HasanainOdharthesis Lapinski Rule

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Identiﬁcation of novel scaffolds for Monoamine oxidase B inhibitors.
Thesis · May 2014

DOI: 10.13140/RG.2.2.30232.44800
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Identification of novel scaffolds for Monoamine
oxidase B inhibitors
A thesis submitted
to Kent State University in partial
Fulfillment of the requirements for the
Degree of Master of Science
By
Hasanain Odhar
May, 2014
Thesis written by
Hasanain Odhar
B.S., University of Baghdad, 2007
M.S., Kent State University, 2014
Approved by
Werner Geldenhuys
__________________________, Chair, Master’s Thesis Committee
Altaf Darvesh
__________________________, Member, Master’s Thesis Committee
Richard Carroll
__________________________, Member, Master’s Thesis Committee
Eric Mintz
__________________________, Director, School of Biomedical Sciences
Janis Crowther
__________________________, Dean, College of Arts and Sciences
ii
Table of Contents
List of figures ..................................................................................................................... vi
List of tables ....................................................................................................................... ix
Acknowledgments............................................................................................................... x
Chapter one: Introduction ............................................................................................... 1
1.1- Parkinson's disease (PD) .......................................................................................... 1
1.2- Monoamine oxidase enzyme (MAO)....................................................................... 3
1.3- Structure of MAO-B ................................................................................................ 4
1.3.1- membrane binding domain ................................................................................ 6
1.3.2- Substrate binding domain .................................................................................. 6
1.3.3- Flavin binding domain ...................................................................................... 7
1.3.4- Structural comparison between human MAO-B and human MAO-A .............. 9
1.4- Reaction mechanism of MAO enzyme .................................................................. 11
1.5- Functional role of aromatic cage............................................................................ 18
1.6- MAO inhibitors with a possible or approved clinical application ......................... 20
1.7- MAO-B inhibitors with a possible disease modifying effect ................................. 22
iii
Chapter two: Materials and Methods ........................................................................... 28
2.1- Prospective candidates ........................................................................................... 28
2.2- Docking studies ...................................................................................................... 36
2.3- Monoamine oxidase inhibition assay ..................................................................... 36
2.4- Bovine serum albumin (BSA) binding assay ......................................................... 40
2.5- Parallel Artificial Membrane Permeability Assay (PAMPA) ................................ 41
2.6- Oxygen Radical Absorbance Capacity (ORAC) assay .......................................... 43
2.7- Data analysis .......................................................................................................... 44
Chapter three: Results .................................................................................................... 45
3.1- Selection of the most efficient candidates ............................................................. 45
3.2- Docking studies results .......................................................................................... 47
3.3- Monoamine oxidase inhibition assay results ......................................................... 71
3.4- Bovine serum albumin (BSA) binding assay results ............................................. 76
3.5- Parallel artificial membrane permeability assay (PAMPA) results ....................... 82
3.6- Oxygen radical absorbance capacity (ORAC) assay results .................................. 84
3.7- Structure activity relationship (SAR) study results................................................ 87
Chapter four: Discussion.............................................................................................. 105
iv
References ...................................................................................................................... 138
v
List of figures
Figure 1.1 - Three dimensional structure of human MAO-B enzyme (monomer A) ......... 5
Figure 1.2 - A schematic representation for catalytic reaction pathway followed by MAO
enzyme. .......................................................................................................... 13
Figure 1.3 - A schematic representation for the proposed polar nucleophilic mechanism
of MAO enzyme catalysis ............................................................................. 16
Figure 1.4 - A schematic representation for the proposed single electron transfer (SET)
mechanism of MAO catalysis ........................................................................ 17
Figure 1.5 - A schematic representation for the effect of dipole moments of Tyr398 and
Tyr435 in MAO-B on the electron lone pair of the substrate amine ............. 20
Figure 2.1 - Conversion of kynuramine into 4-Hydroxyquinoline as catalyzed by both
MAO-A and MAO-B ..................................................................................... 39
Figure 3.1 - A scatter plot of MAO-B inhibition potential (percentage) against antioxidant
potential (percentage) for our chemicals. ...................................................... 46
Figure 3.2 - Docking of compound 5140311 into human MAO-B crystal (2D and 3D
images) ........................................................................................................... 49
images) ........................................................................................................... 51
images) ........................................................................................................... 53
vi
images) ........................................................................................................... 55
images) ........................................................................................................... 57
images) ........................................................................................................... 59
images) ........................................................................................................... 63
images) ........................................................................................................... 66
Figure 3.10 - MAO-B inhibition potential as produced by the selected compounds ....... 73
Figure 3.11 - The potency of the inhibitory effect against MAO-A as produced by the
tested compounds........................................................................................... 74
Figure 3.12 - BSA binding potential of the tested chemicals ........................................... 77
Figure 3.13 - Relative BSA binding curves for the selected compounds ......................... 78
Figure 3.14 - Percentage of antioxidant power for the selected chemicals ...................... 85
Figure 3.15 - MAO-B inhibition potential, antioxidant power, chemical structural formula
and MAO-B docking images for compounds 5140311 and 5140321 (SAR
study). ............................................................................................................ 89
Figure 3.16 - MAO-B inhibition potential, antioxidant power and chemical structural
formula for compounds 5143886, 5144779, 5108394 and 5108400 (SAR
vii
study). MAO-B docking images for compounds 5108394 and 5108400 are
also shown ..................................................................................................... 96
Figure 3.17 - MAO-B inhibition potential, antioxidant power and chemical structural
formula for compounds 7935085 and 5476423 (SAR study) ...................... 102
Figure 4.1 - Chemical structural formula, IUPAC name and MAO-B docking images for
our first prospective scaffold (scaffold A) ................................................... 114
our second prospective scaffold (scaffold B) .............................................. 117
Figure 4.3 - Chemical structural formula, IUPAC name and MAO-B docking image for
the modified scaffold (scaffold C) ............................................................... 124
the modified scaffold (scaffold D) ............................................................... 126
the modified scaffolds E and F (L-alanine derivatives)............................... 130
our novel design (scaffold G) ...................................................................... 134
viii
List of tables
Table 1.1 - A brief summary about MAO-inhibitors with a possible or approved clinical
application...................................................................................................... 21
Table 2.1 - Prospective candidates' specifications. ........................................................... 29
Table 3.1 - Docking results for BSA crystal. .................................................................... 70
Table 3.2 - Inhibition selectivity toward MAO isozymes................................................. 75
Table 3.3 - EC50 values for (antioxidant capacity – concentration) curve of each tested
compound. ..................................................................................................... 86
Table 4.1 - Classification of PAMPA-BBB results. ....................................................... 111
Table 4.2 - The obedience degree to Lipinski’s rule of five (scaffolds A, B, C and D). 122
Table 4.3 - The obedience degree to Norinder’s rules for CNS permeability. ............... 123
Table 4.4 - The application of Lipinski’s rule of five to scaffolds E, F and G. .............. 136
ix
Acknowledgments
I would like to express my deep gratefulness to my parents, brother and sisters for
their endless support, help and encouragement throughout my life journey.
I would like to thank my advisor (mentor) Dr. Werner Geldenhuys for his
priceless support and substantial guidance throughout research course and thesis writing.
I would also like to express my gratefulness to Dr. Altaf Darvesh and Dr. Richard Carroll
for serving on my thesis committee and for their vital contribution to my project and
thesis.
Finally, I would like to extend my gratitude to Dr. Eric Mintz and Mrs. Judith
Wearden for their precious support.
x
Chapter one: Introduction
1.1- Parkinson's disease (PD)
It is an age related neurodegenerative disease that affects about one million
persons in the United States [1]. The prevalence of Parkinson's disease usually increases
from 1-2% at age 50 to about 5% at age 85 [2, 3]. Pathologically the disease is
characterized by a progressive degeneration of dopaminergic neurons in the substantia
nigra pars compacta, this depletion of dopaminergic neurons will result in four major
motor related hallmarks including tremor, rigidity, bradykinesia and postural imbalance
[4]. These manifestations usually appear when about 60-70% of dopaminergic neurons
are lost [5]. Non-motor symptoms (like depression and dementia) may also appear as the
disease progress over time [6]. Another characteristic pathological feature of Parkinson's
disease is the presence of proteinaceous inclusions called Lewy bodies within the
surviving dopaminergic neurons, these inclusions consist mainly of α-synuclein which is
highly ubiquitinated [7]. Although the exact cause of Parkinson's disease remains unclear,
several factors may play a role in increasing susceptibility to develop the disease. These
factors include: age, environmental factors (ex: Paraquat PQ) [8, 9] and genetic factors
(autosomal dominant and recessive genes like α-synuclein, parkin, DJ-1, LRRK2) [10].
Current therapeutic approach focuses on symptoms attenuation only [11] through
dopamine replacement therapy by using either L-dopa (the immediate
1
2
metabolic precursor of dopamine) or dopamine receptor agonist [12, 13]. Dopamine
replacement therapy does not halt or slow down the pathological degeneration process
and as the disease is advancing, the efficiency of such therapeutic approach is falling
down [14]. In this situation, higher medication dose is required to maintain adequate
therapeutic benefit. Such decision is usually restricted by the fact that the tolerated
levodopa dose is limited by several side effects associated with long term therapy such as
dyskinesia and behavioral disturbances [15, 16]. Drugs that can lower L-dopa dose and/
or delay the need for L-dopa are considered essential for Parkinson's disease therapy
approach [17, 18]. These adjunct drugs involve the following categories: Monoamine
oxidase B (MAO-B) inhibitors, Catechol-O-methyltransferase inhibitors and amantadine
[11]. No drug with neuroprotective activity has been approved by FDA for Parkinson's
disease treatment till this point [19, 20].
Parkinson's disease is a complex neurologic condition with multiple underlying
etiological pathways and treating such disorder with a molecule that target a single
pathway is inefficient, a drug molecule that target different pathways seems to be more
convenient [21-28]. A possible candidate for such multi-targeted drug design is MAO-B
inhibitors, by inhibiting MAO-B enzyme; these compounds can elevate the level of both
endogenous and exogenous dopamine [29, 30]. By blocking the activity of MAO-B
enzyme, these compounds can also lower the production of hydrogen peroxide (a
byproduct of oxidative deamination reaction) and by this way oxidative stress can be
minimized [31]. The rationale for using MAO-B inhibitors as a basic model for the
development of a new antiparkinsonian therapy is encouraged by several facts. The first

3
one is that the activity level of MAO-B enzyme inside the brain increases with age [32-
34] while MAO-A level is not affected by age [87]. Also in patients with Parkinson's
disease, the level of MAO-B is elevated as a result of gliosis. Finally, the activity level of
MAO-B in human basal ganglia is higher than that of MAO-A and since both isozymes
metabolize dopamine with the same level of efficiency, then it is more reasonable to
target MAO-B and not MAO-A [36].
1.2- Monoamine oxidase enzyme (MAO)
MAO is an outer mitochondrial membrane bound flavin containing enzyme with a
molecular weight of about 60 kDa, it plays an important role in the metabolism of
endogenous and exogenous amines [35, 45]. Intraneuronal MAO terminates the action of
endogenous amine neurotransmitters, regulates intraneuronal amine stores and protects
cells from dietary amines (false transmitters) [36].
This enzyme exists in two isoforms (MAO-A) and (MAO-B), these isoforms
shares about 70% sequence identity and represent separate X chromosome related gene
products [37]. These isoforms have different substrate specificities in that MAO-A
catalyzes the oxidation of serotonin (5-HT) while MAO-B catalyzes the oxidation of both
benzylamine and 2-phenylethylamine. Other substrates like dopamine, epinephrine,
norepinephrine, tyramine and tryptamine are oxidized by both isoforms [38]. During
embryonic development, MAO-A is expressed first but the expression level of MAO-B
inside the brain is increased dramatically after birth [32, 39, 40]. MAO enzyme level of
activity is different among various regions of human brain, where the hypothalamus and
4
basal ganglia show the highest activity while cerebellum and neocortex show the lowest
activity [41]. Radio labeled inhibitors were used to identify the distribution of MAO
isozymes throughout the brain, it was found that basal ganglia contains mainly MAO-B
[42].
Immunohistochemical analysis reveals that serotonergic neurons contain
predominantly MAO-B isoform while catecholaminergic neurons contain MAO-A
mainly [43]. These immunohistochemical analysis data would suggest two conclusions:
first, the role of MAO-B in serotonergic neurons is to eliminate any foreign amines and
prevent them from reaching the synaptic cleft while the second conclusion is that MAO-
A in glial cells should be responsible for degradation of serotonin in brain regions rich in
serotonergic neurons. Noradrenergic neurons contain MAO-A and MAO-B, both
isoforms can catalyze the oxidative deamination of norepinephrine efficiently but because
the uptake of norepinephrine into synaptic vesicle is strongly favorable over binding to
MAO therefore the substrate can escape metabolic degradation to some extent [44].
1.3- Structure of MAO-B
MAO-B enzyme consists mainly from protein (520 amino acids) and FAD (redox
cofactor), it crystallizes as a dimer and each monomer involves the following subunits:
membrane binding domain, substrate binding domain and flavin binding domain [46, 47].
Figure 1.1 shows a three dimensional representation for human MAO-B monomer.
MAO-B undergoes post translational modification (acetylation) where the amino terminal
methionine is cleaved and the resultant N-terminal serine is acetylated [48, 49].
5
Figure 1.1- Three dimensional structure of human MAO-B enzyme (monomer A). The
membrane binding domain is in green color (residues 489–500), the substrate binding
domain is in red color (residues 80–210, 286–390 and 454–488) and the flavin binding
domain is in blue color (residues 4–79, 211–285 and 391–453). This figure was generated
by using PyMOL v.1.3 (www.schrodinger.com) [154] and human MAO-B crystal with
the code 2BK3; the sequences of amino acids for the designated domains were obtained
from reference [55].

6
1.3.1- membrane binding domain
C-terminal truncation experiments together with sequence analysis had suggested
that the C-terminal region can function as the membrane binding domain (C-terminal α
helix), this 27 residue transmembrane helix has apolar surface which would encourage its
insertion into mitochondrial membrane [50, 51]. Truncation experiments showed that C-
terminal truncated MAO-B can retain some affinity towards membranes but not as strong
or as specific as intact enzyme and this would suggest the presence of other membrane
binding regions. The electrostatic surface maps show positively charged domains that can
form electrostatic interactions with the anionic membrane surface [54].
1.3.2- Substrate binding domain
In order to access covalent FAD and undergo metabolism, substrates must cross a
specific path with a distance of about 20 Å [52]. The journey starts by negotiation with a
flexible loop (residue 99-112) which function as a gating switch at the surface of outer
mitochondrial membrane, this loop must be moved so that the substrate can reach active
site cavities [53]. The substrate then must pass through two hydrophobic cavities; the first
one is called the entrance cavity with a volume of ~290 Å³, the second larger cavity is
called the substrate cavity with a volume of ~400 Å³ [47, 54]. These two cavities are
separated by an Isoleucine 199 side chain which can function as a gate; depending on the
nature of the substrate, the Isoleucine (Ile) 199 side chain can exhibit either an open
conformation (fuse both cavities) or closed conformation (separate both cavities) [53].
7
Tyr326 residue also plays a role in separating the active site cavities of MAO-B; the side
chain of Tyr326 undergoes conformational changes upon substrate binding [72]
The outer mitochondrial membrane is covered with negative charges and these
charges will attract amine substrates (positively charged) into MAO-B enzyme [52]. FAD
cofactor is located at the end of substrate cavity where it is covalently bound to Cysteine
397 through thioether linkage [46].
1.3.3- Flavin binding domain
Flavin adenine dinucleotide (FAD) is considered as an essential entity for MAO
enzyme catalytic activity (redox cofactor); it is bound covalently to MAO-B apoenzyme
through a thioether linkage which is formed between cysteine 397 residue and the 8α-
methylene of isoalloxazine ring [46, 37]. This binding is located near the C-terminus of
MAO enzyme [55].
FAD coenzyme is incorporated in an extended conformation into a hydrophobic
environment within MAO-B, specific interactions are found between FAD and MAO-B
apoenzyme [55]. These interactions are mainly hydrogen bonds between FAD and either
amino acids side chains or amide linkages in the protein. Mutagenesis experiments had
shown that if Arg42 was replaced by Ala or Lys then this would adversely influence FAD
incorporation into MAO-B, These results showed the requirement of electrostatic
interaction between anionic pyrophosphate group of FAD and the positively charged
guanidino group of Arg42 [56]. The amino acid residues that interact with FAD in MAO-
8
B enzyme are conserved in MAO-A enzyme and this suggests that FAD binding site is
identical in both enzymes [55]. The only difference between these two enzymes is that
Ser59 is replaced with Ala in MAO-A and since the peptide bond (not amino acid side
chain) at this position is involved in hydrogen bonding with the flavin C-4 carbonyl
oxygen, then such amino acid substitution is not thought to cause a significant difference
in FAD binding domain between the these two isozymes. Flavin coenzyme analogs
analysis had shown that both MAO-A and MAO-B have a similar FAD incorporation
mechanism and a similar FAD binding sit [59, 60]. Analogs analysis results also provide
additional support for covalent flavinylation mechanism (quinone methide); it is believed
that both MAO-A and MAO-B follow this mechanism for FAD incorporation [61].
Structural data, obtained through diffraction analysis, had shown that the amide
bond between Cys397 and Tyr398 is in cis rather than the favorable trans configuration
[53]. This energetically unfavorable configuration (cis configuration) of the peptide bond
is necessary to make the phenolic side chain of Tyr398 in a perpendicular orientation to
the flavin ring, this orientation is essential for catalytic activity (aromatic cage) [58]. Two
main steric constraints that affect flavin reactivity had been recognized, first one is the
bending about (N5)-(N10) axis of isoalloxazine ring so that it will deviate 30˚ from
normal planar conformation while the second one is being puckered about the pyrimidine
portion of the ring. These steric deformations will make the reactive sites of flavin ring
(N-5 and C-4) assuming a more sp3 rather than a planar sp2 configuration and this will
enhance MAO enzyme reactivity [53].

9
To determine the effect of covalent binding of FAD coenzyme on the activity and
general stability of MAO enzyme, two mutagenesis experiments were performed. First
experiment had shown that replacement of Cys397 with Ser would result in an inactive
enzyme, it is clear that the hydroxyl group of serine does not have enough nucleophilicity
to form covalent bond with FAD [62]. The second experiment involved the expression of
Cys406Ala mutant MAO-A in riboflavin dependent strain of S. cerevisiae. Mutant MAO-
A was expressed in the absence of riboflavin and it did not show any activity until FAD
was added to the medium. The level of MAO-A activity was dependent on the
concentration of FAD added. This experiment suggested that enzyme activity is not
absolutely dependent on covalent FAD linkage and it also showed the importance of this
covalent linkage in structural stabilization of the enzyme against detergents solubilization
[63].
1.3.4- Structural comparison between human MAO-B and human MAO-A
The most important structural difference between these two isozymes is that
human MAO-B crystallizes as a dimer while human MAO-A crystallizes as a monomer
[64, 47, 58]. It was suggested that in human MAO-A, a Glu-151 is replaced by a Lys
residue. This selective mutation at the dimeric interface may be responsible for
destabilization of the dimeric form in human MAO-A [65]. This shift in the oligomeric
state of human MAO-A mainly affects the temperature stability of the enzyme; it was
found that rat MAO-A (dimer) is more thermally stable than human MAO-A (monomer)
[66]. Dipole moment calculations show a significant change in the direction of

10
monomeric MAO-B dipole moment upon dimerization. In dimeric MAO-B, the
orientation of positively charged end is directed towards the anionic membrane surface
which will provide addition interaction. Such alteration in dipole direction is less
pronounced in MAO-A [54]. New results of distance measurements by pulsed EPR-
DEER (electron paramagnetic resonance-double electron-electron resonance)
experiments showed that human MAO-A is present in dimeric form in the mitochondrial
membrane, it is believed that solubilization of human MAO-A by using detergent will
result in a mixture of both monomeric and dimeric forms of human MAO-A and the
monomeric form will be crystallized more readily [71]
Other significant differences are also present between human MAO-B and human
MAO-A regarding active site shape and volume. The active site in human MAO-B is
bipartite with a total volume of ~700Å³ (entrance cavity: 290Å³ and substrate cavity:
~400Å³) while in human MAO-A, the active site is monopartite with a volume of ~550Å³
[67, 64]. The shape of active site in human MAO-B is longer and narrower than that of
human MAO-A. This difference in active site shape and volume may be in part as a result
of variation in the conformation of cavity shaping loop, this six residue loop is present in
a more compact conformation in human MAO-B as compared with human MAO-A [64,
68]. Active site in both isozymes also show important amino acid replacements for
example human MAO-A contains Phe-208 (Ile-199 in human MAO-B) and Ile-335 (Tyr-
326 in human MAO-B) [64]. A combination of both conformational changes and residue
replacements affect the shape and size of the active site and this in turn will modify the
substrate specificity of the isozyme.

11
Rat MAO-A is about 92% identical with human MAO-A in terms of sequence
identity [64]. Rat MAO-A crystallizes as a dimer with active site cavity volume of about
450Å³, its cavity shaping loop conformation is very similar to that of human MAO-B [64,
70]. These structural data about rat and human MAO-A would suggest a possible
relationship between dimer formation and the conformation of cavity shaping loop
through a long range effect [64]. Such long range effect hypothesis had been observed in
a number of other enzymes like dihydrofolate reductase [69]. Kinetic studies showed that
rat MAO-A metabolizes human MAO-B substrates (like benzylamine) more efficiently
than does human MAO-A [66]. Based on the facts mentioned above, the approach of
using rat MAO-A as a laboratory model for the development of human MAO-A specific
inhibitor should be re-evaluated [64].
1.4- Reaction mechanism of MAO enzyme
The enzyme catalyzes the oxidative deamination of primary, secondary and some
tertiary amines, this reaction will result in the formation of hydrogen peroxide, an
aldehyde and either ammonia (if the substrate is primary amine) or substituted amine (if
the substrate is secondary amine) [36, 52]. For MAO-B, the reaction pathway depends on
the type of substrate. In this regard the oxidation of benzylamine follows ternary complex
mechanism where the imine product dissociates from the oxidized enzyme. On the other
hand the binary complex mechanism is usually followed by phenethylamine and in this
case the imine product will dissociate from reduced enzyme before oxidation of the
reduced enzyme-imine complex takes place [73, 74]. Figure 1.2 shows the catalytic
12
reaction pathway for MAO enzyme. The existence of these two pathways (binary and
ternary) depends on the relationship between the rate of imine dissociation from reduced
enzyme and the rate of reduced enzyme-imine complex oxidation [75]. For ternary
complex mechanism (benzylamine), the oxidation rate of reduced enzyme-imine complex
is faster than the rate of imine dissociation from reduced enzyme. In binary complex
mechanism (phenethylamine), the rate of imine dissociation is faster than the rate of
reduced enzyme-imine complex oxidation [73, 75]. Catalysis pathway for MAO-A had
been found to follow ternary complex mechanism only [76].

13
Figure1.2- A schematic representation for catalytic reaction pathway followed by MAO
enzyme. This figure was adapted from reference [52].

14
MAO enzyme catalytic mechanism involves the cleavage of Cα-H bond in amine
substrate with the transfer of two reducing equivalents to the enzyme (flavin ring) and
this will result in formation of imine product and flavin hydroquinone [68]. It is believed
that the amine substrate should be in the deprotonated form so that it can enter the active
site and undergo metabolism, however the exact mechanism of substrate deprotonation at
physiological condition is not known yet [68, 77, 86]. Kinetic isotope studies had
demonstrated that Cα-H bond cleavage is the rate limiting step in oxidative deamination
reaction [77, 78]. Stereochemical analysis showed that only pro-R hydrogen can be
transferred in this catalytic reaction (undergoes abstraction) [73, 79, 80]. The abstraction
of pro-R H can happen in one of three possibilities: as a proton, a hydride ion or a
hydrogen atom. To identify hydrogen form that undergo abstraction, the effect of electron
withdrawing group in para substituted substrate on reaction rate was tested. It was found
that increasing electron withdrawing power will increase reaction rate and this fact
supports the possibility of proton abstraction [77, 78].
Two hypotheses had been suggested for the transfer of reducing equivalents in
MAO catalyzed reaction. The first and more acceptable hypothesis is called polar
nucleophilic mechanism. In this mechanism, substrate amine will attack flavin C4a and
this will form flavin-substrate adduct. Once this adduct is formed, N5 position of flavin
ring becomes a strong base that can abstract proton from Cα [77]. Figure 1.3 shows a
schematic representation for polar nucleophilic mechanism of MAO catalysis. NMR
studies had observed that the pKa value of N5 proton for flavin hydroquinone to be ~24
and this support the idea of N5 sufficient basicity to perform abstraction [81]. As
15
mentioned previously in FAD binding domain structure, the isoalloxazine ring is in a bent
conformation about (N5-N10) axis which deviates about 30˚ from normal planarity [53].
This bending will make N5 atom closer to sp3 than sp2 conformation and this will lead to
a higher electron density at N5 position (better nucleophile) and a lower electron density
at C4a position (better electrophile). This will facilitate the nucleophilic attack by a
suitable substrate at C4a position [68].
The other and less acceptable hypothesis is called single electron transfer (SET)
mechanism; this mechanism involves one electron oxidation of substrate amine nitrogen
leading to the formation of aminium cationic radical and flavin anionic radical. The
production of aminium radical will lower pKa value of Cα-H and this will facilitate
proton abstraction by a base in the active site of MAO enzyme [82-84]. Figure 1.4 shows
a schematic picture for single electron transfer hypothesis. Different structural data
showed that no amino acid within MAO active site can function as an active base or acid
[52]. Experimental monitoring failed to identify intermediate radical and thus it is
difficult to provide any support for SET hypothesis [85]. The formation of aminium
cationic radical is expected to interact with tyrosine residues in aromatic cage (π-cationic
interaction) and this can halt any further reaction [52, 75].
16
Figure 1.3- A schematic representation for the proposed polar nucleophilic mechanism of
MAO enzyme catalysis. This figure was adapted from reference [52].
17
Figure 1.4- A schematic representation for the proposed single electron transfer (SET)
mechanism of MAO catalysis. This schematic figure was adapted from reference [52].
18
1.5- Functional role of aromatic cage
Previous structural analyses had observed that the active site of MAO enzyme
contains two tyrosine residues (Tyr398 and Tyr435 in MAO-B), these residues are
situated in front of the re face of flavin ring and they exhibit an approximately
perpendicular conformation relative to the flavin ring. The phenolic side chains of these
two residues are separated from each other by about 7.8Å [47]. A number of mutagenesis
analyses targeting Tyr435 in MAO-B were performed to identify possible functional
roles of the aromatic cage. Targeting Tyr398 in these analyses was excluded because this
residue is involved in a cis bonding with Cys397 which binds covalently through
thioether linkage with flavin. It is believed that any change in Tyr398 may adversely
affect covalent flavin binding [68, 88].
The aromatic cage does not seem to perform any important structural role within
the active site of MAO enzyme but mutant enzymes show lower stability upon
solubilization by detergents as compared to wild enzyme [88]. Other flavin dependent
amine oxidase enzymes contain aromatic amino acids in a conformation similar to that
observed in the aromatic cage of MAO enzyme and this observation suggests a possible
functional role for aromatic cage in catalytic activity [89, 90]. One of the expected roles
of aromatic cage is to polarize the electron single pair of substrate amine and this will
enhance substrate nucleophilicity. As mentioned above, the distance between phenolic
side chains of aromatic cage was calculated to be 7.8Å which is believed to be optimal
for substrate amine polarization [88]. Aromatic cage may also play a steric role through
19
dipole moment effect of Tyrosine side chain. These dipolar effects can simulate the
presence of electron withdrawing group at para position of substrate leading to a
reduction in the distance between amine nitrogen and flavin C4a, this will facilitate the
transfer of charge from amine to flavin and encourage the formation of flavin C4a adduct
[91]. Figure 1.5 shows us a schematic representation for the role of aromatic cage in
substrate oxidation.
The dipole effect of aromatic cage may also contribute to substrate specificity of
MAO-B, it is suggested that using a substrate having a dipole moment in the same
direction as do the aromatic cage dipoles will result in repulsive interaction and a change
in substrate orientation within active site cavity [88]. All these facts about aromatic cage
structure and function strengthen polar nucleophilic mechanism of MAO-B catalysis
[88].
20
Figure 1.5- A schematic representation for the effect of dipole moments of Tyr398 and
Tyr435 residues in MAO-B on the electron lone pair of the substrate amine before the
nucleophilic attack takes place. This figure was adapted from reference [88].
1.6- MAO inhibitors with a possible or approved clinical application
A summary about these chemicals is shown in the below table (table 1.1) which
was adapted from reference [134].

21
Table 1.1- A brief summary about MAO-inhibitors with a possible or approved clinical
application.
MAO inhibitor Selectivity Binding Applications
Irreversible, Antidepressant and

Phenelzine MAO-A/ MAO-B
covalent. anxiolytic.
Irreversible, Antidepressant and

Tranylcypromine MAO-A/ MAO-B
covalent. anxiolytic.
Selegiline Irreversible,
MAO-B Antiparkinsonian
(L-deprenyl) covalent.
Irreversible,
Rasagiline MAO-B Antiparkinsonian
covalent.
Development as
Reversible, antiparkinsonian was
Lazabemide MAO-B
covalent. discontinued due to
toxicity issues.
Currently undergo phase
Reversible,
Safinamide MAO-B III clinical trials as
non-covalent.
antiparkinsonian.
Antiepileptic and it is
Reversible,
Zonisamide MAO-B approved in Japan as
non-covalent.
antiparkinsonian.
Antidepressant and
Reversible,
Moclobemide MAO-A anxiolytic. It is not
non-covalent.
approved in USA.
22
1.7- MAO-B inhibitors with a possible disease modifying effect
Traditional drug design approach focuses on a single target within disease
pathologic pathway; this discovery tactic is based on the idea of minimizing adverse
effects by designing a highly selective ligand that will avoid interaction with other
unwanted biological targets (cross reactivity) [22, 23]. Such method for drug design and
discovery proved to be incompetent when dealing with diseases that involve complex
pathological picture; a good example for such conditions is the neurodegenerative
diseases. New attempts, to address an effective therapeutic approach for
neurodegenerative diseases, concentrate on designing a ligand that targets multiple
abnormalities within disease pathway. This novel pharmaceutical instrument shows some
successful results regarding disease process modification with no significant elevation in
the incidence of side effects [21, 92]. Below we will mention some compounds that were
designed using designed multiple ligand approach.
A- Rasagiline:
It is an irreversible and selective inhibitor of MAO-B [93]. It was approved by
FDA for Parkinson's disease treatment in 2006 either as an early monotherapy or as an
adjunct treatment to levodopa [11]. Rasagiline exhibits a disease modifying capability
when given in a dose of 1 mg/ day [94]. It seems that the propargylamine moiety is
responsible for the neuroprotective feature of rasagiline; it was observed that the S-
enantiomer is more than 1000 times weaker than rasagiline (R-enantiomer) as a MAO
inhibitor however the S-enantiomer is still able to show neuroprotective activity [95-98].
23
The main metabolite of rasagiline (aminoindan) also shows neuroprotective effect in vitro
studies [98, 99].
The neuroprotective mechanism of propargylamine moiety depends on defending
the mitochondria through several means like activation of protein kinase C (PKC), Bcl-2,
phosphoinositide kinase 3 and mitogen activated protein kinase (MAPK). It also down
regulates pro-apoptotic proteins like Bax, Bad and Fas [93]. Rasagiline also stimulates
the liberation of the soluble form of amyloid precursor protein α (sAPPα) through PKC-
MAP mediated activation of α-secretase [27, 28]. Rasagiline seems to possess
neurorescuing ability through induction of brain derived neurotrophic factors (BDNF)
and glial derived neurotrophic factors (GDNF) [100, 155].
B- Ladostigil:
This drug was designed by combining the propargylamine moiety of rasagiline
with the carbamate cholinesterase inhibitory moiety of rivastigmine. The resultant drug
can inhibit acetylcholine esterase, butyrylcholine esterase, MAO-A and MAO-B [101].
The drug shows a neuroprotective effect similar to that of rasagiline [101, 102].
Ladostigil also shows antidepressant activity through inhibition of MAO-A inside the
brain leading to an elevation in the level of dopamine, norepinephrine and serotonin
[108]. This compound is believed to be effective in the treatment of Alzheimer's disease
and depression.
24
C- HLA20:
This lead compound was prepared by combining the antioxidant chelator moiety
of 8-hydroxyquinoline derivative of the iron chelator VK-28 with the propargylamine
moiety of rasagiline or selegiline [103-105]. This compound exhibits a moderate
selectivity for MAO-B enzyme and also acts as a free radical scavenger. The rationale
behind combining iron chelation effect with MAO-B inhibition emerges from the fact
that brain level of iron and MAO-B increases with age and it also increases in
neurodegenerative diseases as a result of gliosis. The rise in MAO-B level will elevate
the level of oxidative deamination leading to an increase in hydrogen peroxide production
as a byproduct. The resultant hydrogen peroxide will undergo Fenton reaction in the
presence of elevated iron content leading to exaggerated hydroxyl radical level and
inevitable oxidative stress [95, 106].
Based on this scenario M30 compound was introduced, this compound is a potent
inhibitor of MAO-A and MAO-B (inhibits both of them equally) and it has iron chelation
characteristics similar to desferoxamine. The neuroprotection features of M30 are like
those of rasagiline and ladostigil [103, 107].
D- Istradefylline (KW-6002):
It is MAO-B enzyme inhibitor and Adenosine A2a receptor blocker [92].
Adenosine A2a receptor is a G-protein coupled receptor which will stimulate adenylate
cyclase upon activation leading to an increase in cAMP level [109]. This receptor subtype
25
is found mainly in the striatum (GABAergic striatopallidal neurons) where it is co-
localized with dopamine D2 receptor [110-112]. It is believed that activation of
adenosine A2a receptor can produce a functional antagonistic effect on dopamine D2
receptor through reduction in G-protein coupling activity of dopamine D2 receptor and
lowering the binding affinity of dopamine D2 agonist [113, 114]. Because both dopamine
D2 receptor and adenosine A2a receptor are coupled to adenylate cyclase and have some
similarities in signaling system, it is possible that the functional antagonistic effect can
also be mediated at the downstream of signaling pathway (second messenger level) [115].
Based on the localization of adenosine A2a receptor and their functional
relationship with dopamine D2 receptor, it is obvious that the blockade of adenosine A2a
receptor can be used as a non-dopaminergic approach to treat Parkinson's disease.
Blockade of adenosine A2a receptor can improve motor activity in animal models of
Parkinson's disease through strengthening dopamine D2 neurotransmission [116-118].
Some studies showed that adenosine A2a receptor antagonism can also produce a
beneficial motor effect in dopamine D2 receptor knockout mice suggesting that
adenosine A2a receptor antagonism can produce such effect through dopamine D2
receptor independent pathway [119].
Many epidemiological studies had shown that blockade of adenosine A2a
receptors can provide a protective effect against dopaminergic neurons degeneration
observed in Parkinson's disease and MPTP induced neurotoxicity [115, 120, 121].
Caffeine ingestion (non-selective adenosine A1/ A2a receptor blocker) is shown to be

26
associated with a lower tendency to develop Parkinson's disease in men [122-125]. The
neuroprotective effect produced by adenosine A2a receptor antagonists is not fully
understood, a possible explanation for such effect involves the reduction of glutamate
release through adenosine A2a receptor blockade and this can lower neurodegeneration
induced excitotoxicity [115, 120].
E- Pioglitazone and rosiglitazone:
These drugs are used for the treatment of type II diabetes (rosiglitazone is
subjected to usage restrictions due to possible cardiotoxic effects) [126, 127]. These
drugs (Thiazolidinedione TZD) reduce insulin resistance through targeting peroxisome
proliferator activated receptor gamma (PPARγ) [128]. Recently these compounds receive
a lot of attention for a possible use as neuroprotective agents in stroke and Parkinson's
disease [129]. The neuroprotective effect is attributed to three main effects: modulation
of inflammation, MAO-B inhibition and activation of mitoNEET.
These compounds can manipulate inflammatory pathways leading to a reduction
in reactive oxygen species (ROS) production through inhibition of matrix
metallopeptidase 9 and Inhibition of iNOS [92]. They can also increase the level of
CuZn-superoxide dismutase and this can enhance the neuroprotective effect [130]. It had
been shown that pioglitazone can lower the liberation of cytokines in cultured cells upon
lipopolysaccharide treatment [131]. Both rosiglitazone and pioglitazone can inhibit the
deposition of beta amyloid in the brain and lower the release of inflammatory mediator
induced by beta amyloid [132, 156, 157].

27
The neuroprotective effect of pioglitazone and rosiglitazone can be explained
partially through the inhibition of MAO-B enzyme and this will prevent the conversion of
MPTP to the neurotoxic form MPP+ in MPTP Parkinsonian mouse model [133].
Finally pioglitazone can function as a ligand for a mitochondrial protein named
mitoNEET and through this pathway pioglitazone can stabilize mitoNEET and change
the functional state of mitochondria leading to a neuroprotective output. Binding of
ligands to mitoNEET protein can result in depolarization of mitochondrial membrane,
alteration in mitochondrial oxidative condition and attenuation of rotenone toxic effect
[92].
Chapter two: Materials and Methods
2.1- Prospective candidates
Twenty four chemical compounds were purchased from Chembridge online
chemical store (www.hit2lead.com) as possible candidates for MAO-B inhibition. The
chemical structures for these compounds were predicted using similarity analysis
approach which depends on molecular shape comparison using Gaussian function for
volume matching [135]. Some specifications about these candidates are shown in table
2.1. Each compound was dissolved in an appropriate volume of dimethyl sulfoxide
(DMSO) so that a stock solution with 10 mM concentration was produced.
Initially, virtual approaches (docking studies) were used to build an early
impression about the binding tendency of these compounds to both MAO-B and bovine
serum albumin. Based on docking results, these compounds were screened in the
laboratory for both inhibition potential and selectivity against MAO-A and MAO-B. In
order to obtain a better understanding of these compounds' pharmacokinetic profile,
bovine serum albumin (BSA) binding assay and parallel artificial membrane permeability
assay (PAMPA) were performed. Finally in our hope to discover a possible
neuroprotective activity, these compounds were tested for antioxidant effect using
oxygen radical absorbance capacity (ORAC) assay. All these experimental procedures
can facilitate drawing the complete picture of possible scaffolds for potent, selective and
28
29
centrally acting MAO-B inhibitors with disease modifying feature for future use in the
treatment of Parkinson's disease.
Table 2.1- Prospective candidates' specifications.
Mol.
ID code Chemical structure Chemical name Weight
g/ mole
4-[3-(6-chloro-2-hydroxy-
4-phenyl-3-quinolinyl)-5-
7909619 (2-pyridinyl)-4, 5-dihydro- 500.9
1H-pyrazol-1-yl]-4-
oxobutanoic acid.
7, 7'-[1,4-piperazinediylbis
5476423 (methylene)] di (8- 400.5
quinolinol).
4-[(2, 4-dimethyl-1, 3-
thiazol-5-yl) carbonyl]-3-
hydroxy-5-[4-(methylthio)
7747412 451.6
phenyl]-1-(3-pyridinyl
methyl)-1, 5-dihydro-2H-
pyrrol-2-one.
30
Table 2.1- (Continued) Prospective candidates' specifications.
Mol.
g/ mole
2-methyl-5-(3-methyl-4-
oxo-3, 4-dihydro-1-
7655826 phthalazinyl)-N-[2-(4- 442.5
morpholinyl) ethyl]
benzene sulfonamide.
1-[3-(dimethyl amino)
propyl]-4-(2-furoyl)-3-
6656215 hydroxy-5-(3-nitrophenyl)- 399.4
1, 5-dihydro-2H-pyrrol-2-
one.
6-methoxy-N-[3-(4-
6405277 morpholinyl) propyl]-2- 364.5
naphthalene sulfonamide.
1-(4, 5-dimethyl-1, 3-
thiazol-2-yl)-5-(2-furyl)-3-
hydroxy-4-[(7-methoxy-1-
7917584 450.5
benzofuran-2-yl)
carbonyl]-1, 5-dihydro-
2H-pyrrol-2-one.
31
Mol.
g/ mole
5-(2-fluorophenyl)-3-
hydroxy-4-[(4-methyl-2-
phenyl-1, 3-thiazol-5-yl)
7730595 507.6
carbonyl]-1-[2-(4-
morpholinyl) ethyl]-1, 5-
dihydro-2H-pyrrol-2-one.
1-(3, 4-dimethoxybenzyl)-
3-hydroxy-3-[2-(2-
7839510 467.5
naphthyl)-2-oxoethyl]-1,
3-dihydro-2H-indol-2-one.
3-hydroxy-4-[(7-methoxy-
1-benzofuran-2-yl)
7917586 carbonyl]-1-(5-methyl-3- 430.4
isoxazolyl)-5-phenyl-1, 5-
dihydro-2H-pyrrol-2-one.
32
Mol.
g/ mole
3-{5-[4-(dimethylamino)
benzylidene]-4-oxo-2-
5140311 336.4
thioxo-1, 3-thiazolidin-3-
yl}propanoic acid.
5-(2, 4-dimethoxy
benzylidene)-2-
5108394 340.4
(phenylimino)-1, 3-
thiazolidin-4-one.
5-(4-chlorobenzylidene)-2-
5108400 (phenylimino)-1, 3- 314.8
thiazolidin-4-one.
1-(3, 4-dihydroxy phenyl)-
2-{[1-(1-naphthyl)-1H-
7919469 378.4
tetrazol-5- yl]
thio}ethanone.
33
Mol.
g/ mole
5-[(5-phenyl-2-furyl)
5143886 methylene]-2-thioxo-1, 3- 287.4
thiazolidin-4-one.
[5-(4-methoxybenzylidene
)-4-oxo-2-thioxo-1,3-
5140321 309.4
thiazolidin-3-yl]acetic
acid.
5-(1, 3-benzodioxol-5-
ylmethylene)-3-(4-
5144779 355.4
methylphenyl)-2-thioxo-1,
3-thiazolidin-4-one.
34
Mol.
g/ mole
Ethyl N-(1, 1-dioxido-1, 2-
7542216 benzisothiazol-3-yl)-N- 282
methylglycinate.
5, 6-dimethyl-4-{[(2-
methyl-1, 3-thiazol-4-yl)
7738249 307
methyl]thio}thieno[2,3-d]
pyrimidine.
[(1-methyl-1H-pyrrol-2-
yl)methyl](3-
7929249 238
pyridinylmethyl) amine
hydrochloride.
3-methyl-2-{[2-(4-methyl-
1, 3-thiazol-5-yl)
7954103 317
ethyl]thio}-4(3H)-
quinazolinone.
35
Mol.
g/ mole
N-[(2-hydroxy-8-methyl-
3-quinolinyl) methyl]-N-
7935085 372.4
(4-methoxyphenyl)
methane sulfonamide.
[5-(anilinomethylene)-4-
oxo-2-thioxo-1, 3-
5124176 294.4
thiazolidin-3-yl] acetic
acid.
Methyl [(4-methyl-5, 6, 7,
7697413 8-tetrahydro-2- 252
quinazolinyl)thio] acetate.
36
2.2- Docking studies
The 2D and 3D structures of the tested compounds were prepared using
MarvinSketch 6.0.3 (www.chemaxon.com). Human MAO-B crystal (2BK3) and bovine
serum albumin (BSA) crystal (4F5S) were obtained from RCSB Protein Data Bank
(www.rcsb.org). Both MAO-B and BSA crystals were prepared for docking using UCSF
Chimera 1.8 (www.cgl.ucsf.edu/chimera) [150] and AutoDockTools (ADT) 1.5.6
(http://mgltools.scripps.edu) [151, 152]. ADT 1.5.6 was also used to prepare the tested
compounds (ligands) for docking operation. Docking of the ligands into these targets
(crystals) was done using AutoDock 4.2 (http://autodock.scripps.edu) [151]. The final
results were analyzed using AutoDockTools (ADT) 1.5.6, LigPlot+ v.1.4.5 [153], UCSF
Chimera 1.8 and PyMOL v.1.3 (www.schrodinger.com) [154].
2.3- Monoamine oxidase inhibition assay
This assay measure the compound's inhibition strength and degree of selectivity
toward MAO-A and MAO-B. The basic concept of this assay involves the ability of both
MAO-A and MAO-B to convert the non-fluorescent and non-selective substrate
(kynuramine) into the intermediate 3-(2-aminophenyl)-3-oxo-propionaldehyde which will
undergo spontaneous intramolecular condensation leading to the formation of the
fluorescent product 4-Hydroxyquinoline (4-HQ) [136]. Based on this concept, the level
of MAO activity can be determined through measuring the fluorescence level of 4-
Hydroxyquinoline (4-HQ). In the presence of the tested compound, any reduction in

37
fluorescence level will reflect the inhibition capacity of that compound. Figure 2.1 shows
the basic principle of this assay.
In this test, KPO4 solution (0.1 M, pH 7.4) was used as a buffer which was
warmed to 37˚ C prior to performing the test. Falcon black 96 well plates (Cat#353241)
were also used in this test. Kynuramine was purchased from Sigma-Aldrich (Cat#: k-
3250) and a stock solution with a concentration of 25 mM was prepared by dissolving
kynuramine in the appropriate volume of distilled water. This stock solution was further
diluted with KPO4 buffer to yield a final concentration that is close to the Km value for
the reaction catalyzed by either MAO-A or MAO-B (the final concentration of
kynuramine solution after dilution was 40 µM for MAO-A and 20 µM for MAO-B).
Human MAO-A and human MAO-B were purchased from Fisher Scientific
(www.fishersci.com). These enzymes were used in this assay as 5 mg/ ml stock solutions
and in order to minimize thermal induced inactivation of MAO, both enzymes were
stored at -80˚ C and they even kept on ice till their employment in the assay. The stock
solutions of MAO-A and MAO-B were further diluted with KPO4 buffer to obtain final
solutions with concentrations of 0.006 mg/ ml for MAO-A and 0.015 mg/ ml for MAO-
B. 2 N NaOH was used as a stopping reagent by causing protein denaturation.
In this assay, the tested compounds were serially diluted with KPO4 buffer (100
µl/ well in duplicate) across the plate to produce eight points dose inhibition curve. As
mentioned previously, the tested compounds were dissolved in DMSO and the final
concentration of DMSO in the assay was less than 0.5%. It is reported that DMSO shows
38
the least MAO inhibitory effect. The enzyme activity (fluorescence level) in the absence
of any inhibitor (only DMSO) was considered as a control (100% MAO activity). After
the compounds were serially diluted, the plate was covered and incubated at 37˚ C for 10
minutes so that both the compounds and the plate can reach a temperature of 37˚ C. Then
the substrate (kynuramine) was added as 50 µl/ well after that the enzyme (either MAO-
A or MAO-B) was added as 50 µl/ well in order to start the reaction. The plate was then
covered and incubated at 37˚ C for 20 minutes in order to obtain the ultimate level of
activity for the enzyme. The final assay volume was 200 µl per well. After incubation
period, 75 µl of 2 N NaOH solution was added per well to stop the reaction. The
fluorescence level of 4-Hydroxyquinoline was measured at 310/ 380 nm wavelength pair
by using a Flourometer (top reader, BMG-FlouStar).

39
Figure 2.1- Conversion of kynuramine into 4-Hydroxyquinoline as catalyzed by both
MAO-A and MAO-B, this figure was adapted from reference [137].
40
2.4- Bovine serum albumin (BSA) binding assay
This assay measures the binding affinity of the tested compounds toward serum
bovine albumin. Fluorescence quenching represents the main principle of this assay and it
involves the reduction in fluorescence level (quantum yield) of a fluorophore upon
interaction with a quencher molecule [138, 139]. Bovine serum albumin (BSA) shows
intrinsic fluorescence activity (fluorophore) due to the presence of two types of aromatic
amino acids (tryptophan and tyrosine). When BSA is excited at 280 nm, it will show a
strong fluorescence emission at 342 nm and this emission is mainly attributed to
tryptophan [140-142]. This test is based on the idea that higher binding of the tested
compound to BSA will result in greater quenching of the fluorescence activity of BSA
and this can be measured experimentally.
In this assay, KPO4 solution (0.1 M, pH 7.4) was used as a buffer. Bovine serum
albumin was obtained from Boston BioProducts (Ashland, MA, USA). A stock solution
of BSA (1 mg/ ml) was prepared by dissolving BSA in KPO4 buffer just before starting
the test. Falcon black 96 well plates (Cat# 353241) were used for this test. The final assay
volume was 200 µl/ well. The tested compounds were serially diluted (100 µl/ well in
duplicate) with KPO4 buffer across the plate so that the desired range of concentrations
(an eight points data curve) was obtained. As mentioned before, dimethyl sulfoxide
(DMSO) was used to dissolve the tested compounds and the fluorescence level of BSA in
the absence of any tested compound (only DMSO) was considered as a control (no BSA
binding). The stock solution of BSA was added as 100 µl/ well (the final concentration of
41
BSA in each well was 7.5 µM) after that the plate was covered and incubated at room
temperature for 30 minutes. After incubation, the plate was read at 280/ 340 nm
wavelength pair using a Flourometer (BMG-FlouStar).
2.5- Parallel Artificial Membrane Permeability Assay (PAMPA)
This assay represents a high throughput approach to predict passive, transcellular
permeability of the tested compounds in early stages of drug development process. This
assay is cheap, highly successful and reproducible [143]. It uses a synthetic lipid layer
immobilized on filter support to simulate the passive permeability of the compound
across different biological barriers (blood brain barrier, skin and gastrointestinal tract)
[143-146]. This assay can't determine active transport of the compound across biological
membranes [144].
The Hexadecane Method of PAMPA (HDM-PAMPA) measures the passive
diffusion of the tested compound from the donor compartment (which contains the
desired concentration of the compound dissolved in buffer medium) to an acceptor
compartment (compound free compartment, contains only buffer medium). These two
compartments are separated by a hexadecane liquid layer on a polycarbonate membrane
support. The concentration of the tested compound can be determined in these two
compartments by measuring the fluorescence level for the compound.
For this assay, 5% DMSO in phosphate buffered saline pH 7.4 was used as a
buffer system. Hexadecane and hexane were obtained from commercial sources and a 5%
42
(v/v) hexadecane in hexane mixture was prepared. Compound under investigation
(5140311) was diluted with buffer system (5% DMSO in BPS) to yield a final solution
with a concentration of 500 µM. A 96 well MultiScreen filter plate for permeability
(donor plate) was ordered from Millipore Corporation (Billerica, MA, USA, Cat#
MAPBMN310). The 96 well acceptor plate was also obtained from Millipore (Cat#
MSSACCEPTOR).
The assay was initiated by the addition of 15 µl/ well of 5% (v/v) hexadecane in
hexane solution into the donor plate; the plate was then placed uncovered in a fume hood
for one hour in order to ensure that hexane was completely evaporated and a fine layer of
hexadecane was generated on polycarbonate film. This was followed by the addition of
buffer solution (5% DMSO in PBS) into the acceptor plate as 300 µl per each well. The
donor plate was then placed on the acceptor plate so that the underside of the membrane
for donor plate is in contact with the buffer solution in each well of the acceptor plate.
The compound (5140311) solution was added to the donor plate as 150 µl/ well in
triplicate. After that, the donor plate was covered with its lid and incubated at room
temperature for a period of four hours. During incubation period, equilibrium solution for
compound (5140311) was prepared by simply mixing donor compartment volume (150
µl of compound solution) with acceptor compartment volume (300 µl of buffer solution).
After incubation period, 250 µl was transferred from each well of the acceptor plate into
Falcon black 96 well plate (Cat# 353241). A similar volume of equilibrium solution for
compound (5140311) was transferred into the same 96 well black plate. Also compound
(5140311) solution was serially diluted with assay buffer across the black plate to yield a
43
suitable eight points fluorescence-concentration curve. The generation of such curve will
help us to predict the concentration of the compound in the acceptor compartment based
on the observed fluorescence (interpolation). The fluorescence level of the compound
(5140311) was measured at 540/ 620 nm wavelength pair by using FLX 800 microplate
fluorescence reader (Biotek instruments).
2.6- Oxygen Radical Absorbance Capacity (ORAC) assay
This assay measures the ability of the tested compounds to scavenge peroxyl
radicals that are generated by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH),
thereby it can screen these compounds for any possible antioxidant activity [147, 148].
The assay principle is based on the idea that the presence of peroxyl radicals can lower
the fluorescence level of a fluorophore (fluorescein) as a result of oxidative degradation.
A compound with antioxidant power can preserve the fluorescence activity by reducing
the oxidative degradation of fluorescein [149].
For this assay, KPO4 solution (0.1 M, pH 7.4) was used as a buffer, Falcon black
96 well plates (Cat# 353241) were also used and the final volume for this assay was 200
µl per each well. AAPH was obtained from Sigma-Aldrich (St. Louis, MO, USA) and it
was stored in a dry environment. Before starting the assay, AAPH was dissolved in an
appropriate volume of KPO4 buffer to yield a stock solution with a concentration of 72
mM. This stock solution was kept on ice until assay initiation. Fluorescein was also
purchased from Sigma-Aldrich (St. Louis, MO, USA) and prior to assay initiation, it was
also dissolved in KPO4 buffer to prepare a stock solution with a concentration of 112
44
nM. Vitamin C (L-ascorbic acid) was also ordered from Sigma-Aldrich (St. Louis, MO,
USA); before starting the test, it was dissolved in KPO4 buffer to obtain a solution with a
concentration of 10 mM. Vitamin C was used in this assay as a reference substance
(positive control). As mentioned previously, DMSO was used as a vehicle for the tested
compounds, the fluorescence level of fluorescein in the absence of any tested compound
(only DMSO) was considered as a negative control.
The compounds under investigation were serially diluted with KPO4 buffer across
the assay plate (20 µl/ well in duplicate) to yield the required range of concentrations.
Fluorescein solution was then added as 80 µl per each well. This was followed by the
addition of peroxyl radical generator (AAPH) as 100 µl per each well. The plate was then
covered and incubated at room temperature for one hour. After that, the fluorescence
level was read at 485 nm excitation and 528 nm emission wavelengths through using
FLX 800 microplate fluorescence reader (Biotek instruments).
2.7- Data analysis
Most of the results for the previously mentioned experiments were displayed as
(Mean ± SEM). Prism 5 (www.graphpad.com) was used for columns generation, curves
fitting (nonlinear regression) and lines fitting (linear regression).

Chapter three: Results
3.1- Selection of the most efficient candidates
Based on the results of our experiments, a scatter plot was generated by plotting
MAO-B enzyme inhibition potential (capacity) against antioxidant potential (capacity) of
the prospective candidates. The scatter plot is shown in figure 3.1 and it was generated by
using 100µM concentration of each compound in MAO-B inhibition assay and 20µM
concentration of each compound in antioxidant (ORAC) assay.
By careful examination of this plot, we can easily choose the most efficient
candidates that display a perfect combination of both MAO-B inhibition capacity and
antioxidant effect. According to the scatter graph, six compounds show such efficient
combination and the identification codes for these chemicals are: 5143886, 5140311,
7730595, 5140321, 7919469 and 7747412. In this section we will focus only on the
results of these compounds since they represent encouraging candidates for possible
antiparkinsonian agents with disease modifying capability. We will also mention the
results for additional two chemicals and these are: 5476423 and 7954103. According to
the plot, compound 5476423 shows excellent antioxidant behavior with minor MAO-B
inhibition ability. On the other hand, compound 7954103 shows a potent MAO-B
inhibition effect with minor antioxidant activity.
45
46
Figure 3.1- A scatter plot of MAO-B inhibition potential (percentage) against antioxidant
potential (percentage) for our chemicals. For antioxidant potential, vitamin C was used as
a reference (100% potential).

47
3.2- Docking studies results
In order to obtain preliminary expectations about the pharmacodynamics and
pharmacokinetics characteristics of our candidates, docking analyses were performed by
targeting human MAO-B crystal and bovine serum albumin crystal respectively. Docking
studies can also give us a virtual insight into compound's orientation within the active site
of our target.
Based on our results of docking studies, it is obvious that compounds: 5140311,
5140321, 5143886, 7919469 and 7954103 exhibit an extended conformation that will
occupy both the entrance cavity and the substrate cavity within the active site of MAO-B
crystal. Such orientation will force Ile 199 residue to gain an open configuration leading
to the fusion of both entrance cavity and substrate cavity. The results of docking studies
for these five compounds are shown in figure 3.2, 3.3, 3.4, 3.5 and 3.6 respectively.
According to these figures, we can easily notice that these five candidates are situated in
the active site with a position that directly facing the FAD moiety. In figure 3.2, we can
recognize that there is a hydrogen bond between the oxygen atom attached to position
four of the 2-thioxo-1, 3-thiazolidine moiety of compound 5140311 and the hydroxyl
group of phenyl moiety in Tyr 435 (residue of aromatic cage). Another important finding
to mention is the presence of two hydrogen bonds in the docking results of compound
7919469 as can be seen in figure 3.5. The first hydrogen bond is formed between the
nitrogen atom at position three of tetrazole moiety of compound 7919469 and the
hydroxyl group in carboxylic acid moiety of Phe 168. The second and most important
48
hydrogen bond is formed between the p-hydroxyl group of dihydroxy phenyl moiety in
compound 7919469 and N (5) in the isoalloxazine ring system of FAD moiety. For these
five compounds, hydrophobic interactions with residues of active site contribute
significantly to the binding energy.
The results for the three remaining compounds: 5476423, 7730595 and 7747412
are shown in figure 3.7, 3.8 and 3.9 respectively. From these results, we can see clearly
that these compounds do not enter to the active site of MAO-B crystal. These three
compounds occupy a position close to the gating switch (loop 99-112) and through
hydrophobic interactions with the amino acid residues in this loop (mainly Phe 103 and
Pro 102); these compounds can hinder the entrance of substrates into the entrance cavity
of MAO-B enzyme. The values for energy of binding and dissociation constant (KI) for
the last three compounds are higher than those for the first five compounds and this may
be attributed to the difference in the location of binding.

49
Figure 3.2(A)- Two dimensional representation for the docking of compound 5140311
into MAO-B crystal. This image was generated by LigPlot+ v.1.4.5 [153]. Carbon,
oxygen, nitrogen and sulfur atoms are represented by black, red, blue and yellow colors
respectively. Hydrogen bonds are represented by green dashed line while hydrophobic
interactions are drawn as small multiple red lines.

50
Figure 3.2(B)- Three dimensional representation for the docking of compound 5140311
into MAO-B crystal. This image was generated by PyMOL v.1.3
(www.schrodinger.com) [154]. Our ligand is colored by red and the hydrogen bond is
shown as a green line.

51
respectively. Hydrophobic interactions are drawn as small multiple red lines.

52
(www.schrodinger.com) [154]. Our ligand is colored by red.

53

54

55
respectively. Hydrogen bonds are represented by green dashed line while hydrophobic
interactions are drawn as small multiple red lines.

56

57

58

59
oxygen and nitrogen atoms are represented by black, red and blue colors respectively.
Hydrophobic interactions are drawn as small multiple red lines.

60

61
Figure 3.7(C)- This figure shows the location of binding for compound 5476423 (red
color) according to docking studies in MAO-B crystal. This compound occupies a
location close to the gating loop and may interfere with substrates accessibility to the
active site. This figure was generated by using UCSF Chimera 1.8
(www.cgl.ucsf.edu/chimera) [150].
62
Figure 3.7(D)- A close view for the previous figure, Here we can see how compound
5476423 (red color) is located near the gating loop and may interact with residues located
in that loop like Pro 102 and Phe 103. This figure was generated by using UCSF Chimera
1.8 (www.cgl.ucsf.edu/chimera) [150].

63
oxygen, nitrogen, fluoride and sulfur atoms are represented by black, red, blue, green and
yellow colors respectively. Hydrophobic interactions are drawn as small multiple red
lines.
64

65
Figure 3.8(C)- A close view for the binding position of compound 7730595 (red color) as
indicated by docking studies using MAO-B crystal as a target. As shown here the
compound is located close to the gating loop and may be involved in interactions with
residues located within the gating loop like Pro 102 and Phe 103. This figure was
generated by using UCSF Chimera 1.8 (www.cgl.ucsf.edu/chimera) [150].

66
oxygen, nitrogen, fluoride and sulfur atoms are represented by black, red, blue, green and
yellow colors respectively. Hydrophobic interactions are drawn as small multiple red
lines. The green dashed line represents hydrogen bond between the amine group at
position two of Threonine 478 residue and the hydroxyl group attached to position three
at the 1, 5- dihydro-pyrrol-2-one moiety of compound 7747412.

67

68
Figure 3.9(C)- A close view for the binding position of compound 7747412 (red color) as
indicated by docking studies using MAO-B crystal as a target. As shown here the
compound is located close to the gating loop and may be involved in hydrophobic
interactions with residues located within the gating loop like Pro 102 and Phe 103. The
green line represents hydrogen bond between Thr 478 residue and our compound. This
figure was generated by using UCSF Chimera 1.8 (www.cgl.ucsf.edu/chimera) [150].

69
Because the main binding sites in serum albumin for aromatic and heterocyclic
compounds are subdomain IIA (site I) and subdomain IIIA (site II) [158,159], docking
studies in BSA crystal were targeted toward these two binding sites only. By taking the
virtual binding parameters (energy of binding and dissociation constant) into our
consideration, it is clear that our candidates bind efficiently with nearly equal affinity to
site I and site II. The only compound that deviates from this established principle is
7919469, which shows some preference for binding to site I over site II. Table 3.1 shows
virtual binding parameters for our selected compounds in BSA docking studies.
70
Table 3.1- Docking results for BSA crystal.
Subdomain IIA Subdomain IIIA
Compound ID Energy of binding KI Energy of binding KI
(kcal/mol) (µM) (kcal/mol) (µM)
5140311 -5.53 89.11 -5.12 177.88
5140321 -5.78 58.01 -5.07 192.7
5143886 -6.35 22.03 -7.01 7.31
7919469 -7.52 3.1 -4.78 311.84
7954103 -5.97 42.09 -5.66 70.47
5476423 -5.98 41.7 -5.71 65.74
7730595 -5.29 131.79 -5.76 60.34
7747412 -4.96 232.32 -4.7 361.79

71
3.3- Monoamine oxidase inhibition assay results
The results of this assay provide a clear insight into the potential and selectivity of
inhibition produced by our candidates against MAO enzyme isoforms. Our results for this
test are summarized in figure 3.10, figure 3.11 and table 3.2. Figure 3.10 describes the
strength of MAO-B inhibition (potency of inhibition) produced by using 100µM
concentration of each selected compound. According to this figure, the highest inhibition
is produced by compound 5143886 where MAO-B activity falls down to a percentage
value of (2.148 ± 0.2697) and statistically this value represents (Mean ± SEM). The
lowest inhibition is produced by compound 5476423 where the activity of MAO-B
enzyme only falls down to a percentage value of (78.99 ± 0.1677). The remaining
compounds produce a strong inhibitory effect which generally lowers the activity of
MAO-B to below 50% level.
On the other hand, figure 3.11 shows the potency of the inhibitory effect against
MAO-A for only four compounds (5140311, 5140321, 5143886 and 5476423). This
potency of effect was generated by using a concentration of 100µM for each tested
compound. In this figure, the strongest inhibitory effect is produced by compound
5143886 with a reduction in MAO-A level of activity into a percentage value of (42.37 ±
1.737) while the weakest effect is produced again by compound 5476423 with a
reduction in MAO-A activity into a percentage value of (78.81 ± 0.4983). The other two
compounds appear to be able to lower enzyme activity to around 60%. By comparing
data from figure 3.10 with those in figure 3.11, we can obtain some information about the
72
selectivity in action of the tested compounds against MAO enzyme isoforms. It is very
clear that compound 5476423 shows no preference in its inhibitory effect against MAO
enzyme isoforms. Compounds 5140311 and 5143886 appear to be highly selective for
MAO-B inhibition while compound 5140321 shows a slight preference for the inhibition
of MAO-B over MAO-A.
Table 3.2 gives us information about the degree of inhibition selectivity toward
MAO-B and MAO-A isozymes. This table compares the IC50 value for MAO-B
inhibitory effect against that value for MAO-A inhibitory effect. Unfortunately, we were
only able to determine MAO-A IC50 for compounds 5140311 and 5476423 due to
technical restrictions. Compound 5140311 seems to be highly selective for inhibiting
MAO-B enzyme since the MAO-B IC50 value is about 16 times lower than IC50 value
for MAO-A.
73
Figure 3.10- MAO-B inhibition potential as produced by our compounds. Values are
presented as (Mean ± SEM). The concentration used for each compound was 100µM.
74
Figure 3.11- The potency of the inhibitory effect against MAO-A as produced by the
tested compounds. Results are presented as (Mean ± SEM); the concentration used to
generate such effect is 100µM.

75
Table 3.2- Inhibition selectivity toward MAO isozymes.
Compound ID MAO-B IC50 µM MAO-A IC50 µM
5140311 9.71 155
5140321 105 N/A
5143886 2.64 N/A
7919469 1.19 N/A
7730595 62.8 N/A
7747412 94.3 N/A
5476423 N/A 242
7954103 6.27 N/A

76
3.4- Bovine serum albumin (BSA) binding assay results
This assay provides a general view about compounds' degree of binding to serum
albumin and the influence of such interaction on the pharmacokinetics features of these
selected compounds. The binding potentials for these chemicals are shown in figure 3.12.
The results in this figure were generated by using 100µM concentration for each tested
compound; the outcomes in this figure were reported as (Mean ± SEM).
Examination of this figure reveals that these compounds, in general, show high
tendency for BSA binding. The lowest binding percentage is produced by compound
5476423 with a binding value of (44.16 ± 5.205) while the highest binding percentage is
produced by compound 5143886 with a value of (97.57 ± 0.1944). With the exception of
compound 5140311, all the remaining compounds show high degree of BSA binding that
is greater than 70%.
By using different concentrations for each compound (serial dilution), we were
able to generate a relative BSA binding curve (nonlinear regression). This curve was
utilized to obtain an estimation of EC50 value. Figure 3.13 shows the BSA binding curve
for each compound together with its calculated EC50. With some exceptions these EC50
values can be correlated with those results presented in figure 3.12. For instance, the
smallest EC50 values (highest binding) were calculated for compounds 5143886 and
5140321 while the largest value (least binding) was reported for compound 5476423.
77
Figure 3.12- BSA binding potential of the tested chemicals. These results were generated
by using a concentration of 100µM for each compound and the outcomes were presented
as (Mean ± SEM).
78
Figure 3.13- Relative BSA binding curves for the selected compounds. The calculated
EC50 values are also presented with these curves.

79
Figure 3.13- (Continued) Relative BSA binding curves for the selected compounds. The
calculated EC50 values are also presented with these curves.

80

81

82
3.5- Parallel artificial membrane permeability assay (PAMPA) results
This assay gives us an early prediction about the ability of the compound (under
investigation) to pass the blood brain barrier and reach CNS. This assay was only applied
to compound 5140311 because it was the only one that fluoresced at the excitation wave
length range provided by the fluorescence reader. By the end of this assay, the
fluorescence level was determined for both the acceptor compartment and equilibrium
solution as this level of fluorescence reflects the concentration of compound 5140311 in
these solutions. The results of fluorescence reader were then applied to the following
equation (provided by the manufacturer of assay apparatus) in order to calculate the rate
of permeation or effective permeability (Pe):
[𝑑𝑟𝑢𝑔]𝑎𝑐𝑐𝑒𝑝𝑡𝑜𝑟
log 𝑃𝑒 = log {𝐶 ∗ −ln(1 − )}
[𝑑𝑟𝑢𝑔]𝑒𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑢𝑚
𝑉𝐷 ∗𝑉𝐴
Where 𝐶 = ( )
(𝑉𝐷 +𝑉𝐴 ) 𝐴𝑟𝑒𝑎∗𝑡𝑖𝑚𝑒
𝑉𝐷 = Volume of donor compartment= 0.15 cm³.
𝑉𝐴 = Volume of acceptor compartment= 0.3 cm³.
Area= Active surface area of membrane= 0.048 cm².
Time= Incubation time for the assay and in our case it was four hours= 14400 seconds.
83
By applying all of the previously mentioned variables into our equation we get the
following:
0.15∗0.3
𝐶 = ((0.15+0.3) )
0.048∗14400
C = 1.446 × 10−4 cm s −1
611
log 𝑃𝑒 = log {(1.446 × 10−4 ) ∗ −ln(1 − )}
17013
Log Pe = -5.276 cm s −1
Pe = 5.2966× 10−6 cm s −1
According to the classification of PAMPA-BBB results (based on Pe ranges)
[160], it is very clear that our compound 5140311 is highly permeable through blood
brain barrier since our calculated Pe value is greater than 4.0 × 10−6 cm s −1. As we
mentioned previously in the materials and methods chapter (chapter two), compound
5140311 was serially diluted to generate a standard curve (eight points fluorescence-
concentration curve). We can estimate the concentration of our compound in the acceptor
compartment based on the measured level of fluorescence (interpolation using the
standard curve). In our case the concentration of compound 5140311 in the acceptor
compartment was 2.098125µM.

84
3.6- Oxygen radical absorbance capacity (ORAC) assay results
This assay predicts the antioxidant capacity of compound under investigation
through screening the ability of that compound to scavenge peroxyl radicals that are
generated by AAPH (radical initiator) [147, 148]. By scavenging these radicals, our
compound will be able to prevent the degradation of fluorescein thereby preserving the
fluorescence level of this fluorophore [149]. According to this principle, the fluorescence
level will reflect the antioxidant capacity of the tested compound.
The results for this assay are summarized in figure 3.14; these results were
obtained by using 20µM concentration of each tested compound. The outcomes are
presented as (Mean ± SEM); vitamin C (Ascorbic acid) was used as a positive control
(100% activity). In this figure, all the tested compounds show excellent antioxidant
potentials that are either equivalent or higher than that reported for Ascorbic acid (with
the exception of compound 7954103). The highest radical scavenging capacity was
reported for compound 7730595 with a percentage value of (205.2 ± 1.952) while the
least scavenging capacity was observed in compound 7954103 with a percentage value of
(31.24 ± 0.2169).
In this assay, serial dilution was used for each compound in order to generate
eight points fluorescence – concentration curve (nonlinear regression). The generation of
such curve helped us to estimate the value of EC50 for each compound. Unfortunately,
we were unable to correlate the calculated EC50 values (shown in table 3.3) with results
85
in figure 3.14. This may be due to technical restrictions within curve fitting (nonlinear
regression).
Figure 3.14- Percentage of antioxidant power for the selected chemicals. This figure was
generated by using 20µM concentration for each compound. The outcomes are presented
as (Mean ± SEM); Ascorbic acid was used as a positive control (100% activity).
86
Table 3.3- EC50 values for (antioxidant capacity – concentration) curve of each tested
compound.
Compound ID EC50 µM
Ascorbic acid 5.570
5140311 0.5748
5140321 3.698
5143886 N/A
7919469 12.56
7730595 0.9124
7747412 0.6416
5476423 3.256
7954103 3.491
87
3.7- Structure activity relationship (SAR) study results
We established a basic structure activity relationship (SAR) study as an attempt to
build a hypothetical scaffold for the development of potent, selective and centrally active
MAO-B inhibitors with a possible neuroprotective effect that can be applied for the
treatment of Parkinson's disease. Through comparing the structure and activity for
compounds with related building blocks, we tried to identify the structural units that are
essential for MAO-B inhibition and those that are critical for peroxyl radical quenching.
For simplicity in presentation, the data of the scatter plot in figure 3.1 are re-
introduced here in the form of columns (only for compounds under investigation in SAR
study). Figures 3.15, 3.16 and 3.17 show the relative activity for the concerned
compounds (MAO-B inhibition activity and antioxidant potential) together with the
structural formula of these compounds.
Based on figure 3.15, it is very easy to notice that both compounds 5140311 and
5140321 have almost the same antioxidant potential, compound 5140311 appears to have
a stronger MAO-B inhibition effect. This variation in MAO-B inhibition potential can be
attributed in part to the difference in the substitution type at position four of the
benzylidene moiety in these compounds. The dimethyl amino group in compound
5140311 has more electron donating effect than does the methoxy moiety in compound
5140321; this may provide a more favorable position and electron density for compound
5140311 within MAO-B active site so that a hydrogen bond can be formed between
oxygen atom (attached to position four of the 2-thioxo-1, 3-thiazolidine ring) and the
88
hydroxyl group of phenyl moiety in Tyr 435 residue. The presence of such hydrogen
bonding was confirmed for compound 5140311 by using docking analysis. Compounds
5140311 and 5140321 also show difference in substitution length at position three of the
2-thioxo-1, 3-thiazolidin-4-one ring; longer substitution (propanoic acid in 5140311
versus acetic acid in 5140321) may have a positive impact on the potential and selectivity
of MAO-B inhibition effect by providing a more extended conformation.

89
Figure 3.15(A)- The potency of the inhibitory effect against MAO-B as produced by
compounds 5140311 and 5140321; the data are displayed as (Mean ± SEM).
90
Figure 3.15(B)- Percentage of peroxyl radical quenching potential (antioxidant) for
compounds 5140311 and 5140321, values are presented as (Mean ± SEM) and Ascorbic
acid was used as positive control (100% activity).

91
Figure 3.15(C)- Chemical structural formula for compounds 5140311 and 5140321.
92
Figure 3.15(D)- Two dimensional representation for the docking of compound 5140311
into human MAO-B crystal, this image was generated by using LigPlot+ v.1.4.5 [153].
93
Figure 3.15(E)- Two dimensional representation for the docking of compound 5140321
94
In figure 3.16(A), only compounds 5143886 and 5108394 show high MAO-B
inhibition potential. The other two compounds 5144779 and 5108400 have a minor
MAO-B inhibitory effect. Compounds 5108394 and 5108400 are very similar in terms of
structural formula but they show significant difference regarding MAO-B inhibition
effect (in vitro). Structural comparison for these two compounds show that the only
difference between them is the presence of two electron donating groups (methoxy
group) attached to positions two and four of the benzylidene moiety in compound
5108394 while compound 5108400 has an electron withdrawing group (chlorine atom)
attached to position four of the benzylidene moiety. This variation in substituent type will
affect the orientation and the electron density of these compounds within MAO-B active
site. Docking studies showed that compound 5108394 occupies a position opposite in
direction to that occupied by compound 5108400 within MAO-B crystal; such orientation
will favor the formation of hydrogen bond between oxygen atom attached to position four
of 1, 3-thiazolidine ring in compound 5108394 and the sulfhydryl group attached to
position three of Cysteine 172 residue within MAO-B active site. The reversed
orientation in compound 5108400 will facilitate the formation of hydrogen bond between
the sulfur atom at position one of 1, 3-thiazolidin-4-one ring and the sulfhydryl group in
Cysteine 172 residue, this hydrogen bond is weaker than that formed in case of
compound 5108394 and this may account for the difference in MAO-B inhibition
potential.
Previous studies showed that nitrogen atom in position three and oxygen atom
attached to position four of the thiazolidinedione ring within pioglitazone molecule are
95
involved in hydrogen bonding with conserved active site water molecules within the
aromatic cage of Tyr398 and Tyr435 [128]. We believe that such hydrogen bonding can
also exist between the conserved active site water molecules and (nitrogen atom in
position three and oxygen atom attached to position four of the 1, 3-thiazolidine ring).
We expect that attachment of 4-methylphenyl group to the nitrogen atom at position three
of the 2-thioxo-1, 3-thiazolidin-4-one ring in compound 5144779 can hinder the
formation of hydrogen bond with conserved active site water molecule and this may
adversely impact MAO-B inhibitory effect.
In figure 3.16(B), we can clearly notice that only compound 5143886 has peroxyl
radical quenching activity, the other three compounds (5144779, 5108400 and 5108394)
have very low or almost no antioxidant activity. Chain breaking antioxidants produce
their radicals scavenging activity through donating hydrogen atoms (radicals reduction)
and formation of stable radicals that are unable to continue chain reaction [149, 182]. We
believe that nitrogen atom at position three of 2-thioxo-1, 3-thiazolidin-4-one ring
represents a good hydrogen donor, and we also expect that the presence of thioxo group
at position two of 1, 3-thiazolidin-4-one ring is considered essential for antioxidant
activity. Such criteria are readily available in compound 5143886. Similar structural
criteria for antioxidant activity can be also observed in compounds 5140311 and 5140321
by taking into consideration that the nitrogen atom at position three of 2-thioxo-1, 3-
thiazolidin-4-one is attached to carboxylic acid moiety that can still donate hydrogen
atom.
96
compounds 5143886, 5144779, 5108394 and 5108400; the data are displayed as (Mean ±
SEM).
97
compounds 5143886, 5144779, 5108394 and 5108400, values are presented as (Mean ±
SEM) and Ascorbic acid was used as positive control (100% activity).
98
Figure 3.16(C)- Chemical structural formula for compounds 5108394, 5108400, 5144779
and 5143886.
99
Figure 3.16(D)- Two dimensional representation for the docking of compound 5108394
100
Figure 3.16(E)- Two dimensional representation for the docking of compound 5108400
101
Figure 3.17 clearly shows that compounds 7935085 and 5476423 have a very
weak MAO-B inhibition activity. Compound 5476423 appears to be a potent radical
quencher; on the other hand compound 7935085 shows a weaker potency for radical
reduction. This variation in antioxidant power can be attributed in part to the difference in
the substitution type and position within quinoline moiety. Previous researches showed
that attachment of hydroxyl group to position eight in quinoline moiety can greatly
enhance the radical scavenging activity [182].Such structural requirement for antioxidant
potential is clearly evident in compound 5476423 with the presence of two 8-
hydroxyquinoline moieties; the weaker scavenging activity of compound 7935085 can be
partially explained by the presence of methyl group attached to position eight of
quinoline moiety and the attachment of hydroxyl group to position two of quinoline
system.
102
compounds 7935085 and 5476423; the data are displayed as (Mean ± SEM).
103
compounds 5476423 and 7935085, values are presented as (Mean ± SEM) and Ascorbic
acid was used as positive control (100% activity).

104
Figure 3.17(C)- Chemical structural formula for both compounds 5476423 and 7935085.
Chapter four: Discussion
Parkinson's disease is a progressive neurodegenerative disorder that mainly
affects elderly people; it holds the second rank after Alzheimer's disease in terms of
neurodegenerative disorders prevalence among this segment of population [161].
Parkinson's disease is characterized by a progressive loss (degeneration) of dopaminergic
neurons in the substantia nigra pars compacta region of the midbrain, this gradual damage
will negatively impact nigrostriatal pathway leading to the emergence of classical motor
related symptoms. As the disease progresses in course, non-motor symptoms (like
depression and dementia) may also appear due to sporadic degeneration within
cholinergic, noradrenergic and serotonergic pathways in the brain [162].
Parkinson's disease is a complex neurologic condition with multiple etiological
factors being involved in guiding the pathophysiological course of this disorder. Current
therapeutic approaches for this disorder focus only on symptomatic mitigation through
dopamine replacement therapy [11-13]. No treatment is currently available that can
hinder or slow down the progression of the neurodegenerative process [19, 20]. The
application of designed multiple ligands (DMLs) approach seems to be the most
convenient tool to produce a molecule capable of targeting more than one pathway within
the pathological process with a possible disease course modifying effect [21-28].
105
106
Monoamine oxidase B (MAO-B) enzyme appears to be a promising target for
such therapeutic approach. MAO-B enzyme catalyzes the oxidative deamination of
dopamine and several other amines [38] and by blocking the effect of this enzyme we can
elevate the level of both endogenous and exogenous dopamine [29, 30]. The inhibition of
MAO-B enzyme can also lower the production of hydrogen peroxide as a byproduct of
oxidative deamination reaction [31]. It is believed that the produced hydrogen peroxide
can undergo Fenton reaction in the presence of ferrous iron to yield hydroxyl radicals
[163]. By lowering the level of hydrogen peroxide, we can minimize the activity of
Fenton reaction and protect neurons from detrimental oxidative stress status.
𝐅𝐞𝟐+ → 𝐇𝟐 𝐎𝟐 → 𝐅𝐞𝟑+ + 𝐇𝐎. + 𝐎𝐇 − (Fenton reaction)
It is important to mention that the expression level of antioxidant enzymes
decreases with age leading to a reduction in the antioxidant capacity of the brain [164,
165]. The cellular iron level inside the brain is elevated with age and with
neurodegenerative conditions [92]. MAO-B but not MAO-A expression level inside the
brain is increased with age [32, 33, 34, 87]. MAO-B is expressed in glial cells; the level
of MAO-B inside the brain is elevated in patients with Parkinson's disease as a direct
result of gliosis. Human basal ganglia shows high level of activity for MAO-B as
compared with MAO-A and since both isozymes catalyze the oxidative deamination
(termination) of dopamine neurotransmitter with equal efficiency then it is very logical
from pharmacological perspective to target MAO-B selectively [36].

107
Based on monoamine oxidase inhibition assay results, it is very obvious that
compounds 5143886 and 5140311 are highly potent and selective in terms of inhibition
effect against MAO-B enzyme. This potency and selectivity in action can be explained
through docking studies results; it appears that these two compounds can exhibit an
extended conformation that traverses both the entrance cavity and substrate cavity of the
active site in MAO-B enzyme. Such conformation will force Ile 199 residue to acquire an
open configuration that lead to the fusion of these two cavities. It is believed that the
configuration of Ile 199 residue plays a critical role in determining substrate and inhibitor
binding specificity for MAO-B [167]. When small molecule (substrate or inhibitor) enters
the active site of MAO-B, Ile 199 residue will obtain a closed conformation and this will
produce the bipartite configuration of the active site. On the other hand, Isoleucine 199
residue will obtain an open conformation when long, planar molecule (extended
conformation) enters the active site and this will result in the fusion of both entrance
cavity and substrate cavity.
Another interesting finding in MAO-B inhibition assay results is that both
compounds 7730595 and 7747412 seem to be potent inhibitors, however docking studies
predict that these compounds don't enter the active site of MAO-B. The images generated
by docking programs reveal that these two compounds occupy a position that is very
close to the gating switch (loop 99-112), it is very clear to notice that these compounds
are involved in hydrophobic interactions with amino acid residues in the gating loop
(mainly Phe 103 and Pro 102). Based on these images, we can build an assumption that
these compounds produce their inhibitory effect through retarding the accessibility of
108
substrates to the active site of MAO-B enzyme. This difference in docking position
between compounds (5140311, 5143886) and compounds (7747412, 7730595) can be
attributed to the difference in structural geometry, the structures of compounds 7747412
and 7730595 are bulkier (four to five rings system) than those for compounds 5140311
and 5143886 (two to three rings system). Although the chemical structure of compound
7919469 is composed of four rings, this compound appears to be capable of entering the
active site of MAO-B enzyme as shown in docking images. According to MAO-B
inhibition assay results, compound 7919469 seems to be a potent inhibitor. We expect
that the four rings in this compound are arranged in a way that can provide a planar and
an extended conformation and this will eventually allow compound 7919469 to access
MAO-B active site and produce a potent inhibitory effect.
A recent literature proposed the presence of a conformational relationship
between Ile 199 residue and Phe 103 residue [166]. When Ile 199 residue exhibits an
open conformation, the side chain of Phe 103 residue will be forced to acquire a
conformation that makes the gating switch (loop) closes the entrance into the active site
of MAO-B. On the other hand, when Ile 199 residue is in closed conformation then the
side chain of Phe 103 will obtain a conformation that favors opening of the gating loop.
In the ligand free condition, these two conformations are rapidly interchangeable and this
will provide accessibility to the active site of MAO-B enzyme.

109
Albumin is the most abundant form of plasma protein in human beings; it acts as
a vehicle for the transport of many endogenous and exogenous compounds through
reversible binding [142, 168]. Exploring the profile of drug binding to serum albumin is
considered an important step in drug development process; such binding can influence
pharmacokinetics and pharmacodynamics parameters of drug under investigation [169-
173]. Binding of drug molecules to plasma albumin can change the volume of
distribution; lower the rate of clearance and increase plasma half-life for that drug [174,
175]. There are two main binding regions in albumin molecule for aromatic and
heterocyclic compounds, these binding regions are hydrophobic in nature and are located
in subdomain IIA (site I) and subdomain IIIA (site II) [158,159]. Drug molecules usually
bind to plasma albumin in a dynamic and reversible style with a very fast dissociation
rate [176].
We have used bovine serum albumin (BSA) as an alternative for human serum
albumin (HSA) for the binding assay because BSA is readily available and because of its
structural homology with HSA [177]. Our results for BSA binding assay clearly show
that most of the tested compounds have a high tendency for BSA binding; this may
negatively affects the distribution of these compounds throughout body tissues and limits
the accessibility to site of action. The only exceptions are compounds 5476423 and
5140311 which show moderate tendency to bind BSA.

110
One of the main challenges in producing a successful neurotherapeutic agent is to
design a molecule capable of passing through the blood brain barrier (BBB) and reaching
the required site of action [178]. Blood brain barrier (BBB) is the main site for molecular
exchange between the brain and blood stream; it functions to maintain a stable
microenvironment for the brain. This barrier is composed mainly from endothelial cells
that line cerebral microvesseles; it represents a combination of both physical and
metabolic barriers. The physical barrier involves the presence of intercellular tight
junctions between the endothelial cells with a very limited vesicular transport. The blood
brain barrier also contains active transporters; some of these transporters transfer
nutrients and other materials from blood to brain (influx) while the other transporters
mediate the clearance of waste products and some drugs from brain to blood (efflux). The
metabolic barrier involves phase I and phase II metabolic enzymes that can limit the
ability of lipophilic compounds to penetrate BBB by converting them into a more
hydrophilic products [179-181]. Most CNS active drugs penetrate BBB through
transcellular passive diffusion process [143].
Parallel artificial membrane permeability assay (PAMPA) is used to predict BBB
permeability potential for compound under investigation. We performed this assay only
for compound 5140311; this compound was the only one that fluoresced at the excitation
wave length range produced by the fluorescence reader. We calculated the value of
effective permeability (Pe) as mentioned previously and then we used the following table
(classification of PAMPA-BBB results based on Pe ranges provided by reference [160])
to interpret our results.

111
Table 4.1-Classification of PAMPA-BBB results.
BBB permeability Pe (𝟏𝟎−𝟔 cm 𝐬 −𝟏 )
High (CNS+) >4.0
Low (CNS-) <2.0
Uncertain (CNS+/-) 4.0-2.0
The calculated Pe value for compound 5140311 was equal to 5.2966× 10−6 cm
s−1 and according to this table, our compound is highly permeable.
Oxidation reaction can produce free radicals; these radicals can start a chain
reaction that involves several steps (initiation, propagation, branching and termination of
these radicals). Such chain of oxidative events can eventually damage cellular contents
like lipid membranes, DNA and proteins. In general, antioxidants can block such reaction
through two major mechanisms: They can either inhibit the formation of free radicals
from their unstable precursors (preventive antioxidants) or they can break the reaction
chain through inhibiting the propagation and branching steps (chain breaking
antioxidants). Most antioxidants are classified as chain breaking agents; they exert their
effect through scavenging free radicals. The chain breaking antioxidants should be able to
act as good hydrogen donors in order to reduce free radicals. Once they donate hydrogen
atom, they should form stable radicals that are unable to continue the chain reaction or
they continue it but with very low efficiency [149, 182].

112
Based on our results for oxygen radical absorbance capacity (ORAC) assay, it is
very clear that all of the tested compounds show high antioxidant capacity that is either
equivalent or higher than that reported for Ascorbic acid (positive control). The only
exception is compound 7954103 which shows minor antioxidant effect.
It is evident from all these virtual and in vitro assays that candidate 5140311
represents a potential lead compound for the development of new antiparkinsonian
agents. This compound is a potent and selective inhibitor for MAO-B, it is highly
permeable through BBB and shows moderate tendency for BSA binding. Finally this
compound shows excellent potential for peroxyl radicals quenching.
As a conclusion for our basic structure activity relationship (SAR) study, it is very
clear that 2-thioxo-1, 3-thiazolidin-4-one ring plays an essential role as a structural
requirement for both MAO-B inhibition activity and radicals quenching potential. For
MAO-B inhibitory effect, we believe that the oxygen atom attached to position four of 2-
thioxo-1, 3-thiazolidine ring can be involved in hydrogen bonding with a certain amino
acid residue or a conserved water molecule within the active site. The nitrogen atom in
position three of the 2-thioxo-1, 3-thiazolidin-4-one ring can be also involved in the
formation of hydrogen bond with a conserved active site water molecule. For antioxidant
potential, we assume that amine group at position three of the 2-thioxo-1, 3-thiazolidin-4-
one ring can act as a good hydrogen donor. The presence of thioxo group at position two
of the 1, 3-thiazolidin-4-one ring appears to be essential for antioxidant activity.

113
8-hydroxyquinoline has been shown to exhibit a strong iron chelating activity and
an antioxidant potential [183, 184]. This molecule has been also shown to be capable of
passing through blood brain barrier [57]. 8-hydroxyquinoline has been successfully used
as a building block for the design and synthesis of several novel bi-functional neuroactive
compounds like HLA20 and M30 [103, 104, 105, 107]. Based on these facts and our
results, it is very clear that 8-hydroxyquinoline represents a promising building block for
iron chelating activity and radical scavenging potential.
By using 2-thioxo-1, 3-thiazolidin-4-one ring and 8-hydroxyquinoline as building
blocks, we were able to design two possible hypothetical scaffolds for MAO-B inhibitors.
We hope that the combination of these two building blocks can help us in producing
potent, selective and centrally acting MAO-B inhibitors with a promising iron chelating
effect and a possible antioxidant potential. Figure 4.1 and figure 4.2 show the chemical
formula for our scaffolds (scaffolds A and B) together with initial results of docking
analysis. Docking results for scaffold A in figure 4.1 confirm the presence of two
hydrogen bonds; the first bond is between the oxygen atom attached to position four of 2-
thioxo-1, 3-thiazolidine ring and the hydroxyl group of phenyl moiety in Tyr 435 (residue
of aromatic cage) while the second hydrogen bond is between the hydroxyl group
attached to position eight of the quinoline moiety and the sulfhydryl group of Cysteine
172 residue. In figure 4.2, docking images for scaffold B confirm the presence of one
hydrogen bond between the oxygen atom attached to position four of 2-thioxo-1, 3-
thiazolidine ring and the sulfhydryl group of Cysteine 172 residue.

114
Scaffold A
Molecular weight =288.34 g/mole
5-[(8-hydroxyquinolin-7-yl) methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one
Figure 4.1(A)- Chemical structure together with IUPAC name for our first prospective
scaffold (scaffold A).

115
Scaffold A
Figure 4.1(B)- Two dimensional representation for the docking of scaffold A into MAO-
B crystal, this image was generated by using LigPlot+ v.1.4.5 [153]. Hydrogen bonds are
represented by green dashed lines.

116
Figure 4.1(C)- Three dimensional representation for the docking of scaffold A into
MAO-B crystal. This image was generated by PyMOL v.1.3 (www.schrodinger.com)
[154]. Our ligand is colored by red and the hydrogen bonds are shown as cyan lines.
117
Scaffold B
5-[(8-hydroxyquinolin-5-yl) methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one
Figure 4.2(A)- Chemical structure together with IUPAC name for our second prospective
scaffold (scaffold B).

118
Scaffold B
Figure 4.2(B)- Two dimensional representation for the docking of scaffold B into MAO-

119
Figure 4.2(C)- Three dimensional representation for the docking of scaffold B into MAO-
B crystal. This image was generated by PyMOL v.1.3 (www.schrodinger.com) [154].
Our ligand is colored by red and the hydrogen bonds are shown as cyan lines.
120
As part of our attempts to refine the overall structure of these scaffolds and to
optimize their expected pharmacokinetic profile, virtual evaluation of oral bioavailability
and CNS permeability was performed for both scaffolds. For this purpose, a number of
physicochemical parameters were initially calculated by using ChemAxon’s calculators
and calculator plugins (www.chemaxon.com). These parameters were then assessed for
their degree of obedience to Lipinski’s rule of five for oral bioavailability [185] and
Norinder’s two rules (AstraZeneca’s rules) for CNS permeability [186]. For simplicity in
presentation, we have listed the standards for the above rules together with our calculated
parameters in tables 4.2 and 4.3.
Based on data in table 4.2, we can clearly notice that both scaffolds A and B will
probably show a good oral activity profile. These scaffolds are in complete agreement
with Lipinski’s rule of five. In table 4.3, on the other hand, these two scaffolds deviate
from the last two standards for CNS permeability (parameters with red color). Such
deviation may cause a future problem in CNS accessibility during lead optimization.
From our point of view, we expect that such probable obstacle can be defeated by
reducing the size of polar mass in these scaffolds. We believe that removal of nitrogen
atom from 8-hydroxyquinoline part can enhance the value of {log P - (N+O)} and this
will eventually increase the value of log BB. Such modification in the structure of these
scaffolds will also lower the value of PSA (Polar surface area) and by this way the
modified scaffolds will obey all CNS permeability rules. Based on this assumption, we
have modified the structure of scaffolds A and B by using hydroxynaphthalene moiety
instead of 8-hydroxyquinoline and this gave us two new scaffolds, namely scaffold C and
121
scaffold D. To avoid redundancy, we have listed the physicochemical parameters for
these modified scaffolds in table 4.2 and 4.3 also. Based on these parameters, we can
easily observe that these modified scaffolds (C and D) will probably have a good oral
absorption profile and a satisfactory CNS permeation capacity.
Docking studies in figure 4.3 show that scaffold C exhibits a very similar
configuration to that of scaffold A within MAO-B crystal. In figure 4.4, MAO-B docking
studies for scaffold D show that the thiazolidine ring occupies a reversed orientation as
compared to that of scaffold B. This reversed direction can be attributed to rotation of the
single bond between 4-hydroxynaphthalene moiety and methylidene group (the only
rotatable bond in our design); this rotation will lead to the loss of hydrogen bond between
the oxygen atom attached to position four of 2-thioxo-1, 3-thiazolidine ring and the
sulfhydryl group of Cysteine 172 residue.
Such proposed structural modification (removal of nitrogen atom from 8-
hydroxyquinoline) may not adversely impact MAO-B inhibition capacity of our design. It
is believed that hydroxynaphthalene moiety can quench radical species through single
electron transfer process. This will convert hydroxynaphthalene molecule into a
stabilized radical form which in turn may undergo dimerization (coupling) reaction [189].
In addition some scientific literatures have shown that phenolic compounds can form
complex with iron [190]. Based on these facts, we hope that both scaffolds C and D can
still meet our goals to produce a potent, selective and centrally acting MAO-B inhibitor
with a possible antioxidant activity and a promising iron chelating ability.

122
Table 4.2- The obedience degree to Lipinski’s rule of five. It is important to mention that
the number of rotatable bonds condition in this table was obtained from Veber’s rules for
oral bioavailability of drug candidates [187].
Physicochemical parameter Standard value Scaffolds A&B Scaffolds C&D
Molecular Weight ≤ 500 g/mol 288.34 g/mol 287.36 g/mol
Log P ≤5 2.59 3.43
H-bond donors (OH + NH) ≤5 2 2
H-bond acceptors (O + N) ≤ 10 2 1
Number of rotatable bonds ≤ 10 1 1

123
Table 4.3- The obedience degree to Norinder’s rules (AstraZeneca’s rules) for CNS
permeability. It is important to notice that the third condition for PSA value in this table
was obtained from reference [188].
Physicochemical parameter Standard value Scaffolds A&B Scaffolds C&D
N + O atoms count ≤5 4 3
Log P – (N + O) value > 0 (positive) -1.41 +0.43
Polar surface area (PSA) < 60 – 70 Å² 62.22 Å² 49.33 Å²

124
Scaffold C
5-[(1-hydroxynaphthalen-2-yl) methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one
Figure 4.3(A)- Chemical structure together with IUPAC name for the modified scaffold
(scaffold C).
125
Figure 4.3(B)- Two dimensional representation for the docking of scaffold C into MAO-

126
Scaffold D
5-[(4-hydroxynaphthalen-1-yl) methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one
(scaffold D).
127
Figure 4.4(B)- Two dimensional representation for the docking of scaffold D into MAO-
B crystal, this image was generated by using LigPlot+ v.1.4.5 [153].

128
As a final attempt to defeat the blood brain barrier and to improve the overall
kinetic profile of our design, we found that it is more convenient to target the transporters
system within this barrier. Several previous researches have shown that attachment of L-
alanine residue to certain drugs can enhance the CNS accessibility of these drugs. The
addition of such neutral amino acid residue will make the whole resultant molecule a
good substrate for large neutral amino acid transporter (uptake system L). Such strategy
was employed successfully for the selective delivery of several drugs into the brain and
good examples in our case are L-dopa and the experimental drug M10 [107]. The
attachment of L-alanine residue to scaffold A or scaffold B will make these chemicals
more hydrophilic (elevates polar surface area) and this will lower their passive
permeation into tissues other than the brain. Through such addition, we can enhance the
selective delivery of our chemicals into the brain and minimize the unwanted side effects
of our design. We expect that such modification in our design will not negatively impact
MAO-B inhibition effect, iron chelation capacity or radicals quenching potential. The
structural formula and docking images for the two new scaffolds (L-alanine derivatives)
can be seen in figure 4.5. The application of Lipinski’s rule of five shows that these two
new scaffolds (E and F) may be still able to pass gastrointestinal tract as can be seen in
table 4.4.
In order to expand the scope of our possible opportunities, we are hereby
presenting a new design for another possible scaffold. The new design (scaffold G)
combines three essential building blocks and these are: 2-thioxo-1, 3-thiazolidin-4-one
ring (for MAO-B inhibitory effect and antioxidant capacity), catechol moiety (for iron
129
chelation activity and radical quenching potential) and L-alanine residue (for selective
delivery to the brain). We believe that the core of our research is represented in this new
design (scaffold G). The docking image and virtual pharmacokinetic parameters for this
scaffold can be seen in figure 4.6 and table 4.4.

130
Scaffold E
Molecular weight= 375.42 g/mol
2-amino-3-(8-hydroxy-7-{[4-oxo-2-sulfanylidene-1,3-thiazolidin-5-
ylidene]methyl}quinolin-4-yl)propanoic acid
(scaffold E).
131
Figure 4.5(B)- Two dimensional representation for the docking of scaffold E into MAO-

132
Scaffold F
2-amino-3-(8-hydroxy-5-{[4-oxo-2-sulfanylidene-1,3-thiazolidin-5-
ylidene]methyl}quinolin-2-yl)propanoic acid
Figure 4.5(C)- Chemical structure together with IUPAC name for the modified scaffold
(scaffold F).
133
Figure 4.5(D)- Two dimensional representation for the docking of scaffold F into MAO-

134
Scaffold G
2-amino-3-(2,3-dihydroxy-4-{[4-oxo-2-sulfanylidene-1,3-thiazolidin-5-
ylidene]methyl}phenyl)propanoic acid
Figure 4.6(A)- Chemical structure together with IUPAC name for our novel design
(scaffold G).
135
Figure 4.6(B)- Two dimensional representation for the docking of scaffold G into MAO-

136
Table 4.4- The application of Lipinski’s rule of five to scaffolds E, F and G. The last two
conditions (number of rotatable bonds and polar surface area) were obtained from
Veber’s rules for oral bioavailability of drug candidates [187].
Physicochemical parameter Standard value Scaffolds E&F Scaffold G
Molecular Weight ≤ 500 g/mol 375.42 g/mol 340.37 g/mol
Log P ≤5 1.7 1.24
H-bond donors (OH + NH) ≤5 4 5
H-bond acceptors (O + N) ≤ 10 3 2
Number of rotatable bonds ≤ 10 4 4
Polar surface area (PSA) ≤ 140 Å² 125.54 Å² 132.88 Å²

137
Our preliminary data indicate that compounds with a close structural formula to
that of both scaffolds A and B are effective in MAO-B inhibition assay; some of these
compounds were able to display an IC50 value within micro Molar range. One of these
compounds, the one that closely resembles scaffold A, was superior in MAO-B inhibition
to Zonisamide. It is important to mention that Zonisamide is a well-known anticonvulsant
agent with antiparkinsonian effect (reversible MAO-B inhibitor); it was used as a positive
control in our MAO-B inhibition assay. Based on these recent data, we are currently
trying to synthesize both scaffolds A and B in our lab. Characterization of these two
scaffolds and refining their structural formula appears to be a promising future plan to
produce and optimize an antiparkinsonian lead compound. The newly proposed structural
modification (generation of scaffolds C, D, E and F) may also expand the scope of our
future plans, goals and opportunities. We believe that scaffold G is a novel design for a
multifunctional antiparkinsonian agent and we hope that such design can see the light in
the nearby future.

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Identiﬁcation of novel scaffolds for Monoamine oxidase B inhibitors.

Thesis · May 2014

The user has requested enhancement of the downloaded file.

to Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Science

B.S., University of Baghdad, 2007

M.S., Kent State University, 2014

List of figures ..................................................................................................................... vi

List of tables ....................................................................................................................... ix

Chapter one: Introduction ............................................................................................... 1

1.1- Parkinson's disease (PD) .......................................................................................... 1

1.2- Monoamine oxidase enzyme (MAO)....................................................................... 3

1.3- Structure of MAO-B ................................................................................................ 4

1.3.1- membrane binding domain ................................................................................ 6

1.3.2- Substrate binding domain .................................................................................. 6

1.3.3- Flavin binding domain ...................................................................................... 7

1.4- Reaction mechanism of MAO enzyme .................................................................. 11

1.5- Functional role of aromatic cage............................................................................ 18

1.6- MAO inhibitors with a possible or approved clinical application ......................... 20

1.7- MAO-B inhibitors with a possible disease modifying effect ................................. 22

2.1- Prospective candidates ........................................................................................... 28

2.2- Docking studies ...................................................................................................... 36

2.3- Monoamine oxidase inhibition assay ..................................................................... 36

2.4- Bovine serum albumin (BSA) binding assay ......................................................... 40

2.5- Parallel Artificial Membrane Permeability Assay (PAMPA) ................................ 41

2.6- Oxygen Radical Absorbance Capacity (ORAC) assay .......................................... 43

2.7- Data analysis .......................................................................................................... 44

Chapter three: Results .................................................................................................... 45

3.1- Selection of the most efficient candidates ............................................................. 45

3.2- Docking studies results .......................................................................................... 47

3.3- Monoamine oxidase inhibition assay results ......................................................... 71

3.4- Bovine serum albumin (BSA) binding assay results ............................................. 76

3.5- Parallel artificial membrane permeability assay (PAMPA) results ....................... 82

3.6- Oxygen radical absorbance capacity (ORAC) assay results .................................. 84

3.7- Structure activity relationship (SAR) study results................................................ 87

Chapter four: Discussion.............................................................................................. 105

of MAO enzyme catalysis ............................................................................. 16

mechanism of MAO catalysis ........................................................................ 17

Figure 2.1 - Conversion of kynuramine into 4-Hydroxyquinoline as catalyzed by both

MAO-A and MAO-B ..................................................................................... 39

potential (percentage) for our chemicals. ...................................................... 46

Figure 3.12 - BSA binding potential of the tested chemicals ........................................... 77

formula for compounds 5143886, 5144779, 5108394 and 5108400 (SAR

also shown ..................................................................................................... 96

our first prospective scaffold (scaffold A) ................................................... 114

our second prospective scaffold (scaffold B) .............................................. 117

the modified scaffold (scaffold C) ............................................................... 124

the modified scaffold (scaffold D) ............................................................... 126

the modified scaffolds E and F (L-alanine derivatives)............................... 130

our novel design (scaffold G) ...................................................................... 134

Table 2.1 - Prospective candidates' specifications. ........................................................... 29

Table 3.1 - Docking results for BSA crystal. .................................................................... 70

Table 3.2 - Inhibition selectivity toward MAO isozymes................................................. 75

Table 4.1 - Classification of PAMPA-BBB results. ....................................................... 111

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1.1- Parkinson's disease (PD)

It is an age related neurodegenerative disease that affects about one million

characterized by a progressive degeneration of dopaminergic neurons in the substantia

surviving dopaminergic neurons, these inclusions consist mainly of α-synuclein which is

Current therapeutic approach focuses on symptoms attenuation only [11] through

dopamine replacement therapy by using either L-dopa (the immediate