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

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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ACCEPTED MANUSCRIPT

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 Sıcak^2,3, Tuğba Taşkın-Tok¹, Mehmet Öztürk³, Ayşegül Karaküçük-İyidoğan¹,

Miris Dikmen⁴, Bedia Koçyiğit-Kaymakçıoğlu⁵, Emine Elçin Oruç-Emre^1*

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

³Department 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^-1except 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^-1and 1643–1667

cm^-1, respectively. Broad peaks at 3150–3370 cm^-1indicated N–H stretching vibrations of hydrazone; where

sharp peaks at 3285–3319 cm^-1indicated 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, H_Aprotons 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; H_Awas 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 H_Bshifted more to the downfield then H_C. This was seen also in the NOESY

and ROESY spectra of compound 1 and 11 (Fig. 1 and 2) that far interaction between H_Band H_Awas

observed. Due to the effect of strongly withdrawing groups; nitro and trifluoromethyl for the compounds 5, 6,

7, 15, 16 and 17, H_Cwas deshielded to the downfield. This effect was observed clearly for the compounds 7

and 17. When the coupling constants were analyzed, for compound 7, H_Bwas resonated as doublet by H_C;

where the H_Cwas affected by both H_Band H_Jand gives doublet of doublet at relatively low field region than

the H_B. Except from the fluorine atom containing compounds H_Band H_Cwere resonated as doublets. For the

fluorine containing compounds H_Csplitted both by the H_Band the F atom and also H_Cobserved as triplet

because of the spin number of fluorine atom (1/2). Similarly, H_Balso 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,

H_Ifor 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, H_Ahad far interaction with H_Dor H_Eand also with H_G. These interactions described by the

structure of the molecule that hydrazone group twisted towards one side of the piperidine ring. So, H_G

resonates as multiplet at 1.17–1.22 ppm for compounds 8 and 9 because of the far interactions of F atoms;

where H_Gresonated at 1.21 as triplet for the other compounds. H_Dand H_Ewere 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. H_Fand H_F'also resonated as multiplet at

3.38–3.57 ppm; but for compound 5 H_F'gave triplet at 3.53 ppm. For compound 7, H_Fand H_F'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 H_Aand H_G; H_Aand H_D; H_Aand H_B. H_Dand H_D'affected by the H_Eand H_E'and resonated

as doublets at 3.16–3.20 ppm and 2.56–2.69 ppm where H_Eand H_E'splitted into triplets by H_D,D'and H_F,F'

hydrogens at 2.06–2.37 ppm and 2.38–2.45 ppm, respectively. Similarly, H_Fand H_F'splitted into doublets by

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H_Eand H_E'at 3.85–3.89 ppm and 3.94–3.97 ppm, respectively. By the far interactions observed between H_F

and H_J, H_F'and H_J'; the chemical shifts of the H_Jand H_J'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 H_I, H_I', H_J, H_J', were clearly separated and

identified. Chemical shifts of H_I, H_I'and H_K, H_K'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 H_Bcorrelated with 128.12 ppm belonged to

C12 and C16; similarly, H_Cinteracted 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); H_I, H_I'correlated with the peaks (128.74, 128.67, 128.62, 128.60 ppm) belonged to C9

and C11; H_K, H_K'correlated with the peaks (127.65, 127.59, 127.24 ppm) belonging to C10; H_J, H_J'

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 CF₃group at 4 positions. In the CUPRAC assay, A_0.5

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

IC₅₀values 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 IC₅₀values 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

A_0.50(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-Tocopherol^b

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

IC₅₀values were lower than 100 µM. In AChE assay, only the IC₅₀values 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 IC₅₀values between 50 and 100 µM.

Table 2 Acetyl- and butyryl-cholinesterase inhibitory activities of compounds (1–19).

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

Galantamine^b

^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 (IC₅₀64.77 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

ACCEPTED MANUSCRIPT

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

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

Pi-Orbitals

To Chem.

H-Acceptor

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

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

To

Electrostat

ic

4.36531

Pi-Anion

Hydrophob

ic

A:TRP286 - B11

Name-BChE -11

5.41047

Pi-Pi Stacked

Types

A:TRP286 Pi-Orbitals

Hydrophob

4.10229

ic

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

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

ACCEPTED MANUSCRIPT

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 F₂₅₄aluminum 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-d₆or CDCl₃as 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, H_G); 2.45–2.55 (m, 4H, H_D, H_E); 3.50–3.56 (m,

4H, H_{F, Fʹ}); 4.08 (q, 2H, J = 7.2 Hz, H_I); 7.56 (d, 2H, J = 8.0 Hz, H_C); 7.86 (d, 2H, J = 7.6 Hz, H_B); 10.81 (s, 1H,

H_A); 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, H_G); 2.45–2.55 (m, 4H, H_D, H_E); 3.50–3.56 (m,

4H, H_F); 4.08 (q, 2H, J₁= 6.8 Hz, J₂= 7.2 Hz, H_I); 7.32 (t, 2H, J = 8.8 Hz, H_C); 7.91 (s, 2H, H_B); 10.75 (s, 1H,

H_A); 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, H_G); 2.44–2.54 (m, 4H, H_D, H_E); 3.50–3.57 (m, 4H, H_F); 4.08 (q,

2H, J = 7.2 Hz, H_I); 7.01 (d, 2H, J = 8.8 Hz, H_C); 7.83 (d, 2H, J = 8.4 Hz, H_B); 10.59 (s, 1H, H_A); 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, H_G); 2.37 (s, 3H, H_J); 2.45–2.55 (m, 4H, H_D, H_E); 3.50–3.56 (m, 4H, H_F); 4.08

(q, 2H, J₁= 6.8 Hz, J₂= 7.2 Hz, H_I); 7.29 (d, 2H, J = 7.6 Hz, H_C); 7.75 (d, 2H, J = 7.6 Hz, H_B); 10.66 (s, 1H,

H_A); 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, H_G); 2.47 (t, 2H, J₁= 6.0 Hz, J₂= 5.6 Hz, H_E); 2.55 (t, 2H, J = 5.6

Hz, H_D); 3.53 (t, 2H, J = 6.0 Hz, H_Fʹ); 3.57 (t, 2H, J = 5.6 Hz, H_F); 4.08 (q, 2H, J₁= 6.8 Hz, J₂= 7.2 Hz, H_I);

7.87 (d, 2H, J = 8.0 Hz, H_B); 8.03 (d, 2H, J = 8.0 Hz, H_C); 10.95 (s, 1H, H_A); 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, H_G); 2.47–2.57 (m, 4H, H_D, H_E); 3.51–3.57 (m, 4H,

H_I, H_J); 4.08 (q, 2H, H_F); 8.06 (d, 2H, H_B); 8.33 (d, 2H, H_C); 11.05 (s, 1H, H_A); 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, H_G); 2.59 (t, 2H, J = 6.0 Hz, H_E); 2.64 (t, 2H, J = 6.0 Hz, H_D); 3.58

(t, 2H, J = 5.6 Hz, H_F’); 3.63 (t, 2H, J = 6.0 Hz, H_F); 4.08 (q, 2H, J = 7.2 Hz, H_I); 7.86 (d, 1H, J = 9.6 Hz, H_B);

8.38 (dd, 1H, J₁= 2.8 Hz, J₂= 9.6 Hz, H_C,); 8.86 (d, 1H, J = 2.8 Hz, H_J); 10.92 (s, 1H, H_A); 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, H_G); 2.16 (t, 1H, H_E); 2.44–2.57 (m, 3H, H_D, H_Eʹ); 3.47–3.56 (m,

4H, H_{F, Fʹ}); 4.04–4.10 (m, 2H, H_I); 7.64–7.86 (m, 3H, H_{B, C, J}); 10.98 (s, 1H, H_A); 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, H_G); 2.11 (t, 1H, J = 6.0 Hz, H_E); 2.43–2.55 (m, 3H, H_{D, Eʹ});

3.38–3.57 (m, 4H, H_{F, Fʹ}); 4.02–4.10 (m, 2H, H_I); 7.50–7.62 (m, 1H, H_C); 7.63–7.72 (m, 1H, H_J); 7.74–7.77

(dd, 1H, H_B); 10.94 (s, 1H, H_A); 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, H_E); 2.43 (t, 1H, J₁=

12.0 Hz, J₂= 12.8 Hz, H_Eʹ); 2.61 (d, 1H, J = 12.8 Hz, H_Dʹ); 2.88 (s, 1H, H_G); 3.18 (d, 1H, J = 13.2 Hz, H_D); );

3.88 (d, 1H, J = 10.8 Hz, H_F); 3.96 (d, 1H, J = 10.8 Hz, H_Fʹ); 7.26–7.30 (m, 2H, H_{K, Kʹ}); 7.34–7.39 (m, 4H, H_I,Iʹ);

7.46–7.55 (m, 7H, H_C, H_{J, Jʹ}, H_L); 7.83 (d, 2H, J = 7.2 Hz, H_B); 10.86 (s, 1H, H_A); 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, H_E); 2.43 (t, 1H, J₁

= 12.0 Hz, J₂= 13.2 Hz, H_Eʹ); 2.60 (d, 1H, J = 12.0 Hz H_Dʹ); 2.88 (s, 1H, H_G); 3.18 (d, 1H, J = 12.0 Hz, H_D);

3.87 (d, 1H, J = 10.8 Hz, H_F); 3.95 (d, 1H, J = 10.8 Hz, H_Fʹ); 7.28–7.30 (m, 2H, H_{K, Kʹ}); 7.35–7.38 (m, 4H, H_I,Iʹ);

7.53–7.56 (m, 6H, H_C, H_{J, Jʹ}); 7.86 (d, 2H, J = 8.0 Hz, H_B); 10.89 (s, 1H, H_A); 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, H_E); 2.43 (t, 1H, J₁= 12.0 Hz,

J₂= 12.4 Hz, H_Eʹ); 2.58–2.61 (m, 1H, H_Dʹ); 2.88 (s, 1H, H_G); 3.16 (d, 1H, J = 13.2 Hz, H_D); 3.87 (d, 1H, J =

18

ACCEPTED MANUSCRIPT

11.2 Hz, H_F); 3.96 (d, 1H, J = 11.2 Hz, H_Fʹ); 7.28–7.39 (m, 8H, H_{I, Iʹ K, Kʹ, C}); 7.49–7.54 (m, 4H, H_J, H_Jʹ,); 7.88–

7.92 (m, 2H, H_B); 10.83 (s, 1H, H_A); 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, H_E); 2.41 (t, 1H, J =

12.4 Hz H_Eʹ); 2.59 (d, 1H, J = 12.8 Hz, H_Dʹ); 2.88 (s, 1H, H_G); 3.16 (d, 1H, J = 13.2 Hz, H_D); 3.81 (s, 3H, H_L);

3.86 (d, 1H, J = 11.6 Hz, H_F); 3.95 (d, 1H, J = 10.8 Hz, H_Fʹ); 7.01 (d, 2H, J = 8.8 Hz, H_C); 7.28–7.30 (m, 2H,

H

_{K, Kʹ}); 7.34–7.39 (m, 4H, H_I,Iʹ); 7.52–7.55 (m, 4H, H_{J, Jʹ}); 7.83 (d, 2H, J = 8.8 Hz, H_B); 10.71 (s, 1H, H_A); 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, H_E); 2.36 (s, 3H, H_L); 2.42 (t, 1H,

J₁= 12.4 Hz, J₂= 13.2 Hz, H_Eʹ); 2.59 (d, 1H, J =12.4 Hz, H_Dʹ); 2.88 (s, 1H, H_G); 3.16 (d, 1H, J = 13.6 Hz, H_D);

3.87 (d, 1H, J = 11.2 Hz, H_F); 3.95 (d, 1H, J = 10.8 Hz, H_Fʹ); 7.27–7.30 (m, 4H, H_{C, K, Kʹ}); 7.34–7.39 (m, 4H,

H_I,Iʹ); 7.54 (d, 4H, H_{J, Jʹ}); 7.74 (d, 2H, J = 8.0 Hz, H_B); 10.77 (s, 1H, H_A); 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, H_E); 2.45 (t, 1H, J₁= 12.0 Hz, J₂

= 12.8 Hz, H_Eʹ); 2.61 (d, 1H, J = 13.4 Hz, H_Dʹ); 2.89 (s, 1H, H_G); 3.19 (d, 1H, J = 13.6 Hz, H_D); 3.88 (d, 1H, J =

11.12 Hz H_F); 3.96 (d, 1H, J = 10.8 Hz H_Fʹ); 7.26–7.30(m, 2H, H_{K, Kʹ}); 7.34–7.39(m, 4H, H_{I, Iʹ}); 7.54 (d, 4H, J =

7.2 Hz, H_{J, Jʹ}); 7.85 (d, 2H, J = 8.0 Hz, H_B); 8.02 (d, 2H, J = 8.0 Hz, H_C); 11.03 (s, 1H, H_A); ¹³C-NMR (100

19

ACCEPTED MANUSCRIPT

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, H_E); 2.46

(t, 1H, J₁= 12.0 Hz, J₂= 12.8 Hz, H_Eʹ); 2.63 (d, 1H, J = 12.8 Hz, H_Dʹ); 2.89 (s, 1H, H_G); 3.20 (d, 1H, J = 13.6

Hz, H_D); 3.88 (d, 1H, J = 10.4 Hz, H_F); 3.97 (d, 1H, J = 10.4 Hz, H_Fʹ); 7.28–7.38(m, 6H, H_{K, Kʹ, I, Iʹ}); 7.54 (d, 4H,

J = 7.2 Hz, H_{J, Jʹ}); 8.07 (d, 2H, J = 8.4 Hz, H_B); 8.31 (d, 2H, J = 8.8 Hz, H_C); 9.90 (s, 1H, H_A); 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, H_E); 2.54–2.57 (m, 1H, H_Eʹ); 2.69 (d, 1H, J = 12.4 Hz, H_Dʹ);

2.97–3.00 (m, 2H, H_D, H_G); 3.95–.01 (m, 2H, H_{F, Fʹ}); 7.31–7.34 (m, 2H, H_{K, Kʹ}); 7.36–7.42 (m, 4H, H_I,Iʹ); 7.54–

7.57 (m, 4H, H_{J, Jʹ}); 7.90 (d, 1H, J = 9.6 Hz, H_B); 8.39 (dd, 1H, J₁= 9.6 Hz, J₂= 2.8 Hz H_C); 8.86 (d, 1H, J =

2.8 Hz, H_L); 11.05 (s, 1H, H_A); 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, H_E); 2.43 (t, 1H,

J₁= 12.4 Hz, J₂= 12.8 Hz, H_Eʹ); 2.58 (d, 1H, J = 12.8 Hz, H_Dʹ); 2.91 (s, 1H, H_G); 3.18(d, 1H, J = 13.6 Hz, H_D);

3.85 (d, 1H, J = 12.0 Hz, H_F); 3.96(d, 1H, J = 11.6 Hz, H_Fʹ); 7.24–7.31 (m, 2H, H_{K, Kʹ}); 7.33–7.45 (m, 4H, H_{I, Iʹ});

7.52–7.56 (m, 4H, H_{J, Jʹ}); 7.65–7.85 (m, 3H, H_{B, C, L}); 11.04 (s, 1H, H_A); 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

ACCEPTED MANUSCRIPT

(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, H_E); 2.41 (t, 1H, J₁= 12.0 Hz, J₂= 12.8

Hz, H_Eʹ); 2.56 (d, 1H, J = 13.2 Hz, H_Dʹ); 2.90 (s, 1H, H_G); 3.17(d, 1H, J = 13.6 Hz H_D); 3.11–3.84 (m, 1H, H_F);

3.95(d, 1H, J = 10.8 Hz, H_Fʹ); 7.23–7.42 (m, 6H, H_{K, Kʹ, I, Iʹ}); 7.50–7.58 (m, 4H, H_{J, Jʹ}); 7.60–7.65 (m, 1H, H_C);

7.69–7.78 (m, 2H, H_B,L); 10.99 (s, 1H, H_A); 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

21

ACCEPTED MANUSCRIPT

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 3x10³cells 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 IC₅₀values 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.

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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

Article Doi

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

DOI: 10.1016/j.ejmech.2016.08.037

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