Research Article | Open Access
Zainab Ngaini, Wan Sharifatun Handayani Wan Zulkiplee, Ainaa Nadiah Abd Halim, "One-Pot Multicomponent Synthesis of Thiourea Derivatives in Cyclotriphosphazenes Moieties", Journal of Chemistry, vol. 2017, Article ID 1509129, 7 pages, 2017. https://doi.org/10.1155/2017/1509129
One-Pot Multicomponent Synthesis of Thiourea Derivatives in Cyclotriphosphazenes Moieties
In this study, hexasubstituted thiourea was carried out via reaction of isothiocyanato cyclophosphazene intermediates with a series of aromatics amines and amino acids in a one-pot reaction system. The reaction was not as straightforward as typical thiourea synthesis. Six unexpected thiourea derivatives 3a–f were formed in the presence of cyclotriphosphazene moieties in good yields (53–82%). The structures of 3a–f were characterized by elemental analysis and FTIR, 1H, 13C, and 31P NMR spectroscopies. The occurrence of reverse thioureas formation in a one-pot reaction system is discussed. The possible binding interaction of the synthesised thiourea 3a-b in comparison to the predicted phenyl thiourea 5a-b and the targeted 4a with enzyme enoyl ACP reductase (FabI) is also discussed. Molecular docking of the targeted hexasubstituted thiourea 4a is able to give higher binding affinity of −7.5 kcal/mol compared to 5a-b (−5.9 kcal/mol and −6.3 kcal/mol) and thiourea 3a-b (−4.5 kcal/mol and −4.7 Kcal/mol).
Thiourea is widely studied and claimed to be used in many applications such as herbicides, pharmaceutical agents, pesticides, rodenticides, vulcanization accelerator, and scaffolds in organic synthesis . In the synthesis of thiourea, isothiocyanate is formed as a reactive intermediate and easily converted to other side product during isolation . Many studies reported on the direct reaction of isothiocyanate intermediate with amines after isolation of KCl to produce thiourea in good purity .
Several studies reported on monosubstituted thiourea which consists of one thiourea moiety either as a ligand bearing aromatic, halogen, or alkyl substituents  or as a complex compound coordinated with heavy metal center . Multisubstituted thioureas have gained more interest among researchers due to the increase of their pharmaceutical properties. Our recent studies on thiourea reported that compounds that consist of more than one thiourea moiety possess better antimicrobial activities [6–8]. It was due to the presence of more active sites of thiourea moieties containing C=S, C=O, and N-H groups, which are easily protonated under acidic condition and interacted with the carboxyl and phosphate groups of the bacterial surfaces, thus enhancing the biological activities . Various methods have been reported to make this versatile group of thiourea derivatives easily accessible with excellent yields [2, 9–12].
Hexakisphosphazenes bearing thioureas moieties have been reported from the stepwise reaction of the isolated isothiocyanate intermediates with a series of aliphatic amines via P-Cl substitution of hexachlorocyclotriphosphazene . Hexachlorocyclotriphosphazene, a cyclic inorganic compound with alternating phosphorus and nitrogen atoms, has sparked great interest among researchers for an excellent candidate in constructing hexasubstituted molecules [14, 15]. The substitution of P-Cl bonds with various types of nucleophiles allowed the construction of phosphazenes-based ligands with different types of physical and chemical properties . A wide range of hexasubstituted phosphazene derivatives with various substituents such as hydroxyl, amino, and many other functional groups had been reported [13, 15, 16].
To the best of our knowledge, no studies reported on the synthesis of hexasubstituted thiourea onto cyclotriphosphazene moieties bearing six units of amino acid or aromatic amines. Our previous studies reported on thiourea bearing aromatic amine with excellent antibacterial properties [6, 17]. In continuation to our previous work, in this article, we report on the synthesis of thiourea compounds with hexachlorocyclotriphosphazene as a hexasubstituted precursor in a typical one-pot reaction system . The plausible mechanism which leads to the unexpected final products is discussed. The binding interaction of the synthesised thiourea via molecular docking interaction in comparison to predicted phenyl thiourea and the targeted compound with enzyme enoyl ACP reductase (FabI) is also thoroughly discussed.
2. Materials and Methods
Hexachlorocyclotriphosphazene (99%) was purchased from Aldrich. Potassium thiocyanate, aniline, ρ-toluidine, ρ–anisidine, glycine, L-alanine, and L-phenyl alanine were obtained from Merck and used without purification. Acetone was distilled over magnesium sulphate anhydrous. All other reagents and solvent were used as received.
Physical Measurement. Melting points were determined by the open tube capillary method and were uncorrected. Infrared spectra (/cm−1) were recorded as KBr pellets on a Perkin Elmer 1605 FTIR spectrophotometer. 1H and 13C NMR spectra were recorded on a JEOL ECA 500 spectrometer at 500 MHz (1H) and 125 MHz (13C), respectively, with the chemical shifts (ppm) being reported relative to DMSO-d6 as standard. The chemical shifts for 31P NMR are relative to the internal standard of 85% phosphoric acid. CHNS microanalyses were performed by use of a FLASHEA 1112 CHNS analyser.
2.1. General Procedure for the Synthesis of 3a–f
A mixture of hexachlorocyclotriphosphazene (0.35 g, 1.0 mmol) in dry acetone (15.0 mL) was added dropwise into a solution of potassium thiocyanate (0.87 g, 9.0 mmol) in dry acetone (15.0 mL). The mixture was stirred for 1 h at room temperature to form intermediate 2. The white potassium chloride (KCl) was filtered. The filtrate was added to amine (6.0 mmol) in dry acetone (15.0 mL) and heated under reflux for 18 h. The mixture was cooled to room temperature and filtered. The filtrate was evaporated in vacuum to form a yellowish powder. The crude was recrystallized in EtOH : CH3CN (1 : 1). The general procedure for the preparation of 3a–f utilised a different type of amines (g, mmol) and yields as follows.
2-(Carbamothioylamino) Propanoic Acid (3e). L-alanine (0.534 g, 6 mmol). (62% yield) as a yellow powder, m.p: 138.8–139.5°C; (KBr/cm−1) 3229 (OH) 3010 (NH), 2968 (CH), 1698 (COOH) 1226 (C=S). (500 MHz, DMSO-d6) 1.59 (3H, d, J = 6.8, CH3), 5.10 (1H, q, CH), 10.11 (2H, s, NH2), 10.54 (1H, s, NH). (125 MHz, DMSO-d6) 17.0 (CH3), 61.9 (CH), 173.5 (COOH), 180.6 (C=S). Calculated for C4H8N2O2S: C, 32.40; H, 5.40; N, 18.90; S, 21.60%, found C, 31.74; H, 4.98; N, 18.79; S, 21.64%.
2-(Carbamothioylamino)-3-phenyl-propanoic Acid (3f). L-phenyl alanine (0.990 g, 6 mmol). (61% yield) as a yellow crystal, m.p: 198.3–198.9°C; (KBr/cm−1) 3172 (OH) 3100 (NH), 2911 (CH), 1740 (COOH) 1452 (Ar-C), 1249 (C=S). (500 MHz, DMSO-d6) 3.89 (2H, d, J = 13.8, CH2), 5.42 (1H, q, CH) 7.05 (2H, d, J = 6.3, Ar-H), 7.28 (3H, m, Ar-H), 10.19 (2H, s, NH2), 10.63 (1H, s, NH). (125 MHz, DMSO-d6) 35.9 (CH2), 67.0 (CH), 127.8, 128.9, 129.8, 134.5 (Ar-C), 172.5 (COOH), 179.9 (C=S). Calculated for C10H12N2O2S: C, 53.60; H, 5.40; N, 12.50; S, 14.30%, found: C, 53.35; H, 5.31; N, 12.17; S, 14.02%.
2.2. Antibacterial Screening
Antibacterial activities of 3a–f were analysed against E. coli (ATCC 8739) using the turbidimetric kinetic method. The Gram-negative E. coli were cultured on a Luria-Bertani plate agar at 37°C. Then a colony of the inoculums was transferred and allowed to grow in media containing nutrient broth at 37°C with permanent stirring at 250 rpm for overnight. 0.2 mL of inoculums was inoculated with 10 mL of culture medium that has been added with increasing concentration of synthesised compounds dissolved in DMSO. The mixture was shaken at 180 rpm at 37°C. The negative control was medium broth of inoculums with solvent. The aliquots of each replicate were taken on every 1 h interval for 6 h. The transmittance () was recorded using UV-Visible Spectrophotometer Optima SP-300. The antibacterial activity was determined by plotting a graph of versus time. The value represents the number of colony forming units/mL which followed the expression of .
2.3. Molecular Docking
Molecular docking studies on the series of 3a-b, 4a, and 5a-b were carried out using AutoDock Vina 1.1.2 program . The polar hydrogens of the synthesised compounds and protein were added with AutoDock Tools 1.5.6  before docking using Auto-Dock Vina program. In Auto-Dock Vina program, the cubic grid box of 60°A sizes (, , and ) with a spacing of 0.375°A was centered to the active site of the protein. The X-ray crystal structure of the enzyme enoyl ACP reductase (FabI) of E. coli (PDB entry: 1C14) was obtained from Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) [7, 24].
3. Results and Discussion
The synthesis of the proposed hexasubstituted thioureas 4a–f was prepared via reaction of hexachlorocyclotriphosphazene with potassium thiocyanate to form isothiocyanates phosphazene intermediates, followed by typical thiourea reaction with a series of amines derivatives in a one-pot reaction system. All compounds were subjected to IR spectroscopy and showed the disappearance of (NCS) at 2140–1990 cm−1 and the formation of (N-H) at 3276–3010 cm−1. The formation of thiourea was evidenced by the strong absorption peak at 1265–1227 cm−1 corresponding to (C=S) which shifted to the lower frequency due to the attachment of more electronegative nitrogen atoms . The absorbance peak attributed to the formation of (P=N) asymmetric vibration at 1400–1200 cm−1 [26, 27] but, however, was not observed. This phenomenon was also transpired in 31P NMR spectra where no phosphorus moieties were present.
Further characterization of the synthesised compounds via 1H NMR showed the presence of thiourea (-NHCSNH-) represented by two NH peaks at 9.80–9.30 ppm and 3.33–3.30 ppm. The higher resonance of NH peaks in 3d–f at 10.84–10.53 ppm and 10.18–10.01 ppm was due to downfield effect resulting from the formation of intramolecular hydrogen bond between the hydrogen atom from thiourea moieties with oxygen atom from the carboxylic acid group . 13C NMR spectra of compound 3a–f showed good agreement with the corresponded structures with the presence of C=S peak at 181.2–180.9 ppm [28–30].
Elemental analysis of the synthesised compounds afforded low carbon percentage in each compound which indicated the formation of 3a–f. Based on the IR, 1H, 13C, and 31P NMR spectra, it was suggested that 3a–f were synthesised in one-pot reaction system and not the targeted molecule 4a–f (Scheme 1).
The presence of hexachlorocyclotriphosphazene in a one-pot reaction system is envisaged, not only forming isothiocyanate intermediate 2 via P-Cl substitution but also generating Cl− from the partially soluble KCl in acetone . The free chlorine ions deprotonate amines in the reaction system and form HCl and anionic amines, which in turn reacted with hydrogen thiocyanate  and formed 3a–f. The plausible mechanism for the formation of 3a–f is shown in Scheme 2.
3.2. Antibacterial Activity
Compounds 3a–f were further investigated for antibacterial activities by plotting the graph of versus time. Compounds 3a–f were examined at the concentration of 50 ppm, 80 ppm, and 100 ppm against wild-type E coli at 37°C. The result indicated that compounds 3a–f showed poor inhibition against E. coli. The MIC graph for compounds 3a–f as shown in Figure 1 was determined by extrapolating the concentration at the zero-growth rate of E. coli (μ = 0) . The MIC values for all compounds 3a–f were observed to exceed 220 ppm. Compounds with MIC value up to 400 ppm are considered to have inhibition activity against growth of Gram-negative bacteria, but only compounds with MIC value smaller than 220 ppm can be suggested for clinical purposes .
Like other typical Gram-negative bacteria, the cell wall of E. coli is made up from thin layer of peptidoglycan and an outer membrane constituted of lipopolysaccharide, lipoprotein, and phospholipids . In view of this, the large molecular weight compound is required to coat the cell surface and prevent the leakage of intercellular components of the bacteria .
3.3. Molecular Docking Design and Optimisation
For a better understanding of the interaction between thiourea derivatives and Gram-negative bacteria E. coli, molecular docking studies were carried out and optimised by comparing 3a-b with the predicted phenyl thiourea 5a-b and the targeted 4a. The studies were carried out via molecular docking to the active site of the enzyme enoyl ACP reductase (FabI) of E. coli (PDB entry: 1C14) using AutoDock Vina 1.1.2 program [7, 22–24]. The compounds and binding interactions are shown in Table 1. The binding affinity of the compounds was evaluated based on binding free energies (, kcal/mol) .
The binding model of thiourea and the predicted phenyl thiourea 5a-b is depicted in Table 1. Compounds 3a-b showed binding free energy of −4.5 kcal/mol and −4.7 kcal/mol, respectively. Based on the importance properties of the aromatic group in earlier studies [6–8], the optimisation study via molecular docking was carried out to evaluate the binding free energy of 3a-b in comparison to the predicted phenyl thiourea 5a-b. The presence of another aromatic group in 5a-b demonstrated for a higher binding affinity with the free energy of −5.9 kcal/mol and −6.3 kcal/mol, respectively. The additional aromatic group in 5a-b is strongly bound to enzyme enoyl ACP reductase (FabI) of E. coli through - bond interactions (yellow colour cylindrical wireframe) with hydrophobic pockets of Phe 1251. The hydrophobic interaction between phenyl rings has increased the lipophilicity of the compound [7, 33]. The binding affinity of 5b is slightly higher than 5a due to the electron donating inductive effect of the substituted methyl group, which provides better interactions network with the active site residues . The absence of aromatic ring was accountable for lesser binding affinity resulting in less activity in 3a-b .
Due to the importance of phenyl groups for a better binding affinity, it is noteworthy to analyse the significance of hexasubstituted thiourea moieties onto cyclotriphosphazene 4a. Based on Table 1, the presence of six thiourea moieties in 4a showed the highest binding affinity with a free energy of −7.5 kcal/mol. Apart from the - bond interactions with Phe 1251, 4a was observed to interact with the enzyme via two hydrogen bonds (green colour sphere). The NH groups in 4a are forming hydrogen bonding with C=O and NH of Ala 1152. The bonding provides specificity and stabilisation of binding between 4a and enzyme active site which consequently enhanced the binding affinity [38, 39]. Other basic residues such as Pro 1154, Ile 1153, Val 1213, Ala 1254, Hoh 2087, Hoh 2067, Arg 171, and Gly 242 were observed in the vicinity of compound 4a, which suggested that a strong electrostatic interaction was also involved in the binding process .
In summary, the thiourea derivatives 3a–f were unexpectedly synthesised from the reaction of amines with excess thiocyanates groups in a one-pot reaction system. The isolation of isothiocyanato cyclophosphazene intermediates could be the best method to give hexasubstituted thioureas. The formation of HCl in the reaction condition was envisaged to be responsible for the deprotonation of amines, thus reducing the possible formation of hexasubstituted thioureas. Biological activities of thiourea 3a–f showed poor inhibitions towards E. coli. Molecular docking interaction study thoroughly explained the binding interactions of the selected thiourea 3a-b compared to the binding affinity with the predicted 5a-b and the targeted 4a. Based on the molecular docking study, it can be concluded that the targeted hexasubstituted thiourea as in 4a is envisaged to give better binding affinity compared to monothiourea 3a–f.
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors would like to acknowledge Universiti Malaysia Sarawak and the Ministry of Higher Education for financial support through C09/SpSTG/1359/16/1 and FRGS/ST0l(0l)/1298/2015(15). They acknowledge Universiti Malaysia Terengganu, Malaysia, for providing CHNS elemental analysis services.
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