Conformational Properties of New Thiosemicarbazone and Thiocarbohydrazone Derivatives and Their Possible Targets

Abstract

The structure assignment and conformational analysis of thiosemicarbazone KKI15 and thiocarbohydrazone KKI18 were performed through homonuclear and heteronuclear 2D Nuclear Magnetic Resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-HSQC, and 2D-HMBC) and quantum mechanics (QM) calculations using Functional Density Theory (DFT). After the structure identification of the compounds, various conformations of the two compounds were calculated using DFT. The two molecules showed the most energy-favorable values when their two double bonds adopted the E configuration. These configurations were compatible with the spatial correlations observed in the 2D-NOESY spectrum. In addition, due to the various isomers that occurred, the energy of the transition states from one isomer to another was calculated. Finally, molecular binding experiments were performed to detect potential targets for KKI15 and KKI18 derived from SwissAdme. In silico molecular binding experiments showed favorable binding energy values for all four enzymes studied. The strongest binding energy was observed in the enzyme butyrylcholinesterase. ADMET calculations using the preADMET and pKCSm software showed that the two molecules appear as possible drug leads.

1. Introduction

The framework 1,3-diphenylprop-2-en-1-one (Figure 1A) is well known by the generic term ‘‘chalcone’’, a name coined by Kostanecki and Tambor [1]. Chalcones belong to the flavonoid family, and they contain conjugated double bonds with absolute delocalization and two aromatic rings. They act as synthons by which a range of analogs and novel heterocycles with pharmaceutical structures can be targeted [2]. Chalcones can be used to obtain several heterocyclic rings through ring closure reactions [3]. Various chalcone derivatives show antimicrobial [4], antifungal [5], antimalarial [6], antiviral [7], anti-inflammatory [8], antileishmanial [9] anti-tumor, and anticancer [10] properties.

Thiosemicarbazones (TCSs) (Figure 1B) are an important class of compounds possessing remarkable biological properties making them of interest to structural and medicinal chemists. A wide variety of TSCs containing an appropriate structural framework were found to have antineoplastic [11], antibacterial [12], and antifungal [13] properties. Furthermore, their extraordinary complexing capacity with metal ions such as iron, copper, and zinc provides additional versatility as potential candidates for the preparation of coordinate complexes [14]. Thiosemicarbazones are well known to exhibit both syn and anti isomeric forms [15].

In recent years, there has been growing interest in the coordination chemistry of thiocarbohydrazones, compounds that share the general formula depicted in Figure 1C and that can be considered the higher homologues of thiosemicarbazones. The first synthesis of these systems is dated in 1925 and described the condensation of ketones and aldehydes with thiocarbohydrazide [16,17].

Compounds combining these structural features (KKI15: chalcone-thiosemicarbazone) (KKI18: chalcone-thiocarbohydrazone) have never been studied, and the elucidation of their structure is of utmost interest in the understanding of their biological results. Finally, these compounds have great potential for research activities as they are potential lead drugs for diseases to be discovered.

2. Results and Discussion

2.1. Structure Assignment

As a convenient starting point for the structure assignment of KKI15, the readily assigned H-7, which resonates at 6.79 and 7.20 ppm, was used. These two signals are due to the two conformations of KKI15. Through 2D-COSY, H-6 resonates at 6.46 ppm. Through 2D HSQC, the H-7 and H-6 show ¹J_C-H coupling with the C7 and C6, respectively, and therefore, C7 and C6 are assigned unambiguously at 118.0 and 143.0 ppm, respectively. Through 2D-NOESY, H-9 is identified at 7.83 and 8.31 ppm due to its correlation with H-7. H-10 and H-11 are then identified due to their correlation with H-9, through 2D-COSY. Furthermore, it is observed that H-7 has two more correlations with protons H-10 and H-13. Through 2D-HSQC, H-9, H-10, and H-11 show ¹J_C-H coupling with C-9, C-10, and C-11, respectively, and, therefore, C-9, C-10, and C-11 are assigned unambiguously at 119.10, 129.37, and 129.65 ppm. Through 2D-COSY, H-13, H-14, and H-15 are identified, and through 2D-HSQC, C-13, C-14, and C-15 are therefore assigned unambiguously at 127.39, 128.76, and 131.27 ppm. Moreover, it is observed in one correlation between H-6 and H-14. Protons attached to nitrogen are increasingly deshielded. NH(3) resonates at 11.10 ppm. Through 2D-NOESY, NH₂ resonates at 7.80 ppm. Through a two-dimensional ¹³C-¹H spectrum of the KKI15, all the carbons were identified except for the quaternary and carbonyl carbons. These carbons have been identified through 2D-HMBC. Specifically, H-7 shows ³J_C-H with C-5, H-6 shows ³J_C-H with C-8, H-13 shows ²J_C-H with C-12, and finally, NH shows ²J_C-H with C-2. Based on this strategy, the complete identification of all the proton and carbon atoms of the KKI15 molecule was achieved.

A similar procedure was carried out for thiocarbohydrazone KKI18. Two signals were observed for H-8 due to the two conformations of KKI18. The only difference is that KKI18 has one more amine group than KKI15. For that reason, NH(4) is resonated to 11 and 9.62 ppm, while through 2D-NOESY, NH2 is identified at 3.33 and 5.02 ppm. Finally, through 2D-NOESY, NH(2) is identified as it is associated with NH2. The NH makes sense to be increasingly deshielded because it is next to two electronegative individuals. Moreover, there were observed correlations similar to KKI15 between H-8 and H-10, H-11 and H-14, and H-7 with H-15.

All the calculations were carried out in DMSO because it is considered to be a solvent that simulates the amphoteric environment and is suitable for the observation of NOE effects [18,19].

The two identification strategies are shown in Supplementary Material Figure S3A,B. The two proton spectra of KKI15 (Figure 2 and Figure 3) and KKI18 (Figure 2 and Figure 3) are shown below. Moreover,

Table 1. Assignment of the experimental ¹H NMR spectra of ΚΚΙ15 and KKI18 in DMSO-d6.

Peak KKI15	Chemical Shift (ppm)	Peak	Chemical Shift (ppm)	Peak	Chemical Shift (ppm)
6	6.45	15	7.65	3	11.10
7	6.79	10	7.65
7′	7.20	13	7.73
11	7.35	1	7.80
14	7.42	9	7.82
Peak KKI18	Chemical Shift (ppm)	Peak	Chemical Shift (ppm)	Peak	Chemical Shift (ppm)
1	3.34	15	7.40	2′	9.60
1′	4.99	14	7.72	4	9.38
7	6.29	16	7.45	4′	11.07
7′	6.75	11	7.66	12	7.33
8	6.42	10	7.78
8′	7.20	2	8.65

with the chemical shifts of these two compounds is shown below.

2.2. Conformational Analysis

DFT was used to predict the lowest energy conformations for KKI15 and KKI18 as this method offers the highest accuracy of the existing ones. Various initial structures were geometry optimized, and all geometry calculations resulted in eight conformers for KKI15 and for KKI18, see Figure 4. Then their frequencies were calculated. No imaginary frequencies have been found confirming that there are true minima structures.

Four dihedral angles were selected for each molecular structure. Specifically, the angles of KKI15 that were selected for DFT are formed by the following atoms: 5-6-7-8 (τ1), 3-4-5-6 (τ2), 3-4-5-12 (τ3), and 8-7-6-5 (τ4) and for KKI18: 6-7-8-9 (τ1′), 4-5-6-7 (τ2′), 4-5-6-13 (τ3′) and 9-8-7-6 (τ4′). The relative energies are given in

Table 2. Results of energy minimization calculations. The values are given in Hartree, while their difference from the stable structure MS1 for KKI15 and MS5 for KKI18 in kcal/mol (top). Values in degrees of the dihedral angles, of the minimized structures (MS) for KKI15 and KKI18 (bottom).

KKI15	Difference in Energy between the Various Conformers and the Conformer at the Minimum Energy	Double Bond Isomerism	Amide Bond Isomerism	Dihedral Angle (Degrees)	Dihedral Angle (Degrees)
MS1	0	Ε	Ε	τ1 = 179.56	τ2 = −6.97
MS2	0.79	Ε	Ζ	τ1 = 179.12	τ3 = −4.53
MS3	3.04	Ζ	Ε	τ4 = −3.93	τ2 = 1.69
MS4	6.72	Z	Z	τ4 = −6.54	τ2 = −5.76
KKI18	Difference in Energy between the Various Conformers and the Conformer at the Minimum Energy	Double Bond Isomerism	Amide Bond Isomerism	Dihedral Angle (Degrees)	Dihedral Angle (Degrees)
MS5	0	Ε	Ε	τ1′ = −179.07	τ3′ = 4.38
MS6	0.81	Ε	Ζ	τ4′ = −4.22	τ2′ = 2.14
MS7	2.98	Ζ	Ε	τ4′ = 0.95	τ2′ = −177.57
MS8	6.66	Z	Z	τ4′ = 0.95	τ2′ = −177.57

with the values of dihedrals angles for KKI15 and KKI18.

In Table 2, the values of the dihedral angles differ in each isomer. Although the theoretical value of the dihedral angle should be 0° or 180°, there are some small deviations from these values. This may be due to stereochemical hindrances.

Table 2 clearly shows the relationship between the energy values and the structures, with the E isomers having lower energies values than the Z. From the three-dimensional structures, it is observed that the E configurations reduce the stereochemical repulsions in relation to the Z. Figure 5 shows the hydrogen bonds between the hydrogen of amines and nitrogens for (a) KKI15 and (b) KKI18. The same hydrogen bonds were observed for all the conformations of each compound.

Considering the predicted energy values, the structures of the compounds, and the correlations that were observed in 2D-NOESY spectra (Figure 6), the structures MS1 and MS5 are taken as the most probable configurations for KKI15 and KKI18, respectively, as shown in Figure 7 (

Table 3. The most important comparative distances in the complex obtained through 2D NOESY and DFT MD calculations for KKI15 (top) and KKI18 (bottom).

Spatial Interactions	MD Distances (Å)	Qualitative Characterization of the Spatial Interactions
H7-H13	2.23	S
H7-H10	4.65	W
H7-H9	2.32	S
H6-H14	4.81	W
H8-H14	2.72	S
H8-H11	4.65	W
H8-H10	2.30	S
H7-H15	4.80	W

).

2.3. Energy of Transition States

In order to calculate the transition state structures, an initial conformation with dihedral angles τ₁ or τ₂ of 90° was used, which subsequently was geometry optimized. For the calculation, the geometries of the isomers were considered as implemented in the STQN methodology. The values of the energy of transition states for KKI15 are shown in

Table 4. Kinetic isomerization between cis-trans for KKI15.

Conformation	Energy (kcal/mol)
MS4	6.72
ts1	50.09
MS3	3.04
ts2	48.79
MS1	0.00
ts3	63.60
MS2	0.79

. Figure 8 and Figure 9 show the diagrams for the cis-trans isomerization energy barrier for compound KKI15.

The values of the energy of transition states for KKI18 are shown in

Table 5. Kinetic isomerization between cis-trans for KKI18.

Conformation	Energy(kcal/mol)
MS8	6.66
ts1	75.30
MS7	2.98
ts2	42.91
MS5	0.00
ts3	13.91
MS6	0.81

. Figure 10 and Figure 11 show the diagrams for the cis-trans isomerization energy barrier for compound KKI18.

The first two energy gaps for KKI15 have very high values (larger than 20 kcal/mol), and for this reason, these conformations are not observed in the NMR spectrum. On the contrary, for KKI18, the energy gap between MS5 and MS6 is less than 20 kcal/mol. This explains the experimental result of observing two conformations. Moreover, the stabilization of the transition states due to the explicit inclusion of the solvent has been evaluated. Specifically, the addition of a solvent molecule in the reaction path MS1→TS3→MS2 of KKI15 results in further stabilization of about 10 kcal/mol, and the energy gap was found to be 53.8 kcal/mol. For the remaining transition states structures, the explicit inclusion of the solvent does not further stabilize them.

2.4. Population Calculation

To make the identification of molecules easier, charge calculations were performed in double bond protons C6=C7 and C7=C8 for compounds KKI15 and KKI18, respectively. This will determine which carbon and proton are the most deshielded in the NMR spectrum. For the protons, the calculations showed similar charges. Specifically, CM5 showed a slightly increased positive charge for proton 7 than 6, while the NMO showed 6 to be more positive than 7. On carbons, CM5 showed a more positive charge on C7 by 0.01, while NBO and Mulliken showed carbon 6 to be more negative than 7 by 0.08 and 0.14, respectively. Due to such small differences, a firm conclusion cannot be derived. The charges of carbons range from 0.01 to 0.1, depending on the method. The detailed results are shown in the Supporting Information (Supplementary Material Table S2A,B).

In the next step, molecular orbitals were calculated in each conformation. For both compounds, the HOMO molecular orbital is localized in the sulfocarbonyl group, and LOMO molecular orbital is localized in the sulfocarbonyl group and in the aromatic ring next to the double bond. The figures with the molecular orbitals are also shown in Supporting Information.

2.5. ECD Results

Absorption UV-Vis spectra of all conformers are given in SI. It was found that there are triplet excitations in the visible area only for KKI15, specifically in the region 625–750 nm. Absorption spectra of KKI18 conformers show no triplet excitation in the visible region. Finally, both compounds present intense single excitations in the UV region at about 230 nm.

2.6. Circular Dichroism (CD)

As it is observed from the CD spectra, in KKI15, there is a distinct difference between the two isomers; in contrast with KKI18, where this difference appears very small. That explains the computational results in which KKI18 was found to show a very low energy gap between the two conformations (See Figures in SI).

2.7. Molecular Binding

SwissADME [20] target was utilized to discover possible targets for the two molecules [21]. Four targets were detected, and more specifically, 5DYW for Butyrylcholinesterase, 4EY7 for Acetylcholinesterase, 5UEN for Adenosine A1 receptor, and 1YK8 for Cathepsin K. In all these crystal files the macromolecule was crystallized with a target. Molecular docking [22] has been applied for all targets. The grid parameters used were the same for all the substrates.: X = 40, Y = 40, Z = 40 (default) and the distance of the dots: 0.375 Å (default).

Then the coordinates from the co-crystallized ligand which were used for the active center of each macromolecule were: 5DYW [23]: X = 14.209, Y = 26.367, Z = −41.477 4EY7 [24]: X = −18,53, Y = −41.928, Z= 24.258, 5UEN [25]: X = 52.632, Y = 56.381, Z = 141.281, 1ΥΚ8 [26] X = 71.977, Y = 12.669, Z = 131.026

All docking scores are shown below in

Table 6. Binding energies BE (kcal/mol) and Inhibition constants between the compounds and macromolecules.

5UEN (Adenosine A1 Receptor)	BE	INHIBITION CONSTANT
KKI15	−7.46 ± 0.5	3.38 ± 0.5 μM
KKI18	−7.46 ± 0.5	3.38 μM ± 0.5
5DYW (Butyrylcholinesterase)
KKI15	−8.24 ± 0.5	0.917 ± 0.5 μΜ
KKI18	−7.75 ± 0.5	2.1 μM ± 0.5
1YK8 (Cathepsin K)
KKI15	−6.29 ± 0.5	24.36 ± 0.5 μΜ
KKI18	−6.05 ± 0.5	0.037 μΜ ± 0.5
4EY7 (Acetylcholinesterase)
KKI15	−8.6 ± 0.5	0.499 ± 0.5 μΜ
KKI18	−9.08 ± 0.5	0.221 μΜ ± 0.5

.

Regarding the 5UEN macromolecule, the binding energy values are the same for both compounds (ΔG = −7.46 kcal/mol). Thus, they strongly bind to Adenosine A1 receptors. The same results apply to the macromolecular 5DYW. The binding energy is −7.75 kcal/mol for thiocarbohydrazone KKI18 and −8.24 kcal/mol for thiosemicarbazone KKI15. Thus, they strongly bind to butyrylcholinesterase.

Proceeding to the 4EY7 macromolecule, it was observed that both KKI15 and KKI18 bind strongly to acetylcholinesterase, with binding energy values of −8.6 and −9.08 kcal/mol, respectively.

In the latter macromolecule, the compounds do not bind strongly. Specifically, they gave values of −6.29 kcal/mol and −6.05 kcal/mol for KKI15 and KKI18, respectively.

The in silico experiments are shown below. The binding energy and the inhibition constants were calculated computationally via the Autodock program.

Following the above conclusions, a thiosemicarbazone (test2) molecule and a thiocarbohdrazone (test1) molecule carrying an electronegative substituent in both rings were designed using the ChemDraw platform to control the influence of electronegativity on both rings and to examine the additive effect. The results are shown in

Table 7. Molecular binding results of compound test1 with four macromolecules.

test1	Binding Energy (kcal/mol)	Inhibition Constant
5UEN (Adenosine A1 receptor)	−8.89 ± 0.5	0.304 ± 0.5 μM
5DYW (Butyrylcholinesterase)	0.06 ± 0.5	-
4EY7 (Acetylcholinesterase)	−9.62 ± 0.5	0.089 ± 0.5 μΜ
1YK8 (Cathepsin K)	−5.78 ± 0.5	57.93 ± 0.5 μΜ

and

Table 8. Molecular binding results of compound test2 with four macromolecules.

test2	Binding Energy (kcal/mol)	Inhibition Constant
5UEN (Adenosine A1 receptor)	−8.89 ± 0.5	0.305 ± 0.5 μM
5DYW (Butyrylcholinesterase)	−0.24 ± 0.5	670.000 ± 0.5 μM
4EY7 (Acetylcholinesterase)	−9.75 ± 0.5	71.11 ± 0.5 μΜ
1YK8 (Cathepsin K)	−5.58 ± 0.5	81.85 ± 0.5 μΜ

below.

In summary, both classes of compounds provide a strong binding at the macromolecules 5UEN and 4EY7. That is, the addition of electronegative substituents to both rings can also lead to compounds that bind strongly to these enzymes but with no additional binding energy. With respect to the 5DYW enzyme, the compounds are almost non-binding, so the addition of electronegative substituents to both rings attenuates the binding to butyrylcholinesterase. This may be attributed to the size of the compounds. In the latter macromolecule, the compounds do not bind strongly; a similar result was observed obtained with KKI15 and KKI18 compounds. The interactions of the test1 compound with acetylcholinesterase differ from KKI15. It forms three hydrogen bonds with SER293 amino acid and two p-p interactions with aminoacid TRP286. Furthermore, test2 compound forms one hydrogen bond with aminoacidARG296 in contrast with KKI18 in that it does not form any bond with acetylcholinesterase. Secondly, in the adenosine A1 receptor, the test compounds form more interactions than KKI15 and KKI18. All in all, the additions of substituents in three of the four macromolecules do not affect the binding energy.

2.8. Results of the Pharmacokinetics and Toxicity Properties of the Two Compounds

Table 9. The physicochemical parameters for compounds KKI15 and KKI18.

Properties	Compound KKI15	Compound KKI18
Molecular Weight	281.384	296.399
LogP	2.9373	2.4419
Rotable bonds	4	4
Hydrogen Bond Acceptors	2	3
Hydrogen Bond Donors	2	3
Surface Area	123.373 (Å2)	128.893 (Å2)
Water solubility	−3.556 (mol/L)	−3.215 (mol/L)

shows the Drug-Likeness Properties of KKI15 and KKI18.

According to pkCSM and preADMET, thiosemicarbazone KKI15 has a molecular weight < 500 g/mol, hydrogen bonding donors < 5, number of hydrogen bonding acceptors < 10, and lipophilicity less than five. As a result, it obeys Lipinski’s Rules of Five. Because lipophilicity is less than five, it can be easily absorbed from the body. Also, Veber’s Rule is qualified because the rotable bonds are less than seven. The same conclusions apply to thiocarbohydrazone KKI18 (

Table 10. The ADME results of KKI15 and KKI18 according to preADMET.

	Compound KKI15	Compound KKI18
BBB	0.66358	1.08871
Buffer_solubility_mg_L	148.736	878.339
Caco2	21.0168	20.112
CYP_2C19_inhibition	Non	Non
CYP_2C9_inhibition	Non	Non
CYP_2D6_inhibition	Non	Non
CYP_2D6_substrate	Non	Non
CYP_3A4_inhibition	Non	Non
CYP_3A4_substrate	Weakly	Weakly
HIA	95.243578	93.484318
MDCK	167.329	153.511
Pgp_inhibition	Non	Inhibitor
Plasma_Protein_Binding	100	99.274409
Pure_water_solubility_mg_L	0.429393	0.471067
Skin_Permeability	−2.03257	−2.76463

).

According to preADMET, the BBB value is less than one. As a result, KKI15 is classified as inactive in Central Nervous System (CNS). In contrast, KKI18 has a BBB value of more than one, and it might affect the Central Nervous System (CNS). The value for Human intestinal absorption is high for both compounds, and this signifies that these compounds might be better absorbed from the intestinal tract on oral administration. Moreover, KKI18 was found to be an inhibitor of Pgp, and this means that it can increase oral bioavailability. According to pKCSM, both compounds might have the ability to penetrate the blood-brain barrier and thus be potential drugs for the Central Nervous System (CNS). Both compounds are not inhibitors of CP isoenzymes and therefore are not toxic or do not exert other unwanted adverse effects.

According to pKCSM, KKI15 and KKI18 have not been predicted to be hepatotoxic (

Table 11. Toxicity results of KKI15 and KKI18 according to pKCSm.

Properties	Compound KKI15	Compound KKI18
Toxicity
AMES toxicity	No	No
Max. tolerated dose (human)	0.243 (log mg/kg/day)	0.317 (log mg/kg/day)
Herg I inhibitor	No	No
Herg II inhibitor	Yes	Yes
Oral Rat Acute Toxicity (LD50)	2.68 (mol/kg)	2.629 (mol/kg)
Oral Rat Chronic Toxicity	0.667 (log mg/kg_bw/day)	1.506 (log mg/kg_bw/day)
Hepatotoxicity	No	No
Skin Sensitisation	Yes	Yes

). Moreover, AMES toxicity is negative, and that means that the compounds are not mutagenic. Finally, they are inhibitors of Herg II and, according to preADMET, present a low risk for Herg II.

3. Materials and Methods

3.1. Synthesis

Chalcone A was obtained using Claisen–Schmidt aldolic condensation and standard conditions [27]. Briefly, 55 mmoles of NaOH were dissolved in 95% EtOH/H₂O 1:1 (250 mL), then 50 mmoles of acetophenone were added, and the mixture was cooled to 0 °C. Benzaldehyde, 50 mmoles, dissolved in 95% EtOH (50 mL), was added dropwise, and the reaction mixture was left to reach room temperature gradually for 24 h. The solid formed was filtered, washed with 95% EtOH/H₂O 1:1, and used for the next preparations after drying in a desiccator over P₂O₅.

The corresponding thiosemicarbazone KKI15 and thiocarbohydrazone KKI18 were synthesized from chalcone A, which was subjected to a condensation reaction with thiosemicarbazide or thicarbohydrazide, respectively (Scheme 1). The reactant ratio was a controlled condition to avoid the formation of bis-thiocarbohydrazone, and it gave the mono-adduct KKI18 or KKI15 exclusively.

Experimental

(E)-2-((E)-1,3-diphenylallylidene)hydrazine-1-carbothioamide, KKI15

To a stirred solution of chalcone A (0.50 g, 2.40 mmoles) in 95% EtOH (60 mL), thiosemicarbazide (0.22 g, 2.40 mmoles) is added, followed by 3 drops of 6N HCl. The reaction mixture was stirred at room temperature for 24 h, then concentrated in vacuo, and the remaining was purified by column chromatography using PΕ(40–60)/AcOEt:8/2 as eluent to give thiosemicarbazone KKI15 as yellowish solid in 66% yield (0.45 g), Rf = 0.73 (PE/AcOEt 8/2).

N’-((1E,2E)-1,3-diphenylallylidene)hydrazinecarbothiohydrazide, KKI18

To a stirred solution of thiocarbohydrazide (0.20 g, 1.92 mmoles) in a mixture of 95% EtOH (58 mL) and H₂O (20 mL), chalcone A (0.20 g, 0.96 mmoles) is added followed by 3 drops of 6N HCl. The reaction mixture was stirred at 80 °C for 3 h and then left to crystalize at room temperature for 24 h. The formed solid was filtered, washed with EtOH/H₂O 1:1, and recrystallized from EtOH 95% to give KKI18 in the form of a slightly ochre solid in 55% yield (0.31 g).

3.2. Structure Assignment

The two molecules under study have been structurally identified using an 800 MHz spectrometer (Bruker Avance spectrometer-Bruker Biospin GmbH, Reinsteten, Germany) installed in the NMR Centre in the National Institute of Chemistry in Slovenia using 1D and 2D homonuclear and heteronuclear experiments. The pulse sequences were obtained from the library of the spectrometer. The spectra were processed and analyzed using the MestreNova software.

3.3. Computational Details: DFT Calculations

Conformation analysis: Conformational analysis of the compounds has been carried out to find the lowest energy minimum structures employing the density functional theory (DFT) [28] at the B3LYP [29,30]/6-311 + G(d,p) [31] level of theory. This methodology is considered suitable for conformational analysis as it provides consistent results [32]. All conformers were fully optimized, and subsequently, their frequencies were computed in order to confirm that they are true minima. All calculations were carried out in DMSO solvent employing the polarizable continuum model (PCM) [33,34]. This model is divided into a solute part lying inside a cavity, surrounded by the solvent part represented as a material without structure, characterized by its macroscopic properties, i.e., dielectric constants and solvent radius. Furthermore, a solvent DMSO molecule has been added explicitly in all minimum structures and transitions states; see below. Finally, all theoretical data are compared with the experimental results obtained from the NMR spectra.

Activation Energy and transitions states: The paths of the isomerization process were calculated. The transition states connecting the various minima structures were computed using the synchronous transit-guided quasi-Newton (STQN) method [35].

Population analysis: The charges of the atoms were calculated using three population analyses, i.e., Mulliken, NBO and CM5 [36]. The B3LYP [28], M06-2X [37], ωB97-ΧD [38], and 3 basis sets, i.e., 6-311G(d,p) [without diffuse functions], 6-311 + G(d,p) [with diffuse functions for all atoms but H], and 6-311 ++ G(d,p) [with diffuse functions for all atoms]. All calculations were conducted using the Gaussian16 program [39].

Spectra calculations: UV-vis absorption and ECD spectra were calculated for the lowest forty excited states, using the TD-DFT methodology at the B3LYP/6-311 + G(d,p) level of theory. [40]

Circular Dichroism: Circular Dichroism was performed with a Jasco J-815model, featuring a peltier model PTC-423S/15 thermo stabilizing system. The cell used was 1 mm Quartz Suprasil cell. Concentrations used for the two samples were about 2 × 10⁻⁴ g/mL.

3.4. Molecular Binding

AutoDock [41] software was used for the molecular binding calculations and, more specifically, the Lamarckian Genetic algorithm. The crystal structures of the proteins were used by the online database “Protein Data Bank—PDB” and downloaded directly to the AutoDock program for study. The compounds used as ligands were designed with the help of the ChemOffice program, and using the same program, their energy was minimized with an MM2 force field.

3.5. ADMET Calculations

Both compounds were sketched in Chem Draw, and they were converted into SMILES in both web application tools preADMET [42] and pKCSm [43]. Both programs were used to examine the drug-likeness (Lipinski’s Rule of Five [44] and Veber’s rule [45]). Moreover, several toxicity parameters were examined via these tools. This procedure is very important for computational drug design because some potential biological compounds fail to reach the clinical trials because of their unfavorable (ADME) parameters.

4. Conclusions

This study focuses on structure assignment and conformational analysis of thiosemicarbazone adduct KKI15 and thiocarbohydrazone KKI18 using a combination of NMR spectroscopy and computational studies (QM methods).

The conformational analysis showed that both molecules could obtain four configurations, but the most favorable is when the two double bonds adopt the E relationship. Through 2D-NOESY and DFT, the conformations that are most probably to agree are MS1 and MS2 for KKI15 and MS5 and MS6 for KKI18. The calculation of transition proves that the molecules obtain these two conformations.

In silico experiments were performed with four macromolecules to find some possible biological targets. The results showed that the compounds bind strongly to acetylcholinesterase, butyrylcholinesterase, and adenosine A1 receptors. In cathepsin K, they bind weakly. Both derivatives are not hepatoxic, and they obey Lipinski’s Rule of Five.

It appears that both molecules can be safe and bioactive, and putative for biological targets. This is very useful to synthetic chemists who wish to investigate new structures for various targets. The ultimate goal is through in silico molecular modeling screening to synthesize molecules that act selectively on certain biological targets.