Novel Multitarget Directed Triazinoindole Derivatives as Anti-Alzheimer Agents

Article abstract of DOI:10.1021/acschemneuro.9b00226

The multifaceted nature of Alzheimer's disease (AD) demands treatment with multitarget-directed ligands (MTDLs) to confront the key pathological aberrations. A novel series of triazinoindole derivatives were designed and synthesized. In vitro studies revealed that all the compounds showed moderate to good anticholinesterase activity; the most active compound 23e showed an IC₅₀ value of 0.56 ± 0.02 μM for AChE and an IC₅₀ value of 1.17 ± 0.09 μM for BuChE. These derivatives are also endowed with potent antioxidant activity. To understand the plausible binding mode of the compound 23e, molecular docking studies and molecular dynamics simulation studies were performed, and the results indicated significant interactions of 23e within the active sites of AChE as well as BuChE. Compound 23e successfully diminished H₂O₂-induced oxidative stress in SH-SY5Y cells and displayed excellent neuroprotective activity against H₂O₂ as well as Aβ-induced toxicity in SH-SY5Y cells in a concentration dependent manner. Furthermore, it did not show any significant toxicity in neuronal SH-SY5Y cells in the cytotoxicity assay. Compound 23e did not show any acute toxicity in rats at doses up to 2000 mg/kg, and it significantly reversed scopolamine-induced memory deficit in mice model. Additionally, compound 23e showed notable in silico ADMET properties. Taken collectively, these findings project compound 23e as a potential balanced MTDL in the evolution process of novel anti-AD drugs.

Full text of DOI:10.1021/acschemneuro.9b00226

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Article
Novel Multi-Target Directed Triazinoindole
Derivatives as Anti-Alzheimer Agents
Dushyant V Patel, Nirav R Patel, Ashish M Kanhed, Sagar P Patel, Anshuman Sinha, Deep
D Kansara, Annie R Mecwan, Sarvangee B Patel, Pragnesh N Upadhyay, Kishan B Patel,
Dharti B Shah, Navnit K Prajapati, Prashant R. Murumkar, Kirti V Patel, and Mange Ram Yadav
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00226 • Publication Date (Web): 16 Jul 2019
Downloaded from pubs.acs.org on July 17, 2019
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Novel Multi-Target Directed Triazinoindole Derivatives as Anti-Alzheimer
Agents
Dushyant V. Patel, Nirav R. Patel, Ashish M. Kanhed, Sagar P. Patel, Anshuman Sinha, Deep D.
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Kansara, Annie R. Mecwan, Sarvangee B. Patel, Pragnesh N. Upadhyay, Kishan B. Patel, Dharti
B. Shah, Navnit K. Prajapati, Prashant R. Murumkar, Kirti V. Patel, Mange Ram Yadava*
Faculty of Pharmacy, Kalabhavan Campus,
The Maharaja Sayajirao University of Baroda, Vadodara- 390001
Gujarat, India
Abstract: The multifaceted nature of Alzheimer's disease (AD) demands treatment with
multitarget-directed ligands to confront the key pathological aberrations. A novel series of
triazinoindole derivatives were designed and synthesized. In vitro studies revealed that all
the compounds showed moderate to good anti-cholinesterase activity; the most active compound
2
3e showing IC value of 0.56 ± 0.02 M for AChE and IC value of 1.17 ± 0.09 M for BuChE.
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These derivatives are also endowed with potent anti-oxidant activity. To understand the plausible
binding mode of the compound 23e, molecular docking studies and molecular dynamics
simulation studies were performed and the results indicated significant interactions of 23e within
the active sites of AChE as well as BuChE. Compound 23e successfully diminished H O -
2
2
induced oxidative stress in SH-SY5Y cells and displayed excellent neuroprotective activity
against H O as well as A-induced toxicity in SH-SY5Y cells in a concentration dependent
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manner. Furthermore, it did not show any significant toxicity in neuronal SH-SY5Y cells in the
cytotoxicity assay. Compound 23e did not show any acute toxicity in mice at doses up to 2000
mg/kg, and significantly reversed scopolamine-induced memory deficit in mice model.
Additionally, compound 23e showed notable in silico ADMET properties. Taken collectively,
these findings project compound 23e as a potential balanced MTDL in the evolution process of
novel anti-AD drugs.
Keywords: Alzheimer’s disease, MTDL, anticholinesterase, antioxidant, neuroprotection,
triazinoindole
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Introduction
Alzheimer’s disease (AD) is an irrevocable age-related neurodegenerative disorder
clinically identified by progressive deterioration in memory, cognitive deficit, abnormal behavior
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and incoherent language. It is the most prominent form of dementia. More than 50 million people
are suffering from it worldwide, and the number will significantly rise up to 152 million by 2050
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if no cure or preventive measures are found. This burgeoning number of people suffering from
AD, in both developed and developing countries, has drawn the attention of medicinal chemists to
accelerate research on drug discovery in this area.
The etiology of AD is still enigmatic. Different factors, like low levels of neurotransmitter
acetylcholine (ACh),3 aggregation of the -amyloid peptide,4,5 accumulation of
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hyperphosphorylated tau protein, dyshomeostasis of biometals, oxidative stress, mitochondrial
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dysfunction and neuroinflammation
are proposed to play pivotal roles in the pathogenesis of
AD. To combat AD like diseases having a complex etiology, development of multitarget-directed
ligands (MTDLs) is recognized as one of the most assuring drug discovery approaches.14-17 Drugs
acting on a single target even though having high affinity and selectivity for their targets might not
influence the mysterious etiology of the disease satisfactorily. An MTDL having moderate, but
balanced affinities for the targets can still exert more beneficial effects compared to a single-
targeted molecule. Concurrent effects on several therapeutic targets make MTDLs superior in
altering the complex equilibrium of the cellular network.18,19 A mild and balanced activity on
multiple therapeutic targets might secure higher safety, and reduce the risk of therapeutic
resistance.20
After perusal of current research in this field, we focused our attentions on inhibition of
cholinesterase and anti-oxidant potential of new chemical entities (NCEs) as the core objectives
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of anti-AD drug design. Two types of cholinesterase enzymes (ChEs) namely acetylcholinesterase
(AChE) (EC 3.1.1.7) and butyrylcholinesterase (BuChE) (EC 3.1.1.8), are found in the central
nervous system. Both these enzymes belong to the carboxylesterase family of enzymes and
play an important role in cholinergic transmission through hydrolysis of the neurotransmitter
ACh. Although AChE and BuChE are produced by different genes they are highly homologous
with more than 65 % similarity in their active sites.21,22 AChE has two major binding sub-sites, a
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peripheral anionic site (PAS) and the other a catalytic active site (CAS). The CAS of the enzyme
is actively involved in the maintenance of cholinergic neurotransmission. PAS is involved in the
formation of -amyloid fibrils that are associated with plaque deposition.24,25 AChE inhibitors
blocking both CAS and PAS simultaneously could alleviate the cognitive defect in AD patients by
elevating ACh levels and have also been endowed with disease modifying ability by inhibiting the
amyloid plaque formation.26 In healthy brains, AChE is more active than BuChE and can
hydrolyze about 80 % of ACh. Current studies have demonstrated that as the disease progresses,
the ability of BuChE increases by 40-90 %, and that of AChE declines in the hippocampus and
temporal cortex areas of the brain.27-29 BuChE plays several roles both in neural and non-neural
functioning. Clinical data suggested that the high cortical levels of BuChE were associated with
some important AD hallmarks, such as extracellular deposition of the A and aggregation of
hyper-phosphorylated tau protein.21,30,31 This reflects the important role played by both the
cholinesterases and the necessity to develop MTDLs which could act on both these ChEs.32
Recent research has emphasized the significance of oxidative stress in the fundamental
molecular mechanism of AD.8,9 Oxidative stress occurs when there is an inequity between the
formation and quenching of free radicals formed from oxygen species. These reactive oxygen
species (ROS) are regarded as the other major etiological factor of AD, since the role of the ROS
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in the formation of both amyloid plaques and neurofibrillary tangles is confirmed. Through
pathological oxidation-reduction steps, ROS can denature biomolecules like proteins, lipids and
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nucleic acids. This can cause tissue damage through necrosis and apoptosis. Thus, oxidative
stress plays a central role in the pathogenesis of AD leading to neuronal dysfunction and cell
death.35
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Considering the facts that inhibition of AChE provides a symptomatic treatment to AD,
increase in levels of BuChE are observed in the brains of AD patients, and the ROS system plays
a vital role in neurodegeneration, a series of triazinoindole derivatives were designed, synthesized
and evaluated in the current report for their multifactorial anti-AD activities, including
cholinesterase inhibitory activity, antioxidant activity, cytoprotective effect against H O /A-
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induced cell injury and acute toxicity in animal models. The in silico ADMET properties of the
synthesized derivatives were predicted and the structure-activity relationship of these novel
derivatives has been discussed. Molecular modeling studies were performed to offer an insight
into the binding mode of the derivatives with the target proteins, and these findings were supported
by the time dependent stability analysis of the ligand-receptor complex of the most active ligand
and the receptor under study, by means of molecular dynamics simulations.
Designing aspects
To combat a disease like AD having complex etiology, development of MTDLs has been
recognized as one of the most assuring drug discovery approaches. In spite of considerable
research on new targets available for AD treatment, the cholinesterase inhibitors still remain the
drugs of choice, although they provide symptomatic and transient benefits to the
patients. Oxidative stress plays an important role in pathogenesis of AD. However antioxidant
molecules all alone might not be enough to treat such highly complex pathological disease
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like AD. So dual cholinesterase inhibitors endowed with additional anti-oxidant and
neuroprotective properties could increase the chances of combating AD successfully.
Indole ring is prodigiously present in many natural compounds possessing invaluable
medicinal and biological properties.37,38 Melatonin is an indole ring containing pineal
neurohormone whose levels decrease during aging, especially in AD patients.39-41 It is endowed
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with strong free radical scavenging properties. The high reactivity of melatonin with ROS is
apparently due to its electron-rich indole ring, acting as an electron donor. Recent studies have
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also shown that melatonin offers protective effects against A-induced apoptosis, glutamate-
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induced excitotoxicity, nitric oxide toxicity, decreases neurofilament hyperphosphorylation
and augments learning and memory in rats. In recent years, many indole based hybrids e.g. tacrine-
melatonin hybrids,46 donepezil-chromone-melatonin hybrids,47 melatonin-N,N-dibenzyl(N-
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methyl)amine hybrids, and carbamate derivatives of indolines, have been designed to act as
multifunctional agents for the treatment of AD. Furthermore, the indole moiety is present in
several CNS-active drugs like rizatriptan and oxypertine. Thus indole ring offers a privileged
scaffold for CNS-active drugs which could augment the search for new therapeutics for AD. 1,2,4-
Triazine nucleus is another important structural system present in several biologically active
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their potential anti-AD,51-54 antianxiety, antiepileptic and anti-depressant activities. Some
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triazine derivatives have been reported to be potent neuroprotective agents against H O -
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induced cell death in PC12 cell line. In recent past our research group had reported substituted
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diaryltriazines as potential anti-AD entities. Reports of remarkable biological activities
associated with the indole ring and the 1,2,4-triazine nucleus prompted us to fuse these two
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privileged scaffolds into a single moiety which could potentially offer multiple favorable activities
for the treatment of AD.
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ChE inhibition
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SH
NH2
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R
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(III)
Figure 1. Molecular hybridization approach to design triazinoindole derivatives.
Different 5H-[1,2,4]triazino[5,6-b]indole-3-thiol and 5H-[1,2,4]triazino[5,6-b]indol-3-
amine derivatives were designed by following a rational hybridization approach (Figure 1). The
molecular interactions of the designed derivatives were evaluated theoretically by means of
docking the designed compounds within the active site of AChE and BuChE. Both the designed
scaffolds showed promising binding affinities within the active sites of AChE as well as BuChE
(Figure 2). The 5H-[1,2,4]triazino[5,6-b]indole-3-thiol scaffold showed stability within the CAS
of AChE (docking score: -7.96) by forming - interactions with Trp84 and Phe330 and hydrogen
bonding with His440. While, the same scaffold in the active site of BuChE (docking score: -7.21)
exhibited - interactions with Trp82, Trp430 and His438 along with hydrogen bonding with
His438. Another designed scaffold 5H-[1,2,4]triazino[5,6-b]indol-3-amine showed similar -
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interactions but offering higher binding affinities with AChE (docking score: -8.89) and BuChE
(docking score: -7.22) enzymes due to additional strong hydrogen bonding caused by the 3-amino
group. During the in vitro enzyme inhibition study, 5H-[1,2,4]triazino[5,6-b]indole-3-thiol showed
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1.26 µM and 55.81 µM inhibitory activity (IC ) against AChE and BuChE respectively, whereas
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H-[1,2,4]triazino[5,6-b]indol-3-amine was found to possess IC values of 9.76 µM and 51.25
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µM against AChE and BuChE, respectively. On the basis of these moderately promising activities
of the designed scaffolds, modifications were carried out by incorporating various alkyl/benzyl
substituents to understand the SAR and to come up with some potential leads. Accordingly,
compounds of the four series (I-IV) as shown in Figure 1, were synthesized and discussed here in
this work.
Figure 2. Interaction of 5H-[1,2,4]triazino[5,6-b]indole-3-thiol with (A) AChE, (B) BuChE, and 5H
[1,2,4]triazino[5,6-b]indol-3-amine with (C) AChE and (D) BuChE.
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RESULTS AND DISCUSSION
Chemistry
Compounds 2, 3a-3l and 5a-5k were synthesized as depicted in Scheme 1. Compound 2
was obtained by condensation of commercially available isatin (1) with thiosemicarbazide in
aqueous potassium carbonate solution at reflux conditions. The clear liquid obtained was acidified
by glacial acetic acid to get the condensed product 2. Alkylation or benzylation of compound 2
with the corresponding alkyl/benzyl halides gave thio-substituted compounds 3a-3l. Similarly, N-
substituted 1,2,4-triazino-[5,6-b]indole-3-thiol derivatives 5a-5k were synthesized by
condensation of the N-substituted isatins 4a-4k with thiosemicarbazide. N-Alkylation or N-
benzylation of the isatins were achieved with the corresponding alkyl/benzyl halides in the
presence of potassium carbonate in DMF.
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Scheme 1. Synthesis of compounds 2, 3a-3l and 5a-5k.
N N
O
O
SH
(i)
(ii)
N
O
O
N
N
N
R
4a-4k
H
H
2
1
(ii)
(i)
N N
R
R
N N
S
3a
SH
Methyl
3b Ethyl
c isoPropyl
3d Butyl
5a Methyl
5b Ethyl
5c isoPropyl
R
N
N
3
N
N
R
H
5d
Butyl
5e Benzyl
3
e Benzyl
3a-3l
5a-5k
3f 2-Methylbenzyl
5f 2-Bromobenzyl
5g 3-Fluorobenzyl
5h 4-Chlorobenzyl
5i 4-tertButylbenzyl
5j 4-Methoxybenzyl
5k 4-Methylbenzyl
3g 3-Fluorobenzyl
3h 4-Bromobenzyl
3i 4-Cyanobenzyl
3j 4-Methoxybenzyl
3k 4-Methylbenzyl
3l
Phenethyl
Reagents and conditions. (i) Thiosemicarbazide, K
CO , DMF.
2
CO
3
, H
2
O, reflux, overnight; (ii) alkyl/substituted benzyl halides,
K
2
3
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When the substituted isatins were subjected to cyclization reaction with aminoguanidine
instead of thiosemicarbazide, uncyclized N-substituted isatin-3-aminoguanylhydrazones were
obtained instead of the 5-substituted [1,2,4]triazino[5,6-b]indol-3-amine derivatives. All efforts
made to cyclize these N-substituted isatin-3-aminoguanylhydrazones to get 5-substituted
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5
5
5
5
5
5
5
5
6
0
1
2
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7
8
9
0
1
2
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9
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[1,2,4]triazino[5,6-b]indol-3-amine derivatives failed and only the non-cyclized starting
compounds were obtained. In one attempt, cyclization of N-benzylisatin-3-aminoguanylhydrazone
in the presence of a strong base and diethylene glycol as a polar protic solvent, the hydrolyzed
product, i.e. 2-(benzylamino)benzoic acid was isolated.
In an alternative approach, the desired amino derivatives were synthesized from the thiol
derivatives. The thiol derivatives were first methylated and the methylthio derivatives were treated
6
0
with various amines. Unfortunately the thiomethyl derivatives failed to react with the amines. So
the thiomethyl group at the C-3 position was converted to sulfone, as it has more electron
withdrawing ability compared to the parent thiomethyl group. This was achieved by oxidizing the
thiomethyl group by mCPBA to sulfone. When these sulfone derivatives were reacted with amines
in THF under refluxing conditions, the sulfone group was substituted by the amino group offering
the desired substituted [1,2,4]triazino[5,6-b]indol-3-amine derivatives 7a-7j. Similarly,
compounds 5 were methylated and further oxidized to the corresponding sulfone compounds 9a-
9
j. These sulfone compounds were reacted with ammonia to obtain the desired 5-substituted
[1,2,4]triazino[5,6-b]indol-3-amine derivatives 10a-10j (Scheme 2).
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5
5
5
5
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Scheme 2. Synthesis of compounds 7a-7j and 10a-10j.
O
N
N N
O
N
N
N
S
S
NH
R
(i)
(ii)
Me
Me
N
N
N
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
H
3a
6
7a-7j
R
R
7a Propyl
7f 3,4-Dimethoxybenzyl
7g 2-Picolyl
7h 2-Furanylmethyl
7i 2-(1-Piperidinyl)ethyl
7
7
7
7
b isoButyl
c Benzyl
d 4-Chlorobenzyl
e 4-Methoxybenzyl 7j 2-(4-Morpholinyl)ethyl
O
N
N
N N
N
N
O
SH
SMe
S
(iii)
(i)
Me
N
N
N
N
N
N
R
R
R
5
3a, 8a-8j
6, 9a-9j
R
(ii)
8
8
8
8
8
8
8
8
8
3
a 9a 10a Methyl
b 9b 10b Ethyl
c 9c 10c Propyl
d 9d 10d Butyl
e 9e 10e Benzyl
N
N
NH2
N
f
9f 10f 2-Methylbenzyl
N
R
g 9g 10g 4-Methylbenzyl
h 9h 10h 4-Chlorobenzyl
i
9i 10i
3-Fluorobenzyl
H
1
0a-10j
a 6 10j
Reagents and conditions. (i) mCPBA, DCM, 0 °C to RT; (ii) Amines/ammonia, THF, reflux; (iii) MeI, K
2
CO
3
, DMF.
The aminoalkylamines 18a-20h required for the synthesis of compounds 21a-23h were
prepared through Gabriel synthesis using phthalimide as the starting material. Phthalimide (11)
was reacted with dibromoalkanes to form N-(bromoalkyl)phthalimides 12-14. The desired basic
1
2
amines (a-h; R R NH) were reacted with N-(bromoalkyl)phthalimides 12-14 to yield compounds
1
5a-17h. Hydrazinolysis of compounds 15a-17h in methanol offered the desired aminoalkyl-
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amines 18a-20h in 67–87 % yields. These aminoalkylamines 18a-20h were reacted with
compound 6 as discussed previously to obtain the desired final products 21a-23h (Scheme 3).
Scheme 3. Synthesis of compounds 21a-23h.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
O
O
O
R1
N R2
N R2
n
(i)
Br
n
(ii)
(iii)
NH
N
N
H N
2
n
O
O
O
11
12, n = 4
15a-15h, n = 4
16a-16h, n = 5
17a-17h, n = 6
18a-18h, n = 4
19a-19h, n = 5
20a-20h, n = 6
13, n = 5
14, n = 6
O
S
N N
O
N N
R1
R1
N
(iv)
NH
N R2
n
Me
N
N
R2
H N
2
n
N
N
H
H
6
18a-18h, n = 4
19a-19h, n = 5
20a-20h, n = 6
21a-21h, n = 4
22a-22h, n = 5
23a-23h, n = 6
1
2
R R N =
Me
Me
N
N
N
Me
Me
N
Me
Me
Me
Me
a
b
c
d
N
N
N
N
O
N
Me
e
f
g
h
1
2
Reagents and conditions. (i) Br(CH
2
)
n
Br, K
2
3
CO , TEBAC, acetone, RT; (ii) HNR R , TEA, MeOH, reflux; (iii)
NH NH .H O, MeOH, reflux; (iv) THF, reflux.
2
2
2
Biological evaluation
Inhibition studies on AChE and BuChE
The potential of the synthesized compounds to inhibit cholinesterases (ChEs) was
evaluated in vitro using a spectrophotometric method of Ellman et al. using donepezil and tacrine
as reference drugs as previously reported by our group.59,61,62 The obtained IC values of the
5
0
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2
2
2
2
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6
compounds for the two enzymes and their selectivity over each other were summarized in Table
1
-3. All the tested compounds showed IC50 values for both the enzymes in micromolar to
submicromolar ranges. Compounds 23e and 23f exhibited the highest inhibition of AChE (IC50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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4
5
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values of 0.56 µM and 0.67 µM, respectively) and BuChE (IC values of 1.17 µM and 0.84 µM,
5
0
respectively).
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Table 1. In vitro Inhibition of AChE, BuChE and Selectivity Index (SI) of compounds 2, 3a-
3
l and 5a-5k.
N N
N
N
S
SH
R
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
H
R
(2, 3a-3l)
(5a-5k)
IC50 ± SEM (µM)
IC50± SEM(µM)
AChEa BuChEb
SIc
Compd
2
R
SIc
Compd
R
AChEa
BuChEb
H
11.26 ± 1.24
12.62 ± 1.45
55.81 ± 2.12
4.96
4.37
5a
5b
Me
7.33 ± 0.82 58.61 ± 1.23
7.99
10.9
3
a
Me
55.24 ± 2.04
48.40 ± 1.93
8.50 ± 0.77
6.16 ± 0.53
91.62 ± 2.76
35.28 ± 3.01
Me
Me
Me
3
b
16.01 ± 1.42
3.02
5c
5.72
Me
Me
Me
3c
10.03 ± 0.83
12.28 ± 1.66
33.69 ± 1.76
40.08 ± 1.51
3.35
3.26
5d
5e
5.36 ± 0.46
8.05 ± 0.78
14.26 ± 1.29
75.29 ± 2.82
2.66
9.35
Me
3
d
Me
Br
3
e
20.33 ± 1.47
9.88 ± 0.75
19.16 ± 1.43
42.05 ± 1.68
76.50 ± 2.51
84.96 ± 2.02
2.07
7.74
4.43
5f
5g
5h
15.35 ± 1.02 22.01 ± 1.34
1.43
12.7
1.49
Me
F
3f
5.52 ± 0.40
70.22 ± 2.56
F
3
g
Cl
15.73 ± 0.98 23.47 ± 1.76
Me
3
h
Br
CN
17.49 ± 1.32
19.02 ± 1.65
9.64 ± 0.87
15.52 ± 1.37
9.93 ± 0.92
51.93 ± 2.95
75.99 ± 2.77
46.74 ± 2.69
49.34 ± 2.85
61.42 ± 2.13
2.97
3.99
4.84
3.18
6.18
5i
5j
Me
Me
13.97 ± 0.93 18.24 ± 1.11
13.21 ± 1.52 19.02 ± 1.34
1.30
1.44
0.52
0.14
81.3
3i
OMe
3
j
OMe
Me
5k
Me
5.73 ± 0.51
2.97 ± 0.90
3
k
Tacrine
0.056 ± 0.01 0.008 ± 0.00
3
l
Donepezil
0.023 ± 0.01
1.87 ± 0.08
a
AChE from human erythrocytes; IC50, 50% inhibitory concentration (means ± SEM of three experiments).
BuChE from equine serum.
Selectivity Index = IC50 (BuChE)/IC50 (AChE).
b
c
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2
2
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4
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4
4
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5
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Table 2. In vitro Inhibition of AChE, BuChE and Selectivity Index (SI) of compounds 7a-7j
and 10a-10j.
N N
N N
NH
R
NH2
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
R
H
(
7a-7j)
(
10a-10j)
IC50± SEM (µM)
IC50± SEM (µM)
AChEa BuChEb
Compd
R
SI
Compd
10a
R
SI
AChEa
BuChEb
7
a
11.07 ± 0.86 52.19 ± 2.01 4.71
Me
7.81 ± 1.01
8.13 ± 1.32
52.23 ± 1.76 6.69
29.42 ± 1.21 3.62
Me
Me
7
b
9.21 ± 0.54
61.23 ± 1.12 6.64
56.41 ± 1.65 6.99
10b
Me
Me
7
c
8.06 ± 0.75
10c
10.12 ± 1.16 32.78 ± 1.47 3.24
Me
7
d
Cl
41.22 ± 1.83
9.09 ± 0.65
0.22
10d
10e
9.26 ± 0.82
16.02 ± 1.10 1.73
Me
7
e
OMe
36.63 ± 1.22 67.32 ± 1.32 1.84
10.75 ± 0.62 13.12 ± 1.02 1.22
20.57 ± 1.34 24.87 ± 1.38 1.21
17.23 ± 1.22 32.72 ± 1.32 1.90
OMe
OMe
Me
7
f
10f
7
g
14.71 ± 1.37 26.08 ± 1.21 1.77
20.22 ± 1.21 33.02 ± 1.65 1.63
10g
10h
Me 22.09 ± 1.78 22.64 ± 1.49 1.02
Cl 24.63 ± 2.01 29.45 ± 1.62 1.20
N
O
7
h
F
7
i
6.16 ± 0.53
6.61 ± 0.69
20.53 ± 2.01 3.33
10i
10j
20.22 ± 1.25 25.32 ± 1.23 1.25
N
7
j
9.14 ± 0.54
1.38
H
9.76 ± 1.25
51.25 ± 1.32 5.25
N
O
a
b
c
AChE from human erythrocytes; IC50, 50% inhibitory concentration (means ± SEM of three experiments).
BuChE from equine serum.
Selectivity Index = IC50 (BuChE)/IC50 (AChE).
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Table 3. In vitro Inhibition of AChE, BuChE and Selectivity Index (SI) of compounds 21a-
2
3h.
N N
R1
NH
N
N
R2
n
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H
(21a-23h)
IC50± SEM (µM)
IC50± SEM (µM)
Compd
1
2
SI
Compd
1
2
SI
n
R R N
n
R R N
AChEa
BuChEb
AChEa
BuChEb
Me
N
N
N
2
1a
4
4
33.70 ± 1.12 6.39 ± 0.22
0.19
22e
22f
5
5
0.85 ± 0.05
17.01 ± 0.40 20.0
Me
N
Me
Me
2
1b
6.31 ± 0.43 11.09 ± 0.27 1.75
0.96 ± 0.04
2.77 ± 0.05
6.74 ± 0.21
2.89
0.36
Me
Me
N
N
O
21c
4
4
4
4
2.47 ± 0.11
8.05 ± 0.51
3.26
0.02
2.19
22g
22h
23a
5
5
6
6
18.58 ± 0.41
1.65 ± 0.11
1.25 ± 0.09
N
Me
Me
N
2
1d
20.70 ± 0.76 0.47 ± 0.03
29.19 ± 1.10 17.7
N
Me
Me
N
N
2
1e
2.69 ± 0.08
3.17 ± 0.10
5.89 ± 0.70
4.43 ± 0.27
3.54
Me
N
N
Me
Me
21f
18.01± 1.31 5.68
23b
2.40 ± 0.11
12.70 ± 0.42 5.29
Me
Me
N
N
21g
21h
4
4
5
5
15.01 ± 0.54 1.92 ± 0.15
0.13
23c
23d
23e
6
6
6
6
1.48 ± 0.08
1.43 ± 0.07
0.56 ± 0.02
3.43 ± 0.24
4.01 ± 0.32
1.17 ± 0.09
2.32
2.80
2.09
O
N
N
Me
Me
2.61 ± 0.07 32.19 ± 1.02 12.3
3.58 ± 0.28 23.24 ± 0.67 6.49
N
Me
Me
N
N
N
2
2a
Me
N
Me
Me
22b
6.79 ± 0.22
0.34 ± 0.03
0.05
23f
0.67 ± 0.02
0.84 ± 0.03
1.25
Me
Me
N
N
22c
22d
5
5
2.77 ± 0.07
2.76 ± 0.18
0.48 ± 0.03
0.38 ± 0.05
0.17
0.14
23g
23h
6
6
4.16 ± 0.15
0.79 ± 0.04
23.65 ± 1.38 5.69
O
N
Me
Me
N
3.92 ± 0.21
4.96
N
Me
a
AChE from human erythrocytes; IC50, 50% inhibitory concentration (means ± SEM of three experiments).
BuChE from equine serum.
Selectivity Index = IC50 (BuChE)/IC50 (AChE).
b
c
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Structure-activity relationships
To validate our design rationale, initially compound 2 was prepared and evaluated
initially for its cholinesterase inhibitory potential. To our delight, as shown in Table 1. Compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
exhibited good inhibitory activity (AChE, IC = 11.26 µM; BuChE, IC = 55.81 µM). This
50 50
encouraging result of the lead scaffold prompted us to explore various substituents on thiol sulphur
and the nitrogen of the indole ring to frame a structure-activity relationship. Introducing alkyl
substituents on the indole ring as in compounds 5a-5d increased the inhibitory activity. Among
them, compound 5d with the butyl chain showed the most potent inhibitory activity (AChE, IC50
=
5.36 µM; BuChE, IC = 14.26 µM). Incorporation of benzyl and substituted benzyls at NH of
50
indole ring decreased the AChE inhibitory activity except for compounds 5g and 5k. Compound
g and 5k having 3-fluorobenzyl and 4-methylbenzyl groups, respectively showed potent
inhibitory activities. The thio-substituted compounds 3a-3l offered IC values for AChE in the
5
5
0
range of 9-20 µM. Presence of various substituents on thiol group did not influence the activity
significantly in comparison to the lead compound 2. The inhibitory activity was retained as such
when thiol group was replaced with amino group (Table 2). When the N-propyl moiety in
compound 7a was replaced with 2-(1-piperidinyl)ethyl (compound 7i) and 2-(4-morpholinyl)ethyl
(compound 7j) moieties, inhibitory activities against both the enzymes got increased notably.
Compounds 7i and 7j showed inhibitory activities (AChE; IC =6.16 µM, 6.61 µM, respectively)
5
0
and (BuChE; IC = 20.53 µM, 9.14 µM, respectively).
50
Joining of the tricyclic triazinoindole moiety with cyclic amines like piperidine and
morpholine through two carbon atom spacers improved AChE inhibition dramatically compared
to the simple alkyl/benzyl substituted triazinoindole derivatives. Based on this observation, we
planned to study the effect of the attached basic amines and the length of the linker on the
1
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cholinesterase inhibitory activity of the resulting compounds. As indicated in Table 3, all the
compounds 21a-23h exhibited good inhibitory activity against both the enzymes ranging from IC50
values of 0.564 to 36.7 μM for AChE and from 0.341 to 32.19 μM for BuChE. These results
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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suggested that the presence of a heterocyclic amines is particularly important for the ChE
inhibitory activity as all the compounds bearing pyrrolidino, piperidino, morpholino and N-
methylpiperazino moieties exhibited strong ChE inhibition. Pyrrolidino moiety appeared to be a
better choice over other amines, as compounds 21e, 22e and 23e bearing pyrrolidino moiety
exhibited higher activity than the rest of the compounds having other amines as attachments
(Figure 3).
It has been observed that changing the length of the carbon chain also potentially affected
the inhibitory activity. A comparative analysis of the inhibitory potential of compounds 21e, 22e
and 23e having a pyrrolidine ring as heterocyclic amine, revealed that compound 23e (n = 6, IC50
value of 0.564 μM) exhibited the highest AChE inhibitory activity while compound 21e (n = 4,
IC value of 2.690 μM) and compound 22e (n = 5, IC value of 0.853 ± 0.02 μM) showed 5-fold
5
0
50
and 1.5-fold decrease in AChE inhibitory activities in comparison to compound 23e (Figure 3).
However, their inhibitory activity against BuChE was slightly lower than that against AChE, which
might be due to the conformational differences between the structures of these two enzymes.63
Compound 23e (IC value of 1.178 μM) exhibited an acceptable level of BuChE inhibitory
5
0
activity while compound 21e (IC value of 5.893 μM) and compound 22e (IC value of 17.01
5
0
50
μM) showed 5-fold and 14-fold decrease in BuChE inhibitory activities. Compounds 23e (SI value
of 2.09) and 23f (SI value of 1.25) have more balanced activity on both the cholinesterase enzymes
than donepezil (SI value of 81.3) and tacrine (SI value of 0.14).
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Figure 3. Important structural features of compound 23e.
Antioxidant activity [1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity]
The DPPH radical scavenging assay is commonly used as a rapid and reliable method to
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assess the antioxidant/free radical scavenging potential of compounds. DPPH is a stable free
radical that can accept a hydrogen radical or an electron to become a stable molecule. The
antioxidant activity of the selected compounds was estimated by their ability to reduce DPPH.
radical (purple color) to DPPHH (yellow) and the corresponding radical-scavenging potential was
65
evaluated by the decrease in the absorbance at 517 nm. Only those compounds having IC values
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0
(
AChE) less than 5 μM were selected for this study. Ascorbic acid was used as the positive control
in this assay. All the test compounds exhibited notable free radical scavenging activity ranging
from 40−55 % and 56-70 % at 10 μM and 20 μM concentrations respectively (Table 4).
Compound 23e showed better free radical scavenging activity (54.9 %, 64.3 % at 10 μM
and 20 μM concentrations, respectively) compared to ascorbic acid (36.5 %, 61.8 % at 10 μM and
2
0 μM concentrations, respectively) where as tacrine and donepezil were found to be devoid of
significant free radical scavenging activity at these concentrations.
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Table 4. DPPH Radical Scavenging Activity of the Compounds.a
RP of DPPH (%)b
RP of DPPH (%)b
Compd
Compd
10 μM
20 μM
10 μM
20 μM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
21c
21e
21f
45.5 ± 3.1
52.1 ± 2.4
51.5 ± 1.6
53.7 ± 2.7
43.2 ± 3.3
44.3 ± 2.4
46.7 ± 1.9
53.2 ± 2.4
52.8 ± 2.9
54.1 ± 1.7
44.1 ± 3.4
57.3 ± 2.9
63.1 ± 2.3
62.7 ± 1.7
65.4 ± 1.3
60.2 ± 2.7
59.4 ± 3.1
60.7 ± 3.3
62.4 ± 2.5
60.7 ± 2.9
65.1 ± 2.1
61.3 ± 2.3
23b
23c
43.7 ± 2.5
42.7 ± 3.7
47.3 ± 2.0
54.9 ± 1.8
54.3 ± 2.1
56.7 ± 1.6
55.1 ± 3.4
3.4 ± 0.4
60.3 ± 3.4
58.4 ± 3.1
59.6 ± 2.2
64.3 ± 2.8
66.4 ± 2.4
67.3 ± 1.7
64.2 ± 1.9
6.4 ± 0.3
4.9 ± 1.2
61.8 ± 3.2
23d
21h
22a
22c
22d
22e
22f
23e
23f
23g
23h
Tacrine
Done.
4.5 ± 0.6
Ascorbic
acid
22h
23a
36.5 ± 2.9
a
Data are expressed as Mean ± SE (three independent experiments)
RP of DPPH (%) = reduction percentage of DPPH.
b
Assessment of Cytotoxicity and Neuroprotection offered by the synthesized compounds
To ascertain the therapeutic potential of the synthesized compounds, their effect on cell
viability and neuroprotective ability against oxidative stress were assessed using the human
neuroblastoma SH-SY5Y cell line. Only those compounds having IC values (AChE) less than 5
5
0
μM were selected for these studies. For the assessment of cytotoxicity of the test compounds, cells
were exposed to significantly high concentrations of the test compounds (40 μM and 80 μM) for
2
4 hours, followed by determination of the cell viability using MTT assay. Even at such high
concentrations, all the test compounds caused negligible cell death (Table 5).
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Table 5. Cell viability, and neuroprotective action of the selected test compounds against
a
H O and A induced toxicity in the human neuroblastoma SH-SY5Y cell line.
2
2
1-42
Neuroprotection (%)
against H
Neuroprotection (%)
(against A1-42
Cell viability (%)
(
2
O
2
)
)
Compd
0
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4
0 μM
80 μM
10 μM
20 μM
10 μM
20 μM
2
2
1c
1e
92.4 ± 2.2
93.0 ± 1.5
91.9 ± 2.4
91.3 ± 3.2
92.2 ± 3.1
92.4 ± 2.5
90.4 ± 3.3
93.1 ± 2.7
93.9 ± 3.2
90.5 ± 3.7
92.4 ± 3.2
93.2 ± 2.1
94.3 ± 2.2
91.4 ± 2.8
94.9 ± 3.2
94.5 ± 2.1
92.1 ± 2.7
91.4 ± 3.2
90.1 ± 1.8
92.4 ± 2.5
90.3 ± 1.8
91.2 ± 2.1
87.1 ± 3.3
85.7 ± 2.4
90.5 ± 1.9
89.8 ± 3.1
87.1 ± 2.2
90.6 ± 1.5
92.2 ± 1.9
86.4 ± 2.9
89.3 ± 2.5
91.7 ± 1.9
91.1 ± 3.2
87.4 ± 2.3
92.3 ± 1.5
91.8 ± 2.8
89.7 ± 1.9
85.4 ± 2.7
88.1 ± 2.4
90.7 ± 1.2
34.4 ± 2.7
39.8 ± 1.9
41.7 ± 1.3
37.2 ± 2.8
30.4 ± 3.6
31.8 ± 3.5
33.5 ± 2.9.
41.8 ± 3.5
43.4 ± 2.2
41.2 ± 2.7.
32.7 ± 3.4
31.2 ± 2.5
35.3 ± 1.4
34.2 ± 3.5
42.5 ± 2.1
44.8 ± 1.7
40.3 ± 1.8
41.2 ± 1.4
51.0 ± 3.4
53.4 ± 3.1
48.9 ± 2.3
53.2 ± 3.2
55.4 ± 1.9
50.3 ± 2.9
46.5 ± 2.3
48.4 ± 2.2
48.7 ± 3.1
54.3 ± 1.7
55.6 ± 2.1
50.2 ± 2.8
43.5 ± 2.6
40.4 ± 3.6
46.1 ± 2.0
49.5 ± 2.7
53.7 ± 1.3
54.5 ± 2.4
52.3 ± 2.2
51.6 ± 2.5
70.4 ± 3.2
72.1 ± 2.7
37.5 ± 2.1
40.3 ± 2.5
42.1 ± 1.9
41.5 ± 2.7
39.4 ± 3.1
38.1 ± 2.8
38.5 ± 2.5
43.7 ± 3.1
41.4 ± 2.4
40.3 ± 1.9
37.1 ± 2.3
37.7 ± 3.1
39.1 ± 2.6
38.8 ± 1.9
44.5 ± 2.1
43.2 ± 2.5
45.3 ± 2.2
42.7 ± 3.1
40.5 ± 2.3
47.2 ± 2.1
56.2 ± 1.9
57.5 ± 2.2
60.7 ± 1.4
59.0 ± 1.5
57.3 ± 2.2
56.2 ± 2.9
59.5 ± 2.3
60.1 ± 3.4
62.4 ± 1.9
58.7 ± 2.1
59.3 ± 3.3
57.2 ± 3.4
57.5 ± 2.3
58.3 ± 2.9
62.6 ± 1.9
63.6 ± 2.2
61.4 ± 3.3
62.5 ± 2.5
58.7 ± 2.7
65.1 ± 1.8
2
1f
1h
2a
2c
2d
2e
2f
2h
3a
3b
3c
3d
3e
3f
3g
3h
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Tacrine
Donep.
aData are expressed as Mean ± SE (three independent experiments)
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The neuroprotective potential of the selected compounds against the oxidative stress
induced by exogenous toxins (H O or A ) was evaluated. H O -induced toxicity is due to the
2
2
1-42
2
2
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oxidative damage to the neuronal cells while the A-induced toxicity is more complex, involving
generation of reactive oxygen species, interleukin-1, interleukin-6, TNF- like damaging
cytokines’ release, and mitochondrial dysfunction.33,67
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In this study, when the cells were exposed to H O (100 μM) and A
(25 μM)
1-42
2
2
separately, notable toxicities to cells were observed and the cell viability got declined to nearly
55% and 52%, respectively. To assess the neuroprotective potential of the test compounds against
these toxic insults, the cells were pretreated with the test compounds (10 μM, and 20 μM) for 2 h
followed by treatment with the insults for 24 h. The selected derivatives exhibited a significant
neuroprotective effect at 10 μM and 20 μM concentrations. For comparison, the cells were co-
incubated with compound 23e, 23f, donepezil and tacrine at different concentrations. Compound
2
3e offered significant protection to the cells against the toxic insults (Figure 4). The results
suggested that these compounds possessed the ability to protect neuronal cells against oxidative-
stress-associated cell death.
Figure 4. Protective effects of compounds 23e, 23f, donepezil and tacrine against H
cytotoxicity in SH-SY5Y cells. Determination of the viability of SH-SY5Y cells by the MTT assay after treatment
with (A) H (100 μM) and (B) A1-42 (25 μM) in the absence or presence of the indicated concentrations of
2 2
O and A1-42-induced
2
O
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compounds 23e, 23f and the standard drugs. All the data were expressed as mean ± SD of three experiments and each
included triplicate sets. #p < 0.05 vs control; **p < 0.01, ***p < 0.001 vs H O /A1-42 alone.
2 2
In vitro Blood-Brain Barrier Permeation Assay
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The BBB permeability is a primary criterion for the development of novel CNS active
agents. The ability of triazinoindole derivatives to penetrate into the brain was assessed using a
parallel artificial membrane permeation assay (PAMPA), in a similar manner as described by Di
et al.68,69 This assay is used to predict the passive diffusion of a molecule through the BBB. The
BBB permeability (P ) of the most active compounds 23e and 23f was determined through a
e
porcine brain lipid. The assay was validated by comparing the experimental permeability values
[P (exp)] of seven commercial drugs with the reported permeability values [P (ref)], offering a
e
e
2
linear relationship i.e., P (exp.) = 1.16 P (ref.) + 0.1668 (R = 0.9781) (see the Supporting
e
e
Information, Figure S1). From this equation and considering the limits for BBB permeation
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established by Di et al., it was concluded that compounds with P (exp) greater than 4.8 × 10
e
-1
cm s (see the Supporting Information, Table S2) were capable of crossing the BBB. Both the
compounds 23e and 23f showed permeability values above this limit (Table 6). Therefore,
Pe(exp) suggested a high potential of the compounds to cross the BBB by passive diffusion.
-6
-1
Table 6. Permeability (P 10 cm s ) of compounds 23e, 23f and donepezil in the PAMPA-
e
BBB permeation assay with their predicted penetration into the CNS.
-6
-1
Compd.
3e
(P 10 cm s )
Prediction
e
2
11.3 ± 1.8
12.5 ± 1.2
14.3 ± 1.7
CNS+
CNS+
CNS+
23f
Donepezil
Data are expressed as mean ± SEM of three independent experiments.
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Computational studies
Docking studies
To have an idea of the binding mode of compound 23e with the cholinesterase enzymes,
docking studies were performed within the active sites of Torpedo Californica AChE (TcAChE)
and Human BuChE (hBuChE). To validate the generated grids for docking studies, the co-
crystallized molecules in the 3D structures of TcAChE and hBuChE (PDB Code: 2CKM and
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BDS respectively) were initially knocked out of the binding sites. The knocked out molecules
were constructed a fresh, energy minimized and re-docked into the active sites of the grids. Very
similar interactions were observed between the re-docked molecules and the enzymes as was the
case with the original co-crystallized ligands. The RMSD values of the re-docked ligands with
those of the original orientations in co-crystallized forms in 2CKM and 4BDS were observed to
be 0.40 Å and 0.26 Å, respectively.
It is well known that AChE has a dumbbell shaped active site gorge composed of two active
sites: the CAS at the bottom and the PAS at the lip. The CAS pocket looks like a vessel on its
bottom lies Trp84 which is crucial for binding of both the substrates to the inhibitors. Moreover,
halfway up the gorge is a narrow tunnel where some crucial amino acid residues such as Phe330
and Tyr-334 stabilize the enzyme-inhibitor complex. At the entrance of the gorge exists the PAS,
which is mainly built up by Trp279 and Tyr70 residues. The docking interactions of compound
2
3e were studied in the active site of the AChE of TcAChE, and then TcAChE was humanized
with hAChE to know the human sequence interacting with compound 23e.
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Figure 5. Docking interactions of compound 23e in the active sites of (A) AChE and (B) BuChE.
The interaction view of compound 23e with AChE is represented in Figure 5A. As
presented, compound 23e was stacked well in the groove formed by Trp84, Trp279 and Phe330
(hAChE: Trp86, Trp284 and Tyr337) amino acid residues. It has orientation along the active site
gorge similar to the reference compound donepezil, extending from the active site amino acid
residue Trp84 to the peripheral site amino acid residue Trp279. Ligand interactions with these two
amino acids are important to elicit a strong inhibitory effect by the dual binding site inhibitors.
Hydrophobic planar nature of the tricyclic scaffold allows making of a more favorable sandwich
type - stacking with Trp84 and Phe330 (hAChE: Trp86 and Tyr337). The -NH group of the
indole ring exhibited hydrogen bonding with Gly80 (hAChE: Gly82). The -NH group at the 3rd
position of the triazine ring showed hydrogen bonding with Ser122 (hAChE: Ser125). The
nitrogen atom of pyrrolidine ring could be protonated at physiological pH. The protonated nitrogen
showed strong cation- interaction with Trp279 (hAChE Trp284). This interaction is particularly
important as Trp279 plays a prominent role in deposition of beta-amyloid plaques.24
The binding mode of the compound 23e with BuChE revealed that it also occupied the
large catalytic cavity of BuChE (Figure 5B). The aromatic ring of the tricyclic moiety was
observed to be interacting with Trp231 and Phe329 residues by - stacking. -Alkyl interaction
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was observed between the aromatic ring of the scaffold and Leu286. Further, the -NH group on 3rd
position added stability to the ligand-receptor complex by forming hydrogen bond with Ser287.
Additionally, salt bridge between -NH of the pyrrolidine and Asp70, and cation- interaction
between the nitrogen of pyrrolidine and Trp82 provided stability to the ligand-receptor complex.
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Molecular dynamics simulations
In the molecular docking studies, compound 23e exhibited very good interactions within
the active sites of the AChE and BuChE enzymes. Thus to confirm, validate and understand the
time dependent interactions of the active compound 23e with the AChE and BuChE enzymes and
stability of ligand-receptor complex, molecular dynamics study was performed. The binding
stability of the ligand-receptor complex was studied for a period of 20 ns duration to check its time
dependent stability. In order to observe the binding stability of the complex over a period of time,
certain parameters like RMSD-P, RMSF-P and RMSD-L (P = Protein; L = Ligand) were
scrutinized to support the results of docking study. Initial pose of the ligand-receptor complex was
considered as the reference frame to calculate these values. The RMSD-P is essentially studied to
understand the level of movements of various atoms/groups in the enzyme when the ligand is
present in the active site of the receptor. This provides insight into the structural conformations of
the enzyme over a given period of simulation. The RMSD-P for AChE in the ligand -receptor
complex was found to be in the range of 0.7 to 2.0 Å. This finding explained that the presence of
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3e in the active site of the AChE has not prejudiced the stability of the protein backbone all the
way throughout the simulation period. To recognize the stability of the ligand with respect to the
receptor and its active binding site, the RMSD-L of 23e was determined. The ‘Lig fit’ on Prot
RMSD-L for ligand was observed in the range of 0.8 to 3.2 Å. The ‘Lig fit Prot’ is the RMSD of
a ligand when the protein-ligand complex is first aligned on the reference protein backbone and
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then the RMSD of the ligand heavy atoms is determined. Here, despite having a large number of
rotatable bonds in the ligand, the RMSD value is not observed to be significantly higher than the
protein RMSD, suggesting that the compound 23e is stable within the binding site and it does not
diffuse away from the active site during the entire course of simulation period. Additionally the
Lig fit on Lig RMSD was calculated to comprehend the internal fluctuation of the ligand atoms,
and it was observed in the acceptable range of 0.4 to 2.4 Å (Figure 6A).
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The structural integrity of the receptor and the residual mobility of the ligand were
quantified in terms of RMSF-P (Figure 6B). For all the residues, including the loop as well as the
terminal residues of the protein, with the compound 23e in the active site, the RMSF-P was below
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.2 Å. The protein-ligand stability interaction study was also performed over a period of time to
evaluate the interaction stability. In the docking studies, the H-bond was observed between –NH
of indole and –NH of 3-amino group of compound 23e with Gly80 and Ser122 (hAChE: Gly82
and Ser125) residues respectively. From the MD simulation study it was established that the two
–NH groups of the compound 23e formed H-bonds with Gly80 and Ser122. These were observed
to be stable over 54 % and 91 % of the simulation time with Gly80 and Ser122 respectively.
Further, the salt bridge between the protonated pyrrolidine and Asp72 (hAChE: Asp74) was
observed to be stable for around 24 % of the simulation time. All these H-bond strengths were
with H-bond distance of 2.5 Å or less and donor angle of ≥ 120° and acceptor angle of ≥ 90°. The
cation- interactions between the protonated pyrrolidine and Trp279 and Tyr334 (hAChE: Trp284
and Tyr341) were also found to be stable over 76 % and 29 % of entire simulation time
respectively. Further, strong hydrophobic interactions of the compound 23e with Trp-84, Trp-279
and Phe-330 played a vital role in providing stability to the ligand-receptor complex where all
these hydrophobic interactions were observed for more than 60 % of the total simulation time
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(
Figure 6C). The other ligand parameters like rGyr, MolSA, SASA and PSA were observed in the
acceptable range as shown in Figure 6D.
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Figure 6. (A) RMSD-P and RMSD-L plots for AChE with 23e; (B) RMSF-P plot for AChE with 23e; (C) Ligand and
receptor residue contact diagram for AChE with 23e; (D) rGyr; MolSA; SASA and PSA for 23e with AChE.
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Similarly, the molecular dynamics study of compound 23e with BuChE was also performed
and this also indicated good stability of ligand-receptor complex. The RMSD-P was in the range
of 0.8 to 1.9 Å. Whereas the RMSD-L ‘Lig fit’ on Prot was observed in between 1.2 to 3.2 Å which
was not alarmingly higher than the RMSD-P. The ‘Lig fit’ on the ligand was observed in 0.4 to
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(Figure 7B). The ligand-receptor interaction analysis elucidated the stability of ligand-receptor
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complex by supporting the H-bonding between -NH of indole and -NH at 3 position of the
compound 23e with protein residues Leu286 and Ser287, respectively. These were observed to be
stable over 34 % and 87 % of simulation time respectively. The protonated pyrrolidine formed a
stable salt bridge with Asp70 over 96 % of the simulation time period. The residues Trp82, Trp231,
Leu286, Phe329 and Tyr332 were positively contributing towards the stability of the complex by
hydrophobic interactions (Figure 7C). The other ligand parameters like rGyr, MolSA, SASA and
PSA were also observed in the acceptable range (Figure 7D). These observations from the
molecular dynamics study strongly supported the observations made in the docking studies.
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Figure 7. (A) RMSD-P and RMSD-L plots for BuChE with 23e; (B) RMSF-P plot for BuChE with 23e; (C) Ligand
and receptor residue contact diagram for BuChE with 23e; (D) rGyr; MolSA; SASA and PSA for 23e with BuChE.
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