kolandaivel, who demonstrated greater stability in the polar environment of the bound tautomer with respect to the corresponding unsubstituted barbituric acid tautomer [17]. Temperini et al. proved that tautomeric forms determined the strength of interaction with the active site of an enzyme [18]. They showed that the complex of carbonic anhydrase II with chlorthalidone is bound in the compound lactimic form, instead of the amide form. Many studies focused on keto-enol tautomerism in order to rationalize the biological activity of the tested compounds, assuming that the keto and enol forms may differ in pharmacological activity [4].

Another common type of tautomerism is thione-thiol tautomerism, which occurs in the compounds bearing a thione group (Scheme 1). Jayaram et al. reported that antithyroid drugs are mainly in the form of the thione tautomer [19]. They demonstrated that the tautomeric thione form was related to the presence of the NH group in the structures they studied, which is crucial for the inhibitory effect on lactoperoxidase.

Scheme 1
scheme 1

Exemplary tautomeric forms of thiosemicarbazides

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Thiosemicarbazides are a privileged scaffold in medicinal chemistry [20]. Their easy synthesis and broad spectrum of biological activity make them a promising group of therapeutic compounds [21,22,23,24]. From the point of view of biological activity, it is very important to study the tautomeric stability of thiosemicarbazides [25,26,27,28]. Knowledge of the preferred tautomeric form enables the correct interpretation of the structure–activity relationship (SAR) and molecular docking studies to determine ligand–protein interactions. In order to determine the most stable tautomeric structure, quantum-chemical calculations are often used [29, 30] and the most frequently used method for determining tautomeric stability is DFT [6, 8, 31,32,33,34]. Therefore, the use of computational chemistry and structural bioinformatics techniques is of key importance in the contemporary process of drug design and discovery [8, 35]. On the other hand, X-ray crystallography and NMR are also used to determine the structure of chemical compounds [8, 36,37,38,39,40] and to confirm the results of quantum-chemical calculations [16, 35, 41, 42]. Therefore, in our research, we perform a comparison of computational and experimental data.

The aim of our work is to study the phenomenon of tautomerism of pyridine carbonyl thiosemicarbazide derivatives using quantum-chemical calculations in the gas phase and in the solvent environment as IR and NMR spectroscopy and X-ray studies for selected compounds.

Experimental

IR spectra were recorded using a Thermo Nicolet 6700 FTIR spectrometer, with the ATR Diamond Orbit stage. 1H and 13C NMR spectra using DMSO-d6 as a solvent were recorded using the Bruker AVANCE III 600-MHz, Z-gradient BBO probe spectrometer. The solvent was used as received from a commercial supplier. Tetramethylsilane was applied as an internal standard. B3LYP DFT (a variant of the DFT method using Becke’s three-parameter hybrid functional (B3) [43], with a correlation functional such as the one proposed by Lee, Yang, and Parr (LYP) [44], using 6–311 ++ G(3df, 3pd) basis set as included in Gaussian09 [45], was used to optimize the 9 tautomeric structures of compounds 1–9 in the ground state and in DMSO using the Polarizable Continuum Model (PCM) [46, 47]. This approach relies on the overlapping of spheres to form a cavity of the solute. The stabilization energy of the tautomers 01–09 was also calculated for the isolated molecules (gas phase) and molecules in DMSO solutions for all the compounds. When calculating the stabilization energy of the tautomers, the reference was as the least stable tautomer for each compound. The Continuous Set of Gauge Transformations (CSGT) approach [48,49,50] was used to compute 1H and 13C NMR chemical shifts in DMSO at the same theory level. Next, vibrational frequencies and infrared intensities were also computed. The IR spectra were rescaled by 0.9608 in accordance with the recommendations for this level of theory [51]. B3LYP DFT method and the 6–311 ++ G(3df, 3pd) basis set of Gaussian09 [17] software were used to calculate the energy of HOMO and LUMO orbitals. GaussView v. 6.0 was applied to visualize HOMO and LUMO orbital shapes. Electrostatic potential distribution was computed and visualized with ArgusLab v. 4.0.1 [52]. Non-covalent interaction maps were calculated with NCIPlot v. 3.0 [53] and visualized with VMD v. 1.9.4 [54], as reported earlier [55].

X-ray data of 9 were collected on the KUMA Diffraction KM-4 CCD diffractometer (MoKα (λ = 0.71073 Å) radiation, ω scans, T = 296(2) K; crystal sizes 0.50 × 0.30 × 0.05 mm, absorption correction: multi-scan CrysAlisPro [56], Tmin/Tmax of 0.7711/1.0000). The structure was solved by direct methods using SHELXS97 [56] and refined by full-matrix least squares with SHELXL-2014/7 [57]. The N-bound H atoms were located by difference Fourier synthesis and refined freely. The remaining H atoms were positioned geometrically and treated as riding on their parent C atoms with C-H distances of 0.93 Å (aromatic). All H atoms were refined with isotropic displacement parameters taken as 1.5 times those of the respective parent atoms. Electron density associated with an additional disordered solvent molecule was removed with the SQUEEZE procedure in PLATON [58] (the solvent-accessible volume of 316 Å3 with 76 electrons in the cavities). All calculations were performed using the WINGX version 1.64.05 package [59]. CCDC-2189746 for 9 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: + 44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk).

Crystal data of C1: C13H10N4OS, M = 341.21, monoclinic, space group P21/c, a = 12.7027(9), b = 9.9754(6), c = 13.5119(7) Å, β = 90.851(6), V = 1711.96(18) Å3, Z = 4, dcalc = 1.324 Mg m−3, F(000) = 696, μ(Mo Kα) = 0.503 mm−1, T = 296 K, 7580 measured reflections (θ range 2.54–29.07o), 3861 unique reflections, final R = 0.048, wR = 0.121, S = 1.027 for 2773 reflections with I > 2σ(I) (Supplement 1).

Results and discussion

Thiosemicarbazides are a very important group of compounds used in organic synthesis to obtain biologically active systems, e.g., triazoles, thiadiazoles, and oxadiazoles [60, 61]. For several years, researchers have focused on thiosemicarbazide derivatives as privileged structures for biological activity. Their antiviral and anthelmintic properties are widely described in the literature [62, 63]. Many compounds from this group exhibit antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli comparable to standard antibacterial drugs [64]. Thiosemicarbazide derivatives containing a thiazole ring were screened for inhibitory activity against Mycobacterium tuberculosis H37Ra and Mycobacterium bovis strains [65].

Our group obtained a series of 1- (2-, 3-, 4-pyridinecarbonyl)-4-substituted thiosemicarbazide derivatives and evaluated their antimicrobial and antitumor activity (Scheme 2) [21, 22].

Scheme 2
scheme 2

Synthesis of 1-pyridinecarbonyl-4-substituted thiosemicarbazide derivatives (1–9)

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The title compounds were synthesized in the reaction of 2- or 3- or 4-pyridinecarboxylic acid hydrazide reaction with 4-methylphenyl or 4-nitrophenyl or 2,4-dichlorophenyl isothiocyanate according to the previously described procedure [21, 22]. Biological studies have shown that some of these compounds (1, 2, 4–6) display antibacterial activity against Staphylococcus epidermidis, Streptococcus mutans, and Streptococcus sanguinis with MIC values in the range of 7.81–62.5 mg/mL and show a therapeutic index higher than that of ethacridine lactate. Furthermore, compound (3) potently inhibits the proliferation of HepG2 (human hepatocellular carcinoma) and MCF-7 (human breast adenocarcinoma) cells with an IC50 = 2.09 ± 0.11 μM and 8.63 ± 1.75 μM, respectively, in a concentration-dependent manner [21] and (2) inhibits A549 (lung adenocarcinoma cell line) with an IC50 = 4.96 ± 1.96 μg/mL [22].

Due to the key importance of the influence of physicochemical parameters on biological activity, we decided to investigate the phenomenon of tautomerism for the obtained thiosemicarbazide derivatives. The phenomenon of tautomerism, especially of the proton transfer, plays an important role in modern organic chemistry, biochemistry, drug chemistry, pharmacology, and molecular biology.

Related to the possibility of migration of labile protons from NH groups to carbonyl (C = O) or thione (C = S) groups, the thiosemicarbazide derivatives studied by us may exist in nine tautomeric forms (Fig. 1). We showed in our earlier work that in the solid state, thiosemicarbazides exist in the keto-thione form [22]. Here, we present experimental and computational studies of the tautomerism phenomenon for new biologically active compounds 1–9.

Fig. 1
figure 1

Possible tautomeric forms of the 1,4-disubstituted thiosemicarbazide derivatives

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B3LYP DFT approach and 6–311 ++ G(3df, 3pd) basis set as included in Gaussian09 were used to optimize energy and geometry of compounds 1–9 in the ground state (in vacuum) and in DMSO. The stabilization energy values were calculated as the difference between the value of the most stable tautomer and the corresponding one. The population analysis of tautomeric forms was estimated using a non-degenerate Boltzmann distribution. The results of the calculations are presented in Table 1.

Table 1 Stabilization energy E [kcal/mol] and population pi [%] of tautomers (T) 01–09 of compounds 1–9 in vacuum and DMSO
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The obtained values of energy stabilization indicated that tautomer 01 is the most stable in both the gas phase and DMSO for all the studied compounds, which shows that the presence of ketone and thione groups in the carbonyl thiosemicarbazide system stabilized molecules 1–9 in both considered environments. In the case of compounds 4–6 and 5–9 with pyridyn-3-yl and pyridin-4-yl substituents, respectively, two tautomeric forms 01 and 02 can coexist both in the gas phase and in the solution, wherein the population of them is according to the relation 01 >> 02, with the highest participation of tautomeric form 02 observed for 5 of 1.30% and 8 of 1.28% in the gas phase. In other analyzed cases, the population of the tautomeric forms 02–09 in considered environments is below the threshold of the detectability of conventional analytical methods with the highest stabilization energy for the tautomeric form 09, ranging from 42.80 kcal/mol for 2 to 54.80 kcal/mol for 6 in the gas phase and from 48.75 kcal/mol for 2 to 57.74 kcal/mol for 8 in DMSO.

Molecular orbitals and their properties, i.e., energy, are useful for explaining the electronic properties of the compounds. Frontier electron density is often applied for predicting the most reactive position in π-electron systems and to explain a number of reactions in conjugated systems [66].

HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) energy is often used to determine the chemical reactivity of molecules. During molecular interactions, the LUMO accepts electrons while the HOMO represents electron donors [67]. Energies of HOMO and LUMO as well as LUMO–HOMO gap for the most stable tautomer 01 of compounds 1–9 are presented in Table 2. HOMO and LUMO orbitals for tautomer 01 of the most active compounds 2 and 3 are shown in Fig. 2. It can be seen that for 2, both HOMO and LUMO orbitals are present on the thiosemicarbazide and phenyl part of the molecule, making it most reactive, in contrast to the pyridyl moiety which is not reactive. The reason for this is the large accumulation of electronegative atoms on one side of the molecule. However, for 3, the LUMO orbital is stretched into the entire molecule and the distribution of the HOMO orbital is the same as previously described for 2.

Table 2 Energies of HOMO and LUMO orbitals for the most stable tautomer of compounds 1–9
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Fig. 2
figure 2

HOMO (A, C) and LUMO (B, D) orbitals for the most stable tautomer of compounds 2 (A, B) and 3 (C, D)

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Investigation of protein and ligand electrostatic potential aimed at optimizing electrostatic complementarity is of great importance in drug design [68]. The distribution of electrostatic potential maps for the most stable tautomer 01 of compounds 2 and 3 is presented in Fig. 3. It can be seen that pyridyl nitrogen, the nearby carbonyl oxygen, and nitro group oxygen atoms are the most electronegative part of compound 2, similarly as for compound 3, which, however, does not possess a nitro group.

Fig. 3
figure 3

Molecular structures of the most stable tautomer of compounds 2 (A) and 3 (B). Electrostatic potential surface for the most stable tautomer of compounds 2 (C) and 3 (D)

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Non-covalent interactions are crucial for the explanation of a number of chemical, biological, and technological problems [53]. A thorough description of these interactions, in particular their positions in real space, is the starting point for decoupling the complex balance of forces that define the interactions [53]. Non-covalent interaction maps for the most stable tautomer 01 of compounds 2 and 3 are presented in Fig. 4. A number of week attracting interactions (green) have been identified for the compounds studied. Furthermore, we studied experimental and computed NMR spectra of the most stable tautomer 01 of compounds 1–9. 1H NMR spectra for all the derivatives show peaks of N7-H, N9-H, and N10-H at 9.58–10.18 ppm, 9.73–0.27 ppm, and 10.74–11.00 ppm, respectively, which is supported by the literature [55, 69, 70]. The signals in the range of 7.22–9.12 ppm confirmed the presence of two phenyl rings. These signals differed depending on the position of the heteroatom on the phenyl ring and on the type of the second substituent [55, 71].

Fig. 4
figure 4

Non-covalent interaction maps for the most stable tautomer of compounds 2 (A) and 3 (B). Green spots depict attractive interactions while red spots depict repulsive interactions

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13C NMR spectra confirmed the presence of carbonyl group (C = O) signal in the range of 163–165 ppm and thione group (C = S) signal in the range of 181–189 ppm. This correlates with the chemical shifts obtained for these atoms by several authors [21, 22, 53, 72,73,74]. Related to the presence of many highly electronegative atoms, the chemical shifts differ slightly. The most stable tautomer has a carbonyl and a thione group, which is confirmed by these studies. Experimental and computed 1H and 13C NMR spectra are presented in Table 3 and they correspond well with each other.

Table 3 Experimental and computed 1H and 13C NMR chemical shifts for the most stable tautomer 01 of compounds 1–9 View full size image
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We also conducted a detailed analysis of experimental and computed IR spectra of the most stable tautomer 01 for compounds 1–9 (Table 4), as they can provide valuable structural information about the compounds. The presence of a band in the absorption range 3328–2937 cm−1 indicates the presence of NH groups. Analysis of the IR spectra confirms the presence of strong absorption band characteristic of the group C = O in the range of 1701–1653 cm−1. Another structural feature of the tested derivatives of thiosemicarbazide is a thione group, which occurs in the spectra in the range of 1307–1296 cm−1 [69, 74]. Moreover, the IR spectra of compounds 1–9 did not reveal a C-SH stretching band in the ~ 2600 cm−1 range [69], which confirms our assumptions regarding the most likely tautomeric state. The experimental and computed scaled IR frequencies are in good accordance and confirm the energetical preference of tautomer 01 in case of all compounds studied.

Table 4 Experimental and computed (raw and scaled) IR frequencies for the most stable tautomer 01 of compounds 1–9
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In order to confirm the synthesis pathway, the assumed molecular structures, and identification of the tautomeric form in the crystalline state for the compounds obtained, X-ray studies were performed and structures were described for compounds 3, 4, and 6 [73]. Here, we presented the crystal and molecular structure of the next, compound 9, in the investigated series of carbonyl thiosemicarbazides. The molecular structure of 9 in the conformation observed in the crystal is shown in Fig. 5.

Fig. 5
figure 5

The molecular structure of 9 with atom labeling and displacement ellipsoids (30% probability level)

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The molecule occurs in N1-amino/S3-thione/N4-amino/N5-amino/O7-keto, 01 (Fig. 1), tautomeric form, which is confirmed by the C3–S3 and C5–O5 bond lengths of 1.683(2) and 1.218(3) Å, respectively, typical for the thione and carbonyl groups [75], and the positions of the amino H-atoms at the difference electron-density map in the immediate vicinity of the N1, N4, and N5 atoms. The torsion angles C21–N1–C2–N4, N1–C2–N4–N5, C2–N4–N5–C6, and N4–N5–C6–C31 of − 178.6(2), 5.4(3), 92.7(3), and 176.70(19)o, respectively, show that the carbonyl thiosemicarbazide chain adopts a trans–cis-gauche-trans conformation. The 2,4-dichlorophenyl and pyridyl substituents with respect to the carbonyl thiosemicarbazide system have the gauche and cis conformations, respectively, as shown by the torsion angles C22–C21–N1–C2 of 110.6(3)o and N5–C6–C31–C32 of − 13.8(3)o. The thione C2–S3 group adopts a trans conformation with respect to N4–N5 with the torsion angle N5–N4–C2–S3 of − 176.11(17)o, while the carbonyl C6–O7 group has the cis position with respect to this bond and is practically coplanar with the pyridine ring, as evidenced by the torsion angles N4–N5–C6–O7 and C32–C31–C6–O7 of − 4.9(3) and 167.8(2)o, respectively. In the crystal structures of 9, the molecular packing is influenced by the net of strong intermolecular hydrogen bonds N1–H1…N34i [N1–H1 = 0.79(3), H1…N34 = 2.22(3), N1…N34 = 2.993(3) Å, N1–H1…N34 = 167(3)o, (i) = 1-x, 1-y, -z], N4–H4…S3ii [N4–H4 = 0.82(3), H4…S3 = 2.52 (3), N4…S3 = 3.312(2) Å, N4–H4…S3 = 166(3)o, (ii) = 1-x, 1-y, 1-z] and N5–H5…O7iii [N5–H5 = 0.76(3), H5…O7 = 2.21(3), N5…O7 = 2.893(3) Å, N5–H5…O7 = 151(3)o, (iii) = 1-x, ½ + y, ½-z], which stabilized the tautomeric form observed in crystal. Moreover, the π…π interaction between pyridine rings within the molecular dimer formed by inversion-related molecules is observed; the centroid-to-centroid separation and the angle between the overlapping planes of these rings are 3.4947(14) Å and 0.03(12)o, respectively. It is worth noting that the intermolecular interactions observed in the crystal are in good agreement with the predicted non-covalent interactions shown in Fig. 4. In our previous research on thiosemicarbazide derivatives, we presented the crystal and molecular structures of several compounds containing this system, e.g., 4-cyclohexyl-1-(4-nitrophenyl)carbonyl thiosemicarbazide [21], 4-(2,4-dichlorophenyl)-1-(pyridin-2-yl)carbonyl thiosemicarbazide (3), 4-(4-methylthiophenyl)-1-(pyridin-3-yl)carbonyl thiosemicarbazide (4), 4-(2,4-dichlorophenyl)-1-(pyridin-3-yl)carbonyl thiosemicarbazide (6), 4-(2-fluorophenyl)-1-(pyridin-4-yl)carbonyl thiosemicarbazide, and 4-(2-chlorophenyl)-1-(pyridin-4-yl)carbonyl-thiosemicarbazide [76]. All these compounds exist in the crystalline state in the tautomeric N1-amino/N3-amino/N4-amino/S2-thione/O5-keto form the same as compound 9.

A search of the Cambridge Structural Database (CSD; version 5.43; November 2021, [77]) for the presence of the carbonyl thiosemicarbazide system in organic molecules (restrictions applied: R ≤ 0.1, only non-disordered, no errors, not polymeric, no ions) revealed 102 crystal structures with this system. In all these structures, the carbonyl thiosemicarbazide system is in the N-amino/S-thione/O-keto 01 tautomeric form (Fig. 1). It should be noted that there are three crystal structures in the CSD with the carbonyl thiosemicarbazide system in the ionic structure: imidazolium N-(naphthalen-1-ylcarbamothioyl)-3,5-dinitrobenzenecarbohydrazonate, hexamethylenetetraminium N-(naphthalen-1-ylcarbamothioyl)-3,5-dinitrobenzenecarbohydra zonate, and triethylammonium N-(naphthalen-1-ylcarbamothioyl)-3,5-dinitrobenzene carbohydrazonate with the Refcodes MATLIE, MATLOK, and MATLUQ, respectively, and only in these structures the carbonyl thiosemicarbazide system appears in a different tautomeric form, namely, N1-amino/S3-thione/N4-amino/N5-imino/O7-hydroxy, 02 tautomeric form (Fig. 1).

In summarizing the results of X-ray studies of the crystal structures of compounds containing the carbonyl thiosemicarbazide system, it can be stated that the organic compounds containing this system occur in the 01 tautomeric form in the crystalline state. Only in the case of ionization of the oxygen atom of the carbonyl thiosemicarbazide system, the tautomeric equilibrium shifts towards the tautomeric form 02.

Conclusion

The experimental and theoretical studies of 1- (2-, 3- and 4-pyridinecarbonyl)-4-substituted thiosemicarbazide derivatives have shown that the most stable tautomeric form contains a carbonyl and thione group. The obtained data broaden the knowledge of the tautomerism of thiosemicarbazide derivatives and can be used for the rational design of new therapeutic compounds.