Synthesis, characterization, antimicrobial, antioxidant and computational evaluation of N-acyl-morpholine-4-carbothioamides

Article abstract of DOI:10.1007/s11030-020-10054-w

Abstract: The present research paper reports the convenient synthesis, successful characterization, in vitro antibacterial, antifungal, antioxidant potency and biocompatibility of N-acyl-morpholine-4-carbothioamides (5a–5j). The biocompatible derivatives were found to be highly active against the tested bacterial and fungal strains. Moreover, some of the screened N-acyl-morpholine-4-carbothioamides exhibited excellent antioxidant potential. Docking simulation provided additional information about possibilities of their inhibitory potential against RNA. It has been predicted by in silico investigation of the binding pattern that compounds 5a and 5j can serve as the potential surrogate for design of novel and potent antibacterial agents. The results for the in vitro bioassays were promising with the identification of compounds 5a and 5j as the lead and selective candidate for RNA inhibition. Results of the docking computations further ascertained the inhibitory potential of compound 5a. Based on the in silico studies, it can be suggested that compounds 5a and 5j can serve as a structural model for the design of antibacterial agents with better inhibitory potential. Graphic abstract: Binding mode of compound 5j inside the active site of RNA in 3D space. 5j displayed highest antibacterial potential than the reference drug ampicillin with ZOI 10.50?mm against Staphylococcus aureus. 5j also displayed highest antifungal potential than the reference drug amphotericin B with ZOI 18.20?mm against Fusarium solani.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s11030-020-10054-w

Molecular Diversity
https://doi.org/10.1007/s11030-020-10054-w
ORIGINAL ARTICLE
Synthesis, characterization, antimicrobial,
antioxidant and computational evaluation
of N‑acyl‑morpholine‑4‑carbothioamides
Hamid Aziz1 · Aamer Saeed1 · Muhammad Aslam Khan2 · Shakeeb Afridi2 · Farukh Jabeen3,4
Received: 16 October 2019 / Accepted: 15 February 2020
© Springer Nature Switzerland AG 2020
Abstract
The present research paper reports the convenient synthesis, successful characterization, in vitro antibacterial, antifungal,
antioxidant potency and biocompatibility of N-acyl-morpholine-4-carbothioamides (5a–5j). The biocompatible derivatives
were found to be highly active against the tested bacterial and fungal strains. Moreover, some of the screened N-acyl-mor-
pholine-4-carbothioamides exhibited excellent antioxidant potential. Docking simulation provided additional information
about possibilities of their inhibitory potential against RNA. It has been predicted by in silico investigation of the binding
pattern that compounds 5a and 5j can serve as the potential surrogate for design of novel and potent antibacterial agents.
The results for the in vitro bioassays were promising with the identifcation of compounds 5a and 5j as the lead and selective
candidate for RNA inhibition. Results of the docking computations further ascertained the inhibitory potential of compound
5a. Based on the in silico studies, it can be suggested that compounds 5a and 5j can serve as a structural model for the design
of antibacterial agents with better inhibitory potential.
Graphic abstract
Binding mode of compound 5j inside the active site of RNA in 3D space. 5j displayed highest antibacterial potential than the
reference drug ampicillin with ZOI 10.50 mm against Staphylococcus aureus. 5j also displayed highest antifungal potential
than the reference drug amphotericin B with ZOI 18.20 mm against Fusarium solani.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10054-w) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
Keywords Multidrug-resistant · Antibiotics · Antimicrobial · DNA · Disk difusion
in the construction of anion receptors, metal complexes and
organocatalysts [10]. The presence of electron-donating or
electron-withdrawing groups imparts TU derivative electro-
chemical properties [8]. Aroyl TU are successfully employed
in environmental control, as ionophores and in ion selective
electrodes [11, 12]. In synthetic chemistry, TU derivatives
are used in the synthesis of triazines, isoxazoles and oxa-
zoles [13, 14]. TU have received central interest in recent
past owing to their pharmacological activities like anti-
diabetic, antituberculosis, anti-infammatory, insecticidal,
rodenticidal, antiviral and antimicrobial [15]. A plethora
of examples from the literature are available emphasizing
the antibacterial and antifungal potential of TU derivatives.
Quinazolinone-based TU possess analgesic, anticonvulsant,
analgesic, cytotoxic and antimicrobial activities [16, 17]. TU
incorporating a hippuric acid moiety exhibit antimicrobial
activity comparable to the reference standard ciprofoxacin
[18]. 5,5-Diphenylpyrrolidine N-aroylthiourea heterocycles
display a broader spectrum of superior antibacterial and anti-
mycobacterial activity [19]. Cui et al. reported the synthesis
and antibacterial potential of thiouracil-based thioureas. The
synthesized thioureas displayed good inhibitory potential
against the tested bacterial strains [20]. TU derivatives such
as thiophene-ethyl thiourea exhibited more efcient efects
on multidrug-resistant HIV-1 strains than the standards com-
monly used such as trovirdine, delavirdine, MKC-442 and
AZT [21]. Some literature-reported potent, biocompatible,
antioxidant and antimicrobial thiourea is shown in Fig. 1.
Introduction
The infectious diseases caused by drug-resistant microbial
strains are a growing threat for scientifc community to deal
with [1]. Systemic and dermal fungal infections have been
increased especially in immuno-suppressed individuals like
cancer and AIDS [2]. Moreover, the available antifungal
drugs being biochemically like human cells are associated
with severe side efects. Another major problem is the lack
of development of antibacterial agents against drug-resistant
bacterial pathogens [3, 4]. Except for lipopeptides, most of
the currently used antibiotics belong to those discovered
before 1980s. Besides, most of the advances that have been
made since 1980s are achieved through modifcations to the
existing antibiotics. Consequently, research has been focused
toward the development of potent antimicrobial agents
against novel targets to overcome the problems associated
with the drug resistance [5, 6]. Accordingly, the develop-
ment of potent antimicrobial agents or the expansion of pre-
vious antimicrobial drugs is required. In this regard, litera-
ture survey reveals thiourea-based heterocyclic analogues as
potent and biocompatible antimicrobial agents [7].
Thioureas (TU) are organosulfur compounds having
general formula of SC (NH2)2. These are versatile building
blocks for the synthesis of biologically relevant heterocycles
[8, 9]. The presence of hard O- and N- and soft S-donor
atoms impart TU the ability to develop efcient H-bonding
interactions. The H-bonding ability of TU has been exploited
Cl
Cl
S
S
O
HN
N
H
O
H
N
H
OCH3
N
N
N
N
O
H
H3CO
CH3
N
S
H
N
O
S
O
N
N
N
CN
N
N
Cl
NH
S
O
Br
H3CO
H3CO
N
O
CH3
O
S
S
HN
O
OH
N
N
N
H
N
H
HN
O
N
S
H
HN
S
HN
Fig. 1 Some literature-reported potent, biocompatible, antioxidant and antimicrobial thiourea derivatives
1 3

Molecular Diversity
Table 1 Synthesis of diferent N-acyl-morpholine-4-carbothioamide
(5a–5j) derivatives
Considering the global spread of antibiotic resistance and
the emergence of novel antibiotics-resistant microbes cou-
pled with the lack of efective antibiotics, there is an urgent
need for the design and synthesis of potent antimicrobial
agents. In this context, the current research presents the fac-
ile synthesis, antimicrobial evaluation and docking analysis
of some morpholine-based TU derivatives. The synthetic
work proceeds in the presence of dry distilled acetone with
higher yields.
S. no. Compound
R
S. no. Compound R
1
2
3
4
5
5a
5b
5c
5d
5e
2-NO2Ph
Hydrogen atom (H)
3-NO2 Ph
2,4-diClPh
4-CH3Ph
6
7
8
9
10
5f
5g
5h
5i
2-CH3Ph
C3H7
C4H9
PhCH2
C6H13
5j
Results and discussion
at 3259.53–3339.75 cm−1 (SI Figs. 5, 9). The H NMR
spectra present peaks for NH protons at 8.60–8.48 ppm
(SI Figs. 6, 10). FT-IR spectra have new peaks ranging
from 1662.19 to 1683.90 cm−1 for the amide linkages (SI
Figs. 5, 9). 13C NMR spectra of the synthesized N-acyl-mor-
pholine-4-carbothioamide (5a–5j) derivatives display sharp
signals at 161–163 ppm for the resulting carbonyl carbon of
the amides (SI Figs. 8, 12). 1H NMR spectra display double
doublets at 7.75–7.29 ppm for the 1,4-substituted pattern
of the synthesized N-acyl-morpholine-4-carbothioamide
(5a–5j) derivatives (SI Fig. 11). The aliphatic chain displays
strong absorption peaks in the range of 2995–2867 cm−1 in
the FT-IR spectra. The aliphatic chain display multiplets at
2.19–0.90 and 13–38 ppm in the 1H and 13C NMR spectra,
respectively. The characteristic peak for thiocarbonyl carbon
(C=S) is present as intense peak at 1259–1264 (SI Figs. 5, 9)
in the FT-IR and at 177–179 ppm in 13C NMR spectra (SI
Figs. 8, 12) of the synthesized compounds.
1
Synthesis
The synthetic route adopted for the synthesis of N-acyl-
morpholine-4-carbothioamides (5a–5j) derivatives is shown
in Scheme 1. Briefy, aryl and alkyl chlorides (2a–2j) are
synthesized from variously substituted acid derivatives at
room temperature using dry DMF and thionyl chloride. The
freshly obtained acid chlorides were added to a solution of
potassium thiocyanate in dry distilled acetone to prepare
corresponding aroyl/alkyl isothiocyanates (3a–3j). The latter
were treated in situ with morpholine in equimolar ratios to
furnish the corresponding the N-acyl-morpholine-4-carboth-
ioamide (5a–5j) derivatives in excellent yields (70–90%).
The synthesized compounds (5a–5j) (Table 1) were puri-
fed by recrystallization using DCM as a solvent at room
temperature [9, 15].
Spectroscopic characterizations
In vitro antimicrobial assays
Structures of the synthesized N-acyl-morpholine-4-car-
bothioamide (5a–5j) derivatives were assigned from spec-
Antibacterial assay
1
troscopic technique (FT-IR, H/13C NMR) and elemental
The antibacterial potential of the synthesized N-acyl-mor-
pholine-4-carbothioamide (5a–5j) derivatives was assessed
from agar disk difusion method using two gram-positive
(Staphylococcus aureus and Bacillus subtilis) and three
gram-negative (Pseudomonas aeruginosa, Escherichia
analysis (CHNS). The synthesized N-acyl-morpholine-
4-carbothioamide (5a–5j) derivatives provided satisfac-
tory elemental analysis results for the elements C, H,
N and S aligned with the proposed structural formulae.
FT-IR spectra displayed broader peaks for the NH protons
Scheme 1 Synthetic route employed for the synthesis of N-acyl-morpholine-4-carbothioamide (5a–5j) derivatives
1 3

Molecular Diversity
Table 3 Antifungal activity of the screened N-acyl-morpholine-4-car-
bothioamide (5a–5j) derivatives
coli and Klebsiella pneumoniae) strains. The antibacterial
potential of the screened derivatives in terms of their zone
of inhibition (ZOI) at a moderate concentration of 70 µg/
disk is summarized in Table 2. The screened derivatives
showed varied response against the various screened bacte-
rial strains. Among the tested strains, the screened deriva-
tives (5a–5j) were found to be more active against gram-
positive bacterial strains than the gram-negative strains.
The screened derivatives displayed highest potential against
Staphylococcus aureus with 5j and 5a inducing the highest
ZOI, i.e., 10.50 mm and 9.90 mm higher than the refer-
ence drug ampicillin. The same trend was found among the
derivatives screened against Bacillus subtilis with 5j and 5a
inducing the highest ZOI, i.e., 11.20 mm and 9.0 mm. In
the case of gram-negative bacterial strains, Pseudomonas
aeruginosa was found to be more resistant to the screened
derivatives with highest ZOI displayed by 5j and 5i. How-
ever, derivatives 5c, 5e and 5f were lacking any antibac-
terial potential. Moreover, the Escherichia coli were com-
paratively more prone to the screened derivatives where the
compounds displayed highest antibacterial potential than
the rest of the gram-negative bacterial strains. The screened
derivative 5f displayed the highest ZOI, i.e., 8.20 mm fol-
lowed by 5j and 5b with almost similar ZOI, i.e., 8.0 mm.
Furthermore, 5e was found to be the least active derivative
with no activity against Pseudomonas aeruginosa and Kleb-
siella pneumoniae.
S. no.
Compound
Zone of inhibition (in mm at 70 ug/
disk)
A. favus
A. niger
F. solani
1
2
3
4
5
6
7
8
5a
10.1
7.1
–
17.7
8.0
7.0
7.9
6.9
5.8
9.1
10.1
5.8
10.3
16.0
–
12.1
5.8
–
5b
5c
5d
6.0
7.5
6.2
9.5
10.3
5.8
12.6
15.6
–
7.0
8.0
6.1
9.2
11.3
5.9
18.2
15.4
–
5e
5f
5g
5h
9
5i
10
11
12
5j
Amphotericin B
DMSO
The sample concentration was 70 µg/mL. Values (mean SD) are
average of triplicate
– no observed/calculated activity, DMSO negative control
summarized in Table 3. Among the screened compounds,
5j was found to be the most potent against the tested fun-
gal strains as the most prominent zones of inhibition were
observed against Fusarium solani, i.e., 18.20 mm higher
than the reference drug amphotericin B at moderate con-
centration of 70 µg/disk. Derivative 5j was found to be
highly active against the remaining fungal strains with ZOI
close to the standard reference drug used. Compound 5a
was found to be the most active one against Aspergillus
niger with ZOI higher than amphotericin B. In general,
Aspergillus favus was found to be the slightly resistant
strain against the screened strains. Derivative 5b was
Antifungal assay
The synthesized N-Acyl-morpholine-4-carbothioamide
(5a–5j) derivatives were further evaluated to confer
their antifungal potential against spore-forming strains
via agar disk difusion method. The results obtained are
Table 2 Antibacterial activity
S. no.
Compound
Zone of inhibition (mm)
of the screened N-acyl-
morpholine-4-carbothioamides
(5a–5j) derivatives
E. coli
P. aeruginosa
S. aureus
B. subtilis
K. pneumoniae
1
2
3
4
5
6
7
8
5a
7.2
8.0
7.0
6.9
6.8
8.2
7.5
6.9
6.5
8.0
20
8.70
6.20
–
7.50
–
9.9
8.0
6.7
7.8
6.2
7.0
8.8
9.7
6.0
10.5
9.8
–
9.0
6.0
5.8
9.5
5.8
6.9
8.0
8.0
5.8
11.2
35
8.0
5.3
7.5
8.0
–
5.3
7.0
8.3
8.4
9.5
22
5b
5c
5d
5e
5f
–
5g
7.50
8.0
9.20
13.0
35.0
–
5h
9
5i
10
11
12
5j
Ampicillin
DMSO
–
–
–
The sample concentration was 70 µg/mL. Values (mean SD) are average of triplicate
– no observed/calculated activity, DMSO negative control
1 3

Molecular Diversity
found to be inactive against the Aspergillus favus and
Fusarium solani and did not induce any prominent zone
of inhibition.
mL, respectively. From the results summarized, it can be
suggested that compounds 5f, 5i, 5g and 5j exhibited excel-
lent antioxidant potential and have the potential to be further
analyzed for in vivo antioxidant potency (Table 4).
Antioxidant assays
Biocompatibility against human red blood cells (hRBCs)
Total antioxidant capacity (TAC), total reducing power
(TRP) and DPPH free radical scavenging assays were per-
formed to probe the antioxidant potential of the synthe-
sized compounds at a screening concentration of 200 μg/
mL. TAC is based on the conversion of Mo (VI) to Mo (V)
and indicates the quenching ability of the tested substance
toward ROS [22]. In general, the screened compounds
displayed varied antioxidant ability. Maximum value for
total antioxidants in terms of ascorbic acid equivalents of
the test compound was found as 123.47 1.20 μg AAE/
mg for compound 5i followed by 5h, 5f and 5j with TAC
of 119.82 1.21 μgAAE/mg, 112.63 1.14 μgAAE/mg
and 107.21 0.82 μgAAE/mg, respectively. Furthermore,
to assess the antioxidant capacity of the synthesized com-
pounds TRP was performed. A compound possessing redox
property neutralizes and absorbs free radicals by transforma-
tion of Fe+3 to Fe+2 ion. Compounds possessing reducing
power have the capacity to reduce ferric ion into ferrous
ion [23]. Highest reducing power potential was observed
for 5i as 158.71 0.40 μgAAE/mg followed by 5f, 5g, 5j
as 141.47 0.64 μgAAE/mg, 132.46 0.22 μgAAE/mg and
129.51 1.0 μgAAE/mg, respectively. Subsequently, DPPH
free radical scavenging assay was performed to augment the
TAC and TRP. The spectrophotometric method is based on
quenching of stable colored radicals of DPPH indicating the
scavenging ability of antioxidants [24]. Likewise, free radi-
cal scavenging activity varied among the compounds with
some exhibited excellent scavenging potential. Signifcant
FRSA was observed for 5f and 5i with 64.21 0.32% and
58.81 0.44 at 200 μg/mL with IC50 154 µg/mL and 180 µg/
To evaluate the biosafe nature, the synthesized N-acyl-mor-
pholine-4-carbothioamide (5a–5j) derivatives were screened
for biocompatibility against human red blood cells (hRBCs).
In the experiment, freshly isolated human red blood cells
(hRBCs) and test compounds were co-incubated in phos-
phate bufer saline (PBS) that mimic extracellular environ-
ment [25]. The bioassay is based on the release and measure-
ment of hemoglobin from red blood cells (RBCs) which can
be prompt by the tested compound if it could rupture RBCs
[26]. The compounds were tested for hemolytic activity at
high concentration, i.e., 400 µg/mL. In generally, hemoly-
sis≥25% is considered hemolytic, while hemolysis≤10%
is considered non-hemolytic. In the study, the tested com-
pounds exhibited remarkable hemocompatibility even at
the highest concentration as shown in Table 5. The study
thus concludes that the synthesized compounds are highly
compatible; however, detailed in vivo studies are required
to verify and augment the in vitro results.
Molecular docking
Molecular docking analysis was opted to get deeper insights
into the RNA binding afnity in silico for the screened com-
pounds. In order to study the binding interaction of the series
of synthesized compounds, investigators relied on molecular
docking simulations to complement the experimental results
and to acquire the binding mode [27–31]. The volume occu-
pied by the cognate ligand in the receptor was defned as
the binding site of the minimized eubacterial ribosomal
Table 4 Antioxidant potential
S. no.
Compound
TAC (µg AAE/mg)
TRP (µg AAE/mg)
DPPH (% FRSA)
DPPH
(IC50) µg/
mL
of the screened N-acyl-
morpholine-4-carbothioamide
(5a–5j) derivatives at 200 µg/
mL
1
2
3
4
5
6
7
8
9
10
5a
5b
5c
5d
5e
5f
91.66 1.14
73.71 1.32
52.43 0.84
81.72 0.71
86.78 1.22
112.63 0.22
97.23 1.13
119.82 1.32
123.47 1.20
107.21 1.43
121.32 1.24
96.71 0.98
74.23 0.92
93.87 0.95
93.23 1.13
141.47 0.64
132.46 0.22
152.91 1.43
158.71 0.40
129.51 1.0
33.5 1.38
28.11 1.2
23.32 1.74
27.0 0.97
29.37 1.22
58.81 0.44
51.33 0.82
53.85 0.71
64.21 0.32
50.93 0.47
>200
>200
>200
>200
>200
180
200
200
154
200
5g
5h
5i
5j
Values (mean SD) are average of triplicate
1 3

Molecular Diversity
Table 5 % Hemolysis of the screened N-acyl-morpholine-4-carboth-
ioamide (5a–5j) derivatives
S. no.
Compound
% Hemolysis
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
0.842 0.22
0.25 0.18
0.72 0.20
1.28 0.22
0.66 0.24
2.04 0.08
0.53 0.12
2.17 0.22
0.88 0.38
0.72 0.12
0.19 0.08
9
10
11
DMSO
The sample concentration was 400 µg/mL. Values (mean SD) are
average of triplicate
decoding A site (E. coli 16S rRNA A site) and an input site
sphere was defned within a radius of 5 Å located inside
the major groove of receptor. The ligands with optimized
geometries were docked into the active site of the minimized
eubacterial ribosomal decoding A site (E. coli 16S rRNA
A site, PDB codes:1j7t). van der Waals forces, H bonds,
electrostatic and hydrophobic interactions are the acting
forces found between a small molecule and macromolecule
found to anchor the ligands within the gorge of active site.
The variants with receptor–inhibitor complex having lowest
energy were selected, for further studies. Among the docked
conformations for each ligand, the most favorable conforma-
tion was retained to perform further analysis of the interac-
tions. Docking poses having lowest binding energy, and a
maximum number of docking interactions were ranked as
the most favorable pose. The software computed their bind-
ing energy as a negative value. The most preferred dock-
ing poses among all the docked conformations was used to
probe the interactions of the docked conformations with the
nitrogen bases.
Fig. 2 Superimposition of docked ligands inside the active site of
RNA. Ligands are presented in thick tube mode, heteroatoms in ele-
mental color and carbon in cyan color, while receptor is shown in rib-
bon format in yellow color, and bases are presented in wires. (Color
fgure online)
This H-bonding displayed by the screened compounds is
responsible for imparting stability to the screened com-
pounds inside the pocket of RNA.
The docked compounds showed sufciently strong inter-
actions especially, 5a which was stabilized by H-bonding
with the key bases including A17 and C30. Ligand 5a also
formed π–H interaction which provided further strength
inside the major groove of RNA. Similarly, compound
5j was anchored inside the RNA pocket via tremendous
signifcant H-bonding and aromatic H-bonding with the
nitrogen bases. Figure 3a, b shows binding pattern of the
compound inside the major groove of the duplex. Ligand
5j stabilized itself in the binding pocket by establishing
copious electrostatic contacts with the nitrogen bases
including hydrogen bonding, G15, A31, A17 (Fig. 4a,
b) and aromatic H-bonding, C30. Moreover, ligands 5a
and 5j showed good binding energy, − 8.54 kCal/mol and
− 8.52 kCal/mol further ascertain the strong binding and
inhibitory potential of ligands 5a and 5j.
Consequently, the screened compounds were found
to be nicely stacked within the active site of the major
groove of the RNA complex (Fig. 2). H-bonding signif-
cantly infuences the binding of the screened compounds
with RNA. Detailed investigation of the interaction of
screened compounds with the RNA duplex of RNA was
performed. The docked poses of the screened compounds
were observed to be signifcantly surrounded by nitrogen
bases A16, A17, A29, C30, G15, G18, U19 infuencing the
interaction and in turn driving inhibitory potential of the
antibacterial compounds. Based on the observations, the
carbonyl oxygen of the screened compounds was found to
have displayed H-bonding with the key nitrogen bases and
anchored the molecular geometries within the active site.
The docked ligands exhibited fairly well binding with
energy values ranging from − 9.62 to − 7.63 KCal/mol.
The higher binding energy values stabilized the ligands
within the gorge of the active site through various interac-
tions with RNA pocket. The lowest energy docked poses
was associated with signifcant binding features mainly
based on interaction due to numerous interacting moieties
of the druglike ligands including the versatile structure of
the docked ligands.
1 3

Molecular Diversity
Fig. 3 Binding mode of compound 5a inside the active site of RNA
in 3D space. a, b Docking poses of compound 5a in 3D space.
Ligands are presented in thick tube mode with elemental and cyan
color, while key residues are presented in wire mode, elemental color
and the helix of duplex is shown in yellow-colored ribbon format. a
Aromatic H-bonding and H-bonding of ligand atoms and bases. b
Docked pose of ligand 5a within the active site in 2D space. (Color
fgure online)
Fig. 4 Binding mode of compound 5j inside the active site of RNA
in 3D space. a, b 3D docked poses of compound 5j. Ligand is pre-
sented in tube-and-ball mode in elemental and cyan color, interacting
residues are shown in tube mode elemental color, while key residues
are shown in wire mode, elemental color and the helix of duplex is
shown in yellow-colored ribbon format. a H-bonding of ligand atoms
and bases. b Docked pose of ligand 5j within the active site in 2D
space. (Color fgure online)
derivatives. In silico results revealed the synthesized N-acyl-
morpholine-4-carbothioamide (5a–5j) showed compliance
to the Lipinski’s Ro5 [32] and Veber’s Ro3 [33], cutof lim-
its, which pave their way to be used as RNA inhibitors with
no derivative found to show any violation.
Drug likeness
Descriptors such as number of rotatable bonds, hydrogen
bond donor (NOR), hydrogen bond acceptor (HBA), Lipin-
ski’s acceptors (lip_acc), Lipinski’s donors (lip_don), Lipin-
ski’s violation (lip_viol), Lipinski’s drug likeness (lip_drug),
logP, molecular weight and total polar surface area (TPSA)
were computed to check the druglike properties of the
synthesized N-acyl-morpholine-4-carbothioamide (5a–5j)
Accordingly, the druglike molecules must have molecular
weight≤500, log P (logarithm of the octanol/water partition
coefcient) ≤ 5, total polar surface area (TPSA) < 140 Å2,
number of hydrogen bond donors (HBD)≤5 and hydrogen
1 3

Molecular Diversity
bond acceptor (HBA)≤10 as per Lipinski’s Ro5 [32]. Veber
et al. ofered further modifcations in the Ro5 [33]. Accord-
ing to Veber, number of rotatable bonds (NOR) of a druglike
molecule should be fewer or equal to 10 [32]. Molecules
may have problem with bioavailability, which violate more
than one of these criteria. Detailed results of drug likeness
of the N-acyl-morpholine-4-carbothioamides (5a–5j) are
tabulated in Table 6.
Materials and methods
Experimental
The reactions involved were performed in a fame-dried
glassware under a positive pressure of nitrogen gas using
dry distilled solvents. Dried and purifed commercial-grade
solvents and reagents were purchased directly from chemi-
cal suppliers and were used without any further purifcation.
Thin-layer chromatography was performed using silica gel
60 F254 precoated plates (0.25 mm) to judge the progress
of the reaction by TLC analysis (single spot/two solvent sys-
Conclusions
tems) using UV lamp for detection purposes. H and 13C
1
The present paper presents the synthesis of a series of
N-acyl-morpholine-4-carbothioamide (5a–5j) derivatives.
The synthesized compounds were investigated against path-
ogenic bacterial and fungal strains. Our results concluded
that the compounds exhibited excellent growth inhibitory
potential against gram-positive and gram-negative bacteria
as well as against certain spore-forming fungi. Antioxidant
studies revealed that some of the derivatives such as 5i and
5f possess considerable antioxidant properties. Moreover,
the derivatives were found to be highly hemocompatible that
make the derivatives biosafe. To ascertain the possible infu-
ence of inhibitory potential of compounds, molecular dock-
ing was performed. On the basis of docking computations,
compounds 5a and 5j showed good binding mode. Thus, we
suggest that the electrostatic and hydrophobic interactions
existing between the target and compounds, particularly
5a and 5j, are well corroborated by the experimental data.
Docking simulation also elucidated the valuable information
to understand the mechanistic pathway of inhibition and can
contribute to the molecular design and synthesis of potent
antibacterial and fungal agents to quite a good level. These
results further demonstrate that compounds 5a and 5j can
serve as the structural model for design of novel antibacte-
rial agents.
NMR spectra were recorded on a 400 and 100 MHz Bruker
Avance AVIII spectrometer for compounds 5a–5j except
compound 5f, where 1H and 13C NMR spectra were recorded
on a 300 and 75 MHz Bruker spectrometer. TMS was used
as an internal standard and deuterated chloroform (CDCl3)
as a solvent for compounds 5a–5j except compound 5f,
where deuterated acetone was used. Proton chemical shifts
are reported as δ values in ppm relative to residual signals
from deuterated solvents, while coupling constants are indi-
cated by J in Hz. PerkinElmer spectrum 1000 series Fourier
transform infrared ATR spectrometer was used to record the
infrared spectra, and the spectra are measured in wave num-
bers (cm−1). The synthesized compounds were subjected to
elemental analysis to determine % of each element.
Synthesis of N‑acyl‑morpholine‑4‑carbothioamide
(5a–5j)
The synthetic work was accomplished as reported elsewhere,
but with minor modifcations. Briefy, acid chlorides were
synthesized from substituted acid derivatives (10 mmol) and
thionyl chloride (10 mmol) at 50 °C using dry DMF as a
catalyst. The freshly prepared acid chloride (10 mmol) dis-
solved in dry distilled acetone (10 mL) was added dropwise
Table 6 Physicochemical descriptors of the N-acyl-morpholine-4-carbothioamide (5a–5j) derivatives
Code
HBA
HBD
NOR
h_pKb
lip_acc
lip_don
lip_drug
lip_viol
logP (o/w)
logS
TPSA
Weight
5a
5b
5c
5d
5e
5f
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
3.00
2.00
3.00
2.00
2.00
2.00
3.00
4.00
3.00
6.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
14.00
7.00
4.00
7.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.78
0.84
0.82
2.06
1.14
1.14
0.10
0.54
0.93
1.43
−4.20
−3.41
−4.20
−4.88
−3.89
−3.89
−2.37
−2.88
−3.47
−3.91
119.48
73.66
119.48
73.66
73.66
73.66
73.66
73.66
73.66
73.66
295.32
250.32
295.32
319.21
264.34
264.34
216.30
230.33
264.34
258.38
5g
5h
5i
5j
1 3

Molecular Diversity
to a suspension of potassium thiocyanate (10 mmol) in ace-
tone (05 mL), and the reaction mixture was refuxed at 50 °C
for 30 min under nitrogen. After cooling to room tempera-
ture, a solution of the morpholine (10 mmol) in dry distilled
acetone (10 mL) was added to the resulting mixture and
was refuxed for 30 further min. After the required time, the
reaction mixture was poured into cold water and the products
precipitated were recrystallized from DCM [9, 15].
1H, Ar–H), 8.48 (d, 1H, J =8.6 Hz, Ar–H), 8.36 (d, 1H,
J=8.6 Hz, Ar–H), 7.79 (t, 1H, J=7.8 Hz, Ar–H), 4.22 (b,
s, 2H, O–CH2), 3.86 (b, s, 4H, N–CH2, O–CH2), 3.75 (b, s,
2H, N–CH2); 13C NMR (CDCl3): δ=180.91 (C=S), 169.63
(C=O), 148.54 (Ar), 135.14 (Ar), 133.64 (Ar), 129.34 (Ar),
124.58 (Ar), 122.44 (Ar), 66.30 (O–CH2), 53.08 (b, N–CH2),
52.14 (b, N–CH2); Anal. Calc. for C12H13N3O4S: C: 48.81%;
H: 4.44%; N: 14.23%; S: 10.86%; Found: C: 48.70%; H:
4.45%; N: 14.29%; S: 10.93%.
N‑(2‑Nitrobenzoyl)morpholine‑4‑carbothioamide (5a)
N‑(2,4‑Dichlorobenzoyl)morpholine‑4‑carbothioamide (5d)
Mol. Wt. 327.31, Yield 76%, Rf = 0.48 (pet-ether: ethyl
acetate 4:1), Yellow powder, m.p 130 °C. IR (ATR, cm−1)
ν: 3289.24 (NH), 2973.5 (sp2 C–H), 2958.23, 2876.58
(sp3 C–H), 1683.29 (NCO), 1601.37, 1580.19, 1519.60
(Ar–C=C), 1440.97 (–C–H bending), 1354.23 (–C–H
Mol. Wt. 319.21, Yield 85%, Rf = 0.42 (pet-ether: ethyl
acetate 4:1), m.p 136 °C, Yellow powder, IR (ATR, cm−1)
ν: 3259.53 (NH), 2985.49 (sp2 C–H), 2930.628, 286.90
(sp3 C–H), 1683.90 (NCO), 1582.82, 1526.52, 1456.94
(Ar–C=C), 1435.81 (–C–H bending), 1371.94 (–C–H
1
bending), 1245.75 (C=S); H NMR (CDCl3): δ = 8.63 (b,
1
s, 1H, NH), 8.43 (d, 1H, J =8.29 Hz, Ar–H), 8.24 (d, 1H,
J=8.29 Hz, Ar–H), 7.86 (t, 1H, J=8.2 Hz, Ar–H), 7.79 (t,
1H, J=8.2 Hz, Ar–H), 4.20 (b, s, 2H, O–CH2), 3.85 (b, s,
4H, N–CH2, O–CH2), 3.76 (b, s, 2H, N–CH2); 13C NMR
(CDCl3): δ = 180.71 (C=S), 169.83 (C=O), 147.24 (Ar),
135.43 (Ar), 133.40 (Ar), 128.34 (Ar), 128.13 (Ar), 121.24
(Ar), 66.34 (O–CH2), 52.82 (b, N–CH2), 52.24 (b, N–CH2);
Anal. Calc. for C12H13N3O4S: C: 48.81%; H: 4.44%;
N: 14.23%; S: 10.86%; Found: C: 48.75%; H: 4.48%; N:
14.29%; S: 10.88%.
bending), 1262.24 (C=S); H NMR (CDCl3): δ = 8.60 (b,
s, 1H, NH), 7.62 (d, 1H, J =8.4 Hz, Ar–H), 7.476 (d, 1H,
J=2.0 Hz, Ar–H), 7.365 (dd, 1H, J=8.4 Hz, Ar–H), 4.19 (b,
s, 2H, O–CH2), 3.84 (b, s, 4H, N–CH2, O–CH2), 3.72 (b, s,
2H, N–CH2); 13C NMR (CDCl3): δ=177.94 (C=S), 161.37
(C=O), 138.39 (Ar), 132.15 (Ar), 131.62 (Ar), 131.34 (Ar),
130.55 (Ar), 127.85 (Ar), 66.21 (O–CH2), 52.80 (b, N–CH2),
51.54 (b, N–CH2); Anal. Calc. for C12H12Cl2N2O2S: C:
45.15%; H: 3.79%; N: 8.78%; S: 10.05%; Found: C: 45.05%;
H: 3.89%; N: 8.83%; S: 10.0%.
N‑Benzoylmorpholine‑4‑carbothioamide (5b)
N‑(4‑Methylbenzoyl)morpholine‑4‑carbothioamide (5e)
Mol. Wt. 282.0, Yield 80%, Rf =0.38 (pet-ether: ethyl ace-
tate 4:1), Light yellow powder, m.p 154 °C. IR (ATR, cm−1)
ν: 3237.48 (NH), 2970.84 (sp2 C–H), 2924.72, 2848.35
(sp3 C–H), 1663.62 (NCO), 1520.27, 1451.88, 1441.58
(Ar–C=C), 1425.58 (–C–H bending), 1357.40 (–C–H bend-
ing), 1222.82 (C=S); 1H NMR (CDCl3): δ=8.46 (b, s, 1H,
NH), 7.83 (m, 2H, Ar–H), 7.60 (m, 1H, Ar–H), 7.49 (m,
2H, Ar–H), 4.24 (b, s, 2H, O–CH2), 3.845 (b, s, 4H, N–CH2,
O–CH2), 3.667 (b, s, 2H, N–CH2); 13C NMR (CDCl3):
δ=179.23 (C=S), 163.22 (C=O), 133.23 (Ar), 132.32 (Ar),
129.03 (Ar), 127.79 (Ar), 66.26 (O–CH2), 52.80 (b, N–CH2),
51.70 (b, N–CH2); Anal. Calc. for C12H14N2O2S: C: 57.58%;
H: 5.64%; N: 11.19%; S: 12.81%; Found: C: 57.55%; H:
5.63%; N: 11.21%; S: 12.85%.
Mol. Wt. 264.34, Yield 79%, Rf = 0.52 (pet-ether: ethyl
acetate 4:1), m.p 152 °C, White powder, IR (ATR, cm−1) ν:
3339.75 (NH), 3031.0, 2962.12 (sp2 C–H), 2924.34, 2866.24
(sp3 C–H), 1662.19 (NCO), 1610.17, 1589.20, 1521.52,
1452.78 (Ar–C=C), 1428.95 (–C–H bending), 1389.94
1
(–C–H bending), 1259.47 (C=S); H NMR (CDCl3):
δ=8.48 (b, s, 1H, NH), 7.31 (d, 2H, J=8.0 Hz, Ar–H), 7.282
(d, 2H, J=8.0 Hz, Ar–H), 4.23 (b, s, 2H, O–CH2), 3.83 (b, s,
4H, N–CH2, O–CH2), 3.65 (b, s, 2H, N–CH2), 2.431 (s, 3H,
C–H); 13C NMR (CDCl3): δ=179.41 (C=S), 163.20 (C=O),
144.09 (Ar), 129.67 (Ar), 127.87 (Ar), 127.70 (Ar), 66.26
(O–CH2), 52.80 (b, N–CH2), 51.54 (b, N–CH2), 21.639
(C–H); Anal. Calc. for C11H8N4O3S: C: 59.07%; H: 6.10%;
N: 10.60%; S: 12.13%; Found: C: 59.01%; H: 6.11%; N:
10.63%; S: 12.16%.
N‑(3‑Nitrobenzoyl)morpholine‑4‑carbothioamide (5c)
N‑(2‑Methylbenzoyl)morpholine‑4‑carbothioamide (5f)
Mol. Wt. 327.31, Yield 90%, Rf = 0.48 (pet-ether: ethyl
acetate 4:1), m.p 146 °C; Yellow powder, IR (ATR, cm−1)
ν: 3350.65 (NH), 2958.73 (sp2 C–H), 2962.75, 2866.98
(sp3 C–H), 1676.39 (NCO), 1578.49, 1531.0 (Ar–C=C),
1409.67 (–C–H bending), 1384.24 (–C–H bending), 1239.18
(C=S); 1H NMR (CDCl3): δ=8.56 (b, s, 1H, NH), 8.93 (s,
Mol. Wt. 264.34, Yield 70%, Rf = 0.45 (pet-ether: ethyl
acetate 4:1), m.p 280 °C, White powder, IR (ATR, cm−1)
ν: 3259.45 (NH), 3044.09 (sp2 C–H), 2896.16 (sp3 C–H),
1634.08 (NCO), 1583.34, 1543.71 (Ar–C=C), 1430.55
(–C–H bending), 1363.91 (–C–H bending), 1281.21 (C=S);
1 3

Molecular Diversity
1
1H NMR (CD3COCD3): δ=9.68 (b, s, 1H, NH), 7.56 (d, 1H,
J=7.50 Hz, Ar–H), 7.41 (t, 1H, J=8.4 Hz, Ar–H), 7.28 (m,
2H, Ar–H), 4.20 (b, s, 2H, O–CH2), 3.78 (b, s, 6H, N–CH2,
O–CH2), 2.90 (s, 3H, C–H); 13C NMR (CD3COCD3):
δ = 179.55 (C=S), 165.75 (C=O), 136.93 (Ar), 134.60
(Ar), 130.99 (Ar), 130.71 (Ar), 127.81 (Ar), 125.60 (Ar),
65.93 (O–CH2), 51.83 (b, N–CH2), 50.87 (b, N–CH2),
19.31 (C–H); Anal. Calc. for C13H16N2O2S: C: 59.07%; H:
6.10%; N: 10.60%; S: 12.13%; Found: C: 59.0%; H: 6.17%;
N: 10.64%; S: 12.10%.
bending), 1229.08 (C=S); H NMR (CDCl3): δ = 8.29 (b,
s, 1H, NH), 7.19 (t, 2H, J = 8.2 Hz, Ar–H), 7.08 (t, 1H,
J=8.2 Hz, Ar–H), 7.06 (d, 2H, J=8.2 Hz, Ar–H), 4.20 (b, s,
2H, O–CH2), 3.84 (b, s, 4H, N–CH2, O–CH2), 3.73 (b, s, 2H,
N–CH2), 3.47 (s, 2H, C–H); 13C NMR (CDCl3): δ=180.93
(C=S), 172.63 (C=O), 135.77 (Ar), 129.73 (Ar), 129.33
(Ar), 127.84 (Ar), 66.18 (O–CH2), 52.60 (b, N–CH2), 51.58
(b, N–CH2), 40.53 (C–H); Anal. Calc. for C13H16N2O2S: C:
59.07%; H: 6.10%; N: 10.60%; S: 12.13%; Found: C: 59.0%;
H: 6.18%; N:10.63%; S: 12.19.
N‑Butyrylmorpholine‑4‑carbothioamide (5g)
N‑Heptanoylmorpholine‑4‑carbothioamide (5j)
Mol. Wt. 216.30, Yield 80%, Rf = 0.47 (pet-ether: ethyl
acetate 4:1), m.p>300 °C, Light Yellow powder, IR (ATR,
cm−1) ν: 3290.85 (NH), 3091.05 (sp2 C–H), 2964.81,
2852.11 (sp3 C–H), 1716.34 (NCO), 1540.32, 1498.98,
1466.72 (Ar–C=C), 1444.72 (–C–H bending), 1371.96
Mol. Wt. 258.14, Yield 70%, Rf = 0.54 (pet-ether: ethyl
acetate 4:1), m.p 93.20 °C, Yellow powder, IR (ATR, cm−1)
ν: 3267.60 (NH), 2914.88, 2849.45 (sp3 C–H), 1669.13
(NCO), 1514.42, 1459.74 (–C–H Bending), 1430.18 (C–N),
1238.52 (C=S); 1H NMR (CDCl3): δ=8.27 (b, s, 1H, NH),
4.13 (b, s, 2H, O–CH2), 3.80 (b, s, 4H, N–CH2, O–CH2),
3.70 (b, s, 2H, N–CH2), 2.16 (t, 2H, J = 7.50 Hz, C–H),
1.80 (q, 2H, J = 7.20 Hz, C–H), 1.34 (m, 6H, C–H), 0.90
(t, 3H, J = 7.50 Hz, C–H); 13C NMR (CDCl3): δ = 180.93
(C=S), 175.40 (C=O), 66.15 (O–CH2), 52.50 (b, N–CH2),
51.52 (b, N–CH2), 36.44 (C–H), 31.96 (C–H), 28.90 (C–H),
26.23 (C–H), 22.63 (C–H), 14.73 (C–H); Anal. Calc. for
C12H22N2O2S: C: 55.78%; H: 8.58%; N: 10.84%; S: 12.41%;
Found: C: 55.71%; H: 8.88%; N: 10.37%; S: 11.73%.
1
(–C–H bending), 1246.27 (C=S); H NMR (CDCl3):
δ=8.32 (b, s, 1H, NH), 4.15 (b, s, 2H, O–CH2), 3.80 (b, s,
4H, N–CH2, O–CH2), 3.70 (b, s, 2H, N–CH2), 2.19 (t, 2H,
J=7.90 Hz, C–H), 1.68 (sixtet, 2H, J=7.90 Hz, C–H), 0.95
(t, 3H, J = 7.90 Hz, C–H); 13C NMR (CDCl3): δ = 180.47
(C=S), 175.47 (C=O), 66.21 (O–CH2), 52.70 (b, N–CH2),
51.75 (b, N–CH2), 38.44 (C–H), 19.12 (C–H), 13.93
(C–H); Anal. Calc. for C9H16N2O2S: C: 49.98%; H: 7.46%;
N: 12.95%; S: 14.82%; Found: C: 49.89%; H: 7.53%; N:
12.91%; S: 14.85%.
In vitro antimicrobial assays
N‑Pentanoylmorpholine‑4‑carbothioamide (5h)
Antibacterial assay
Mol. Wt. 230.33, Yield 77%, Rf =0.50 (pet-ether: ethyl ace-
tate 4:1), m.p 260 °C, Light yellow powder, IR (ATR, cm−1)
ν: 3359.75 (NH), 3059.25 (sp2 C–H), 2974.30, 2865.51 (sp3
C–H), 1713.74 (NCO stretch), 1564.12, 1479.88 (–C–H
Bending), 1460.62 (C–N), 1434.67 (–C–H Bending),
1232.92 (C=S); 1H NMR (CDCl3): δ=8.30 (b, s, 1H, NH),
4.15 (b, s, 2H, O–CH2), 3.80 (b, s, 4H, N–CH2, O–CH2),
3.69 (b, s, 2H, N–CH2), 2.17 (t, 2H, J=7.50 Hz, C–H), 1.63
(m, 4H, C–H), 0.94 (t, 3H, J = 7.50 Hz, C–H); 13C NMR
(CDCl3): δ=180.47 (C=S), 175.47 (C=O), 66.20 (O–CH2),
52.72 (b, N–CH2), 51.64 (b, N–CH2), 38.44 (C–H), 19.12
(C–H), 13.93 (C–H); Anal. Calc. for C12H22N2OS: C:
52.15%; H: 7.88%; N: 12.16%; S: 13.92%; Found: C:
52.08%; H: 7.96%; N: 12.15%; S: 13.96%.
The antibacterial potential of the synthesized N-acyl-mor-
pholine-4-carbothioamide (5a–5j) derivatives was evalu-
ated using agar disk difusion method in a well-maintained
sterile environment. Two gram-negative bacterial strains
(Escherichia coli (ATCC25992) and Pseudomonas aer-
uginosa (ATCC-15442)) and three gram-positive bacterial
strains (Staphylococcus aureus (ATCC-6538), Bacillus
subtilis (ATCC-6633) and Klebsiella pneumoniae (ATCC-
1705)) were cultured in a nutrient broth at 37 °C for 24 h.
According to the test specifcation (1×106 CFU/mL), seed-
ing density of the bacterial culture was adjusted. To prepare
lawn on the nutrient agar plates, 50 µL of refreshed cultures
was poured on the agar media and swapped. 7 µL of the
test compound (with fnal concentration of 70 µg/disk) was
poured on the sterile flter paper disks (5 mm) and placed
on nutrient ager plates, respectively. DMSO and ampicillin
were used as negative and positive controls, respectively.
The assay was performed in duplicate, and the plates were
incubated at 37 °C for 24 h. The antibacterial activity of the
compounds was determined by measuring the diameter of
N‑(2‑Phenylacetyl)morpholine‑4‑carbothioamide (5i)
Mol. Wt. 264.36, Yield 75%, Rf =0.51 (pet-ether: ethyl ace-
tate 4:1), m.p 231 °C, Light yellow powder, IR (ATR, cm−1)
ν: 3104.90 (b, s 1H, NH), 2963.05 (sp2 C–H), 2950, 2856.13
(sp3 C–H), 1605.59 (NCO), 1538.83, 1549.20, 1495.68
(Ar–C=C), 1450.85 (–C–H bending), 1354.07 (–C–H
1 3

Molecular Diversity
zones (mm) showing complete inhibition with the help of
vernier caliper [34].
respectively. The IC50 was calculated by twofold serial dilu-
tion method [36].
Antifungal assays
Total antioxidant capacity (phosphomolybdenum assay)
The antifungal potential of the synthesized N-acyl-mor-
pholine-4-carbothioamide (5a–5j) derivatives against
Aspergillus favus (FCBP 0064), Aspergillus niger (FCBP
0198) and Fusarium solani (FCBP 0291) was assessed as
reported elsewhere. Stock cultures of fungal strains were
cultured on sterile plates containing SDA (20–25 mL) and
then swabbed/streaked through cotton buds. For spore for-
mation, the plates were incubated at 28 °C for 7 days. After
incubation, fungal spores were taken, and spore suspension
was made in 0.02% v/v sterile Tween 20 solution in distilled
water. Finally, turbidity was modifed as per McFarland 0.5
turbidity standard. Standard agar disk difusion method was
employed for the analysis of possible antifungal potential
of the compounds. 100 µL volume of spore suspension was
taken for each fungal strain from Tween 20 solution (0.02%
v/v) and swabbed on separate petri plates containing sterile
SDA media. Compounds (7 µL) were applied to the steri-
lized flter paper disks (5 mm) which were then retained
on the seeded SDA plates. Paper disks of amphotericin B
(5 µL) served as a positive control, while the disk soaked
with DMSO (7 µL) acted as a negative control. For fungal
growth, the plates were then incubated at 28 °C for 24–48 h.
After incubation, each loaded disk with compounds and con-
trols were observed for the appearance of zone of inhibition.
The diameter of ZOI was measured by using a vernier cali-
per to the nearest mm [35].
Total antioxidant capacity of the synthesized N-acyl-mor-
pholine-4-carbothioamide (5a–5j) derivatives was deter-
mined by using phosphomolybdenum assay as reported
previously. A reagent solution (900 µL) comprising of 4 mM
ammonium molybdate, 28 mM sodium phosphate and 0.6 M
sulfuric acid was mixed with 100 µL of the test compound.
The mixture was then incubated at 95 °C for 90 min, fol-
lowed by cooling at room temperature. Absorbance was
measured at 630 nm using a microplate reader. Ascorbic
acid was used as a standard (positive control) and DMSO
as blank (negative control). The test was performed in trip-
licate, and the resultant TAC was expressed as µg ascorbic
acid equivalent per mg dry weight of the compound (µg
AAE/mg compound) [37].
Total reducing power estimation
The reducing power/capacity of the synthesized N-acyl-
morpholine-4-carbothioamide (5a–5j) derivatives was esti-
mated following previously reported potassium ferricyanide
colorimetric assay, but with slight modifcations. The assay
was performed by taking 200 µL of each sample, 250 µL
each of 0.2 M phosphate bufer and 200 µL of 1% (w/v)
potassium ferricyanide. The reaction mixture was then incu-
bated at 50 °C for 20 min. After incubation, 200 µL of 10%
(w/v) TCA solution was added to the mixture followed by
centrifugation at 3000 rpm for 10 min. An aliquot of 150 µL
was taken from the supernatant of each mixture and shifted
to corresponding well in the microwell plate. Lastly, 50 µL
0.1% (w/v) ferricyanide solution was added to each well
and absorbance was measured at 630 nm. Ascorbic acid
was used as a positive standard, while DMSO was used as
a negative control. The assay was performed in triplicate,
and reducing power of the test samples was expressed as µg
ascorbic acid equivalent (AAE) per mg dry weight of the
compounds (µg AAE/mg compound) [37, 38].
Antioxidant assays
Free radical scavenging activity (FRSA)
Free radical scavenging activity of the synthesized N-acyl-
morpholine-4-carbothioamide (5a–5j) derivatives was
determined using 2,2-diphenyl 1-picrylhydrazyl (DPPH)
assay. Briefy, an aliquot of 20 μl from test sample (4 mg/
mL DMSO) was mixed with 180 μL of DPPH solution
(9.20 mg/100 mL methanol) in a 96-well plate. After incu-
bation at 37 °C for 30 min, the absorbance was measured
at 515 nm using microplate reader. Free radical scavenging
activity of the tested samples was calculated using the fol-
lowing formula
Biocompatibility against human red blood cells (hRBCs)
To investigate the biocompatible nature of the synthesized
N-acyl-morpholine-4-carbothioamide (5a–5j) derivatives
against isolated human red blood cells (hRBCs), a hemolytic
assay was performed by a previously established protocol
with minor modifcation. Briefy, fresh blood of about 3 mL
was collected from a healthy individual and dispensed into a
sterile EDTA tube. 1 mL blood was centrifuged (10000 rpm
for 10 min) for the separation of RBCs. The suspension was
washed thrice with PBS via centrifugation (2000 rpm for
% Inhibition
(absorbance of the control − absorbance of the sample)
=
× 100.
absorbance of the controle
The assay was performed in triplicate. Ascorbic acid
and DMSO were used as positive and negative controls,
1 3

Molecular Diversity
10 min). For the preparation of erythrocytes suspension in
phosphate-bufered saline (PBS), 200 µL of the washed pel-
leted erythrocyte was added to 4.8 mL of PBS (pH: 7.4)
followed by gentle shaking. In separate Eppendorf tubes,
erythrocyte suspension (180 µL) and test 20 µL sample were
gently mixed and incubated for 1 h at 35 °C. The mixture
was the centrifuged at 2000 rpm for 10 min, and superna-
tant was collected. The hemoglobin release was monitored
using microplate reader (BIOTEK) at 540 nm after dispens-
ing the supernatant in the 96-well plate. Triton X-100 and
distilled water were used as positive and negative controls,
respectively. Results were calculated as percentage hemoly-
sis induced by compounds calculated through the following
equation [26]
2D docking pose of the ligand protein complex having the
lowest energy was used to visualize the 2D ligand–protein
interactions.
Molecular docking
The MOE-Dock module was used to dock the optimized
ligands with the RNA. For this purpose, 30 total independ-
ent computations were carried out through MOE simula-
tions. Among the docked conformations, the conformation
with highest score for each ligand was studied in detail. The
lowest energy docking pose predicted by MOE program for
the ligand–RNA complex was analyzed to visualize the 2D
ligand–protein interaction.
[
]
ABs − ABnc
Drug likeness
% Hemolysis =
× 10
ABpc − ABnc
This study was performed using descriptor calculation
module of the MOE 2018. The ligand property calculation
function of MOE was used to perform the computations of
the molecular descriptors for all the compounds (Table 6).
Energy-minimized geometries of compounds 5a–5j stored
in molecular database (mdb) file of the MOE software
were used to calculate the selected descriptors by using the
descriptor calculation module of the MOE program.
where ABs is the absorbance of the tested sample and ABnc
and ABpc depict absorbance of negative and positive con-
trols, respectively.
Methods
Preparation of ligand fles
Files of the ligand were prepared using building module of
the Molecular Operating Environment (MOE-2018) program
by Chemical Computing Group (CCG) [39]. The ligand’s
geometries were drawn in building panel of MOE and were
optimized at a standard MMFF94 force feld level, having
0.0001 kcal/mol energy gradient convergence criterion [40].
The optimized 3D geometries were saved for further studies
in a molecular database (mdb) fle.
Acknowledgements The author Mr. Hamid Aziz is highly grateful
to the Higher Education Commission (HEC), Pakistan, for providing
indigenous scholarship as the fnancial support for the research work
performed.
Compliance with ethical standards
Conflict of interest All authors declare that they have no confict of
interest.
Preparation of receptor
References
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the RNA, and 30 independent computations were performed
in total for each ligand. The highest docking score conforma-
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Afliations
Hamid Aziz1 · Aamer Saeed1 · Muhammad Aslam Khan2 · Shakeeb Afridi2 · Farukh Jabeen3,4
3
* Hamid Aziz
hamidazizwazir@gmail.com
Department of Biology, Laurentian University, 935 Ramsey
Lake Road, Sudbury, ON P3E 2C6, Canada
4
Computation, Science, Research and Development
Organization, 1401, 2485 Hurontario Street, Mississauga,
ON L5A2G6, Canada
1
Department of Chemistry, Quaid-I-Azam University,
Islamabad 45320, Pakistan
2
Department of Biotechnology, Quaid-I-Azam University,
Islamabad 45320, Pakistan
1 3