Synthesis and molecular design of mono aspirinate thiourea-azo hybrid molecules as potential antibacterial agents

Article abstract of DOI:10.1080/10426507.2020.1828885

New mono aspirinate thiourea-azo derivatives 2a–f were synthesized using aspirin as a natural product-based precursor, with 4-aminophenylazophenol derivatives in excellent 70–90% yields. Overall, 2a–f gave excellent antibacterial activity against Staphylo

Full text of DOI:10.1080/10426507.2020.1828885

Phosphorus, Sulfur, and Silicon and the Related Elements
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Synthesis and molecular design of mono
aspirinate thiourea-azo hybrid molecules as
potential antibacterial agents
Zainab Ngaini , Ferlicia Rasin , Wan Sharifatun H. Wan Zullkiplee & Ainaa
Nadiah Abd Halim
To cite this article: Zainab Ngaini , Ferlicia Rasin , Wan Sharifatun H. Wan Zullkiplee & Ainaa
Nadiah Abd Halim (2020): Synthesis and molecular design of mono aspirinate thiourea-azo hybrid
molecules as potential antibacterial agents, Phosphorus, Sulfur, and Silicon and the Related
Elements, DOI: 10.1080/10426507.2020.1828885
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2020.1828885
Synthesis and molecular design of mono aspirinate thiourea-azo hybrid
molecules as potential antibacterial agents
Zainab Ngainia , Ferlicia Rasina , Wan Sharifatun H. Wan Zullkipleeb, and Ainaa Nadiah Abd Halima
b
aFaculty of Resource Science and Technology, Kota Samarahan, Malaysia; Centre for Pre-University Studies, Universiti Malaysia Sarawak,
Kota Samarahan, Malaysia
ABSTRACT
ARTICLE HISTORY
Received 15 June 2020
Accepted 23 September 2020
New mono aspirinate thiourea-azo derivatives 2a–f were synthesized using aspirin as a natural
product-based precursor, with 4-aminophenylazophenol derivatives in excellent 70–90% yields.
Overall, 2a–f gave excellent antibacterial activity against Staphylococcus aureus S48/81 and 2-(((3-
((2-bromophenyl)diazenyl)-4-hydroxyphenyl)carbamothioyl)carbamoyl)phenyl acetate (2d) exhib-
ited a larger zone of inhibition 10 mm, comparable to the standard ampicillin. The molecular dock-
ing analysis of 2d against S. aureus tyrosyl-tRNA synthetase protein displayed a free energy
interaction of ꢀ7.7 kcal/mol compared to aspirin (ꢀ6.4 kcal/mol).
KEYWORDS
Aspirin; molecular docking;
S. aureus; E. coli;
antimicrobial
GRAPHICAL ABSTRACT
Introduction
as ulceration and gastrointestinal bleeding.[9] Many studies
have been reported on the chemical modification of aspirin to
enhance the pharmacological properties via incorporation of
active moieties to the aspirin network such as thiourea
(NHC ¼ SNH)[7,10] which are known for their excellent anti-
gastrointestinal,[11] antibacterial,[6] antihrombotic,[12] and
anticancer[13,14] properties. Thiourea is a compound with an
efficient bactericidal property.[15] The presence of active moi-
eties in thiourea namely N–H, C ¼ O, and C ¼ S groups has
been recognized to form strong binding interactions with
receptors.[16–18] Several commercially available drug-based
thioureas such as carbimazole, propyl and methyl-thioura-
cil[19] shown remarkable potential as anticancer drugs.[20]
We have earlier reported on aspirin derivatives bearing
thiourea[20] and azo dyes moieties[21] via formation of iso-
thiocyanate and azo coupling reaction, respectively, as
potential antibacterial agents. Azo dyes are recognized for
their diverse applications in numerous fields. The active
moiety (–N ¼ N–) in azo can be easily protonated under
The occurrence of antimicrobial resistance among pathogenic
bacteria has triggered alarming health concerns to man-
kind.[1–3] Utilizing natural product-based compounds as
potential drugs to overcome antimicrobial resistance has
gained a great interest among researchers. The presence of
various active functional groups available in natural product
moieties offers strong binding interactions with the target
receptors for inhibition. A major drawback in the naturally
occurring plant extracts, however, is the mismatch of the pre-
existing and complex functional groups in the compounds
that reduce the efficiency of the drugs to the targeted recep-
tors.[4] Chemical modification of naturally derived com-
pounds such as aspirin has been intensively studied by
targeting at the pre-designed position of the active groups to
form specific interaction with the targeted receptor.[5–7]
Aspirin is a natural product compound resource from wil-
low bark with analgesic properties.[8] Some drawbacks have
been reported on the long-term consumption of aspirin such acidic conditions, resulting in a wide spectrum of biological
CONTACT Zainab Ngaini
nzainab@unimas.my
Faculty of Resource Science and Technology, Kota Samarahan 94300, Malaysia.
Supplemental data for this article is available online at https://doi.org/10.1080/10426507.2020.1828885.
ß 2020 Taylor & Francis Group, LLC

2
Z. NGAINI ET AL.
Scheme 1. Synthetic pathway of 1a–f and 2a–f in (i) NaOH, NaNO2, HCl, 0–5 ꢁC, (ii) EtOH, HCl, reflux, (iii) KSCN, CH3CN.
properties[22–24] such as antibacterial,[25,26] antioxidant,[27]
In this study, we report the synthesis of halogenated thio-
anti-inflammatory,[28] antifungal,[29] and antitumor.[30] The urea-azo series onto a natural product based on aspirin and
azo group can form two geometric isomers, which are trans comparatively analyzed for potential antibacterial properties
(thermally stable) and cis (meta-stable) configuration against Escherichia coli (ATCC 25922) and S. aureus (S48/
depending on the external energy applied such as UV light 81). The structure–activity relationship and molecular inter-
or photolysis. Nevertheless, N ¼ N is energetically stable in action study of the synthesized compounds to an enzyme
the trans conformation.[31–33] In drug discovery and devel-
were demonstrated using molecular docking analysis on S.
aureus tyrosyl-tRNA synthetase protein. The series of
molecular hybrid compounds could be potentially developed
in pharmaceutical industries as new drugs with bacteriostatic
property.
opment, molecular hybridization has become an auspicious
approach to cure diseases using a single compound to act
on multiple targets. The synergistic permutation of multi-
functional pharmacophore (i.e. –N ¼ N–, NH, C ¼ S, C ¼ O,
and halogens) in a single molecule is envisaged to form
strong interaction to several targets and produce stronger
therapeutic activity. Many studies reported on the biological
properties of thiourea and azo compounds.[5,20,21] However,
less study reported on the incorporation of both active
groups (i.e. thiourea and azo) in a single compound. It is
envisaged that the compound could be able to modulate
Results and discussion
The series of aspirinate thiourea-azo compounds (2a–f)
were prepared from the reaction of aspirin with aminated
azo dyes (1a–f).[39] The azo dyes 1a–f were initially pre-
pared via azo coupling reaction of 4-acetaminophenol (para-
cetamol) with halogenated anilines (i.e. ortho-, meta-, para-
position) in cold condition (0–5 ꢁC), followed by acid
hydrolysis to afford 1a–f in 43–83% yield (Scheme 1). The
instability of diazonium ions has commonly produced a low
yield of azo products in the acid catalyzed condition.[40] The
synthesis of 2a–f commenced with the initial formation of
acetoxybenzoyl isothiocyanate from a reaction of potassium
thiocyanate and acetylsalicyloyl chloride,[6] followed by
nucleophilic substitution of azobenzenes 1a–f to yield 2a–f
in 70–90%.[7]
The characterization of the compounds was carried out
using spectroscopic analysis namely FTIR, 1H, and 13C
NMR. FTIR spectra of azo dyes 1a–f showed the peaks pre-
sent at 3449–3439 cmꢀ1 which corresponded to ꢀOH. The
disappearance of ꢀC ¼ O amide bond at 1663–1651 cmꢀ1 in
1a–f indicates complete loss of the acetanilide group via
multiple targets of interest for
interaction.
a
stronger binding
The treatment of antimicrobial resistance requires active
compounds with suitable active sites to achieve strong inter-
action with the receptor. Choosing the right molecular tar-
gets for new antibiotics is crucial in the drug development
process. Staphylococcus aureus is one of many bacteria that
contributed to the increasing issues on the microbial infec-
tions that also lead to antimicrobial resistance.[34] Their
antibacterial activity is widely dependent on the interaction
between drugs and their molecular targets. S. aureus enzyme
tyrosyl-tRNA synthetase belongs to the aminoacyl-tRNA
synthetases (aaRSs) which are responsible for the translation
of nucleic acid sequence information into proteins.[35,36] The
ability of a drug to inhibit the enzyme will consequently
inhibit S. aureus’s cell growth.[37,38] In recent years, the
search for inhibitors of novel targets has been intensively hydrolysis.[6] A strong peak observed at 1779–1771 cmꢀ1
studied due to the upsurge of new diseases and drug-resist- and 1667–1662 cmꢀ1 corresponded to ꢀC ¼ O of ester and
ant bacteria. In this regard, suitable therapeutics and rapid amide, respectively, which indicates a successful formation
diagnostics of potential drugs are highly required.
of thiourea in 2a–f. The peaks identified as ꢀN ¼ N were

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
Table 1. Molecular docking of compounds 1d and 2d, in comparison to aspirin and ampicillin.
Compound
Affinity (kcal/mol)
H-bond residue
Residues
1d
2d
Aspirin
Ampicillin
ꢀ6.6
ꢀ7.7
ꢀ6.4
ꢀ7.1
–
629-421, HIS-50, HIS-47
629-421, HIS-50, HIS-47
629-421, HIS-50, HIS-47, THR-272
629-421, HIS-50, HIS-47
PHE-27
ASN-109, LYS-30, GLU-302
629-421, HIS-50, HIS-47, ASN-109, LYS-30, GLU-302
–: H-bond interaction not observed.
observed at 1452–1447 cmꢀ1. The peaks attributed to ꢀC–N excellent antibacterial activity via zone inhibition compared
and ꢀC ¼ S were present at 1284–1279 cmꢀ1 and to aspirin alone[6,48] and comparable to ampicillin, as a ref-
1159–1154 cmꢀ1, respectively.
erence drug. Among all the aspirinate thiourea-azo deriva-
The formation of 1a–f and 2a–f was further proven using tives
(2a–f),
2-(((3-((2-bromophenyl)diazenyl)-4-
1H and 13C NMR spectroscopy. The 1H NMR spectra of hydroxyphenyl)carbamothioyl) carbamoyl)phenyl acetate
1a–f displayed resonances at dH 11.37–10.93 and dH (2d) showed a larger zone of inhibition of 10 mm. The stra-
8.24–7.22 corresponding to NH and aromatic groups, tegic position of the active groups in the molecular network
respectively. The 13C NMR spectra showed the disappear- has significantly bound to the active sites of the bacterial
ance of –CH3 peak at dC 23.8 indicated the successful for- cells.[49] The presence of active pharmacophore in thiourea
mation of 1a–f. The formation of 2a–f was in agreement moieties such as N–H, C ¼ S, and C ¼ O has formed strong
1
with the H NMR spectra by the presence of resonance at binding interactions with the phosphate and carboxyl groups
dH 12.37–12.36 and dH 11.84–11.81 attributed to CSNH and of the bacterial shells during inhibition activity. A combin-
CONH, respectively. Both protons experience anisotropic ation of lipophilic and hydrophilic characteristic presence in
effect and are observed downfield due to the H-bonding for- the hybrid molecules of aspirinate thiourea-azo (2a–f) has
mation between NH (amine) and oxygen or sulfur from the significantly contributed to the antimicrobial activities.[50]
thiocarbonyl group.[41] The peaks corresponded to aromatic The lipophilicity of the aromatic group is believed to form
were observed at dH 8.27–6.51 while –CH3 from acetyl interaction with the hydrophobic sections in the enzyme[42]
group indicated as a singlet peak at dH 2.33–2.32. The 13C and produced good binding interaction with the bacter-
NMR spectra further confirmed the formation of thiourea ial receptor.
(C ¼ S) with the resonance presence at dC 179.4–179.2. The
peaks referred to carbonyl and aromatic carbon observed at
dC 168.9–167.0 and dC 155.7–107.5, respectively.
Molecular docking study
Compound 2d with inhibition zone 10 mm was further
study for molecular docking interaction and affinity against
the parent azo dyes 1d, aspirin and standard drug ampicillin
Antibacterial study
All the synthesized compounds (1a–f and 2a–f) were eval- as shown in Table 1. Molecular docking interaction and
uated against S. aureus (S48/81) and E. coli (ATCC 25922) affinity on 1d, 2d and aspirin were performed in compari-
using turbidimetry assay.[42,43] Nevertheless, the compounds son to ampicillin, as a reference drug. S. aureus tyrosyl-
were precipitated upon introduction into the media, which tRNA synthetase was selected as a binding protein due to its
abstained the evaluation of the microbial activities. The importance in the protein biosynthesis process on S. aureus’s
molecular structure of 1a–f and 2a–f is believed to be highly cell growth.[38]
lipophilic that reduced the solubility in the culture
The molecular docking interaction of 1d (Figure 1a)
media.[44,45] Alternatively, Kirby Bauer assay via disk diffu- shows no formation of H-bond interaction, which is sup-
sion method was utilized. The bacteriostatic activities of ported by the absence of inhibition activity against S. aureus
1a–f and 2a–f were tabulated and compared with aspirin[6] (Table 1). The presence of H-bond interactions is important
as control and standard antimicrobial drug, ampicillin (sup- in biochemistry and biological recognition processes as they
plementary Table S1).
help in promoting interactions with cell membranes.[51,52]
The azo dyes bearing amines groups 1a–f showed no Significantly, the molecular docking interaction of 2d
inhibition against both bacteria (Table 1). All the synthe- showed good binding energy (ꢀ7.7 kcal/mol) as compared
sized compounds 1a–f and 2a–f were found to be unsuscep- to ampicillin (ꢀ7.1 kcal/mol) and aspirin (ꢀ6.4 kcal/mol)
tible toward E. coli. This phenomenon could be due to the (Figures 1 and 2). Aspirin showed no inhibition activity
structural differences in the bacterial cell structures. Gram- against S. aureus[6] which could be due to low binding
positive bacteria (S. aureus) cell wall is made up from sugars energy (Figure 2a) compared to 2d.
and amino acid polymer known as peptidoglycan (mur-
The docking of 2d via PyMOL able to visualize the pres-
ein).[42,44] In gram-positive bacteria, there are various pep- ence of hydrogen bond interactions (indicated by the yellow
tide arrangements among peptidoglycan derived from line) against ampicillin as a reference drug, which firmly
lipopolysaccharide, lipoprotein, and phospholipids which supported similar inhibition activity. Compound 2d showed
influenced its thickness[46] and diffusion of drugs into the the formation of H-bond interaction with amino acid resi-
cell wall.[47]
dues of the binding protein, S. aureus tyrosyl-tRNA synthe-
Interestingly, the incorporation of aspirin into azo dyes tase. Similar to ampicillin, which formed H-bond interaction
2a–f via thiourea (NHSNH) as a linker demonstrated between C ¼ O and ASN109, –OH and LYS305, and –NH

4
Z. NGAINI ET AL.
Figure 1. Docking visualization of (a) 1d and (b) 2d using PyMOL modeling.
Figure 2. Docking visualization of (a) aspirin and (b) ampicillin using PyMOL modeling.
with GLU302, 2d demonstrated good molecular docking
interaction in the binding pockets of S. aureus tyrosyl-tRNA
synthetase and demonstrated hydrogen bonding interaction
with HIS50 and HIS47 (Figures 1 and 2). The molecular
docking interaction further supported the inhibition activity
of 2d against S. aureus tyrosyl-tRNA synthetase via inter-
action with the amino acid residues with good binding affin-
ity as compared to the natural product-based precursor
of aspirin.
Experimental
All reagents and solvents were obtained from Merck without
further purification. Solvent such as acetonitrile was distilled
over calcium hydride. Melting points were analyzed using
1
open tube capillary (Stuart SMP3) and uncorrected. H and
13C NMR spectra were recorded on JEOL ECA 500 operat-
ing at 500 MHz (1H) and 125 MHz (13C). All NMR spectra
were recorded in DMSO-d6 and the chemical shifts were
reported in d ppm. The FTIR spectra (ꢀ/cmꢀ1) were ana-
lyzed on Perkin Elmer 1605 spectrophotometer using KBr
pellet. The elemental CHNS analyses were recorded using
Thermo ScientificTM FlashSmartTM Elemental Analyzer. The
Supplemental Materials contain sample IR, 1H, and 13C
NMR spectra of products 1 and 2 (Figures S1–S36).
Conclusion
A series of a molecular hybrid with multifunctional groups
namely aspirinate thiourea-azo derivatives, 2a–f has been
successfully synthesized and showed excellent antibacterial
activity against S. aureus. Among these target compounds,
mono aspirinate thiourea-azo 2d gave comparable inhibition
rate as the standard drug ampicillin. The presence of active
pharmacophore (i.e. –N ¼ N–, NH, C ¼ S, C ¼ O, and halo-
gens) in the aspirin network has significantly contributed to
excellent interaction against S. aureus. The structure–activity
relationship of the active compound via molecular inter-
action visualization (PyMOL) supported the inhibition and
interaction activity of 2d toward the protein receptor of S.
aureus. This study is potentially served as a suitable bac-
teriostatic tool in searching for competent lead compounds
in the drug development process.
General procedure for the synthesis of 4-amino phenyl
azo phenol derivatives (1a–f)
A solution of halogenated aniline (10 mmol) with HCl in
water (10 mL) was cooled at 0–5 ꢁC. Sodium nitrite solution
(10 mL, 1 M) was added to form a diazonium salt solution
and directly used for the following reaction. A solution of 4-
acetamidophenol (10 mmol) in NaOH (20 mL, 1 M) was
introduced into diazonium salt solution until the precipitate
was formed. The mixture was filtered and HCl (2 M) was
added into the filtrate to induce precipitation. The crude

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
5
solid was filtered and dissolved in ethanol:conc. HCl (1:1 v/ J ¼ 6.3 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.67 (s, 1H), 7.75 (d,
v). The mixture was heated at reflux for 3 h and cooled to J ¼ 8.0 Hz, 1H), 8.02 (d, J ¼ 7.4 Hz, 1H), 8.24 (s, 1H). dC
room temperature. Water was added and the aqueous layer (125 MHz, DMSO-d6) 112.5 (Ar–C), 119.7 (Ar–C), 122.7
was extracted with DCM (20.0 mL). The organic layer was (Ar–C), 123.2 (Ar–C), 123.8 (Ar–C), 124.5 (C–N ¼ N–),
128.6 (Ar–C), 131.4 (Ar–C), 133.9 (Ar–C), 138.4 (C–NH2),
152.9 (C–OH), 155.7 (C–N ¼ N–).
separated, dried (MgSO4), filtered, and evaporated in vacuo
to give compounds 1a–f with yields as follows.
4-Amino-2-((4-bromophenyl)diazenyl)phenol
(1f)
4-Amino-2-((2-chlorophenyl)diazenyl)phenol
(1a)
(1.60 g, 4.8 mmol). (0.80 g, 57.2%) as orange solid, m.p:
222–223 ꢁC; (Found: C, 49.1; H, 3.1; N, 13.9. C12H10BrN3O,
Requires C, 49.3; H, 3.5; N, 14.4%); ꢀmax (KBr/cmꢀ1) 3439
(OH), 1602 (Ar–C), 1499 (N ¼ N). dH (500 MHz, DMSO-d6)
7.22 (d, J ¼ 8.6 Hz, 1H), 7.46 (dd, J ¼ 8.6, 2.3 Hz, 1H), 7.69
(d, J ¼ 2.3 Hz, 1H), 7.81 (d, J ¼ 8.6 Hz, 2H), 7.96 (d,
J ¼ 8.6 Hz, 2H). dC (125 MHz, DMSO-d6) 113.0 (Ar–C),
119.6 (Ar–C), 123.8 (C–N ¼ N–), 124.9 (Ar–C), 125.1
(Ar–C), 128.3 (Ar–C), 132.5 (Ar–C), 138.6 (C–NH2), 150.7
(C–OH), 155.4 (C–N ¼ N–).
(0.28 g, 0.9 mmol). (0.20 g, 83.6%) as brick red solid, m.p:
189–190 ꢁC; (Found: C, 52.2; H, 3.9; N, 16.8. C12H10ClN3O,
Requires C, 52.8; H, 4.1; N, 17.0%); ꢀmax (KBr/cmꢀ1) 3451
(OH), 1599 (Ar–C), 1498 (N ¼ N). dH (500 MHz, DMSO-d6)
7.23 (d, J ¼ 9.2 Hz, 1H), 7.46 (dd, J ¼ 8.9, 2.6 Hz, 1H),
7.50–7.53 (m, 1H), 7.59 (td, J ¼ 7.4, 1.7 Hz, 1H), 7.73–7.75
(m, 2H), 7.92 (dd, J ¼ 8.0, 1.7 Hz, 1H), 11.31 (s, 1 ꢂ NH). dC
(125 MHz, DMSO-d6) 115.1 (Ar–C), 118.1 (Ar–C), 119.6
(Ar–C), 124.3 (C–N ¼ N–), 128.2 (Ar–C), 128.6 (Ar–C),
130.7 (Ar–C), 133.0 (Ar–C), 133.9 (Ar–C), 138.6 (C–NH2),
147.3 (C–OH), 154.7 (C–N ¼ N–).
4-Amino-2-((3-chlorophenyl)diazenyl)phenol
(1b)
General procedure for the synthesis of aspirinate
(0.26 g, 0.8 mmol). (0.11 g, 49.5%) as bright orange solid,
m.p: 210–211 ꢁC; (Found: C, 52.0; H, 4.0; N, 16.5.
C12H10ClN3O, Requires C, 52.8; H, 4.1; N, 17.0%); ꢀmax
(KBr/cmꢀ1) 3445 (OH), 1596 (Ar–C), 1499 (N ¼ N). dH
(500 MHz, DMSO-d6) 7.22–7.25 (m, 1H), 7.48 (dd, J ¼ 8.6,
2.9 Hz, 1H), 7.61–7.69 (m, 3H), 7.90–7.99 (m, 1H), 8.10 (s,
1H). dC (125 MHz, DMSO-d6) 112.7 (Ar–C), 119.8 (Ar–C),
120.6 (Ar–C), 123.8 (Ar–C), 124.1 (C–N ¼ N–), 128.7
(Ar–C), 131.2 (Ar–C), 131.3 (Ar–C), 134.4 (Ar–C), 138.6
(C–NH2), 152.9 (C–OH), 155.8 (C–N ¼ N–).
thiourea-azo derivatives (2a–f)
A solution of o-acetylsalicyloyl chloride (0.4 mmol) in dry
acetonitrile (10 mL) was added into a suspension of KSCN
(0.4 mmol) in acetonitrile (10 mL). The mixture was stirred
at room temperature for 1 h to form precipitates of KCl and
filtered off. The filtrate was used in the subsequent reaction.
In another flask, triethylamine (0.4 mmol) was added into a
solution of 4-amino phenyl azo phenol derivatives 1a–f
(0.4 mmol) in acetonitrile (10 mL) and stirred at room tem-
perature for 3 h. The reaction mixture was then added to
the filtrate, stirred at room temperature for 18 h, filtered and
dried in vacuo. The reaction mixture dissolved in dichloro-
methane (5 mL) and water (5 mL). The organic layer was
separated, dried over MgSO4, filtered, and evaporated in
vacuo. The crude was purified by recrystallization from hot
ethanol which affords aspirinate thiourea-azo 2a–f.
4-Amino-2-((4-chlorophenyl)diazenyl)phenol
(1c)
(0.97 g, 3.3 mmol). (0.62 g, 74.8%) as orange solid, m.p:
220–221 ꢁC; (Found: C, 52.1; H, 4.0; N, 16.9. C12H10ClN3O,
Requires C, 52.8; H, 4.1; N, 17.0%); ꢀmax (KBr/cmꢀ1) 3445
(OH), 1602 (Ar–C), 1499 (N ¼ N). dH (500 MHz, DMSO-d6)
7.22 (d, J ¼ 8.6 Hz, 1H), 7.46 (dd, J ¼ 8.6, 2.9 Hz, 1H),
7.65–7.69 (m, 3H), 8.02–8.04 (m, 2H), 10.35 (s, 1 ꢂ OH),
10.93 (s, 1 ꢂ NH). dC (125 MHz, DMSO-d6) 113.1 (Ar–C),
119.6 (Ar–C), 123.7 (C–N ¼ N–), 124.7 (Ar–C), 128.3
(Ar–C), 129.6 (Ar–C), 136.2 (Ar–C), 138.6 (C–NH2), 150.4
(C–OH), 155.4 (C–N ¼ N–).
2-(((3-((2-Chlorophenyl)diazenyl)-4-hydroxyphenyl)car-
bamothioyl)carbamoyl)phenyl
acetate
(2a)
(0.10 g,
0.5 mmol). (0.17 g, 74%) as reddish orange solid, m.p:
235–236 ꢁC; (Found: C, 56.3 H, 3.5; N, 11.8; S, 6.8.
C22H18ClN4O4S, Requires C, 56.4; H, 3.6; N, 11.9; S, 6.8%);
ꢀmax (KBr/cmꢀ1) 3360 (NH), 1775 (C ¼ O ester), 1667
(C ¼ O amide), 1520 (Ar–C), 1452 (N ¼ N), 1281 (C ¼ S),
1154 (C–N). dH (500 MHz, DMSO-d6) 2.32 (s, 3H), 7.13 (d,
J ¼ 9.2 Hz, 1H), 7.30 (d, J ¼ 8.0 Hz, 1H), 7.39 (dd, J ¼ 28.4,
7.2 Hz, 2H), 7.51 (d, J ¼ 9.7 Hz, 1H), 7.65 (q, J ¼ 7.6 Hz, 3H),
7.75 (d, J ¼ 7.4 Hz, 1H), 7.95–8.00 (m, 2H), 11.09 (s, 1H),
11.62 (s, 1H), 12.19 (s, 1H). dC (125 MHz, DMSO-d6) 20.9
(CH3), 116.8 (Ar–C), 117.2 (Ar–C), 117.4 (Ar–C), 118.0
(Ar–C), 118.4 (Ar–C), 123.4 (Ar–C), 125.2 (C–N ¼ N–),
125.9 (Ar–C), 126.8 (Ar–C), 130.2 (Ar–C), 130.4 (Ar–C),
131.4 (Ar–C), 133.3 (Ar–C), 133.5 (Ar–C), 138.5 (Ar–C),
139.6 (Ar–C), 148.4 (Ar–C), 154.2 (C–N ¼ N–), 166.4
(C ¼ O), 169.0 (C ¼ O), 179.3 (C ¼ S).
4-Amino-2-((2-bromophenyl)diazenyl)phenol
(1d)
(1.00 g, 3.0 mmol). (0.42 g, 47.1%) as mustard yellow solid,
m.p: 212–213 ꢁC; (Found: C, 49.0; H, 3.3; N, 14.0.
C12H10BrN3O, Requires C, 49.3; H, 3.5; N, 14.4%); ꢀmax
(KBr/cmꢀ1) 3445 (OH), 1597 (Ar–C), 1499 (N ¼ N). dH
(500 MHz, DMSO-d6) 7.24 (d, J ¼ 9.2 Hz, 1H), 7.48–7.56 (m,
3H), 7.76 (d, J ¼ 2.3 Hz, 1H), 7.89 (q, J ¼ 4.2 Hz, 2H), 10.35
(s, 1 ꢂ OH), 11.37 (s, 1 ꢂ NH). dC (125 MHz, DMSO-d6)
115.5 (Ar–C), 118.7(Ar–C), 120.2 (Ar–C), 124.5
(C–N ¼ N–), 128.7 (Ar–C), 129.3 (Ar–C), 131.3 (Ar–C),
133.6 (Ar–C), 134.4 (Ar–C), 139.2 (C–NH2), 147.8 (C–OH),
155.5 (C–N ¼ N–).
4-Amino-2-((3-bromophenyl)diazenyl)phenol
(1e)
(0.29 g, 0.8 mmol). (0.11 g, 43.4%) as greenish yellow solid,
m.p: 214–215 ꢁC; (Found: C, 49.0; H, 3.1; N, 14.0.
2-(((3-((3-Chlorophenyl)diazenyl)-4-hydroxyphenyl)car-
C12H10BrN3O, Requires C, 49.3; H, 3.5; N, 14.4%); ꢀmax bamothioyl)carbamoyl)phenyl
acetate
(2b)
(0.12 g,
(KBr/cmꢀ1) 3454 (OH), 1597 (Ar–C), 1497 (N ¼ N). dH 0.5 mmol). (0.16 g, 70%) as orange solid, m.p: 231–232 ꢁC;
(500 MHz, DMSO-d6) 7.22 (d, J ¼ 9.2 Hz, 1H), 7.46 (d, (Found: C, 56.3 H, 3.5; N, 11.8; S, 6.8. C22H18ClN4O4S,

6
Z. NGAINI ET AL.
Requires C, 56.4; H, 3.6; N, 11.9; S, 6.8%); ꢀmax (KBr/cmꢀ1
)
J ¼ 8.0 Hz, 1H), 7.66 (q, J ¼ 8.2 Hz, 2H), 7.74 (d, J ¼ 8.0 Hz,
2H), 7.98 (s, 1H), 8.03 (d, J ¼ 7.4 Hz, 1H), 8.26 (s, 1H),
10.83 (s, 1H), 11.63 (s, 1H), 12.20 (s, 1H). dC (125 MHz,
DMSO-d6) 20.9 (CH3), 107.5 (Ar–C), 107.7 (Ar–C), 115.4
(Ar–C), 118.0 (Ar–C), 121.6 (Ar–C), 123.4 (Ar–C), 126.0
(C–N ¼ N–), 126.8 (Ar–C), 130.2 (Ar–C), 130.4 (Ar–C),
131.4 (Ar–C), 133.3 (Ar–C), 138.1 (Ar–C), 148.4 (Ar–C),
153.6 (Ar–C), 154.6 (Ar–C), 161.9 (C–N ¼ N–), 166.5
(C ¼ O), 169.1 (C ¼ O), 179.3 (C ¼ S).
3370 (NH), 1772 (C ¼ O ester), 1665 (C ¼ O amide), 1524
(Ar–C), 1451 (N ¼ N), 1284 (C ¼ S), 1154 (C–N). dH
(500 MHz, DMSO-d6) 2.33 (s, 3H), 7.13 (d, J ¼ 9.2 Hz, 1H),
7.29 (s, 1H), 7.42 (d, J ¼ 6.0 Hz, 1H), 7.61–7.68 (m, 4H),
7.74 (d, J ¼ 6.3 Hz, 1H), 7.98 (s, 2H), 8.13 (s, 1H), 10.83 (s,
1H), 11.63 (s, 1H), 12.20 (s, 1H), dC (125 MHz, DMSO-d6)
20.6 (CH3), 107.2 (Ar–C), 107.3 (Ar–C), 115.1 (Ar–C), 117.7
(Ar–C), 121.3 (Ar–C), 123.1 (Ar–C), 125.7 (C–N ¼ N–),
126.5 (Ar–C), 129.9 (Ar–C), 130.1 (Ar–C), 131.1 (Ar–C),
132.9 (Ar–C), 137.7 (Ar–C), 148.1 (Ar–C), 153.2 (Ar–C),
154.2 (Ar–C), 161.6 (C–N ¼ N–), 166.2 (C ¼ O), 168.7
(C ¼ O), 178.9 (C ¼ S).
2-(((3-((4-Bromophenyl)diazenyl)-4-hydroxyphenyl)car-
bamothioyl)carbamoyl)phenyl
acetate
(2f)
(0.14 g,
0.5 mmol). (0.23 g, 90%) as mustard yellow solid, m.p:
235–236 ꢁC; (Found: C, 50.8; H, 3.2; N, 10.5; S, 6.0.
C22H18BrN4O4S, Requires C, 51.4; H, 3.3; N, 10.9; S, 6.2%);
ꢀmax (KBr/cmꢀ1) 3369 (NH), 1773 (C ¼ O ester), 1664
(C ¼ O amide), 1524 (Ar–C), 1452 (N ¼ N), 1283 (C ¼ S),
1156 (C–N). dH (500 MHz, DMSO-d6) 2.29 (s, 3H), 7.09 (d,
J ¼ 9.2 Hz, 1H), 7.27 (d, J ¼ 8.0 Hz, 1H), 7.38 (t, J ¼ 7.4 Hz,
1H), 7.60–7.63 (m, 2H), 7.71 (d, J ¼ 7.4 Hz, 1H), 7.77 (d,
J ¼ 9.2 Hz, 2H), 7.93–7.96 (m, 3H), 10.82 (s, 1H), 11.60 (s,
1H), 12.16 (s, 1H). dC (125 MHz, DMSO-d6) 21.3 (CH3),
116.3 (Ar–C), 123.8 (Ar–C), 125.3 (Ar–C), 126.4
(C–N ¼ N–), 126.6 (Ar–C), 127.2 (Ar–C), 130.6 (Ar–C),
130.7 (Ar–C), 131.9 (Ar–C), 132.9 (Ar–C), 133.7 (Ar–C),
134.3 (Ar–C), 138.6 (Ar–C), 148.8 (Ar–C), 151.2 (Ar–C),
154.6 (C–N ¼ N–), 166.9 (C ¼ O), 169.4 (C ¼ O),
179.6 (C ¼ S).
2-(((3-((4-Chlorophenyl)diazenyl)-4-hydroxyphenyl)car-
bamothioyl)carbamoyl)phenyl
acetate
(2c)
(0.11 g,
0.5 mmol). (0.17 g, 74%) as orange solid, m.p: 238–239 ꢁC;
(Found: C, 56.1 H, 3.4; N, 11.7; S, 6.6. C22H18ClN4O4S,
Requires C, 56.4; H, 3.6; N, 11.9; S, 6.8%); ꢀmax (KBr/cmꢀ1
)
3355 (NH), 1771 (C ¼ O ester), 1662 (C ¼ O amide), 1523
(Ar–C), 1451 (N ¼ N), 1279 (C ¼ S), 1156 (C–N). dH
(500 MHz, DMSO-d6) 2.32 (s, 3H), 7.12 (d, J ¼ 8.6 Hz, 1H),
7.30 (d, J ¼ 8.0 Hz, 1H), 7.41 (t, J ¼ 7.7 Hz, 1H), 7.63–7.67
(m, 4H), 7.74 (d, J ¼ 6.3 Hz, 1H), 7.99 (d, J ¼ 2.3 Hz, 1H),
8.05 (d, J ¼ 8.6 Hz, 2H), 10.86 (s, 1H), 11.62 (s, 1H), 12.20
(s, 1H). 20.8 (CH3), 115.8 (Ar–C), 116.0 (Ar–C), 117.8
(Ar–C), 118.0 (Ar–C), 118.8 (Ar–C), 124.4 (Ar–C), 124.6
(Ar–C), 125.5 (C–N ¼ N–), 129.4 (Ar–C), 129.6 (Ar–C),
130.2 (Ar–C), 135.8 (Ar–C), 138.0 (Ar–C), 150.4 (Ar–C),
154.0 (Ar–C), 156.6 (C–N ¼ N–), 166.3 (C ¼ O), 168.9
(C ¼ O), 179.1 (C ¼ S).
Bacteriostatic screening
2-(((3-((2-Bromophenyl)diazenyl)-4-hydroxyphenyl)car-
Turbidimetry assay
bamothioyl)carbamoyl)phenyl
acetate
(2d)
(0.14 g,
0.5 mmol). (0.19 g, 74%) as reddish orange solid, m.p:
235–236 ꢁC; (Found: C, 51.0; H, 3.2; N, 10.7; S, 6.0.
C22H18BrN4O4S, Requires C, 51.4; H, 3.3; N, 10.9; S, 6.2%);
ꢀmax (KBr/cmꢀ1) 3359 (NH), 1779 (C ¼ O ester), 1665
(C ¼ O amide), 1529 (Ar–C), 1447 (N ¼ N), 1281 (C ¼ S),
1159 (C–N). dH (500 MHz, DMSO-d6) 2.33 (s, 3H), 7.15 (d,
J ¼ 9.2 Hz, 1H), 7.30 (d, J ¼ 8.6 Hz, 1H), 7.42 (d, J ¼ 8.0 Hz,
1H), 7.49–7.52 (m, 1H), 7.57 (t, J ¼ 7.4 Hz, 1H), 7.64 (d,
J ¼ 9.7 Hz, 2H), 7.75 (d, J ¼ 8.0 Hz, 1H), 7.91 (q, J ¼ 4.2 Hz,
2H), 8.13 (d, J ¼ 2.9 Hz, 1H), 11.48 (s, 1H), 11.65 (s, 1H),
12.22 (s, 1H). dC (125 MHz, DMSO-d6) 20.8 (CH3), 118.2
(Ar–C), 118.3 (Ar–C), 120.1 (Ar–C), 123.3 (Ar–C), 124.5
(Ar–C), 125.9 (C–N ¼ N–), 126.6 (Ar–C), 128.8 (Ar–C),
130.1 (Ar–C), 130.4 (Ar–C), 131.6 (Ar–C), 133.0 (Ar–C),
133.2 (Ar–C), 133.7 (Ar–C), 137.7 (Ar–C), 148.0 (Ar–C),
148.3 (Ar–C), 153.0 (C–N ¼ N–), 168.9 (C ¼ O),
179.2 (C ¼ S).
All of the synthesized compounds were attempted for anti-
microbial activity via turbidimetry assay against E. coli
(ATCC 25922) and S. aureus (S48/81). The inoculums were
prepared by allowing bacteria to grow in Luria–Bertani (LB)
broth at 37 ꢁC for 24 h at 200 rpm. 50, 80, and 100 ppm con-
centrations of compounds with addition of 0.99 mL of inoc-
ulums were incubated at 37 ꢁC and shaken at 180 rpm. Due
to the solubility problem, this method was discontinued.
Kirby–Bauer disk diffusion method
All the compounds were screened for their antimicrobial
activity via disk diffusion method against E. coli (ATCC
25922) and S. aureus (S48/81). Ampicillin was used as a
positive control and DMSO as a negative control. All bac-
teria were cultured in nutrient broth and incubated at 37 ꢁC
for 24 h. The agar media was prepared by pouring nutrient
agar solution onto the sterilized petri dishes. Then, the bac-
teria cultures were spread on the surface of the agar. The
sterilized disks (6 mm diameter) were impregnated with
(10 lL/disk) of the test samples and then placed on the sur-
face of the agar media. Each agar plates were incubated at
37 ꢁC for 24 h. Antimicrobial activity was determined by
measuring the diameter of the inhibition zone and com-
2-(((3-((3-Bromophenyl)diazenyl)-4-hydroxyphenyl)car-
bamothioyl)carbamoyl)phenyl
acetate
(2e)
(0.11 g,
0.5 mmol). (0.20 g, 78%) as orange solid, m.p: 237–238 ꢁC;
(Found: C, 50.9; H, 3.2; N, 10.5; S, 5.9. C22H18BrN4O4S,
Requires C, 51.4; H, 3.3; N, 10.9; S, 6.2%); ꢀmax (KBr/cmꢀ1
)
3376 (NH), 1776 (C ¼ O ester), 1662 (C ¼ O amide), 1524
(Ar–C), 1452 (N ¼ N), 1283 (C ¼ S), 1156 (C–N). dH
(500 MHz, DMSO-d6) 2.32 (s, 3H), 7.13 (d, J ¼ 8.6 Hz, 1H),
7.30 (d, J ¼ 8.6 Hz, 1H), 7.41 (t, J ¼ 7.4 Hz, 1H), 7.57 (t, pared with the positive control.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
7
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AutoDock Tool, and PyMOL software.[53,54] The grid box
(x, y, and z) with size 40 Å and spacing 0.375 Å were posi-
tioned on the protein’s active sites. The structure of S. aur-
eus tyrosyl-tRNA synthetase protein (PDB entry: 1JIJ) was
attained from Protein Data repository (http://www.rcsb.org/
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and targeted protein were performed using AutoDock Tool,
while binding affinity and molecular interaction evaluation
and prediction were performed using Vina software. The
molecular interaction was visualized using PyMOL computer
modeling software.
Conflict of Interest: None.
Funding
The authors would like to thank the Ministry of Education Malaysia [15] Saeed, A.; Shaheen, U.; Hameed, A.; Naqvi, S. Z. H. Synthesis,
for financial support through F07/FRGS/1883/2019.
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Antimicrobial and Anticancer Agents. Eur. J. Med. Chem.
2010, 45, 1323–1331. DOI: 10.1016/j.ejmech.2009.12.016.
ORCID
Zainab Ngaini
Ferlicia Rasin
http://orcid.org/0000-0001-7243-6856
http://orcid.org/0000-0003-1414-5852
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