New piperidine-hydrazone derivatives: Synthesis, biological evaluations and molecular docking studies as AChE and BChE inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.08.037

Hydrazones and the piperidine ring containing compounds were considered as beneficial substrates in drug design. Therefore, this study was aimed at the synthesis of new benzoyl hydrazones derived from ethyl 4-oxopiperidine-1-carboxylate and 2,6-diphenylpiperidin-4-one. The synthesized compounds (1?19) were screened for their antioxidant, anticholinesterase and anticancer activities. The antioxidant capacity of the compounds was evaluated by using four complementary tests. The results showed that compound 7 and 17 have the higher lipid peroxidation inhibitory activity than the other compounds. In DPPH˙ scavenging assay, compounds 5, 6, 10, 14, 17 demonstrated better activity than that of standard BHT, while in ABTS⁺˙ scavenging assay compound 6 and 17 exhibited better activity among the other compounds. The CUPRAC assay disclosed that compound 2 displayed better activity than α-tocopherol. The anticholinesterase activity was performed against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Compound 11 (IC₅₀: 35.30?±?1.11?μM) inhibited BChE better than galantamine (IC₅₀: 46.03?±?0.14?μM). We conclude that the compound 11 can be considered as a candidate for BChE inhibitor. Moreover docking method was applied to elucidate the AChE and BChE inhibitory mechanism of the compound 11. Molecular docking analysis revealed that compound 11 bound to BChE enzyme more efficiently when compared to the AChE due to its orientations and different types of interactions. In addition, the non-cytotoxic properties of the compounds brought them into prominence, although they did not show significant anticancer properties.

Full text of DOI:10.1016/j.ejmech.2016.08.037

Accepted Manuscript
New piperidine-hydrazone derivatives: Synthesis, biological evaluations and
molecular docking studies as AChE and BChE inhibitors
Nurcan Karaman, Yusuf Sıcak, Tuğba Taşkın-Tok, Mehmet Öztürk, Ayşegül
Karaküçük-İyidoğan, Miris Dikmen, Bedia Koçyiğit-Kaymakçıoğlu, Emine Elçin Oruç-
Emre
PII:
S0223-5234(16)30680-8
10.1016/j.ejmech.2016.08.037
EJMECH 8833
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 June 2016
Revised Date: 16 August 2016
Accepted Date: 17 August 2016
Please cite this article as: N. Karaman, Y. Sıcak, T. Taşkın-Tok, M. Öztürk, A. Karaküçük-İyidoğan, M.
Dikmen, B. Koçyiğit-Kaymakçıoğlu, E.E. Oruç-Emre, New piperidine-hydrazone derivatives: Synthesis,
biological evaluations and molecular docking studies as AChE and BChE inhibitors, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.037.
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Antioxidant Activity
Anticholinesterase Activity

ACCEPTED MANUSCRIPT
Title: New Piperidine-Hydrazone Derivatives: Synthesis, Biological Evaluations and Molecular Docking
Studies as AChE and BChE Inhibitors
Nurcan Karaman1, Yusuf Sıcak2,3, Tuğba Taşkın-Tok1, Mehmet Öztürk3, Ayşegül Karaküçük-İyidoğan1,
Miris Dikmen4, Bedia Koçyiğit-Kaymakçıoğlu5, Emine Elçin Oruç-Emre1*
1Department of Chemistry, Faculty of Arts and Sciences, Gaziantep University, Gaziantep, Türkiye
2Department of Herbal and Animal Production, Köyceğiz Vocational School, Muğla Sıtkı Koçman University,
Muğla, Türkiye
3 Department of Chemistry, Faculty of Sciences, Muğla Sıtkı Koçman University, Muğla, Türkiye
4Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskişehir, Türkiye
5Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, İstanbul, Türkiye
*Correspondence Author:
Emine Elçin Oruç-Emre
Address: Gaziantep University Faculty of Arts and Sciences Department of Chemistry 27310 Gaziantep/
Turkey
Phone number: +903423171827
Fax number: +903423601032
e-mail address: oruc@gantep.edu.tr
Keywords: Piperidine, Hydrazone, Anticholinesterase activity, Antioxidant activity, Molecular docking.
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Abstract:
Hydrazones and the piperidine ring containing compounds were considered as beneficial substrates
in drug design. Therefore, this study was aimed at the synthesis of new benzoyl hydrazones derived from
ethyl 4-oxopiperidine-1-carboxylate and 2,6-diphenylpiperidin-4-one. The synthesized compounds (1–19)
were screened for their antioxidant, anticholinesterase and anticancer activities. The antioxidant capacity of
the compounds was evaluated by using four complementary tests. The results showed that compound 7 and
17 have the higher lipid peroxidation inhibitory activity than the other compounds. In DPPH˙ scavenging
assay, compounds 5, 6, 10, 14, 17 demonstrated better activity than that of standard BHT, while in ABTS+˙
scavenging assay compound 6 and 17 exhibited better activity among the other compounds. The CUPRAC
assay disclosed that compound 2 displayed better activity than α-tocopherol. The anticholinesterase activity
was performed against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Compound
11 (IC50: 35.30±1.11 µM) inhibited BChE better than galantamine (IC50: 46.03±0.14 µM). We conclude that
the compound 11 can be considered as a candidate for BChE inhibitor. Moreover docking method was
applied to elucidate the AChE and BChE inhibitory mechanism of the compound 11. Molecular docking
analysis revealed that compound 11 bound to BChE enzyme more efficiently when compared to the AChE
due to its orientations and different types of interactions. In addition, the non-cytotoxic properties of the
compounds brought them into prominence, although they did not show significant anticancer properties.
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1. Introduction
Reactive oxygen species (ROS) are reactive oxygen containing molecules including free radicals.
ROS have important roles in biological systems such as cell signaling, apoptosis, gene expression and ion
transportation. In case of excessive generation of reactive oxygen species, a chain reaction starts by the free
radicals and then oxidative stress occurs in the cells. This situation initiates the cell or genetic material
damages. Such cell damages can cause disorders in physiology of tissues and organs which can be resulted
in many diseases such as cancer, heart diseases, decline in brain function and immune system. Natural and
synthetic antioxidants inhibit the oxidation by stabilizing or deactivating free radicals thus they became
valuable components to protect or support the body against oxidative stress and found use as therapeutics
[1-5]. Antioxidant molecules can protect or treat the human body for many chronic diseases. In some
researches this relationship between the antioxidant properties and anticancer, anticholinesterase properties
of the compounds were mentioned [1, 4, 6].
Hydrazones are potential substrates for drug design. They exhibited various biological activities such
as antioxidant, anticonvulsant, antidepressant, antimicrobial, antitumoral, anticholinesterase, analgesic and
anti-inflammatory activities [6-14]. Wide range of biological activities was also reported for the molecules
containing piperidine ring [1, 10, 15, 16]. With the motivation of obtaining more potent antioxidant, anti-
Alzheimer and anticancer agents for lung cancer and as a continuation of our previous work [17], we
designed hydrazone derivatives of ethyl 4-(2-benzoylhydrazono)piperidine-1-carboxylate and N'-(2,6-
diphenylpiperidin-4-ylidene)benzohydrazide. Different substituted phenyl groups were used in the synthesis
of the compounds to determine the effects of the substituents on activities. In this work, new hydrazones
were synthesized, characterized and then screened for their pharmacological activities. The structure of the
molecules was also elucidated by using the molecular docking application of Discovery Studio (DS) 2016
software to investigate the AChE and BChE inhibitory effects. This approach was applied to model the
interactions between the studied compounds and AChE and BChE at the atomic level. Molecular docking
enables to characterize the behavior of small molecules in the active site of AChE and BChE and explain the
fundamental biochemical processes.
2. Results and Discussion
2.1.Chemistry
In this study, we synthesized nineteen benzoyl hydrazones bearing piperidine ring and screened for
their antioxidant activity, anticholinesterase activity against AChE and BChE and cytotoxic activity against
lung cancer cell lines (A549) and fibroblast (BJ) cell lines. The overall synthetic representation of target
compounds is outlined in Scheme 1. 2,6-diaryl piperidine 4-one (I) was obtained with low yield using the
procedure mentioned in the literature [18, 19]. In order to increase this yield, several different procedures
and catalysts were tried. The results of the trials were reported in our previous work and with this modified
method the yield was improved to 75% by using L-proline catalyst [17]. The benzoyl hydrazides (III) were
obtained according to the literature [12, 20] and then reacted with ethyl 4-oxo-piperidine-1-carboxylate and
2,6-diarylpiperidine-4-one to obtain hydrazone derivatives (compound 1–19). For the synthesis of benzoyl
hydrazones, MW-assisted method was tried as an alternative to the conventional method in order to increase
the yield. As a consequence, approximately twofold increase was achieved in the yields. The compounds
were characterized by FTIR, NMR, mass spectra and elemental analysis.
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Scheme 1. Synthetic pathway of 4-substitutedphenylbenzoyl hydrazones (1–19). Reagents and conditions:
(a) NaOH; (b) NH2NH2, CH3OH (c) EtOH, reflux; (d) EtOH, L-proline, rt.
In the IR spectra of all compounds, the C=N stretching vibration bands were observed at 1630–1667
cm-1 except for the compounds 7 and 17 that they were observed at 1615 and 1613 cm-1, respectively. The
stretching vibrations of C=O due to ester and hydrazones were observed at 1679–1693 cm-1 and 1643–1667
cm-1, respectively. Broad peaks at 3150–3370 cm-1 indicated N–H stretching vibrations of hydrazone; where
sharp peaks at 3285–3319 cm-1 indicated N–H stretching vibrations of piperidine (for compound 10–19). N–H
stretching vibrations of hydrazone of compound 7 and 17 were observed at 3313 and 3323 cm-1,
respectively.
Piperidine ring generally prefer chair conformation [17, 21, 22]. To elucidate the conformation and
correlate the peaks of the hydrogen with the carbons, NOESY, ROESY, HMBC, HSQC spectra were used
(Supplementary Data). All discussions were performed according to the numbering of the atoms in Scheme
2.
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Scheme 2. Atom numbering for 1H NMR and 13C NMR.
In the 1H NMR spectra, HA protons of the compounds appeared at 10.59–11.05 ppm as a singlet. As
methyl and methoxy groups were donating electrons to the ring and chlorine and fluorine atoms sharing the
lone pairs; HA was less deshielded and displayed resonance signals having relatively high field chemical shift
for those substituents, whereas with the electron withdrawing character trifluoromethyl and nitro groups
shifted to the downfield region. Halogens and oxygen atoms strongly withdraw the electrons from the ring but
due to the effect of lone pairs HB shifted more to the downfield then HC. This was seen also in the NOESY
and ROESY spectra of compound 1 and 11 (Fig. 1 and 2) that far interaction between HB and HA was
observed. Due to the effect of strongly withdrawing groups; nitro and trifluoromethyl for the compounds 5, 6,
7, 15, 16 and 17, HC was deshielded to the downfield. This effect was observed clearly for the compounds 7
and 17. When the coupling constants were analyzed, for compound 7, HB was resonated as doublet by HC;
where the HC was affected by both HB and HJ and gives doublet of doublet at relatively low field region than
the HB. Except from the fluorine atom containing compounds HB and HC were resonated as doublets. For the
fluorine containing compounds HC splitted both by the HB and the F atom and also HC observed as triplet
because of the spin number of fluorine atom (1/2). Similarly, HB also affected by the F atom but by the far
interaction peak was unable to splitted into triplet. It was observed as multiplet. Among the compounds 1–9,
HI for compounds 8 and 9 resonated at 4.02–4.10 ppm as multiplet because of the fluorine atom interactions
and for the other compounds resonated at 4.08 ppm as quartet. According to NOESY and ROESY spectra of
compound 1, HA had far interaction with HD or HE and also with HG. These interactions described by the
structure of the molecule that hydrazone group twisted towards one side of the piperidine ring. So, HG
resonates as multiplet at 1.17–1.22 ppm for compounds 8 and 9 because of the far interactions of F atoms;
where HG resonated at 1.21 as triplet for the other compounds. HD and HE were not identical because of the
chair confirmation and generally resonated at 2.47–2.57 ppm as multiplet; where for compounds 5, 7, 8 and
9 they were observed as two or three triplets in the same region. HF and HF' also resonated as multiplet at
3.38–3.57 ppm; but for compound 5 HF' gave triplet at 3.53 ppm. For compound 7, HF and HF' resonated as
triplets at 3.63 and 3.58 ppm, respectively. The NOESY and ROESY spectra of the compound 11 verified
that the hydrazone group was twisted towards the one side of the piperidine ring that; there were far
interactions between HA and HG; HA and HD; HA and HB. HD and HD' affected by the HE and HE' and resonated
as doublets at 3.16–3.20 ppm and 2.56–2.69 ppm where HE and HE' splitted into triplets by HD,D' and HF,F'
hydrogens at 2.06–2.37 ppm and 2.38–2.45 ppm, respectively. Similarly, HF and HF' splitted into doublets by
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HE and HE' at 3.85–3.89 ppm and 3.94–3.97 ppm, respectively. By the far interactions observed between HF
and HJ, HF' and HJ'; the chemical shifts of the HJ and HJ' were appeared as a doublet or multiplet at 7.49–7.58
ppm. In addition, in the spectrum of the compound 17 the peaks of HI, HI', HJ, HJ', were clearly separated and
identified. Chemical shifts of HI, HI' and HK, HK' were observed at 7.33–7.45 ppm and 7.24–7.34 ppm,
respectively, as multiplets.
Fig. 1 NOESY and ROESY spectrum of compound 1
Fig. 2 NOESY and ROESY spectrum of compound 11
In 13C NMR spectra for compounds 1–9, C9 carbons were observed at 15.02–15.06 ppm, where C8
at 61.32–61.37 ppm; C3 and C5 carbons resonated as two separate peaks at 27.62–28.75 ppm and 33.49–
34.15 ppm; similarly, C2 and C6 was observed as two peaks at 40.62–42.15 ppm and 43.42–43.95 ppm. For
compounds 10–19, aliphatic carbons appeared as four separate peaks; C2 and C6 at 60.07–60.53 ppm and
61.26–61.46 ppm and C3 and C5 at 36.57–37.39 ppm and 43.64–43.95 ppm. In order to define the aromatic
carbon peaks, HSQC spectrum of compound 4 (Fig. 3) and HSQC and HMBC spectra of compound 18 (Fig.
4) were used. According to HSQC of compound 4 the peaks of HB correlated with 128.12 ppm belonged to
C12 and C16; similarly, HC interacted with 129.20 ppm C13 and C15. When the 2D NMR spectra of
compound 18 were analyzed, many interactions between the carbons with hydrogens were observed. The
hydrogen peaks of the benzene ring attached to the hydazone correlated with the peaks (114.00, 114.14,
121.84, 131.84 ppm); HI , HI' correlated with the peaks (128.74, 128.67, 128.62, 128.60 ppm) belonged to C9
and C11; HK , HK' correlated with the peaks (127.65, 127.59, 127.24 ppm) belonging to C10; HJ , HJ'
correlated with the peaks (127.18, 127.05 ppm) belonging to C8 and C12. The remaining carbon peaks
belonged to the ipso and the carbonyl carbons. C7 and C7' peaks were observed at 144.34 and 144.19 ppm
in the spectrum of compound 18, similarly, for the other compounds two peaks were observed at 144.16–
144.60 ppm. In the spectra of the compounds 1–9, peaks observed at 155.03–155.13 ppm were attributed to
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C7. Except from fluorine atom substituted compounds, C4 and carbonyl carbon of the hydrazones (C10 for
compounds 1–9, C13 for compounds 10–19) were observed at the paramagnetic region with a largest
chemical shift. For F containing compounds (2, 8, 9, 12, 18, 19) the ipso carbon attached to the fluorine atom
had a chemical shift at 165.67–166.91 ppm when the F atom at 4 position of the benzene ring [23]; where
when the F atom at 2 position by the effect of both carbonyl and the F, ipso carbons more deshielded and
observed at 169.42 and 169.45 ppm.
Fig. 3 HSQC spectrum of compound 4
Fig. 4 HSQC and HMBC spectrum of compound 18
The structures of the benzoyl hydrazones were also confirmed by the mass spectra. The cleavage of
N–N, C–N and C=N bond of hydrazone and the separation of benzene groups were specific fragments of
these compounds. The other peaks were due to the specific fragmentations of piperidine ring seen in the Fig.
5.
Fig. 5 Mass fragmentation pattern of the compounds.
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2.2. Pharmacology
2.2.1. Antioxidant activity of synthesized compounds
The synthesized compounds were evaluated for their antioxidant activity using four different methods
(Table 1), where α-.Tocopherol and butylated hydroxyl toluene (BHT) was used as positive control. Among
the tested compounds, better antioxidant activities were observed generally with compounds having
electron-withdrawing substituent NO2. The effect of the fluorine atom can be analyzed by comparing
compounds 2, 5, 8, 9 and compounds 12, 15, 18, 19. F containing compounds at 4 position of the phenyl
ring had better activity than the compounds having CF3 group at 4 positions. In the CUPRAC assay, A0.5
values of the compounds were found lower than 100 µM. Compound 2 indicated the best reducing effect
among the compounds (1–19). At the same time, it was better than the antioxidant standard α-tocopherol
and close activity to that of BHT. In addition, compounds 4, 6, 16, 1, 9, 8, 17 and 3 exhibited better activity
than the α-tocopherol, respectively. In β-carotene-linoleic acid assay, compounds 7 and 17 exhibited the
higher lipid peroxidation inhibitory activity, followed by 13, 6, 18, 10 and 11, respectively. In DPPH assay, the
IC50 values of the compounds (1–19) were lower than 100 µM. Compounds, 5, 6, 10, 14 and 17 exhibited
better DPPH free radical scavenging activity than BHT (IC50:54.97±0.99 µM). In the ABTS assay, among the
tested compounds, 6, 17 showed the best ABTS cation radical scavenging activity with IC50 values of
10.86±1.80 and 19.25±1.70, respectively. Except compounds 7, 10, 15, 16 this series showed good IC50
values that were lower than 100 µM.
Table 1 Antioxidant activity results of compounds (1–19), BHT and α-tocopherol by the β-carotene-linoleic
acid, DPPH, ABTS and CUPRAC assay.
β-carotene/linoleic
acid assay
DPPH˙
assay
ABTS˙+
assay
CUPRAC
assay
Compounds
R
IC50 (µM)a
IC50 (µM) a
58.95±0.24
74.83±0.46
85.41±0.41
75.50±0.21
33.84±0.48
51.70±1.39
59.95±0.20
86.01±0.15
86.81±0.20
41.99±0.32
96.71±0.28
66.04±0.08
75.81±0.18
53.29±0.57
64.95±0.19
88.14±0.56
19.99±1.03
70.17±0.65
88.28±0.16
54.97±0.99
12.26±0.07
IC50 (µM) a
63.87±1.64
39.21±0.71
38.27±1.05
28.08±1.40
23.50±1.60
10.86±1.80
117.16±0.55
59.45±1.02
41.85±0.39
238.01±1.06
67.87±1.90
83.67±0.41
27.92±0.73
64.14±1.78
152.81±1.33
169.60±0.51
19.25±1.70
63.48±2.24
88.48±1.64
2.91±0.55
A0.50(µM) a
23.52±0.08
4.16±0.04
36.94±0.00
11.11±0.02
51.38±0.09
13.32±0.02
83.13±0.01
27.27±0.01
25.63±0.02
45.42±0.00
49.76±0.01
59.57±0.04
77.73±0.01
50.59±0.01
92.13±0.00
17.83±0.13
29.20±0.02
85.18±0.03
88.20±0.01
3.80±0.02
40.48±1.87
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
4-Cl
4-F
4-OCH3
4-CH3
72.27±1.95
65.55±0.95
-
52.54±0.53
49.33±0.70
40.24±1.09
27.83±0.95
77.44±0.78
68.23±0.72
41.85±0.11
43.56±0.23
56.61±0.80
34.46±0.65
69.03±0.69
55.28±0.95
45.16±1.10
28.15±1.26
41.08±0.39
114.87±0.51
2.34 ± 0.09
4.50 ± 0.09
4-CF3
4-NO2
2,4- NO2
2-F-4-CF3
4-F-2-CF3
H
4-Cl
4-F
4-OCH3
4-CH3
4-CF3
4-NO2
2,4- NO2
2-F-4-CF3
4-F-2-CF3
17
18
19
BHT b
α-Tocopherolb
4.87±0.45
aValues expressed are mean ± SEM of three parallel measurements (p< 0.05).
bReference compounds.
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2.2.2. Acetyl- and butyryl- cholinesterase inhibitory activities of synthesized compounds
The compounds (1-19) were further screened for in vitro inhibitory activity against
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which are the chief enzymes of Alzheimer’
disease (Table 2). Galantamine, used for mild Alzheimer’ disease, was used as positive control. BChE
inhibitory effects of the newly synthesized compounds were better than the AChE inhibitory effect. In BChE
assay, compound 11 (IC50: 35.30±1.11 µM) was a leader compound among the others as it exhibited better
activity than galantamine. In addition, in the same assay, compounds 14, 19, 10 and 1 competed with the
galantamine. Compounds 3, 4, 5, 8, 9, 12, 15, 16, and 17 have also considerable inhibitory values since their
IC50 values were lower than 100 µM. In AChE assay, only the IC50 values of compounds 11 and 19 were
lower than 50 µM. The compounds 1, 4, 12, 13, 14, 15, 16, 17, 18 and 19 had also considerable results
since they exhibited IC50 values between 50 and 100 µM.
Table 2 Acetyl- and butyryl-cholinesterase inhibitory activities of compounds (1–19).
Anticholinesterase Assay
Anticholinesterase Assay
Compounds AChE Assay BChE Assay Compounds
AChE Assay
IC50 (µM) a
41.19±0.40
91.06±0.70
92.23±0.02
74.94±0.20
84.38±0.30
63.93±0.17
70.64±1.02
90.06±1.42
49.54±1.87
4.48±0.78
BChE Assay
IC50 (µM) a
81.76±0.63
128.18±0.66
100.60±0.08
94.82±0.42
135.60±0.46
125.34±0.27
102.04±0.47
122.54±0.23
124.43±0.44
99.74±0.30
IC50 (µM) a
69.49±1.11
113.44±0.74
82.23±1.47
76.93±0.87
91.59±0.93
155.57±0.41
103.15±1.93
95.06±1.56
99.61±0.35
65.96±1.42
IC50 (µM) a
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
19
35.30±1.11
72.81±0.39
110.07±1.02
50.10±1.76
99.14±1.34
86.82±1.62
93.90±1.77
117.29±0.78
52.38±1.40
46.03±0.14
10
Galantamineb
aValues expressed are mean ± SEM of three parallel measurements (p< 0.05).
bReference compounds.
2.2.3. Anticancer activity of synthesized compounds
At the side of the antioxidant, acetyl- and butyryl- cholinesterase inhibitor activities of the
compounds, we investigated their anticancer activities. There was no significant decrease on viability of
A549 and BJ cell lines according to the MTT assay results (supplementary material). Based on the positive
control cisplatin (IC50 64.77 µM), only compound 7 slightly inhibited viability of the A549 lung carcinoma cells.
On the other hand the viability of BJ fibroblast cells was not affected. Due to the non-cytotoxic nature of
these compounds, they may hold promise for other diseases treatment.
2.3.Computational Studies
Biological activity tests on ethyl 4-(2-benzoylhydrazono)piperidine-1-carboxylate and N'-(2,6-
diphenylpiperidin-4-ylidene)benzohydrazide derivatives have been realized by using in vitro method. It was
known that galantamine which was used in the treatment of mild Alzheimer’ disease was a positive control in
this research. So that, we performed molecular docking for galantamine by using DS 2016. Then the
compound 11 was docked with the mentioned enzymes. The detailed information about galantamine with the
enzymes was given in supplementary data. In this study, the aim of the docking was to give a prediction of
the most active compound 11- AChE and BChE complex structures using computation methods. Based on
the mentioned before, it performed to explain why compound 11 demonstrated more BChE efficient inhibition
capacity than AChE’s. It is known that the active sites of AChE includes an anionic binding site, Trp84,
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Glu199, and Phe330; an esteratic binding site that contains the catalytic triad Ser200, His440, and Glu327;
an acyl binding site, Phe288, and Phe299, which bound to the acetyl group of AChE. In addition, peripheral
anionic sites (PAS) of AChE also contained Trp279, Tyr70, Tyr121, Asp72, Glu199 and Phe290 residues of
the enzyme [24-28]. As a result of the docking, the compound 11 displayed multiple binding patterns in
catalytic active sites (CAS) and peripheral active sites (PAS) of AChE, and BChE. In the 1ACJ-11 complex,
oxygen atoms from Ser286 (2.2 Å) and Tyr334 (2.6 Å) into the CAS formed two hydrogen bonds with
nitrogen moieties of the compound 11. The compound 11 formed hydrophobic interactions (π–π and π-alkyl)
withTrp279 (4.7 Å) and Leu282 (5.2 Å) residues of the peripheral anionic sites (PAS), (Fig. 6). In addition,
molecular docking was performed with 4EY6 model which include galantamine for the compound 11. Figure
6 showed that there were three hydrogen bonds (Phe295, Tyr124), two electrostatic (His447 and Asp74),
two hydrophobic (Trp286) interactions between compound 11 and AChE. The most potent BChE inhibitor,
compound 11 showed interactions with the CAS amino acid residues such as Ala328 and Asp70, Ala277,
Pro285 in the PAS of human butyryl cholinesterase (Fig. 7). In addition, compound 11 had hydrophobic
interactions (π–sigma, alkyl, π–alkyl) with the C-H, alkyl and C-H groups of Asp70 (2.7 Å), Ala277 (4.1 Å),
and Ala328 (4.6 Å) in the enzyme. The carbonyl moiety of the compound 11 interacted with Pro285 (2.2 Å).
In addition, the phenyl moiety of the compound 11 interacted with electrostatic interaction with residue
oxygen atom of the Asp70 (4.9 Å). The possible interactions of compound 11 with the related enzymes were
summarized in Table 3.
The generated conformations (117 confs) of the compound 11 were presented in Table S3
(supplementary material). The obtained docking data (the bottom ten binding energy values of the generated
conformations of the compound 11 and the related enzyme complexes) were shown in Table S4
(supplementary material). It was mentioned that compound 11 formed more strong interactions with BChE
(BE: -56.978 kcal/mol) than AChE (BE: -0.91680 kcal/mol). These numerical values revealed why compound
11 showed more BChE efficient inhibition capacity than AChE’s. Furthermore, the compound 11 was
compared with galantamine for the enzymes (AChE and BChE) in Figure 8. The results of the docking
exhibited that the compound 11 in BChE was more appropriate than the compound 11 in AChE based on the
superimposed the galantamine. The molecular docking results showed that hydrogen bond, electrostatic
interaction and hydrophobic interaction of chloride atom of the compound 11 were mainly responsible for
more BChE efficient inhibition capacity than AChE’s with CAS and PAS of the related enzyme, respectively,
given in Fig. 7.
10

ACCEPTED MANUSCRIPT
Fig. 6. Global minimum energy binding mode of the compound 11 at the active site of AChE (1ACJ and
4EY6, respectively).
11

ACCEPTED MANUSCRIPT
Fig. 7. Global minimum energy binding mode of the compound 11 at the active site of BChE.
12

ACCEPTED MANUSCRIPT
Fig. 8. Global minimum energy binding modes of the compound 11 (blue sticks) and GNT(orange sticks) at
the active site of AChE (light blue sphere) and BChE (red sphere). The binding sites residues of the
enzymes, (1ACJ and 1POI) were displayed.
13

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Table 3 The possible interactions of the compound 11 for AChE and BChE at docking study.
From
Chem.
Name-AChE -11
Distance Category
Types
From
To
To Chem.
H-Acceptor
H-Acceptor
Pi-Orbitals
Alkyl
B11:H50 -
A:SER286:O
B11:H51 -
Hydrogen
Conventional
Hydrogen Bond
Conventional
Hydrogen Bond
A:SER2
86:O
A:TYR3
34:O
2.210
Bond
B12:H50
B12:H51
H-Donor
Hydrogen
2.655
Bond
H-Donor
A:TYR334:O
Hydrophob
ic
A:TRP279 - B11
B11 - A:LEU282
4.795
Pi-Pi T-shaped
Pi-Alkyl
A:TRP279 Pi-Orbitals
B11
Hydrophob
A:LEU2
82
5.258
ic
B12
Pi-Orbitals
Name-AChE
(4EY6) -11
A:PHE295:HN -
B11:O14
B11:H50 -
A:TYR124:OH
B11:H10 -
A:TYR124:OH
A:HIS447:NE2 -
B11
A:ASP74:OD2 -
B11
From
Chem.
Distance Category
Types
From
To
To Chem.
H-Acceptor
H-Acceptor
H-Acceptor
Pi-Orbitals
Pi-Orbitals
Pi-Orbitals
Pi-Orbitals
To Chem.
H-Acceptor
Pi-Orbitals
Pi-Orbitals
Alkyl
Hydrogen
2.64444
Conventional
Hydrogen Bond
Conventional
Hydrogen Bond
Carbon Hydrogen
Bond
A:PHE295
:HN
B11:O1
4
A:TYR1
14:OH
A:TYR1
14:OH
H-Donor
H-Donor
H-Donor
Positive
Negative
Bond
Hydrogen
Bond
2.57366
B11:H50
B11:H10
Hydrogen
Bond
2.54344
Electrostat
A:HIS447:
NE2
A:ASP74:
OD2
4.14455
ic
Pi-Cation
B11
B11
B11
B11
To
Electrostat
ic
4.36531
Pi-Anion
Hydrophob
ic
A:TRP286 - B11
A:TRP286 - B11
Name-BChE -11
5.41047
Pi-Pi Stacked
Pi-Pi Stacked
Types
A:TRP286 Pi-Orbitals
A:TRP286 Pi-Orbitals
Hydrophob
4.10229
ic
From
From
Distance Category
Chem.
A:PRO285:HA -
B11:O14
A:ASP70:OD2 -
B11
A:ASP70:HB2 -
B11
A:ALA277 -
B11:Cl49
Hydrogen
Carbon Hydrogen
Bond
A:PRO28
H-Donor
5:HA
A:ASP70:
Negative
OD2
B11:O1
4
2.159
Bond
Electrostat
ic
4.856
Pi-Anion
Pi-Sigma
Alkyl
B11
B11
Hydrophob
A:ASP70:
HB2
2.729
ic
C-H
Hydrophob
ic
B11:Cl4
9
4.137
A:ALA277 Alkyl
Hydrophob
A:ALA3
28
B11 - A:ALA328
4.646
ic
Pi-Alkyl
B11
Pi-Orbitals
Alkyl
3. Conclusion
In this research, ethyl 4-(2-benzoylhydrazono)piperidine-1-carboxylate and N'-(2,6-diphenylpiperidin-
4-ylidene)benzohydrazide derivatives were synthesized.
•
•
The antioxidant activity was studied, most of the compounds (1, 2, 3, 4, 5, 6, 8, 9, 10, 14, 16, 17)
exhibited more activity than positive controls α-Tocopherol and BHT.
The anticholinesterase assay results demonstrate that, BChE assay results were better than the
AChE assay results for the studied compounds. The compound 11 became the most prominent one
among the other compounds. It was found more active than the galantamine as a BChE inhibitor.
According to the molecular docking results, global minimum energy binding mode of compound 11
showed efficient BChE inhibition capacity due to multiple binding patterns in CAS and PAS of BChE.
For anticancer studies; the compounds did not show significant effect on the viability of A549 cells.
Compounds did not decrease the viability of the healthy BJ fibroblast cells.
•
•
•
As a conclusion, because of the non-cytotoxic properties and the good activity results, some of these
compounds can be improved for using as antioxidant agents and potent butyrylcholinesterase inhibitors.
4. Experimental
4.1.General
14

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All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Reactions were carried on
Milestone Start Synth Microwave Synthesis Labstation (Sorisole, İtaly). The reactions and the purities of the
compounds were monitored by thin layer chromatography (TLC) on silica gel 60 F254 aluminum sheets
purchased from Merck (Darmstadt, Germany). Melting points were recorded by open capillaries on EzMelt
melting point apparatus. C, H, N, S percent of the compounds were detected by Thermo Scientific Flash
2000 (Finnegan MAT, USA) elemental analyzer. FTIR spectra were recorded on Perkin Elmer Frontier
spectrometer by attenuated total reflectance (ATR) apparatus (Waltham, Massachusetts, USA). Mass
spectra were recorded on Ab-SciEx 3200 Q-Trap MSMS detector with electronspray ionization probe
(Framingham, MA, USA). NMR spectra were recorded on Brucker Avance-400 MHz spectrometer (Billerica,
MA, USA) by using DMSO-d6 or CDCl3 as a solvent and TMS as an internal standard. The absorbance was
measured for MTT assay by using a Bio-Tek Cytation 3 Cell Imaging Multi-Mode Reader (Winooski, VT,
USA). The other activity tests were done by using Molecular Devices Spectra Max PC340 microplate reader
(Sunnyvale, CA, USA). The NMR and MS spectra of the compounds 1–19 were given in the supplementary
data.
4.2.Synthesis
4.2.1. Synthesis of 2,6-diphenylpiperidine-4-one (I)
Acetone, benzaldehyde and ammonium acetate were reacted by using L-proline catalyst according
to the previously reported procedure [17].
4.2.2. Synthesis of substitutedphenyl benzoate (II)
Substituted benzoyl chloride (0,1 mol) was reacted with phenol (0.1 mol in 100 mL of 10% sodium
hydroxide solution). The crude product was washed with water and recrystallized from ethanol [12, 20].
4.2.3. Synthesis of substitutedbenzoyl hydrazides (III)
The substituted phenyl benzoate (II) (0.01 mol) was reacted with hydrazine hydrate (0.02 mol) in
methanol. The mixture was refluxed and monitored by TLC. The crude product was washed with water and
recrystallized from ethanol [12, 20].
4.2.4. Synthesis of substitutedbenzoyl hydrazones (1–19)
0.01 mol ketone (I or IV) and 0.01 mol substitutedbenzoyl hydrazide in 30 mL ethanol were heated
inside a microwave for 15 minute at 400 W and the product was obtained as reported previously [11, 12, 17].
4.2.4.1. Ethyl 4-(2-(4-chlorobenzoyl)hydrazono)piperidine-1-carboxylate (1)
Yield: 82%; white solid, m.p. 105–107°C; IR ( υ, cm-1): 3200 (N–H); 3031 (aromatic C–H); 2984, 2908
1
(aliphatic C–H); 1690 (ester C=O); 1651 (hydrazone C=O); 1637 (C=N); 1091 (C–Cl); H-NMR (400 MHz)
(DMSO-d6/TMS) δ (ppm): 1.21 (t, 3H, J1 = 6.8 Hz, J2 = 7.2 Hz, HG); 2.45–2.55 (m, 4H, HD, HE); 3.50–3.56 (m,
4H, HF, Fʹ); 4.08 (q, 2H, J = 7.2 Hz, HI); 7.56 (d, 2H, J = 8.0 Hz, HC); 7.86 (d, 2H, J = 7.6 Hz, HB); 10.81 (s, 1H,
HA); 13C-NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 162.96, 162.31 (C4, C10); 155.11 (C7); 136.65, 133,10
(C11, C14); 130.08, 128.8 (C12, C13, C15, C16); 61.37 (C8); 43.93, 42.13 (C2, C6); 34.1, 28.64 (C3, C5);
+
15.04 (C9); MS (m/z) (%): 324.8 [M+H] (8.57); 265.0 (5.71); 247.2 (21.43); 236.2 (11.43); 203.4 (100);
189.4 (41.43); 187.2 (10.00); 175.2 (12.86); 171 (10.00); 169.4 (22.86); 167 (5.71); 165.2 (5.71); 143 (10.00);
140.4 (14.29); 139 (10.00); 137.2 (5.71); 125.4 (85.71); 111.4 (60.00); 101.21 (10.00); 97.4 (27.14); 89.2
15

ACCEPTED MANUSCRIPT
(5.71); 85.2 (5.71); 79.2 (10.00); 71.4 (12.86); 70.6 (8.57); 61 (7.14); 57.4 (22.86); 56.4 (7.14); Anal. calcd.
for C15H18ClN3O3: C 55.64; H 5.60; N 12.98%; found: C 54.26; H 5.53; N 12.93%.
4.2.4.2. Ethyl 4-(2-(4-fluorobenzoyl)hydrazono)piperidine-1-carboxylate (2)
Yield: 60%; Cream solid, m.p. 120–122°C; IR ( υ, cm-1): 3200 (N–H); 3061 (aromatic C–H); 2985, 2935, 2909,
1
2869 (aliphatic C–H); 1707, 1694 (ester C=O); 1650 (hydrazone C=O); 1636 (C=N); 1158 (C–F); H-NMR
(400 MHz) (DMSO-d6/TMS) δ (ppm): 1.21 (t, 3H, J = 7.2 Hz, HG); 2.45–2.55 (m, 4H, HD, HE); 3.50–3.56 (m,
4H, HF); 4.08 (q, 2H, J1 = 6.8 Hz, J2 = 7.2 Hz, HI); 7.32 (t, 2H, J = 8.8 Hz, HC); 7.91 (s, 2H, HB); 10.75 (s, 1H,
HA); 13C-NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 165.67 (C14); 162.95, 161.98 (C4, C10); 155.10 (C7);
130.87 (C11); 115.75, 115.53 (C12, C13, C15, C16); 61.35 (C8); 43.95, 42.14 (C2, C6); 34.10, 28.63 (C3,
C5); 15.04 (C9); MS (m/z) (%): 308.8 [M+H] + (1.51); 231.4 (27.78); 217.4 (8.33); 199.4 (8.08); 171.4 (6.57);
153.4 (55.55); 151.4 (6.06); 139.4 (12.62); 121.4 (23.23); 119.4 (5.30); 105.6 (5.81); 93.4 (7.83); 76.6
(17.68); 75.4 (100); 72.6 (7.07); 71.6 (20.45); 61.4 (18.18); 57.4 (6.31); Anal. calcd. for C15H18FN3O3: C
58.62; H 5.90; N 13.67%; found: C 58.24; H 5.90; N 13.32%.
4.2.4.3. Ethyl 4-(2-(4-methoxybenzoyl)hydrazono)piperidine-1-carboxylate (3)
Yield: 61 %; White solid, m.p. 117–119 °C; IR ( υ, cm-1): 3211 (N–H); 3060 (aromatic C–H); 2984, 2936,
1
2905 (aliphatic C–H); 1695 (ester C=O); 1638 (C=N); 1662 (hydrazone C=O); H-NMR (400 MHz) (DMSO-
d6/TMS) δ (ppm): 1.21 (t, 3H, J = 7.2 Hz, HG); 2.44–2.54 (m, 4H, HD, HE); 3.50–3.57 (m, 4H, HF); 4.08 (q,
2H, J = 7.2 Hz, HI); 7.01 (d, 2H, J = 8.8 Hz, HC); 7.83 (d, 2H, J = 8.4 Hz, HB); 10.59 (s, 1H, HA); 13C-NMR
(100 MHz) (DMSO-d6/TMS) δ (ppm): 162.93, 158.33 (C4, C10, C14); 155.03 (C7); 131.21, 130.14, 114.60
(C11, C12, C13, C15, C16); 61.32 (C8); 56.10 (C17); 43.68, 41.76 (C2, C6); 33.49, 28.02 (C3, C5); 15.02
–
(C9); MS (m/z) (%): 318.4 [M-H] (100); 272.4 (42.45); 229.4 (20.76); 217.6 (42.45); 213.0 (5.66); 201.4
(22.64); 174 (7.55); 173.6 (10.38); 159.2 (5.66); 159.0 (5.66); 120.0 (7.55); 108.2 (10.38); 95.0 (5.66); 93.4
(5.66); 92.4 (22.64); 81.4 (14.15); 42.4 (81.14); Anal. calcd. For C16H21FN3O4: C 60.17; H 6.63; N 13.16 %;
found: C 59.89; H 6.48; N 12.96 %.
4.2.4.4. Ethyl 4-(2-(4-methylbenzoyl)hydrazono)piperidine-1-carboxylate (4)
Yield: 68%; Cream solid, m.p. 120–122°C; IR ( υ, cm-1): 3199 (N–H); 3059 (aromatic C–H); 2976, 2907, 2868
1
(aliphatic C–H); 1698 (ester C=O); 1634 (C=N, hydrazone C=O); H-NMR (400 MHz) (DMSO-d6/TMS) δ
(ppm): 1.21 (t, 3H, J = 7.2 Hz, HG); 2.37 (s, 3H, HJ); 2.45–2.55 (m, 4H, HD, HE); 3.50–3.56 (m, 4H, HF); 4.08
(q, 2H, J1 = 6.8 Hz, J2 = 7.2 Hz, HI); 7.29 (d, 2H, J = 7.6 Hz, HC); 7.75 (d, 2H, J = 7.6 Hz, HB); 10.66 (s, 1H,
HA); 13C-NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 163.76, 161.50 (C4, C10); 155.06 (C7); 141.75, 131.49
(C11, C14); 129.20, 128.12 (C12, C13, C15, C16); 61.32 (C8); 43.94, 42.13 (C2, C6); 34.08, 28.55 (C3,
C5); 21.43 (C17); 15.03 (C9); MS (m/z) (%): 304.8 [M+H] + 228.8 (22.22); 213.4 (12.22); 195.4 (13.11); 167.4
(8.88); 149.6 (46.86); 135.4 (28.47); 117.8 (38.41); 101.8 (9.60); 89.4 (12.53); 72.6 (31.32); 71.6 (100); 57.6
(25.49); Anal. calcd. for C16H21N3O3: C 63.35; H 6.98; N 13.85%; found: C 63.01; H 6.85; N 13.67%.
4.2.4.5. Ethyl 4-(2-(4-(trifluoromethyl)benzoyl)hydrazono)piperidine-1-carboxylate (5)
Yield: 55 %; White solid, m.p. 116–118 °C; IR ( υ, cm-1): 3166 (N–H); 3063 (aromatic C–H); 2987, 2914, 2871
(aliphatic C–H); 1683 (ester C=O); 1658 (hydrazone C=O); 1645 (C=N); 1H-NMR (400 MHz) (DMSO-
d6/TMS) δ (ppm): 1.21 (t, 3H, J = 7.2 Hz, HG); 2.47 (t, 2H, J1 = 6.0 Hz, J2 = 5.6 Hz, HE); 2.55 (t, 2H, J = 5.6
Hz, HD); 3.53 (t, 2H, J = 6.0 Hz, HFʹ); 3.57 (t, 2H, J = 5.6 Hz, HF); 4.08 (q, 2H, J1 = 6.8 Hz, J2 = 7.2 Hz, HI);
7.87 (d, 2H, J = 8.0 Hz, HB); 8.03 (d, 2H, J = 8.0 Hz, HC); 10.95 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-
16

ACCEPTED MANUSCRIPT
d6/TMS) δ (ppm): 162.94, 162.65 (C4, C10); 155.08 (C7);138.29, 131.84, 129.81, 129.09, 125.74, 123.03,
115.67 (C11, C12, C13, C14, C15, C16, C17); 61.36 (C8); 43.92, 42.14 (C2, C6); 34.15, 28.70 (C3, C5);
15.04 (C9); MS (m/z) (%): 356.4 [M-H] – (100); 310.4 (23.81); 267.4 (26.10); 255.4 (45.91); 237.4 (15.05);
145.4 (24.76); 109.4 (5.52); 93.4 (6.86); 81.4 (8.38); 42.4 (59.43); Anal. calcd. For C16H18F3N3O3: C 53.78; H
5.08; N 11.76 %; found: C 53.37; H 5.34; N 11.39 %.
4.2.4.6. Ethyl 4-(2-(4-nitrobenzoyl)hydrazono)piperidine-1-carboxylate (6)
Yield: 56 %; Light yellow solid, m.p. 112–114°C; IR (υ, cm-1): 3198 (N–H); 3040 (aromatic C–H); 2987, 2918,
1
2872, 2829 (aliphatic C–H); 1679 (ester C=O); 1645 (C=N); 1663 (hydrazone C=O); 1516, 1343 (NO2); H-
NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 1.21 (t, 3H, HG); 2.47–2.57 (m, 4H, HD, HE); 3.51–3.57 (m, 4H,
HI, HJ); 4.08 (q, 2H, HF); 8.06 (d, 2H, HB); 8.33 (d, 2H, HC); 11.05 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-
d6/TMS) δ (ppm): 163.21, 162.40 (C4, C10); 155.09 (C7); 149.51, 140.18 (C11, C14); 129.70, 123.87 (C12,
C13, C15, C16); 61.37 (C8); 43.91, 42.15 (C2, C6); 34.15, 28.75 (C3, C5); 15.05 (C9); MS (m/z) (%): 333.4
[M-H] – (100); 287.4 (19.13); 244.4 (25.65); 232.4 (46.52); 214.4 (9.13); 122.4 (38.26); 109.4 (4.78); 93.4
(8.26); 81.4 (6.52); 46.4 (29.57); 42.6 (36.96); Anal. calcd. for C15H18N4O5: C 53.89; H 5.43; N 16.76%;
found: C 53.75; H 5.74; N 16.69%.
4.2.4.7. Ethyl 4-(2-(2,4-dinitrophenyl)hydrazono)piperidine-1-carboxylate (7)
Yield: 62%; yellow solid, m.p. 196–198°C; IR ( υ, cm-1): 3313(N–H); 3089 (aromatic C–H); 3000, 2978, 2931,
1
2880 (aliphatic C–H); 1686 (ester C=O); 1615 (C=N); 1517, 1335 (NO2); H-NMR (400 MHz) (DMSO-
d6/TMS) δ (ppm): 1.22 (t, 3H, J = 7.2 Hz, HG); 2.59 (t, 2H, J = 6.0 Hz, HE); 2.64 (t, 2H, J = 6.0 Hz, HD); 3.58
(t, 2H, J = 5.6 Hz, HF’); 3.63 (t, 2H, J = 6.0 Hz, HF); 4.08 (q, 2H, J = 7.2 Hz, HI); 7.86 (d, 1H, J = 9.6 Hz, HB);
8.38 (dd, 1H, J1 = 2.8 Hz, J2 = 9.6 Hz, HC,); 8.86 (d, 1H, J = 2.8 Hz, HJ); 10.92 (s, 1H, HA); 13C-NMR (100
MHz) (DMSO-d6/TMS) δ (ppm): 159.32 (C4); 155.09 (C7); 145.18, 137.32, 130.57, 129.65, 123.53, 116.41
(C11, C12, C13, C14, C15, C16); 61.37 (C8); 43.91, 42.15 (C2, C6); 34.15, 28.75 (C3, C5); 15.05 (C9); MS
+
(m/z) (%): 352.2 [M+H] 309.0 (8.33); 275.4 (13.33); 231.4 (90.00); 230.2 (30.00); 229.2 (21.67); 228.4
(8.33); 228.0 (13.33); 217.2 (20.00); 215.4 (10.00); 214.2 (8.33); 199.4 (26.67); 197.6 (45.00); 171.2 (10.00);
170.0 (5.00); 153.4 (53.30); 152.2 (11.67); 151.4 (23.33); 150.4 (13.33); 149.4 (25.00); 139.4 (28.3); 137.4
(16.67); 135.6 (11.67); 121.4 (43.3); 119.6 (46.7); 105.4 (10.00); 104.2 (10.00); 93.4 (10.00); 91.4 (10.00);
89.6 (10.00); 76.6 (18.30);75.6 (100); 73.4 (21.67); 72.4 (40.00); 71.6 (90.00); 61.4 (23.30); 60.6 (10.00);57.4
(16.7); Anal. calcd. for C14H17N5O6: C 47.86; H 4.88; N 19.93%; found: C 48.05; H 4.84; N 19.59%.
4.2.4.8. Ethyl 4-(2-(2-fluoro-4-(trifluoromethyl)benzoyl)hydrazono)piperidine-1-carboxylate (8)
Yield: 69 %; White solid, m.p. 148–150 °C; IR ( υ, cm-1): 3264(N–H); 3102 (aromatic C–H); 2983, 2911, 2870
1
(aliphatic C–H); 1693 (ester C=O); 1634 (C=N); 1652 (hydrazone C=O); 1151 (C–F); H-NMR (400 MHz)
(DMSO-d6/TMS) δ (ppm): 1.17–1.22 (m, 3H, HG); 2.16 (t, 1H, HE); 2.44–2.57 (m, 3H, HD, HEʹ); 3.47–3.56 (m,
4H, HF, Fʹ); 4.04–4.10 (m, 2H, HI); 7.64–7.86 (m, 3H, HB, C, J); 10.98 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-
d6/TMS) δ (ppm): 166.91 (C12); 160.67, 160.01 (C4, C10); 155.08 (C7); 131.87, 130.94, 128.35, 128.18,
121.88, 114.29, 114.03 (C11, C13, C14, C15, C16, C17); 61.37 (C8); 43.80, 41.92 (C2, C6); 33.86, 28.40
(C3, C5); 15.04 (C9); MS (m/z) (%): 374.4 [M-H] – (64.81); 354.2 (10); 328.2 (5.56); 265.4 (19.91); 253.4
(99.54); 251.2 (6.48); 201.4 (51.39); 200.2 (83.8); 163.4 (22.69); 145.4 (14.82); 143.4 (60.65); 123.4 (7.87);
117.2 (10.65); 93.4 (25.46); 73.4 (12.04); 42.4 (46.3); Anal. calcd. for C16H17F4N3O3: C 51.20; H 4.57; N
11.20 %; found: C 51.16; H 5.08; N 11.08 %.
17

ACCEPTED MANUSCRIPT
4.2.4.9. Ethyl 4-(2-(4-fluoro-2-(trifluoromethyl)benzoyl)hydrazono)piperidine-1-carboxylate (9)
Yield: 52 %; White solid, m.p. 132–134 °C; IR ( υ, cm-1): 3204(N–H); 3054 (aromatic C–H); 2994, 2975, 2920,
1
2856 (aliphatic C–H); 1705 (ester C=O); 1631 (C=N); 1654 (hydrazone C=O); 1150 (C–F); H-NMR (400
MHz) (DMSO-d6/TMS) δ (ppm): 1.17–1.22 (m, 3H, HG); 2.11 (t, 1H, J = 6.0 Hz, HE); 2.43–2.55 (m, 3H, HD, Eʹ);
3.38–3.57 (m, 4H, HF, Fʹ); 4.02–4.10 (m, 2H, HI); 7.50–7.62 (m, 1H, HC); 7.63–7.72 (m, 1H, HJ); 7.74–7.77
(dd, 1H, HB); 10.94 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 169.42 (C14); 162.99, 160.40
(C4, C10); 155.09 (C7); 132.43, 132.34, 132.05, 131.56, 131.47, 119.87, 119.63 (C11, C12, C13, C15, C16,
–
C17); 61.34 (C8); 43.79, 41.89 (C2, C6); 33.76, 28.30 (C3, C5); 15.03 (C9); MS (m/z) (%): 374.2 [M-H]
(100); 328.2 (21.2); 285.2 (15.89); 273.4 (24.8); 253.2 (18.36); 163.4 (65.21); 143.4 (7.95); 123.4 (13.56);
93.4 (6.71); 73.4 (6.57); 69.4 (14.93); 42.4 (54.25); Anal. calcd. for C16H17F4N3O3: C 51.20; H 4.57; N 11.20
%; found: C 50.97; H 4.64; N 11.12 %.
4.2.4.10. N'-(2,6-diphenylpiperidin-4-ylidene)benzohydrazide (10)[29]
Yield: 51%; Light yellow solid, m.p. 216–218°C; IR (υ, cm-1): 3308 ( seconder amine N–H); 3188 (hydrazone
N–H); 3064, 3032 (aromatic C–H); 2965, 2900, 2845, 2803 (aliphatic C–H); 1646 (hydrazone C=O, C=N);
1H-NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.08 (t, 1H, J1 = 12.4 Hz, J2 = 13.2 Hz, HE); 2.43 (t, 1H, J1 =
12.0 Hz, J2 = 12.8 Hz, HEʹ); 2.61 (d, 1H, J = 12.8 Hz, HDʹ); 2.88 (s, 1H, HG); 3.18 (d, 1H, J = 13.2 Hz, HD); );
3.88 (d, 1H, J = 10.8 Hz, HF); 3.96 (d, 1H, J = 10.8 Hz, HFʹ); 7.26–7.30 (m, 2H, HK, Kʹ); 7.34–7.39 (m, 4H, HI,Iʹ);
7.46–7.55 (m, 7H, HC, HJ, Jʹ, HL); 7.83 (d, 2H, J = 7.2 Hz, HB); 10.86 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-
d6/TMS) δ (ppm): 164.21, 162.49 (C4, C13); 144.57, 144.51 (C7); 134.51, 131.75, 128.78, 128.69, 128.19,
127.61, 127.31, 127.10 (C8, C9, C10, C11, C12, C14, C15, C16, C17, C18, C19, C20); 61.46, 60.20 (C2,
C6); 43.94, 37.37 (C3, C5); MS (m/z) (%): 368.4 [M-H] – (100); 263.6 (59.92); 245.4 (12.06); 235.2 (7.78);
232.4 (15.95); 185.4 (8.56); 157.4 (5.06); 144.4 (5.45); 135.4 (5.45); 120.4 (8.17); 42.4 (21.4); Anal. calcd.
for C24H23N3O: C 78.02; H 6.27; N 11.37%; found: C 77.67; H 6.28; N 11.34%.
4.2.4.11. N'-(2,6-diphenylpiperidin-4-ylidene)-4-chlorobenzohydrazide (11)
Yield: 64%; Yellow solid, m.p. 226–228°C; IR ( υ, cm-1): 3291 (seconder amine N–H); 3174 (hydrazone N–H);
3065, 3030 (aromatic C–H); 2961, 2899, 2795, 2737 (aliphatic C–H); 1646 (hydrazone C=O, C=N); 1084 (C–
Cl); 1H-NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.08 (t, 1H, J1 = 12.0 Hz, J2 = 13.6 Hz, HE); 2.43 (t, 1H, J1
= 12.0 Hz, J2 = 13.2 Hz, HEʹ); 2.60 (d, 1H, J = 12.0 Hz HDʹ); 2.88 (s, 1H, HG); 3.18 (d, 1H, J = 12.0 Hz, HD);
3.87 (d, 1H, J = 10.8 Hz, HF); 3.95 (d, 1H, J = 10.8 Hz, HFʹ); 7.28–7.30 (m, 2H, HK, Kʹ); 7.35–7.38 (m, 4H, HI,Iʹ);
7.53–7.56 (m, 6H, HC, HJ, Jʹ); 7.86 (d, 2H, J = 8.0 Hz, HB); 10.89 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-
d6/TMS) δ (ppm): 163.16, 162.86 (C4, C13); 144.53, 144.48 (C7); 130.15, 129.33, 128.77, 128.68, 127.62,
127.31, 127.10 (C8, C9, C10, C11, C12, C14, C15, C16, C17, C18, C19); 61.44, 60.21 (C2, C6); 43.89,
37.38 (C3, C5); MS (m/z) (%): 402.4 [M-H] – (100); 297.4 (65.09); 279.2 (16.57); 232.4 (14.2); 218.8 (6.51);
178.2 (7.1); 169.2 (8.28); 154.4 (8.28); 111.2 (7.69); 42.4 (20.12); Anal. calcd. for C24H22ClN3O: C 71.37; H
5.49; N 10.4%; found: C 71.5; H 5.18; N 9.96%.
4.2.4.12. N'-(2,6-diphenylpiperidin-4-ylidene)-4-fluorobenzohydrazide (12)
Yield: 50%; Light yellow solid, m.p. 159–160°C; IR (υ, cm-1): 3309 (seconder amine N–H); 3179 (hydrazone
N–H); 3064, 3032 (aromatic C–H); 2970, 2902, 2842 (aliphatic C–H); (); 1646 (hydrazone C=O, C=N); 1155
(C–F); 1H-NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.08 (t, 1H, J = 12.0 Hz, HE); 2.43 (t, 1H, J1 = 12.0 Hz,
J2 = 12.4 Hz, HEʹ); 2.58–2.61 (m, 1H, HDʹ); 2.88 (s, 1H, HG); 3.16 (d, 1H, J = 13.2 Hz, HD); 3.87 (d, 1H, J =
18

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11.2 Hz, HF); 3.96 (d, 1H, J = 11.2 Hz, HFʹ); 7.28–7.39 (m, 8H, HI, Iʹ K, Kʹ, C); 7.49–7.54 (m, 4H, HJ, HJʹ,); 7.88–
7.92 (m, 2H, HB); 10.83 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 163.11, 162.68 (C4,
C13); 144.53, 144.47 (C7); 130.90, 128.79, 128.77, 128.69, 127.68, 127.61, 127.30, 127.10, 115.74, 115.51
(C8, C9, C10, C11, C12, C14, C15, C16, C18, C19); 61.44, 60.53 (C2, C6); 43.87, 37.35 (C3, C5); MS (m/z)
(%): 388.6 [M+H] + (15.77); 283.6 (26.00); 194.8 (100); 167.6 (21.48); 165.6 (13.93); 152.6 (12.42); 144.6
(7.05); 123.4 (68.46); 116.6 (20.47); 103.6 (17.45); 95.4 (23.49); 91.4 (10.24); 89.6 (8.73); 77.6 (10.07); 75.6
(18.46); 71.6 (5.54); 65.6 (10.74); ; Anal. calcd. for C24H22FN3O: C 78.4; H 5.72; N 10.85%; found: C 78.17;
H 5.6; N 10.53%.
4.2.4.13. N'-(2,6-diphenylpiperidin-4-ylidene)-4-methoxybenzohydrazide (13)
Yield: 50 %; White solid, m.p. 151–153 °C; IR ( υ, cm-1): 3319 (seconder amine N–H); 3371 (hydrazone N–H);
3059, 3026 (aromatic C–H); 2978, 2911, 2841, 2819 (aliphatic C–H); 1629 (C=N); 1643(hydrazone C=O);
1H-NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.06 (t, 1H, J1 = 13.2 Hz, J2 = 12.4 Hz, HE); 2.41 (t, 1H, J =
12.4 Hz HEʹ); 2.59 (d, 1H, J = 12.8 Hz, HDʹ); 2.88 (s, 1H, HG); 3.16 (d, 1H, J = 13.2 Hz, HD); 3.81 (s, 3H, HL);
3.86 (d, 1H, J = 11.6 Hz, HF); 3.95 (d, 1H, J = 10.8 Hz, HFʹ); 7.01 (d, 2H, J = 8.8 Hz, HC); 7.28–7.30 (m, 2H,
H
K, Kʹ); 7.34–7.39 (m, 4H, HI,Iʹ); 7.52–7.55 (m, 4H, HJ, Jʹ); 7.83 (d, 2H, J = 8.8 Hz, HB); 10.71 (s, 1H, HA); 13C-
NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 163.62, 162.14, 161.89 (C4, C13, C17); 144.59 (C7); 130.08,
128.78, 128.69, 127.60, 127.30, 127.10, 126.53, 113.92 (C8, C9, C10, C11, C12, C14, C15, C16, C18,
C19); 61.46, 60.20 (C2, C6); 55.83 (C20); 43.95, 37.39 (C3, C5); MS (m/z) (%): 398.4 [M-H] – (100); 293.4
(54.9); 232.4 (18.63); 231.4 (7.84); 174.2 (5.88); 150.4 (8.82); 120.2 (10.78); 119.8 (6.86); 108.4 (15.69);
97.2 (8.82); 96.6 (6.86); 96.2 (5.88); 95.2 (8.82); 93.4 (7.84); 92.4 (13.73); 81.2 (6.86); 42.4 (14.71); Anal.
calcd. for C25H25N3O2: C 75.16; H 6.31; N 10.52 %; found: C 74.85; H 6.47; N 10.19 %.
4.2.4.14. N'-(2,6-diphenylpiperidin-4-ylidene)-4-methylbenzohydrazide (14)
Yield: 54%; Light yellow solid, m.p. 190–193 °C; IR (υ, cm-1): 3285 (seconder amine N–H); 3171 (hydrazone
N–H); 3059, 3032 (aromatic C–H); 2963, 2899, 2851 (aliphatic C–H); 1644 (hydrazone C=O, C=N); 1H-NMR
(400 MHz) (DMSO-d6/TMS) δ (ppm): 2.07 (t, 1H, J1 = 12.4 Hz, J2 = 13.2 Hz, HE); 2.36 (s, 3H, HL); 2.42 (t, 1H,
J1 = 12.4 Hz, J2 = 13.2 Hz, HEʹ); 2.59 (d, 1H, J =12.4 Hz, HDʹ); 2.88 (s, 1H, HG); 3.16 (d, 1H, J = 13.6 Hz, HD);
3.87 (d, 1H, J = 11.2 Hz, HF); 3.95 (d, 1H, J = 10.8 Hz, HFʹ); 7.27–7.30 (m, 4H, HC, K, Kʹ); 7.34–7.39 (m, 4H,
HI,Iʹ); 7.54 (d, 4H, HJ, Jʹ); 7.74 (d, 2H, J = 8.0 Hz, HB); 10.77 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-d6/TMS)
δ (ppm): 163.99, 162.28 (C4, C13); 144.60 (C7); 141.70, 131.64, 129.21, 128.77, 128.68, 128.20, 127.60,
127.30, 127.10 (C8, C9, C10, C11, C12, C14, C15, C16, C17, C18, C19); 61.46, 60.20 (C2, C6); 43.93,
37.37 (C3, C5); 21.46 (C20); MS (m/z) (%): 382.6 [M-H] - (100); 277.4 (63.42); 259.4 (14.63); 249.2 (8.54);
232.4 (24.39); 199.4 (6.1); 194.2 (6.1); 158.2 (7.32); 149.4 (10.98); 134.4 (8.54); 42.4 (15.85); Anal. calcd.
for C25H25N3O: C 78.3; H 6.57; N 10.96%; found: C 77.97; H 6.52; N 10.75%.
4.2.4.15. N'-(2,6-diphenylpiperidin-4-ylidene)-4-(trifluoromethyl)benzohydrazide (15)
Yield: 63 %; White solid, m.p. 188–190 °C; IR ( υ, cm-1): 3305 (seconder amine N–H); 3185 (hydrazone N–H);
3065, 3033 (aromatic C–H); 2959, 2911, 2845, 2823 (aliphatic C–H); 1659 (hydrazone C=O, C=N); 1H-NMR
(400 MHz) (DMSO-d6/TMS) δ (ppm): 2.10 (t, 1H, J1 = 12.4 Hz, J2 = 13.2 Hz, HE); 2.45 (t, 1H, J1 = 12.0 Hz, J2
= 12.8 Hz, HEʹ); 2.61 (d, 1H, J = 13.4 Hz, HDʹ); 2.89 (s, 1H, HG); 3.19 (d, 1H, J = 13.6 Hz, HD); 3.88 (d, 1H, J =
11.12 Hz HF); 3.96 (d, 1H, J = 10.8 Hz HFʹ); 7.26–7.30(m, 2H, HK, Kʹ); 7.34–7.39(m, 4H, HI, Iʹ); 7.54 (d, 4H, J =
7.2 Hz, HJ, Jʹ); 7.85 (d, 2H, J = 8.0 Hz, HB); 8.02 (d, 2H, J = 8.0 Hz, HC); 11.03 (s, 1H, HA); 13C-NMR (100
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MHz) (DMSO-d6/TMS) δ (ppm): 163.16 (C4, C13); 144.50 (C7); 129.15, 128.78, 128.69, 127.63, 127.33,
127.11, 125.71 (C8, C9, C10, C11, C12, C14, C15, C16, C17, C18, C19, C20); 61.43, 60.21 (C2, C6); 43.92,
37.38 (C3, C5); MS (m/z) (%): 436.6 [M-H] – (100); 331.4 (56.71); 313.4 (16.71); 303.4 (5.75); 253.4 (6.85);
203.4 (7.40); 185.4 (5.75); 145.4 (21.92); 42.4 (17.81); Anal. calcd. for C25H22F3N3O: C 68.64; H 5.07; N 9.61
%; found: C 68.29; H 5.31; N 9.21 %.
4.2.4.16. N'-(2,6-diphenylpiperidin-4-ylidene)-4-nitrobenzohydrazide (16)
Yield: 65%; Orange solid, m.p. 208–210°C; IR ( υ, cm-1): 3294 (seconder amine N–H); 3184 (hydrazone N–
H); 3085, 3032 (aromatic C–H); 2959, 2911, 2849, 2826 (aliphatic C–H); 1652 (hydrazone C=O, C=N); 1522,
1342 (NO2); 1H-NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.12 (t, 1H, J1 = 12.4 Hz, J2 = 13.6 Hz, HE); 2.46
(t, 1H, J1 = 12.0 Hz, J2 = 12.8 Hz, HEʹ); 2.63 (d, 1H, J = 12.8 Hz, HDʹ); 2.89 (s, 1H, HG); 3.20 (d, 1H, J = 13.6
Hz, HD); 3.88 (d, 1H, J = 10.4 Hz, HF); 3.97 (d, 1H, J = 10.4 Hz, HFʹ); 7.28–7.38(m, 6H, HK, Kʹ, I, Iʹ); 7.54 (d, 4H,
J = 7.2 Hz, HJ, Jʹ); 8.07 (d, 2H, J = 8.4 Hz, HB); 8.31 (d, 2H, J = 8.8 Hz, HC); 9.90 (s, 1H, HA); 13C-NMR (100
MHz) (DMSO-d6/TMS) δ (ppm): 163.83 (C4, C13); 149.45 (C17); 144.33 (C7); 143.83, 129.72, 129.64,
129.37, 129.18, 128.91, 128.80, 128.71, 127.69, 127.32, 127.09, 124.12, 124.00, 123.86 (C8, C9, C10, C11,
C12, C14, C15, C16, C17, C18, C19); 61.42, 60.36 (C2, C6); 43.76, 37.33 (C3, C5); MS (m/z) (%): 413.6 [M-
H] – (100); 308.4 (51.22); 290.4 (12.20); 185.4 (6.23); 180.4 (7.05); 122.4 (33.6); 46.4 (33.6); 42.4 (13.55);
Anal. calcd. for C24H22N4O: C 69.55; H 5.35; N 13.52%; found: C 69.17; H 5.04; N 13.16%.
4.2.4.17. 1-(2,4-Dinitrophenyl)-2-(2,6-diphenylpiperidin-4-ylidene)hydrazine (17)
Yield: 49%; Yellow solid, m.p. 194–196°C; IR ( υ, cm-1): 3302 (piperidine N–H); 3324 (hydrazone N–H); 3096
1
(aromatic C–H); 2969, 2906, 2821 (aliphatic C–H); 1613 (C=N); 1519, 1332 (NO2); H-NMR (400 MHz)
(DMSO-d6/TMS) δ (ppm): 2.33–2.39 (m, 1H, HE); 2.54–2.57 (m, 1H, HEʹ); 2.69 (d, 1H, J = 12.4 Hz, HDʹ);
2.97–3.00 (m, 2H, HD, HG); 3.95–.01 (m, 2H, HF, Fʹ); 7.31–7.34 (m, 2H, HK, Kʹ); 7.36–7.42 (m, 4H, HI,Iʹ); 7.54–
7.57 (m, 4H, HJ, Jʹ); 7.90 (d, 1H, J = 9.6 Hz, HB); 8.39 (dd, 1H, J1 = 9.6 Hz, J2 = 2.8 Hz HC); 8.86 (d, 1H, J =
2.8 Hz, HL); 11.05 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 160.37 (C4); 145.21, 144.16
(C7); 137.17, 130.51, 129.59, 128.86, 128.80, 127.87, 127.71, 127.23, 127.12, 123.53, 116.39 (C8, C9, C10,
C11, C12, C14, C15, C16, C17, C18, C19); 61.26, 60.07 (C2, C6); 43.64, 36.57 (C3, C5); MS (m/z) (%):
432.6 [M+H] + (13.85); 232.6 (6.33); 154.4 (5.67); 129.6 (5.13); 128.6 (6.76); 115.6 (8.18); 106.8 (100); 105.2
(6.87); 104.6 (12.54); 103.6 (30.75); 91.6 (12.98); 79.6 (22.79); 78.2 (7.63); 77.6 (24.3); Anal. calcd. for
C23H21N5O4: C 64.03; H 4.91; N 16.23%; found: C 64.01; H 4.88; N 15.97%.
4.2.4.18. N'-(2,6-diphenylpiperidin-4-ylidene)-2-fluoro-4-(trifluoromethyl)benzohydrazide (18)
Yield: 45 %; White solid, m.p. 205–207 °C; IR ( υ, cm-1): 3300 (seconder amine N–H); 3178 (hydrazone N–H);
3085, 3063 (aromatic C–H); 2958, 2898, 2804 (aliphatic C–H); 1659 (hydrazone C=O); 1632 (C=N); 1153
(C–F); 1H-NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 2.08 (t, 1H, J1 = 12.8 Hz, J2 = 13.2 Hz, HE); 2.43 (t, 1H,
J1 = 12.4 Hz, J2 = 12.8 Hz, HEʹ); 2.58 (d, 1H, J = 12.8 Hz, HDʹ); 2.91 (s, 1H, HG); 3.18(d, 1H, J = 13.6 Hz, HD);
3.85 (d, 1H, J = 12.0 Hz, HF); 3.96(d, 1H, J = 11.6 Hz, HFʹ); 7.24–7.31 (m, 2H, HK, Kʹ); 7.33–7.45 (m, 4H, HI, Iʹ);
7.52–7.56 (m, 4H, HJ, Jʹ); 7.65–7.85 (m, 3H, HB, C, L); 11.04 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-d6/TMS)
δ (ppm): 166.83 (C15); 161.44 (C4); 160.05 (C13); 144.34, 144.19 (C7); 158.48, 155.50, 131.84, 128.74,
128.67, 128.62, 128.60, 127.65, 127.59, 127.24, 127.18, 127.05, 121.84, 114.14, 114.00 (C8, C9, C10, C11,
C12, C14, C16, C17,C18, C19, C20); 61.42, 60.20 (C2, C6); 43.81, 36.98 (C3, C5); MS (m/z) (%): 456.4
[M+H] + (7.88); 352.0 (15.56); 247.4 (9.93); 204.6 (6.24); 194.8 (100); 191.6 (34.08); 167.8 (20.16); 165.6
20

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(12.28); 163.4 (24.56); 152.6 (14.12); 144.6 (9.83); 143.2 (6.35); 129.6 (8.60); 116.6 (21.6); 106.6 (6.45);
103.6 (33.5); 91.6 (12.39); 89.6 (7.27); 77.6(12.79); 65.6 (9.62); Anal. calcd. for C25H21F4N3O: C 65.93; H
4.65; N 9.23 %; found: C 65.57; H 5.03; N 9.22 %.
4.2.4.19. N'-(2,6-diphenylpiperidin-4-ylidene)-4-fluoro-2-(trifluoromethyl)benzohydrazide (19)
Yield: 47 %; White solid, m.p. 177–178 °C; IR ( υ, cm-1): 3295 (seconder amine N–H); 3150 (hydrazone N–H);
3061, 3033 (aromatic C–H); 2975, 2904, 2841, 2819 (aliphatic C–H); 1667 (hydrazone C=O, C=N); 1155 (C–
F); 1H-NMR (400 MHz) (DMSO-d6/TMS) δ (ppm): 1.99–2.09 (m, 1H, HE); 2.41 (t, 1H, J1 = 12.0 Hz, J2 = 12.8
Hz, HEʹ); 2.56 (d, 1H, J = 13.2 Hz, HDʹ); 2.90 (s, 1H, HG); 3.17(d, 1H, J = 13.6 Hz HD); 3.11–3.84 (m, 1H, HF);
3.95(d, 1H, J = 10.8 Hz, HFʹ); 7.23–7.42 (m, 6H, HK, Kʹ, I, Iʹ); 7.50–7.58 (m, 4H, HJ, Jʹ); 7.60–7.65 (m, 1H, HC);
7.69–7.78 (m, 2H, HB,L); 10.99 (s, 1H, HA); 13C-NMR (100 MHz) (DMSO-d6/TMS) δ (ppm): 169.45 (C17);
163.04, 161.07 (C4, C13); 144.49, 144.40 (C7); 154.85, 144.24, 132.44, 132.36, 132.20, 128.78, 128.67,
127.65, 127.61, 127.25, 127.09, 127.04, 119.92, 119.71, 114.57 (C8, C9, C10, C11, C12, C14, C15, C16,
C17, C18, C19, C20); 61.45, 60.22 (C2, C6); 43.83, 36.85 (C3, C5); MS (m/z) (%): 456.6 [M+H] + (8.83);
351.8 (14.50); 227.4 (6.88); 195.4 (100); 191.4 (43.03); 186.4 (9.76); 167.6 (22.77); 165.6 (13.38); 163.4
(33.64); 152.6 (10.41); 144.6 (7.90); 129.4 (5.58); 116.6 (20.35); 113.4 (8.08); 103.6 (21.38); 91.6 (11.25);
89.6 (7.25); Anal. calcd. for C25H21F4N3O: C 65.93; H 4.65; N 9.23 %; found: C 65.80; H 5.01; N 9.14 %.
4.3. Biological Studies
β-Carotene-linoleic acid assay.
The total antioxidant activity was evaluated using the β-carotene-linoleic acid model test system [30,
31]. DMSO was used as negative control while BHT and α-tocopherol were used as positive controls.
DPPH free radical scavenging assay.
The free radical scavenging activity was determined spectrophotometrically using the DPPH radical
commercially available [32, 33]. DMSO was used as negative control while BHT and α-tocopherol were used
as positive controls.
ABTS cation radical scavenging assay.
The spectrophotometric analysis of ABTS.+ scavenging activity was determined according to the
previously described method [34] with slight modifications [32]. DMSO was used as negative control, while
BHT and α-tocopherol were used as positive controls.
Cupric reducing antioxidant capacity (CUPRAC).
The cupric reducing antioxidant capacity was determined according to the previous method [32, 35].
DMSO was used as negative control, while BHT and α-tocopherol were used as positive controls.
Determination of acetylcholinesterase- (AChE) and butyrylcholinesterase- (BChE) inhibitory activity.
AChE and BChE inhibitory activities were measured by the spectrophotometric method using Ellman
method. AChE from electric eel and BChE from horse serum were used as enzymes [36].
Cell culture
The A549 (ATCC® CCL-185™) human lung carcinoma and BJ (ATCC® CRL-2522™) human
fibroblast cell lines were gained from American Type Culture Collection (ATCC®) and stored in 10% fetal
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bovine serum and 1% penicillin/streptomycin containing RPMI (A549) or EMEM (BJ) medium at 37°C in a
humidified incubator with 5% CO2.
Cell viability assay
Cell viability was evaluated by MTT (3, 4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide)
assay [37, 38] which was based on the reduction of the yellow MTT by the mitochondrial dehydrogenase of
intact viable or living cells to a purple formazan product. This reduction happened only when mitochondrial
reductase enzymes were active. Therefore, increased purple color was directly related to the number of
viable cells. The amount of purple formazan produced by cells treated with compounds was compared with
the amount of formazan produced by untreated control cells.
Cellular viability was detected by MTT assay as described previously [39]. In short, cells were grown
in 96-well plates at a density of 3x103 cells per well and subjected to different concentrations of the
compounds (1–19 and cisplatin) (1000, 500, 250, 125, 62,5, 31,2, 15,6 7,8, 3,9, 1,9 µM). After 24 hours
incubation, MTT solution was added to reach a final concentration of 0.5 mg/mL. The cells were incubated
for another 3 h. Then current medium was removed and 100 ꢀl of DMSO solution was added. The
absorbance was measured at 540 nm. Cell survival rates were meant as the percentage of the DMSO
(0.1%) solvent control. The IC50 values of the compounds were calculated according to these rates.
4.4. Computational Studies
Molecular docking method was performed using CDOCKER in Discovery Studio 2016 by BIOVIA to
provide an insight into the mentioned compounds. Firstly, compounds (1–19) and enzymes were prepared
using Gaussian 09 (G09) [40] and Discovery Studio (DS) 2016 [41] softwares. The compounds were drawn
and optimized at DFT/B3LYP/6-31G* level by using G09. In the meantime, their conformational analysis was
performed using DS 2016. The protein crystal structures of AChE and BChE (PDB codes: 1ACJ and 1P0I)
were selected for this study. Hydrogen and missing heavy atoms were added to the protein structure, and
atom types and partial charges were assigned. The enzymes were optimized using CHARMm forcefield and
the adopted-basis Newton-Raphson (ABNR) method [42] in the DS 2016 until the root mean square
deviation (RMSD) gradient was < 0.05 kcal/mol Å2. The active sites were determined by using define and
edit binding site tool of the DS software and literatures.
In docking analysis, the compounds were flexible and enzymes were held rigid. Dock Ligands
(CDOCKER) was performed using the default settings. The docking parameters were default form. The best
orientation of the compounds was determined based on the global minimum energy binding, CDOCKER
energy, CDOCKER interaction energy and RMSD values [43].
5. Acknowledgements
This study was supported by the Gaziantep University Scientific Research Projects Governing Unit
(BAPYB) (Project no: FEF.11.03 ), Turkiye.
6. Conflict of Interest
The Authors declare that they have no conflicts of interest to disclose.
7. References
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•
•
•
•
•
A series of benzoyl hydrazones bearing piperidine ring were synthesized.
Antioxidant, anticholinesterase and anticancer activities were studied.
Most of the compounds were found more active than the antioxidant reference standards.
The compound 11 was found more active than the galantamine as a BChE inhibitor.
Molecular docking studies confirmed the anticholinesterase assay results.

ACCEPTED MANUSCRIPT
Compound
1
InChI
1S/C15H18ClN3O3/c1-2-22-15(21)19-9-7-13(8-10-19)17-18-14(20)11-3-5-12(16)6-4-11/h3-
6H,2,7-10H2,1H3,(H,18,20)
1S/C15H18FN3O3/c1-2-22-15(21)19-9-7-13(8-10-19)17-18-14(20)11-3-5-12(16)6-4-11/h3-
6H,2,7-10H2,1H3,(H,18,20)
1S/C16H21N3O4/c1-3-23-16(21)19-10-8-13(9-11-19)17-18-15(20)12-4-6-14(22-2)7-5-12/h4-
7H,3,8-11H2,1-2H3,(H,18,20)
2
3
1S/C16H21N3O3/c1-3-22-16(21)19-10-8-14(9-11-19)17-18-15(20)13-6-4-12(2)5-7-13/h4-7H,3,8-
11H2,1-2H3,(H,18,20)
4
1S/C16H18F3N3O3/c1-2-25-15(24)22-9-7-13(8-10-22)20-21-14(23)11-3-5-12(6-4-
11)16(17,18)19/h3-6H,2,7-10H2,1H3,(H,21,23)
5
1S/C15H18N4O5/c1-2-24-15(21)18-9-7-12(8-10-18)16-17-14(20)11-3-5-13(6-4-11)19(22)23/h3-
6H,2,7-10H2,1H3,(H,17,20)
6
1S/C14H17N5O6/c1-2-25-14(20)17-7-5-10(6-8-17)15-16-12-4-3-11(18(21)22)9-
13(12)19(23)24/h3-4,9,16H,2,5-8H2,1H3
7
1S/C16H17F4N3O3/c1-2-26-15(25)23-7-5-11(6-8-23)21-22-14(24)12-4-3-10(9-
13(12)17)16(18,19)20/h3-4,9H,2,5-8H2,1H3,(H,22,24)
8
1S/C16H17F4N3O3/c1-2-26-15(25)23-7-5-11(6-8-23)21-22-14(24)12-4-3-10(9-
13(12)17)16(18,19)20/h3-4,9H,2,5-8H2,1H3,(H,22,24)
9
1S/C24H23N3O/c28-24(20-14-8-3-9-15-20)27-26-21-16-22(18-10-4-1-5-11-18)25-23(17-21)19-
12-6-2-7-13-19/h1-15,22-23,25H,16-17H2,(H,27,28)
1S/C24H22ClN3O/c25-20-13-11-19(12-14-20)24(29)28-27-21-15-22(17-7-3-1-4-8-17)26-23(16-
21)18-9-5-2-6-10-18/h1-14,22-23,26H,15-16H2,(H,28,29)
1S/C24H22FN3O/c25-20-13-11-19(12-14-20)24(29)28-27-21-15-22(17-7-3-1-4-8-17)26-23(16-
21)18-9-5-2-6-10-18/h1-14,22-23,26H,15-16H2,(H,28,29)
1S/C25H25N3O2/c1-30-22-14-12-20(13-15-22)25(29)28-27-21-16-23(18-8-4-2-5-9-18)26-24(17-
21)19-10-6-3-7-11-19/h2-15,23-24,26H,16-17H2,1H3,(H,28,29)
1S/C25H25N3O/c1-18-12-14-21(15-13-18)25(29)28-27-22-16-23(19-8-4-2-5-9-19)26-24(17-
22)20-10-6-3-7-11-20/h2-15,23-24,26H,16-17H2,1H3,(H,28,29)
1S/C25H22F3N3O/c26-25(27,28)20-13-11-19(12-14-20)24(32)31-30-21-15-22(17-7-3-1-4-8-
17)29-23(16-21)18-9-5-2-6-10-18/h1-14,22-23,29H,15-16H2,(H,31,32)
1S/C24H22N4O3/c29-24(19-11-13-21(14-12-19)28(30)31)27-26-20-15-22(17-7-3-1-4-8-17)25-
23(16-20)18-9-5-2-6-10-18/h1-14,22-23,25H,15-16H2,(H,27,29)
1S/C23H21N5O4/c29-27(30)19-11-12-20(23(15-19)28(31)32)26-25-18-13-21(16-7-3-1-4-8-
16)24-22(14-18)17-9-5-2-6-10-17/h1-12,15,21-22,24,26H,13-14H2
1S/C25H21F4N3O/c26-21-13-18(25(27,28)29)11-12-20(21)24(33)32-31-19-14-22(16-7-3-1-4-8-
16)30-23(15-19)17-9-5-2-6-10-17/h1-13,22-23,30H,14-15H2,(H,32,33)
1S/C25H21F4N3O/c26-18-11-12-20(21(13-18)25(27,28)29)24(33)32-31-19-14-22(16-7-3-1-4-8-
16)30-23(15-19)17-9-5-2-6-10-17/h1-13,22-23,30H,14-15H2,(H,32,33)
10
11
12
13
14
15
16
17
18
19