Design, Synthesis, Molecular Docking, and Cholinesterase Inhibitory Potential of Phthalimide-Dithiocarbamate Hybrids as New Agents for Treatment of Alzheimer's Disease

Article abstract of DOI:10.1002/cbdv.201900370

A novel series of phthalimide-dithiocarbamate hybrids was synthesized and evaluated for in vitro inhibitory potentials against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The anti-cholinesterase results indicated that among the synthesized compounds, the compounds 7g and 7h showed the most potent anti-AChE and anti-BuChE activities, respectively. Molecular docking and dynamic studies of the compounds 7g and 7h, respectively, in the active site of AChE and BuChE revealed that these compounds as well interacted with studied cholinesterases. These compounds also possessed drug-like properties and were able to cross the BBB.

Full text of DOI:10.1002/cbdv.201900370

DOI: 10.1002/cbdv.201900370
FULL PAPER
Design, Synthesis, Molecular Docking, and Cholinesterase Inhibitory
Potential of Phthalimide-Dithiocarbamate Hybrids as New Agents for
Treatment of Alzheimer’s Disease
Mehdi Asadi,a Mostafa Ebrahimi,a Maryam Mohammadi-Khanaposhtani,b Homa Azizian,c
Saghi Sepehri,d Hamid Nadri,e Mahmood Biglar,f Massoud Amanlou,a Bagher Larijani,f
Roghieh Mirzazadeh,*g Najmeh Edraki,h and Mohammad Mahdavi*f
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, 1417653761 Tehran, Iran
Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences,
b
Babol 4717647745, Iran
c
Department of Medicinal Chemistry, School of Pharmacy-International Campus, Iran University of Medical
Sciences, Tehran 14665354 Iran
Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil
d
5618953141, Iran
e
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shahid
Sadoughi University of Medical Sciences, Yazd 8915173160, Iran
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute,
f
Tehran University of Medical Sciences, Tehran 1417653761, Iran,, e-mail: momahdavi@tums.ac.ir
g
Department of Biochemistry, Pasteur Institute of Iran, Tehran 1316943551, Iran,,
e-mail: rmirzazadeh@pasteur.ac.ir
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz
h
7134853734, Iran
A novel series of phthalimide-dithiocarbamate hybrids was synthesized and evaluated for in vitro inhibitory
potentials against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The anti-cholinesterase results
indicated that among the synthesized compounds, the compounds 7g and 7h showed the most potent anti-
AChE and anti-BuChE activities, respectively. Molecular docking and dynamic studies of the compounds 7g and
7h, respectively, in the active site of AChE and BuChE revealed that these compounds as well interacted with
studied cholinesterases. These compounds also possessed drug-like properties and were able to cross the BBB.
Keywords: phthalimide, dithiocarbamate, Alzheimer’s disease, acetylcholinesterase, butyrylcholinesterase,
inhibitory activity.
progression.[2] Despite many efforts of pharmaceutical
companies and academic institutions, there is no
Introduction
Alzheimer’s disease (AD) is a complex and age-related effective treatment for Alzheimer’s disease. Therefore,
neurodegenerative progressive and fatal disorder.[1] It the discovery of new efficient agents for the treatment
is estimated that people suffering dementia in 2018 of AD is a big challenge for the healthcare community.
were 50 million worldwide and this statistic is in
Alzheimer’s disease is characterized by shortage of
neurotransmitter acetylcholine (ACh), accumulation of
amyloid plaques, and tau-phosphorylation in the
brain.[3] Therefore, increase of the level of ACh,
decomposition of amyloid plaques, and inhibition of
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.201900370
Chem. Biodiversity 2019, 16, e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
Figure 1. Design strategy of novel phthalimide-dithiocarbamate hybrids 7 as new anti-Alzheimer agent.
tau-phosphorylation and aggregation in the brain are types of ACh degrading enzymes may be beneficial in
the most important therapeutic targets for AD. AD treatment.[7]
Furthermore, several studies have suggested that
The phthalimide is a bicyclic heterocyclic scaffold
increase levels brain derived neurotrophic factor and its derivatives have diverse range of biologically
(BDNF) and nerve growth factor (NGF) by experimental activities such as anti-inflammatory, anticonvulsant,
drugs such as J147 can be useful for treatment of hypolipidemic, analgesic, and immunomodulatory
AD.[4] The activity of acetylcholine in the brain is activities.[8–13] Recently, several derivatives of phthal-
terminated by acetylcholinesterase (AChE) and there- imide with high inhibitory activity against AChE have
fore inhibition of this enzyme led to ameliorate signs been reported (Figure 1A and B).[14,15] On the other
and symptoms of AD.[5] Today, AChE inhibitors such as hand, two dithiocarbamate derivatives C and D
donepezil and rivastigmine are the main drugs for the exhibited good inhibitory activity against cholinester-
treatment of AD.[6]
ases (Figure 1).[16,17] Keeping in view of the above
Another one of ACh degrading enzymes is butyr- mentioned importance of phthalimide and dithiocar-
ylcholinesterase (BuChE). Recent studies showed that bamate pharmacophores, herein, we designed, synthe-
activity of BuChE in the brain of AD patients with the sized, and evaluated a novel series of phthalimide-
progression of the AD increased while AChE activity in dithiocarbamate hybrids as new anti-cholinesterase
these patients declines. Therefore, inhibition of both agents (Figure 1, compounds 7a–7i and 7j–7o).
www.cb.wiley.com
(2 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
°
Scheme 1. Synthesis of phthalimide containing dithiocarbamate derivatives 7a–7o. Reagents and conditions: a) Acetonitrile, 60 C,
°
°
6 h; b) Potassium hydroxide 10%, DMF, 50 C, 3 h; c) DMF, 50 C, 12 h.
Results and Discussion
Chemistry
The general procedure for the synthesis of phthalimide
containing dithiocarbamate derivatives 7a–7o is pre-
sented in Scheme 1. It was started from the reaction of
potassium 1,3-dioxoisoindolin-2-ide 1 and appropriate
Table 1. Anti-ChE activities of compounds 7a–7o.
°
dibromoalkanes 2a–2c in acetonitrile at 60 C to give
Compound
IC50 [μM] (AChE)[a]
IC50 [μM] (BuChE)[a]
2-(bromoalkyl)isoindoline-1,3-dione derivatives 3a–3c.
On the other hand, carbon disulfide (4) and primary
amines 5a–5c or secondary amines 5d–5e reacted in
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7 l
7m
7n
7o
�100
�100
27.28�0.61
�100
11.7�0.41
14.95�0.52
�100
°
presence of 10% potassium hydroxide in DMF at 50 C
�100
to produce various sodium dithiocarbamate deriva-
tives 6a–6e. Finally, title compounds 7a–7o were
obtained from the reaction of 2-(bromoalkyl)isoindo-
line-1,3-dione derivatives 3a–3c and sodium dithiocar-
�100
23.36�0.59
�100
�100
4.6�0.18
11.72�0.21
28.25�0.29
�100
20.37�0.37
8.8�0.24
�100
°
bamate derivatives 6a–6e in DMF at 50 C.
20.6�0.33
�100
�100
�100
30�0.46
�100
Pharmacology
�100
�100
�100
ChE Inhibitory Activity. The phthalimide-dithiocarba-
mate hybrids 7a–7o were screened against AChE/
BuChE by the Ellman’s method.[18] Inhibitory activity of
these compounds was compared to standard drugs
rivastigmine and donepezil (Table 1). As can be seen in
Table 1, compounds 7b and 7g–7i were potent
�100
13.6�0.36
7.79�0.06
7.72�0.02
Donepezil
Rivastigmine
0.027�0.003
11.07�0.01
[a]
Data are expressed as Mean � SE (three independent
experiments).
www.cb.wiley.com
(3 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
inhibitors against AChE and among them, 7g was the activity against BuChE (compound 7e vs. compounds
most potent inhibitor (IC50 =4.6�0.18 μM). This com- 7d and 7f).
pound was 2.5-fold more active than rivastigmine
For the tertiary amine derivatives 7j–7o, phenyl-
(IC50 =11.07�0.01 μM). Other synthesized compounds piperazine derivative 7o with propylene linker exhib-
did not show any activity against AChE at 100 μM. ited the highest inhibitory activity against BuChE. The
Based on the IC50 values for BuChE, most of the rest phenylpiperazine derivatives 7j–7k did not show
title compounds exhibited satisfactory inhibitory activ- any activity against BuChE. In this series, two piper-
ity against BuChE in the range of 8.8�0.24–30� idine derivatives 7j and 7 l with methylene and
0.46 μM compared with rivastigmine (IC50 =7.72� ethylene linkers showed moderate activity while their
0.02 μM) and donepezil (IC50 =7.79�0.06). Among propylene analog 7k was inactive against BuChE.
them, compounds 7b, 7c, 7h, and 7o were found to
Molecular Docking and Dynamic Studies. Docking
be more potent inhibitors. Six compounds 7a, 7d, 7f, study was performed using by AutoDock 4.2 package
7k, 7m, and 7n showed no activity against BuChE at to find the binding modes of the most potent
100 μM.
compounds 7g and 7h in the active site of AChE and
Structurally, the synthesized compounds are div- BuChE, respectively. For this purpose, the X-ray crystal
ided into two series: secondary amine derivatives 7a– structure of AChE (PDB code: 6O4 W) and BuChE (PDB
7i and tertiary amine derivatives 7j–7o. In each series, code: 4BDS) were retrieved of RCSB protein data bank
the amine groups and length of the linker between (http://www.rcsb.org/pdb/home/home.do).
phthalimide and dithiocarbamate moieties were al-
tered to optimize the anti-ChE inhibitory activity.
The applied docking procedure reliability was
validated by re-docking of standard inhibitors donepe-
The inhibitory activity of secondary amine deriva- zil and tacrine over AChE and BuChE, respectively. The
tives 7a–7i against AChE demonstrated that benzylpi- docked conformations corresponding to the lowest
peridine derivative 7g with methylene linker showed docking score were selected as the most possible
the most potent activity. Replacement of the meth- binding modes. The root mean square deviation
ylene linker to ethylene and propylene led to (RMSD) was calculated for each ligand to measure the
decreases of 2.5 and 6 fold inhibitory activity as docking prediction accuracy. The pose was counted
observed in the compounds 7h and 7i, respectively. In optimal if its RMSD found to be less than 2 Å. The
this series, cyclohexane derivative 7b with ethylene RMSD of the re-docked conformations of donepezil
linker showed a moderate inhibitory activity against and tacrine over 6O4 W and 4BDS was 0.40 Å and
AChE while it analogs 7a and 7c with methylene and 0.29 Å, respectively, which is considered as successfully
propylene linkers showed no activity against AChE. docked.[19,20] As a result, the validity of docking
Moreover, phenyl derivatives 7d–7f showed no anti- parameters are reasonable in order to predict the
AChE activity. Our obtained results were also shown related co-crystalized structures.
that tertiary amine derivatives 7j–7o were not active
against AChE.
Docking of donepezil over AChE active site showed
that the ring center of the benzyl and the indene
In the term of BuChE inhibitory activity in secon- moiety interacted with Trp86 and Trp286 via π–π
dary amine series 7a–7i, benzylpiperidine derivative interactions, respectively. Also, the quaternary amine
7h with ethylene linker showed most potent activity of piperidine ring interacts by two π-cation interac-
while its methylene and propylene analogs 7g and 7i tions with the ring center of Trp86 and Phe338, as well
showed moderate and no activity against BuChE, the indene carbonyl group forms H-bond interaction
respectively. Interestingly, for cyclohexane derivatives with the backbone NÀ H of Phe295 (2.3 Å) (Figure 2a)
7a–7c and phenyl derivatives 7d–7f, similar to
Furthermore, docking of tacrine over BuChE
benzylpiperidine derivatives 7g–7i, the most potent showed the prominent role of Trp82 and His438 in
compounds were compounds containing ethylene stabilizing the ligand in the binding pocket by
linker (compounds 7b and 7e, respectively). In this constructing π–π and π-cation interactions with the
regard, changing ethylene linker to propylene linker in aromatic part of acridine moiety and H-bond with the
compound 7b, as in compound 7c, slightly diminished quaternary amine at the distance of 2.5 Å, respectively
the activity while replacement of ethylene linker with (Figure 2b).
methylene linker, as in compound 7a, led to loss of
In order to understand the criteria for rational
anti-BuChE activity. Moreover, for phenyl derivatives designing of ChEs inhibitors, it is necessary to uncover
7d–7f, replacement of the ethylene linker to meth- the structural perturbations incurred by the most
ylene and propylene linkers led to loss of inhibitory potent compounds over ChEs and the effect of these
www.cb.wiley.com
(4 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
Figure 2. Close-up representation of binding interactions of the superposed docked (green) and co-crystalized donepezil and
tacrine (cyan) over AChE (a) and BuChE (b), respectively, structural waters and their H-bond interaction render in stick.
Figure 3. RMSD of ligand (red) and protein Cα (blue) for 7g-AChE (a), donepezil-AChE (b), 7h-BuChE (c), and 7h-BuChE (d) over
20 ns MD simulation time.
compounds on the active site environment. Here, the tively, with higher fluctuation stabilizing at an average
stability of protein-ligand complex is defined by RMSD of 2.2 Å for both systems (Figure 3a and 3b), while the
of the protein’s backbone from its initial to final complex of rivastigmine and 7h complexed with
conformation over 20 ns MD simulation time. The BuChE displayed longer equilibration time (about the
RMSD simulation showed that AChE complexed with last 15 ns) with lower fluctuations (Figure 3c and 3d).
7g and donepezil maintained an overall stability The RMSD value of each protein-ligand complex
throughout the last 2 and 5 ns of MD time, respec- indicates that the employed simulation time has been
www.cb.wiley.com
(5 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
Figure 4. The variation of interaction energy of the compounds over the AChE and BuChE during 20 ns of MD simulation. Coulomb,
Van der Waals (Vw), and total energy are in red, purple, and green, respectively.
enough to obtain an equilibrium structure over the mine (Figure 4c and 4d) show almost the same anti-
simulation time. Thus, the structures at the MD BuChE activity which highlight the possibility of
equilibrium state used to investigate the structural correlation between interaction and inhibitory activity.
specificity of the ligand-protein complexes.
Figure 5a and 5c show the detailed ligand atom
Interaction energies were calculated in order to interactions that occurred more than 30.0% of the
reveal ligand-protein stability of AChE/7g, donepezil simulation and Figure 5b and 5d represent
or BuChE/7h, rivastigmine (Figure 4). The average total proteinÀ ligand contact bar occupancy during the
interaction energy for the compound 7g over AChE 20 ns simulations for 7g and donepezil. The interaction
was À 10/À 20 kcal/mol and for donepezil over AChE analysis suggests the piperidine NH+ group in 7g
was À 20/À 30 kcal/mol. Comparing the total interac- stabilized by π-cation interaction with Trp86 of
tion energy obtained from MD for the 7g with catalytic anionic site (CAS) and Tyr341 at the periph-
donepezil, it was observed that donepezil has more eral anionic site (PAS), and form H-bond interaction
average total interaction energy (more stable) with Tyr124 of PAS with high occupancy during the
throughout 20 ns of simulation time as compared to simulation time. In addition, dithiocarbamate moiety
that of 7g (Figure 4a and 4b). As the total energy of a stabilized through water mediated H-bond interac-
system is the sum of coulomb and Van der Waals (Vw) tions with Phe295 and Arg296 from the acyl-binding
energies, this difference originally is due to the more pocket of CAS. Furthermore, the acyl-binding pocket
coulomb energy of donepezil over AChE than com- of CAS interacts with phthalimide moiety through
pound 7g. According to the experimental assay, high- Ser293 and Phe338 with H-bond and hydrophobic
er AChE inhibition activity of donepezil rather than 7g interactions (Figure 5a).
may be related to the more stability and higher
Comparison between the binding interactions of
interaction energy of donepezil in comparison with donepezil and 7g with AChE revealed some differ-
compound 7g. On the other hand, compound 7h with ences between the two ligands interactions. The first
higher Vw and total interaction energy than rivastig- Asp74 formed a strong and stable ionic interaction
www.cb.wiley.com
(6 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
Figure 5. Detailed ligand atom interactions and protein-ligand contact bar analyzed at 20 ns simulations of 7g (a, b) and donepezil
(c, d) over AChE. Interactions that occur more than 30.0% of the simulation time are shown. H-bond, hydrophobic, ionic, and water
bridge are in green, purple, pink, and blue, respectively.
between piperidine NH+ group in donepezil with 99% played π-π interaction with Trp231 and Phe329 from
time of simulation (Figure 5c, purple arrow), while the the acyl-binding pocket of CAS. Furthermore, there is a
same NH+ group in 7g stabilized with Trp86, Tyr341, strong ionic bridge between piperidine NH+ group
and Tyr124 as previously mentioned. Although these and Asp70 (Figure 6a, straight red and blue line) with
interactions have specific role in stabilizing 7g, they 77% time of simulation that such strong interaction
are not as strong as ionic interaction participate in has been observed through donepezil and AChE
donepezil by the residue Asp74. Furthermore, by (Figure 5c). The comparison between the binding
comparing Figure 5b and 5d the outstanding contribu- interactions of rivastigmine and 7h over BuChE
tion of Trp268 related to the PAS residues can be represented that although the number of binding
observed which stabilized the 1-oxoindane group of interactions with more that 30% occurrence among
donepezil through π–π stacking interactions with 7h and BuChE are remarkably more than existing
77% time of simulation (Figure 5c, straight green lines). binding interactions between rivastigmine and BuChE,
Consequently, donepezil is regarded as a more stable both of them show remarkable ionic interaction with
ligand in the binding pockets of the AChE, and Asp70 at the PAS (Figure 6c). Therefore, it is expected
subsequently more active compound to inhibit AChE that 7h to be as active as rivastigmine against BuChE.
than compound 7g.
Our molecular dynamics study suggests that the
Regards to compound 7h, the benzyl ring and the persistent ionic interaction of ligand with the PAS
dithiocarbamate moiety established π-π and H-bond residue Asp (Asp74 in AChE and Asp70 in BuChE) has a
interactions with Trp82 and Asn68 at PAS over the significant contribution for ChEs inhibition.
BuChE, respectively. Also, phthalimide moiety dis-
www.cb.wiley.com
(7 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
Figure 6. Detailed ligand atom interactions and protein-ligand contact bar analyzed at 20 ns simulations of 7h (a, b) and
rivastigmine (c, d) over BuChE. Interactions that occur more than 30.0% of the simulation time are shown. H-bond, hydrophobic,
ionic, and water bridge are in green, purple, pink, and blue, respectively.
For analyzing the fluctuation of ChEs residues over their structural and the integrity during the simula-
ligand binding interaction, the root mean square tions.
fluctuation (RMSF) was calculated from MD trajecto-
Drug Likeliness Parameters. The physicochemical
ries. Considering the RMSF plot of 7g and 7h, it was properties and BBB penetration of the compounds 7g
verified that they are in a good agreement with the and 7h as the most potent compounds respectively
corresponding plots of donepezil and rivastigmine, against AChE and BuChE were predicted in silico. The
respectively (Figure 7). The residues that are bonded to physicochemical properties of these compounds and
the ligand were very stable along the simulations (< standard drugs rivastigmine and donepezil were
2 Å). In the case of BuChE, a larger fluctuation (�2 Å) obtained using MarvineSketch 5.8.3, ChemDrawUltra
was observed for the region relative to residues 281– 12.0, and Autodock Tools (ver.1.5.4) to assess their
283 which are located over the loop at the interface compliance with the Lipinski’s rule of five (a molecular
between the catalytic domain of BuChE and 7h/ weight (MW)�500, a number of H-bond acceptors
rivastigmine binding site. This loop does not interact (HBA)�10, a number of H-bond donors (HBD)�5,
with the complexed ligand at the BuChE active site, so octanol-water partition coefficients (log P)�5, and a
observing higher fluctuation is unavoidable. The number of rotatable bonds (RBC)�10) and the Veber
regions showing high fluctuation in AChE-ligand rule (polar surface area (tPSA)�140 Å2).[21,22] As can be
complex are residues 259 to 264 and residues 493 to seen in Table 2, obtained data revealed that studied
498 that are located to the two side loops far from the compounds follow the Lipinski’s rule of five and the
active site and have high solvent exposure. RMSF Veber rule. The BBB penetration of the compounds 7g,
results suggested that the complexes of compound 7g 7h, rivastigmine, and donepezil was predicted by the
and 7h with the ChE enzyme were able to maintain admetSAR server.[23] This software predicted that
agents with SVM_MACCSFP BBB score >0.02 were
www.cb.wiley.com
(8 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
Figure 7. RMSF values of ChE enzyme from all different systems. 7g-AChE (a), donepezil-AChE (b), 7h-BuChE (c), rivastigmine-BuChE
(d). Peaks indicate areas of the protein that fluctuated the most during the simulation. the background pink, blue and white strips
represented the α-helix and β stands and the loop secondary structures of the enzymes, the ligand binding site represented in
green bar at the bottom of the RMSF plot.
Table 2. Physicochemical properties and BBB penetration of the compounds 7g, 7h, Rivastigmine, and Donepezil.
Entry
Rule
7g
7h
Rivastigmine
Donepezil
MW
HBA
HBD
LogP
�500
�10
�5
425
3
1
3.69
7
439
3
1
3.69
7
250.17
2
0
2.41
6
379.21
3
0
4.21
6
�5
RBC
�10
–
Lipinski’s violations
tPSA
Veber violation
BBB score[a]
BBB penetration
0
0
0
0
�140 Å2
52.65
0
0.056
+
52.65
0
0.053
+
32.78
0
0.042
+
38.77
0
0.103
+
–
>0.02
–
[a]
SVM_MACCSFP BBB Score.
able to cross the BBB (Table 2). Obtained results Amyloid β-Protein Peptide (APP) based substrate (Rh-
revealed that studied synthesized compounds similar EVNLDAEFK-quencher) was applied and OM99-2
to standard drugs were able to cross the BBB.
(IC50 =0.014 μM) was used as the reference agent.
β-Secretase Inhibitory Activity. One of the most Obtained results showed that the compound 7g is a
important approaches for treatment of AD is inhibition weak inhibitor against β-secretase (Table 3).
of β-secretase (BACE-1) in order to prevent the
formation of Aβ plaques.[24] Thus, β-secretase inhib-
itory activity of compound 7g as the most active
Conclusions
compound against AChE was obtained via
a
fluorescence resonance emission transfer (FRET) meth- In conclusion, we introduced a novel series of
od. For this propose, the kit consisted of β-site amyloid phthalimide-dithiocarbamate hybrids as new cholines-
precursor protein cleaving enzyme 1 (BACE-1) and terase inhibitors. Two series of secondary amine
www.cb.wiley.com
(9 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
General
Procedure
for
the
Preparation
of
Table 3. β-Secretase inhibitory activity of compound 7g.
2-(Bromoalkyl)isoindoline-1,3-dione Derivatives 3
Compound
% Inhibition
at 10 μM
% Inhibition
at 50 μM
IC50
[μM][a]
Potassium 1,3-dioxoisoindolin-2-ide (1; 1 mmol) and
acetonitrile was put into a round-bottom flask and
7g
OM99-2
24�12.7
39�14.3
ND
–
–
0.014�0.003
°
stirred at 60 C. Then, appropriate dibromoalkanes 2a–
[a]
Mean�S.E.; values are means of three independent
2c (2 mmol) were added dropwise to this mixture for
experiments.
30 min. After that, the reaction mixture was stirred at
°
60 C for 6 h. After completion of the reaction (moni-
tored by TLC), acetonitrile was removed under
derivatives 7a–7i and tertiary amine derivatives 7j–7o vacuum. The obtained precipitate was filtered off and
were synthesized from appropriate dibromoalkanes, washed with diethyl ether to give pure compounds 2-
carbon disulfide, and various amine derivatives. The (bromoalkyl)isoindoline-1,3-dione derivatives 3a–3c.
results of cholinesterase inhibitory activity revealed
that secondary amine derivatives 7g and 7h were the
General Procedure for the Preparation of Sodium Dithio-
most potent compounds against AChE and BuChE,
carbamates 6a–6e
respectively. Therefore, we made attempt to under-
stand dynamics behavior and the detailed interactions A mixture of different amines 5a–5e (1 mmol) and
of the compounds 7g and 7h against AChE and 10% potassium hydroxide (1 mmol) in DMF (5 ml) was
°
BuChE, respectively, and compare them with the FDA dissolved and cooled until 0 C. Then, carbon disulfide
approved donepezil and rivastigmine. Our molecular (4; 1.5 mmol) was added dropwise to this mixture for
dynamics study revealed that the complex of these 30 min. The reaction mixture was stirred and heated in
°
compounds with the studied ChE was able to maintain 50 C for 3 h. The reaction progress was monitored by
their structural and integrity during the simulations TLC. After completion of the reaction, the mixture was
based on RMSD, RMSF, and energy evaluation study. cooled to room temperature and a mixture of water/
Furthermore, docking study revealed the persistent ice was added and the obtained mixture was filtered
ionic interaction of the selected ligands with Asp in to give sodium dithiocarbamate 6a–6e.
the PAS (Asp74 in AChE and Asp70 in BuChE) has a
significant contribution for ChEs inhibition. In silico
General Procedure for the Preparation of 2-(1,3-Dioxoiso-
pharmacokinetic assay and BBB penetration prediction
indolin-2-yl)alkyl Piperidine-1-carbodithioates 7a–7o
demonstrated that compounds 7g and 7h have
satisfactory pharmacokinetics and BBB penetration as A mixture of 2-(bromoalkyl)isoindoline-1,3-dione deriv-
anti-Alzheimer drug candidates.
atives 3a–3c (1 mmol) and sodium dithiocarbamates
°
6a–6e (1 mmol) in DMF was stirred at 50 C for 12 h.
Then, the reaction mixture was allowed to cool at
room temperature and poured into crushed ices, and
subsequently, the pure white precipitates were filtered
off. The pure products 7a–7o were obtained by
Experimental Section
Chemistry
Melting points of phthalimide-dithiocarbamate deriva- recrystallization from ethanol.
tives 7a–7o were measured with a Kofler hot stage
apparatus and are uncorrected. 1H- and 13C-NMR
Biological Evaluation
spectra of the title compounds were recorded with
Bruker FT-400, using TMS as an internal standard. IR In Vitro AChE and BChE Inhibition Assay. The inhibitory
spectra of these compounds were obtained with a potency of the phthalimide-dithiocarbamate hybrids
Nicolet Magna FT-IR 550 spectrophotometer (KBr 7a–7o on AChE and BuChE was evaluated using
disks). MS were recorded with an Agilent Technology Ellman’s method.[18] All the synthesized compounds
(HP) mass spectrometer operating at an ionization were dissolved in 1 mL DMSO and 9 mL ethanol and
potential of 70 eV. Elemental analysis of the derivatives then four different concentrations (56.3, 11.3, 2.2, and
7a–7o was performed with an Elementar Analyses 0.45 μM per well) of each compound were tested to
system GmbH VarioEL CHNS mode.
obtain the range of 20–80% enzyme inhibition for
AChE/BuChE. The reaction mixture included 50 μL
phosphate buffer (0.1 M, pH=8.0), 125 μL of 0.1 M
www.cb.wiley.com
(10 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
DTNB, 25 μL of enzyme (2 U/mL of AChE (E.C. 3.1.1.7,
Before the MD simulation, the appropriate pose for
Type VÀ S, lyophilized powder, from electric eel) or the compounds (7g and 7h) over AChE and BuChE
BuChE (E.C. 3.1.1.8, from equine serum), and 25 mL of was determined by Glide docking protocol.[33,34] Glide
inhibitor solution. The changing of the absorbance application was used for docking investigations using
was measured at 412 nm for 2 min (30 s intervals) after the standard-precision followed by extra-precision
addition of 10 μL substrate (acetylthiocholine iodide or method with flexible ligand sampling. Post-docking
butyrylthiocholine iodide, 0.15 M) to the reaction minimization was performed by applying strain correc-
mixture by a multi-well plate reader (Gen5, Power tion terms. The best docking score poses were used
wave xs2, BioTek, America). The IC50 values were for conducting MD simulation. In order to build system
determined graphically from inhibition curves (log for md simulation, the protein-ligand complexes were
inhibitor concentration vs. percent of inhibition). All solvated with SPC explicit water molecules and placed
experiments were performed in 24-well plates on a in the center of an orthorhombic box of appropriate
Synergy HTX microplate reader in quadruplicates.
size in the Periodic Boundary Condition. Sufficient
counter-ions and a 0.15 M solution of NaCl were also
utilized to neutralize the system and to simulate the
real cellular ionic concentrations, respectively. The MD
Molecular Docking Study
In order to find out the interactions mode of designed protocol involved minimization, pre-production, and
molecules over ChE enzyme, Maestro Molecular Mod- finally production MD simulation steps. In the mini-
eling platform (version 11.5) by Schrödinger, LLC was mization procedure, the entire system was allowed to
performed.[25] Initially, the crystal structures of AChE relax for 2500 steps by the steepest descent approach.
and BuChE enzymes were retrieved from the Protein Then, the temperature of the system was raised from 0
Data Bank (PDB: 6O4 W and 4BDS, respectively) (http:// to 300 K with a small force constant on the enzyme in
www.rcsb.org).[26,27] These pdb structures were se- order to restrict any drastic changes. MD simulations
lected based on criteria like; highest resolution (Å), were performed via NPT (constant number of atoms,
being related to Homo sapiens specious, wild type constant pressure, i.e., 1.01325 bar and constant
with no modified residue, and the existence of co- temperature, i.e., 300 K) ensemble. The Nose-Hoover
crystalized ligand. As the prosthetic group and the co- chain method was used as the default thermostat with
factors are not directly involved in ChE inhibition, so 1.0 ps interval and Martyna-Tobias-Klein as the default
they were totally removed before docking investiga- barostat with 2.0 ps interval by applying isotropic
tion. Except for the four structural waters molecule in coupling style. Long-range electrostatic forces were
the AChE and BuChE active site, which bridge the calculated based on Particle-mesh-based Ewald ap-
receptor important residues by way of H-bonds and proach with the he cut-off radius for coulombic forces
conserve among other specious, the rest of the water set to 9.0 Å. Finally, this system was subjected to
molecules were removed from the enzymes crystallo- production MD simulations for 20 ns for each protein-
graphic structures.[28] The 2D structures of the selected ligand complex. During the simulation, every 1000 ps
compounds were drawn in Marvin 15.10.12.0 program of the actual frame was stored. The dynamic behavior
(http://www.chemaxon.com) and converted into pdb. and structural changes of the systems were analyzed
The Protein Preparation Wizard and the LigPrep by the calculation of energy and the root mean square
module were used to prepare protein and ligand deviation (RMSD). Subsequently, the energy-minimized
structure properly.[29–31]
structure calculated from the equilibrated trajectory
system was evaluated for investigation of each ligand-
protein complex.
Molecular Dynamic Simulation
Molecular simulations of this study were performed
using the Desmond v5.3 using Maestro interface (from
In Silico Molecular Properties Prediction
Schrödinger 2018-4 suite).[32] We evaluated the stabil- The HBA, HBD, and log P values of the compounds 7g,
ity of the best drug candidate from the experimental 7h, rivastigmine, and donepezil were calculated using
study; 7g and 7h over AChE and BuChE, respectively, the MarvineSketch 5.8.3. The tPSA and RBCs of these
and compared their perturbation with the standard compounds were calculated using ChemDrawUltra
inhibitor donepezil and rivastigmine, respectively.
12.0 and Autodock Tools (ver.1.5.6), respectively.
Prediction of BBB penetration of the test compounds
www.cb.wiley.com
(11 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
Lomlim, ‘Synthesis of 2-(2-oxo-2H-chromen-4-yl) acet-
was performed by online BBB predictor (www.cbli-
gand.org).
amides as potent acetylcholinesterase inhibitors and
molecular insights into binding interactions’, Arch. Pharm.
Chem. Life Sci. 2019, e1800310.
[8] U. Sharma, P. Kumar, N. Kumar, B. Singh, ‘Recent advances
in the chemistry of phthalimide analogs and their
therapeutic potential’, Mini-Rev. Med. Chem. 2010, 10, 678–
704.
[9] A. M. Alanazi, A. S. El-Azab, I. A. Al-Suwaidan, K. E. H.
ElTahir, Y. A. Asiri, N. I. Abdel-Aziz, A. M. Alaa, ‘Structure-
based design of phthalimide derivatives as potential cyclo-
oxygenase-2 (COX-2) inhibitors: anti-inflammatory and
analgesic activities’, Eur. J. Med. Chem. 2015, 92, 115–123.
[10] P. Ahuja, A. Husain, N. Siddiqui, ‘Essential amino acid
incorporated GABA-phthalimide derivatives: synthesis and
anticonvulsant evaluation’, Med. Chem. Res. 2014, 23,
4085–4098.
BACE1 Enzymatic Assay
β-Secretase inhibitory assay of the compound 7g was
performed exactly according to the manufacturer’s
instruction for BACE1 (β-Secretase) FRET Assay Kit
(Invitrogen.
http://tools.invitrogen.com/content/sfs/
manuals/L0724.pdf).
Acknowledgements
This research has been supported by a grant from the
Research Council of Tehran University of Medical
Sciences (Grant No. 97-01-108-3787).
[11] S. P. Assis, T. G. Araújo, V. L. Sena, M. T. J. Catanho, M. N.
Ramos, R. M. Srivastava, V. L. Lima, ‘Synthesis, hypolipidem-
ic, and anti-inflammatory activities of arylphthalimides’,
Med. Chem. Res. 2014, 23, 708–716.
[12] R. Antunes, H. Batista, R. M. Srivastava, G. Thomas, C. C.
Araújo, R. L. Longo, H. Magalhães, M. B. Leão, A. C. Pavão,
‘Synthesis, characterization and interaction mechanism of
new oxadiazolo-phthalimides as peripheral analgesics’, J.
Mol. Struct. 2003, 660, 1–13.
[13] C. Pessoa, P. M. P. Ferreira, L. V. C. Lotufo, M. O. de Moraes,
S. M. Cavalcanti, L. C. D. Coêlho, M. Z. Hernandes, A. C. L.
Leite, C. A. De Simone, V. M. Costa, V. M. Souza, ‘Discovery
of phthalimides as immunomodulatory and antitumor
drug prototypes’, ChemMedChem: Chem. Enabl. Drug
Discov. 2010, 5, 523–528.
[14] N. Guzior, M. Bajda, M. Skrok, K. Kurpiewska, K. Lewiński, B.
Brus, A. Pišlar, J. Kos, S. Gobec, B. Malawska, ‘Development
of multifunctional, heterodimeric isoindoline-1,3-dione de-
rivatives as cholinesterase and β-amyloid aggregation
inhibitors with neuroprotective properties’, Eur. J. Med.
Chem. 2015, 92, 738–749.
[15] Z. Sang, K. Wang, H. Wang, L. Yu, H. Wang, Q. Ma, M. Ye, X.
Han, W. Liu, ‘Design, synthesis and biological evaluation of
phthalimide-alkylamine derivatives as balanced multifunc-
tional cholinesterase and monoamine oxidase-B inhibitors
for the treatment of Alzheimer’s disease’, Bioorg. Med.
Chem. Lett. 2017, 27, 5053–5059.
[16] N. Jiang, Q. Huang, J. Liu, N. Liang, Q. Li, Q. Li, S. S. Xie,
‘Design, synthesis and biological evaluation of new
coumarin-dithiocarbamate hybrids as multifunctional
agents for the treatment of Alzheimer’s disease’, Eur. J.
Med. Chem. 2018, 146, 287–298.
[17] N. Jiang, J. Ding, J. Liu, X. Sun, Z. Zhang, Z. Mo, X. Li, H. Yin,
W. Tang, S. S. Xie, ‘Novel chromanone-dithiocarbamate
hybrids as multifunctional AChE inhibitors with β-amyloid
anti-aggregation properties for the treatment of Alz-
heimer’s disease’, Bioorg. Chem. 2019, 103027.
Author Contribution Statement
Mahmood Biglar, Bagher Larijani, and Roghieh Mirza-
zadeh contributed the reagents and materials, and
analyzed the data. Saghi Sepehri and Maryam Moham-
madi-Khanaposhtani performed the in silico studies.
Mehdi Asadi and Mostafa Ebrahimi performed the
synthesis of compounds. Hamid Nadri and Najmeh
Edraki performed the biological assay. Massoud Aman-
lou and Mohammad Mahdavi conceived and designed
the experiments.
References
[1] K. P. Kepp, ‘Alzheimer’s disease due to loss of function: A
new synthesis of the available data’, Prog. Neurobiol. 2016,
143, 36–60.
[2] C. Patterson, ‘The World Alzheimer Report 2018’, ‘The State
of the Art of Dementia Research: New Frontiers’, Alz-
heimer’s Disease International (ADI), London, 2018.
[3] A. Kumar, A. Singh, ‘A review on Alzheimer’s disease
pathophysiology and its management: an update’, Phar-
macol. Rep. 2015, 67, 195–203.
[4] M. Prior, R. Dargusch, J. L. Ehren, C. Chiruta, D. Schubert,
‘The neurotrophic compound J147 reverses cognitive
impairment in aged Alzheimer’s disease mice’, Alzheimer’s
Res. Ther. 2013, 5, 25.
[5] B. M. McGleenon, K. B. Dynan, A. P. Passmore, ‘Acetylcholi-
nesterase inhibitors in Alzheimer’s disease’, Br. J. Clin.
Pharmacol. 1999, 48, 471.
[18] G. L. Ellman, K. D. Courtney, V. Andres Jr., R. M. Feather-
stone, ‘A new and rapid colorimetric determination of
acetylcholinesterase activity’, Biochem. Pharmacol. 1961,
88–95.
[6] G. G. Osborn, A. V. Saunders, ‘Current treatments for
patients with Alzheimer Disease’, J. Am. Osteopath. Assoc.
2010, 110, S16–S26.
[19] J. A. Erickson, M. Jalaie, D. H. Robertson, R. A. Lewis, M.
Vieth, ‘Lessons in molecular recognition: the effects of
[7] J. Kara, P. Suwanhom, C. Wattanapiromsakul, T. Nualnoi, J.
Puripattanavong, P. Khongkow, V. S. Lee, A. Gaurav, L.
www.cb.wiley.com
(12 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2019, 16, e1900370
ligand and protein flexibility on molecular docking accu-
[28] T. Rosenberry, X. Brazzolotto, I. Macdonald, M. Wand-
hammer, M. Trovaslet-Leroy, S. Darvesh, F. Nachon,
‘Comparison of the binding of reversible inhibitors to
human butyrylcholinesterase and acetylcholinesterase: A
crystallographic, kinetic and calorimetric study’, Molecules
2017, 22, 2098.
racy’, J. Med. Chem. 2004, 47, 45–55.
[20] J. W. M. Nissink, C. Murray, M. Hartshorn, M. L. Verdonk,
J. C. Cole, R. Taylor, ‘New test set for validating predictions
of protein-ligand interaction’, Proteins Struct. Funct. Genet.
2002, 49, 457–471.
[21] C. A. Lipinski, ‘Drug-like properties and the causes of poor
solubility and poor permeability’, J. Pharmacol. Toxicol.
Methods 2000, 44, 235–249.
[22] D. F. Veber, S. R. Johnson, H. Y. Cheng, B. R. Smith, K. W.
Ward, K. D. Kopple, ‘Molecular properties that influence the
oral bioavailability of drug candidates’, J. Med. Chem. 2002,
45, 2615–2623.
[23] F. Cheng, W. Li, Y. Zhou, J. Shen, Z. Wu, G. Liu, P. W. Lee, Y.
Tang, ‘AdmetSAR: a comprehensive source and free tool
for evaluating chemical ADMET properties’, J. Chem. Inf.
Model. 2012, 52, 3099–3105.
[29] Marvin 15.10.12.0, 2015, ChemAxon http://www.chemax-
on.com.
[30] Schrödinger Release 2018–4: Schrödinger Release 2018–4
Protein Preparation Wizard; Epik, Schrödinger, LLC, New
York, NY, 2016; Impact, Schrödinger, LLC, New York, NY,
2016; Prime, Schrödinger, LLC, New York, NY, 2019.
[31] Schrödinger Release 2018–4: LigPrep, Schrödinger, LLC,
New York, NY, 2018.
[32] Schrödinger Release 2018–4: Desmond Molecular Dynam-
ics System, D. E. Shaw Research, New York, NY, 2019.
Maestro-Desmond Interoperability Tools, Schrödinger, New
York, NY, 2018.
[33] T. A. Halgren, R. B. Murphy, R. A. Friesner, H. S. Beard, L. L.
Frye, W. T. Pollard, J. L. Banks, ‘Glide: A New Approach for
Rapid, Accurate Docking and Scoring. 2. Enrichment
Factors in Database Screening’, J. Med. Chem. 2004, 47,
1750–1759
[24] R. Yan, R. Vassar, ‘Targeting the β secretase BACE1 for
Alzheimer’s disease therapy’, Lancet Neurol. 2014, 13, 319–
329.
[25] L. L. C. Schrodinger, ‘Schrödinger Suite 2012 Induced Fit
Docking protocol’, Glide version 5.8, New York, NY,
Schrodinger, LLC, 2009.
[26] O. Gerlits, K. Y. Ho, X. Cheng, D. Blumenthal, P. Taylor, A.
Kovalevsky, Z. A. Radic, ‘New crystal form of human
acetylcholinesterase for exploratory room-temperature
crystallography studies’, Chem.-Biol. Interact. 2019, 309,
108698–108698.
[27] F. Nachon, E. Carletti, C. Ronco, M. Trovaslet, Y. Nicolet, L.
Jean, P. Renard, ‘Crystal Structures of Human Cholinester-
ases in Complex with Huprine W and Tacrine: Elements of
Specificity for Anti-Alzheimer’s Drugs Targeting Acetyl- and
Butyrylcholinesterase’, Biochem. J. 2013, 453, 393–399.
[34] R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J.
Klicic, D. T. Mainz, M. P. Repasky, E. H. Knoll, D. E. Shaw, M.
Shelley, J. K. Perry, P. Francis, P. S. Shenkin, ‘Glide: A New
Approach for Rapid, Accurate Docking and Scoring. 1.
Method and Assessment of Docking Accuracy’, J. Med.
Chem. 2004, 47, 1739–1749.
Received July 4, 2019
Accepted September 9, 2019
www.cb.wiley.com
(13 of 13) e1900370
© 2019 Wiley-VHCA AG, Zurich, Switzerland