A novel cyclobutane-derived thiazole–thiourea hybrid with a potency against COVID-19 and tick-borne encephalitis: synthesis, characterization, and computational analysis

In the present contribution, a novel cyclobutane-derived thiazole–thiourea hybrid 1-(4-(3-methyl-3-phenylcyclobutyl)thiazol-2-yl)−3-(p-tolyl)thiourea (1), which was readily fabricated from addition of p-isothiocyanatotoluene to 4-(3-methyl-3-phenylcyclobutyl)thiazol-2-amine, is reported. The formation of 1 was firmly confirmed by the means of elemental analysis, and IR and 1H NMR spectroscopy. Theoretical DFT-based computations were additionally applied to reveal the structure and electronic features of the title compound. The chemical activity of 1 was estimated by the reactivity descriptors and MEP surface. ADMET properties of the reported compound were predicted in silico using online services. Potential inhibition of a series of the SARS-CoV-2 and tick-borne encephalitis proteins by 1 was studied using molecular docking, which, in turn, allowed to reveal the ligand efficiency scores for the resulting protein–1 complexes. It was established that 1 exhibits the best inhibition activity against Nonstructural protein 14 (N7-MTase) and tick-borne encephalitis virus (TBEV) glycoprotein amongst the studied SARS-CoV-2 and TBE proteins, respectively. GRAPHICAL ABSTRACT


Introduction
Throughout human history, different viruses caused deadly outbreaks, some of which turned to pandemics posing a global threat [1].Of pandemics, the most notable are, likely, the Russian flu at the end of nineteenth century [2]; the Spanish flu, which happened right after World War I [3]; HIV (human immunodeficiency viruses), a causative of AIDS (acquired immunodeficiency syndrome), which was identified 40 years ago [4][5][6][7][8].The latest most deadly outbreak was defined as COVID-19 (coronavirus disease 2019) pandemic, which is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus) [9,10].This pandemic has caused a serious negative impact on the global economics [11].The other disease with crucial negative effects for human health, involving the central nervous system, is TBE (tick-borne encephalitis), which is caused by tick-borne encephalitis virus (TBEV) [12].The situation with TBE is further exacerbated due to the avalanche growth of victims over the past few decades [13].Furthermore, since SARS-CoV-2 can significantly weaken the immune system treatment of the TBE-affected patients becomes a serious challenge, especially during the activity period of ticks (Ixodes Ricinus ticks), which are the main vector for TBE [14][15][16].Thus, synthesis and detailed studies of novel compounds with potential complex activity against SARS-CoV-2 and TBE are of great importance.
Of a great number of chemical fragments, heterocycles, likely, play a pivotal role in the fabrication of medications [17][18][19][20][21][22][23].One of the prominent class of heterocycles is the socalled azoles -five-membered aromatic heterocycles, which contain a nitrogen atom and at least one additional heteroatom (nitrogen, oxygen, or sulfur) [24].One of representatives of azoles is thiazole -a compound with one nitrogen and one sulfur atoms, which are 1,3positioned in the structure of a cycle.The thiazole ring is of importance as a fragment of medications and biologically active compounds.To be said, the thiazole ring can be recognized in the structure of Vitamin B 1 , an important micronutrient [25,26], and Bleomycin, which is used to treat cancer [27,28].
One of the most intriguing cycloalkanes is, likely, cyclobutane due to strained intracyclic bond angles.Its derivatives are of particular interest and importance due to their biological properties and medications [29][30][31][32].On the other hand, thiourea and its derivatives are also in the focus of modern medicine [33][34][35][36].Notably, the thiazole-thiourea hybrids have also been in the limelight of researchers [37][38][39].Furthermore, both the thiourea and thiazole derivatives have also been considered against SARS-CoV-2 [40,41].Thus, using thiazole and thiourea fragments in a molecule can potentially induce biological activities of interest, which might be of value against certain diseases.
Nowadays, computational studies have become one of the most powerful tools to reveal potential properties of a molecule [42][43][44][45].Besides, theoretical calculations allow to decrease time for the design and development of efficient strategy toward synthesis of a molecule of interest with desirable properties.

Physical measurements
The FTIR spectrum in the KBr pellet was obtained with a PerkinElmer Spectrum 100 FT-IR spectrometer.The NMR spectra in CDCl 3 were recorded with a Varian-Mercury-Plus 200 MHz spectrometer.Melting point was determined using the Gallenkamp melting point apparatus.Microanalysis was performed using a LECO 932 CHNS-O elemental analyzer.

DFT calculations
The structure of 1 was optimized without symmetry restrictions using the DFT/B3LYP/6-311++G(d,p) method [64][65][66] by the GaussView 6.0 molecular visualization program [67] and the Gaussian 09, Revision D.01 program package [68].The vibration frequencies were calculated for the optimized structure and no imaginary frequencies were obtained.The HOMO, LUMO and MEP surfaces were generated from the optimized structure.

Molecular docking
Molecular docking was performed using the CB-Dock2 online tool [69,70] with the implemented algorithm of AutoDock Vina [71].The structures of the applied proteins, which were downloaded from the RCSB PDB database [72], were prepared for docking by removing water molecules and inserting hydrogen atoms and missing residues and charges.The obtained results were visualized in BIOVIA Discovery Studio 2020 [73].

Results and discussion
The addition reaction of 4-(3-methyl-3-phenylcyclobutyl)thiazol-2-amine with p-isothio cyanatotoluene yielded a novel cyclobutane-derived thiazole-thiourea hybrid 1-(4-(3methyl-3-phenylcyclobutyl)thiazol-2-yl)−3-(p-tolyl)thiourea (1) (Scheme 1).The experimental FTIR spectrum of 1 contains a set of bands at about 2800-3300 cm -1 corresponding to N-H and C-H stretching vibrations (Figure 1).The bands for the C = C and C = N groups were observed at about 1500-1650 cm -1 , while the thiocarbonyl fragment was shown as a band at about 1400 cm -1 (Figure 1).The 1 H NMR spectrum of 1 (Figure S1) contains two singlet bands at 1.56 and 2.33 ppm, which correspond to the hydrogen atoms of the methyl groups attached to the cyclobytane and benzene fragments, respectively.The cyclobutane methylene and methine hydrogen atoms were shown as two multiplets at 2.45-2.65 and 3.59-3.76ppm, respectively.A singlet signal at 6.41 ppm was assigned to the thiazole hydrogen atom, while the phenyl and phenylene hydrogen atoms were observed in the spectrum as a multiplet at 7.00-7.43ppm.Finally, the NH hydrogens were shown as a broad singlet at 10.42 ppm.The 13 C 1 H NMR spectrum of 1 (Figure S1) exhibits signals for the methyl and cyclobutane fragments at 23.0-42.8ppm, while the signal for the thiocarbonyl carbon atom was found at 178.9 ppm.The thiazole and benzene carbons were shown in the spectrum as a set of signals at 106.2-163.8ppm.
The structure of 1 was optimized using the DFT calculations (Table S1).It was established that the most favorable conformation of a molecule is described by the thiourea and thiazole sulfur atoms being oriented in the same direction, while the benzene and toluene fragments are directed to the opposite side (Figure 2).The geometrical parameters in the optimized structure are of typical values for certain functionalities (Table S2).Notably, the thiazole-thiourea fragment is essentially planar, while the mean planes formed by the carbon atoms of the phenyl and toluene fragments are about 89°and 56°rotated with respect to the mean plane formed by the non-hydrogen atoms of the thiazole-thiourea fragment (Figure 2, Table S2).Furthermore, the mean planes of the six-membered aromatic rings are about 35°with respect to each other (Figure 2, Table S2).All the hydrogen atoms in the optimized structure of 1 are positively charged with the highest values corresponding to the NH hydrogens (Figure 3).Of non-hydrogen atoms, the phenyl ipso-carbon atom is the most positively charged, followed by the CH carbon atom of the thiazole fragment and the quaternary carbon atom of the cyclobutane fragment (Figure 3).Notably, the thiazole nitrogen atom is not charged, while the thiourea nitrogens are positively charged, although the nitrogen atom located between the thiazole and thiocarbonyl fragments carries about 2.4 times higher charge than the other one (Figure 3).The most negatively charged atoms are the thiazole and methyl carbon atoms, both linked to the cyclobutane fragment, and both secondary carbons of the cyclobutane fragment (Figure 3).Both sulfur atoms are also negatively charged, although the thiocarbonyl sulfur is about 1.7 more negatively charged (Figure 3).
Since the molecule of 1 is constructed from 50 atoms, it has 144 normal modes in the IR spectrum.The calculated spectrum is in agreement with the experimental one; however, some discrepancies are also revealed (Figure 1).The latter can be explained by the gas phase optimization of the structure of 1.In the calculated spectrum, the N-H and C-H stretching vibrations are shown as a set of bands at 2900-3090 cm -1 and a band at 3415 cm -1 (Figure 1).The bands for the C = C and C = N groups in the calculated spectrum were observed at about 1480-1680 cm -1 , while the thiocarbonyl fragment was found as a band at about 1395 cm -1 (Figure 1).
The optimized molecule of 1 exhibits a dipole moment of 4.5152 Debye.The HOMO and LUMO for 1 are -6.05100 and -1.43050 eV, respectively, yielding the energy gap  of 4.62050 eV (Table 1).It was found that the HOMO is mainly located on the thiazole-NH-C(S) fragment and CH 2 -CH-CH 2 fragment of the cyclobutane (Figure 4).The LUMO is mainly found on the thiazole, thiourea and toluene fragments (Figure 4).According to the values of I, A, and µ, the optimized structure of 1 is a pronounced electron donor.Furthermore, 1 tends to exchange its electron cloud with surrounding environment as established from a relatively low value of chemical hardness and a relatively high value of chemical softness (Table 1).The value of the electrophilicity index of 1 is characteristic for strong electrophiles [77].
Analysis of the molecular electrostatic potential (MEP) surface allowed to highlight the electrophilic and nucleophilic sites in 1.The most pronounced nucleophilic sites are located on the thiocarbonyl sulfur atom, followed by the thiazole sulfur and nitrogen atoms (Figure 5).As the most remarkable electrophilic centers, the NH and toluene hydrogen atoms can be revealed (Figure 5).
Compound 1 was predicted to belong to the third class of toxicity with the estimated LD 50 value of 250 mg/kg and is hepatotoxic (Figure 6).The bioavailability radar indicates  that 1 is preferred in five parameters and is slightly less preferred in lipophilicity.Furthermore, 1 was predicted to be an inhibitor of protease, nuclear receptor, family A G protein-coupled receptor, kinase, and voltage-gated ion channel (Figure 6).The BOILED-Egg model demonstrates that 1 exhibits negative human blood-brain barrier (BBB) penetration and positive gastrointestinal absorption (GA), and is potentially effluated from the central nervous system by the P-glycoprotein (Figure 6).
Compound 1 was further probed in silico as a potential inhibitor of a series of the SARS-CoV-2 and TBEV proteins (Table 2).The target proteins (Table 2) were primarily selected in accordance with the structural features of the viruses as well as based on biological mechanisms and functions that can be utilized to reduce, prevent or treat the viruses [78][79][80][81][82][83].The obtained results were compared with Remdesivir and Favipiravir, which are used to treat COVID-19 [84,85], and with iodoantipyrine, which is used for prevention and combinational treatment of TBE [86].
To further shed light on the potential bioactivity of 1 toward the discussed proteins, we have additionally computed the so-called ligand efficiency scores [87][88][89][90][91][92].The target parameters for some of these scores are as follows: the K i value must fall in the 0.1-1 μM range for a compound to be considered as a Hit, and < 10 nM for a drug [91]; the LE, FQ, and LELP are recommended as ≥ 0.3, ≥ 0.8 and from -10 to 10, respectively, for a Hit [91].For complex TBEV MTase-1, the ligand efficiency scores are within the range for a Hit, while for complex Nsp14_N7-MTase-1, the same parameters are close to be for a drug (Table 2).However, the LELP parameter for both complexes is somewhat out of the recommended range (Table 2).

Conclusions
A novel cyclobutane-derived thiazole-thiourea hybrid 1-(4-(3-methyl-3-phenylcyclo butyl)thiazol-2-yl)−3-(p-tolyl)thiourea (1), which was readily synthesized from the addition of p-isothiocyanatotoluene to 4-(3-methyl-3-phenylcyclobutyl)thiazol-2-amine, is reported.The formation of the obtained compound was testified by means of elemental analysis, IR and 1 H NMR spectroscopy.The DFT/B3LYP/6-311G++(d,p) computations were applied to reveal the optimized structure and electronic properties of 1.According to the global chemical reactivity descriptors and MEP surface, 1 is a strong electrophile with the most pronounced nucleophilic sites located on both sulfur atoms and the thiazole nitrogen atom, while the NH and toluene hydrogen atoms are the most remarkable electrophilic centers.
According to the in silico ADMET studies, 1 belongs to the third class of toxicity and is, likely, hepatotoxic.Furthermore, 1 exhibits negative BBB penetration and positive GA, and is potentially effluated from the central nervous system by the P-glycoprotein.As evidenced from the molecular docking simulations, 1 is a potential inhibitor of all the applied SARS-CoV-2 and TBEV proteins.Furthermore, 1 exhibits a remarkably higher activity toward all the studied proteins in comparison to Favipiravir and iodoantipyrine, and for a majority of the proteins it exhibits a superior activity in comparison to Remdesivir.The best activity of 1 was revealed against Nonstructural protein 14 (Nsp14_N7-MTase) and TBEV methyltransferase (TBEV MTase), respectively.The estimated ligand efficiency scores for the complex of TBEV MTase with 1 are within the range for a Hit, while for complex of Nsp14_N7-MTase with 1 the same parameters are close to be for a drug.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.The experimental (bottom) and calculated (top) IR spectra of the optimized structure of 1.

Figure 2 .
Figure 2. Different views on the optimized structure of 1.

Figure 3 .
Figure 3. Mulliken atomic charges in the optimized structure of 1 (see Figure 2 for atoms labeling).

Figure 4 .
Figure 4. Energy levels and views on the electronic isosurfaces of the HOMO and LUMO of the optimized structure of 1.

02851 Figure 5 .
Figure 5.View of the molecular electrostatic potential surface of the optimized structure of 1.

Figure 6 .
Figure 6.Toxicity results, calculated by ProTox-II, druggability predictions, BOILED-Egg model and the bioavailability radar within the domain borders of ADME properties, calculated by SwissADME, of 1 (the colored zone of the radar is the suitable physicochemical space for oral bioavailability).

Table 1 .
Dipole moment, frontier molecular orbitals, gap value and descriptors for the optimized structure of 1.

Table 2 .
Ligand efficiency scores for iodoantipyrine, Remdesivir, Favipiravir and 1 inside the binding sites of the listed proteins.