Anticholinesterase activity screening of some novel dithiocarbamate derivatives including piperidine and piperazine moieties

Article abstract of DOI:10.1080/10426507.2016.1259228

The present study was undertaken to synthesize some novel lipophilic piperazine and piperidinedithiocarbamates and investigate their inhibitory potencies against cholinesterase enzymes. In the synthetic studies, 44 new compounds were isolated. The structu

Full text of DOI:10.1080/10426507.2016.1259228

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Anticholinesterase Activity Screening of some Novel Dithiocarbamate Derivatives
Including Piperidine and Piperazine Moieties
Serkan Levent1,2, Ulviye Acar Çevik1,2, Begüm Nurpelin Sağlık1,2, Yusuf Özkay1,2*, Özgür
D. Can3, Ümide Demir Özkay3, Ümit Uçucu1
1Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470,
Eskişehir, Turkey
2Anadolu University, Faculty of Pharmacy, Doping and Narcotic Compounds Analysis
Laboratory, 26470, Eskişehir, Turkey
3Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470, Eskişehir,
Turkey
*Author to whom correspondence should be addressed; E-Mail: yozkay@anadolu.edu.tr
Tel.: +90-222-335-0780 (ext. 3779); Fax: +90-222-335-0750.
Abstract
The present study was undertaken to synthesize some novel lipophilic piperazine and piperidine
dithiocarbamates and investigate their inhibitory potencies against cholinesterase enzymes. In the
synthetic studies, 44 new compounds were isolated. The structures of the synthesized compounds
were confirmed by spectroscopic analyses. Enzymatic studies were carried out using modified
Ellman’s assay against Acetylcholinesterase (AChE) and Butrylcholinesterase (BChE) enzymes,
and it was observed that some of the compounds selectively inhibit AChE. Theoretical ADME
predictions were calculated for selected compounds in the series. Enzyme kinetics and molecular
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docking studies were performed for the most active compound C41 and nature of inhibition and
interactions between enzyme and ligand were explained.
Keywords
Alzheimer’s disease; Dithiocarbamate; Piperazine; Piperidine; AChE; BChE
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Introduction
Alzheimer's disease (AD) is a chronic, irreversible, neurodegenerative disorder characterized by
a progressive deterioration of intellectual functions, including memory, language, visuospatial
skills, problem-solving ability, basic activities of daily living and ultimately causing death 1-3. It
is connected with a selective loss of cholinergic neurons, which occurs due to various
neuropathological conditions such as amyloid plaques, neurofibrillary tangles and reduced levels
4
of acetylcholine neurotransmitter . One rational way to treat the AD’s symptoms, is raising the
ACh through the inhibition of acetylcholinesterase (AChE) that is responsible for hydrolysis of
ACh in pre-synaptic areas 5,6. Many efforts have been spent in the search for potent AChE
inhibitors and four AChE inhibitors belonging to different chemical groups have been developed
for the symptomatic treatment of mild to moderate stages of AD. These are tacrine 7, donepezil 8,
rivastigmine 9 and galantamine 10, which have been approved by FDA 11, 12
.
In the development of new anticholinesterase agents, carbamate is one of the most used
moieties13, 14. The major reason for this approach is rivastigmine, a carbamate based
cholinesterase inhibitor used widely for the treatment of AD. Dithiocarbamate, an isoster of
carbamate, is also an important pharmacophore for anticholinesterase agents 15,16. Replacement
of carbamate with dithiocarbamate increases the lipophilicity, which is crucial for the delivery of
central nervous system drugs to their site of action through the blood-brain barrier 17-19
.
Functionalized piperidine scaffolds are found to form a very crucial core in many natural
20,21
products, synthetic pharmaceuticals, and a wide variety of biologically active compounds
.
Many researchers have synthesized piperidine-carrying compounds and investigated them for
their anti-Alzheimer potential 22-28. The chemical structure of donepezil, an important and widely
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used drug for the treatment of AD patients in neurology clinics, is also based on a 1,4-
disubstituted piperidine ring. Like piperidine, its bioisoster, piperazine has as well as been
subjected to the development of novel anticholinesterase agents 29-38
.
In the light of above knowledge, we herein introduce new compounds, which include a
dithiocarbamate moiety along with piperidine/piperazine ring to assess their potential against
cholinesterases.
Results and discussion
Chemistry
In the present study, some piperidine or piperazine-dithiocarbamate compounds were
synthesized and evaluated for their inhibitory potency against cholinesterase enzymes 39,40. The
target compounds were gained in three reaction steps. Sodium dithiocarbamates (A1-A11) were
prepared by the reaction of carbon disulfide and cyclic secondary amine in the presence of
sodium hydroxide 41. Secondly, halogenated derivatives (B1-B4) were synthesized via
acetylation of anilines by chloroacetyl chloride to obtain the compounds B1-B3 and bromination
of 3,4-dichloroacetophenone gave the 2-bromo-3’,4’-acetophenone (B4) 42,43. Finally, the
compounds synthesized in initial steps were reacted in acetone to achieve target compounds (C1-
C44). The synthetic route of the compounds was outlined in Scheme 1.
[Insert Scheme 1]
The structures of the obtained compounds were elucidated from their spectral data. In the IR
spectra, significant stretching bands belonging to N-H were observed at 3225-3437 cm-1. The
stretching bands for C=O and C=S were observed between 1625-1695 cm-1 and 1200-1246 cm-1.
In the 1H-NMR spectra, methylene protons between carbonyl and dithioate group were recorded
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as a singlet peak between 4.20-4.93 ppm. Trisubstituted benzene peaks were observed as two
doublets and one doublets of the doublet with varying shifting values depending on the
substituted group. Amide protons on the dihalogenated and dimethoxy substituted derivatives
gave a singlet peak between 10.51-10.80 ppm and 10.18-10.21 ppm, respectively. In the 13C-
NMR spectra, all compounds have thiocarbonyl peaks between 192 and 198 ppm. While
carbonyl peaks for compound C1-C33 was recorded at 162 ppm -168 ppm, peaks of C34-C44
observed between 190 ppm and 193 ppm. All other aromatic and aliphatic protons and carbons
were recorded on near to the expected values. HRMS was also performed, and all measured mass
and isotope scores were compatible with calculated values.
Cholinesterase Inhibitory Activity
The synthesized compounds C1-C44 were assessed as AChE and BChE inhibitors by using in
vitro modified Ellman’s spectrophotometric method 44. Donepezil was used as the reference
drug.
Generally, it was understood that the synthesized compounds have more potent inhibitory
activity against AChE enzyme as regards BChE enzyme. The compounds C8, C9, C19, C30,
C31, C41 and C42 exhibited significant inhibition profiles against AChE enzyme at 10-3 M
concentration (Table 1). On the other hand, none of the compounds displayed remarkable
inhibitor activity on BChE enzyme. Compound C41 was the most active compound against
BChE with a 22.67 % inhibition potency at 10-3 M concentration (data not shown). These results
show that the synthesized compounds are selective AChE inhibitors.
By looking at the inhibition potency (>50%) at 10-3 M, the compounds C8, C9, C19, C30, C31,
C41 and C42 were investigated in further concentrations (10-5-10-9 M) to calculate IC50 values
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(Table S 1 Supplemental Materials). According to enzyme inhibition studies, compound C41 is
the most active derivative due to its inhibitory profile on AChE with an IC50 value of 11.82 µM.
Enzyme inhibition results gave a chance to evaluate the structure activity relationships. Once the
structures of synthesized compounds were examined, it was striking that dimethylaminoethyl and
dimethylaminopropyl substituents on 4th position of piperazine ring have a great impact on
enzyme inhibitory activity.
Kinetics Study
The kinetics of this series of AChE agents were studied in detail using the most effective
compound C41. The nature of AChE inhibition, caused by this compound, was evaluated by the
graphical analysis of steady-state inhibition data. Lineweaver - Burk plots determined the
compound C41 as a mix-typed inhibitor, because of the different intercepts on both y- and x-
axes (Figure 1). The values of Km and Vmax were investigated by nonlinear regression were
calculated as 6.6869 and 0.2853, respectively.
[Insert Figure 1]
Theoretical Determination of ADME Properties
The theoretical prediction of ADME properties (molecular weight, log P, TPSA, number of
hydrogen donors and acceptors, volume) for the active compounds C8, C9, C19, C30, C31, C41
and C42 were determined and presented in Table S 2 (Supplemental Materials) along with
45
violations of Lipinski’s rule . This rule suggests that, an orally active drug has no more than
one violation. As seen in Table S 2, all calculated physicochemical parameters for the
45
compounds are compatible with Lipinski’s rule . Furthermore, the most active compound C41
has an ideal lipophilic character (logP=3.63) which is required to cross the central nervous
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system (CNS) 46. Besides, TPSA, described to be a predictive indicator of membrane penetration,
is positive (26.79) and as AChE inhibitors have to pass different membranes and reach the CNS,
this supports the potential of compound C41.
Molecular Docking
In order to designate the binding modes between the ligand and the receptor, docking studies
were carried out using the X-ray crystal structure of Homo sapiens AChE (hAChE PDB ID:
4EY7), which is very similar to Electrophorus electricus AChE (EeAChE) include E2020 as
47
ligand . The compound C41 was docked into the active site of hAChE. To identify the most
likely interaction with the receptor, low energy docked coordinates were selected.
The best docking poses, showing interactions with the active site, are viewed in Figure S1. When
the docking studies are analyzed, it is clearly understood that the compound C41 is very
compatible with the pocket of the active site. It interacts with the amino acids, Phe295, Tyr72,
Tyr337, Trp86, His447, Ser203, Glu202. Compound C41 settles down formation of H bond
between the oxygen atom of the carbonyl group and the amino group of the Phe295 residue. The
formation of π-π interactions between piperazine moiety and phenyl ring of Tyr337 amino acid is
also sighted in the gorge. Due to the formation of H bond between aliphatic nitrogen atom and
the carboxyl group of Glu202 residue, there are increased interactions with the active site. The
ethyl group, among the rest of the piperazine moiety and aliphatic nitrogen atom sets up van der
Waals interactions with the amino acids of the binding side. These interactions equipoise the
ligand in the active site of the enzyme.
Conclusion
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In the present study, cholinesterase inhibitory potency of some 44 novel piperazine and
piperidine dithiocarbamate derivatives investigated. 2-(4-(2-Dimethylaminoethyl)piperazin-1-yl-
dithiocarbamoyl)-3’,4’-dichloroacetophenone (C41) was found as the most active compound
AChE with a IC50 value of 11.82 µM. Enzyme kinetic studies revealed this compound as a mixed
type inhibitor. Molecular modeling studies indicated the importance of 4-(2-dimethylaminoethyl)
and ketonic carbonyl for interaction between AChE and ligand. Consequently, findings of this
study may have an impact on chemists to synthesize similar compounds that may have higher
potency against cholinesterase enzymes.
Experimental
Chemistry
The chemicals used in the syntheses were purchased from Sigma-Aldrich Chemicals (Sigma-
Aldrich Corp., St. Louis, MO, USA) or Merck Chemicals (Merck KGaA, Darmstadt, Germany).
Melting points of the synthesized compounds were recorded on a MP90 digital melting point
apparatus (Mettler Toledo, Ohio, USA) and were uncorrected. 1H NMR and 13C NMR spectrums
were recorded on a Bruker 500 MHz digital FT-NMR spectrometer and Bruker Fourier 300
(Bruker Bioscience, Billerica, MA, USA) respectively, in DMSO-d6. Operating frequencies of
1
the experiments were 500 MHz for H NMR and 75 MHz for 13C NMR. The IR spectra was
obtained on a Shimadzu, IR Prestige-21 (Shimadzu, Tokyo, Japan). LCMS studies were
performed on Shimadzu LCMS-IT-TOF (Shimadzu, Tokyo, Japan). The purities of compounds
were checked by TLC on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany).
Materials and methods
General procedure for the synthesis of sodium dithiocarbamate derivatives A1-A11
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Substituted piperazine or piperidine derivative (15 mmol) was dissolved in EtOH (200 mL), and
sodium hydroxide (15 mmol) was added. The mixture was cooled in an ice bath, and CS2 (150
mmol) was added in portions. The reaction was allowed to stir for 1 h at room temperature. The
solvent and excess of CS2 were removed under reduced pressure 48. The residue was washed with
dry ether, and the raw product was recrystallized from ethanol.
General procedure for the synthesis of 2-chloro-N-(3,4-disubstitutedphenyl)acetamide
derivatives B1-B3
The appropriate 3,4-disubstituted aniline (50 mmol) and TEA (60 mmol, 8.45 mL) in THF (150
mL) were mixed on ice bath. Chloroacetyl chloride (60 mmol, 4,81 mL) in THF (20 mL) was
added dropwise to this solution. After completion, the reaction mixture was stirred at room
temperature for 1 h. The precipitated product was filtered washed with water, dried and then
recrystallized from ethanol.
Synthesis of 3’,4’-dichloro-2-bromo acetophenone B4
3,4-Dichloroacetophenone (50 mmol, 9.45 g) was dissolved in AcOH (250 mL) and catalytic
amount of HBr was added. This solution was placed on an ice bath and bromine (60 mmol, 3,09
mL) was added dropwise. The reaction was routinely checked by TLC. After completion of
reaction, the mixture was poured into the iced-water, precipitated products was filtered, washed
with water, dried and then recrystallized from ethanol.
General synthesis procedure for target compounds
The corresponding sodium dithiocarbamate derivative (A1-A11) (5 mmol) and halogenated
compound (B1-B4) (5 mmol) were dissolved in acetone and refluxed for 2 h. After TLC control,
the solvent was evaporated. The residue was washed with water, dried and then recrystallized
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from ethanol to afford final compounds (C1-C44). Complete characterization data for C2-C44
1
are presented in the Supplemental Materials and representative H NMR spectra are given in
Figures S 1 – S 2.
2-(4-Methylpiperazin-1-yl-dithiocarbamoyl)-N-(3,4-dimethoxyphenyl)acetamide C1
Yield: 79 %, M.P. = 110.4 - 111.8 °C, FTIR (ATR, cm-1 ): 3256 (N-H), 1663 (C=O), 1229
1
(C=S), 1051, 843, 729. H-NMR (500 MHz, DMSO-d6): δ = 2.23 (3H, s, –CH3), 3.08-3.14 (4H,
m, piperazine –CH2-), 3.72 (6H, s, –OCH3), 4.08 (2H, s, piperazine –CH2-), 4.28 (2H, s, –SCH2-
), 4.61 (2H, s, piperazine –CH2-), 6.89 (1H, d, J=8.50 Hz, aromatic –CH-), 7.10 (1H, d, J=8.50
Hz, aromatic –CH-), 7.29 (1H, s, aromatic –CH-), 10.18 (1H, s,–NH-). 13C-NMR (75 MHz,
DMSO-d6): δ = 45.2, 48.6, 50.1, 51.8, 52.4, 55.7, 56.3, 105.1, 111.5, 113.3, 132.4, 136.6, 144.3,
149.9, 166.2, 195.8. HRMS (m/z): [M+H]+ calcd for C16H23N3O3S2: 370.1254; found 370.1245.
Enzyme Inhibition Assay
Inhibition potency of the compounds against AChE and BuChE has been determined using
Ellman’s method 44. Enzyme solutions were prepared in gelatin solution (1%), at a concentration
of 2.5 units/mL. Synthesized compounds and donepezil were prepared at 10-3 M and 10-4 M
concentrations using 2% DMSO. AChE or BuChE solution (20µL/well) and compound solution
(20µL/well) were added to phosphate buffer (140 µL/well, pH 8± 0.1) and incubated at 25°C for
5 min. The reaction was started by adding the chromogenic reagent 5,5-dithio-bis(2-nitrobenzoic
acid) (DTNB) (20 µL/well, 10 mM) and the substrates acetylthiocholine iodine (ATCI) or
butrylthiocholine iodine (BTCI) (10 µL/well, 75 mM) to the enzyme-inhibitor mixture. The
production of the yellow anion was recorded for 10 min at 412 nm. As a control, an identical
solution of the enzyme without the inhibitor was processed. Control and inhibitor readings were
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corrected with blank-reading. All processes were assayed in four independent wells. The same
procedure was followed for further concentrations (10-5-10-9 M, 20 μL/well) of donepezil and
selected compounds indicating ≥50% inhibition at the concentration of 10-3 M. The IC50 value
was calculated from the plots of enzyme activity against concentrations by applying regression
analyses on GraphPad Prism Version 5.
Enzyme Kinetics
The same materials were used in the cholinesterase inhibition assay. The compound C41 was
prepared at IC50 concentration that was calculated in enzyme assay and then added to the wells
(20 μL/well). AChE was added to the plate (20 μL/well) and enzyme inhibitor mixture was
incubated at 25°C for 5 min. The reaction was started by adding DTNB (20 µL/well) and the
various concentrations (150, 75, 37.5, 18.75, 9.375, 4.6875, 2.3437, 1.1718, 0.5859 and 0.2929
µM) of substrate (ATCI) (10 μL/well). The production of the yellow anion was recorded for 10
min at 412 nm. A parallel control without inhibitor was used for comparison. All processes were
assayed in four independent wells. The results were analyzed as Lineweaver-Burk plots using
Microsoft Office Excel 2013.
Theoretical Calculation of ADME Parameters
In order to evaluate pharmacokinetic profiles (ADME) of the active compounds (C8, C9, C19,
C30, C31, C41 and C42), some physicochemical parameters were predicted using the
Molinspiration property calculation program.
Molecular Docking
Docking studies were performed using Autodock Vina 49. The coordinates of Homo sapiens
AChE (hAChE PDB ID: 4EY7) were obtained from the Protein Data Bank (PDB) 50. For
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docking studies premier protein was prepared by removing all non-protein molecules, including
water, ions, any co-crystallized solvent and ligands. Autodock Tolls (ADT, version 1.5.6) 51, was
used to prepare the ligand and the receptor, which were saved in pdbqt format and also to add
hydrogens and fractional changes for protein and ligands. Autodock Vina was used to dock the
ligand into the active site of hAChE. The parameters of the docking were formed as follows:
center_x=3.102, center_y=-40.295, center_z=30.604, size_x=60, size_y=72, size_z=74. Docking
of the compound C41 was performed in a limited grid box described by Autodock tools (ADT,
version 1.5.6). Docked ligand was analyzed, and the results were visualized by PyMOL 1.6.X 52
molecular graphics system, version 1.8.
Conflicts of Interest
The authors declare no conflict of interest
Acknowledgments
This study was financially supported by Anadolu University Scientific Research Projects Funds.
Project No: 1305S83.
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Figure 1: Lineweaver–Burk plots for compounds C41 (IC50 = 11.82 μM). Substrate (ATCI-
Acetylthiocholin iodine) concentrations used: 150, 75, 37.5, 18.75, 9.375, 4.6875, 2.3437,
1.1718, 0.5859 and 0.2929 mM. 1/V: 1/velocity of reaction [1/(absorbance/1 min)], 1/S:
1/substrate concentration (1/µM ATCI).
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CH3,Ph,Bn,4-CH3-Bn, 4-OCH3Bn,4-F-Bn,4-CF3-Bn,2-(NCH3)2C2H4, 3-
CH3, Bn
CH
R1:
(NCH3)2C3H6
N
X:
F, Cl, OCH3
R2:
Scheme 1: Synthesis way for target compounds
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