One-Pot Multicomponent Synthesis of Thiourea Derivatives in Cyclotriphosphazenes Moieties

Article abstract of DOI:10.1155/2017/1509129

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).

Full text of DOI:10.1155/2017/1509129

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Hindawi
Journal of Chemistry
Volume 2017, Article ID 1509129, 7 pages
https://doi.org/10.1155/2017/1509129
Research Article
One-Pot Multicomponent Synthesis of Thiourea Derivatives in
Cyclotriphosphazenes Moieties
Zainab Ngaini,1 Wan Sharifatun Handayani Wan Zulkiplee,2
and Ainaa Nadiah Abd Halim1
1Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
2Centre for Pre-University Studies, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Correspondence should be addressed to Zainab Ngaini; nzainab@unimas.my
Received 15 March 2017; Revised 21 April 2017; Accepted 23 April 2017; Published 3 July 2017
Academic Editor: Liviu Mitu
Copyright © 2017 Zainab Ngaini et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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. e 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
1
(53–82%). e structures of 3a–f were characterized by elemental analysis and FTIR, H, 13C, and 31P NMR spectroscopies.
e occurrence of reverse thioureas formation in a one-pot reaction system is discussed. e 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).
1. Introduction
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 [7]. Various meth-
ods 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 [13].
Hexachlorocyclotriphosphazene, a cyclic inorganic com-
pound with alternating phosphorus and nitrogen atoms, has
sparked great interest among researchers for an excellent can-
didate in constructing hexasubstituted molecules [14, 15]. e
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 [16]. A
wide range of hexasubstituted phosphazene derivatives with
iourea is widely studied and claimed to be used in many
applications such as herbicides, pharmaceutical agents, pes-
ticides, rodenticides, vulcanization accelerator, and scaffolds
in organic synthesis [1]. In the synthesis of thiourea, isoth-
iocyanate is formed as a reactive intermediate and easily
converted to other side product during isolation [2]. Many
studies reported on the direct reaction of isothiocyanate
intermediate with amines afer isolation of KCl to produce
thiourea in good purity [3].
Several studies reported on monosubstituted thiourea
which consists of one thiourea moiety either as a ligand
bearing aromatic, halogen, or alkyl substituents [4] or as
a complex compound coordinated with heavy metal center
[5]. Multisubstituted thioureas have gained more interest
among researchers due to the increase of their pharmaceu-
tical 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

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2
Journal of Chemistry
various substituents such as hydroxyl, amino, and many other
functional groups had been reported [13, 15, 16].
(4-Methoxyphenyl) iourea (3c) [18]. p-Anisidine (0.739 g,
6 mmol). (68% yield) as grey powder, m.p: 172.4–173.2∘C (lit
[18] 193∘C).
To the best of our knowledge, no studies reported on
the synthesis of hexasubstituted thiourea onto cyclotriphos-
phazene 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 hexachlorocy-
clotriphosphazene as a hexasubstituted precursor in a typical
one-pot reaction system [11]. e plausible mechanism which
leads to the unexpected final products is discussed. e
binding interaction of the synthesised thiourea via molecu-
lar docking interaction in comparison to predicted phenyl
thiourea and the targeted compound with enzyme enoyl ACP
reductase (FabI) is also thoroughly discussed.
2-(Carbamothioylamino) Acetic Acid (3d) [20]. Glycine
(0.451 g, 6 mmol). (53% yield) as a yellowish powder, m.p:
133.1–134.5∘C (lit [20] 176–179∘C).
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; ]max (KBr/cm−1) 3229 (OH) 3010 (NH), 2968
(CH), 1698 (COOH) 1226 (C=S). ꢁ (500 MHz, DMSO-
d ) 1.59 (3H, d, J = 6.8, CH ), 5.10 (H1H, q, CH), 10.11 (2H,
6
3
s, NH ), 10.54 (1H, s, NH). ꢁ (125 MHz, DMSO-d ) 17.0
2
C
6
(CH ), 61.9 (CH), 173.5 (COOH), 180.6 (C=S). Calculated for
3
C H N O S: C, 32.40; H, 5.40; N, 18.90; S, 21.60%, found C,
4
8
2
2
31.74; H, 4.98; N, 18.79; S, 21.64%.
2. Materials and Methods
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; ]max (KBr/cm−1) 3172 (OH) 3100
Hexachlorocyclotriphosphazene (99%) was purchased from
Aldrich. Potassium thiocyanate, aniline, ꢀ-toluidine, ꢀ–
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.
(NH), 2911 (CH), 1740 (COOH) 1452 (Ar-C), 1249 (C=S). ꢁ
H
(500 MHz, DMSO-d ) 3.89 (2H, d, J = 13.8, CH ), 5.42 (1H,
6
2
2
q, CH) 7.05 (2H, d, J = 6.3, Ar-H), 7.28 (3H, m, Ar-H), 10.19
(2H, s, NH ), 10.63 (1H, s, NH). ꢁ (125 MHz, DMSO-d )
2
C
6
35.9 (CH ), 67.0 (CH), 127.8, 128.9, 129.8, 134.5 (Ar-C), 172.5
2
(COOH), 179.9 (C=S). Calculated for C H N O S: C, 53.60;
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
10 12
2
H, 5.40; N, 12.50; S, 14.30%, found: C, 53.35; H, 5.31; N, 12.17;
S, 14.02%.
1
a Perkin Elmer 1605 FTIR spectrophotometer. H and 13C
NMR spectra were recorded on a JEOL ECA 500 spectrom-
eter at 500 MHz (1H) and 125 MHz (13C), respectively, with
the chemical shifs ꢁ (ppm) being reported relative to DMSO-
2.2. Antibacterial Screening. Antibacterial activities of 3a–f
were analysed against E. coli (ATCC 8739) using the tur-
bidimetric kinetic method. e Gram-negative E. coli were
cultured on a Luria-Bertani plate agar at 37∘C. en 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. e mixture was shaken at 180 rpm at
37∘C. e negative control was medium broth of inoculums
with solvent. e aliquots of each replicate were taken on
every 1 h interval for 6 h. e transmittance (ꢃ) was recorded
using UV-Visible Spectrophotometer Optima SP-300. e
antibacterial activity was determined by plotting a graph of
ln ꢄꢀ versus time. e ln ꢄꢅ value represents the number of
colony forming units/mL which followed the expression of
ln ꢄꢀ = 27.1 − 8.56ꢃ [21].
d as standard. e chemical shifs for 31P NMR are relative
6
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). e mixture was stirred for 1 h at room temperature
to form intermediate 2. e white potassium chloride (KCl)
was filtered. e filtrate was added to amine (6.0 mmol)
in dry acetone (15.0 mL) and heated under reflux for 18 h.
e mixture was cooled to room temperature and filtered.
e filtrate was evaporated in vacuum to form a yellowish
powder. e crude was recrystallized in EtOH : CH CN (1 : 1).
3
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 [22]. e polar hydrogens of the synthe-
sised compounds and protein were added with AutoDock
Tools 1.5.6 [23] before docking using Auto-Dock Vina pro-
gram. In Auto-Dock Vina program, the cubic grid box of
60∘A sizes (ꢆ, ꢇ, and ꢈ) with a spacing of 0.375∘A was
centered to the active site of the protein. e X-ray crystal
structure of the enzyme enoyl ACP reductase (FabI) of E.
e general procedure for the preparation of 3a–f utilised a
different type of amines (g, mmol) and yields as follows.
Phenylthiourea (3a) [18]. Aniline (565.0 ꢂL, 6 mmol). (73%
yield) as a white crystal, m.p: 153.2–153.5∘C (lit [18] 163∘C).
p-Tolylthiourea (3b) [19]. p-Toluidine (0.643 g, 6 mmol).
(82% yield) as a white crystal, m.p: 167.8–168.8∘C (lit [19]
182–186∘C).

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Journal of Chemistry
3
Cl Cl
SCN NCS
P
S
N
P
N
P
P
RꢀNH2
Rꢀ
KSCN
Acetone
N
P
N
P
N
H
NH2
Cl
Cl
Acetone
reꢀux
SCN
NCS
N
N
Cl
Cl
SCN
NCS
3a–f
1
2
Rꢀ a = C6H5
b = C6H5CH3
RꢀNH2
Acetone
reꢀux
c = C6H5OCH3
d = CH2(COOH)
e = (CH3)CH(COOH)
f = (C6H5)CH2CH(COOH)
NHCSNHRꢀ NHCSNHRꢀ
P
N
P
N
P
NHCSNHRꢀ
NHCSNHRꢀ
N
NHCSNHRꢀ
NHCSNHRꢀ
4
Scheme 1: e synthesis of 3a–f.
coli (PDB entry: 1C14) was obtained from Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do) [7, 24].
synthesised in one-pot reaction system and not the targeted
molecule 4a–f (Scheme 1).
e 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 [31].
e free chlorine ions deprotonate amines in the reaction
system and form HCl and anionic amines, which in turn
reacted with hydrogen thiocyanate [18] and formed 3a–f. e
plausible mechanism for the formation of 3a–f is shown in
Scheme 2.
3. Results and Discussion
3.1. Chemistry. e synthesis of the proposed hexasubstituted
thioureas 4a–f was prepared via reaction of hexachloro-
cyclotriphosphazene with potassium thiocyanate to form
isothiocyanates phosphazene intermediates, followed by typ-
ical thiourea reaction with a series of amines derivatives
in a one-pot reaction system. All compounds were sub-
jected to IR spectroscopy and showed the disappearance of
V(NCS) at 2140–1990 cm−1 and the formation of V(N-H) at
3276–3010 cm−1. e formation of thiourea was evidenced by
the strong absorption peak at 1265–1227 cm−1 corresponding
to V(C=S) which shifed to the lower frequency due to the
attachment of more electronegative nitrogen atoms [25]. e
absorbance peak attributed to the formation of V(P=N) asym-
metric vibration at 1400–1200 cm−1 [26, 27] but, however, was
not observed. is 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. e 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 hydro-
gen bond between the hydrogen atom from thiourea moieties
with oxygen atom from the carboxylic acid group [5]. 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].
3.2. Antibacterial Activity. Compounds 3a–f were further
investigated for antibacterial activities by plotting the graph
of ln ꢄꢀ 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. e result indicated that compounds 3a–f
showed poor inhibition against E. coli. e 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 (ꢂ = 0) [32]. e 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 [33].
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 [34]. 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 [32].
Elemental analysis of the synthesised compounds
afforded low carbon percentage in each compound which
3.3. Molecular Docking Design and Optimisation. For a better
understanding of the interaction between thiourea deriva-
tives and Gram-negative bacteria E. coli, molecular docking
indicated the formation of 3a–f. Based on the IR, H, 13C,
1
and 31P NMR spectra, it was suggested that 3a–f were

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Journal of Chemistry
N
N
N
Cl
Cl
(excess)
N=C=S + KCl +
S=C=N−
K+ S=C=N−
+
+
−
P
P
+
+
K
Cl
N
Cl
2
1
H∙ ∙
∙ ∙
Rꢀ
N
Rꢀ
∙N
∙
−
H
+
Cl
Cl−
H
H
+
Amines
−Cl−
−N=C=S
S
H+
∙ ∙
Rꢀ
Rꢀ
∙
−
∙N
N
H
+
H-N=C=S
H2N
H
3a–f
Scheme 2: Mechanism on the formation of 3a–f.
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
free energy of −5.9 kcal/mol and −6.3 kcal/mol, respectively.
e additional aromatic group in 5a-b is strongly bound to
enzyme enoyl ACP reductase (FabI) of E. coli through ꢋ-
ꢋ bond interactions (yellow colour cylindrical wireframe)
with hydrophobic pockets of Phe 1251. e hydrophobic
interaction between phenyl rings has increased the lipophilic-
ity of the compound [7, 33]. e 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 [36]. e
absence of aromatic ring was accountable for lesser binding
affinity resulting in less activity in 3a-b [37].
0
50
100
150
200
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. Apar t f rom the ꢋ-ꢋ bond interactions with
Phe 1251, 4a was observed to interact with the enzyme via two
hydrogen bonds (green colour sphere). e NH groups in 4a
are forming hydrogen bonding with C=O and NH of Ala 1152.
e 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 [40].
(ppm)
3a
3b
3c
3d
3e
3f
Figure 1: MIC graph for 3a–f.
studies were carried out and optimised by comparing 3a-b
with the predicted phenyl thiourea 5a-b and the targeted 4a.
e 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]. e compounds and binding interactions are shown in
Table 1. e binding affinity of the compounds was evaluated
based on binding free energies (Δꢉꢊ, kcal/mol) [35].
e 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 aro-
matic 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. e presence of another aromatic group in
5a-b demonstrated for a higher binding affinity with the
4. Conclusions
In summary, the thiourea derivatives 3a–f were unexpectedly
synthesised from the reaction of amines with excess thio-
cyanates groups in a one-pot reaction system. e isolation of
isothiocyanato cyclophosphazene intermediates could be the
best method to give hexasubstituted thioureas. e formation
of HCl in the reaction condition was envisaged to be respon-
sible for the deprotonation of amines, thus reducing the