Thiourea derivatives containing 4-arylthiazoles and d-glucose moiety: design, synthesis, antimicrobial activity evaluation, and molecular docking/dynamics simulations

Abstract

Some substituted glucose-conjugated thioureas containing 1,3-thiazole ring, 4a–h, were synthesized by the reaction of the corresponding substituted 2-amino-4-phenyl-1,3-thiazoles 2a–h with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isocyanate. The antibacterial and antifungal activities of these thiazole-containing thioureas were estimated using a minimum inhibitory concentration protocol. Among these compounds, 4c, 4g, and 4h were better inhibitors with MIC = 0.78–3.125 μg mL⁻¹. These three compounds were also tested for their ability to inhibit S. aureus enzymes, including DNA gyrase, DNA topoisomerase IV (Topo IV), and dihydrofolate reductase, and compound 4h was found to be a strong inhibitor with IC₅₀ = 1.25 ± 0.12, 67.28 ± 1.21, and 0.13 ± 0.05 μM, respectively. Induced-fit docking and MM-GBSA calculations were performed to observe the binding efficiencies and steric interactions of these compounds. The obtained results showed that compound 4h is compatible with the active site of S. aureus DNA gyrase 2XCS with four H-bond interactions with residues Ala1118, Met1121, and F:DC11 and also three interactions with F:DG10 (two interactions) and F:DC11 (one interaction). Molecular dynamics simulation in a water solvent system showed that ligand 4h had active interactions with enzyme 2XCS through residues Ala1083, Glu1088, Ala1118, Gly1117, and Met1121.

Substituted thioureas with 1,3-thiazole and d-glucose were gained from 2-amino-1,3-thiazoles and glucopyranosyl isocyanate. They had antimicrobial activity, some inhibiting S. aureus DNA gyrase, DNA Topo IV, and dihydrofolate reductase. IFD, MM-GBSA and MD were performed.

Introduction

1,3-Thiazole is an important five-membered heterocyclic ring. It contains one nitrogen and one sulphur in ring.¹ This heterocycle is present in many of the structures of substances widely used in the fields of agriculture^2–4 and medicine.^5–7 In particular, aminothiazole is a useful structural motif and is present in numerous compounds possessing diverse biological properties, including antibacterial,⁸ antifungal,⁹ anticancer,¹⁰ anticonvulsant,^9,11 antitumor,¹² and antidiabetic¹³ among many others. The aminothiazole scaffold has been used widely as a privileged pharmacophore in the discovery of new chemical entities with diverse biological activities.^6,14 There are various drugs containing the aminothiazole scaffold with important therapeutic effects, such as sulfathiazole (antimicrobial drug),¹⁵ thiamine (vitamin B1, reduces intracellular protein glycation),¹⁶ cefcapene (cephalosporin antibiotic),¹⁷ talipexole (antifungal),¹⁸ meloxicam (anti-inflammatory),¹⁹ and many others (Fig. 1).

Fig. 1. Representative drugs with a thiazole unit.

Thioureas or thiocarbamides are a class of organic compounds that contain sulphur, with the general formula (R₁R₂N)(R₃R₄N)C S. These organosulfur compounds have widely diverse applications in almost every branch of chemistry, commercially, industrially, and academically. They are synthesized by several different pathways, such as by the reaction of carbon disulphide with various amines,²⁰ organo-isothiocyanates with (hetero)aromatic amines,^21,22 isocyanides with aliphatic amines in the presence of elemental sulphur.²³ In addition to being used as agents in many chemical transformations, thioureas possess many important biological activities, including antifungal,²⁴ antibacterial,^24–26 antitubercular,²⁷ anti-inflammatory,^26,27 and herbicidal²⁸ activities. For example, thiourea derivatives of 1,3-thiazole A exhibit inhibitory activity in vitro against several microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans, as well as both methicillin-resistant and standard strains of S. epidermidis.²⁹ 4-Nitro-N-(thiazol-2-ylcarbamothioyl)benzamide B has remarkable antifungal activity against Aspergillus flavus, Aspergillus niger, Curvularia lunata, Rhizopus spp., and Penicillium spp.³⁰ Thiazole analogue C has been investigated as a potential development inhibitor of cytomegalovirus.³¹ Thiourea D shows high antimicrobial activity in vitro against Streptococcus pneumoniae and Bacillus subtilis (Gram-positive bacteria), Pseudomonas aeruginosa and Escherichia coli (Gram-negative bacteria), as well as antifungal potential against fungal strains, including Aspergillus fumigatus and Candida albicans.³²

From the brief reviews on the synthesis of the thiourea derivatives containing the 1,3-thiazole ring and also their biological activity against bacteria and fungi as mentioned above, we came up with the design for the target molecule as shown in Fig. 2. The d-glucose moiety acts as the polar part of the target molecule, which can contribute to increasing the number of active hydrophilic interactions of this molecule. The thiazole ring makes its own biologically active contribution to the overall molecular framework of the target molecule. This heterocyclic component plays the role of the hydrophobic moiety, in addition to its biological effects. The substituents located on the benzene ring in position 4 of the thiazole ring contribute to the increase or decrease of the inhibitory activity against the microorganisms. The thiourea bridge group acts as a linker group, as well as a pharmacophore.³³ In the rigid body docking, the bond angles, bond lengths and torsion angles of the components are not modified at any stage of complex generation. The problem is whether or not rigid-body docking is sufficient for most docking. In the induced-fit docking protocol, the docking simulation for the receptor–ligand complex is based on the belief that the receptor adjusts significantly to the presence of the ligand by performing constrained minimization of the receptor, continuing the initial Glide docking of the ligands using a softened potential, followed by Prime side-chain prediction and minimization, the best receptor structures for each ligand are passed back to Glide for the redocking of the ligand. Therefore, we have chosen the induced-fit docking method for the simulation of the ligand–receptor interaction. The construction of the 1,3-thiazole heterocycle as well as its connection to the d-glucose moiety are shown in Scheme 1.

Fig. 2. Representative drugs with the thiourea unit, as well as the thiazole–thiourea hybrid and the rationale for the molecular design of the thiazole–glucose-conjugated thiourea scaffold, A as inhibitor against microorganisms, B as antifungal inhibitor, C as potential candidate inhibitor of cytomegalovirus, and D as antimicrobial inhibitor.

Scheme 1. Synthesis of thiourea derivatives containing 4-arylthiazoles and the d-glucose moiety.

Results and discussion

Chemistry

Synthesis

According to this synthetic route, substituted 4-aryl-2-aminothiazoles (2a–h) were synthesized by the reaction of the corresponding substituted acetophenones (1a–h) with thiourea in the presence of iodine.^34,35 This process took place in the solid phase. The derivatives of 1-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-arylthiazol-2-yl) thioureas (4a–h) were easily synthesized by the addition of the corresponding amino compounds 2a–h to peracetylated glucopyranosyl isothiocyanate 3. We performed this reaction in a microwave oven for several minutes. We found that the nucleophilic addition of 2-amino-4-arylthiazoles to 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate has taken place fairly easily. Reaction yields were rather high in this method. All these obtained thioureas could be dissolved in a mixture of ethanol and toluene (1 : 1 in volume) solvent, and could not be dissolved in ethanol and water. Their structures have been confirmed by NMR and mass spectroscopic data (see ESI†). The ¹H-NMR spectra of these thioureas show resonance signals for protons in thiourea N–H groups at δ 11.95 and 2.08 ppm. C–H protons in the pyranose ring of monosaccharides have chemical shifts from δ 5.93 ppm to 3.98 ppm, which are usually observed in the ¹H-NMR spectra of monosaccharide compounds. Proton H-1 of the glucopyranose ring has a chemical shift in the region of δ 5.907 ppm (in triplet) with coupling constant J = 9.0 Hz. The resonance signal of proton H-2 appeared as a triplet in the region of δ 5.03 ppm with coupling constant J = 5.0 Hz. The values of coupling constants were correlated with trans-H–H coupling interactions and indicated the β-anomer configuration of the NH-thiourea group.

Pharmacology

Antibacterial assays

All synthesized glucose-conjugated thioureas 4a–h were evaluated for their biological activity against some bacteria, such as Bacillus subtilis, Clostridium difficile, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae (Gram-positive bacteria), Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella typhimurium (Gram-negative bacteria). Ciprofloxacin and vancomycin were used as reference drugs for Gram-positive bacteria, and vancomycin for Gram-negative bacteria. Their MIC values were as follows: ciprofloxacin, 3.125 μg mL⁻¹ (for Gram-positive bacteria), 1.56 μg mL⁻¹ (for Gram-negative bacteria); vancomycin: 0.78–3.12 μg mL⁻¹ (for Gram-positive bacteria).

The results in Table 1 show that almost all novel hybrid molecules of thioureas 4a–h exhibited remarkable antibacterial activity against the tested bacteria. Their activities were comparable to the MIC values of the reference drugs. Some compounds had strong inhibitory effects on both Gram-positive and Gram-negative bacteria in the MIC value range of 0.78–3.125 μg mL⁻¹. Compounds 4b, 4d, 4g, and 4h exhibited stronger inhibitory activity against almost all of the tested Gram-positive and Gram-negative bacteria in this range. Compound 4b with a 3-nitro group on the benzene ring of thiazole most strongly inhibited Gram-positive bacteria C. difficile and S. pneumoniae with MIC values of 0.78 μg mL⁻¹, as well as Gram-negative bacteria E. coli, K. pneumonia, P. aeruginosa, and S. typhimurium with the MIC value range of 0.78–3.125 μM. However, this compound had low inhibitory activity against S. aureus, which is the most pathogenic. It often causes skin infections and sometimes pneumonia, endocarditis, and osteomyelitis, and often leads to the formation of abscesses. Some strains cause complex toxins that cause gastroenteritis, desquamation syndrome, and toxic shock syndrome. Compound 4d with the 4-bromine group had good inhibition against bacterium B. subtilis, E. coli, and S. typhimurium with MIC values of 3.125, 1.56, and 0.78 μM, respectively, whereas the substitution of the chlorine group at this position in compound 4c causes the target molecule to show no activity towards these microorganisms. This compound exhibited weak activity against S. epidermidis with MIC = 3.125 μg mL⁻¹. This phenomenon was also observed for alkyl substitution in the para position for compounds 4e and 4f. While compound 4f with p-ethyl substitution had strong inhibitory activity against B. subtilis with MIC = 0.78 μM, compound 4e did not inhibit this bacterium. Compound 4e also showed no inhibition toward other tested bacteria. Compound 4h, with the presence of both the electron-withdrawing group (3-nitro) and the electron-donating group (4-methoxy), showed an abnormal change in inhibitory activity when compared to compounds 4b and 4g, respectively. Both compounds 4g and 4h exhibited excellent inhibitory activity against the Gram-positive bacterium S. aureus with IC₅₀ values of 1.56 and 0.78 μM, respectively. The comparison also showed that compound 4h did not inhibit almost all Gram-positive bacteria, as well as most Gram-negative bacteria, except for S. aureus and two other Gram-negative bacteria K. pneumonia and P. aeruginosa with all MIC values of 0.78 μM.

Antibacterial activity of glucose-conjugated thioureas 4a–h.

Entry	R	Micro-organisms/MIC (μg mL−1)
		Gram-positive bacteria					Gram-negative bacteria
		B.s.	C.d.	S.a.	S.e.	S.p.	E.c.	K.p.	P.a.	S.t.
4a	H	25	50	200	0.78	25	100	25	6.25	400
4b	3-NO2	6.25	0.78	6.25	12.5	0.78	0.78	3.125	3.125	3.125
4c	4-Cl	12.5	12.5	3.125	3.125	400	50	12.5	200	12.5
4d	4-Br	3.125	6.25	12.5	25	50	1.56	6.25	100	0.78
4e	4-CH3	100	25	25	400	12.5	6.25	200	50	25
4f	4-C2H5	0.78	400	400	6.25	100	12.5	50	25	100
4g	4-OCH3	400	3.125	1.56	100	3.125	3.125	1.56	12.5	6.25
4h	4-OCH3–3-NO2	50	200	0.78	50	6.25	200	0.78	0.78	50
	Ciprofloxacin	3.125	6.25	3.125	3.125	6.25	1.56	1.56	1.56	1.56
	Vancomycin	1.56	1.56	1.56	0.78	1.56	—	—	—	—

In vitro inhibition of DNA gyrase, topoisomerase IV, and dihydrofolate reductase from S. aureus

DNA gyrase is a type II topoisomerase. It can introduce negative supercoils into DNA at the expense of ATP hydrolysis. This is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death.³⁶ Dihydrofolate reductase (DHFR) is a member of the reductase enzyme family that is ubiquitously expressed in all organisms. This enzyme is required to maintain bacterial growth, and hence inhibitors of DHFR have been proven as effective agents for treating bacterial infections.³⁷ Based on the results of antibacterial activity tests obtained above in Table 1, among thiazole-containing thioureas 4a–h (inhibitors), compounds 4c (R = 4-Cl), 4g (4-OCH₃), and 4h (4-OCH₃–3-NO₂) exhibited the best inhibitory activity against the tested Gram-positive bacteria, especially S. aureus, with MIC values of 3.125, 1.56, and 0.78 μg mL⁻¹, respectively. To understand the preliminary mechanism of these compounds with potent antibacterial activity and to confirm the results of the docking study (see section ‘Induced-fit docking’), an enzyme inhibitory assay, including S. aureus DNA gyrase and topoisomerase IV, was performed towards compounds 4c, 4g, and 4h.^38,39 The obtained IC₅₀ results are displayed in Table 2. Two standard drugs, ciprofloxacin and methotrexate, were used as suitable positive controls.

Inhibitory assessment (IC₅₀ in μM) of the most active inhibitors 4b and 4g on S. aureus DNA gyrase, topoisomerase IV, and dihydrofolate reductase enzymes.

Compound	IC50 (μM)
	S. aureus DNA gyrase	Topoisomerase IV	Dihydrofolate reductase
4c (R = 4-Cl)	2.82 ± 0.13	68.68 ± 1.21	0.23 ± 0.06
4g (R = 4-OCH3)	1.37 ± 0.11	79.15 ± 1.23	0.17 ± 0.03
4h (4-OCH3–3-NO2)	1.25 ± 0.12	67.28 ± 1.21	0.13 ± 0.05
Ciprofloxacin	1.15 ± 0.13	25.42 ± 1.21	—
Methotrexate	—	—	0.36 ± 0.02

The results in Table 2 showed that inhibitor 4h (with 4-methoxy and 3-nitro substituents) expressed equipotent inhibitory activity towards DNA gyrase (IC₅₀ = 1.25 ± 0.12 μM in comparison with IC₅₀ = 1.15 ± 0.13 μM for the reference drug ciprofloxacin). Among the three tested inhibitors, this compound also expressed better inhibitory activity against topoisomerase IV with an IC₅₀ value of 45.28 ± 1.21 μM, but weaker than the reference drug (with IC₅₀ = 25.42 ± 1.21 μM). Compound 4g (with a 4-methoxy substituent) had weaker activity (IC₅₀ = 1.37 ± 0.11 μM) than the standard drug as well as compound 4h. Compound 4c exhibited the weakest inhibition toward DNA gyrase with an IC₅₀ value of 2.82 ± 0.13 μM. For enzyme topoisomerase IV, compounds 4c and 4g exhibited weaker inhibitory activities than 4h (with IC₅₀ values of 68.68 ± 1.21 and 78.17 ± 1.22 μM vs. 45.28 ± 1.21), and their inhibitions were weaker when compared to ciprofloxacin (IC₅₀ = 25.42 ± 1.21 μM). These compounds displayed an increase in the suppression effect towards dihydrofolate reductase as compared with methotrexate: the tested compounds and 4h (IC₅₀ = 0.17 ± 0.03, 0.13 ± 0.03, and 0.23 ± 0.05 μM, respectively, vs. 0.36 ± 0.02 μM). These compounds also exhibited good inhibitory activity against the enzyme dihydrofolate reductase. Compound 4h showed an increase in inhibitory effects on dihydrofolate reductase when compared to methotrexate, with IC₅₀ values of 0.23 ± 0.05 μM vs. IC₅₀ = 0.36 ± 0.02 μM of the reference drug. The other two compounds showed weaker inhibitory activity for this enzyme.

Antifungal assays

The antifungal activities of thioureas 4a–h were also evaluated. Some fungi, such as Aspergillus niger, Aspergillus flavus, Candida albicans, Saccharomyces cerevisiae, and Fusarium oxysporum, were used for these evaluations. Miconazole and fluconazole were used as reference drugs. The obtained results are presented in Table 3.

Antifungal activities of compounds 4a–h.

Entry		Fungi/MIC (μg mL−1)
	R	A. niger	A. flavus	C. albicans	S. cerevisiae	F. oxysporum
4a	H	0.78	50	3.125	1.56	6.25
4b	3-NO2	3.125	0.78	100	6.25	50
4c	4-Cl	25	25	25	3.125	3.125
4d	4-Br	50	3.125	50	25	0.78
4e	4-CH3	12.5	12.5	400	0.78	200
4f	4-C2H5	200	200	6.25	400	400
4g	4-OCH3	6.25	6.25	0.78	12.5	12.5
4h	4-OCH3–3-NO2	100	1.56	12.5	200	25
	Miconazole	1.56	1.56	3.125	3.125	3.125
	Fluconazole	1.56	0.78	0.78	0.78	0.78

From this table, it is clear that electron-withdrawing groups were resistant to all tested fungi, such as miconazole and fluconazole, with MIC values of 0.78–6.25 μg mL⁻¹. Particularly, compound 4a (R = H) exhibited excellent inhibitory activity against A. niger, C. albicans and S. cerevisiae with MIC values of 0.78, 3.125, and 1.56 μg mL⁻¹, respectively. Compound 4b (with 3-nitro-substitition) showed strong inhibition toward A. flavus (MIC = 0.78 μg mL⁻¹) and remarkable inhibitory activity against A. niger with MIC = 3.125 μg mL⁻¹. Compounds 4e (R = 4-CH₃) and 4g (R = 4-C₂H₅) exhibited strong inhibitory activity against S. cerevisiae and C. albicans with MIC values of 0.78 μM mL⁻¹, respectively. Halogen substituents (Cl and Br) gave different inhibitory activities for the tested fungi. Both compounds inhibited the fungus F. oxysporum, however, compound 4d (R = 4-Br) showed stronger inhibition than compound 4c (R = 4-Cl). In addition, 4c inhibited S. cerevisiae, while 4d inhibited A. flavus. The simultaneous presence of 3-nitro and 4-methoxy substituents also significantly altered the inhibitory activity of the target compound 4h. Fig. 5 shows the comparison of the inhibitory activities of potent synthesized compounds 4a–h with the reference drug Vancomycin against S. aureus and MRSAs.

Fig. 5. Comparison of the inhibitory activities of potent synthesized compounds 4a–h with the reference drug vancomycin against S. aureus and MRSAs.

Structure–activity relationship (SAR) study for antibacterial and fungal activity

Regarding the SAR, in the MIC range of 0.78–6.25 μg mL⁻¹, the inhibitory activity against the tested bacteria by electron-withdrawing substituents (such as nitro and halogen groups) is stronger than that of electron-donating substituents (such as methyl, ethyl, and methoxy). The electron-withdrawing substituents increased the inhibition for bacteria, and the electron-donating substituents decreased this activity. This phenomenon was also observed in antifungal activity in Fig. 3–5. Compounds 4a–h could exhibit a wide spectrum of antibacterial activity (Gram-positive and Gram-negative) as well as antifungal activity. Such molecules together with negatively charged ones (due to the presence of electron-donating groups) were shown to be unable to cross the protective outer membrane of Gram-negative bacteria.⁴⁰ This explains the effects of the impaired inhibitory activities of these compounds (Fig. 6).

Fig. 3. Comparison of the inhibitory activity of synthesized compounds 4a–h with the reference drugs ciprofloxacin and vancomycin against Gram-positive bacteria.

Fig. 4. Comparison of the inhibitory activity of synthesized compounds 4a–h with the reference drug ciprofloxacin against Gram-negative bacteria.

Fig. 6. The structure–activity relationship of the bioactive compounds 4a–h.

The structure–activity relationship (SAR) results for antibacterial activity are as follows (Fig. 3 and 4):

+ The alkyl substitution reduced activity;

+ The nitro substitution increased the inhibitory activity against all tested Gram-positive and Gram-negative bacteria;

+ In the presence of the methoxy group, the inhibitory activity against Gram-positive bacteria decreased;

+ The methoxy group itself made the compound moderately active against Gram-negative bacteria and inactive against Gram-positive bacteria.

From both in vitro antibacterial (Table 1) and kinase assessment results (Table 2), we can conclude that the thiazole ring was the essential moiety for the antibacterial activity against Gram-positive bacteria. The addition of d-glucose to form a hybrid scaffold increased the antibacterial activity. Moreover, the presence of chloro and/or nitro groups significantly increased the activity in comparison with unsubstituted compound 4a.

Induced-fit docking

From the bioactive test results in the section above on ‘Antibacterial assays’, we realized that most newly synthesized d-glucose-conjugated hybrid compounds with substituted thiazole-containing thioureas exhibited good antibacterial activity, in particular against the Gram-positive bacterium S. aureus, which is one of the most dangerous bacteria among the strains of bacteria tested.⁴¹ On the other hand, the obtained results above on the inhibitory activity in vitro against some S. aureus enzymes, such as DNA gyrase, topoisomerase IV, and dihydrofolate reductase showed that these compounds were the stronger inhibitors of this bacterium. To consider the inhibitory activity of these inhibitors on the above enzymes, an induced fit docking (IFD) study was carried out for these compounds on the S. aureus gyrase 2XCS enzyme to see their correct binding mode in the active pocket of this enzyme.

Induced fit docking (IFD) was performed to accurately predict the correct binding mode of the studied better inhibitors 4c, 4g, and 4h within the catalytic pocket of 2XCS (Table 4). The worst inhibitor 4f (with 4-ethyl substituent) was also included in these IFD calculations for comparison with the better inhibitors above. The Glide-score and IFD score values of inhibitors 4c, 4f, 4g, and 4h, as well as co-crystal GSK299423, are presented in Table 4. The obtained IFD results for the settlement of inhibitors 4c, 4f, 4g, and 4h displayed in bioactive interactions of these ligands with residues in the active pocket of enzyme 2XCS are shown in Fig. 7A (4c), 7B (4f), 7C (4g), and 7D (4h), respectively. IFD calculations for the settlement of these inhibitors in the active pocket of 2XCS suggest that they were better settled when compared to co-crystal GSK299423, reflected in the more negative values of their G_score and IFD scores. Compound 4h had higher negative values for both the G_score and IFD score, which indicates that this inhibitor settles better in the active pocket of enzyme 2XCS. Because compound 4h has better antibacterial activity and molecular docking characteristics, we have focused on analysing its molecular docking energy value terms (Table 5).

IFD calculations for inhibitors 4c, 4f, 4g, and 4h in comparison with the co-crystal GSK299423 on enzyme 2XCS.

Entry	Groups	G score (kcal mol−1)	IFD score (kcal mol−1)
4c	5-Cl	−8.571	−1809.532
4f	4-Et	−7.556	−1809.956
4g	4-OMe	−9.076	−1808.679
4h	4-OMe-3-NO2	−10.943	−1814.744
	GSK299423	−7.282	−1803.088

Fig. 7. Three-dimensional diagram of the ligand interactions of 4c (A), 4f (B), 4g (C), 4h (D) and GSK299423 (E), respectively, in the active site of 2XCS showing active ligand–receptor interactions. Hydrogen bonding distances are indicated; ligands are in element-colored thick tubes in the active region surrounding the active site of residues. Residues that had active interactions are represented by thin tubes. (F) Superimpositions of 4c (in cyan colour), 4f (in green colour), 4g (in magenta colour), and 4h (red colour) with GSK299423 (in orange colour).

Induced-fit docking score and binding free energy (MM-GBSA) between the selected most active inhibitor 4b and co-crystal GSK299423 on enzyme 2XCS.

Contributions	IFD (kcal mol−1)		Contributions	MM-GBSA (kcal mol−1)
	4h	GSK299423		4h	GSK299423
G score a	−10.943	−7.282	ΔGBindi	−26.486	−31.474
G Lipo b	−3.636	−3.915	ΔGCoulj	−28.447	−7.131
G Hbond g	−0.864	−0.160	ΔGCovk	6.971	3.087
G evdW c	−63.857	−51.923	ΔGH-bondl	−0.824	−0.337
G eCoul d	−18.569	−3.558	ΔGLipom	−9.005	−13.585
G emodel e	−135.299	−74.950	ΔGPackingn	−0.290	−0.096
G energy f	−82.426	−55.481	ΔGSolvo	65.332	45.518
IFDscoreh	−1814.744	−1803.088	ΔGvdWp	−60.222	−58.930

^aGlide score.

^bGlide lipophilic contact plus phobic attractive term in the glide score.

^cGlide van der Waals energy.

^dGlide Coulomb energy.

^eGlide model energy.

^fGlide energy.

^gGlide hydrogen-bonding.

^hInduced fit docking score.

ⁱFree energy of binding.

^jCoulomb energy.

^kCovalent energy (internal energy).

^lHydrogen bonding.

^mLipophilic energy (nonpolar contribution estimated by solvent accessible surface area).

ⁿGeneralized Born electrostatic solvation energy.

^ovan der Waals energy.

^pπ–π packing energy.

This table shows that the negative value of G_emodel (−135.299 kcal mol⁻¹) had a significant weighting of the electrostatic energy components and van der Waals interactions in the OPLS4 force field. This value indicates that inhibitor 4h was located stably in the catalytic pocket of this enzyme through the high binding affinity of this inhibitor 4h with 2XCS. On the other hand, the high binding affinity of inhibitor 4h at the active site of enzyme 2XCS was also expressed through the high negative values of the G_score (−10.943 kcal mol⁻¹) and IFD score (−1814.744 kcal mol⁻¹). These values indicated that the inhibitor had more active interactions with residues in the active pocket of 2XCS than co-crystal ligand GSK299423 (Table 5). The fairly strong hydrophobic interactions of inhibitor 4h with binding sites of this enzyme were also indicated by hydrophobic forces as well as van der Waals forces, with G_Lipo = −3.636 kcal mol⁻¹ and G_evdW = −63.857 kcal mol⁻¹, respectively. This also favoured the settlement of this inhibitor in the active pocket of 2XCS. Additionally, the small value of hydrophilic force (with G_Hbond = −0.864 kcal mol⁻¹) showed that the polar interaction had a small weight in the G_score value.

The high negative value of G_emodel (−135.299 kcal mol⁻¹), which had a significant weighting of electrostatic and van der Waals energy components of the force field OPLS4, indicated that the inhibitor 4h was located stably within the catalytic pocket of this enzyme. The high negative values of the G_score (−10.943 kcal mol⁻¹) and IFD score (−1814.744 kcal mol⁻¹) also showed the high binding affinity of inhibitor 4h with 2XCS (Table 4). The favourable hydrophobic and van der Waals forces (with G_Lipo = −3.636 kcal mol⁻¹ and G_evdW = −63.857 kcal mol⁻¹, respectively) and also the hydrophilic force (with G_Hbond = −0.864 kcal mol⁻¹) showed that inhibitor 4h interacted with the 2XCS binding pocket through hydrophilic and hydrophobic interactions with residues in the active pocket of 2XCS on chains B, E, and F.

The hydrophobic interactions took place with the highly conserved residues Asp1083, Ser1084, Tyr1087, Glu1088, Val1091, GLy1117, Ala1118, Ala1119, Ala1120, Met1121, and Arg1122 on chain B. Other hydrophobic interactions were also established with residues DG10, DC11, and DC13 on chain E as well as with residues DG10, DC11, and DC12 on chain F. Of these hydrophobic interactions, three π–π stacking interactions took place from residue F:DG10 through two interactions with distances of = 3.68 and 4.15 Å, and another one from residue F:DC11 with = 3.51 Å. There were four hydrophilic interactions through H-bond interactions of ligand 4h with residues on chains B and F. One H-bond interaction between residue Ala1118 on chain B with a distance of = 2.02 Å, and another from residue Met1121 on chain B with = 1.95 Å. On chain F, residue DC11 made two H-bond interactions with distances of = 1.75 and 2.02 Å. The two-dimensional diagram of ligand interactions of 4h in the active site of 2XCS in Fig. 8B showed active ligand–receptor interactions in these cases. The hydrogen binding interactions helped this compound to be located in the active site of the receptor, including one H-bond interaction between the carbonyl bond (>C O) of the 4-acetoxy group and the N–H bond of residue Ala1118, and another one between the carbonyl bond of the 6-acetoxy group and the N–H bond of residue Arg1122. Additionally, the phosphate group of residue DC11 on chain F made two H-bond interactions with two N–H bonds of the thiourea linkage through the negatively charged oxygen atom of the P–O⁻ link to each hydrogen atom of thiourea. Residues DG10 and DC11 on chain F made three π–π stacking interactions with the thiazole ring. Residue DG10 had two interactions between imidazole and pyrimidine moieties of guanine base in residue DG10 and thiazole ring with distances of = 3.68 and 4.15 Å, respectively. Residue DC11 had one H-bond interaction between the pyrimidine ring of the cytosine base and the thiazole ring with a distance of = 3.51 Å (Table 6).

Fig. 8. (A) Docking sites of ligand 4h along with co-crystal ligand GSK299423 in the active site of S. aureus gyrase enzyme 2XCS. (B) A two-dimensional diagram of the ligand interaction of 4h in the active site of 2XCS showing active ligand–receptor interactions.

Molecular docking analysis of protein target 2XCS with ligands 4c and GSK299423.

Ligands	Hydrogen bonding interactions	Aromatic H-bond interactions	π–π stacking interactions
4c	Four interactions: from residue Met1121 (chain B, one interaction, = 2.06 Å), from residue Arg1122 (chain B, one interaction, = 2.43 Å), and from residue DG10 (chain F, two interactions, = 1.76 and 2.02 Å)	One interaction: from residue E:DC11 (one interaction with = 3.75 Å)	Four interactions: from residue E:DG10 (one interaction, = 3.99 Å), and from residue F:DG10 (two interactions, = 3.75 and 3.96 Å), and from residue E:DC11 (one interaction, = 4.08 Å)
4f	Two interactions: from residue Met1121 (chain B, one interaction, = 2.11 Å), and from residue Arg1122 (chain B, one interaction, = 2.45 Å)	Two interactions: from residue E:DC11 (two interactions with = 3.25 and 3.36 Å)	Three interactions: from residue E:DG10 (one interaction, = 4.15 Å), from residue F:DG10 (one interaction, = 4.09 Å), and from residues F:DC11 (one interaction, = 3.56 Å)
4g	Five interactions: from residue Ala1120 (chain B, one interaction, = 2.12 Å), from Arg1122 (chain B, one interaction, = 2.31 Å), from residue DA7 (chain E, one interaction, = 2.61 Å), from residue DC11 (chain F, two interactions, = 1.63 and 2.18 Å)	None	Four interactions: from residue E:DG10 (one interaction, = 4.00 Å), from residue E:DC11 (one interaction, = 3.98 Å), from residue F:DG10 (one interaction, = 4.11 Å), and from residues F:DC11 (one interaction, = 3.76 Å)
4h	Four interactions: from residue Ala1118 (chain B, one interaction, = 2.02 Å), from Met1121 (chain B, one interaction, = 1.95 Å), from residue DC11 (chain F, two interactions, = 1.75 and 2.02 Å)	None	Three interactions: from residue F:DG10 (two interactions, = 3.68 and 4.15 Å), and from residue F:DC11 (one interaction, = 3.51 Å)
GSK299423	One interaction: from residue DG10 (one interaction to 2° amino group (chain F, = 1.96 Å))	Two interactions: from residue Tyr1087, two interactions to the oxygen atom in the oxathiole moiety of [1,3]oxathiolo[5,4-c]pyridine ring of GSK299423 (chain B, = 3.44 and 3.46 Å), and another from residue Glu1088 to oxathiole C–H (chain B, = 3.72 Å)	One interaction: one interaction from residue E:DC11 ( = 3.70 Å)

To validate this docking protocol, molecular induced fit docking calculation of co-crystal ligand, GSK299423, was also performed on the active site of S. aureus gyrase 2XCS. The obtained IFD results for this ligand and inhibitor 4h are displayed in Table 5, Fig. 7 and 8. The results received showed a good correlation between their G_score and IFD score values (−10.943 vs. −7.282 kcal mol⁻¹ and −1814.744 vs. −1803.088 kcal mol⁻¹ for 4h and GSK299423, respectively), as well as component energy contributions. This shows that the 4h ligand had docked as well as the co-crystal ligand GSK299423. However, because ligand 4h contained more polar functional groups, the energy components that characterized hydrophilic interactions contributed more to the G_score value as compared to those of ligand GSK299423; for example, G_evdW = −63.857 kcal mol⁻¹vs. −51.923 kcal mol⁻¹, G_eCoul = −18.569 kcal mol⁻¹vs. −3.558 kcal mol⁻¹, G_emodel = −135.299 kcal mol⁻¹vs. −74.950 kcal mol⁻¹ for 4h and GSK299423, respectively. From Table 4, the lower G_score and IFD score values of ligand 4h when compared to GSK299423 showed that the hydrogen-binding interactions with residue Asp1083 were the decisive interactions for the docking of these ligands in the active site of 2XCS. The active pocket of 4h was also that of GSK299423 (Fig. 8A).

Binding free energy (MM-GBSA) analysis

The binding free energy values of inhibitors 4h and GSK299423 were calculated by using the MM-GBSA protocol.⁴² These values showed how these inhibitors were binding to the active site on the 2XCS enzyme. The obtained free energy of binding with a ΔG_bind value of −26.486 kcal mol⁻¹ was correlated with a negative Glide energy(G_energy) value (Table 5). The association of 4h with receptors was favored by the major contributions such as Coulomb (ΔG_Coul = −28.447 kcal mol⁻¹), lipophilic (non-polar solvation, ΔG_Lipo = −9.005 kcal mol⁻¹), and van der Waals (ΔG_vdW = −60.222 kcal mol⁻¹) energies. However, the inhibitor binding of 4h to the 2XCS enzyme was disfavoured by other energy terms, including covalent interactions with the ΔG_Cov value of 6.971 kcal mol⁻¹ and electrostatic solvation with ΔG_Solv value of 65.332 kcal mol⁻¹. Hydrogen bonding energy with ΔG_H-bond value of −0.824 kcal mol⁻¹ and π–π packing energy with ΔG_Packing value of −0.290 kcal mol⁻¹ had little favourable contributions. Furthermore, the ΔG_vdW contribution was much stronger than ΔG_Coul and ΔG_Lipo contributions. This showed that the van der Waals interaction was the driving force for the inhibitor binding to the 2XCS enzyme. This conclusion was in agreement with the IFD results (Table 5), where the G_vdW energy term with a value of −63.857 kcal mol⁻¹ also strongly favoured the inhibitor binding to this enzyme. Moreover, high negative values of ΔG_vdW and ΔG_Lipo energies represented the massive hydrophobic interaction between 2XCS and inhibitor 4h. However, π–π packing energy terms had insignificant contributions to the binding of this inhibitor to enzyme 2XCS.

MM-GBSA calculations for ligand GSK299423 were also included to compare the interaction energies of the activity of inhibitor 4h with this co-crystal. The calculation results showed that the ΔG_Bind values of both ligands are equivalent, although the component energy terms had different contribution values (Table 5). It appears that the co-crystal had polar interactions with residues in the active pocket that were inferior to inhibitor 4h, including Coulomb and H-bond interactions, with ΔG_Coul values of −28.447 kcal mol⁻¹ (4h) vs. −7.131 kcal mol⁻¹ (GSK299423), and ΔG_H-bond values of −0.824 kcal mol⁻¹ (4h) vs. −0.337 kcal mol⁻¹ (GSK299423); however, GSK299423 had a stronger hydrophobic interaction than inhibitor 4h, with ΔG_Lipo values of −9.005 kcal mol⁻¹ (4h) vs. −13.585 kcal mol⁻¹ (GSK299423). The solvation interaction of 4h was also inferior to that of GSK299423, with ΔG_Solv values of 65.332 kcal mol⁻¹ (4h) vs. 45.518 kcal mol⁻¹ (GSK299423).

Molecular dynamics simulations and analysis

Molecular dynamics simulation was applied to look at the active interactions that took place over time as inhibitors 4h approached and interacted with residues in the active pocket of enzyme 2XCS, and also to evaluate the stability of the 4h/2XCS complex of the ligand 4h and 2XCS receptor. The molecular dynamics simulation interval was 300 ns. The RMSD results of the protein (enzyme) and ligand 4h obtained were also analysed. The solvent model chosen for application was water.

The protein–ligand RMSD profile in Fig. 9A, describing the backbone complex and C-α chain, indicated the stability of this complex in about the first 130 ns in the MD 300 ns; there was a sudden increase at about 130 ns and a decrease at 170 ns and it remained stable until 300 ns. This phenomenon showed that the entire 4h/2XCS complex system was in equilibrium in this range of 170–300 ns. The records obtained, including RMSF protein and protein–ligand contact in Fig. 9B, showed that the protein had active interactions with ligand 4h through certain residues. These residues have several active interactions with ligand 4h using different hydrophilic (polar) and hydrophobic (apolar) interactions.

Fig. 9. Plots representing (A) the protein–ligand RMSD 4h/2XCS complex during MD simulation; (B) protein–ligand contacts of this complex during MD simulation; (C) frame C shows the protein–ligand profile in an interval of 200–300 ns of molecular dynamics simulation.

Some residues interacted with ligand 4h only using hydrophobic interactions, including Tyr1078 (stronger interaction, indicated by the value of fraction interactions) and Val1091 (weaker interaction), while residues Glu1088, Gly1115, Asp1116, and Arg1122 were exposed to this ligand only by water-bridge interactions. Several residue–ligand interactions occurred through both H-bond and water-bridge interactions, including the residues Gly1117, Ala1118, Ala1119, and Met1121; of these, residue Ala1118 had the strongest interactions. Residue Asp1083 interacted with ligand 4h through both water-bridge and ionic interactions, while residue Ser1084 interacted with this ligand through three types of interactions, water bridge, hydrogen bond, and ionic. Fig. 10 displays the active interactions that took place between ligand 4h and residues in the receptor of enzyme 2XCS in the interval of the 200–300 ns MD simulation.

Fig. 10. The active interactions of ligand 4h with residues in the receptor of enzyme 2XCS in an interval of 200–300 ns MD simulation.

Experimental

Melting points were determined by the open capillary method on a STUART SMP3 (BIBBY STERILIN, UK). ¹H and ¹³C NMR spectra were recorded on an Avance AV500 Spectrometer (Bruker, Germany) at 500 MHz and 125 MHz, respectively, using DMSO-d₆ as solvent and TMS as an internal standard. In the NMR spectra of most of the compounds, the residual solvent peaks were observed at δ_H = 2.50 ppm and δ_C = 39.5 ppm along with moisture peaks. ESI-mass spectra were recorded on an LC–MS LTQ Orbitrap XL, and ESI/HR-mass spectra were recorded on a Thermo Scientific Exactive Plus Orbitrap spectrometer (ThermoScientific, USA) in methanol using the ESI method. Analytical thin-layer chromatography (TLC) was performed on silica gel 60 WF₂₅₄S aluminum sheets (Merck, Germany) and was visualized with UV light or by iodine vapor. Chemical reagents with high purity were purchased from the Merck Chemical Company (in Viet Nam). All materials were of reagent grade for organic synthesis.

General procedure for the synthesis of 4-aryl thiazoles (2a–h)

Substituted 2-amino-4-phenyl-1,3-thiazoles (2a–h) were prepared by modifying the literature synthetic procedure.⁴³ Thiourea (3.04 g, 0.04 mol) and I₂ (5.08 g, 0.02 mol) were triturated and mixed with the appropriate substituted acetophenones (0.02 mol). The mixture was heated in a water bath with occasional mixing for 8 h. The obtained solid was triturated with Et₂O to remove unreacted acetophenone, washed with aqueous sodium thiosulfate to remove excess iodine and then with water. The crude product was dissolved in hot water, filtered to remove the sulphone by-product, and the desired 2-amino-4-phenylthiazoles were precipitated by the addition of 35% ammonium solution to the filtrate and crystallized from 96% EtOH to afford the titled compounds (2a–h).

General procedure for the synthesis of thioureas (4a–h)

The corresponding 2-amino-4-arylthiazoles 2a–h (1 mmol) and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate 3 (1 mmol, 389 mg) were mixed well and irradiated for about 5 min with microwave power of 750 W. The mixture became dark-yellow. It was cooled to room temperature, and water was added and the reaction mixture was stirred well. The precipitate was filtered, washed with water, and recrystallized from a mixture of 96% ethanol and toluene (1 : 1 in volume) to afford the titled thioureas 4a–h.

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-phenylthiazol-2-yl)thiourea (4a)

White solid from 2-amino-4-phenylthiazole 2a (R = H, 1 mmol, 176 mg). MW irradiation time: 30 min. Yield: 407 mg (72%). M.p. 200–202 °C (toluene-96% ethanol, 1 : 1 v/v); [α]²⁵_D + 69.0 (c = 0.25, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 11.97 (s, 1H, NH thiourea), 9.32 (s br, 1H, NH thiourea), 7.78 (d, J = 7.5 Hz, 2H, H-2 & H-6 phenyl), 7.58 (s, 1H, H-5 thiazole), 7.54 (t, J = 7.5 Hz, 2H, H-3 & H-5 phenyl), 7.34 (t, J = 7.5 Hz, 1H, H-4 phenyl), 5.92 (t, J = 9.0 Hz, 1H, H-1 glucopyranose), 5.48 (t, J = 9.5 Hz, 1H, H-3 glucopyranose), 5.05 (t, J = 9.2 Hz, 1H, H-2 glucopyranose), 5.01 (t, J = 9.5 Hz, 1H, H-4 glucopyranose), 4.19 (dd, J = 4.9, 12.4 Hz, 1H, H-6a glucopyranose), 4.15–4.13 (m, 1H, H-5 glucopyranose), 4.02 (dd, J = 2.3, 12.4 Hz, 1H, H-6b glucopyranose), 2.01, 2.00, 1.99, 1.98 (s, 4 × 3H, 4 × COCH₃ acetate); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 169.9, 169.4, 169.3, 169.3 (4 × COCH₃ acetate), 179.2 (C S thiourea), 160.1 (C-2 phenyl), 148.7 (C-4 thiazole), 107.5 (C-5 thiazole), 133.8 (C-1 phenyl), 128.7 (C-3 & C-5 phenyl), 127.9 (C-4 phenyl), 125.6 (C-2 & C-6 phenyl), 81.0 (C-1 glucopyranose), 70.5 (C-2 glucopyranose), 72.3 (C-3 glucopyranose), 67.9 (C-4 glucopyranose), 72.5 (C-5 glucopyranose), 61.7 (C-6 glucopyranose), 20.5, 20.4, 20.3, 20.2 (4 × CH₃CO acetate); EI-HRMS: C₂₄H₂₇N₃O₉S₂, calc. for [M]⁺ 597.1078 Da, found: m/z 597.1094 ([M]⁺˙).

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-(3-nitrophenyl)thiazol-2-yl)thiourea (4b)

Pale-yellow solid from 2-amino-4-(3-nitrophenyl)-1,3-thiazole 2b (R = 3-NO₂, 1 mmol, 221 mg). MW irradiation time: 30 min. Yield: 403 mg (66%). M.p. 215–216 °C (toluene-96% ethanol, 1 : 1 v/v); [α]²⁵_D + 67.0 (c = 0.25, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 12.07 (s, 1H, NH thiourea), 9.09 (s br, 1H, NH thiourea), 8.71 (d, J = 1.4 Hz, 1H, H-2 phenyl), 8.33 (d, J = 7.9 Hz, 1H, H-4 phenyl), 8.17 (dd, J = 1.8, 8.8 Hz, 1H, H-6 phenyl), 7.88 (s, 1H, H-5 thiazole), 7.74 (t, J = 8.0 Hz, 1H, H-5 phenyl), 5.88 (t, J = 9.1 Hz, 1H, H-1 glucopyranose), 5.47 (t, J = 9.5 Hz, 1H, H-3 glucopyranose), 5.05 (t, J = 9.3 Hz, 1H, H-2 glucopyranose), 5.01 (t, J = 9.5 Hz, 1H, H-4 glucopyranose), 4.20 (dd, J = 5.3, 12.5 Hz, 1H, H-6a glucopyranose), 4.13 (m, 1H, H-5 glucopyranose), 4.01 (dd, J = 2.5, 12.5 Hz, 1H, H-6b glucopyranose), 2.01, 2.00, 1.99, 1.97 (s, 4 × 3H, 4 × COCH₃ acetate); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 169.8, 169.4, 169.3, 169.8 (4 × COCH₃ acetate), 179.2 (C S thiourea), 160.4 (C-2 phenyl), 148.3 (C-4 thiazole), 109.7 (C-5 thiazole), 131.6 (C-1 phenyl), 130.2 (C-3 phenyl), 135.4 (C-5 phenyl), 146.1 (C-4 phenyl), 119.9 (C-2 phenyl), 122.2 (C-6 phenyl), 81.0 (C-1 glucopyranose), 70.5 (C-2 glucopyranose), 72.3 (C-3 glucopyranose), 68.0 (C-4 glucopyranose), 72.4 (C-5 glucopyranose), 61.6 (C-6 glucopyranose), 20.3, 20.3, 20.2, 20.2 (4 × CH₃CO acetate); EI-HRMS: C₂₄H₂₆N₄O₁₁S₂, calc. for [M + H]⁺ = 611.1243 Da, found: m/z 611.1208 ([M + H]⁺˙).

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)thiourea (4c)

White solid from 2-amino-4-(4-chlorophenyl)-1,3-thiazole 2c (R = 4-Cl, 1 mmol, 211 mg). MW irradiation time: 30 min. Yield: 348 mg (58%). M.p. 238–240 °C (toluene-96% ethanol, 1 : 1 v/v); [α]²⁵_D + 67.5 (c = 0.25, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 11.96 (s, 1H, NH thiourea), 9.31 (s br, 1H, NH thiourea), 7.82 (d, J = 8.6 Hz, 2H, H-2 & H-6 phenyl), 7.63 (s, 1H, H-5 thiazole), 7.62 (d, J = 8.6 Hz, 2H, H-3 & H-5 phenyl), 5.89 (t, J = 9.2 Hz, 1H, H-1 glucopyranose), 5.45 (t, J = 9.5 Hz, 1H, H-3 glucopyranose), 5.04 (t, J = 9.3 Hz, 1H, H-2 glucopyranose), 5.00 (t, J = 9.5 Hz, 1H, H-4 glucopyranose), 4.20 (dd, J = 5.0, 12.9 Hz, 1H, H-6a glucopyranose), 4.11 (m, 1H, H-5 glucopyranose), 3.99 (dd, J = 2.1, 12.9 Hz, 1H, H-6b glucopyranose), 2.30, 2.02, 2.00, 1.97 (s, 4 × 3H, 4 × COCH₃ acetate); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 169.9, 169.4, 169.4, 169.28 (4 × COCH₃ acetate), 179.2 (C S thiourea), 160.2 (C-2 phenyl), 147.5 (C-4 thiazole), 108.2 (C-5 thiazole), 132.6 (C-1 phenyl), 128.7 (C-3 & C-5 phenyl), 132.4 (C-4 phenyl), 127.3 (C-2 & C-6 phenyl), 81.0 (C-1 glucopyranose), 70.4 (C-2 glucopyranose), 72.3 (C-3 glucopyranose), 68.0 (C-4 glucopyranose), 72.5 (C-5 glucopyranose), 61.7 (C-6 glucopyranose), 20.4, 20.3, 20.2, 20.2 (4 × CH₃CO acetate); Elemental Anal. for C₂₄H₂₆ClN₃O₉S₂: C, 48.04; H, 4.37; N, 7.00%; found: C, 48.31; H, 4.55; N, 7.21%.

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-(4-bromophenyl)thiazol-2-yl)thiourea (4d)

White solid from 2-amino-4-(4-bromophenyl)-1,3-thiazole 2d (R = 4-Br, 1 mmol, 255 mg). MW irradiation time: 30 min. Yield: 399 mg (62%). M.p. 237–238 °C (toluene-96% ethanol, 1 : 1 v/v); [α]²⁵_D + 65.3 (c = 0.22, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 11.96 (s, 1H, NH thiourea), 9.28 (s br, 1H, NH thiourea), 7.89 (d, J = 8.6 Hz, 2H, H-2 phenyl), 7.63 (s, 1H, H-5 thiazole), 7.59 (d, 2H, J = 8.6 Hz, H-3 & H-5 phenyl), 5.89 (t, J = 9.2 Hz, 1H, H-1 glucopyranose), 5.46 (t, J = 9.5 Hz,1H, H-3 glucopyranose), 5.04 (t, J = 9.3 Hz, 1H, H-2 glucopyranose), 5.00 (t, J = 9.7 Hz, 1H, H-4 glucopyranose), 4.19 (dd, J = 3.0, 12.3 Hz, 1H, H-6a glucopyranose), 4.11–4.13 (m, 1H, H-5 glucopyranose), 3.99 (dd, J = 2.1, 12.3 Hz, 1H, H-6b glucopyranose), 2.01, 1.99. 1.97, 1.97 (s, 4 × 3H, 4 × COCH₃ acetate); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 170.0, 169.7, 169.5, 169.4 (4 × COCH₃ acetate), 179.2 (C S thiourea), 160.1 (C-2 phenyl), 147.5 (C-4 thiazole), 108.2 (C-5 thiazole), 132.4 (C-1 phenyl), 128.8 (C-3 & C-5 phenyl), (C-4 phenyl), 127.4 (C-2 & C-6 phenyl), 81.1 (C-1 glucopyranose), 70.4 (C-2 glucopyranose), 72.3 (C-3 glucopyranose), 68.0 (C-4 glucopyranose), 72.5 (C-5 glucopyranose), 61.7 (C-6 glucopyranose), 20.5, 20.4, 20.3, 20.3 (4 × CH₃CO acetate); Elemental Anal. for C₂₄H₂₆BrN₃O₉S₂: C, 44.73; H, 4.07; N, 6.52%; found: C, 44.91; H, 4.29; N, 6.34%.

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-(4-tolyl)thiazol-2-yl)thiourea (4e)

White solid from 2-amino-4-(4-tolyl)-1,3-thiazole 2e (R = 4-CH₃, 1 mmol, 190 mg). MW irradiation time: 30 min. Yield: 394 mg (68%). M.p. 231–233 °C (toluene-96% ethanol, 1 : 1 v/v); [α]²⁵_D + 66.3 (c = 0.24, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 11.95 (s, 1H, NH thiourea), 9.26 (s br, 1H, NH thiourea), 7.75 (d, J = 8.0 Hz, 2H, H-2 & H-6 phenyl), 7.59 (s, 1H, H-5 thiazole), 7.24 (d, J = 8.0 Hz, 2H, H-3 & H-5 phenyl), 5.91 (t, J = 9.1 Hz, 1H, H-1 glucopyranose), 5.46 (t, J = 9.5 Hz, 1H, H-3 glucopyranose), 5.03 (t, J = 9.3 Hz, 1H, H-2 glucopyranose), 4.99 (t, J = 9.7 Hz, 1H, H-4 glucopyranose), 4.19 (dd, J = 4.8, 12.4 Hz, 1H, H-6a glucopyranose), 4.11–4.13 (m, 1H, H-5 glucopyranose), 3.99 (dd, J = 1.8, 12.4 Hz, 1H, H-6b glucopyranose), 2.02, 1.99, 1.97, 1.97 (s, 4 × 3H, 4 × COCH₃ acetate), 2.33 (s, 3H, 4-CH₃ phenyl); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 170.0, 170.0, 169.5, 169.4 (4 × COCH₃ acetate), 179.2 (C S thiourea), 160.1 (C-2 phenyl), 148.9 (C-4 thiazole), 106.7 (C-5 thiazole), 137.4 (C-1 phenyl), 129.4 (C-3 & C-5 phenyl), 137.4 (C-4 phenyl), 125.6 (C-2 & C-6 phenyl), 81.1 (C-1 glucopyranose), 70.5 (C-2 glucopyranose), 72.4 (C-3 glucopyranose), 68.0 (C-4 glucopyranose), 2.5 (C-5 glucopyranose), 61.7 (C-6 glucopyranose), 20.8, 20.5, 20.4, 20.3 (4 × CH₃CO acetate), 18.5 (4-CH₃ phenyl); Elemental Anal. for C₂₅H₂₉N₃O₉S₂: C, 51.80; H, 5.04; N, 7.25%; found: C, 51.67; H, 5.31; N, 7.51%.

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-(4-ethylphenyl)thiazol-2-yl)thiourea (4f)

White solid from 2-amino-4-(4-ethylphenyl)-1,3-thiazole 2f (R = 4-C₂H₅, 1 mmol, 204 mg). MW irradiation time: 30 min. Yield: 415 mg (70%). M.p. 202–203 °C (toluene-96% ethanol, 1 : 1 v/v); [α]²⁵_D + 68.3 (c = 0.25, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 11.95 (s, 1H, NH thiourea), 9.34 (s br, 1H, NH thiourea), 7.79 (d, J = 8.0 Hz, 2H, H-2 & H-6 phenyl), 7.50 (s, 1H, H-5 thiazole), 7.27 (d, J = 8.0 Hz, 2H, H-3 & H-5 phenyl), 5.92 (t, J = 9.1 Hz, 1H, H-1 glucopyranose), 5.47 (t, J = 9.5 Hz, 1H, H-3 glucopyranose), 4.04 (t, J = 9.3 Hz, 1H, H-2 glucopyranose), 5.02 (t, J = 9.6 Hz, 1H, H-4 glucopyranose), 4.12 (dd, J = 5.0, 12.1 Hz, 1H, H-6a glucopyranose), 4.13 (m, 1H, H-5 glucopyranose), 4.00 (dd, J = 1.9, 12.1 Hz, 1H, H-6b glucopyranose), 2.02, 1.99, 1.97, 1.97 (s, 4 × 3H, 4 × COCH₃ acetate), 2.50 (q, J = 7.0 Hz, 2H, 4-CH₂CH₃ phenyl), 1.22 (t, J = 7.0 Hz, 3H, 4-CH₂CH₃ phenyl); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 169.9, 169.9, 169.4, 16.3 (4 × COCH₃ acetate), 179.2 (C S thiourea), 160.0 (C-2 phenyl), 148.8 (C-4 thiazole), 106.7 (C-5 thiazole), 131.3 (C-1 phenyl), 128.9 (C-3 & C-5 phenyl), 143.6 (C-4 phenyl), 125.6 (C-2 & C-6 phenyl), 81.0 (C-1 glucopyranose), 70.5 (C-2 glucopyranose), 72.3 (C-3 glucopyranose), 68.0 (C-4 glucopyranose), 72.4 (C-5 glucopyranose), 61.7 (C-6 glucopyranose), 20.5, 20.4, 20.3, 20.3 (4 × CH₃CO acetate), 27.5 (4-CH₂CH₃ phenyl), 15.4 (4-CH₂CH₃ phenyl); Elemental Anal. for C₂₆H₃₁N₃O₉S₂: C, 52.60; H, 5.26; N, 7.08%; found: C, 52.87; H, 5.51; N, 7.35%.

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-(4-methoxyphenyl)thiazol-2-yl)thiourea (4g)

Light-purple solid from 2-amino-4-(4-methoxyphenyl)-1,3-thiazole 2g (R = 4-OCH₃, 1 mmol, 206 mg). MW irradiation time: 30 min. Yield: 524 mg (88%). M.p. 216–218 °C (toluene-96% ethanol, 1 : 1 v/v); [α]²⁵_D + 68.2 (c = 0.25, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 11.94 (s, 1H, NH thiourea), 9.38 (s br, 1H, NH thiourea), 7.81 (d, J = 8.6 Hz, 2H, H-2 & H-6 phenyl), 7.51 (s, 1H, H-5 thiazole), 6.91 (d, J = 8.6 Hz, 2H, H-3 & H-5 phenyl), 5.92 (t, J = 9.0 Hz, 1H, H-1 glucopyranose), 5.47 (t, J = 9.3 Hz, 1H, H-3 glucopyranose), 5.04 (t, J = 9.1 Hz, 1H, H-2 glucopyranose), 5.00 (t, J = 9.5 Hz, 1H, H-4 glucopyranose), 4.19 (dd, J = 4.9, 12.6 Hz, 1H, H-6a glucopyranose), 4.12–4.14 (m, 1H, H-5 glucopyranose), 4.00 (dd, J = 2.3, 12.6 Hz, 1H, H-6b glucopyranose), 2.02, 1.99, 1.97, 1.97 (s, 4 × 3H, 4 × COCH₃ acetate), 3.79 (s, 3H, 4-OCH₃ phenyl); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 169.9, 169.9, 169.4, 169.3 (4 × COCH₃ acetate), 179.2 (C S thiourea), 159.1 (C-2 phenyl), 149.0 (C-4 thiazole), 105.5 (C-5 thiazole), 131.4 (C-1 phenyl), 127.0 (C-3 & C-5 phenyl), 161.0 (C-4 phenyl), 114.1 (C-2 & C-6 phenyl), 81.1 (C-1 glucopyranose), 70.4 (C-2 glucopyranose), 72.3 (C-3 glucopyranose), 70.0 (C-4 glucopyranose), 72.5 (C-5 glucopyranose), 61.9 (C-6 glucopyranose), 20.5, 20.4, 20.3, 20.3 (4 × CH₃CO acetate), 55.1 (4-OCH₃ phenyl); Elemental Anal. for C₂₅H₂₉N₃O₁₀S₂: C, 50.41; H, 4.91; N, 7.05%; found: C, 50.69; H, 4.67; N, 7.36%.

1-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-3-(4-(4-methoxy-3-nitrophenyl)thiazol-2-yl)thiourea (4h)

Yellow solid from 4-(4-methoxy-3-nitrophenyl)-1,3-thiazol-2-amine 2h (R = 4-OCH₃–3-NO₂, 1 mmol, 206 mg). MW irradiation time: 30 min. Yield: 410 mg (64%). M.p. 230–232 °C (toluene-96% ethanol, 1:1 v/v); [α]²⁵_D + 67.8 (c = 0.25, CHCl₃). ¹H NMR (500 MHz, DMSO-d₆), δ (ppm): 12.01 (s, 1H, NH thiourea), 9.11 (s br, 1H, NH thiourea), 8.38 (d, J = 0.9 Hz, 1H, H-2 phenyl), 8.16 (dd, J = 2.1, 8.3 Hz, 1H, H-6 phenyl), 7.66 (s, 1H, H-5 thiazole), 7.54 (d, J = 8.9 Hz, 1H, H-5 phenyl), 5.89 (t, J = 9.1 Hz, 1H, H-1 glucopyranose), 5.48 (t, J = 9.6 Hz, 1H, H-3 glucopyranose), 5.05 (t, J = 9.5 Hz, 1H, H-2 glucopyranose), 5.01 (t, J = 9.5 Hz, 1H, H-4 glucopyranose), 4.20 (dd, J = 5.2, 12.6 Hz, 1H, H-6a glucopyranose), 4.13–4.14 (m, 1H, H-5 glucopyranose), 4.02 (dd, J = 2.4, 12.4 Hz, 1H, H-6b glucopyranose), 2.01, 2.00, 1.99, 1.98 (s, 4 × 3H, 4 × COCH₃ acetate), 3.98 (s, 3H, 4-OCH₃ phenyl); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 169.8, 169.4, 169.3, 169.1 (4 × COCH₃ acetate), 179.2 (C S thiourea), 160.3 (C-2 phenyl), 146.2 (C-4 thiazole), 107.7 (C-5 thiazole), 131.1 (C-1 phenyl), 126.6 (C-3 phenyl), 139.3 (C-5 phenyl), 151.5 (C-4 phenyl), 114.7 (C-2 phenyl), 121.9 (C-6 phenyl), 81.0 (C-1 glucopyranose), 70.4 (C-2 glucopyranose), 72.3 (C-3 glucopyranose), 68.0 (C-4 glucopyranose), 72.4 (C-5 glucopyranose), 61.6 (C-6 glucopyranose), 20.3, 20.2, 20.2, 20.1 (4 × CH₃CO acetate), 56.8 (4-OCH₃ phenyl); Elemental Anal. for C₂₅H₂₈N₄O₁₂S₂: C, 46.87; H, 4.41; N, 8.75%; found: C, 46.94; H, 4.60; N, 8.46%.

Biological assays

In vitro antimicrobial activity

All synthesized thioureas 4a–h were evaluated for their in vitro antibacterial activity against Gram-positive and Gram-negative bacteria. The Gram-positive bacteria were B. subtilis, C. difficile, S. aureus, S. epidermidis, and S. pneumoniae; Gram-negative bacteria were Escherichia coli, K. pneumoniae, P. aeruginosa, and S. typhimurium. The minimum inhibitory concentration (MIC) was used for the above evaluation. The microbroth dilution technique was applied using Mueller–Hinton broth.⁴⁴ The drug references were ciprofloxacin, methicillin and vancomycin. The solutions with concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μg mL⁻¹ were prepared by further diluting the test compounds and standard drugs prepared above. The inoculum was prepared using a 4–6 h broth adjusted to the turbidity equivalent of an 0.5 McFarland standard, diluted in broth media to give a final concentration of 5 × 10⁵ CFU mL⁻¹ in the test tray. The plates were incubated at 35 °C for 18–20 h. The MIC was defined as the lowest concentration of the compound giving complete inhibition of visible growth. All the experiments were performed three times.

In vitro antifungal activity

All synthesized compounds 4a–h were examined for their in vitro antifungal activity against the following fungi: A. niger, A. flavus, S. cerevisiae, C. albicans, S. cerevisiae, and F. oxysporum. The agar dilution method with Saburoud's dextrose agar (Hi-Media) was used. The drug references were miconazole and fluconazole. Solutions with concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μg mL⁻¹ of each tested compound and standard drugs, were prepared. Suspensions of each microorganism were prepared to contain 10 CFU mL⁻¹ and applied to agar plates that had been serially diluted with the compounds to be tested. The plates were incubated at 35 °C. After 72 h, the MICs were determined.⁴⁴ Minimal inhibitory concentrations for each compound were investigated against standard fungal strains. All the experiments were performed three times.

In vitro inhibition of DNA gyrase, topoisomerase IV, and dihydrofolate reductase from S. aureus

The in vitro enzyme inhibitory activities of compounds 4c, 4g, and 4h against S. aureus DNA gyrase, topoisomerase IV, and dihydrofolate reductase enzymes were determined using ciprofloxacin and methotrexate as reference drugs according to previously reported methods.^45,46

Molecular docking

Ligand and protein preparation

The two-dimensional structures (.mae) of compounds (ligands) 4c, 4f, 4g, and 4h were drawn and the structure was analysed by using the 2D sketcher and 3D builder of Maestro 12.8 (Schrödinger, LLC, New York, NY, USA).⁴⁷ The three-dimensional structures of these compounds (ligands) were generated from three-dimensional structures prepared by a conformational search tool using the OPLS4 force field for geometrical minimization with MacroModel 13.2 followed by conformational analysis using the MMFFs force field. The Monte Carlo Multiple Minimum (MCMM) conformational search was used with 2500 iterations and a convergence threshold of 0.05 kJ mol⁻¹. Water was chosen as the solvent. Truncated Newton Conjugate Gradient minimization was used with 2500 iterations and a convergence threshold of 0.05 kJ mol⁻¹. Other parameters were used as default. The crystal structures of two enzymes, including S. aureus gyrase complexed with GSK299423 and DNA, were retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/structure/2XCS, PDB ID: 2XCS). These structures were solved by X-ray crystallography at the resolution of 2.10 Å. Coordinates of the protein–ligand complexes were fixed for errors in atomic representations and optimized using the Protein Preparation Wizard (v. 12.8). The crystallographic water molecules W2035, W2120, and W2162, which are close to the native ligand and hydrogen bonded, were retained, while the remaining water molecules were removed. All Mn²⁺ ions and 5UA (5′-O-carboxy-2′-deoxyadenosine) were also removed. The bond orders were assigned and the protein structure was protonated at pH 7.0 ± 2.0. The missing side chain atoms were added and breaks present in the protein structure were repaired with Prime. Hydrogens of altered species were minimized with PROPKA at pH 7.0 and a restrained energy minimization was carried out on the full atomic model with the OPLS4 force field until the root mean square deviation (RMSD) of heavy atoms converged to 0.30 Å.⁴⁸

Induced fit docking

The two-dimensional structures (.mae) of compounds (ligands) 4c, 4f, 4g, and 4h were drawn and the structure was analyzed by using a 2D sketcher and 3D builder tools of Maestro 12.8 and optimized with the LigPrep module in the Schrodinger 2021-2 suite.⁴⁷ The OPLS4 force field was applied and other parameters were used as default; the specified chirality was retained and possible states were generated with Epik (v. 5.6).⁴⁸ The energy minimizations were carried out with this force field till the root mean square deviation (RMSD) of 0.01 Å was reached. The generated low-energy conformation thus obtained was used for the modelling studies. To perform molecular docking calculations, the crystal structure of the S. aureus gyrase complex with GSK299423 and DNA (PDB ID: 2XCS, resolution 2.1 Å)^49,50 was retrieved. Protein was prepared by the Protein Preparation Wizard tool. The all-crystallographic water molecules and four Mn²⁺ ions were removed. The bond orders were assigned and the protein structure was protonated at pH 7.0 ± 2.0 using the Epik tool. The missing side chain atoms were added and breaks present in the protein structure were repaired with Prime.⁴² Hydrogens of altered species were minimized with PROPKA at pH 7.0. Restrained energy minimization was carried out on the full atomic model with the OPLS4 force field⁴⁸ until the root mean square deviation (RMSD) of heavy atoms converged to 0.30 Å. A 10 Å distance around the center of the co-crystallized ligand was used to define the active site and a 3D grid box was generated.

The accurate prediction of the three-dimensional arrangement of 4h inhibitors in the 4h/2XCS complex was carried out by IFD⁵¹ using the Refinement module in Prime.⁵² IFD accounts for both receptor and ligand flexibility and uses reduced van der Walls radii and an increased Coulomb–van der Waals cutoff, and highly flexible side chains are temporarily removed in the course of the docking process. The low energy conformation of prepared ligands 4h, respectively, was docked into the study model with standard sampling. The scaling factor was kept at 0.5 to soften the potentials of the receptor and ligand and a maximum of 40 poses was saved. The IFD scores, which account for both the protein–ligand interaction energy and the total energy of the system, were calculated to rank the IFD poses.

MM-GBSA binding free energy calculation

The free energy of binding was evaluated by the MMGBSA method⁵² using Prime (v5.4)⁵³ implemented in Schrodinger 2021-2. This method includes a combination of the OPLS4 force-field⁴⁸ molecular mechanics energies, a VSGB 2.1 prime energy polar solvation model, and a non-polar solvation term comprised of the non-polar solvent-accessible surface area and van der Waals interactions. The IFD best poses of the 4h/2XCS complex were subjected to energy minimization with a local optimization feature in Prime and then simulation was performed using the VSGB 2.0 energy model⁵⁴ with input ligand partial charges without applying any constraint on flexible residues. The energies of these complexes were calculated with an OPLS4 force field using the GBSA implicit solvation model.

Conclusions

In summary, some thioureas containing thiazole and d-glucose moieties were designed and synthesized. These compounds exhibited remarkable antibacterial and antifungal activity. The obtained results showed that they displayed significant inhibition in vitro against most of the tested bacteria and fungi; of these compounds, 4c, 4g, and 4h were the best inhibitors. These three compounds were also tested for their inhibition against S. aureus enzymes, including two enzymes of bacterial topoisomerase type II, DNA gyrase and DNA topoisomerase IV (Topo IV). Induced-fit docking and MM-GBSA calculation studies were performed to observe the binding efficiency and steric interactions of these compounds. The obtained results showed that compound 4h is compatible with the active site of S. aureus DNA gyrase 2XCS with four H-bond interactions with residues Ala1118, Met1121, and F:DC11, and also three interactions to F:DG10 (two interactions) and F:DC11 (one interaction). Molecular dynamics simulation in a water solvent system showed that ligand 4h had active interactions with enzyme 2XCS through residues Ala1083, Glu1088, Ala1118, Gly1117, and Met1121.

Author contributions

All authors have the same contributions for preparation this article.

Conflicts of interest

There are no conflicts to declare.