Microwave-assisted synthesis of (Piperidin-1-yl)quinolin-3-yl) methylene)hydrazinecarbothioamides as potent inhibitors of cholinesterases: A biochemical and in silico approach

Article abstract of DOI:10.3390/molecules26030656

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, characterized by central cognitive dysfunction, memory loss, and intellectual decline poses a major public health problem affecting millions of people around the globe. Despite several clinically approved drugs and development of anti-Alzheimer’s heterocyclic structural leads, the treatment of AD requires safer hybrid therapeutics with characteristic structural and biochemical properties. In this endeavor, we herein report a microwave-assisted synthesis of a library of quinoline thiosemicarbazones endowed with a piperidine moiety, achieved via the condensation of 6/8-methyl-2-(piperidin-1-yl)quinoline3-carbaldehydes and (un)substituted thiosemicarbazides. The target N-heterocyclic products were isolated in excellent yields. The structures of all the synthesized compounds were fully established using readily available spectroscopic techniques (FTIR,¹H-and¹³C-NMR). Anti-Alzheimer potential of the synthesized heterocyclic compounds was evaluated using acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The in vitro biochemical assay results revealed several compounds as potent inhibitors of both enzymes. Among them, five compounds exhibited IC₅₀ values less than 20 μM. N-(3-chlorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine carbothioamide emerged as the most potent dual inhibitor of AChE and BChE with IC₅₀ values of 9.68 and 11.59 μM, respectively. Various informative structure–activity relationship (SAR) analyses were also concluded indicating the critical role of substitution pattern on the inhibitory efficacy of the tested derivatives. In vitro results were further validated through molecular docking analysis where interactive behavior of the potent inhibitors within the active pocket of enzymes was established. Quinoline thiosemicarbazones were also tested for their cytotoxicity using MTT assay against HepG2 cells. Among the 26 novel compounds, there were five cytotoxical and 18 showed proliferative properties.

Full text of DOI:10.3390/molecules26030656

molecules
Article
Microwave-Assisted Synthesis of (Piperidin-1-yl)quinolin-3-yl)
methylene)hydrazinecarbothioamides as Potent Inhibitors of
Cholinesterases: A Biochemical and In Silico Approach
Rubina Munir 1,
* ,
, Muhammad Zia-ur-Rehman 2,*, Shahzad Murtaza 3, Sumera Zaib 4, Noman Javid 1,5
Sana Javaid Awan 6,7, Kiran Iftikhar 3, Muhammad Makshoof Athar 1 and Imtiaz Khan 8,
*
1
Institute of Chemistry, University of the Punjab, Lahore 54590, Pakistan; noumanjavid@gmail.com (N.J.);
atharmakshoof@gmail.com (M.M.A.)
Applied Chemistry Research Centre, PCSIR Laboratories Complex, Lahore 54600, Pakistan
Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan; shahzad.murtaza@uog.edu.pk (S.M.);
kiran.iffi@gmail.com (K.I.)
Department of Biochemistry, Faculty of Life Sciences, University of Central Punjab, Lahore 54590, Pakistan;
sumera.biochem@gmail.com
Department of Chemistry (C-Block), Forman Christian College, Ferozepur Road, Lahore 54600, Pakistan
Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore 54000, Pakistan;
sana.javaidawan@yahoo.com
2
3
4
5
6
7
8
Department of Zoology, Kinnaird College for Women, Lahore 54000, Pakistan
Department of Chemistry and Manchester Institute of Biotechnology, The University of Manchester,
131 Princess Street, Manchester M1 7DN, UK
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
*
Correspondence: organist94@gmail.com (R.M.); rehman_pcsir@yahoo.com (M.Z.-u.-R.);
imtiaz.khan@manchester.ac.uk (I.K.)
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Munir, R.; Zia-ur-Rehman,
M.; Murtaza, S.; Zaib, S.; Javid, N.;
Awan, S.J.; Iftikhar, K.; Athar, M.M.;
Khan, I. Microwave-Assisted
Abstract: Alzheimer’s disease (AD), a progressive neurodegenerative disorder, characterized by
central cognitive dysfunction, memory loss, and intellectual decline poses a major public health
problem affecting millions of people around the globe. Despite several clinically approved drugs and
development of anti-Alzheimer’s heterocyclic structural leads, the treatment of AD requires safer
hybrid therapeutics with characteristic structural and biochemical properties. In this endeavor, we
herein report a microwave-assisted synthesis of a library of quinoline thiosemicarbazones endowed
with a piperidine moiety, achieved via the condensation of 6/8-methyl-2-(piperidin-1-yl)quinoline-
3-carbaldehydes and (un)substituted thiosemicarbazides. The target N-heterocyclic products were
isolated in excellent yields. The structures of all the synthesized compounds were fully established
using readily available spectroscopic techniques (FTIR, 1H- and 13C-NMR). Anti-Alzheimer potential
of the synthesized heterocyclic compounds was evaluated using acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) enzymes. The in vitro biochemical assay results revealed several com-
pounds as potent inhibitors of both enzymes. Among them, five compounds exhibited IC50 values less
Synthesis of (Piperidin-1-yl)quinolin-3-
yl)methylene)hydrazinecarbothioamides
as Potent Inhibitors of Cholinesterases:
A Biochemical and In Silico
Approach. Molecules 2021, 26, 656.
https://doi.org/10.3390/molecules
26030656
Academic Editor: Jacek Nycz
Received: 7 January 2021
Accepted: 24 January 2021
Published: 27 January 2021
than 20
carbothioamide emerged as the most potent dual inhibitor of AChE and BChE with IC50 values of
9.68 and 11.59 M, respectively. Various informative structure–activity relationship (SAR) analyses
were also concluded indicating the critical role of substitution pattern on the inhibitory efficacy
of the tested derivatives. In vitro results were further validated through molecular docking anal-
ysis where interactive behavior of the potent inhibitors within the active pocket of enzymes was
established. Quinoline thiosemicarbazones were also tested for their cytotoxicity using MTT assay
against HepG2 cells. Among the 26 novel compounds, there were five cytotoxical and 18 showed
proliferative properties.
µM. N-(3-chlorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
µ
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Keywords: quinoline; piperidine; thiosemicarbazone; carbothioamide; Alzheimer’s disease; neu-
rodegeneration; cholinesterases; molecular docking; ADME properties; HYDE assessment
Molecules 2021, 26, 656. https://doi.org/10.3390/molecules26030656
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 656
2 of 31
1. Introduction
Alzheimer’s, also known as senile dementia, is a chronic neurodegenerative disease
and is a global health problem due to its limited available treatments. The development
mechanism of Alzheimer’s disease (AD) still remains elusive though multiple factors have
been proposed for its induction out of which cholinergic hypothesis explains in the best
manner [1]. According to the hypothesis, the diminution of acetylcholine level causes
cognitive deficit and memory loss. The inhibition of acetylcholinesterase (AChE; EC 3.1.1.7)
and butyrylcholinesterase (BChE; EC 3.1.1.8) is supposed to be beneficial for the treatment
of AD [
and choline resulting in reduction of its levels that is the main cause of AD [
hydrolyzes butyrylcholine (BuCh). Histologically, AChE is mostly of neuronal origin, while
BChE is primarily present in the blood and glial cells [ ]. To prevent the cholinesterase
2]. The AChE enzyme hydrolyzes the neurotransmitter (acetylcholine) to acetic acid
3] while BChE
4–8
enzyme from hydrolyzing the neurotransmitters, several cholinesterase inhibitors such
as donepezil, rivastigimine, tacrine, ensaculin, and galantamine have been designed,
and used for the treatment of cognitive dysfunction and memory loss of AD patients.
However, several adverse side effects such as nausea, vomiting, decreased appetite, weight
loss, and hepatotoxicity associated with these drugs necessitate the development of new
cholinesterase inhibitors for the effective treatment of AD [9–11].
Among nitrogen-containing heterocycles, quinoline is considered as an important scaf-
fold due to its marvelous pharmacological potential [12
as Primaquine®, Plasmoquine®, Mefloquine®, Chloroquine®, OSI-930, and Saquinavir®
incorporate quinoline pharmacophore [14 15]. A range of quinoline derivatives have been
reported for their analgesic [16], antifungal [17], antibacterial [18], antioxidant [19], anti-
cancer [20 21], anti-inflammatory [22], antiviral [23 24], anti-Alzheimer [25], cytotoxic [26],
antileishmanial [27 28], and anti-hypertensive activities [29 30]. Similarly, piperidine ring
,13]. Several commercial drugs such
,
,
,
,
,
is also prevalent in numerous naturally occurring alkaloids [31]. According to FDA Or-
ange Book, piperidine ring falls among the top classified scaffolds in the list of 100 most
exploited simple ring systems in drug design and synthesis [32]. Piperidine derivatives
also demonstrate various biological functions such as antimalarial [33], anticonvulsant [34],
anticancer [35], and antidepressant [36] activities. Crizotinib®
Risperidone® 39], and Methyl phenidate®
based drugs (Figure 1). Likewise, thiosemicarbazones are emerging scaffolds of medicinal
interest and their antimicrobial [41 42], anti-inflammatory [43], antioxidant [44 45], anti-
cancer [46 48], anti-Alzheimer [49], and alpha-glucosidase inhibitory [50] activities have
[
37], Donepezil®
[38],
[
[40] are the well-known examples of piperidine-
,
,
–
been reported in recent years.
In view of our continued interest in the development of heterocyclic leads for the
treatment of AD [51
featuring more than one biologically potent moiety [57
–
56], and emerging trend in the exploration of hybrid molecules
59], we herein report the successful
–
integration of piperidine and thiosemicarbazone pharmacophores with quinoline scaffold
to develop a library of new hybrid heterocyclic molecules for the treatment of Alzheimer’s
disease (AD). The target compounds were synthesized successfully under microwave
irradiation conditions and were evaluated for their AChE and BChE inhibitory potential.
Quinoline thiosemicarbazones were also tested for their cytotoxicity using MTT assay
against HepG2 cells. Moreover, the binding affinities of the potent inhibitors in the active
site of both enzymes were elucidated using molecular docking approach.

Molecules 2021, 26, 656
3 of 31
Figure 1. Medicinal importance of quinoline, piperidine, and thiosemicarbazone motifs and rationale of current study.
2. Results and Discussion
2.1. Synthetic Chemistry
A library of new piperidine containing quinolinyl thiosemicarbazones 5(a
–
m) and
6(a–m) was synthesized using a facile synthetic approach as illustrated in Scheme 1. Acety-
lation of the commercial anilines 1(a,b) produced acetanilides 2(a,b) which underwent
Vilsmeier–Haack formylation using dimethylformamide and phosphoryl chloride affording
2-chloroquinoline-3-carbaldehydes 3(a,b) in 65–75% yield [60]. Subsequently, cetyltrimethy-
lammonium bromide (CTAB) catalyzed nucleophilic aromatic substitution of 3(a,b) with
piperidine in polyethylene glycol-400 (PEG-400) produced 2-(piperidin-1-yl)quinoline-3-
carbaldehydes 4(a,b) in 97–98% yield [61]. Finally, microwave-assisted condensation of
4(a,b) with a range of thiosemicarbazides afforded the desired hybrid compounds 5(a–m)

Molecules 2021, 26, 656
4 of 31
and 6(a
–
m) in excellent yields in 3–5 min (Table 1). The target compounds were also
prepared using the conventional approach, however, the isolated yields were lower com-
pared to microwave-assisted methodology. The comparative yields are given in Table 1.
Two-point structural diversity was introduced in the target compounds. Quinoline phar-
macophore inherits 6- and 8-substitutions (R1 and R2) from commercially available anilines
whereas electron-rich and electron-deficient groups were introduced at the aromatic ring
(R3) in the thiosemicarbazides. Benzyl and morpholinoethyl were also found as successful
substituents as R3 on the thiosemicarbazide moiety.
Scheme 1. Synthesis of 2-((6/8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazinecarbothi-
oamides. Reagents and Conditions: (
a
) glacial acetic acid, o-phosphoric acid, reflux, 6–7 h, 80–85%;
80 ◦C, 16–18 h, 65–75%; ( ) piperidine, PEG-400, CTAB, 135 ◦C, 2.5 h, 97–98%
) substituted thiosemicarbazide, glacial acetic acid, EtOH, MWI, 3–5 min; ( ) substituted thiosemi-
(b
d
) DMF, POCl3, 0
→
c
;
(
e
carbazide, glacial acetic acid, EtOH, reflux, 0.5–2 h.
Table 1. Comparative synthetic yields and anti-cholinesterase (AChE and BChE) potential of synthe-
sized compounds 5(a–m) and 6(a–m).
Conventional
Approach
Microwave-Assisted
Approach
Acetylcholinesterase
(AChE)
Butyrylcholinesterse
(BChE)
Compound
Yield (%)
IC50 ± SEM (µM)
5a
5b
5c
5d
5e
5f
80
86
85
90
85
94
93
91
92
92
95
91
97
97
27.6 ± 0.08
25.1 ± 0.25
42.3 ± 0.99
25.9 ± 0.96
35.2 ± 0.54
23.9 ± 0.25
19.85 ± 0.14
35.3 ± 0.01
33.1 ± 0.09
37.2 ± 0.83
29.8 ± 1.2
40.26 ± 0.17
24.6 ± 0.57
23.1 ± 0.11
5g

Molecules 2021, 26, 656
5 of 31
Table 1. Cont.
Conventional
Approach
Microwave-Assisted
Approach
Acetylcholinesterase
(AChE)
Butyrylcholinesterse
(BChE)
Compound
Yield (%)
IC50 ± SEM (µM)
5h
80
90
83
94
91
78
81
89
85
92
89
88
92
92
87
83
90
82
82
—
90
96
90
97
95
89
90
93
91
98
93
93
95
96
90
91
95
91
90
—
31.28 ± 0.37
24.09 ± 0.43
28.21 ± 0.94
30.65 ± 0.56
35.09 ± 1.2
62.3 ± 0.68
36.25 ± 0.36
25.89 ± 0.45
39.12 ± 0.27
12.89 ± 0.33
32.11 ± 0.15
9.68 ± 0.21
13.85 ± 0.78
40.23 ± 0.25
15.8 ± 1.3
25.12 ± 0.99
36.23 ± 0.02
42.36 ± 0.44
32.01 ± 0.87
45.02 ± 0.38
59.35 ± 0.13
60.02 ± 0.04
28.77 ± 0.63
24.59 ± 0.09
17.86 ± 0.35
37.06 ± 0.59
11.59 ± 1.2
18.56 ± 0.22
45.12 ± 0.19
51.11 ± 0.28
51.03 ± 0.52
32.01 ± 0.31
41.02 ± 0.89
35.14 ± 0.77
7.21 ± 0.39
5i
5j
5k
5l
5m
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
56.66 ± 0.41
39.91 ± 0.85
57.25 ± 0.03
21.01 ± 2.3
2.98 ± 0.62
6k
6l
6m
Donepezil
2.2. Spectroscopic Characterization
The condensation reaction between a carbaldehyde and a thiosemicarbazide was
confirmed by the appearance of a singlet around 8.77–9.07 ppm attributable to imine
(N=C
formation of desired product. The =N-N
around 11.74–12.27 ppm, while C-N proton was observed around 9.17–10.28 ppm. An
exception was observed in the spectral data of compounds 5a and 6a regarding C-N
H
) proton. Two downfield singlets of secondary thioamide protons endorsed the
H
proton, being more deshielded exhibited signal
H
H
signal. Two –NH2 protons appeared as two discrete broad singlets at 8.14 and 8.33–8.37
ppm. This is attributed to the existence of thiosemicarbazone in different stereochemical
forms and is in accordance with the previous theoretical and stereochemical studies of
1
carbothioamides [62
–
64]. In the H NMR spectra of compounds 5h and 6h, the C-N
H
proton signal appeared downfield due to the electron-withdrawing inductive effect of
fluoro group attached to the phenyl ring ortho to thioamide functionality. Along with
electron-withdrawing inductive effect, the electrostatic attraction between the electronega-
tive fluorine and the electropositive hydrogen may also develop resulting in the downfield
shifting of the peak (Figure 2).
Figure 2. Deshielding of Ar-NH in compounds 5h and 6h.

Molecules 2021, 26, 656
6 of 31
Another distinct pattern was observed in compounds 5(l,m) and 6(l,m) in which the
C-N
The upfield chemical shift can be ascribed to the aliphatic carbon atom succeeding –N
that shields this proton as compared to the C-N of the rest of the compounds in this series.
The triplet spin multiplicity of –N peak is due to the coupling of this proton with –CH2
H signal emerged comparatively upfield as a triplet (J = 6.0 Hz) instead of a singlet.
H
H
H
protons in its immediate vicinity (Figure 3).
Figure 3. Upfield shifting and splitting of –NH signal in 5(l,m) and 6(l,m).
The peaks of aromatic protons, depending upon their chemical environment, were
seen in a range of 7.05–8.37 ppm. Among aromatic protons, the singlet for H4 of quinoline
ring showed the highest chemical shift around 8.29–8.37 ppm. In the spectral data of com-
pounds 5e, 5h, and 6h, the aromatic protons appeared downfield referring to the presence
of electronegative fluoro and chloro group at ortho position of thioamide functionality. In
the spectral data of compounds (5,6)m, the six methylene protons referring to four –OCH2
protons of morpholine ring and two protons of thioamide NH-CH2 emerged as a multiplet
around 3.89–3.97 ppm while the six N–CH2 protons connected to nitrogen of morpholine
ring gave a multiplet peak around 2.90–2.70 ppm. In compounds (5,6)l, the methylene of
benzyl appeared as a triplet at 4.90 ppm, the higher chemical shift and splitting of signal
owing to the vicinity of the –N
H (Figure 3). Likewise, the piperidine ring protons appeared
as two multiplet peaks near 1.70 and 3.25 ppm, respectively.
13C NMR spectra further confirmed the newly formed structures by showing the peak
for C=S carbon as the most deshielded signal around 175.5–178.5 ppm. The peaks of the
aromatic and imine carbon atoms appeared between 112 and 163 ppm. The elemental
analyses were also in good agreement with the proposed structures.
Furthermore, the phenomenon of geometric as well as conformational isomerism
was observed in the 1H NMR as well as 13C NMR of the derivatives in less polar solvents
like chloroform. The compounds 5(a–m) and 6(a–m) could exist in either E or Z isomeric
form because of azomethine (-CH=N-) linkage while C-N bond rotation may give rise
to rotamers. Compound 5g was selected as a test compound and its NMR spectral data
were recorded in CDCl3 as well as DMSO. The spectra in CDCl3 showed additional peaks
referring to various isomers. However, 1H NMR spectra in DMSO exhibited no additional
peaks regarding NH or N=CH indicating the presence of a single isomeric form (see
Supplementary Materials).
2.3. In Vitro Cholinesterase Inhibition and Structure–Activity Relationship Analyses
The newly synthesized (piperidin-1-yl)quinolin-3-yl)methylene)hydrazinecarbothioa-
mides were screened for the identification of robust and potent inhibitors of cholinesterase
(AChE and BChE) enzymes. The results presented in Table 1 indicate the potential of this
hybrid scaffold to serve as a template for future investigations while making some key
structural variations to obtain the cholinesterase inhibitors of desirable impact. Herein, we
demonstrate some key structure–activity relationships based on the in vitro biochemical as-
say results. The effect of methyl substitution at the 6- (R1) and 8-position (R2) of quinoline was
studied in the first instance while keeping the thiosemicarbazone chain unchanged. Donepezil
was employed as a standard drug. Several compounds showed IC50 values less than 20 µM.
2-((6-Methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazinecarbothioamides showed
moderate results with relatively higher IC50 values as compared to their (8-methyl-2-

Molecules 2021, 26, 656
7 of 31
(piperidin-1-yl)quinolin-3-yl) analogues. Only compound 5g demonstrated good inhibitory
efficacy with IC50 value of 19.85
this series were less active (IC50 > 20
±
0.14
µ
M against AChE while rest of the compounds in
µ
M). Compound 5g incorporates a 4-chlorophenyl
ring as a R3 substituent on the thiosemicarbazone moiety. A dual and enhanced inhibition
was noticed when the position of methyl substituent was switched from 6 to 8 on the
quinoline ring (compound 6g). Replacing the 4-chloro substituent with a more electronega-
tive 3-fluoro group at the aryl ring (R3) produced diminished inhibition (5i), however, a
combination of 3-fluoro and 8-methyl substituents produced the lead selective inhibitor of
AChE (6i; IC50 = 15.8 ± 1.3 µM) (Figure 4).
Figure 4. Structure–activity relationship analysis of compounds 5g, 5i, 6g, and 6i.
The compounds with 8-methyl substitution exhibited good results with three com-
pounds (6d
Among them, 6f emerged as the most potent inhibitor against both enzymes with IC50
values of 9.68 0.21 M (AChE) and 11.59 1.2 M (BChE). Compound 6f incorporates a
, 6f, and 6g) acting as dual inhibitors with IC50 < 20 µM against AChE and BChE.
±
µ
±
µ
3-chlorophenyl ring as R3 substituent. The introduction of a di-substitution (2,6-dimethyl)
caused a detrimental effect on the inhibitory potency (compound 6d). Similar trend was
also noticed when chlorine substituent was moved to position 4 at the phenyl ring (com-
pound 6g) (Figure 5). The effect on the anti-cholinergic activity by different substituents
such as chloro, fluoro, and methyl on the phenyl group of hydrazine carbothioamide
moiety was also evaluated [65]. Anti-cholinesterase potential was predominant for the
meta substituted chloro and fluoro molecules when 8-position of quinoline is occupied by
a methyl substituent.
In case of dimethyl substituted inhibitors, 2,6-disubstituted analogues showed re-
markably good results as compared to compounds bearing 2,4-dimethyl substitution
on the aromatic ring (R3), no matter whether the quinoline ring is substituted at 6- or
8-position. Compounds bearing a benzyl substituent as R3 were ranked among the least ac-
tive inhibitors. Moreover, morpholine substituted compound 6m showed better inhibitory
activity than 5m.

Molecules 2021, 26, 656
8 of 31
Figure 5. Structure–activity relationship analysis of dual inhibitors 6d, 6f, and 6g.
2.4. Molecular Docking Studies
For docking studies, X-ray structures of human AChE (PDB ID: 4BDT) [66] and BChE
(PDB ID: 4BDS) [66] were selected as templates, because structures of electric eel AChE
were available at low crystallographic resolutions (>4 Å) and a structure of equine BChE
was not available. Molecular docking analysis of potent compounds was performed against
AChE and BChE for the identification of possible binding modes. The orientation of the
most potent and selective compounds 5i and 6i and crystallographic inhibitor huprine W
were shown in the active site of AChE (Figure 6), whereas, the binding modes of 5h and 6c
,
the most potent and selective compounds and cognate ligand tacrine were shown in the
active site of BChE (Figure 7). The orientation of dual inhibitors (5f, 5g, 6f, and 6g) were
shown in Figures 8 and 9.
2.4.1. Molecular Docking Studies of Acetylcholinesterase (AChE)
The active pocket of AChE was surrounded by amino acid residues Tyr124, Gly122,
Tyr337, Phe297, Leu289, Val340, Ser298, Arg296, Phe338, Trp286, Ser125, Leu76, Tyr341,
Tyr72, Ala204, Ser203, and His447. The hydrogen bonds and π-π interactions were formed
by the potent inhibitor 5i as well as by huprine W, as reported previously [66]. The
cognate ligand (huprine W) showed two conventional hydrogen bonds with Ser203 (2.33 Å)
and Gly122 (2.96 Å) and multiple
Additionally, 2-alkyl linkages (4.18 and 4.87 Å) and an alkyl linkage (4.52 Å) were seen with
Pro446. Moreover, Tyr337 formed two stacked bonds (3.54 and 4.47 Å), one -alkyl
bond (4.45 Å), and -donor hydrogen bond (4.01 Å) with huprine W. Other interactions
like one -alkyl with Tyr449 (5.38 Å), one -alkyl with Met443 (4.89 Å) and two -alkyl with
π-π stacking (4.00, 4.41, 5.30, and 3.69 Å) with Trp86.
π-
π
π
π
π
π
π
Trp439 (3.80 and 3.46 Å) and a carbon-hydrogen bond (3.53 Å) with His447 were observed.
The most potent compound 5i displayed several important interactions with amino acids
in the active pocket like
π
-alkyl bond (5.14 Å) with methyl substituent and
π-π T shaped
(5.06 Å) with phenyl ring of Trp286. Moreover, two
π-π
T shaped with phenyl ring (4.66
and 5.42 Å) and a conventional hydrogen bond with carbothioamide (2.32 Å) by Tyr341
and a carbon hydrogen bond (2.42 Å) with Asp74 were noted. Likewise, the compound
showed
with 3-fluoro substituent by Trp439 and two
fluorophenyl ring with Trp86. The amino acid Tyr337 showed
π
-
π
T shaped interactions (5.46 Å) with phenyl ring Phe338,
stacked (4.98 and 4.95 Å) formed by
T shaped (3.92 Å) with
π-lone pair (2.95 Å)
π-π
π-π

Molecules 2021, 26, 656
9 of 31
fluorophenyl ring and a conventional hydrogen bond (2.37 Å) with carbothioamide along
with a
selective compound 6i docked inside the AChE represented several important interactions
including stacking (5.67 Å) with methylquinoline ring by His287, T shaped (5.51 Å)
with fluorophenyl ring by Tyr124 and a conventional hydrogen bond (3.56 Å) with fluorine
substituent by Tyr337. Other interactions are T shaped (4.75 Å) with fluorophenyl
ring and -alkyl (5.24 Å) with piperidine ring by Tyr341, conventional hydrogen bonds
(3.61 and 2.95 Å, respectively) by Arg296 and Ser293 with sulfur of thiocarbonyl moiety.
Compound 6i also formed three stacked interactions with methylquinoline ring (4.30,
5.24, and 4.86 Å), a -sigma bond (2.74 Å), and two -alkyl bonds with methyl (5.25 Å)
and piperidine (5.20 Å) by Trp286 and a
π-alkyl bond (4.64 Å) with piperidine ring by Tyr124 (Figure 6). Another potent and
π
-π
π-π
π-π
π
π-π
π
π
π-alkyl (5.07 Å) with piperidine and one carbon
hydrogen bond (2.41 Å) by Tyr72 (Figure 6).
(b)
(a)
(c)
Figure 6. 3D binding modes of 5i
AChE, hydrogen bonding is shown by green dashed line, hydrophobic interactions are shown by
light purple color, dark pink dashed lines show -T shaped, light pink color shows stacked
interactions, and purple color dashed lines show π-sigma type interactions.
(a), 6i (b), and Huprine W (c) with the amino acid residue of
π
π-π

Molecules 2021, 26, 656
10 of 31
(b)
(a)
(c)
Figure 7. 3D binding modes of 5h (a), 6c (b), and tacrine (c) with the amino acid residue of BChE,
hydrogen bonding is shown by green dashed line, hydrophobic interactions are shown by light
purple color, dark pink dashed lines show -T shaped, light pink color shows stacked interactions,
π
π-π
and purple color dashed lines show π-sigma type interactions.
2.4.2. Molecular Docking Studies of Butyrylcholinesterase (BChE)
The active pocket of BChE was surrounded by amino acid residues Tyr332, Ser198,
Met437, Glu197, Ala328, Asp70, Trp430, Phe73, Gly121, Thr120, Trp82, Ile442, Gly115,
Gly117, Trp231, Ser79, His438, Tyr332, Tyr440, and Val331 [53]. The detailed analysis of
tacrine (tetrahydroacridin-9-amine) suggested the presence of two
π-alkyl (4.32 and 4.97 Å)
and four stacked bonds (4.25, 4.23, 5.61, and 3.59 Å) with Trp82 and a π
π
-
π
-alkyl linkage
(4.56 Å) with Ala328. All the interactions were presented by amino acid residues Trp82 and
Ala328 as shown in Figure 7.
The potent and selective compound 5h formed a conventional hydrogen bond (2.49 Å)
with quinoline ring and a carbon-hydrogen bond (2.81 Å) with piperidine ring by Ser198.
Similarly, a conventional hydrogen bond (3.78 Å) by Tyr332 with sulfur atom and same
bond (1.84 Å) by Asp70 with NH of carbothioamide moiety were observed. A halogen
bond (2.90 Å) and a conventional hydrogen bond (3.37) with 2-fluoro group by Asn83, and
a halogen bond (3.66 Å) with 2-fluoro group by Ser79 were noted. Moreover, two π-alkyl
linkage with piperidine (4.41 and 5.01 Å) and a halogen bond with 2-fluoro group (3.28 Å)
by Trp82, and two carbon-hydrogen bonds (2.35 and 3.06 Å) with piperidine ring by Glu197
and a π-π T shaped (5.63 Å) with quinolone ring by Phe329 were noticed. Additionally, two

Molecules 2021, 26, 656
11 of 31
π-alkyl linkages (4.01 and 4.73 Å) with 8-methyl and two
π
-
π
T shaped linkages (5.03 and
4.73 Å) with quinoline ring in addition to a π-sigma bond (2.67 Å) with the same by Trp231
were formed. The alkyl linkages by Leu286 (4.07 Å) and Val288 (5.07 Å) with 8-methyl
substituent were observed by the same compound (Figure 7).
Another compound 6c produced two
5.10 Å) by Trp82, a conventional hydrogen bond (1.81 Å) with carbothioamide moiety
by Asp70, and a stacked (3.60 Å) interaction with 2,4-dimethyphenyl ring and two
-alkyl (4.70 Å) interactions with 2- and 4-methyphenyl (4.67 and 3.79 Å, respectively)
by Tyr332. Moreover, amide- stacked interactions (4.01 Å) were noticed by Gly116
and a -donor (3.71 Å) by Gly117 with quinoline ring, while, a -alkyl (4.19 Å) with
4-methyphenyl and another -alkyl (5.26 Å) with 8-methyl group and a T shaped
linkage with quinoline ring (5.64 Å) were formed by Phe329. An additional alkyl linkage
(4.70 Å) with 4-methyphenyl by Pro285, two -alkyl linkages via piperidine ring (4.52 Å),
and another by 8-methyl of quinoline ring (4.81 Å) by His438 and a -alkyl linkage by
8-methyl of quinoline ring (4.07 Å) by Phe398 were observed. Two
noticed by 8-methyl of quinoline ring (4.06 and 4.08 Å) and two
π-alkyl linkages via piperidine ring (4.96 and
π-π
π
π
π
π
π
π-π
π
π
π
-alkyl linkages were
π-π T shaped linkages
with quinoline ring (5.39 and 5.31 Å) by Trp231, while, two
quinoline ring (5.46, 5.40 Å) by Leu286 (Figure 7).
π
-alkyl linkages by 8-methyl of
(a)
(b)
(d)
(c)
Figure 8. 3D binding modes of 5f
hydrogen bonding is shown by green dashed line, hydrophobic interactions are shown by light
purple color, dark pink dashed lines show -T shaped, light pink color shows stacked interactions,
and purple color dashed lines show π-sigma type interactions.
(a), 5g (b), 6f (c), and 6g (d) with the amino acid residue of AChE,
π
π-π

Molecules 2021, 26, 656
12 of 31
(b)
(a)
(c)
(d)
Figure 9. 3D binding modes of 5f
hydrogen bonding is shown by green dashed line, hydrophobic interactions are shown by light
purple color, dark pink dashed lines show -T shaped, light pink color shows stacked interactions,
(a), 5g (b), 6f (c), and 6g (d) with the amino acid residue of BChE,
π
π-π
and purple color dashed lines show π-sigma type interactions.
2.4.3. Dual Inhibitors of AChE and BChE
Compound 5f being the dual inhibitor of both enzymes showed some interesting
interactions inside the active site of AChE that includes two stacked bonds (4.42 and
5.67 Å) with methylquinoline and a -alkyl linkage (4 Å) with methyl group through Tyr337,
-alkyl linkage (5.38 Å), and a carbon H bond (2.61 Å) with piperidine ring by Tyr124 and
alkyl bonds with His447 (4.51 Å) and Ser203 (3.84 Å) by 3-chloro substituent. Additionally,
a carbon-hydrogen bond (2.01 Å) with piperidine ring by Asn87, -anion (3.99 Å) with
a 3-chloro group by Glu202, -sulfur (5.59 Å) with sulfur atom and two conventional
π-π
π
π
π-
π
π
hydrogen bonds with sulfur (3.64 Å) and NH (2.76 Å) by Tyr133 and a conventional
hydrogen bond (2.30 Å) and carbon-hydrogen bond (2.84 Å) with carbothioamide moiety
by Ser125. However, a
bonds with methylquinoline (4.15 and 4.29 Å) and additionally a
methyl group (5.20 Å) by Trp86 and two
by Trp439 were formed (Figure 8).
π
-
π
T shaped with 3-chloro group (5.15 Å) and two
-alkyl linkage with
-alkyl bonds (4.06 and 4.08 Å) with methyl group
π-π stacked
π
π
Similarly, compound 5g showed a conventional hydrogen bond (2.58 Å) and
linkage (4.80 Å) with 4-chloro group by Tyr337, and -alkyl linkage (5.01 Å) with piperidine,
T shaped (4.69 Å) with chlorophenyl ring and -alkyl (4.35 Å) with 4-chloro group
by Tyr341. Other important amino acids contributed significantly by different types of
interactions like three stacked bonds with methylquinoline (4.89, 4.77, and 4.03 Å) and
-alkyl bond (5.49 Å) with piperidine by Trp286, a carbon H bond (4.72 Å) and -alkyl
linkage (5.17 Å) with piperidine by Tyr72 and T shaped (5.32 Å) with chlorophenyl ring
π-alkyl
π
π-
π
π
π-π
a
π
π
π-π

Molecules 2021, 26, 656
13 of 31
and
π-alkyl (5.42 Å) with 4-chloro group by Tyr124. Moreover, a conventional hydrogen
bond (1.94 Å) with carbothioamide moiety by Arg296 and same bond (3.34 Å) with sulfur
atom by Ser293 and a stacked bond (5.70 Å) with methylquinoline and -alkyl bonds
π
-π
π
(4.19 Å) with methyl group by His287 were noticed (Figure 8).
Another dual and the most potent inhibitor 6f, formed one
π
-
π
stacked (5.65 Å)
-alkyl
T shaped (4.64 Å) with 3-chlorophenyl
-alkyl (5.32 Å) with piperidine ring by Tyr341 and -alkyl linkage (4.98 Å)
interaction with 8-methylquinoline ring by His287, one
(4.98 Å) with 4-chlorophenyl ring by Tyr124, one
ring and one
π-π T shaped (5.60 Å) and a π
π-π
π
π
with chloro group by Phe338. More interactions like two conventional hydrogen bonds
with sulfur (3.58 Å) and NH (1.71 Å) of carbothioamide by Arg296, a hydrogen bond
(2.95 Å) with sulfur of thiocarbonyl by Ser293, and a
group by Phe297 were noted. Three stacked interactions (4.37, 5.33, and 4.95 Å) were
observed with methyl quinoline ring, a -sigma bond (2.74 Å), and a -alkyl bond with
-alkyl (5.06 Å) with piperidine ring by Tyr72
π-alkyl linkage (4.40 Å) with chloro
π-π
π
π
piperidine (5.23 Å) by Trp286 in addition to a
(Figure 8).
π
The next dual inhibitor was 6g that formed
methylquinoline ring by His287, a T shaped (5.48 Å) with 4-chlorophenyl ring by Tyr124,
and a conventional hydrogen bond (2.71 Å) and -alkyl bond (4.83 Å) with chloro group
by Tyr337. Other interactions like T shaped (4.75 Å) with 4-chlorophenyl ring and one
-alkyl (4.06 Å) with chloro group and another with piperidine ring (5.34 Å) by Tyr341 were
π-π stacked (5.67 Å) interaction with 8-
π-π
π
π-π
π
also observed. Additionally, two conventional hydrogen bonds (3.60 Å) with sulfur and
another with NH (1.68 Å) of the carbothioamide by Arg296 and a conventional hydrogen
bond (2.94 Å) with sulfur of the carbothioamide by Ser293 were noticed. Moreover, three
π
-
π
stacked interactions (4.28, 5.24, and 4.85 Å) with methylquinoline ring, a
bond (2.70 Å), and two -alkyl bonds with methyl (5.24 Å) and piperidine ring (5.17 Å) by
Trp286 and a -alkyl (5.05 Å) and one carbon-hydrogen bond (2.38 Å) with piperidine ring
by Tyr72 were noticed (Figure 8). These multiple interactions especially and strong
π-sigma
π
π
π-π
hydrogen bonds of potent compounds binding in the center of the active pocket may be
the possible reason for the inhibitory profile of these derivatives.
Similarly, compound 5f being a dual inhibitor showed several interactions inside the
active pocket of BChE such as a conventional hydrogen bond (2.52 Å) with nitrogen atom
of quinoline ring by Ser198, a
by Phe329, two more T shaped linkages (4.74 and 5.05 Å) with 6-methyl quinoline
ring, -sigma bond with the same ring (2.69 Å), and -alkyl bonds (4.04 and 4.74 Å) with
π-π T shaped linkage (5.04 Å) with 6-methyl quinoline ring
π-π
π
π
6-methyl of quinoline ring by Trp231. One alkyl bond (4.08 Å) by Leu286 and another by
Val288 (5.05 Å) with 6-methyl of quinoline ring were noted. Asp70 formed a conventional
hydrogen bond (1.82 Å) with NH of carbothioamide moiety. Moreover, an alkyl bond
(4.36 Å) with 3-chloro of phenyl by Pro84, a carbon halogen bond (3.27 Å) with 3-chloro of
phenyl by Asn83, an alkyl bond (4.54 Å) with 3-chloro of phenyl by Ile69 and two more
π-alkyl linkages (5.00 and 4.40 Å) with piperidine ring by Trp82 were noticed (Figure 9).
Compound 5g showed an alkyl linkage (4.97 Å) by Val288 and Leu286 (4.12 Å) with
6-methyl quinoline ring, additionally a
and a conventional H-bond (1.91 Å) with NH of carbothioamide by Asp70. A halogen
bond (2.30 Å) was shown by 4-chloro with Asn68. Moreover, two -alkyl linkages (4.97
and 4.31 Å) with 6-methyl and two T shaped linkages (5.13 and 4.88 Å) with 6-methyl
quinoline ring, in addition to -sigma bond with the same ring (2.75 Å) were formed by
Trp231. Similarly, T shaped linkage (5.57 Å) with 6-methylquinoline ring by Phe329 was
π-donor bond (3.58 Å) with 4-chlorophenyl ring
π
π-π
π
π-π
noted. A conventional hydrogen bond (2.48 Å) with quinoline ring and a carbon-hydrogen
bond (2.80 Å) with piperidine ring by Ser198 were observed. Two carbon-hydrogen bonds
(2.24 and 3.00 Å) by Glu197 and two
π-alkyl linkage (5.03 and 4.38 Å) by Trp82 with
piperidine ring and a -alkyl linkage (5.30 Å) with piperidine ring by His438, an alkyl bond
π
(4.76 Å) with 4-chloro by Pro84, carbon-hydrogen bond (3.21 Å) with 4-chloro and an alkyl
bond (4.12 Å) with the same by Ile69 were also formed (Figure 9).

Molecules 2021, 26, 656
14 of 31
Compound 6f formed
(4.01 and 4.06 Å) with methyl and two
by Trp231, -alkyl with methyl (3.91 Å) by Phe398,
Leu286, -alkyl (5.39 Å) with 8-methyl of quinoline ring and two π-π
π
-alkyl (4.63 Å) with methyl substituent by His438, two
T shaped (5.46 and 5.71 Å) with quinoline ring
-alkyl (4.43 Å) with quinoline ring by
T shaped linkages
π-alkyl
π-π
π
π
π
(4.68 and 5.37 Å) with quinoline ring by Phe329 were observed. Moreover, a conventional
hydrogen bond (1.97 Å) with carbothioamide by Asp70, an alkyl linkage (4.59 Å) with
chloro group by Pro285, and a
and a π-alkyl (4.24 Å) with chloro group by Tyr332 were also present (Figure 9).
Compound 6g showed -alkyl bond (5.48 Å) by His438 and two -alkyl bonds (3.75
and 3.96 Å) by Trp82 with 8-methyl substituent. A -alkyl bond (5.99 Å) with piperi-
dine ring by Phe329, a conventional hydrogen bond (2.02 Å) with carbothioamide by
Pro285, a -anion (4.46 Å) with 4-chlorophenyl by Asp70, a stacked (5.39 Å) interac-
tion with 4-chlorophenyl by Tyr332 and a -sigma (3.88 Å) and a -alkyl (4.40 Å) with
8-methylquinoline ring by Ala328 were also formed (Figure 9).
All the compounds were found to bind deep inside the active pocket of BChE and
were involved in forming -sigma, stacked, -anion interactions with different amino
π-π stacked (3.82 Å) interaction with 3-chlorophenyl ring
π
π
π
π
π-π
π
π
π
π
-
π
π
acid residues. The docking studies were in parallel to the in vitro results. Taken to-
gether, the results presented herein showed that the novel 6- and 8-methyl-2-(piperidin-
1-yl)quinolin-3-yl)methylene)hydrazine-1-carbothioamide derivatives are promising in-
hibitors of cholinesterases.
2.5. HYDE Assessment of Selective Compounds against Cholinesterases (AChE and BChE)
HYDE visual affinity of all the ligands was carried out using LeadIT [67] software for
top 30 ranked docked conformers within the active site of the human AChE and BChE.
The binding energy and docking score by FlexX for the all the synthetic derivatives (6-
and 8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine-1-carbothioamides) are
given in Table 2. The FlexX docking score depicted that the selective derivatives have
lower energy scores as compared to nonselective inhibitors. Moreover, the binding free
energies ∆G given in Table 2 showed that the potent inhibitors exhibited higher affinity
towards the respective target (AChE and BChE). The docking scores of all the compounds
revealed the similar pattern as was suggested by in vitro analysis. The compounds that
were inactive exhibited lower docking scores while active and potent inhibitors showed
significant docking scores with the significant free binding energy values.
Table 2. Docking score of the top pose of selected compounds and their ranks after HYDE visual inspection in the AChE
and BChE.
Acetylcholinesterase
Docking Score by FlexX Free Energy of Binding
Butyrylcholinesterase
Docking Score by FlexX Free Energy of Binding
Compounds
−1
−1
−1
−1
)
for Top Pose (kcal mol
)
∆G (kJ mol
)
for Top Pose (kcal mol
)
∆G (kJ mol
5a
5b
5c
5d
5e
5f
−23.1892
−27.3759
−15.1888
−34.0828
−21.2287
−30.9532
−15
−18
−15
−17
−19
−23
−27.4250
−33.8532
−38.2179
−36.4835
−35.5422
−32.4958
−13
−18
−19
−20
−17
−21
5g
5h
5i
−24.9478
−25.3819
−26.3914
−22
−14
−25
−31.3943
−33.4593
−31.8917
−20
−22
−18
5j
−27.7381
−13
−33.3263
−19
5k
−18.8869
−10
−31.3938
−17

Molecules 2021, 26, 656
15 of 31
Table 2. Cont.
Acetylcholinesterase
Docking Score by FlexX Free Energy of Binding
Butyrylcholinesterase
Docking Score by FlexX Free Energy of Binding
Compounds
−1
−1
−1
−1
)
for Top Pose (kcal mol
−23.4789
−25.4734
−25.2746
−24.0306
−14.5216
−21.9230
−26.5830
−25.5518
−25.8729
−24.8538
−26.5146
−30.6048
−19.5944
−22.5508
−28.8842
−16.29
)
∆G (kJ mol
−11
−17
−14
−13
−10
−12
−15
−24
−24
−15
−26
−14
−16
−11
−17
−23
—
)
for Top Pose (kcal mol
−30.7439
−29.6099
−28.9743
−32.3915
−34.1889
−34.6624
−32.5630
−31.0974
−28.6461
−28.7235
−31.4126
−29.3584
−27.7883
−27.0438
−28.7614
—
)
∆G (kJ mol
−14
−13
−17
−14
−22
−20
−19
−20
−19
−14
−18
−17
−11
−16
−15
—
5l
5m
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
Huprine W
Tacrine
—
−17.70
−18
2.6. ADME Properties
ADME properties predict the impact of therapeutic compounds to access the tar-
get considering some parameters. These properties include physicochemical properties,
lipophilicity, water solubility, pharmacokinetics, drug-likeness, and medicinal chemistry.
These properties were evaluated using several prediction tools [68–70]. The properties
help in determining the drug-likeness of compounds being used for drug discovery and
development by sorting out new druggable candidates that are safer and follow the effec-
tive rules used for determination of these parameters. Table 3 represents some important
ADME properties compiled using web service and its underlying methodologies (Swis-
sADME) [68]. The properties suggested that our selective derivatives are safer to use as a
drug and have high probability of gastrointestinal absorption. Moreover, the compounds
showed no violation for Lipinski, Veber, and Egan rules and these filters originate from
analyses by well-known pharmaceutical companies for improving the quality of chemical
entities. Additionally, these recognition methods employ identification of the problematic
fragments in a molecule. PAINS (for pan assay interference compounds) are molecules
containing substructures showing potent response in assays irrespective of the protein
target. Such fragments may yield false positive biological output. Our selected compounds
revealed no alert for PAINS.

Molecules 2021, 26, 656
16 of 31
Table 3. ADMET prediction scores for the selected compounds [68].
Compounds
Properties
5f
5g
5h
5i
6c
6f
6g
6i
Physicochemical properties
Mol. Wt. (g/mol)
No. of atoms
437.99
30
437.99
30
421.53
30
421.53
431.60
31
437.99
437.99
30
421.53
30
30
0
16
No. of aromatic atoms
Fraction Csp3
16
16
16
16
16
16
16
0.26
6
0.26
6
0.26
6
0.26
0.32
6
0.26
6
0.26
6
0.26
6
No. of rotatable bonds
No. of H-bond acceptors
No. of H-bond donors
Molar refractivity
TPSA
6
2
2
3
3
2
2
2
3
2
2
2
2
128.99
2
2
2
2
134.05
84.64
134.05
84.64
128.99
84.64
138.97
84.64
134.05
84.64
134.05
84.64
128.99
84.64
84.64
Lipophilicity
4.25
Log Po/w (iLOGP)
Log Po/w (XLOGP3)
Log Po/w (WLOGP)
Log Po/w (MLOGP)
Log Po/w (SILICOS-IT)
Consensus Log Po/w
4.45
5.72
4.94
4.13
5.84
5.02
4.45
5.72
4.94
4.13
5.84
5.02
4.28
5.19
4.84
4.02
5.62
4.79
4.43
5.82
4.90
4.07
6.26
5.10
3.96
5.72
4.94
4.13
5.84
4.92
4.38
5.72
4.94
4.13
5.84
5.00
4.00
5.19
4.84
4.02
5.62
4.74
5.19
4.84
4.02
5.62
4.78
Water solubility
−5.72
Log S (ESOL)
−6.16
−6.16
−5.72
−6.17
−6.16
−6.16
−5.72
3.05 × 10−4
;
3.05 × 10−4
;
8.00 × 10−4
;
8.00 × 10−4
;
2.93 × 10−4
;
3.05 × 10−4
;
3.05 × 10−4
;
8.00 × 10−4
;
Solubility
(mg/mL; mol/L)
6.95 × 10−7
Poorly soluble
6.95 × 10−7
Poorly soluble
1.90 × 10−6
Moderately soluble
1.90 × 10−6
Moderately soluble
6.79 × 10−7
Poorly soluble
6.95 × 10−7
Poorly soluble
6.95 × 10−7
Poorly soluble
1.90 × 10−6
Moderately soluble
Class
Log S (ALi)
−7.26
−7.26
−6.71
−6.71
−7.37
−7.26
−7.26
−6.71
2.38 × 10−5
;
2.38 × 10−5
;
8.14 × 10−5
;
8.14 × 10−5
;
1.85 × 10−5
;
2.38 × 10−5
;
2.38 × 10−5
;
8.14 × 10−5
;
Solubility
(mg/mL; mol/L)
5.44 × 10−8
Poorly soluble
5.44 × 10−8
Poorly soluble
1.93 × 10−7
Poorly soluble
1.93 × 10−7
Poorly soluble
4.28 × 10−8
Poorly soluble
5.44 × 10−8
Poorly soluble
5.44 × 10−8
Poorly soluble
1.93 × 10−7
Poorly soluble
Class
Log S (SILICOS-IT)
−8.18
−8.18
−7.86
−7.86
−8.35
−8.18
−8.18
−7.86
2.89 × 10−6
;
2.89 × 10−6
;
5.82 × 10−6
;
5.82 × 10−6
;
1.95 × 10−6
;
2.89 × 10−6
;
2.89 × 10−6
;
5.82 × 10−6
;
Solubility
(mg/mL; mol/L)
6.59 × 10−9
Poorly soluble
6.59 × 10−9
Poorly soluble
1.38 × 10−8
Poorly soluble
1.38 × 10−8
Poorly soluble
4.51 × 10−9
Poorly soluble
6.59 × 10−9
Poorly soluble
6.59 × 10−9
Poorly soluble
1.38 × 10−8
Poorly soluble
Class

Molecules 2021, 26, 656
17 of 31
Table 3. Cont.
Compounds
Properties
5f
5g
5h
5i
6c
6f
6g
6i
Pharmacokinetics
GI absorption
BBB permeant
High
No
High
No
High
No
High
No
High
No
High
No
High
No
High
No
P-gp substrate
No
No
No
No
Yes
No
No
No
No
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
No
No
No
No
No
No
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Log Kp (skin
permeation) (cm/s)
−4.91
−4.91
−5.19
−5.19
Drug-likeness
Yes; 0 violation
−4.80
−4.91
−4.91
−5.19
Yes; 1 violation:
MLOGP > 4.15
Yes; 1 violation:
MLOGP > 4.15
Lipinski
Ghose
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
No; 1 violation:
MR > 130
No; 1 violation:
MR > 130
No; 1 violation:
MR > 130
No; 1 violation:
MR > 130
No; 1 violation:
MR > 130
Yes; 0 violation
Yes; 0 violation
Veber
Egan
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
Yes; 0 violation
No; 1 violation:
XLOGP3 > 5
No; 1 violation:
XLOGP3 > 5
No; 1 violation:
XLOGP3 > 5
No; 1 violation:
XLOGP3 > 5
No; 1 violation:
XLOGP3 > 5
No; 1 violation:
XLOGP3 > 5
No; 1 violation:
XLOGP3 > 5
No; 1 violation:
XLOGP3 > 5
Muegge
Bioavailability score
0.55
0.55
0.55
0.55
Medicinal chemistry
0 alert
0.55
0.55
0.55
0.55
PAINS
Brenk
0 alert
0 alert
0 alert
0 alert
0 alert
0 alert
0 alert
2 alerts: imine_1,
thiocar-
bonyl_group
2 alerts: imine_1,
thiocar-
bonyl_group
2 alerts: imine_1,
thiocar-
bonyl_group
2 alerts: imine_1,
thiocar-
bonyl_group
2 alerts: imine_1,
thiocar-
bonyl_group
2 alerts: imine_1,
thiocarbonyl_group
2 alerts: imine_1,
thiocarbonyl_group
2 alerts: imine_1,
thiocarbonyl_group
No; 1 Violation;
XLOGP3 > 3.5
No; 1 Violation;
XLOGP3 > 3.5
No; 1 Violation;
XLOGP3 > 3.5
No; 1 Violation;
XLOGP3 > 3.5
No; 1 Violation;
XLOGP3 > 3.5
No; 1 Violation;
XLOGP3 > 3.5
No; 1 Violation;
XLOGP3 > 3.5
No; 2 Violations;
XLOGP3 > 3.5
Lead-likeness
Synthetic accessibility
3.36
3.35
3.47
3.40
3.62
3.39
3.38
3.43

Molecules 2021, 26, 656
18 of 31
2.7. In Vitro Cytotoxicity Testing
The cytotoxic effects of compounds 5(a–m) and 6(a–m) against HepG2 cells were
evaluated using cisplatin as a standard drug with an IC50 of 20 g/mL. The evaluation
of the acquired data indicated that compounds 5a 5m, and 6j are non-cytotoxic. On
the other hand, the viability values of 5e 5f 6a 6b, and 6m refer them as cytotoxic.
µ
,
,
,
,
The rest of the compounds show proliferation in HepG2 cells hence are indicated as
proliferative ones (Table 4). The graphical representation of the cytotoxicity results is given
in Figures S1 and S2 (see Supplementary Materials).
Table 4. Cytotoxicity results of the newly synthesized compounds.
Cell Viability at Concentrations (µg/mL)
Compound
Control
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
50 µg/mL
95.4
100 µg/mL
88.9
200 µg/mL
92.8
500 µg/mL
95.8
1000 µg/mL
102
Status
5a
5b
5c
5d
5e
5f
Non-cytotoxic
Proliferative
Proliferative
Proliferative
Cytotoxic
106
107
139
169
227
105
109
122
188
262
105
108
110
134
131
94.2
97.2
158
196
76.3
84.8
94.9
93.9
93.2
56.9
Cytotoxic
5g
5h
5i
84.3
96.4
94.1
122
135
Proliferative
Proliferative
Proliferative
Proliferative
Proliferative
Proliferative
Non-cytotoxic
Cytotoxic
113
121
83.8
151
209
118
99.4
97.2
99.4
149
5j
93.4
98.1
107
138
166
5k
5l
111
110
99.6
106
132
101
101
107
109
133
5m
6a
6b
6c
6d
6e
6f
94.0
102
109
107
115
82.5
86.3
80.6
83.3
77.9
89.91
87.46
75.00
102.2
109.7
113.1
100.4
95.55
102.9
103.2
103.2
97.87
95.72
115.7
93.87
112.5
110.1
106.3
110.0
102.5
93.71
104.6
104.6
96.75
92.82
153.7
91.26
167.4
108.8
110.7
125.6
131.4
94.81
107.3
107.3
95.29
112.3
186.7
128.7
179.7
139.0
149.5
151.1
112.2
107.9
137.7
137.7
97.41
69.21
233.1
173.4
212.1
188.7
214.1
201.9
186.2
114.4
156.6
156.6
87.58
Cytotoxic
Proliferative
Proliferative
Proliferative
Proliferative
Proliferative
Proliferative
Proliferative
Non-cytotoxic
Proliferative
Proliferative
Cytotoxic
6g
6h
6i
6j
6k
6l
6m
3. Materials and Methods
3.1. General
The chemicals and solvents used were of analytical grade and obtained from com-
mercial suppliers (Scharlau (Barcelona, Spain), Merck (Darmstadt, Germany), and Fluka
(St. Louis, MO, USA), and were used without further purification. Thin layer chromatog-
raphy was performed using aluminum plates coated with silica gel 60F254 (Merck) in

Molecules 2021, 26, 656
19 of 31
an appropriate eluent. The spots were visualized using ultraviolet irradiation. Melting
points were recorded on Gallenkamp melting point apparatus (Sigma-Aldrich Chemie
1
GmbH, Taufkirchen, Germany) and were uncorrected. H NMR spectra were recorded
in CDCl3 and DMSO-d6 solvents on a Bruker Avance NMR (300 MHz) spectrometer
(Karlsruhe, Germany) while 13C NMR spectra were recorded at 75 MHz. Chemical shifts
are reported as
δ values in parts per million (ppm) relative to tetramethylsilane as internal
standard. Coupling constant (J) is given in Hertz. FTIR spectra were recorded on an Agilent
Technologies Cary 630 FTIR (Santa Clara, CA, USA). Elemental analysis was performed
on a LECO 630-200-200 TRUSPEC CHNS micro analyzer (St. Joseph, MI, USA) and the
values observed were within
synthesized following the literature procedures [60].
±
0.4% of the calculated results. Compounds 2 and 3 were
3.2. Preparation of Piperidinyl Quinoline-3-carbaldehydes (4a,b)
Compounds 4a and 4b were prepared following literature procedure [61].
3.2.1. Preparation of 2-Chloroquinoline-3-carbaldehydes 3(a,b)
2-Chloroquinoline-3-carbaldehydes 3(a,b) were prepared by using method reported
by Meth-Cohn and coworkers. POCl3 (65.3 mL, 107.45 g, 0.70 mol) was added dropwise to
DMF (19.3 mL, 18.26 g, 0.25 mol) with constant stirring while maintaining the temperature
◦
of the flask at 0 C. To the resulting Vilsmeyer reage◦nt, acetanilide
2 (0.10 mol) was
added and the reaction mixture was heated at 70–80 C. The progress of the reaction
was monitored thin layer chromatography (TLC). The reaction mixt◦ure was then poured
on crushed ice (500 g) cautiously and stirred vigorously at 0–10 C. The precipitated
2-chloroquinoline-3-carbaldehyde
recrystallized from ethyl acetate.
3 was filtered, washed with excess water, dried, and
3.2.2. Preparation of Piperidinyl Quinoline-3-carbaldehydes (4a,b)
Piperidine (11 mmol) was added to a stirred solution of 2-chloro-6-methylquinoline-3-
carbaldehyde 3a or 2-chloro-8-methylquinoline-3-carbaldehyde 3b (10 mmol) and catalytic
amount of cetyltrimethylammonium bromide (CTAB) in PEG-400 (10 mL). The resulting
◦
reaction mixture was heated at 135 C for 2.5 h. After cooling to room temperature,
the reaction mixture was poured onto crushed ice and stirred overnight. The yellow
precipitates were filtered, washed with water, dried and recrystallized from ethanol.
3.2.3. 6-Methyl-2-(piperidin-1-yl)quinoline-3-carbaldehyde (4a) Yield 98%
◦
◦
Yellow solid. Mp 90–92 C (lit. 91–93 C). FTIR (cm−1) 3030 (CH-aromatic), 2936 (CH),
1
2852 (CH-formyl), 1691 (C=O), 1572 (C=N, aromatic), 1053 (C-N); H NMR (CDCl3,
300 MHz)
δ = 1.66–1.73 (m, 2H, piperidinyl-CH2), 1.77-1.84 (m, 4H, piperidinyl-CH2),
2.51 (s, 3H, CH3), 3.41-3.45 (m, 4H, piperidinyl-N-CH2), 7.52 (d, J = 1.8 Hz, 1H, ArH),
7.54–7.56 (m, 1H, ArH), 7.75 (d, J = 8.4 Hz, 1H, ArH), 8.42 (s, 1H, ArH), 10.18 (s, 1H,
O=CH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 21.2 (Q-CH3), 24.5 (piperidinyl-CH2), 25.8 (2C,
piperidinyl-CH2), 52.2 (piperidinyl-N-CH2), 122.5 (C-3), 123.9 (C-10), 127.1 (C-8), 128.7 (C-
5), 134.0 (C-6), 135.0 (C-7), 142.0 (C-4), 147.5 (C-9), 159.0 (C-2), 190.8 (C=O); Anal. Calcd.
for C16H18N2O: C, 75.56; H, 7.13; N, 11.01%, Found: C, 75.79; H, 4.19; N, 11.12%.
3.2.4. 8-Methyl-2-(piperidin-1-yl)quinoline-3-carbaldehyde (4b)
◦
◦
Yield 97%. Yellow solid. Mp 82–84 C (lit. 83–85 C). FTIR (cm−1): 3023 (CH-aromatic),
1
2928 (CH), 2851 (CH-formyl), 1687 (C=O), 1569 (C=N, aromatic), 1050 (C-N); H NMR
(CDCl3, 300 MHz) δ = 1.69–1.76 (m, 2H, piperidinyl-CH2), 1.80–1.85 (m, 4H, piperidinyl-
CH2), 2.70 (s, 3H, CH3), 3.48–3.51 (m, 4H, piperidinyl-N-CH2), 7.26 (t, J = 7.5 Hz, 1H, ArH),
7.56 (d, J = 6.9 Hz, 1H, ArH), 7.64 (d, J = 8.1 Hz, 1H, ArH), 8.47 (s, 1H, ArH), 10.18 (s,
1H, O=CH); 13C NMR (CDCl3, 75 MHz)
δ
= 17.7 (Q-
CH3), 24.6 (piperidinyl-CH2), 25.9
(2C, piperidinyl-
C
H2), 52.5 (2C, piperidinyl-N- H2), 121.7 (C-3), 123.6 (C-6), 124.0 (C-10),
C

Molecules 2021, 26, 656
20 of 31
127.1 (C-5), 132.3 (C-7), 135.6 (C-8), 141.6 (C-4), 148.2 (C-9), 158.9 (C-2), 190.8 (C=O); Anal.
Calcd. for C16H18N2O: C, 75.56; H, 7.13; N, 11.01%, Found: C, 75.83; H, 4.32; N, 11.19%.
3.3. General Procedure for the Preparation of (Piperidin-1-yl)quinolin-3-yl)methylene)
hydrazinecarbothioamides (5,6)
3.3.1. Method A: Conventional Synthesis
To a stirred solution of 4a or 4b (254 mg, 1 mmol) in absolute ethanol (20 mL) was
added N-substituted thiosemicarbazide (1 mmol) and catalytic amount of glacial acetic
acid. The reaction mixture was heated to reflux for 0.5–2 h. The precipitated product was
filtered, washed with ethanol, and dried to afford 5(a–m) and 6(a–m).
3.3.2. Method B: Microwave-Assisted Synthesis
The equimolar quantity of 4a or 4b (1 mmol) and N-substituted thiosemicarbazide
(1 mmol) with catalytic amount of glacial acetic acid in absolute ethanol (20 mL) was
exposed to microwave irradiation for 3–5 min. The precipitated solid was filtered, washed
with hot ethanol and dried to afford 5(a–m) and 6(a–m).
Yields of 5(a–m) and 6(a–m) reported herein were observed by using method B.
2-((6-Methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazinecarbothioamide (5a
)
◦
Yield 91%. Yellow fluffy solid. Mp 220–222 C. FTIR (cm−1): 3476 (NH), 3329 (NH),
1
2927 (CH), 2803 (CH-imine), 1609 (C=N), 1119 (C=S); H NMR (DMSO-d6, 300 MHz)
δ = 1.62–1.76 (m, 6H, piperidinyl-CH2), 2.46 (s, 3H, CH3), 3.23–3.26 (m, 4H, piperidinyl-N-
CH2), 7.53 (d, J = 8.7 Hz, 1H, ArH), 7.60 (s, 1H, ArH), 7.74 (d, J = 8.1 Hz, 1H, ArH), 8.14
(br s, 1H, NH2), 8.20 (s, 1H, ArH), 8.37 (br s, 1H, NH2), 8.92 (s, 1H, N=CH), 11.79 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
(2C, piperidinyl- H2), 52.4 (2C, piperidinyl-N-
C-5, C-8), 133.1 (C-6), 134.6 (C-7), 139.0 (C-9), 145.7 (N=
δ
= 21.4 (Q-
H2), 122.0 (C-3), 124.9 (C-10), 127.5 (2C,
H), 159.4 (C-2), 178.5 (C=S); Anal.
CH3), 24.3 (piperidinyl-CH2), 25.8
C
C
C
Calcd. for C17H21N5S: C, 62.36; H, 6.46; N, 21.39; S, 9.79%. Found: C, 62.52; H, 6.71; N,
21.63; S, 9.91%.
2-((6-Methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)-N-phenylhydrazinecarboth-
◦
ioamide (5b) Yield 92%. Yellow fluffy solid. Mp 210–212 C. FTIR (cm−1): 3281 (NH), 3128
(NH), 3061 (CH-aromatic), 2913 (CH), 2809 (CH-imine), 1596 (C=N), 1125 (C=S); 1H NMR
(CDCl3, 300 MHz) δ =1.63–1.69 (m, 2H, piperidinyl-CH2), 1.75–1.82 (m, 4H, piperidinyl-
CH2), 2.51 (s, 3H, CH3), 3.27–3.30 (m, 4H, piperidinyl-N-CH2), 7.42–7.55 (m, 4H, ArH), 7.29
(t, J = 7.2 Hz, 1H, ArH), 7.73–7.79 (m, 3H, ArH), 8.15 (s, 1H, ArH), 8.41 (s, 1H, N=CH), 9.32
(s, 1H, NH), 10.14 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 21.4 (Q-CH3), 24.5
(piperidinyl-
C
H2), 25.09 (2C, piperidin0yl-
C
H2), 52.4 (2C, piperidinyl-N- H2), 121.6 (C-3),
C
0
0
0
125.2 (C-10), 125.9 (C-4 ), 126.5 (2C, C-2 , C-6 ), 127.3 (C-8), 127.5 (C-5), 128.6 (2C, C-3 , C-5 ),
132.7 (C-6), 134.2 (C-7), 135.4 (C-4), 139.5 (C-1 ), 140.4 (C-9), 145.7 (N=
0
CH), 160.3 (C-2), 176.4
(C=S); Anal. Calcd. for C23H25N5S: C, 68.46; H, 6.24; N, 17.35; S, 7.95%. Found: C, 68.55; H,
6.39; N, 17.47; S, 8.01%.
N-(2,4-Dimethylphenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (5c) Yield 92%. Light yellow solid. Mp 218–220 C. FTIR (cm−1): 3329
(NH), 3118 (NH), 2917 (CH), 2831 (CH-imine), 1617 (C=N), 1124 (C=S); 1H NMR (CDCl3,
300 MHz)
δ = 1.66–1.70 (m, 2H, piperidinyl-CH2), 1.75–1.82 (m, 4H, piperidinyl-CH2), 2.38
(s, 6H, CH3), 2.50 (s, 3H, CH3), 3.27–3.30 (m, 4H, piperidinyl-N-CH2), 7.10–7.13 (m, 2H,
ArH), 7.4–7.54 (m, 3H, ArH), 7.78 (d, J = 8.4 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 8.39 (s, 1H,
N=CH), 9.00 (s, 1H, NH), 10.07 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 18.3
H2), 25.9 (2C, piperidinyl- H2),
H2), 121.8 (C-3), 125.3 (C-10), 127.0 (C-8), 127.3 (C-6 ), 127.5 (C-5),
(Ar-
C
H3), 21.1 (Ar-
C
H3), 21.4 (Q-
C
H3), 24.5 (piperidinyl-
C
C
0
52.4 (2C, piperidinyl-N-
C
0
0
0
0
129.1 (C-5 ), 131.1 (C-3 ), 132.6 (C-6), 134.1 (C-7), 135.2 (C-4), 135.8 (C-1 ), 135.9 (C-2 ), 136.4
(C-40), 139.7 (C-9), 145.7 (N=
C
H), 160.3 (C-2), 177.4 (C=S); Anal. Calcd. for C25H29N5S: C,
69.57; H, 6.77; N, 16.23; S, 7.43%. Found: C, 69.85; H, 6.92; N, 16.51; S, 7.73%.
N-(2,6-Dimethylphenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-◦yl)methylene)hy-
drazine carbothioamide (5d) Yield 95%. Yellow solid. Mp 244–246 C. FTIR (cm−1):

Molecules 2021, 26, 656
21 of 31
3332 (NH), 3128 (NH), 2937 (CH), 1604 (C=N), 1125 (C=S); 1H NMR (CDCl3, 300 MHz)
δ = 1.67–1.71 (m, 2H, piperidinyl-CH2), 1.77–1.81 (m, 4H, piperidinyl-CH2), 2.39 (s, 6H,
CH3), 2.50 (s, 3H, CH3), 3.28–3.31 (m, 4H, piperidinyl-N-CH2), 7.07–7.27 (m, 3H, ArH),
7.47–7.55 (m, 2H, ArH), 7.77–7.80 (m, 1H, ArH), 8.10 (s, 1H, ArH), 8.40 (s, 1H, ArH), 8.76 (s,
1H, NH), 9.86 (s, 1H, =N-NH); 13C NMR (CDCl3, 75 MHz)
21.4 (Q- H3), 24.5 (piperidinyl- H2), 26.1 (2C, piperidinyl-
H2), 120.5 (C-3), 124.8 (C-40), 125.1 (C-10), 127.0 (C-8), 128.3 (C-5), 12.5 (2C, C-30, C-50),
δ
= 18.5 (Ar-
CH3), 18.8 (Ar-CH3),
C
C
C
H2), 52.5 (2C, piperidinyl-N-
C
0
0
0
132.8 (C-6), 134.6 (C-7), 135.2 (C-4), 136.7 (2C, C-1 ), 137.8 (2C, C-2 , C-6 ), 140.3 (C-9), 146.2
(N= H), 160.3 (C-2), 177.4 (C=S); Anal. Calcd. for C25H29N5S: C, 69.57; H, 6.77; N, 16.23; S,
C
7.43%. Found: C, 69.29; H, 6.58; N, 16.07; S, 7.27%.
N-(2-Chlorophenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (5e) Yield 91%. Yellow solid. Mp 208–210 C. FTIR (cm−1): 3246 (NH), 3109
(NH), 2915 (CH), 2810 (CH-imine), 1593 (C=N), 1124 (C=S); 1H NMR (CDCl3, 300 MHz)
δ
= 1.63–1.68 (m, 2H, piperidinyl-CH2), 1.76–1.83 (m, 4H, piperidinyl-CH2), 2.51 (s, 3H,
CH3), 3.27–3.30 (m, 4H, piperidinyl-N-CH2), 7.20 (td, J = 7.8, 1.2 Hz, 1H, ArH), 7.38 (td,
J = 8.1, 1.2 Hz, 1H, ArH), 7.47–7.52 (m, 3H, ArH), 7.78 (d, J = 8.4 Hz, 1H, ArH), 8.18 (s,
1H, ArH), 8.46 (s, 1H, N=CH), 8.74 (dd, J = 8.4, 0.9 Hz, 1H), 9.91 (s, 1H, NH), 10.21 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
piperidinyl- H2), 52.5 (2C, piperidinyl-N-
δ
= 21.4 (Q-
C
H3), 24.5 (piperidinyl-CH2), 25.9 (2C,
C
C
H2), 121.6 (C-3), 125.2 (C-10), 127.3 (C-8), 127.5
0
0
0
0
0
(C-5), 127.7 (C-5 ), 128.5 (C-3 ), 129.8 (C-2 ), 130.8 (C-4 ), 131.6 (C-6 ), 132.7 (C-6), 134.2 (C-7),
135.2 (C-4), 137.0 (C-10), 140.4 (C-9), 145.8 (N=
CH), 160.3 (C-2), 177.2 (C=S); Anal. Calcd.
for C23H24ClN5S: C, 63.07; H, 5.52; N, 15.99; S, 7.32%. Found: C, 63.19; H, 5.61; N, 16.11;
S, 7.52%.
N-(3-Chlorophenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (5f) Yield 97%. Yellow solid. Mp 216–218 C. FTIR (cm−1): 3325 (NH), 3127
(NH), 2931 (CH), 2813 (CH-imine), 1600 (C=N), 1125 (C=S); 1H NMR (DMSO-d6, 300 MHz)
δ
= 1.61–1.76 (m, 6H, piperidinyl-CH2), 2.44 (s, 3H, CH3), 3.17–3.20 (m, 4H, piperidinyl-N-
CH2), 7.29 (dd, J = 8.1, 0.9 Hz, 1H, ArH), 7.40–7.50 (m, 2H, ArH), 7.62–7.67 (m, 3H, ArH),
7.81 (t, J = 2.1 Hz, 1H, ArH), 8.36 (s, 1H, N=CH), 8.92 (s, 1H), 10.26 (s, 1H, NH), 12.78 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 21.4 (Q-
C
H3), 24.4 (piperidinyl-
C
H2), 25.9 (2C,
0
0
piperidinyl-
CH2), 52.4 (2C, piperidinyl-N-
CH2), 121.5 (C-3), 124.8 (C-6 ), 125.2 (C-2 ), 125.6
0
0
0
(C-4 ), 125.7 (C-10), 127.4 (C-8), 127.5 (C-5), 130.1 (C-5 ), 132.7 (C-3 ), 132.8 (C-6), 134.3 (C-7),
135.5 (C-4), 140.9 (C-9), 141.0 (C-10), 145.8 (N=
CH), 160.3 (C-2), 176.2 (C=S); Anal. Calcd.
for C23H24ClN5S: C, 63.07; H, 5.52; N, 15.99; S, 7.32%. Found: C, 63.39; H, 5.81; N, 16.15;
S, 7.60%.
N-(4-Chlorophenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
carbothioamide (5g) Yield 97%. Yellow solid. Mp 217–219 ◦C. FTIR (cm−1): 3295 (NH),
1
3137 (NH), 2936 (CH), 2826 (CH-imine), 1608 (C=O), 1124 (C=S); H NMR (DMSO-d6,
300 MHz)
piperidinyl-N-CH2), 7.44–7.50 (m, 3H, ArH), 7.61–7.68 (m, 4H, ArH), 8.36 (s, 1H, ArH), 8.92
(s, 1H, N=CH), 10.23 (s, 1H, NH), 12.22 (s, 1H, =N-NH); 13C NMR (CDCl3, 75 MHz)
= 21.4
(Q- H3), 24.5 (piperidinyl- H2), 26.0 (2C, piperidinyl- H2), 52.5 (2C, piperidinyl-N- H2),
121.6 (C-3), 125.2 (C-10), 127.3 (C-8), 127.5 (C-5), 127.7 (C-6 ), 128.5 (C-2 ), 129.8 (C-4 ), 130.8
(C-6), 135.1 (C-7), 136.2 (C-4), 137.0 (C-10), 140.4 (C-9), 145.5 (N=
H), 160.3 (C-2), 177.0
δ = 1.61–1.76 (m, 6H, piperidinyl-CH2), 2.44 (s, 3H, CH3), 3.17–3.20 (m, 4H,
δ
C
C
C
C
0
0
0
C
(C=S); Anal. Calcd. for C23H24ClN5S: C, 63.07; H, 5.52; N, 15.99; S, 7.32%. Found: C, 63.23;
H, 5.75; N, 16.08; S, 7.45%.
N-(2-Fluorophenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (5h) Yield 90%. Yellow solid. Mp 207–209 C. FTIR (cm−1): 3282 (NH), 3118
(NH), 2928 (CH), 2826 (CH-imine), 1620 (C=N), 1125 (C=S); 1H NMR (CDCl3, 300 MHz)
δ = 1.68–1.83 (m, 6H, piperidinyl-CH2), 2.51 (s, 3H, CH3), 3.35–3.38 (m, 4H, piperidinyl-N-
CH2), 7.15–7.26 (m, 3H, ArH), 7.53–7.56 (m, 3H, ArH), 8.24 (s, 1H, ArH), 8.42–8.49 (m, 2H,
N=CH, ArH), 9.43 (s, 1H, NH), 10.15 (s, 1H, =N-NH); 13C NMR (CDCl3, 75 MHz)
δ
= 21.4
H2), 52.9 (2C, piperidinyl-N- H2),
115.4 (C-30, d, J = 19.5 Hz), 120.6 (C-3), 124.0 (C-10), 124.1 (C-60), 125.5 (C-8), 126.0 (C-5),
(Q-CH3), 25.1 (piperidinyl-
C
H2), 26.0 (2C, piperidinyl-
C
C

Molecules 2021, 26, 656
22 of 31
126.2 (C-40), 126.7 (C-50), 126.9 (C-10), 127.2 (C-8), 127.6 (C-5), 129.6 (C-4), 133.7 (C-6), 153.4
(N=C
H), 156.7 (C-2), 175.5 (C-20, C=S); Anal. Calcd. for C23H24FN5S: C, 65.53; H, 5.74; N,
16.61; S, 7.61%. Found: C, 65.64; H, 5.90; N, 16.81; S, 7.79%.
N-(3-Fluorophenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (5i) Yield 96%. Yellow solid. Mp 206–208 C. FTIR (cm−1): 3336 (NH), 3133
(NH), 2934 (CH), 2813 (CH-imine), 1602 (C=N), 1163 (C=S); 1H NMR (DMSO-d6, 300 MHz)
δ
= 1.61–1.76 (m, 6H, piperidinyl-CH2), 2.44 (s, 3H, CH3), 3.17–3.20 (m, 4H, piperidinyl-N-
CH2), 7.07 (td, J = 8.4, 1.8 Hz, 1H, ArH), 7.39–7.52 (m, 3H, ArH), 7.62–7.67 (m, 3H, ArH),
8.36 (s, 1H, ArH), 8.92 (s, 1H, N=CH), 10.26 (s, 1H, NH), 12.27 (s, 1H, =N-NH); 13C NMR
(DMSO-d6, 75 MHz) δ = 21.4 (Q-CH3), 24.5 (piperidinyl-CH2), 25.9 (2C, piperidinyl-CH2),
52.4 (2C, piperidinyl-N-C
H2), 112.4 (C-40, d, J = 20.3 Hz), 112.8 (C-20, d, J = 24.8 Hz),
121.5 (C-3), 121.9 (C-60, d, J = 2.3 Hz), 125.2 (C-10), 127.4 (C-8), 127.5 (C-5), 130.1 (C-50, d,
0
J = 9.0 Hz), 132.8 (C-6), 134.3 (C-7), 135.5 (C-4), 140.9 (C-9), 141.2 (C-1 , d, J = 10.5 Hz), 145.8
0
(N=C
H), 160.3 (C-2), 162.0 (C-3 , d, J = 240.0 Hz), 176.1 (C=S); Anal. Calcd. for C23H24FN5S:
C, 65.53; H, 5.74; N, 16.61; S, 7.61%. Found: C, 65.80; H, 5.94; N, 16.79; S, 7.83%.
N-(4-Fluorophenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (5j) Yield 90%. Yellow solid. Mp 213–215 C. FTIR (cm−1): 3332 (NH),
1
2956 (CH), 2830 (CH-imine), 1603 (C=N), 1124 (C=S); H NMR (DMSO-d6, 300 MHz)
δ = 1.61–1.76 (m, 6H, piperidinyl-CH2), 2.44 (s, 3H, CH3), 3.17–3.20 (m, 4H, piperidinyl-
N-CH2), 7.24 (td, J = 7.8, 2.1 Hz, 2H, ArH), 7.48 (dd, J = 8.4, 1.8 Hz, 1H, ArH), 7.56–7.61
(m, 3H, ArH), 7.66 (d, J = 8.4, 1H, ArH), 8.35 (s, 1H, ArH), 8.93 (s, 1H, N=CH), 10.19 (s,
1H, NH), 12.17 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 21.4 (Q-CH3), 24.5
(piperidinyl-
C
H2), 25.9 (2C, piperidinyl-
C
H2), 52.4 (2C, piperidinyl-N- H2), 115.3 (2C,
C
C-30, C-50, d, J = 22.5 Hz), 121.6 (C-3), 125.2 (C-10), 127.3 (C-8), 127.5 (C-5), 128.7 (2C, C-20,
C-60, d, J = 8.3 Hz), 132.7 (C-6), 134.2 (C-7), 135.4 (C-4), 135.9 (C-10, d, J = 2.3 Hz), 140.5
(C-9), 145.7 (N=C
H), 160.2 (C-40, d, J = 240.8 Hz), 160.3 (C-2), 176.8 (C=S); Anal. Calcd.
for C23H24FN5S: C, 65.53; H, 5.74; N, 16.61; S, 7.61%. Found: C, 65.35; H, 5.45; N, 16.40;
S, 7.55%.
N-(4-Ethylphenyl)-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
carbothioamide (5k) Yield 97%. Yellow solid. Mp 218–220 ◦C. FTIR (cm−1): 3274 (NH),
1
3112 (NH), 2964 (CH), 2914 (CH), 2820 (CH-imine), 1604 (C=N), 1125 (C=S); H NMR
(DMSO-d6, 300 MHz)
δ = 1.21 (t, J = 7.5 Hz, 3H, CH3), 1.61–1.76 (m, 6H, piperidinyl-CH2),
2.44 (s, 3H, CH3), 2.63 (q, J = 7.5 Hz, 2H, CH2), 3.17–3.20 (m, 4H, piperidinyl-N-CH2), 7.23
(d, J = 8.4 Hz, 2H, ArH), 7.47–7.50 (m, 3H, ArH), 7.59–7.67 (m, 2H, ArH), 8.34 (s, 1H, ArH),
8.94 (s, 1H, N=CH), 10.12 (s, 1H, NH), 12.11 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 16.2 (Ar-CH2-
CH3), 21.4 (Q-CH3), 24.5 (piperidinyl-CH2), 25.9 (2C, piperidinyl-CH2),
28.2 (Ar-
C
H2-CH3), 52.4 (2C, piperidinyl-N-C
H2), 121.6 (C-3), 125.2 (C-10), 126.5 (2C, C-20,
C-60), 127.3 (C-8), 127.5 (C-5), 127.9 (2C, C-30, C-50), 132.7 (C-40), 134.2 (C-6), 135.3 (C-7),
0
137.1 (C-4), 140.2 (C-1 ), 141.5 (C-9), 145.7 (N=
CH), 160.2 (C-2), 176.4 (C=S); Anal. Calcd. for
C25H29N5S: C, 69.57; H, 6.77; N, 16.23; S, 7.43%. Found: C, 69.61; H, 6.85; N, 16.33; S, 7.50%.
N-Benzyl-2-((6-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazinecarboth-
◦
ioamide (5l) Yield 95%. Yellow solid. Mp 208–210 C. FTIR (cm−1): 3382 (NH), 3116
(NH), 2971 (CH), 2932 (CH), 2841 (CH-imine), 1602 (C=N), 1107 (C=S); 1H NMR (DMSO-d6,
300 MHz)
δ = 1.60–1.74 (m, 6H, piperidinyl-CH2), 2.43 (s, 3H, CH3), 3.15–3.18 (m, 4H,
piperidinyl-N-CH2), 4.90 (d, J = 6.3 Hz, 2H, N-CH2), 7.25 (tt, J = 6.9, 1.5 Hz, 1H, ArH),
7.32–7.41 (m, 4H, ArH), 7.46 (dd, J = 8.4, 1.8 Hz, 1H, ArH), 7.57 (s, 1H, ArH), 7.64 (d,
J = 8.7 Hz, 1H, ArH), 8.29 (s, 1H, ArH), 8.77 (s, 1H, N=CH), 9.17 (t, J = 6.3 Hz, 1H, NH),
11.92 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ = 21.4 (Q-CH3), 24.5 (piperidinyl-
C
H2), 25.9 (2C, piperidinyl-
C
H2), 47.1 (Ar-
C
H2-NH), 52.4 (2C, pip0eridinyl-N-
CH2), 121.8
0
0
0
0
(C-3), 125.1 (C-10), 127.2 (C-8), 127.5 (C-5), 127.7 (3C, C-2 , C-4 , C-6 ), 128.7 (2C, C-3 , C-5 ),
132.6 (C-6), 134.2 (C-7), 134.8 (C-4), 139.8 (C-1 ), 139.9 (C-9), 145.7 (N=
0
CH), 160.2 (C-2), 178.0
(C=S); Anal. Calcd. for C24H27N5S: C, 69.03; H, 6.52; N, 16.77; S, 7.68%. Found: C, 69.15; H,
6.66; N, 16.89; S, 7.75%.

Molecules 2021, 26, 656
23 of 31
2-((6-Methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)-N-(2-mor◦pholinoethyl)hy-
drazine carbothioamide (5m) Yield 89%. Yellow solid. Mp 212–214 C. FTIR (cm−1):
1
3210 (NH), 3124 (NH), 2930 (CH), 2799 (CH-imine), 1603 (C=N), 1111 (C=S); H NMR
(CDCl3, 300 MHz) δ = 1.66–1.69 (m, 2H, piperidinyl-CH2), 1.74–1.81 (m, 4H, piperidinyl-
CH2), 2.49 (s, 3H, CH3), 2.78–2.90 (m, 6H, NCH2 linked to morpholine ring), 3.23–3.26
(m, 4H, piperidinyl-N-CH2), 3.93–3.97 (m, 6H, NCH2, OCH2 of morpholine ring), 7.47 (d,
J = 8.4 Hz, 1H, ArH), 7.52 (s, 1H, ArH), 7.75 (d, J = 8.4 Hz, 1H, ArH), 8.04 (s, 1H, ArH), 8.43
(s, 1H, N=CH), 8.52 (t, J = 6.0 Hz, 1H, NH), 9.71 (s, 1H, =N-NH); 13C NMR (CDCl3, 75 MHz)
δ
= 21.5 (Q-
N(morpholine)), 52.4 (2C, piperidinyl-N-
(-N- H2-CH2-O(morpholine)), 66.5 (N-CH2-
127.0 (C-8), 127.4 (C-5), 132.7 (C-6), 134.5 (C-7), 135.0 (C-4), 140.3 (C-9), 146.2 (N=
C
H3), 24.5 (piperidinyl-
C
H2), 26.1 (2C, piperidinyl-
H2), 53.2 (NH-CH2-
H2-O(morpholine)),120.7 (C-3), 125.0 (C-10),
H),
C
H2), 40.1 (NH-
CH2-CH2-
C
CH2-N(morpholine)), 56.7
C
C
C
160.3 (C-2), 177.1 (C=S); Anal. Calcd. for C23H32N6OS: C, 62.70; H, 7.32; N, 19.07; S, 7.28%.
Found: C, 62.59; H, 7.21; N, 19.00; S, 7.19%.
2-((8-Methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazinecarbothioamide (6a
Yield 90%. Yellow solid. Mp 222–224 ◦C. FTIR (cm−1): 3471 (NH), 3330 (NH), 2929 (CH),
2803 (CH-imine), 1608 (C=N), 1119 (C=S); 1H NMR (DMSO-d6, 300 MHz)
= 1.62–1.76
)
δ
(m, 6H, piperidinyl-CH2), 2.61 (s, 3H, CH3), 3.22–3.24 (m, 4H, piperidinyl-N-CH2), 7.30
(t, J = 7.5 Hz, 1H, ArH), 7.51 (d, J = 6.9 Hz, 1H, ArH), 7.65 (d, J = 7.8 Hz, 1H, ArH), 8.13
(br s, 1H, NH), 8.24 (s, 1H, ArH), 8.33 (br s, 1H, NH), 8.89 (s, 1H, N=CH), 11.74 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
(2C, piperidinyl- H2), 52.2 (2C, piperidinyl-N-
126.3 (C-5), 130.5 (C-7), 135.0 (C-8), 135.9 (C-4), 139.7 (C-9), 145.8 (N=
δ
= 17.8 (Q-
H2), 121.1 (C-3), 124.6 (C-6), 124.9 (C-10),
H), 159.5 (C-2), 178.4
CH3), 24.5 (piperidinyl-CH2), 25.8
C
C
C
(C=S); Anal. Calcd. for C17H21N5S: C, 62.36; H, 6.46; N, 21.39; S, 9.79%. Found: C, 62.51; H,
6.65; N, 21.51; S, 9.91%.
2-((8-Methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)-N-phenylhydrazinecarboth-
◦
ioamide (6b) Yield 93%. Yellow solid. Mp 208–210 C. FTIR (cm−1): 3283 (NH), 3128
(NH), 3060 (CH-aromatic), 2910 (CH), 2807 (CH-imine), 1595 (C=N), 1125 (C=S); 1H NMR
(DMSO-d6, 300 MHz) δ = 1.64–1.79 (m, 6H, piperidinyl-CH2), 2.63 (s, 3H, CH3), 3.25–3.28
(m, 4H, piperidinyl-N-CH2), 7.24 (tt, J = 7.5, 1.2 Hz, 1H, ArH), 7.31 (t, J = 7.5 Hz, 1H, ArH),
7.38–7.43 (m, 2H, ArH), 7.52 (d, J = 6.9 Hz, 1H, ArH), 7.60 (d, J = 7.5 Hz, 2H, ArH), 7.68 (d,
J = 7.8 Hz, 1H, ArH), 8.36 (s, 1H, ArH), 8.99 (s, 1H, N=CH), 10.22 (s, 1H, NH), 12.15 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 17.8 (Q-CH3), 24.5 (piperidinyl-CH2), 25.9 (2C,
piperidinyl-
C
H2), 52.2 (2C, piperidinyl-N-
C
H2), 121.0 (C-3), 124.7 (C-6), 124.9 (C-10), 126.0
0
0
0
0
0
(C-4 ), 126.4 (C-5), 126.6 (2C, C-2 , C-6 ), 128.6 (2C, C-3 , C-5 ), 130.6 (C-7), 135.1 (C-8), 136.3
(C-4), 139.5 (C-9), 140.4 (C-10), 145.9 (N=
C
H), 159.5 (C-2), 176.4 (C=S); Anal. Calcd. for
C23H25N5S: C, 68.46; H, 6.24; N, 17.35; S, 7.95%. Found: C, 68.70; H, 6.49; N, 17.53; S, 8.07%.
N-(2,4-Dimethylphenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hy-
drazine carbothioamide (6c) Yield 91%. Yellow solid. Mp 218–220 ◦C. FTIR (cm−1): 3329
(NH), 3120 (NH), 2927 (CH), 2831 (CH-imine), 1616 (C=N), 1124 (C=S); 1H NMR (DMSO-d6,
300 MHz)
δ = 1.64–1.79 (m, 6H, piperidinyl-CH2), 2.23 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.62
(s, 3H, CH3), 3.25–3.28 (m, 4H, piperidinyl-N-CH2), 7.05 (dd, J = 8.1, 1.5 Hz, 1H, ArH),
7.11–7.17 (m, 2H, ArH), 7.30 (t, J = 7.5 Hz, 1H, ArH), 7.51 (d, J = 6.9 Hz, 1H, ArH), 7.63 (d,
J = 7.8 Hz, 1H, ArH), 8.34 (s, 1H, ArH), 9.02 (s, 1H, N=CH), 10.00 (s, 1H, NH), 12.07 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
24.6 (piperidinyl- H2), 25.9 (2C, piperidinyl-
δ = 17.8 (Q-CH3), 18.3 (Ar-CH3), 21.1 (Ar-CH3),
C
C
H2)0, 52.3 (2C, piperidinyl-N-
CH2), 1210.1
0
(C-3), 124.6 (C-6), 125.0 (C-10), 126.3 (C-5), 127.0 (C-6 ), 129.2 (C-5 ), 130.5 (C-7), 131.1 (C-3 ),
135.1 (C-8), 135.9 (C-4), 136.0 (C-20), 136.1 (C-40), 136.4 (C-10), 139.7 (C-9), 145.9 (N=
C
H),
159.6 (C-2), 177.4 (C=S); Anal. Calcd. for C25H29N5S: C, 69.57; H, 6.77; N, 16.23; S, 7.43%.
Found: C, 69.30; H, 6.59; N, 16.09; S, 7.20%.
N-(2,6-Dimethylphenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hy-
◦
drazine carbothioamide (6d) Yield 98%. Lemon yellow solid. Mp 226–228 C. FTIR
(cm−1): 3333 (NH), 3128 (NH), 2931 (CH), 1606 (C=N), 1124 (C=S); 1H NMR (DMSO-d6,
300 MHz) δ = 1.64–1.79 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 2.23 (s, 6H, CH3),

Molecules 2021, 26, 656
24 of 31
3.25–3.27 (m, 4H, piperidinyl-N-CH2), 7.07–7.15 (m, 3H, ArH), 7.29 (t, J = 7.6 Hz, 1H, ArH),
7.50 (d, J = 6.9 Hz, 1H, ArH), 7.61 (d, J = 7.8 Hz, 1H, ArH), 8.34 (s, 1H, ArH), 9.07 (s, 1H,
N=CH), 9.96 (s, 1H, NH), 12.07 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 17.8
(Q-
CH3), 18.6 (2C, Ar-CH3), 24.5 (piperidinyl-CH2), 25.9 (2C, piperidinyl-CH2), 52.3 (2C,
piperidinyl-N-
C
H2), 121.2 (C-3), 124.6 (C-6), 125.0 (C-10), 126.2 0(C-5)0, 127.5 (C-40), 128.1
0
0
0
(2C, C-3 , C-5 ), 135.1 (C-8), 136.0 (C-4), 136.5 (C-1 ), 137.0 (2C, C-2 , C-6 ), 137.6 (C-7), 139.5
(C-9), 145.8 (N=CH), 159.6 (C-2), 177.1 (C=S); Anal. Calcd. for C25H29N5S: C, 69.57; H, 6.77;
N, 16.23; S, 7.43%. Found: C, 69.69; H, 6.85; N, 16.31; S, 7.50%.
N-(2-Chlorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
carbothioamide (6e) Yield 93%. Lemon yellow solid. Mp 202–204 ◦C. FTIR (cm−1): 3241
(NH), 3108 (NH), 2915 (CH), 2810 (CH-imine), 1593 (C=N), 1125 (C=S); 1H NMR (DMSO-d6,
300 MHz)
δ = 1.63–1.78 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 3.25–3.28 (m, 4H,
piperidinyl-N-CH2), 7.30 (t, J = 7.5 Hz, 1H, ArH), 7.35–7.44 (m, 2H, ArH), 7.51 (d, J = 6.9 Hz
,
1H, ArH), 7.58 (dd, J = 7.8, 1.5 Hz, 1H, ArH), 7.62–7.66 (m, 2H, ArH), 8.36 (s, 1H, ArH), 8.95
(s, 1H, N=CH), 10.18 (s, 1H, NH), 12.27 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ = 17.8 (Q-
CH3), 24.5 (piperidinyl-CH2), 25.9 (2C, piperidinyl-CH2), 52.3 (2C, piperidinyl-
N-
C
H2), 120.9 (C-3), 124.7 (C-6), 124.9 (C-10), 126.3 (C-5), 127.7 (C-50), 128.6 (C-20), 129.9
0
0
0
0
(C-3 ), 130.7 (C-7), 131.0 (C-4 ), 131.8 (C-6 ), 135.1 (C-8), 136.1 (C-4), 137.1 (C-1 ), 140.4 (C-9),
145.9 (N= H), 159.6 (C-2), 177.3 (C=S); Anal. Calcd. for C23H24ClN5S: C, 63.07; H, 5.52; N,
C
15.99; S, 7.32%. Found: C, 63.25; H, 5.77; N, 16.11; S, 7.53%.
N-(3-Chlorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
carbothioamide (6f) Yield 93%. Lemon yellow solid. Mp 198–200 ◦C. FTIR (cm−1): 3321
(NH), 3128 (NH), 2933 (CH), 2813 (CH-imine), 1601 (C=N), 1125 (C=S); 1H NMR (DMSO-
d6, 300 MHz)
δ = 1.63–1.78 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 3.24–3.28 (m,
4H, piperidinyl- N-CH2), 7.28–7.33 (m, 2H, ArH), 7.42 (t, J = 8.1 Hz, 1H, ArH), 7.52 (d,
J = 6.9 Hz, 1H, ArH), 7.63 (d, J = 8.1 Hz, 1H, ArH), 7.69 (d, J = 7.8 Hz, 1H, ArH), 7.79 (t,
J = 2.1 Hz, 1H, ArH), 8.36 (s, 1H, ArH), 8.97 (s, 1H, N=CH), 10.28 (s, 1H, NH), 12.26 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 17.8 (Q-CH3), 24.5 (piperidinyl-CH2), 25.9 (2C,
piperidinyl-
C
H2), 52.3 (2C, piperidinyl-N-
C
H2), 120.8 (C-3), 124.7 (C-6), 124.8 (C-10), 124.9
0
0
0
0
0
(C-6 ), 125.6 (C-2 ), 125.8 (C-4 ), 126.4 (C-5), 130.1 (C-5 ), 130.7 (C-7), 132.7 (C-3 ), 135.1 (C-8),
136.4 (C-4), 140.9 (C-9), 141.0 (C-10), 146.0 (N=
CH), 159.5 (C-2), 176.2 (C=S); Anal. Calcd.
for C23H24ClN5S: C, 63.07; H, 5.52; N, 15.99; S, 7.32%. Found: C, 62.99; H, 5.37; N, 15.88;
S, 7.20%.
N-(4-Chlorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (6g) Yield 95%. Yellow solid. Mp 190–192 C. FTIR (cm−1): 3291 (NH), 3137
(NH), 2937 (CH), 2826 (CH-imine), 1609 (C=O), 1124 (C=S); 1H NMR (DMSO-d6, 300 MHz)
δ
= 1.63–1.77 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 3.25–3.27 (m, 4H, piperidinyl-N-
CH2), 7.30 (t, J = 7.5 Hz, 1H, ArH), 7.43–7.47 (m, 2H, ArH), 7.51 (d, J = 6.9 Hz, 1H, ArH),
7.63–7.69 (m, 3H, ArH), 8.36 (s, 1H, ArH), 8.96 (s, 1H, N=CH), 10.28 (s, 1H, NH), 11.81
(s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 17.8 (Q-
H2), 52.2 (2C, piperidinyl-N- H2), 120.9 (C-3), 124.7 (C-6), 124.9
(C-10), 126.4 (C-5), 128.1 (C-30, C-50), 128.5 (C-20, C-60), 129.9 (C-40), 130.7 (C-7), 135.1 (C-8),
136.3 (C-4), 138.5 (C-10), 140.8 (C-9), 146.0 (N=
H), 159.5 (C-2), 176.4 (C=S); Anal. Calcd.
CH3), 24.5 (piperidinyl-CH2),
25.9 (2C, piperidinyl-
C
C
C
for C23H24ClN5S: C, 63.07; H, 5.52; N, 15.99; S, 7.32%. Found: C, 63.00; H, 5.45; N, 15.91;
S, 7.26%.
N-(2-Fluorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
carbothioamide (6h) Yield 96%. Lemon yellow solid. Mp 202–204 ◦C. FTIR (cm−1): 3280
(NH), 3120 (NH), 2928 (CH), 2826 (CH-imine), 1620 (C=N), 1125 (C=S); 1H NMR (DMSO-d6,
300 MHz)
piperidinyl-N-CH2), 7.25–7.36 (m, 4H, ArH), 7.50–7.52 (m, 2H, ArH), 7.64 (d, J = 7.8 Hz, 1H,
ArH), 8.35 (s, 1H, ArH), 8.97 (s, 1H, N=CH), 10.09 (s, 1H, NH), 12.28 (s, 1H, =N-NH); 13
δ = 1.61–1.78 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 3.24–3.28 (m, 4H,
C
NMR (DMSO-d6, 75 MHz)
δ = 17.8 (Q-CH3), 24.5 (piperidinyl-CH2), 25.9 (2C, piperidinyl-
CH2), 52.3 (2C, piperidinyl-N-CH2), 116.3 (C-30, d, J = 19.5 Hz), 120.90(C-3), 124.6, (C-60, d,
J = 6.8 Hz), 124.9 (C-6), 126.3 (C-5), 127.5 (C-10), 127.7 (C-7), 128.9 (C-1 , d, J = 8.3 Hz), 130.6

Molecules 2021, 26, 656
25 of 31
(C-500), 131.1 (C-40), 135.1 (C-8), 136.2 (C-4), 140.5 (C-9), 145.9 (N=CH), 159.6 (C-2), 158.0
(C-2 , d, J = 245.3 Hz), 177.8 (C=S); Anal. Calcd. for C23H24FN5S: C, 65.53; H, 5.74; N, 16.61;
S, 7.61%. Found: C, 65.70; H, 5.88; N, 16.76; S, 7.71%.
N-(3-Fluorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (6i) Yield 90%. Yellow solid. Mp 200–202 C. FTIR (cm−1): 3334 (NH), 3137
(NH), 2933 (CH), 2813 (CH-imine), 1601 (C=N), 1163 (C=S); 1H NMR (DMSO-d6, 300 MHz)
δ
= 1.63–1.78 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 3.25–3.28 (m, 4H, piperidinyl-
N-CH2), 7.07 (td, J = 8.4, 1.5 Hz, 1H, ArH), 7.31 (t, J = 7.5 Hz, 1H, ArH), 7.39–7.53 (m, 3H,
ArH), 7.62–7.71 (m, 2H, ArH), 8.37 (s, 1H, ArH), 8.97 (s, 1H, N=CH), 10.28 (s, 1H, NH), 12.26
(s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz)
δ
= 17.8 (Q-
C
C
H3), 24.5 (piperidinyl-
CH2),
25.9 (2C, piperidinyl-
CH2), 52.3 (2C, piperidinyl-N-
H2), 112.4 (C-40, d, J = 21.0 Hz), 112.9
(C-20, d, J = 24.0 Hz), 120.8 (C-3), 122.0 (C-60, d, J = 2.3 Hz), 124.7 (C-6), 124.8 (C-10), 126.4
(C-5), 130.0 (C-50, d, J = 9.0 Hz), 130.7 (C-7), 135.1 (C-8), 136.5 (C-4), 140.9 (C-9), 141.3 (C-10,
d, J = 10.5 Hz), 145.9 (N=C
H), 159.5 (C-2), 162.0 (C-30, d, J = 240.0 Hz), 176.1 (C=S). Anal.
Calcd. for C23H24FN5S: C, 65.53; H, 5.74; N, 16.61; S, 7.61%. Found: C, 65.40; H, 5.61; N,
16.52; S, 7.49%.
N-(4-Fluorophenyl)-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazine
◦
carbothioamide (6j) Yield 91%. Yellow solid. Mp 200–202 C. FTIR (cm−1): 3333 (NH),
1
2954 (CH), 2831 (CH-imine), 1603 (C=N), 1125 (C=S); H NMR (DMSO-d6, 300 MHz)
δ = 1.63–1.78 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 3.24–3.28 (m, 4H, piperidinyl-N-
CH2), 7.21–7.27 (m, 2H, ArH), 7.31 (t, J = 7.5 Hz, 1H, ArH), 7.51–7.59 (m, 3H, ArH), 7.67 (d,
J = 7.8 Hz, 1H, ArH), 8.35 (s, 1H, ArH), 8.98 (s, 1H, N=CH), 10.22 (s, 1H, NH), 12.16 (s, 1H,
=N-NH); 13C NMR (DMSO-d6, 75 MHz)
piperidinyl- H2), 52.2 (2C, piperidinyl-N-
δ
= 17.8 (Q-
C
CH3), 24.5 (piperidinyl-CH2), 25.9 (2C,
C
H2), 115.3 (2C, C-30, C-50, d, J = 22.5 Hz), 120.9
(C-3), 124.7 (C-6), 124.9 (C-10), 126.3 (C-5), 128.9 (2C, C-20, C-60, d, J = 8.3 Hz), 130.6 (C-7),
135.1 (C-8), 135.9 (C-10, d, J = 3.0 Hz), 136.3 (C-4), 140.5 (C-9), 145.9 (N=
C
H), 159.5 (C-2),
0
160.2 (C-4 , d, J = 240.8 Hz), 176.8 (C=S); Anal. Calcd. for C23H24FN5S: C, 65.53; H, 5.74; N,
16.61; S, 7.61%. Found: C, 65.74; H, 5.91; N, 16.82; S, 7.85%.
N-(4-Ethylphenyl)-2-((8-methyl-2-(piperidin-1-yl)qu◦inolin-3-yl)methylene)hydrazine
carbothioamide (6k) Yield 95%. Yellow solid. Mp 198–200 C. FTIR (cm−1): 3272 (NH), 3112
(NH), 2964 (CH), 2914 (CH), 2820 (CH-imine), 1604 (C=N), 1125 (C=S); 1H NMR (DMSO-d6,
300 MHz) δ = 1.20 (t, J = 7.5 Hz, 3H, CH3), 1.63–1.78 (m, 6H, piperidinyl-CH2), 2.59–2.66
(m, 5H, Ar-CH3, -CH2CH3), 3.25–3.27 (m, 4H, piperidinyl-N-CH2), 7.23 (d, J = 8.1 Hz, 2H,
ArH), 7.30 (t, J = 7.5 Hz, 1H, ArH), 7.46–7.52 (m, 3H, ArH), 7.67 (d, J = 8.1 Hz, 1H, ArH),
8.35 (s, 1H, ArH), 8.98 (s, 1H, N=CH), 10.14 (s, 1H, NH), 12.09 (s, 1H, =N-NH); 13C NMR
(DMSO-d6, 75 MHz)
δ
= 16.2 (Ar-CH2-
CH3), 17.8 (Q-CH3), 24.5 (piperidinyl-CH2), 25.9
(2C, piperidinyl- H2), 28.2 (Ar-
C
C
H2-CH3), 52.2 (2C, piperidinyl-N-
C
H2), 121.0 (C-3), 124.6
0
0
0
0
(C-6), 124.9 (C-10), 126.3 (C-5), 126.6 (2C, C-2 , C-6 ), 127.9 (2C, C-3 , C-5 ), 130.6 (C-7), 135.1
(C-8), 136.3 (C-4), 137.1 (C-10), 140.2 (C-9), 141.5 (C-40), 145.9 (N=
C
H), 159.5 (C-2), 176.5
(C=S); Anal. Calcd. for C25H29N5S: C, 69.57; H, 6.77; N, 16.23; S, 7.43%. Found: C, 69.65; H,
6.87; N, 16.31; S, 7.50%.
N-Benzyl-2-((8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hydrazinecarboth-
◦
ioamide (6l) Yield 91%. Yellow solid. Mp 188–190 C. FTIR (cm−1): 3378 (NH), 3119
(NH), 2967 (CH), 2930 (CH), 2843 (CH-imine), 1602 (C=N), 1110 (C=S); 1H NMR (DMSO-d6,
300 MHz)
δ = 1.62–1.76 (m, 6H, piperidinyl-CH2), 2.62 (s, 3H, CH3), 3.23–3.26 (m, 4H,
piperidinyl-N-CH2), 4.91 (d, J = 6.3 Hz, 2H, N-CH2), 7.23–7.42 (m, 6H, ArH), 7.50 (d,
J = 6.9 Hz, 1H, ArH), 7.65 (d, J = 7.8 Hz, 1H, ArH), 8.30 (s, 1H, ArH), 8.82 (s, 1H, N=CH),
9.20 (t, J = 6.3 Hz, 1H, NH), 11.92 (s, 1H, =N-NH); 13C NMR (DMSO-d6, 75 MHz) δ = 17.8
(Q-
piperidinyl-N-
127.7 (2C, C-30, C-50), 128.7 (C-40), 130.5 (C-7), 135.1 (C-8), 135.8 (C-4), 139.9 (C-9, C-10),
145.9 (N= H), 159.5 (C-2), 178.0 (C=S); Anal. Calcd. for C24H27N5S: C, 69.03; H, 6.52; N,
16.77; S, 7.68%. Found: C, 69.17; H, 6.70; N, 16.93; S, 7.77%.
C
H3), 24.5 (piperidinyl-
CH2), 25.8 (2C, piperidinyl-CH2), 47.1 (Ar-CH2-NH), 52.2 (2C,
0
0
CH2), 121.2 (C-3), 124.7 (C-6), 124.8 (C-10), 126.2 (C-5), 127.2 (2C, C-2 , C-6 ),
C

Molecules 2021, 26, 656
26 of 31
2-((8-Methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)-N-(2-mor◦pholinoethyl)hy-
drazine carbothioamide (6m) Yield 90%. Yellow solid. Mp 200–202 C. FTIR (cm−1):
1
3216 (NH), 3122 (NH), 2929 (CH), 2801 (CH-imine), 1601 (C=N), 1111 (C=S); H NMR
(CDCl3, 300 MHz)
CH2), 2.70–2.84 (m, 9H, CH3, NCH2 linked to morpholine nitrogen), 3.29–3.32 (m, 4H,
piperidinyl-N-CH2), 3.89–3.95 (m, 6H, N-CH2, OCH2 of morpholine ring), 7.27 (t, J = 7.5 Hz
δ = 1.65–1.69 (m, 2H, piperidinyl-CH2), 1.78–1.83 (m, 4H, piperidinyl-
,
1H, ArH), 7.49 (d, J = 6.9 Hz, 1H, ArH), 7.58 (d, J = 7.8 Hz, 1H, ArH), 8.06 (s, 1H, ArH), 8.40
(s, 1H, N=CH), 8.54 (t, J = 6.0 Hz, 1H, NH), 9.90 (s, 1H, =N-NH); 13C NMR (CDCl3, 75 MHz)
δ
= 17.7 (Q-
N(morpholine)), 52.3 (2C, piperidinyl-N-
(-N- H2-CH2-O(morpholine)), 66.7 (N-CH2-
124.6 (C-10), 125.8 (C-5), 130.5 (C-7), 135.8 (C-4), 140.3 (C-9), 146.5 (N=
C
H3), 24.6 (piperidinyl-
C
H2), 26.0 (2C, piperidinyl-
H2), 53.2 (NH-CH2-
H2-O(morpholine)), 119.9 (C-3), 124.4 (C-6),
H), 159.3 (C-2),
C
H2), 40.2 (NH-
CH2-CH2-
C
CH2-N(morpholine)), 56.5
C
C
C
177.0 (C=S); Anal. Calcd. for C23H32N6OS: C, 62.70; H, 7.32; N, 19.07; S, 7.28%. Found: C,
62.55; H, 7.19; N, 18.93; S, 7.09%.
3.4. In Vitro Cholinesterase Inhibition Assay
Modified Ellman’s method was adopted to investigate the inhibitory potential of
newly synthesized compounds against cholinesterase enzymes [71]. The assay protocol
involved 96-well plates (100
solution, 10 L of test sample, and 10
or 10 L of 3.4 U/mg of BChE (for BChE inhibition assay). This mixture was incubated at
25 ◦C for 10 min followed by the addition of substrate (10
L). For AChE inhibition assay,
µL per well). Each well contained 20
µL of assay buffer
µ
µL of 0.5 U/mg of AChE (for AChE inhibition assay)
µ
µ
acetylthiocholine iodide (ATCI, 1 mM) while for BChE assay, butyrylthiocholine chloride
◦
(BTCCl, 1 mM), was added. This mixture was incubated for 15 min at 37 C. Ellman reagent
(5,50-dithio-bis(2-nitrobenzoic acid), DTNB, 3 mM) was added. The change in color of the
mixture showed indication of inhibition. The absorbance was measured at 405 nm using
microplate reader BioTek Elx800TM, Inc. USA. Blank assay was also performed. All the
analyses were performed in triplicate. IC50 values were also calculated for the synthesized
compounds in GraphPad prism 5.0. using nonlinear regression method.
3.5. Molecular Docking Studies
Initially 3D structures of synthesized compounds and cognate ligands were drawn
and protonated with the help of molecular operating environment (MOE) [72]. Energy
minimization of selected compounds was performed with the help of MMFF94x forcefield
(New Jersey, USA) through adjustment of hydrogens [73]. The crystal structure of human
AChE (PDB ID: 4BDT) [66] in complex with huprine W and BChE (PDB ID: 4BDS) [66] in
complex with tacrine were obtained from the RCSB Protein Bank. The docking method was
able to reproduce the experimentally bound conformation of ligand in the active site with
an RMSD of <1.0 Å. Active sites within AChE and BChE receptor were carefully chosen
around 9.0 and 10.5 Å radius of cocrystallized ligand, respectively. The solvent handling
and amino acid flip parameters were set as default. With the help of LeadIT program [67],
the best 50 scoring docked poses were nominated for further analysis. For visual analysis,
the Discovery Studio Visualizer v19 was employed [74]. The mode of binding of docked
poses was evaluated with the help of HYDE assessment tool of LeadIT [75]. Binding
free energy (∆G) for each pose was determined to explore the degree of interaction with
receptor. The minimum energy value reflects high stability and affinity of molecules to
bind with receptor.
3.6. Cytotoxicity
3.6.1. Sampling of Cell Lines
HepG2 cell line was obtained from the cell culture laboratory established in The
University of Lahore. These cell lines were preserved in cryo vials present in liquid
nitrogen. Cryo vials were revived for further processing.

Molecules 2021, 26, 656
27 of 31
3.6.2. Culturing of Cell Lines
The cryo vials obtained from liquid nitrogen cylinder were thawed. Then HepG2 cells
were cultured in the culturing flask in which DMEM-HG along with 10% fetal bovine serum
(FBS), supplemented with 100 mg/mL penicillin G (Sigma) and 100 U/mL streptomycin
(Sigma) were added. Cultures were maintained in a humidified incubator supplied with
5% CO2 at 37 ◦C. Experiments were performed in triplicates. When cultured HepG2 cells
achieved 70–80% confluence their subculturing was conducted. For splitting, the cells
attached to the walls of the culturing flask were washed with 1
×
phosphate buffer saline
(PBS) and incubated with 0.05% trypsin-EDTA until cells detached from the surface of
culturing flask. The detachment of the cells was confirmed by observing the flask under
the inverted microscope. A few drops of FBS were added to the flask and mixed well
by stirring. The mixture was centrifuged at 2000 rpm for 5 min. After centrifugation the
supernatant was removed, and pellet was resuspended. HepG2 cells were cultured onto
96-well plates for measurement of cell viability. Treatment was given for both compounds
at a concentration of 0–1000 µg/mL for 24 h.
3.6.3. Cytotoxicity Calculation via MTT Assay
After 24 h of treatment with different concentrations of compounds
were washed with phosphate buffer saline (PBS) (Invitrogen Inc., Carlsbad, CA, USA),
and then incubated with 100 L of DMEM and 25 L MTT solution (Invitrogen Inc.) for
5 and 6, cells
µ
µ
3–4 h. After 4 h formazan crystals were solubilized with 10% sodium dodecylsulphate
(SDS) (Invitrogen Inc.) and absorbance was taken at 570 nm [76]. Percentage viability was
calculated according to the following formula:
% Viable cells = ((abs sample − abs blank) / (abs control − abs blank)) × 100
where abs stands for absorbance.
(1)
Experiments were repeated three times for the average calculations.
4. Conclusions
In summary, the present study described an efficient multistep synthetic route for the
preparation of a library of 2-((6/8-methyl-2-(piperidin-1-yl)quinolin-3-yl)methylene)hy-
drazinecarbothioamides. The synthesized compounds contain a substantial degree of
structural variation in the form of electron-donating as well as electron-withdrawing
groups on different positions of the aromatic rings. Both quinoline motif and thiosemicar-
bazone moiety were endowed with a variety of functional groups. The synthesized hybrid
thiosemicarbazones were evaluated for their inhibitory potential against AChE and BChE
enzymes. Compounds possessing an 8-methyl substituted quinoline ring were found to be
more effective cholinergic inhibitors with relatively lower IC50 values as compared to the
6-substituted analogues. Several compounds displayed good inhibitory activity among
which 6d
,
6f, and 6g were identified as potent dual inhibitors of AChE and BChE with
M. The hybrid thiosemicarbazone 6f was concluded as the most potent dual
M for AChE and IC50 = 11.59 M for
BChE). Moreover, compound 6i appeared to be the selective inhibitor of AChE with an
IC50 value of 15.8 1.3 M. In vitro biochemical assay results were rationalized using
IC50 < 20
µ
inhibitor against both the enzymes (IC50 = 9.68
µ
µ
±
µ
molecular docking approach where the binding site analysis of potent compounds revealed
similar interactions to cognate ligands within the active sites of enzymes. Furthermore,
these compounds have also shown weak to moderate cytotoxic activity. Physicochemical
properties, lipophilicity, water solubility, pharmacokinetics, drug-likeness, and medicinal
chemistry properties were also calculated for the synthesized hybrid scaffold suggesting
the safer profile to be investigated as drug molecules and have high probability of blood–
brain penetration and absorption. Collectively, the identification of these N-heterocyclic
hybrid molecules presents significant implications for the design of new AChE and BChE
inhibitors. Further alterations in the structural framework of these compounds could be a

Molecules 2021, 26, 656
28 of 31
determining factor to improve their anticholinergic potential which may complement the
drug-discovery process against Alzheimer’s disease.
Supplementary Materials: The following are available online, 1H and 13C NMR spectra of all the
synthesized compounds and graphical representation of the cytotoxicity results.
Author Contributions: Conceptualization, R.M. and N.J.; methodology, R.M.; software, S.M. and
S.J.A.; validation, S.Z., S.J.A. and S.M.; formal analysis, N.J. and I.K.; investigation, R.M. (synthesis);
K.I. (bioactivity); S.J.A. (cytotoxicity); S.Z. (molecular docking, ADME properties); resources, M.M.A.;
data curation, R.M.; writing—original draft preparation, R.M.; writing—review and editing, M.Z.-
u.-R. and I.K.; visualization, S.Z.; supervision, M.M.A.; project administration, M.Z.-u.-R.; funding
acquisition, R.M. All authors have read and agreed to the published version of the manuscript.
Funding: Rubina Munir is thankful to Higher Education Commission of Pakistan for financial support
under Indigenous PhD Fellowship Program for 5000 Scholars (Grant No. 112-33715-2PS1-501).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in supplementary material.
Acknowledgments: The authors extend their gratitude towards University of the Punjab, University
of Lahore and University of Gujrat for providing research facilities. S.Z. is grateful to BiosolveIT
(BioSolveIT GmbH, Sankt Augustin, Germany) for the provision of LeadIT license for carrying out
molecular docking studies. I.K. is thankful to “Molecules” for the invitation to contribute in the special
issue and for full APC waiver.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the synthetic compounds are available from the authors on reason-
able request.
References
1.
2.
3.
Laferla, F.M.; Green, K.N.; Oddo, S. Intracellular amyloid-beta in Alzheimer’s disease. Nat. Rev. Neurosci. 2007, 8, 499–509.
[CrossRef] [PubMed]
Munoz-Torrero, D. Acetylcholinesterase inhibitors as disease-modifying therapies for Alzheimer’s disease. Curr. Med. Chem.
2008, 15, 2433–2455. [CrossRef] [PubMed]
Belluti, F.; Bartolini, M.; Bottegoni, G.; Bisi, A.; Cavalli, A.; Andrisano, V.; Rampa, A. Benzophenone-based derivatives: A novel
series of potent and selective dual inhibitors of acetylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation.
Eur. J. Med. Chem. 2011, 46, 1682–1693. [CrossRef] [PubMed]
4.
5.
Wang, L.; Kumar, R.; Pavlov, P.F.; Winblad, B. Small molecule therapeutics for tauopathy in Alzheimer’s disease: Walking on the
path of most resistance. Eur. J. Med. Chem. 2021, 209, 112915. [CrossRef] [PubMed]
Zhou, B.; Li, H.; Cui, Z.; Li, D.; Geng, H.; Gao, J.; Zhou, L. Simple analogues of natural product chelerythrine: Discovery of a
novel anticholinesterase 2-phenylisoquinolin-2-ium scaffold with excellent potency against acetylcholinesterase. Eur. J. Med.
Chem. 2020, 200, 112415. [CrossRef] [PubMed]
6.
7.
8.
9.
Xu, M.; Peng, Y.; Zhu, L.; Wang, S.; Ji, J.; Rakesh, K.P. Triazole derivatives as inhibitors of Alzheimer’s disease: Current
developments and structure-activity relationships. Eur. J. Med. Chem. 2019, 180, 656–672. [CrossRef] [PubMed]
Li, Q.; He, S.; Chen, Y.; Feng, F.; Qu, W.; Sun, H. Donepezil-based multi-functional cholinesterase inhibitors for treatment of
Alzheimer’s disease. Eur. J. Med. Chem. 2018, 158, 463–477. [CrossRef]
Li, Q.; Yang, H.; Chen, Y.; Sun, H. Recent progress in the identification of selective butyrylcholinesterase inhibitors for Alzheimer’s
disease. Eur. J. Med. Chem. 2017, 132, 294–309. [CrossRef]
Dubois, B.; Feldman, H.H.; Jacova, C.; Cummings, J.L.; Dekosky, S.T.; Barberger-Gateau, P. Revising the definition of Alzheimer’s
disease: A new lexicon. Lancet Neurol. 2010, 9, 1118–1127. [CrossRef]
10. Aisen, P.S.; Andrieu, S.; Sampaio, C.; Carrillo, M.; Khachaturian, Z.S.; Dubois, B. Report of the task force on designing clinical
trials in early (predementia) AD. Neurology 2011, 76, 280–286. [CrossRef]
11. Dubois, B.; Hampel, H.; Feldman, H.H.; Scheltens, P.; Aisen, P.; Andrieu, S. Preclinical Alzheimer’s disease: Definition, natural
history, and diagnostic criteria. Alzheimer’s & dementia. J. Alzheimer’s Assoc. 2016, 12, 292–323. [CrossRef]
12. Nainwal, L.M.; Tasneem, S.; Akhtar, W.; Verma, G.; Khan, M.F.; Parvez, S.; Shaquiquzzaman, M.; Akhter, M.; Alam, M.M. Green
recipes to quinoline: A review. Eur. J. Med. Chem. 2019, 164, 121–170. [CrossRef] [PubMed]
13. Chu, X.M.; Wang, C.; Liu, W.; Liang, L.L.; Gong, K.K.; Zhao, C.Y.; Sun, K.L. Quinoline and quinolone dimers and their biological
activities: An overview. Eur. J. Med. Chem. 2019, 161, 101–117. [CrossRef] [PubMed]

Molecules 2021, 26, 656
29 of 31
14. Martin, R.E.; Butterworth, A.S.; Gardiner, D.L.; Kirk, K.; McCarthy, J.S.; Skinner-Adams, T.S. Saquinavir inhibits the malaria
parasite’s chloroquine resistance transporter. Antimicrob. Agents Chemother. 2012, 56, 2283–2289. [CrossRef] [PubMed]
15. Kuang, Y.H.; Patel, J.P.; Sodani, K.; Wu, C.P.; Liao, L.Q.; Patel, A.; Tiwari, A.K.; Dai, C.L.; Chen, X.; Fu, L.W.; et al. OSI-930
analogues as novel reversal agents for ABCG2-mediated multidrug resistance. Biochem. Pharmacol. 2012, 84, 766–774. [CrossRef]
16. Rajanarendar, E.; Reddy, M.N.; Krishna, S.R.; Murthy, K.R.; Reddy, Y.N.; Rajam, M.V. Design, synthesis, antimicrobial, anti-
inflammatory and analgesic activity of novel isoxazolyl pyrimido [4,5-b] quinolines and isoxazolyl chromeno [2,3-d] pyrimidin-4-
ones. Eur. J. Med. Chem. 2012, 55, 273–283. [CrossRef]
17. Musiol, R.; Serda, M.; Hensel-Bielowka, S.; Polanski, J. Quinoline-based antifungals. Curr. Med. Chem. 2010, 17, 1960–1973.
[CrossRef]
18. Hazra, A.; Mondal, S.; Maity, A.; Naskar, S.; Saha, P.; Paira, R.; Sahu, K.B.; Paira, P.; Ghosh, S.; Sinha, C.; et al. Amberlite-IRA-402
(OH) ion exchange resin mediated synthesis of indolizines, pyrrolo [1,2-a] quinolines and isoquinolines: Antibacterial and
antifungal evaluation of the products. Eur. J. Med. Chem. 2011, 46, 2132–2140. [CrossRef]
19. Zhang, Y.; Fang, Y.; Liang, H.; Wang, H.; Hu, K.; Liu, X.; Yi, X.; Peng, Y. Synthesis and antioxidant activities of 2-oxo-quinoline-3-
carbaldehyde Schiff-base derivatives. Bioorg. Med. Chem. Lett. 2013, 23, 107–111. [CrossRef]
20. Solomon, V.R.; Lee, H. Quinoline as a privileged scaffold in cancer drug discovery. Curr. Med. Chem. 2011, 18, 1488–1508.
[CrossRef]
21. Rizvi, S.U.F.; Siddiqui, H.L.; Nisar, M.; Khan, N.; Khan, I. Discovery and molecular docking of quinolyl-thienyl chalcones as
anti-angiogenic agents targeting VEGFR-2 tyrosine kinase. Bioorg. Med. Chem. Lett. 2012, 22, 942–944. [CrossRef] [PubMed]
22. Ratheesh, M.; Sindhu, G.; Helen, A. Anti-inflammatory effect of quinoline alkaloid skimmianine isolated from Ruta graveolens L. J.
Inflamm. Res. 2013, 62, 367–376. [CrossRef] [PubMed]
23. Das, P.; Deng, X.; Zhang, L.; Roth, M.G.; Fontoura, B.M.; Phillips, M.A.; De Brabander, J.K. SAR-based optimization of a
4-quinoline carboxylic acid analogue with potent antiviral activity. ACS Med. Chem. Lett. 2013, 4, 517–521. [CrossRef] [PubMed]
24. Rizvi, S.U.F.; Ahmad, M.; Bukhari, M.H.; Montero, C.; Chatterjee, P.; Detorio, M.; Schinazi, R.F. Anti-HIV-1 screening of
(2E)-3-(2-chloro-6-methyl/methoxyquinolin-3-yl)-1-(aryl) prop-2-en-1-ones. Med. Chem. Res. 2014, 23, 402–407. [CrossRef]
25. Zaib, S.; Rizvi, S.U.F.; Aslam, S.; Ahmad, M.; Abid, S.M.A.; al-Rashida, M.; Iqbal, J. Quinolinyl-thienyl chalcones as monoamine
oxidase inhibitors and their in silico modeling studies. Med. Chem. Res. 2015, 11, 580–589. [CrossRef]
26. Massoud, M.A.; El-Sayed, M.A.; Bayoumi, W.A.; Mansour, B. Cytotoxicity and molecular targeting study of novel 2-chloro-3-
substituted quinoline derivatives as antitumor agents. Lett. Drug Des. Discov. 2019, 16, 273–283. [CrossRef]
27. Tejería, A.; Pérez-Pertejo, Y.; Reguera, R.M.; Carbajo-Andrés, R.; Balaña-Fouce, R.; Alonso, C.; Martin-Encinas, E.; Selas, A.;
Rubiales, G.; Palacios, F. Antileishmanial activity of new hybrid tetrahydroquinoline and quinoline derivatives with phosphorus
substituents. Eur. J. Med. Chem. 2019, 162, 18–31. [CrossRef]
28. Chanquia, S.N.; Larregui, F.; Puente, V.; Labriola, C.; Lombardo, E.; Liñares, G.G. Synthesis and biological evaluation of new
quinoline derivatives as antileishmanial and antitrypanosomal agents. Bioorg. Chem. 2019, 83, 526–534. [CrossRef]
29. Muruganantham, N.; Sivakumar, R.; Anbalagan, N.; Gunasekaran, V.; Leonard, J.T. Synthesis, anticonvulsant and antihypertensive
activities of 8-substituted quinoline derivatives. Biol. Pharm. Bull. 2004, 27, 1683–1687. [CrossRef]
30. Kumar, H.; Devaraji, V.; Joshi, R.; Jadhao, M.; Ahirkar, P.; Prasath, R.; Bhavana, P.; Ghosh, S.K. Antihypertensive activity of a
quinoline appended chalcone derivative and its site specific binding interaction with a relevant target carrier protein. RSC Adv.
2015, 5, 65496–65513. [CrossRef]
31. De Kimpe, N.; Boelens, M.; Contreras, J. Rearrangement of 5-(bromomethyl)-1-pyrrolinium salts into functionalized piperidines.
Tetrahedron Lett. 1996, 37, 3171–3174. [CrossRef]
32. Taylor, R.D.; MacCoss, M.; Lawson, A.D. Rings in drugs: Miniperspective. J. Med. Chem. 2014, 57, 5845–5859. [CrossRef]
[PubMed]
33. Padmanilayam, M.; Scorneaux, B.; Dong, Y.; Chollet, J.; Matile, H.; Charman, S.A.; Creek, D.J.; Charman, W.N.; Tomas, J.S.;
Scheurer, C.; et al. Antimalarial activity of N-alkyl amine, carboxamide, sulfonamide, and urea derivatives of a dispiro-1,2,4-
trioxolane piperidine. Bioorg. Med. Chem. Lett. 2006, 16, 5542–5545. [CrossRef] [PubMed]
34. Kamin´ski, K.; Wiklik, B.; Obniska, J. Synthesis, anticonvulsant properties, and SAR analysis of differently substituted pyrrolidine-2,
5-diones and piperidine-2,6-diones. Arch. Pharm. Chem. Life Sci. 2014, 347, 840–852. [CrossRef] [PubMed]
35. Henderson, N.D.; Plumb, J.A.; Robins, D.J.; Workman, P. Synthesis and anti-cancer activity of 2,6-disubstituted N-
methylpiperidine derivatives and their N-oxides. Anti-Cancer Drug Des. 1996, 11, 421–438. [PubMed]
36. Balsamo, A.; Giorgi, I.; Lapucci, A.; Lucacchini, A.; Macchia, B.; Macchia, F.; Martini, C.; Rossi, A. 3-[(2-Ethoxyphenoxy) methyl]
piperidine derivatives. Synthesis and antidepressant activity. J. Med. Chem. 1987, 30, 222–225. [CrossRef]
37. Shaw, A.T.; Kim, D.W.; Nakagawa, K.; Seto, T.; Crinó, L.; Ahn, M.J.; De Pas, T.; Besse, B.; Solomon, B.J.; Blackhall, F.; et al.
Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N. Engl. J. Med. 2013, 368, 2385–2394. [CrossRef]
38. Ruela, A.L.M.; Carvalho, F.C.; Pereira, G.R. Exploring the phase behavior of monoolein/oleic acid/water systems for enhanced
donezepil administration for Alzheimer disease treatment. J. Pharm. Sci. 2016, 105, 71–77. [CrossRef]
39. Weiser, M.; Levi, L.; Levine, S.Z.; Bialer, M.; Shekh-Ahmad, T.; Matei, V.; Tiugan, A.; Cirjaliu, D.; Sava, C.; Sinita, E.; et al. A
randomized, double-blind, placebo-and risperidone-controlled study on valnoctamide for acute mania. Bipolar Disord. 2017, 19,
285–294. [CrossRef]

Molecules 2021, 26, 656
30 of 31
40. Luethi, D.; Kaeser, P.J.; Brandt, S.D.; Krähenbühl, S.; Hoener, M.C.; Liechti, M.E. Pharmacological profile of methylphenidate-based
designer drugs. Neuropharmacology 2018, 134, 133–140. [CrossRef]
41. Arce, E.R.; Putzu, E.; Lapier, M.; Maya, J.D.; Azar, C.O.; Echeverría, G.A.; Piro, O.E.; Medeiros, A.; Sardi, F.; Comini, M.; et al. New
heterobimetallic ferrocenyl derivatives are promising antitrypanosomal agents. Dalton Trans. 2019, 48, 7644–7658. [CrossRef]
[PubMed]
42. Divar, M.; Khalafi-Nezhad, A.; Zomorodian, K.; Sabet, R.; Faghih, Z.; Jamali, M.; Pournaghz, H.; Khabnadideh, S. Synthesis of
some novel semicarbazone and thiosemicarbazone derivatives of isatin as possible biologically active agents. J. Pharm. Res. Int.
2017, 1–13. [CrossRef]
43. Dawood, D.H.; Batran, R.Z.; Farghaly, T.A.; Khedr, M.A.; Abdulla, M.M. New coumarin derivatives as potent selective COX-2
inhibitors: Synthesis, anti-Inflammatory, QSAR, and molecular modeling studies. Arch. Pharm. Chem. Life Sci. 2015, 348, 875–888.
[CrossRef] [PubMed]
44. Haribabu, J.; Subhashree, G.R.; Saranya, S.; Gomathi, K.; Karvembu, R.; Gayathri, D. Isatin based thiosemicarbazone derivatives
as potential bioactive agents: Antioxidant and molecular docking studies. J. Mol. Struct. 2016, 1110, 185–195. [CrossRef]
45. Bakherad, Z.; Safavi, M.; Fassihi, A.; Sadeghi-Aliabadi, H.; Bakherad, M.; Rastegar, H.; Ghasemi, J.B.; Sepehri, S.; Saghaie, L.;
Mahdavi, M. Anti-cancer, anti-oxidant and molecular docking studies of thiosemicarbazone indole-based derivatives. Res. Chem.
Intermed. 2019, 45, 2827–2854. [CrossRef]
46. Elsayed, H.E.; Ebrahim, H.Y.; Haggag, E.G.; Kamal, A.M.; El Sayed, K.A. Rationally designed hecogenin thiosemicarbazone
analogs as novel MEK inhibitors for the control of breast malignancies. Bioorg. Med. Chem. 2017, 25, 6297–6312. [CrossRef]
47. Hussein, M.A.; Iqbal, M.A.; Asif, M.; Haque, R.A.; Ahamed, M.B.K.; Majid, A.M.A.; Guan, T.S. Synthesis, crystal structures and
in vitro anticancer studies of new thiosemicarbazone derivatives. Phosphorus Sulfur Silicon Relat. Elem. 2015, 190, 1498–1508.
[CrossRef]
48. Pingaew, R.; Prachayasittikul, S.; Ruchirawat, S.; Prachayasittikul, V. Synthesis and cytotoxicity of novel N-sulfonyl-1,2,3,4-
tetrahydroisoquinoline thiosemicarbazone derivatives. Med. Chem. Res. 2013, 22, 267–277. [CrossRef]
49. Palanimuthu, D.; Poon, R.; Sahni, S.; Anjum, R.; Hibbs, D.; Lin, H.Y.; Bernhardt, P.V.; Kalinowski, D.S.; Richardson, D.R. A novel
class of thiosemicarbazones show multi-functional activity for the treatment of Alzheimer’s disease. Eur. J. Med. Chem. 2017, 139,
612–632. [CrossRef]
50. Ali, M.; Khan, K.M.; Salar, U.; Ashraf, M.; Taha, M.; Wadood, A.; Hamid, S.; Riaz, M.; Ali, B.; Shamim, S.; et al. Synthesis, in vitro
alpha-glucosidase inhibitory activity, and in silico study of (E)-thiosemicarbazones and (E)-2-(2-(arylmethylene) hydrazinyl)-4-
arylthiazole derivatives. Mol. Divers. 2018, 22, 841–861. [CrossRef]
51. Khan, I.; Hanif, M.; Hussain, M.T.; Khan, A.A.; Aslam, M.A.S.; Rama, N.H.; Iqbal, J. Synthesis, acetylcholinesterase and alkaline
phosphatase inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole derivatives. Aust. J. Chem. 2012, 65, 1413–1419. [CrossRef]
52. Khan, I.; Ibrar, A.; Zaib, S.; Ahmad, S.; Furtmann, N.; Hameed, S.; Simpson, J.; Bajorath, J.; Iqbal, J. Active compounds from a
diverse library of triazolothiadiazole and triazolothiadiazine scaffolds: Synthesis, crystal structure determination, cytotoxicity,
cholinesterase inhibitory activity, and binding mode analysis. Bioorg. Med. Chem. 2014, 22, 6163–6173. [CrossRef] [PubMed]
53. Khan, I.; Zaib, S.; Ibrar, A.; Rama, N.H.; Simpson, J.; Iqbal, J. Synthesis, crystal structure and biological evaluation of some novel
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines. Eur. J. Med. Chem. 2014, 78, 167–177. [CrossRef]
[PubMed]
54. Khan, I.; Bakht, S.M.; Ibrar, A.; Abbas, S.; Hameed, S.; White, J.M.; Rana, U.A.; Zaib, S.; Shahid, M.; Iqbal, J. Exploration of a
library of triazolothiadiazole and triazolothiadiazine compounds as a highly potent and selective family of cholinesterase and
monoamine oxidase inhibitors: Design, synthesis, X-ray diffraction analysis and molecular docking studies. RSC Adv. 2015, 5,
21249–21267. [CrossRef]
55. Ibrar, A.; Khan, A.; Ali, M.; Sarwar, R.; Mehsud, S.; Farooq, U.; Halimi, S.M.A.; Khan, I.; Al-Harrasi, A. Combined in vitro and in
silico studies for the anticholinesterase activity and pharmacokinetics of coumarinyl thiazoles and oxadiazoles. Front. Chem. 2018
6, 61. [CrossRef]
,
56. Larik, F.A.; Saeed, A.; Faisal, M.; Hamdani, S.; Jabeen, F.; Channar, P.A.; Mumtaz, A.; Khan, I.; Kazi, M.A.; Abbas, Q.; et al.
Synthesis, inhibition studies against AChE and BChE, drug-like profiling, kinetic analysis and molecular docking studies of
N-(4-phenyl-3-aroyl-2(3H)-ylidene) substituted acetamides. J. Mol. Struct. 2020, 1203, 127459. [CrossRef]
57. Ghobadian, R.; Mahdavi, M.; Nadri, M.; Moradi, A.; Edraki, N.; Akbarzadeh, T.; Sharifzadeh, M.; Bukhari, S.N.A.; Amini, M.
Novel tetrahydrocarbazole benzyl pyridine hybrids as potent and selective butryl cholinesterase inhibitors with neuroprotective
and β-secretase inhibition activities. Eur. J. Med. Chem. 2018, 155, 49–60. [CrossRef]
58. Pérez-Areales, F.J.; Turcu, A.L.; Barniol-Xicota, M.; Pont, C.; Pivetta, D.; Espargaró, A.; Bartolini, M.; De Simone, A.; Andrisano,
V.; Pérez, B.; et al. A novel class of multitarget anti-Alzheimer benzohomoadamantane-chlorotacrine hybrids modulating
cholinesterases and glutamate NMDA receptors. Eur. J. Med. Chem. 2019, 180, 613–626. [CrossRef]
59. Najafi, Z.; Mahdavi, M.; Saeedi, M.; Karimpour-Razkenari, E.; Asatouri, R.; Vafadarnejad, F.; Moghadam, F.H.; Khanavi, M.;
Sharifzadeh, M.; Akbarzadeh, T. Novel tacrine-1,2,3-triazole hybrids: In vitro
, in vivo biological evaluation and docking study of
cholinesterase inhibitors. Eur. J. Med. Chem. 2017, 125, 1200–1212. [CrossRef]
60. Meth-Cohn, O.; Narine, B.; Tarnowski, B. A versatile new synthesis of quinolines and related fused pyridines, Part 5. The
synthesis of 2-chloroquinoline-3-carbaldehydes. J. Chem. Soc. Perkin Trans. 1981, 1, 1520–1530. [CrossRef]