Experimental and Computational Evaluation of Piperonylic Acid Derived Hydrazones Bearing Isatin Moieties as Dual Inhibitors of Cholinesterases and Monoamine Oxidases

Accepted Article

Title: Experimental and computational evaluation of some piperonylic

acid derived hydrazones bearing isatin moiety as dual inhibitors

of cholinesterases and monoamine oxidases

Authors: Senthil Raja Ayyannan, Vishnu M S, Pavankumar V, and

Sandeep Kumar

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content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201900277

Link to VoR: http://dx.doi.org/10.1002/cmdc.201900277

A Journal of

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Experimental and computational evaluation of some piperonylic acid derived hydrazones

bearing isatin moiety as dual inhibitors of cholinesterases and monoamine oxidases

Vishnu M S, Pavankumar V, Sandeep Kumar, Senthil Raja A*^[a]

[a]

Vishnu M S, Pavankumar V, Senthil Raja A

Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical Engineering & Technology,

Indian Institute of Technology (Banaras Hindu University)

Varanasi 221005, Uttar Pradesh (India) Fax: (+91)542-2368428

E-mail: asraja.phe@iitbhu.ac.in

Abstract

Aset of piperonylic acid derived hydrazones with variable isatin moiety was synthesized and evaluated

for their inhibitory activity against the enzymes acetylcholinesterase (AChE), butyrylcholinesterase

(BChE), and monoamine oxidase A and B (MAO-A/B). The results of the in vitro studies revealed

compounds possessing IC₅₀values in the micromolar range, with the majority of them showing higher

selectivity towards MAO-B isoform. The compound N-(2-oxo-1-(prop-2-ynyl)indolin-3-

ylidene)benzo[d][1,3]dioxole-5 carbohydrazide (compound 3) was identified as lead AChE inhibitor

with IC₅₀= 0.052 ± 0.006 μM while N‐ [(3E)‐ 5‐ chloro‐ 2‐ oxo‐ 2,3‐ dihydro‐ 1H‐ indol‐ 3‐

ylidene]‐ 2H‐ 1,3‐ benzodioxole‐ 5‐ carbohydrazide (compound 2) proved to be a lead MAO-B

inhibitor with IC₅₀= 0.034 ± 0.007 μM, possessing 50 times more selectivity than MAO-A. The kinetic

studies revealed that compounds 2 and 3 displayed competitive and reversible inhibition of AChE and

MAO-B respectively. The molecular docking studies revealed the significance of hydrophobic

interactions along the active site pocket of the enzymes under inspection. Further lead optimization

studies can favor the development of potential neurotherapeutic agents.

Introduction

Monoamine oxidases (MAOs) are an important family of enzymes which have been extensively

explored in the treatment of depression among patients worldwide. The MAO enzymes comprise 2

isozymes: MAO-A and MAO-B. The former has a high affinity towards substrates serotonin and

norepinephrine, while the latter prefers phenyl ethylamine and benzylamine. These enzymes are

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homologous and share 70% similarity with respect to structure, both containing the cofactor flavin

adenine dinucleotide (FAD) covalently bonded to the amino acid cysteine (Cys406 and Cys397 for

MAO-A and MAO-B respectively)^[1]. MAO-B enzyme is distributed in the CNS, while the MAO-A

isozyme is more profound in the peripheral parts of the body. These enzymes metabolize the substrates

via the oxidative deamination process, resulting in the generation of hydrogen peroxide (H₂O₂),

ammonia (NH₃) and the aldehyde metabolite of the substrate. The generated H₂O₂diffuses into

neighboring cells and takes part in the generation of reactive oxygen species (ROS) which results in

apoptosis and cell death^[2].

The cholinesterase (ChE) family, consisting of acetylcholinesterase (AChE) and butyrylcholinesterase

(BChE), are important enzymes, mainly localized between the synaptic cleft and glial cells^[3]. It serves

the function of hydrolyzing the acetylcholine (ACh) and butyrylcholine (BCh) neurotransmitter

respectively, resulting in the termination of cholinergic neurotransmission from the brain to the

peripheral parts of the body. ACh plays a pivotal role in the management of learning and memory

processing.

Both the MAOs and ChEs are important biomarkers in the diagnosis of advanced stages of

Alzheimer’s disease (AD) and Parkinson’s disease (PD). The MAO-B levels are shown to be elevated

to 3~4 folds on aging neurons, while MAO-A concentration is estimated to be ~6 folds greater in

cardiovascular tissues^[4]. MAO mediated ROS generation results in many pathological conditions

including cardiovascular diseases (MAO-A) and neurodegeneration (MAO-B), some of the leading

causes of death in the present world. The oxidative stress induced disintegration of neurons is an

important model which has guided in the development of some of the important marketed MAO-B

inhibitors, which include selegiline, rasagiline, and safinamide, to treat AD and PD. The availability

of a wealth of co-crystallized structures, more than 40 in number, for the MAO-B isozyme has

tremendously favored research in the development and validation of virtual lead compounds via the

application of high throughput virtual screening and molecular modeling methodologies. It has been

revealed that in patients with AD, the serum levels of AChE and BChE are elevated to a great extent,

leading to an uncontrolled reduction in the levels of ACh, which causes an imbalance in motor

coordination and, problems in cognition and memory. It has been identified that the BChE activity is

increased 40-90% than the normal conditions in the critical parts of the brain in AD diagnosed patients,

while the AChE activity is shown to be either reduced or steady^[5]. This intensifies the symptoms

associated with AD and therefore proves to be a good target to develop potential inhibitors to bring

equilibrium to the induced drastic conditions. At present, four AChE inhibitor drugs are approved to

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treat the symptomatic effects of AD, namely tacrine, galantamine, donepezil, and rivastigmine. Lately,

the enzyme BChE has shown great promise in the development of non-selective AChE/BChE

inhibitors in AD symptomatic treatment as well^[6,7]. The contemporary research has shown an

exponential increase in the drug candidates developed as inhibitors of ChE in the clinical pipeline, but

a significant amount fails due to adverse side effects towards the conclusion of one phase study. As of

now the suggested approach to treat AD with the available drugs is to incorporate individualization

and adjustment in the current treatment regime according to the age and gender.

According to the National Institute of Aging (2016 report), National Institute of Health (NIH), AD is

among the six leading causes of death in the USA ^[8]. It is estimated that approximately 5.7 million

people are suffering from AD in the USA alone. Reports from WHO show an estimate of 47.5 million

people worldwide living with dementia. The total number of individuals with Alzheimer’s dementia

is anticipated to rise number to 75.6 million peoples in 2030 and 135.5 million peoples in 2050, with

the majority of sufferers living in low and middle-income countries (Alzheimer's, 2014)^[9]. Out of all

the major treatment difficulties the “effectiveness of current treatment regimens” poses a major issue

of concern. And for this reason, modification of the disease pathway by means of neuroprotective

therapy is an essential unmet clinical necessity. During the period 2000 to 2015, a staggering 123%

increase in mortality was observed in patients with AD. About 112 drug candidates are being tested in

the clinical pipeline as of 2018, out of which 26 candidates are in Phase III trials^[10]. Though the

number of clinical candidates is soaring high, there is also a substantial decline in the progress to the

next level of evaluation, owing to the rise in toxicity concerns and efficacy issues.

In 2014, the first multi-therapy formulation, Namzaric®, was developed, which is a combination of

NMD Areceptor antagonist, memantine, andAChE inhibitor donepezil hydrochloride^[11]. This regimen

has brought about a major shift in the current treatment methodology with an added benefit of targeting

multiple pathways to AD pathogenesis. Ladostigil, a dual inhibitor of ChE and MAO has recently been

developed and has now entered the Phase IIb clinical pipeline. The molecule is composed of a

carbamate skeleton and a propargylamine chain, resembling rasagiline structure^[12]. It is a selective

inhibitor of the MAO-B enzyme and ChE and has shown to lower 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP) toxicity in PD animal models^[13]. The success of this molecule in the phase

I trials has given an insight into targeting the ChE and MAO pathways to AD and PD.

Science as of today has diversified approaches to treating these diseases and one of the most interesting

approaches being the development of chemical entities to target the particular enzymes responsible for

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the disease condition. From single targeting to dual targeting, the approach has gained a lot of insight

into the world of neurotherapeutics. When the single targeting failed to improve upon the disease state,

a new methodology was adapted so as to tackle this rising problem, which surfaced much earlier than

expected as the now prominent multi target-directed ligands (MTDLs) to treat diseases with multi-

faceted pathophysiology as in the case of AD and PD^[14]. This not only poses a challenge but a great

venture to discover new agents and possible druggable targets. To treat such diseases the viable

macromolecules, which include mainly the enzymes and receptors, are studied in depth to identify

regions of selectivity, activity, and other allosteric sites, in case of explored targets, giving the effect

necessary for possible treatment. All in all, there are more than fifty known target enzymes for

neurodegeneration available till date, having pivotal roles in the disease conditions mentioned above.

The study of these protein targets in great detail has been made possible through the development of

Protein Data Bank (PDB), which provides a library of bound and unbound crystal structures of the

enzymes in all of their known conformations^[15]. Such tools have greatly enhanced the conception of

novel ideas in the development of new molecules with the required features to adapt and target the

enzyme at the right spatial orientation so as to facilitate the much anticipated neuroprotective action.

Figure 1. Some developed MTDLs targeting MAO and ChE.

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Heterocyclic rings are explored extensively in the synthesis of potent compounds with dual inhibitory

properties in the field of neurotherapeutics. Many different naturally occurring scaffolds like

coumarins, pyridines, thiazoles, etc. have utilized in the past few years in developing highly potent

dual inhibitors of MAO and ChE with IC₅₀values within the nanomolar range as shown in Fig

1^{[16,17,18,19,20]}. Isatin is one of the most important scaffolds with a multifaceted pharmacological profile

in the treatment of CNS diseases, especially neurodegenerative disorders like AD, and PD^[21]. The

scaffold has higher selectivity towards MAO-B than MAO-A^[22,23], and its oral administration has

shown to increase the level of ACh and DA in the rat striatum^[24]. Owing to its well-tolerated profile

in human beings, we propose to explore this versatile scaffold into developing lead molecules as multi

target-directed ligands with dual MAO and ChE^[25]inhibition properties.

In the previous work, we have identified potential dual inhibitors of MAO-B and AChE possessing a

3,4-methylenedioxy aniline scaffold and a variable aryl moiety connected by a semicarbazino linker

of the type: R-NH-(C=O)-NH-N=C-R¹(R²)^[25]. The present work attempts to replace the semicarbazino

bridge with a hydrazone linker, by the removal of NH group present at the amido terminal of the

semicarbazino spacer, to further optimize the identified leads. The design approach follows the same

strategy as our previous work, by adopting an electron-rich spacer molecule with a greater affinity

towards forming hydrogen bonds, in this case, a hydrazone bridge, and attaching hydrophobic aryl

groups at the carbimino and amido terminal of the spacer^[26]. The carbimino terminal was introduced

with isatin moiety with varying degree of hydrophobicity and steric properties offered by substituents

at N-1 and 5-position. Thus, a series of 10 compounds have been synthesized in the wet-lab, which

was further taken up for biological and computational studies. All the test compounds were

characterized by FT-IR, 1H, 13C, and mass spectrometry methods. The in vitro biological evaluation

was performed using the AChE, BChE, and MAO-A/B enzyme inhibition assay. Computational

docking methodology was explored to correlate and study in depth the binding characteristics of the

designed ligand with the target proteins ^[27]. The molecular modeling studies were performed using

cheminformatic tools like ChemDraw 12.0 Pro, AutoDock 4.2, Discovery Studios 2017, and Pymol^[28]

Computational prediction of Drug-likeness and Pharmacokinetic properties were carried out using

online platforms such as PKCSM, and Protox servers^[29,30]

.

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Figure 2. The design strategy for piperonylic acid-based hydrazones.

Results and Discussions

Scheme 1. Synthesis of compounds 1-10. Reagents and conditions: a) conc. H₂SO₄/MeOH, Reflux, 80^oC, 2 hrs; b) EtOH,

NH₂NH₂.H₂O, reflux, 80^oC, 2 hrs; c) glacial AcOH/EtOH, reflux, 80^oC, 2-8 hrs.

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Chemistry

Table 1. Physicochemical data for compounds 1-10.

Compd

R

H

R₁

cLogP^[a]

2.186

2.899

3.162

3.836

1.929

3.049

4.83

Yield%

94

MP [^oC]

266-269

286-290

190-195

192-194

295-299

294-296

210-213

199-201

196-199

220-222

R_f^[b]

0.64

0.72

0.53

0.56

0.78

0.69

0.44

0.52

0.40

0.42

H

1

2

Cl

H

C₃H₃

90.5

86.5

3

H

C₃H₅

84.36

88.2

4

NO₂

Br

H

5

H

94.6

6

C₆H₅CH₂

4-F-C₆H₅CH₂

2,4-Cl₂- C₆H₅CH₂

79.1

7

H

4.973

6.256

5.686

81.5

8

Cl

92.81

9

H

87.34

10

^[a]Computational logP value calculated from ChemDraw Pro 12.0.

^[b]Solvent system: Ethylacetate/n-Hexane 3:7.

The synthesis of piperonylic acid derived hydrazones containing substituted isatin moiety is described

in Scheme 1 from 2H-1,3-benzodioxole-5-carbohydrazide^[31]. The reaction of piperonylic acid with

methanol in the presence of concentrated sulphuric acid produced the ester intermediate methyl 2H-

1,3-benzodioxole-5-carboxylate; the hydrazide was prepared by the reaction of the ester with

hydrazine monohydrate using ethanol as a solvent by refluxing. Final products 1-10 were prepared in

good yields (79 – 94%) by acid-catalyzed condensation of the hydrazone with the appropriate N-

substituted and 5-substituted isatin fragments. The physicochemical and spectral data of the final

compounds are in great alignment with the structural composition. The physicochemical

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characterization data of the final compounds 1-10 are given in Table 1 and the spectral data are

described in the experimental section.

In vitro ChE inhibition assay

The in vitro inhibition potential of compounds 1-10 against electric eel AChE (eeAChE) was

determined using a UV-based spectrophotometric method of Ellman et al., with Donepezil as the

reference standard^[25]. The results of AChE and BChE inhibition assays are expressed in terms of IC₅₀

values and are summarized in Table 2. Absorbance readings were taken in triplicate measure for all

the compounds used for the study. The synthesized compounds displayed good activity profile in the

AChE inhibition assay, where the IC₅₀values are obtained in the micromolar range. Compound 2

displayed the highest activity among the series with an IC₅₀value of 0.052 ± 0.006 µM close to the

standard drug Donepezil. The second best inhibition profile was observed in the case of compound 3,

which displayed an IC₅₀of 0.85 ± 0.107 µM, followed by compound 10, with an IC₅₀of 0.928 ± 0.024

µM. Compound 5 was identified with the lowest activity, an IC₅₀of 25.13 ± 2.687 µM.

In the BChE inhibition assay^[20], most of the compounds displayed moderate to excellent results as

compared to the reference standard Donepezil. Compound 3 was identified with the highest activity

among the series, with an IC₅₀value of 0.88 ± 0.007 µM, almost comparable to Donepezil standard.

Compounds 2 and 8 followed as the next best inhibitors possessing IC₅₀values of 0.96 ± 0.005 µM

and 0.89 ± 0.085 µM respectively. Similar toAChE inhibition assay results, compound 5 was identified

with the lowest IC₅₀value among the series, about 13.786 ± 2.163 µM.

Among the series, compounds 8, 9 and 10 have halogen substituents (chloro and fluoro groups) within

the variable aryl region present in the carbimino terminal of the hydrazone spacer. Detailed structure-

activity relationship (SAR) studies have been documented for the series after a thorough analysis of

the inhibitory activity data from Table 2.

Table 2. In vitro and computational results of ChE inhibition studies for compounds 1-10.

Compd

AChE

BChE

IC₅₀[µM]^[a]ΔG [kcal mol^-1]^[b]Ki [µM]^[b]

IC₅₀[µM]^[a]

1.28±0.067

ΔG [kcal mol^-1]^[b]Ki [µM]^[b]

4.72±0.063

-9.91

0.055

-8.08

1.19

1

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0.052±0.006

0.85±0.107

1.925±0.086

25.13±2.687

6.95±0.451

1.272±0.179

2.93±0.84

-9.73

-10.47

-10.37

-9.31

-9.91

-11.55

-11.47

-11.57

-11.51

-

0.073

0.022

0.026

0.151

0.058

0.0034

0.0039

0.0032

0.0036

-

0.96±0.005

0.88±0.007

5.26±0.753

13.786±2.163

8.23±0.068

4.48±0.26

-9.14

0.199

0.288

0.746

0.744

0.198

0.061

0.058

0.2

2

3

-8.92

-8.36

-9.14

-9.84

-9.87

-9.14

-10.06

-

4

5

6

7

0.89±0.085

1.59±0.58

8

5.89±0.223

0.928±0.024

0.021±0.005

9

1.64±0.076

0.78±0.025

0.042

-

10

DNP

[a] Each IC₅₀value is the mean ± SEM. Level of statistical significance: p<.05 versus the corresponding IC₅₀

values obtained against ChEs, as determined by ANOVA/Dunnett’s. Reference inhibitors: DNP – Donepezil.

[b] Computationally predicted values.

SAR for AChE inhibition

In general, substitution at N-1 position in the isatin ring by bulky aromatic rings and aliphatic side-

chains resulted in an overall improvement in the AChE inhibitory activity, as in the case of compounds

3, 4, 7, 8, and 10, in comparison with the compound 1, containing no substitution in the isatin ring. An

exception being compound 9, with para-fluoro benzyl substitution at the R₁position and chloro

substitution at the R₂position, where the activity was lowered as compared to the unsubstituted

derivative, compound 1. An increase in activity was observed when the R₁position was substituted by

aliphatic propargyl and allyl side chains (compounds 3 and 4), where the former showed better profile,

as compared to compounds substituted by halogenated benzyl derivatives. Substitution at the R₁

position by un/substituted benzyl groups led to an increase in activity. Mono- and Di- substitution at

the para and ortho positions of the benzyl groups in the R₁region gave rise to better AChE activity, as

in the case of compounds 8 and 10, where it was observed that the di-substitution with 2-chloro groups

gave a better profile. Substitution at the R₁position by para-substituted benzyl ring with a fluorine

group gave rise to a decrease in AChE inhibitory activity as compared to the other benzyl substituted

derivatives. It was observed that the substitutions at the 2^|and 4^|positions of the benzyl ring

(compound 10) favor a better activity than the mono-substituted and un-substituted benzyl derivatives,

compounds 7, 8 and 9. Introduction of the highly electronegative nitro group at the R₂position of the

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isatin moiety, compound 5, gave rise to a drastic decrease in the AChE inhibitory activity as compared

to bromo and chloro substituted derivatives, compounds 6 and 2 respectively. Substitution by bromo

group (compound 6) at the R₂position gave rise to a decrease in potency as compared to chloro group

(compound 2), which led to a drastic increase in the inhibitory activity, and the un-substituted isatin

derivative compound 1. Substitution at the R₂and R₁position by chloro group and p-fluoro benzyl

group, respectively, led to a decrease in the inhibitory activity as compared to compounds 1-4, 7, 8,

and 10. Chlorine substitution at the R₂position, compound 2, gave the highest activity as compared to

the rest of the series.

SAR for BChE inhibition

The R1 position of the isatin moiety, when substituted with benzyl groups containing halogen atoms,

led to a decrease in the activity as compared to compound 1, except in the case of compound 8, where

it is substituted with a p-fluoro benzyl group. Disubstitution at 2 and 4 positions by chloro group and

mono substitution at the para position by fluoro group in the benzyl ring led to better BChE activity

than the un-substituted derivative, as shown by compounds 8, 9, and 10 compared to compound 7.

Compound 8 surfaced out with better activity as compared to the benzyl substituted derivatives.

Substitution by aliphatic propargyl and allyl group at the R₁position gave rise to mixed results. In the

case of the former, compound 3, the BChE inhibitory activity was found to be greater than that of the

latter, compound 4. Compound 3 possessed greater inhibitory activity than all the compounds of the

series. The R₂position of the isatin moiety when substituted with bromo group, compound 6, led to a

decrease in activity as compared to the chloro substituted derivative, compound 2, but showing better

activity as compared to compound 5. Substitution at R₂position by chloro group and R₁position by

para-substituted fluoro benzyl group, compound 9, led to a decrease in activity as compared to

compound 2 and compound 8, which are monosubstituted each in the positions described above.

Kinetic studies of lead AChE inhibitors

To understand the inhibition mechanism of the lead compounds 2 and 3 on AChE, kinetic studies were

carried out using eeAChE. The Lineweaver–Burk plots for compound 2 and compound 3 (Fig 3A and

Fig 3B) were linear and intersected at the Y-axis. This pattern shows that both the lead compounds

inhibited AChE competitively, and further extrapolation of the results proved that they bind reversibly

to AChE.

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Figure 3. Kinetics and time-dependent inhibition of eeAChE by compound 2 (A and C) and compound 3 (B and D).

Lineweaver-Burk plot of the eeAChE catalyzed oxidation of ACTI in the absence (control) and presence of various

concentrations (2.5 µM, 5 µM, 10 µM) of compound 2 (A) and compound 3 (B). Time-dependent inhibition of AChE

catalyzed oxidation of ACTI by compound 2 (C) and compound 3 (D). Rate data are expressed as nmol product

formed/min/mg protein. data are the mean ± SEM of n=3 determinations.

Determination of K_ivalues for lead AChE inhibitors 2 and 3

The dissociation constant (K_i) was calculated to understand the strength of enzyme-inhibitor

interaction of lead AChE inhibitor compounds 2 and 3. K_ifor the lead compounds 2 and 3 were

calculated using GraphPadPrism, with a value of 6.22 ± 0.002 nM and 37.06 ± 0.014 nM against

eeAChE, respectively. Between the IC₅₀value and Ki value obtained for compounds 2 and 3, an 8-

fold and 23-fold difference were observed, suggesting compact binding of the inhibitor to the enzyme.

Reversibility studies of lead AChE inhibitors 2 and 3

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The most active AChE inhibitors, compounds 2 and 3, were further examined for time-dependent

inhibition studies to understand the reversible/irreversible nature of enzyme inhibition. As depicted in

Fig 3C and Fig 3D, there was no time-dependent decrement in the rates of AChE-catalysed hydrolysis

of acetylthiocholine iodide (ACTI) when both the compounds were pre-incubated with AChE for

various periods of time (0, 15, 30 and 60 min). From this observation, it can be concluded that AChE

inhibition by both the leads is reversible in nature, at least over 60 a min time period. An increase in

the AChE catalytic rates with increased pre-incubation times of both the compounds were observed

with the enzyme.

Molecular docking studies of ChE inhibitors

To understand the binding orientation of the inhibitors, molecular docking studies were performed

using AutoDock 4.2. The X-ray crystal structures of the recombinant human AChE (PDB code: 4EY7)

and human BChE (PDB code: 5DYW) were taken from the Protein Data Bank. The PDB assessment

criteria were used to screen a total of more than 200 structures to select the appropriate PDBs (Bound

configurations) of the ChEs^[27]. Iterative gold standard pose (GSP) analysis was performed in the cross-

validation of docking poses, where troubleshooting was performed in alienating problematic structures

as in the case of BChE structure. The docking results for AChE and BChE were expressed in terms of

theoretical inhibition constants (Ki values) and estimated free energies of binding (ΔG) for each virtual

enzyme-inhibitor complex.

Pose analysis of AChE inhibitors

Examination of the computationally docked binding poses of all the compounds within the AChE

active site resulted in the following observations: All the test set of inhibitors were observed to fit well

into the active site gorge of AChE formed by the catalytic anionic site (CAS) and the peripheral anionic

site (PAS) and were enclosed by the residues: Trp236, Gly121, Asp74, Gly122, Ser125, Tyr124,

Leu289, Trp286, Tyr341, Arg296, Phe297, Phe338, Tyr341 and His447. In all the test inhibitors the

(methylenedioxy)phenyl moiety binds to the catalytic anionic site, while the variable isatin template

binds to the peripheral anionic site. This reveals that almost all the test inhibitors exhibit a completely

reverse orientation as the reference drug Donepezil, except in the case of compounds 2 and 6, which

followed similar binding pose as compared to donepezil. Further, these inhibitors are stabilized by

hydrogen bonding and hydrophobic (π-alkyl) interactions as shown in Fig 4A and Fig 4B.

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Almost all the compounds were shown to undergo hydrogen bonding interaction with the residues

Phe295, Phe296, Gly122, Gly121, Ala204, Asp74, Ser203, and His447. The residue Phe295 is

common in all the compounds undergoing hydrogen bonding. Compound 1 formed hydrogen bonding

with the residues Phe295 and Arg296; compound 2 formed hydrogen bonding interactions with Asp74

and Phe295; residues Phe295, Gly122, Gly121 and Ala204 formed hydrogen bonding with compound

3; compound 4 was involved in hydrogen bond formation with the residues Phe295 and Tyr124;

compound 5 was involved in hydrogen bonding with residues Ser203 and His447; compounds 7, 8,

and 9 were involved in hydrogen bonding with common residues Phe295 and Ser203, with the

exception of two extra interactions with the residues His447 in the case of compound 8 and Asp74 in

the case of compound 9; compound 10 with the residues Phe295, Tyr124 and His447.

In addition, all the compounds showed hydrophobic (π-alkyl and π-sigma) interactions with the

residues Trp286, Leu289, Tyr124, Tyr341, Arg296, Val294, Phe338, Tyr337, and Tyr124. The leads

discovered in the study compounds 2 and 3 were shown to undergo hydrophobic π-sigma interactions

with Val294, Phe338 and Tyr337, and Trp286 respectively as shown in Fig 4D; compound 3 was also

involved in π-alkyl interactions with the residues Leu289, Tyr341, Tyr124, and Arg296.

Binding mode of lead AChE inhibitor compound 2

Assessment of the virtual complex of the lead AChE inhibitor compound 2 and AChE reveals that the

(methylenedioxy)phenyl moiety was bound to the gorge forming the PAS while the variable isatin ring

occupies the CAS. The hydrazone linker was found situated in the middle of the gorge between CAS

and PAS. The phenyl ring associated with the dioxole ring was found to be interacting with Val294

residue via π-sigma hydrophobic interactions at an inter-planar distance of approximately 3.89 Å. The

phenyl ring associated with the isatin template formed π-sigma hydrophobic interactions with the

residues Tyr337 and Phe338 at an inter-planar distance of approximately 3.5 Å and 3.4 Å respectively.

The oxygen atom present in the carbimino terminal was involved in hydrogen bonding interaction

with Phe295 at an inter-planar distance of approximately 2.0 Å. Additionally, the nitrogen atom in the

isatin scaffold interacted with the residue Asp74 via hydrogen bonding at an inter-planar distance of

approximately 2.1 Å. Also, van der Waals interaction was also observed between the isatin ring and

Tyr124 residue at an inter-planar distance of approximately 2.9 Å.

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Figure 4. A) The binding orientation of compound 2 (yellow in color) in the AChE active site, showing hydrophobic

interactions (pink dashed lines) and hydrogen bonding (green dashed line). B) The binding orientation of lead inhibitor

compound 3 (green color) showing hydrophobic interactions (pink dashed lines) and hydrogen bonding (green dashed line)

with the residues lining PAS and CAS of AChE gorge. C) The superimposed orientation of compound 2 (yellow in color),

compound 3 (shown in green), and Donepezil (depicted in purple color) in the AChE active site. D) 2-D interactions

displayed by compound 2 in the AChE active site with the aromatic amino acid residues.

Pose analysis of BChE inhibitors

Examination of the docked poses of all the compounds within the BChE active site resulted in the

following observations: All the test inhibitors were found to fit into the gorge of BChE formed by the

catalytic anionic site (CAS) and peripheral anionic site and were surrounded by the residues Gly116,

Gly117, Ser198, Ala199, Pro285, Val288, Leu286, Ser287, Ala328, Phe329, Tyr332, Phe398, Trp430,

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Met437, Tyr440, and His438. The orientation of the compounds was similar to that of the reference

inhibitor tacrine when docked with the model.

The compounds 1- 10 were found to be stabilized by hydrogen bonding and π- π interactions.

Hydrogen bonding interactions were mainly observed with the residues Gly 116 and Ser 198 in the

case of compound 7; Pro285, Ser117 and Tyr332 residues for compound 1; Pro285, Ser287 and Tyr332

residues for compound 2; Ser198 and Gly116 for compound 3; His438 and Gly117 for compound 4;

Pro285, His438 and Ser287 for compound 5; Gly116 and Ser198 for compound 7; Trp82, Trp430 and

His438 for compound 9; Gly78 and Trp430 for compound 10. Apart from this, π- π interactions rise

to be the most prevalent in these compounds with the compound 2 showing π- π interactions with the

residues His438, Phe398, Leu286, Ala328 and Phe329 as shown in Fig 5A; compound 4 with the

residues Trp231, Leu286, Phe329, Ala328, Tyr332 and Trp82; compound 5 with Trp231, Ala328,

Tyr332 and Val288; compound 7 with Ala328, Trp82, Trp231, Phe329 and Leu286; compound 9 with

Ala328, Met437, Tyr332, Pro285 and Phe329; compound 10 with the residues Ala328, Phe329,

Tyr332 and Leu125.

Binding mode of lead BChE inhibitor compound 3

Assessment of the virtual complex between the lead inhibitor compound 3 and BChE revealed that

compound 3 could perfectly occupy the gorge of BChE simultaneously interacting with the CAS and

PAS of BChE as depicted in Fig 5B. The N-substituted isatin template was observed to bind to the

PAS while the (methylenedioxy)phenyl moiety occupied the CAS, whereas the hydrazone spacer

occupied the middle of the gorge between CAS and PAS. The complex was stabilized by hydrogen

bonding and π- π interactions where the phenyl part of the (methylenedioxy)phenyl moiety was

involved in π- π interactions with the residues Leu286, Phe329, Trp231, and Gly116. The phenyl ring

of the isatin template was involved in π- π interactions with the residues Ala328 and Trp82, where the

propargyl substitution at the N-position of isatin moiety interacted with Trp82 via πalkyl interaction

at an inter-planar distance of approximately 4.7 Å. The oxygen present in the isatin ring was involved

in hydrogen bonding with the residue Gly116 at an inter-planar distance of approximately 3.0 Å. The

NH present in the carbimino terminal was observed to undergo hydrogen bonding formation with

Ser198 at an inter-planar distance of approximately 1.7 Å. The oxygen present in the carbimino

terminal underwent hydrogen bonding with Ser198 residue at an inter-planar distance of

approximately 2.4 Å. Like as in the case of the AChE model, the BChE binding site interactions were

similarly ordered, as in the case of the standard inhibitors.

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Figure 5. A) The binding orientation of compound 2 (orange) in the BChE active site showing hydrophobic interactions

(pink dashed lines) and hydrogen bonding (green dashed line) with the residues lining the cavity. B) The binding orientation

of compound 3 (yellow) within the active site along with the amino acid residues. C) The superimposed orientation of

compound 2 (orange), compound 3 (yellow) and donepezil (blue) in the BChE active site along with the residues lining

the cavity.

In vitro MAO inhibition assay

The in vitro inhibition potency of selected compounds from the series, which showed greater better

results in the ChE inhibition assay, were tested against MAO isozymes using crude rat brain

mitochondrial suspensions by a UV-based spectrophotometric method^[25]. Clorgyline and Selegiline

were used as reference inhibitors for MAO-A and MAO-B, respectively. The results are expressed in

terms of IC₅₀values as presented in Table 3.

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The tested compounds showed moderate to excellent range in terms of IC₅₀values, which is in the

micromolar range, and also indicated that most of them were selective to the MAO-B enzyme. The

IC₅₀values ranged from 1.73 ± 0.087 µM (compound 3) to 11.2 ± 0.276 µM (compound 8) for rat

MAO-A (rMAO-A), and from 0.034 ± 0.007 µM (compound 3) to 4.13 ± 0.092 µM (compound 7) for

rMAO-B. Among the tested compounds, compound 3 was identified as the most active MAO-A

inhibitor, with IC₅₀values of 1.73 ± 0.087 µM, as well as the most active MAO-B inhibitor, where the

IC50 value was found to be 0.034 ± 0.007 µM. It was also observed that compound 3 was 50 folds

more selective towards MAO-B enzyme than MAO-A, and the inhibitory potential for MAO-B

isozyme was close to that of the reference standard Selegiline. The second best MAO-A inhibition

profile was observed in the case of compound 10, which displayed an IC₅₀value of 1.97 ± 0.018 µM,

followed by compound 4. In the case of MAO-B, compound 8 was observed to have the second best

activity, with an IC₅₀value of 0.52 ± 0.093 µM, followed by compound 4, which showed an IC₅₀value

of 0.546 ± 0.032 µM. From the inhibitory data produced in Table 3, SAR has been derived for the

tested compounds.

SAR for MAO-A inhibition

The ortho and para-substituted dichloro benzyl group in the R₁position of the isatin ring gave rise to

better MAO-A activity, as in the case of compound 10 than the para-substituted fluoro benzyl

derivative, compound 8, and benzyl substituted derivative, compound 7. The substitution with para-

fluoro benzyl group at the R₁position, compound 8, led to a drastic decrease in the activity as

compared to the benzyl substituted derivatives. The aliphatic side chain substitution at R₁position by

propargyl and allyl group compounds 3 and 4 respectively, led to an increase in activity, thereby

showing better results than that of the benzyl-substituted derivatives, except in the case of compound

10, which showed comparable results as to that of compound 4. Substitution at the R₂position by a

chlorine group led to a decrease in the MAO-A activity as compared to the R₁substituted compounds,

except in the case of compound 8, which showed the lowest activity among the tested compounds.

Compound 3 was shown to possess overall better MAO-A inhibitory activity than the rest of the test

series.

Table 3. In vitro and computational results of MAO inhibition studies for compounds 1-10.

Compd

MAO-A

MAO-B

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IC₅₀[µM]^[a]

ΔG [kcal mol^-1]^[b]Ki [µM]^[b]

IC₅₀[µM]^[a]

ΔG [kcal mol^-1]^[b]

Ki [µM]^[b]

-

-7.96

-7.57

-5.86

-5.92

-6.27

-6.45

-5.32

-5.28

-5.89

-5.25

-

-10.02

-10.4

-10.65

-10.55

-10.78

-10.55

-12.25

-12.15

-12.32

-10.96

-

1

2

5.164±0.131

1.56

45.76

31.45

16.34

9.86

120.56

142.76

36.32

136.37

-

0.89±0.096

0.17

1.73±0.087

0.034±0.007

0.019

3

2.51±0.058

0.546±0.032

0.217

4

-

5

-

6

3.46±0.009

11.2±0.276

-

4.13±0.092

0.52±0.093

-

1.19

7

0.046

8

-

9

1.97±0.018

0.0043±0.008

-

0.64±0.072

-

0.325

10

CLR

-

0.02±0.0045

-

SGL

[a]

Each IC₅₀value is the mean ± SEM. Level of statistical significance: p<.05 versus the corresponding IC₅₀

values obtained against MAO’s, as determined by ANOVA/Dunnett’s. Reference inhibitors: CLR– Clorgyline,

SGL - Selegiline. ^[b]Computationally predicted values.

SAR for MAO-B inhibition

Substitution at R₁position by para-substituted fluoro or dichloro substituted benzyl groups,

compounds 8 and 10 respectively, led to an increase in MAO-B inhibitory activity as compared to the

unsubstituted benzyl derivative, compound 7. Compounds 8 and 10 displayed, respectively, 22-fold

and 3-fold selectivity than MAO-A, while compound 7 was the least selective of the tested compounds.

Aliphatic side chain substitution at R1 position by propargyl and allyl group led to an increase in

activity as compared to the benzyl-substituted compounds, except in the case of a para-fluoro

substituted benzyl group, compound 8, where it was shown to have comparable activity to that of the

allyl-substituted derivative, compound 4. The substitution at R₂position by a chloro group, in

compound 2, did not show any substantial increase in activity but was shown to possess better

selectivity and activity than compound 7. Compound 3, with propargyl substitution at the R₁position,

was shown to have the highest MAO-B inhibitory activity and selectivity (more than 50 fold as

compared to MAO-A) among the tested compounds.

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Kinetic studies of lead MAO-B inhibitor 2 and 3

The inhibition mode of the discovered lead MAO-B inhibitors, 2 and 3, was examined by performing

Lineweaver-Burk plots. The plots obtained for compound 2 (Fig 6A) showed increased slopes

(decreased V_max), which intercepted (higher K_m) at increasing concentration of the inhibitors. This

implies a mixed-type inhibition by compound 2 and consequently support the dual site binding

property of the compound^[32]. The Lineweaver-Burk plot for Compound 3 (Fig 6B) was linear and

intersected at the Y-axis. This pattern showed that compound 3 inhibited MAO-B competitively, and

further extrapolation of the results proved that they bind reversibly to MAO-B.

Determination of K_ivalue for lead MAO-B inhibitor compound 3

The dissociation constant (K_i) was calculated using GraphPad Prism software to determine the strength

of enzyme-inhibitor interaction of lead MAO-B inhibitor, compound 3. K_ivalue for competitive

inhibitor 3 was calculated to be 12.14 ± 0.026 nM against rMAO. Between the IC₅₀value and K_ivalue

obtained for compound 3, approximately a 3-fold difference was observed, suggesting compact

binding of the inhibitor to the enzyme.

Reversibility studies of lead MAO-B inhibitor compounds 2 and 3

The lead MAO-B inhibitors 2 and 3 were further tested for time-dependent inhibition studies to

determine whether the inhibition was reversible or irreversible. The reversibility test was performed

by adopting a slightly modified method as described by Legoabe et al^[33]. There was no time-dependent

decrease in the rates of rMAO-B catalyzed the oxidation of benzylamine when compound 2 (Fig 6C)

and 3 (Fig 6D) were pre-incubated with the enzyme for various time intervals (0, 15, 30, and 60 min).

From these results, it was concluded that inhibition of rMAO-B by both 2 and 3, was reversible, at

least within a 60 min time period. An increase in rMAO-B catalytic rates was also observed owing to

prolonged incubation times with both compounds.

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Figure 6. Kinetics and time-dependent inhibition of rMAO-B by compound 2 (A and C) and compound 3 (B and D).

Lineweaver-Burk plot of the MAO-B catalyzed oxidation of benzylamine in the absence (control) and presence of various

concentrations (2.5 µM, 5 µM, 10 µM) of compound 2 (A) and compound 3 (B). Time-dependent inhibition of AChE

catalyzed oxidation of benzylamine by compound 2 (C) and compound 3 (D). Rate data are expressed as nmol product

formed/min/mg protein. data are the mean ± SEM of n=3 determinations.

Molecular modeling studies of MAO inhibitors

The Gold Standard Pose (GSP) of the co-crystallized bound protein-ligand complex was produced to

validate the docking procedure, and for the study, hMAO-A, and hMAO-B were procured from the

RCSB PDB database.

According to the PDB analysis criteria adapted for the selection of the appropriate PDB structures for

the modeling studies^[27], human MAO-A (PDB code: 2Z5X) and human MAO-B (PDB code: 2V5Z)

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were selected. The selection was performed out of a test set of more than 40 structures, in the case of

MAO-B structures and around 5 structures for MAO-Astructures (containing both bound and unbound

structures). The GSP was determined to create precision and validate the structure of selected

structures, which was also a major part of the screening analysis for the selection of the appropriate

PDB structure. In cases where the GSP was hard to achieve, structures with Root Mean Square

Deviation (RMSD) < 2.0 Å, the next in line of the most suitable models were chosen to undergo the

validation studies, as what happened in the case of MAO-A enzyme. The results of the docking studies

were expressed theoretically as binding free energies (ΔG) and inhibition constants (Ki values) for

each of the ligand-protein complex and are presented in Table 3.

Binding pose analysis of MAO-A inhibitors

Evaluation of virtual ligand-protein complexes of all the compounds 1-10 within the catalytic site of

MAO-Aresulted in the below-mentioned interpretations: All the tested compounds occupied the active

site cavity of MAO-A and were framed within the binding pocket surrounded by the amino acid

residues Ile180, Asn181, Phe208, Val210, Gln215, Ile325, Tyr407, Tyr444 ad FAD which was almost

similar to the reference MAO-A inhibitor, harmine. Compounds 2 and 4 were grouped with the

reference inhibitor to get the pose, where similar binding orientation was observed within the active

site of MAO-A.

In all the compounds, the (methylenedioxy)phenyl ring occupies the space extending towards the

cavity while the variable isatin group is located towards FAD. Further, the inhibitors are stabilized by

hydrophobic π-alkyl and π- π stacked interactions. None of the selected compounds showed an

inclination for hydrogen bond formation, where the main π- π-alkyl interactions were observed with

the residues Phe208, Ile180, Ile335, Leu337, FAD and Tyr444, while π- π stacked interactions were

observed between the residues Tyr407 and Phe352. The π-sigma interactions were mainly common

with one residue Gln215 with the (methylenedioxy)phenyl moiety of the molecule. Compound 3 was

observed to show π-alkyl interactions, from the propargyl group of the parent to the amino acid

residues Phe208 and Ile180 as shown in Fig 7A and Fig 7B. The isatin moiety was shown to take part

in the majority of the hydrophobic interactions, thus stabilizing inside, near the FAD pocket. Thus in

the more potent compound, preferably π- alkyl and π- π interactions have been found to be responsible

for mediating the inhibitory activity against MAO-A.

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Binding mode of lead MAO-A inhibitor compound 3

Inspection of the virtual complex of the lead MAO-A inhibitor compound 3 and MAO-A reveals that

the hydrazone spacer is aligned in the center of the cavity with the (methylenedioxy)phenyl group

located towards the opening of the receptor and the variable isatin moiety orienting towards FAD. The

benzene ring of the (methylenedioxy)phenyl group is involved in π- alkyl and π- sigma interactions

with the residues Ile335 and Leu337, and Gln215 respectively, with the former interactions at an inter-

plane distance of approximately 4.7 Å and 4.54 Å respectively. Besides this, the molecule is stabilized

by π- alkyl and π- π interactions between the isatin ring and the residues Tyr444, Tyr407, and FAD.

Also, the presence of a π-rich aliphatic sidechain, a propargyl group, gave rise to π-alkyl interactions

with the main residues guarding the cavity, namely Phe208 and Ile180 at a distance of 4.0 Å and 4.54

Å respectively.

Figure 7. A) The superimposed orientation of compound 3 (green) and Harmine (red) in the active site cavity of MAO-A

enzyme along with the co-factor FAD (blue). B) The 2-D binding interactions displayed by compound 3 with the residues

lining the active site.

Pose analysis of MAO-B inhibitors

All the inhibitors are located within the active site of the cavity of MAO-B. The binding orientations

of all the test inhibitors within MAO-B traverse both the entrance cavity surrounded by the residues

Leu171, Ile168, Ile199 and Tyr326; and the substrate cavity composed of the residues Tyr60, Cys172,

Gln206, Phe343, Tyr398 and Tyr435, almost in a similar fashion to that of the posture observed in the

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case of reference MAO-B inhibitor, safinamide. The majority of the test inhibitors showed the same

orientations towards the binding pocket. The amide functional side chain of the safinamide inhibitor

was oriented towards the FAD molecule and the fluoro benzyl group was observed to position towards

the opening of the cavity. The compounds 1-8 possessed similar orientation where the variable isatin

moiety was headed towards the FAD and the (methylenedioxy)phenyl group was positioned towards

the cavity opening. Only compound 10 proved to be an exception where the orientation is reversed

completely. The inhibitors are stabilized by hydrogen bonding and π- alkyl interactions.

All the compounds showed hydrogen bonding interactions with the target residues mainly Ile199,

Tyr326, Gln206, Ile198, Tyr435, Tyr188 and Cys172. The compound 1 and 2 showed hydrogen

bonding interactions with the residues Gln206 and Tyr435, while compound 3 showed hydrogen

bonding with residues Ile199, Tyr326, Gln206 and Ile198. Compound 4 and 6 showed hydrogen

bonding with the common residue Gln206, with compound 4 showing extra interaction with Ile198.

The hydrogen bonding interactions expressed by compound 5 with Tyr188 and Cys172 stands out as

uncommon among the other compounds. The residues Gln206, Cys172 and Ile198 displayed hydrogen

bonding interactions with compound 7. The compounds compound 8 and 9 showed similar hydrogen

bonding interactions with residues Gln206 and Ile198, while compound 10 displayed interactions with

Cys172 and Ile198 residues of the enzyme. Additionally, π- alkyl interactions were shown by all the

compounds, the active residues namely: Cys172, Tyr60 and Tyr435 in the case of compound 3, the

most active inhibitor from the series. In the study, it was concluded that in most of the potent

compounds preferably π- alkyl and hydrogen bonding interactions are responsible for mediating

inhibition against MAO-B.

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Figure 8. A) The binding orientation of compound 2 (blue) in the active site cavity of MAO-B enzyme along with the co-

factor FAD (purple), showing important hydrophobic (pink dashed line) and hydrogen bonding (green dashed line)

interactions. B) The superimposed orientation of compound 2 (blue), compound 3 (green), and safinamide (yellow) in the

active site cavity of MAO-B enzyme along with the co-factor FAD (dark blue). C) The binding orientation displayed by

compound 3 (green) with the amino acid residues within the active site and FAD (violet). D) The 2-D interactions observed

in one of the lead MAO-B inhibitor, compound 2, with the aromatic residues lining the active site.

Binding mode of lead MAO-B inhibitor compound 3

Study of the lead MAO-B inhibitor, compound 3, reveals that the entire molecule is stabilized in both

the cavities with the rigid (methylenedioxy)phenyl moiety extending towards the entrance cavity of

MAO-B, while the variable isatin group is caged into the substrate cavity as displayed in Fig 8C. The

compound is found to exhibit a π-alkyl interaction between propargyl group of the side chain in the

isatin moiety and Tyr60 residue at an inter-planar distance of approximately 4.73 Å. The isatin

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template showed π-sigma and π-alkyl interactions with the residues Leu171, and Tyr435, Cys172 and

Tyr398 respectively at an interplanar distance of approximately 4.3 Å, 5.4 Å, 5.0 Å, and 5.3 Å

respectively. In addition, hydrogen bonding interactions were observed between the oxygen of the

carbimino linker and Tyr326, Ile199 and Gln206 with an inter-planar distance of 2.9 Å, 2.7 Å and 2.4

Å respectively. Additional hydrogen bonding interactions were observed between the nitrogen

involved in the carbimino bond with the residue Ile198 at an inter-planar distance of 2.2 Å. These

interactions in tandem resulted in the firmness of the ligand within the MAO-B active site cavity and

hence confirm the stability of the complex. In most of the compounds, it was seen that the variable

hydrophobic isatin region was involved in strong hydrophobic interactions with the FAD molecule, as

in the case of the standard inhibitor as shown in Fig 8D.

Table 4. Drug-Likeness and Pharmacokinetic properties of compounds 1-10.

Properties

1

2

3

4

5

6

7

8

9

10

Drug Likeness

-Lipinski Rule of 5

pass

Absorption

-WS (log molL^-1)

-IA (%)

-3.403 -3.446 -3.682 -3.943 -3.569 -3.499 -5.268 -4.957 -5.186 -5.159

91.82

92.12

95.68

95.25

83.99

92.05

93.70

-2.76

93.37

-2.74

91.45

92.6

-SP (log K_p)

-2.907 -3.003 -3.262 -2.802 -2.795 -3.002

-2.741 -2.742

Distribution

-BBBP (log BB)

-CNSP (log PS)

-0.083 -0.433 -0.262 -0.288 -0.745 -0.442 -0.231 -0.335 -0.441 -0.508

-2.407 -2.342 -2.462 -2.506 -2.575 -2.319 -2.178 -2.224 -1.944 -2.119

Metabolism

-CYP3A4

inhibitor

No

Yes

No

Yes

No

Yes

No

Yes

No

-CYP2C9 inhibitor

Excretion

-TC (log

0.098 -0.325 0.264

0.36

0.375 -0.347 0.205 -0.055 -0.292 -0.429

ml/min/Kg)

Toxicity

AMES toxicity

MTD (log

mol/Kg/day)

ORAT (mol/Kg)

HT

No

Yes

No

Yes

-0.346 -0.026 -0.366 -0.059 -0.257 -0.035 -0.319 -0.451 -0.493 -0.387

1.962

No

1.957

No

2.524

No

2.499

Yes

No

2.422

Yes

No

1.969

No

2.681

Yes

No

2.452

Yes

No

2.531

Yes

No

2.492

Yes

No

SS

CT

No

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*Abbreviations: LLR = Lead Like Rule, WS = Water Solubility, IA = Intestinal Permeability, SP = Skin

Permeability, BBBP = Blood Brain Barrier Permeability, CNSP = Central Nervous System Permeability, TC =

Total clearance, MTD = Maximum Tolerated Dose, ORAT = Oral Rat Acute Toxicity, HT = Hepatotoxicity,

SS = Skin Sensitization, CT = Cytotoxicity.

In silico drug-likeness and pharmacokinetic prediction

The in silico pharmacokinetic properties were determined for all the compounds using online pkCSM

server and ProTox server^[29,30]. All the compounds showed high water solubility within the range of -

5.268 (log molL^-1), in case of compound 7, to -3.403 (log molL^-1), for compound 1. Intestinal

absorption (IA) was found to be greater than 90% for all the tested compounds except for compound

5, which was shown to have 83% absorption. Compound 3 had the highest IA of 95.68% followed by

compound 4 with 95.25%. All the compounds were skin permeable, showing a range from -3.262 (log

Kp), as observed for compound 3, to -2.74 (log Kp), calculated for compound 8. Compounds 1-10

have the potential to cross the blood-brain barrier (BBB) and exhibit range from -0.745 (log BB), for

compound 5, to -0.083 (log BB), for compound 1. The CNS permeability values for all the compounds

were found to be greater than -2.60 (log PS), with compound 9 displaying the highest value of -1.944,

pertaining to greater penetration into the CNS. The compounds 1-6 are identified to be non-inhibitors

of the metabolizing enzyme CYP3A4, while compounds 7-10 inhibit the enzyme and can potentially

stop the metabolism of xenobiotic agents. All the compounds, 1-10, also did not inhibit the CYP2C9.

The compounds 1-10 showed Total Clearance between -0.429 (log ml/min/Kg) for compound 10 and

0.375 (log ml/min/Kg) as in the case of compound 5. Compounds 1 and 7 gave a negative response to

the AMES test, while the rest gave positive results. The Maximum Tolerated Dose (MTD) for the

compounds 2 and 6 were predicted to be -0.026 (log mol/Kg/day) and -0.035 respectively. The Oral

Rat Acute Toxicity (ORAT) model predicted values of 1.957 mol/kg and 2.524 mol/kg for compounds

2 and 3 respectively. Compounds 4, 5 and 7-10 were hepatotoxic in nature, while compounds 1-3 and

6 were shown to be non-hepatotoxic. None of the compounds were predicted to be skin sensitive and

cytotoxic in nature. All the results of pharmacokinetic predictions are presented in Table 4.

Conclusion

The application of hydrazones has spread extensively in targeting new pathophysiological conditions

with the development of efficient synthetic methodologies. In the present work, we attempted to

identify new MTDLs with dual MAO and ChE inhibitory properties. To bring full justice to the study,

we designed and synthesized a series of piperonylic acid derived hydrazones containing isatin scaffold

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with varying degree of steric and electronic features (compounds 1-10) based on the MTDL approach

and evaluated their pharmacological profile through virtual molecular docking and enzymatic

screening assays (in vitro MAO and ChE inhibition assays) respectively. In silico molecular property

analysis and ADMET, prediction studies were also carried out to predict the pharmacokinetic profile

of the compounds. From the preliminary in vitro MAO and ChE inhibition studies, we have identified

two lead molecules, compounds 2 and 3, from the series possessing dual inhibition potential against

both the enzymes. Most of the compounds were found to be more selective towards MAO-B isozyme

than MAO-A, especially in the case of compound 3, where it was shown to have more than 50 folds

more selectivity towards MAO-B isozyme. Additional Kinetic and reversibility/irreversibility studies

were performed for the lead inhibitors of the study, compounds 2 and 3, to understand the type and

strength of enzyme-inhibitor binding. Compound 2 displayed competitive inhibition for AChE (lead

inhibitor) and a mixed type inhibition for MAO-B, thereby supporting our dual site binding model. It

was also identified to undergo reversible inhibition with both AChE and MAO-B respectively.

Compound 3 was identified as a competitive as well as a reversible inhibitor of MAO-B isozyme as

well as AChE.

The identified leads were found to be more potent than the previously discovered lead compound 22,

whose MAO-B and MAO-A inhibition IC₅₀values were 7.253 ± 0.002 µM and 16.40 ± 0.011 µM

respectively. On comparing the hydrazone lead compound 3 to the semicarbazone based compound

22^[25]from our earlier study, it was observed that the removal of NH group attached to the

(methylenedioxy)phenyl moiety led to an increase in overall activity and selectivity towards MAO-B

enzyme as shown in Fig 9. Also, the docking analysis revealed strong hydrophobic interactions

governing the stability of compound 3 in the active site pocket as compared to compound 22.

Figure 9. The MAO/ChE inhibition data (IC₅₀) for present lead compounds 2 and 3, along with earlier developed

compound 22.

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Most of the compounds of the hydrazone series were found to be potent inhibitors of AChE enzyme,

with the exception of compound 5. The lead AChE inhibitors obtained from the study are compound

2 and 3, while compound 3 emerged as the best BChE inhibitor. The identified leads were found to be

more potent than the previous lead discovered compound 22^[25], whose AChE IC₅₀value was 0.307 ±

0.003 µM. On comparing the hydrazone lead compound 2 to the semicarbazone based compound 22,

it was observed that the removal of the hydrogen-bond donor, NH group associated with the

(methylenedioxy)phenyl moiety led to an increase in overall activity towards AChE enzyme.

The docking results confirmed stronger hydrophobic interactions with the active site gorge for

compound 3 relative to the semicarbazone-isatin leads previously developed.

The present work yielded some lead MAO and ChE inhibitors with interesting activity profile. The

truncation of the semicarbazino linker into a hydrazone linker has yielded far more potent compounds

with superior IC₅₀values in the case of AChE and MAO-B inhibition, with greater selectivity towards

MAO-B. Compound 2 (N'‐ [(3E)‐ 5‐ chloro‐ 2‐ oxo‐ 2,3‐ dihydro‐ 1H‐ indol‐ 3‐ ylidene]‐ 2H‐

1,3‐ benzodioxole‐ 5‐ carbohydrazide) and compound 3 (N'-(2-oxo-1-(prop-2-ynyl)indolin-3-

ylidene)benzo[d][1,3]dioxole-5-carbohydrazide) have emerged as the most promising dual acting lead

compounds which warrant further optimization and preclinical development as neurotherapeutic

agents.

Experimental Section

The specific reactant materials and reagents were procured from Alfa Aesar and Merck (Sigma-

Aldrich) and were consumed without further purification. Melting points (mp) were mapped using a

one-end-closed capillary tube on a Systronics digital melting point apparatus (model IIC327) and are

uncorrected. IR spectra were recorded on an Alpha II Infrared Spectrometer (FT-IR, Bruker) by means

of potassium bromide disc method. ¹H and ¹³C were recorded on a Bruker 500 MHz High-Resolution

FT NMR Spectrophotometer at ambient temperature using deuterated DMSO ([D₆] DMSO) as a

solvent. Chemical shifts are expressed as ð units (ppm) relative to TMS. All exchangeable protons

were confirmed by addition of D₂O. Spin multiplicities are expressed as s (singlet), d (doublet), t

(triplet), dd (doublet of doublet), or m (multiplet). Coupling constants (J) are expressed in hertz (Hz).

Mass spectra were recorded on a Waters Xevo TQD triple Quadrupole mass spectrometer.

10.1002/cmdc.201900277

ChemMedChem

Elemental analyses for C, H, and N were determined with a CE-440 CHN analyzer (ExeterAnalytical),

and the analytical results were within ± 0.4 % purity for all compounds.

Intermediates

Methyl 2H‐1,3‐benzodioxole‐5‐carboxylate (I): 0.027 moles of piperonylic acid was dissolved

in 100 ml of methanol in a 250 ml round- bottom flask. To the solution, 0.072 moles of concentrated

sulfuric acid (3.83 ml) was added and refluxed at 80 ^oC for 2 hours (monitored by TLC). The reaction

mixture was poured into ice cold water to obtain a white colored crystalline precipitate. The precipitate

was filtered out and washed with cold water and vacuum dried. Recrystallization was performed using

ethanol (yield: 90%, mass: 180.16 gmol^-1, R_f: 0.53, mp: 52-54 ^oC): ¹H NMR ([D6] DMSO): δ = 3.76

(s, 3H, CH3), 6.17 (s, 2H, benzodioxole C2), 7.03 (d, J = 7.5, 1H, benzodioxole C4), 7.34 (s, 1H,

benzodioxole C7), 7.48 (d, J = 8.4, 1H, benzodioxole C5); ¹³C NMR ([D6] DMSO): δ = 52.32 (methyl

C), 102.03 (benzodioxole C2), 107.99 ( benzodioxole C4), 112.67 ( benzodioxole C7), 126.43

( benzodioxole C5), 133.59 ( benzodioxole C6), 139.65 ( benzodioxole C3a), 141.08 ( benzodioxole

C7a), 168.31 (C=O); IR (KBr): ΰ = 1413.46 (aromatic C=C med), 1746.12 (C=O str), 1296.76,

1067.22 (C-O str); Anal. for C₉H₈O₄: calcd: C (60.02%), H (4.46%), O (35.52%), found: C (60.08%),

H (4.49%), O (35.43%)

2H‐1,3‐benzodioxole‐5‐carbohydrazide (II): 10ml of hydrazine monohydrate (0.201 mol) was

added to a solution of I (0.0201 mol) in ethanol (60ml) while stirring. The solution was refluxed at 80

^oC with stirring for 2 hrs (monitored by TLC). After completion of the reaction, the mixture was poured

onto ice-cold water to obtain a white crystalline precipitate. The precipitate was filtered and washed

with ice cold water. Recrystallisation was performed using ethanol (yield: 86%, mass: 180.16 gmol^-1,

R_f: 0.38; mp: 170-172 ^oC): ¹H NMR ([D6] DMSO): δ = 1.96 (s, 2H, NH2), 6.14 (s, 2H, benzodioxole

C2), 7.02 (d, J = 8.4, 1H, benzodioxole C4), 7.42 (s, 1H, benzodioxole C7), 7.50 (d, J = 8.3, 1H,

benzodioxole C5), 8.02 (s, 1H, NH); ¹³C NMR ([D6] DMSO): δ = 101.90 (benzodioxole C2), 107.43

( benzodioxole C4), 108.23 ( benzodioxole C7), 126.56 ( benzodioxole C5), 135.12 ( benzodioxole

C6), 138.45 ( benzodioxole C3a), 140.09 ( benzodioxole C7a), 165.43 (C=O); IR (KBr): ΰ = 3320.32

( N-H med), 1420.12 (aromatic C=C med), 1679.84 (C=O str), 1276.53, 1046.08 (C-O str); Anal. for

C₈H₈N₂O₃: calcd: C (53.34%), H (4.47%), N (15.57%), O (26.62%), found: C (53.39%), H (4.42%),

N (15.60%), O (26.59%)

Article Doi

Experimental and Computational Evaluation of Piperonylic Acid Derived Hydrazones Bearing Isatin Moieties as Dual Inhibitors of Cholinesterases and Monoamine Oxidases

DOI: 10.1002/cmdc.201900277

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Article abstract of DOI:10.1002/cmdc.201900277

Full text of DOI:10.1002/cmdc.201900277

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