1. Zakaria Hamdi
Degree project for Bachelor of Science
with a major in Medicinal Chemistry
9 hec
2015:07
Department of Chemistry and Molecular Biology
University of Gothenburg
Dual Acting Ligands as Potential Drugs
for Treatment of Parkinson’s Disease

2. Table of Contents
Parkinson’s disease ............................................................................................................................1
Neurotransmission .............................................................................................................................1
Receptors that are of interest in PD treatment....................................................................................2
The symptomatic treatment of PD today ............................................................................................3
Aim.....................................................................................................................................................6
Method ..............................................................................................................................................6
Dopamine receptors ...........................................................................................................................6
Serotonin receptors ............................................................................................................................7
Adenosine receptors...........................................................................................................................8
The stimulation of multiple receptors with one compound ...............................................................10
The approach to the construction of a dual acting ligand (DAL).........................................................10
DALs targeting the adenosinergic-dopaminergic A2A-D2 heterodimer ................................................11
DALs targeting the A2A receptor and MAO-B......................................................................................16
DALs targeting the serotonergic-dopaminergic 5-HT1A-D2 receptors ..................................................19
Discussion.........................................................................................................................................23
Conclusion........................................................................................................................................24
Acknowledgements ..........................................................................................................................25
References........................................................................................................................................26

3. Abbreviations
5-HT 5-Hydroxytyramine (serotonin)
5-HIAA 5-Hydroxyindoleacetic acid
7-TM 7-Transmembrane
(A)AAD (Aromatic) amino acid decarboxylase
AC Adenylate cyclase
APO Apomorphine
BBB Blood brain barrier
Boc tert-Butyloxycarbonyl
cAMP Cyclic adenosine monophosphate
cLogP Partition coefficient
COMT Catechol-O-methyl transferase
CNS Central nervous system
DA Dopamine
DAG Diacyl glycerol
DAL Dual acting ligand
EC50 Half maximal effective concentration
Emax Efficacy
extAdo Extracellular adenosine
GABA γ-Aminobutyric acid
Gi/o Inhibitory G-protein
GPCR G-protein-coupled receptor
Gs Stimulatory G-protein
HBA Hydrogen bond acceptor
HBD Hydrogen bond donor
IC50 The concentration of an inhibitor required to inhibit an enzyme by 50%
iDAL Integrated-dual acting ligand
IP3 Inositol triphosphate
Kbb Brain:blood ratio
Ki Binding affinity constant
L-DOPA Levodopa
LID Levodopa induced dyskinesia
MAO Monoamine oxidase
MW Molecular weight
NE Norepinephrine
NET Norepinephrine reuptake transporter
NT Neurotransmitter
PD Parkinson’s disease
PEG Polyethylene glycol
RB Rotatable bonds
SAR Structure-activity relationship
SERT Serotonin reuptake transporter
sNC Substantia nigra pars compacta
TPH Tryptophan hydroxylase

4. tPSA Topological surface area
Trp Tryptophan

5. Abstract
Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder, characterized by
rigidity, tremor when resting and general movement difficulties. PD is related to the deficiency of
dopamine in specific parts of the brain. The treatment of the disease is symptomatic and consists of
the use of levodopa, dopamine receptor agonists and anticholinergics. Levodopa and the dopamine
agonists will replenish or mimic the effects of the dopamine neurotransmitter, while the
anticholinergics will block the effects of the neurotransmitter acetylcholine in both the CNS and the
peripheral system, restoring the balance between the activities in the cholinergic and dopaminergic
neurons. Even though these drugs are effective, prolonged use may cause damage to the remaining
neurons, they can also give rise to dyskinesia while slowly losing their efficacy. It has been found that
there are additional targets that when stimulated or inhibited, can make the treatment more
efficient with less side effects. Among these are the adenosinergic- and serotonergic receptors and
the monoamine oxidase B enzyme.
These additional targets are fairly well known and can be affected by administering a drug
combination (two separate compounds, each targeting a different binding site), however this may
lead to undesired pharmacokinetics and pharmacodynamics. The utilization of a single compound
known as a “dual acting ligand” or “bivalent ligand” makes it possible to not only target two different
binding sites in different receptors, but its pharmacokinetic properties become more predictable and
easier to control. This is also something that would be beneficial for patients diagnosed with PD as
they would need to only take a single drug instead of multiple ones.
By combining two active compounds/drugs by either a fusion or by linking them together with a
linker, a potential dual acting compound is created. This study aims to investigate and review
different examples of dual acting ligands that may potentially have a therapeutic effect on PD. The
focus was set on compounds acting on three different combinations of receptors and enzymes;
D2-A2A, D2-5-HT1A and D2-MAO-B. The results of this literature study show that there are a handful of
compounds (6, 9, 12b, 18, 20, 23, 29, 30, 31, and 32), each targeting a different receptor/receptor or
receptor/enzyme combination that showed potential to become novel lead compounds for further
studies.
The A2A-D2 dual acting ligands stand out as the most promising target combination.

6. 1
Parkinson’s disease
Parkinson’s disease (PD) is a commonly encountered neurodegenerative disorder that usually affects
people over 60 years of age.1
PD symptoms usually involve slow movement, an impaired ability to
adjust to the body’s position, as well as rigidity and rhythmic muscle contraction in the form of
tremors when resting.2
The cause for the development of the disease is yet unknown and the only
risk factor seems to be age.3
Neurotransmission
In order to understand how diseases such as PD could be treated, an understanding of the physiology
of the disease is important. The system responsible for people being able to make movement as well
as preventing unwanted movement is the basal ganglia. The basal ganglia comprise a collection of
nuclei that are made up of a network of neurons. It works in such a way that when a neuron receives
signals from other cells in the dendrite network, it generates a depolarization wave that is sent from
one neuron to another via the synapse (Figure 1 and Figure 2).
Figure1. A typical neuron. Left: The dendrite section is one of the main intricate parts of the neuronal cell that forms synaptic
gaps via which the communication with other neurons occurs. Communication with the axon occurs in the form of electric
nerve impulses. The axon’s function is to send out signals that carry information away from the neuron’s cell body. The
soma is the end of a neuron that contains the cell nucleus, which duty is to answer for most of the RNA production in
neurons. The axon terminates at the synapse. This is where the neurotransmitters are released to diffuse across the synaptic
cleft and reach a post-synaptic neuron (the receiving end) in order to generate a physiological response. Right: Synapses
where e.g. glutamate, an activating neurotransmitter or GABA, an inhibitory neurotransmitter, can be released.
When the brain sends out a signal through this network of synapses, it actually communicates
through the neurons. Between each neuron exists a gap called a synapse, and on each side of the gap
is a presynapse (first location) and a postsynapse (arriving destination) which is where they “talk”.
The communication is mediated by chemicals known as neurotransmitters (NT). These NTs are stored
inside vesicles presynaptically. When the signal comes, ion channels are opened and Ca2+
-ions are let
inside the presynaptic neuron, this allows the vesicles containing the NTs to diffuse to the neuronal
membrane where it may release the NTs. Depending on what kind of neurotransmitters that are
released different responses are generated. An example of this is the neurotransmitter γ-
aminobutyric acid (GABA). GABA is the main inhibitory neurotransmitter in the CNS that induces an
inhibitory effect on the postsynaptic neuron. Another NT that can be released is glutamate, showing
Dendrite Axon Terminal
Nucleus
Soma
Axon

7. 2
Figure 1. The serotonergic (5-HT) neuron along with several 5-HT receptor subtypes. Abbreviations: 5-HIAA: 5-
hydroxyindoleacetic acid; TPH: tryptophan hydroxylase; MAO: monoamine oxidase; Trp: tryptophan; AAAD: aromatic amino
acid decarboxylase.
an effect that is opposed to GABA, it excites and increases the activity in the postsynaptic neuron
(Figure 1).
On the presynaptic neuron, there are autoreceptors that detect whether there are sufficient levels of
a specific NT firing or not. If an autoreceptor in for example a serotonergic neuron detects an excess
of extracellular 5-HT, it will induce a negative feedback loop that will stop the firing of the NTs (Figure
2). In order to remove excess amounts of 5-HT from the synaptic cleft, a serotonin reuptake
transporter (SERT) will transport 5-HT into the presynaptic nerve terminal. There it either gets
degraded by the enzyme monoamine oxidase (MAO) or is re-stored into the vesicles (Figure 2).
Depending on the type of neuron, there might exist a combination of several metabolizing enzymes,
in serotonergic and dopaminergic neurons there are both MAO and catechol-O-methyl transferase
(COMT) enzymes.
Receptors that are of interest in PD treatment
For the symptomatic treatment of PD, there are multiple targets that are of interest. Among these
are the dopamine, adenosine and serotonin receptors which all are G-protein coupled receptors
(GPCRs). GPCRs constitute the largest protein family of cell surface receptors, and accounts for ~4%
of the human genome (750 genes in human), accordingly ~30% of all drugs are targeting these kind
of receptors.4-7
The overall structure of GPCRs consists of an extracellular N-terminus that is followed by seven
transmembrane spanning domains (7-TM).When an agonist binds to the binding site of the GPCR a
conformational change is triggered in the cytoplasmic loops and the C-terminus. Hence, the binding
of a ligand from the outside, triggers a response inside the cell.

8. 3
Figure 3. A heterodimer of the A2A-D2 receptor (left) and a general GPCR bound to the G-protein (right: the receptor is shown
in red and its G protein that is bound extracellularly is show in pink and blue). As an NT binds to the binding site of the GPCR,
a conformational change occurs which activates the heterotrimeric G proteins (α, β and γ). As the ligand binds to the
activated GPCR, GDP that is bound to the G-protein is replaced by the active form GTP. This will then trigger a signal cascade
within the cell until the GTP is dephosphorylated into GDP. Modified from Ref 8
During the last 10 years, a substantial number of studies have provided solid evidence that reveals
that the GPCRs actually exist as homo-, hetero- and even higher orders of oligomers (Figure 3).9,10
As
a result, novel therapies based on GPCR oligomerization have recently been proposed, such as the
development of bivalent ligands (compounds with simultaneous affinities for two separate targets),
so called dual acting ligands (DALs).12-15
The reason for this is that there are significant advantages
that can be exploited when targeting heterodimers with bivalent ligands.14, 16
Among these benefits
are an enhanced therapeutic effect, higher ligand affinity, selectivity and a diminished dependence
on multiple drug regimens upon administration. These all add to an enhanced physiological
response.16-19
The symptomatic treatment of PD today
As PD is mainly related to the deficiency of DA in specific parts of the brain, the majority of the
clinical trials focus almost entirely on ways of increasing the levels of DA by stimulation of the
DAergic receptors. Such commonly used drugs today are levodopa (L-DOPA) and DA receptor
agonists like ropinirole (Figure 5). With 40 years of clinical use,20
L-DOPA can be seen as the “gold-
standard” that still remains the most effective symptomatic treatment of PD. Despite its efficacy,
longterm use of L-DOPA is related to the development of side effects, e.g. motor fluctuations such as
the “on-off” phenomenon and dyskinesia.2, 3, 21
These motor complications (“on-off”) which can be
characterized by the slow onset of therapeutic action, dyskinesia and akinesia occurs in ~50% of the
patients treated with L-DOPA for at least five years and in ~80% of those with treatment for ≥10
years.3
With the progressive loss of DAergic neurons and subsequent decreased DA levels in the substantia
nigra pars compacta (SNc), the DAergic- and cholinergic activities gets thrown out of balance.22
This
will cause the cholinergic activities to be more pronounced than the DAergic, which is believed to be
related to the motor symptoms seen in PD.23, 24
In order to restore some balance in the system, an
adequate supply of DA must be applied to the SNc. This can be done by two strategies: i) by a non-
pharmacological approach or ii) a pharmacological approach. The non-pharmacological approach
involves the amelioration of the symptoms by invasive brain surgery and/or by transplanting new

9. 4
stem cells in the brain.26-28
However, none of these methods have so far yielded any convincing
results.23, 28
The pharmacological approach involves the use of exogenously administered xenobiotics
like DA receptor agonists or precursors, or anti-cholinergic agents for symptomatic medications.
Levocar® is an example of a drug that increases the dopaminergic activity. Levocar® consists of a
combination of carbidopa, an amino acid decarboxylase (AAD) inhibitor and L-DOPA, the metabolic
precursor of DA. The reason for using precursors is due to the fact that only L-DOPA (not DA) can
pass the highly selective permeability filter known as the blood brain barrier (BBB). The BBB is what
separates the extracellular fluid of the brain in the central nervous system (CNS) from the circulating
blood. It consists of capillary endothelial cells connected by tight junctions and allows passage of
water, a few gases and lipid soluble molecules by either passive diffusion and/or selective transport
of specific molecules vital to neural functions. Since DA is a polar molecule it will not be able to pass
the BBB and thus not entering the SNc. L-DOPA on the other hand, which is also a polar molecule, is
able to pass the BBB. This is due to it being an amino acid and thus being recognized by transport
proteins like LAT-1 that carries amino acids over the BBB. L-DOPA is a prodrug that once in the
striatum, is converted into DA by decarboxylation. The DAergic neurons in the SNc get stimulated by
the DA formed which will alleviate the symptoms of PD. Carbidopa will not pass the BBB, instead it
will inhibit the extracerebral enzymes from decarboxylating L-DOPA before it has passed the BBB.
Unless carbidopa is there and inhibits or slows down the enzyme (aromatic) amino acid
decarboxylase ((A)AAD), L-DOPA will be decarboxylated into DA (Figure 4). The net result from this
combination is a higher concentration of DA in the SNc and that the gastrointestinal as well as
cardiovascular damage associated with elevated peripheral levels of DA is avoided.
Figure 4. Enzyme catalyzed degradation/inactivation of L-DOPA and DA in striatum. Abbreviations: AAD: amino acid
decarboxylase; COMT: catechol-O-methyl transferase ; MAO: monoamine oxidase.
A continuous infusion of L-DOPA has proved to reduce the “off”-time and dyskinesia associated with
the motor fluctuations, however, prolonged use may also lead to more severe motor fluctuations
and so called L-DOPA induced dyskinesia (LID).29, 30
Treatment of PD today also includes cholinergic antagonists and DA agonists. There are two
subclasses of DA agonists, ergoline and non-ergoline agonists (Figure 5). Both subclasses target the
D2 receptors. The previously mentioned DA agonists ropinirole is a non-ergoline D2, D3 and D4
receptor agonist (with highest affinity for D2) that stimulates the striatal DA receptors which leads to
the alleviation of the parkinsonian symptoms.31, 32
The general concept for the use of DA agonists is

10. 5
that they mimic the endogenous NT DA, hence stimulating the nerve cells and momentarily restore
the balance and deficiency of DA.
These drugs are usually administered alone or together with L-DOPA. The rationale of using the DA
agonists is that it allows a 20-30% reduction in usage of L-DOPA, meaning it would reduce the LID and
the motor complications associated with the use of L-DOPA.32
The reason for this is not yet
completely understood but it is believed that it may be associated with the longer half-life of DA
agonists as well as their somewhat higher receptor selectivity.32
Figure 5. Structures of deprenon (an ergoline) and ropinirole (a non-ergoline), two DA receptor agonists, and the cholinergic
antagonists biperidene and trihexyphenidyl.
A suggested pathway for the symptomatic treatment of PD that would minimize the longterm use of
L-DOPA is to start treating de novo patients with DA agonists and low doses of L-DOPA. If the efficacy
should decrease, instead of increasing the dose of L-DOPA, the dose of DA agonist is increased.32
DA
agonists are thought to be more beneficial for de novo patients that are younger in age and are only
experiencing moderate symptoms related to PD. The opposite is true for older patients with more
severe symptoms.32
While the DA agonists have proved to be just as effective as L-DOPA in the early
stages of the disease, they tend to not be as effective as L-DOPA/carbidopa in treatment of motor
symptoms during the later stages of the disease.21, 32
Alternatives to these two agents are the anticholinergic drugs (like biperidene and trihexyfenidyl)
(Figure 5), that alleviate some of the parkinsonian symptoms such as the involuntary tremors
occurring in a resting state.33
Cholinergic antagonists works in synergy with L-DOPA and can thus be
used as both a monotherapy for de novo patients and/or in conjunction with L-DOPA for those with a
more severe state of the disease.33
The advantages of using these drugs is that they can help delay
the use of L-DOPA for de novo patients, hence further extending the time of which L-DOPA can be
administered and still show good enough efficacy. Biperidene provides a relief for muscle rigidity and
to a lesser extent tremors while trihexyphenidyl binds to the muscarinic receptor and possibly the D2
receptor where it blocks efferent impulses in parasympathetic structures like eyes and muscles.33-35
None of the above mentioned agents are able to cure the disease, they are only able to provide a
symptomatic treatment for PD, meaning they can only slow down the development of the disease to
a certain extent. Newer and more efficient compounds are needed as it is apparent that the agents
used today, while efficient to some extent, have severe side effects or show a loss of efficacy after
prolonged used. PD is as previously stated; related to a deficiency in DA levels and this makes the DA
receptor the most important target to pursue. To address the limited and/or negative effects that
the current remedies exhibit also other targets than DA receptors might be activated or inhibited.
Among these targets, the adenosinergic and serotoninergic receptors as well as the DA degrading
enzymes MAO and COMT show great potential.

11. 6
Figure 6.The dopaminergic GPCR. Modified
from Ref
36
Aim
In this study I have investigated different examples of dual acting ligands (DALs) that may potentially
have a therapeutic effect on PD. The compounds have been evaluated based on:
 Structural properties related to important pharmacophoric features for various receptors
and enzymes of relevance as PD targets. Among these, DALs acting on D2, A2A and the 5HT1A-
receptors as well as the enzyme MAO-B have been of special interest.
 Physiochemical properties mainly related to solubility, permeability, BBB-permeability and
stability of DALs.
The use of DALs for treatment of PD will also be discussed. Attempts will be made to compare the
DALs with drugs used today. The reader should, from this report, have a good understanding of what
PD is and how these new compounds may help in PD treatment.
Method
This report is based on literature published between 1986 and 2015 taken primarily from databases
such as PubMed, SciFinder, Springer and Science Direct, where the keywords: A2A, D2, 5-HT1A,
receptor, dual acting ligands, bivalent ligands, heterobivalent ligands, heterodimers have been used.
Many articles (>2000) were found using these keywords, from them 160 were read and 66 were
chosen for more detailed studies.
Dopamine receptors
There exist five distinct human dopaminergic subtype
receptors (D1-5) that are responsible for mediating the
physiological actions of DA. D1, D4 and D5 are located
postsynaptically while D2 and D3 can be found both pre- and
postsynaptically. These receptors (Figure 6) can be divided into
two groups; D1-like and D2-like, where the D1 subfamily
consists of the D1 and D5 receptors and the D2 subfamily
consists of the D2, D3, D4 receptors. This classification is mainly
based on their different transduction mechanisms. The D1-
like receptors are linked to adenylate cyclase (AC) via coupling to the effector G-protein (Gs)-,
resulting in an increase of cyclic adenosine monophosphate (cAMP) levels.37
In contrast, the D2-like
receptors are negatively linked to AC through the inhibitory G-protein Go/i, which in turn leads to a
lowering of the cAMP levels.
The D2 and D1 receptors are of higher interest in PD therapy since they are not only the primary
targets in PD treatment, they also exist as homomeric complexes with its own subtypes and
heteromeric complexes with the adenosine receptors.16, 38, 39

12. 7
Figure 8. The serotonergic GPCR. Modified
from Ref 41
Figure 7. Pharmacophoric model for dopamine receptors.
Dopamine binds to the D2 receptor through a range of specific interactions (Figure 7). The perhaps
most important interaction is the amino function that after protonation interacts with the binding
site by an ionic interaction to an aspartic acid residue in the receptor. The aromatic ring is involved in
π-π interactions, which is also considered to be important for agonism. The two hydroxyl groups on
the aromatic ring participate in hydrogen bonding (both HBD and HBA) interactions to serine
residues in the receptor, that are considered to enhance the affinity, but are not essential for
agonism.39, 40
Serotonin receptors
The serotonergic receptor family is the largest among the 7-
TM GPCRs. The receptors (Figure 8) are involved in a number
of peripheral actions. Today seven distinct populations of
serotonergic receptors have been identified (5-HT1- 5-HT7)
which in total comprise fourteen receptor subtypes. Among
these receptors, 5-HT3 is not a GPCR but is coupled to a ligand
gated ion channel.42
Much like the previously mentioned
GPCRs, the 5-HT receptors, act through intracellular signaling
pathways where (cAMP), diacyl glycerol (DAG) and inositol triphosphate (IP3) are used to either
depolarize or hyperpolarize their target cells.43, 44, 45
The 5-HT1A, B, D, E, F and 5-HT5A receptors are all
coupled to the Gi/o protein that lowers the cAMP concentration. The subtypes 5-HT2A, B, C increase the
IP3 levels whereas 5-HT6 and 5-HT7A-D increase cAMP.
While each of the 5-HT receptors is localized postsynaptically on target cells, 5-HT1A in particular is
situated on the serotonergic dendrites as well as on the cell bodies within the CNS where its putative
functions include thermoregulation, feeding, stress, pain, mood, emotion, cognition and memory.
What makes the 5-HT1A receptor subtype so interesting in the symptomatic treatment of PD is that
when exposed to an agonist/partial agonist, it possesses the ability to attenuate L-DOPA-induced
dyskinesia (LID) without altering the anti-parkinsonian efficacy. Upon 5-HT1A receptor activation
dopamine release increases in the frontal cortex.46
Consequently, a compound possessing agonistic
properties for both the 5-HT1A and the D2 receptor would be beneficial for alleviating the motor
symptoms related to PD.46, 47

13. 8
Figure 11. The adenosine GPCR. Modified
from: Ref
25
Figure 9. Pharmacophoric model for serotonin receptors.
Serotonin is able to form an ionic interaction through its amino function in the same way as
dopamine (Figure 9). The aromatic system forms strong hydrophobic π-π-interactions while the
hydroxyl groups function both as HBD and HBA. Thus, there are considerable similarities when it
comes to the binding sites of dopamine and serotonin. An ergoline analogue can for example be
specific for the 5-HT1A receptor, but with only slight modifications, it may lose its affinity for the
5-HT1A receptor and gain affinity towards the D2 receptor. For instance, if the O to N distance of an
ergoline agonist is a shorter one (~5.2 Å2
) it will favor binding to the 5-HT1A over the D2 receptor. But
if the O to N distance is larger (~6.5-5.7 Å2
) it will instead favor the D2 receptor. Generally speaking,
an ergoline derivative can bind to the respective receptors, however the functional group that is
positioned on the aromatic group determines its selectivity. According to A HBA function favors 5-
HT1A and a HBD function favors the D2 receptor (Figure 10).39, 40
Figure 10. Left: a pharmacophore model for 5-HT1A where O, OH and OCH3 are possible HBA substituents. Right: a
pharmacophore model for D2 where OH and NH are possible HBD substituents.
39
Adenosine receptors
All four adenosine receptor subtypes (A1A, A2A, A2B, A3) are G-
coupled and can be found throughout the CNS. These GPCRs
(Figure 11) have intricate roles in signal systems related to the
regulation of the heart and the release of NTs such as
dopamine and glutamate.48, 49, 50
Each subtype receptor exhibits
different functions, although some of them might overlap.49, 50
Examples of this are the roles of A1 and A2A in the heart, as both
of them regulate the oxygen consumption and coronary blood
N-Substituent larger
than propyl will not fit.
HBA in 5-HT1A
receptor
HBD in D2
receptor
N-Substituent in this
direction can be large
N-Substituent in this
direction can be large

14. 9
flow, however the A2a receptor displays a wider anti-inflammatory effect throughout the body.51
The A2B and A3 receptors are mainly located peripherally and are involved in processes like
inflammation and/or immune response. A1 and A2A receptors may be characterized by their high
affinity for adenosine in comparison to A2B and A3. In order to activate the A1 and A2A receptors, an
adenosine concentration of 0.3 nM and 1-20 nM, respectively, is required. A2B and A3 on the other
hand require at least an adenosine concentration of 1 µM or higher to get activated.36
Therefore, the
extracellular adenosine (extAdo) which has a concentration of 0.3-1 nM is enough to activate A1 and
A2A but not the other subtypes.36
The A1 receptor is coupled with the inhibiting subfamilies of Gi/o and has been found to be ubiquitous
throughout the brain. When it gets activated it inhibits AC by closing various potassium and calcium
channels. This results in a decrease of the concentration of cAMP.36
A2A and A3 are coupled to the
activating the Gs-protein which leads to an increase in cAMP levels by activation of AC.36
When A3
receptors are activated, it leads to the formation of inositol triphosphate (IP3) which consequently
increases the calcium concentration in the cells.36
The A2A receptors are only found in DAergic regions
where they are often colocalized with D2 receptors (Figure 3).
There is a growing body of work that suggests that the A2A receptors may be of interest for PD
treatment as they are involved in the motor functions in neuronal communication areas within the
basal ganglia.52, 53
It is also known that the activation of the receptor (in animals) results in a
significant decrease in DA affinity for the D2 receptor, which becomes a problem as only small
amounts of extAdo is required to activate the receptors. Through behavioral studies, it has been
determined that the inhibition of the A2A receptor alleviates the motor dysfunctions seen in PD. Thus,
it is of interest to find potent compounds that may be able to at the same time provide an
antagonistic effect for the A2A receptors and an agonistic effect for the D2 receptors.
Figure 12. Pharmacophore model of the A2A antagonists.
The triazolotriazine ZM241385 is an A2A antagonist (Figure 12). Its bicyclic triolotriazine core is
located in the center of the binding pocket where it interacts with the receptor through π-π stacking.
The primary amino function interacts with the binding cavity via an ionic interaction. The compound
is positioned vertically such that the furan moiety is located at the lowest part of the binding cavity
whereas the 4-hydroxyphenethyl substituent is in the upper region of the cavity. These are the
important features of A2A antagonists in order to be effective.11, 52

15. 10
The stimulation of multiple receptors with one compound
As described, the stimulation of other targets in addition to DA is beneficial for PD-patients, this
concept would therefore be of interest to use to improve the treatment. One way would be to co-
administer two already existing conventional drugs, targeting each receptor involved within a
heterodimer complex. The issue with this is that the drugs may exhibit different pharmacokinetics as
well as different bioavailability. This may limit the “time window” the compounds have to induce a
combined therapeutic effect.54
Another proposed way of activating the GPCRs, is by a dual acting ligand (DAL) approach. This either
entails the use of two compounds with different pharmacophores that are linked together by a
spacer, or are fused together (Figure 14 and Figure 19), or compounds that are designed to be dual
target directed. Below an overview of existing DALs with potential use in PD will be presented.
The approach to the construction of a dual acting ligand (DAL)
The most common way to design the DALs is to start with a single molecule which has good activity
towards one receptor and at least some activity to the other desired target. By conducting focused-
and sometimes random screening, a starting compound can be obtained. Then comes the
optimization of the compound where different analogues are synthesized and tested in order to
make sure that the desired activities are balanced.55
The other commonly used design pathway is to
start by two different compounds, each targeting one preferred receptor. Then attempts are made to
incorporate both activities into one compound by either direct fusion, merging the frameworks or by
linking them together. By conducting SAR studies on the compounds, one may hopefully find one
that is can be appropriate for use. It is however not only the efficacy at the respective receptor that
is of importance, also the pharmacokinetic properties of the compound are important for
determining its ”usability”. Among these are the physicochemical properties described by the polar
surface area (PSA), molecular weight (MW), cLogP, the number of rotatable bonds (RB), and the
number of hydrogen bond donors (HBD) and hydrogen bond acceptor (HBA). The guidelines that are
followed are the Lipinski rules and the Veber corrections, which state that in order to have good oral
uptake, a compound must adhere to these rules. Besides this, it is much desired to have a compound
with a low MW and high lipophilicity in order for it to pass the BBB.
While the idea of just linking together two active compounds is an easy concept of thought, the
process of deciding which linker to use and what position to have it attached to is considerably more
complex. If the linker is too short, it would make the simultaneous docking of the compound to the
two binding sites of the GPCR dimer highly unlikely. If it is too long the binding of the compound
could lead to a decrease in entropic gain, thus making the DAL act as a monovalent drug instead.55, 56
Even if there should be enough entropic gain for simultaneous binding, having a long linker would
increase the confinement volume of the compound which ultimately leads to it spending less time
around the binding site. The optimal length must be empirically determined by screening several
bivalent ligands consisting of linkers with different number of atoms.
In addition to the linker length, the composition of the linker as well as its attachment point is crucial
for the activity of the compound. Indeed, the linker may very well interact with the receptor when

16. 11
binding, and depending on its structure it might allow for additional interactions such as hydrogen-
bonding or ionic interactions.
Common linker units used today are shown in Figure 13. The type of linker to use is partially based
on the flexibility of the chain. If it is too rigid, it might not allow the compounds to position
themselves at the two subunits of the receptor dimer. Methylene units make flexible linkers that
have the ability to link two compounds together by one-atom increments. Even though the
polyethylene glycol (PEG) units are most widely used in linkers today, they do not allow the design of
a compound with one-atom incremented spacers. However, this can be circumvented by including a
diamino alkyl chain in PEG linkers (Figure 13).
One also needs to consider the hydrophilic/lipophilic properties of the linker as it also is related to
the ability of the compound to be correctly positioned in the receptor dimer. If the binding sites of
the GPCRs are located extracellularly, they will generally be more receptive towards bivalent ligands
with a hydrophilic linker. The opposite is true for GPCRs with binding sites in the lipophilic
transmembrane domain, and compounds with a lipophilic linker will be more accepted.56
The
lipophilic linkers are also more likely to bridge the two binding sites through the hydrophobic
transmembrane region, whereas the hydrophilic linkers are more likely to cross the extracellular
section.
Figure 13. Examples of various linker units used to combine two pharmacophores.
Lastly, the position on the active compound of which the linker is to be attached needs to be
determined. The attachment point is mainly dictated by the feasibility and compatibility of the
modification to the active compound in relation to its biological activity.56
Depending on the number
and character of chemically reactive groups (such as –NH, -OH, -COOH) being present in the
compound will also be a factor determining the position.
The introduction of the spacer to the active compound must not compromise chirality and affect the
activity negatively by racemization. In many cases an ether bridge is introduced between the
compound and the linker to allow its binding to the active compound. Ultimately, a position that is in
agreement with the SAR data is chosen. The now over 25 year old bivalent ligand approach56
may
have the potential to provide compounds that exhibit unique pharmacological properties. A benefit
to the use of DALs is that they have the potential to produce highly selective compounds that only
target the tissue which co-expresses both receptors.56
DALs targeting the adenosinergic-dopaminergic A2A-D2 heterodimer.
There have been a few examples of studies where the objective has been to produce a DAL capable
to stimulate the A2A-D2 heterodimer. There are, as stated earlier, generally two ways of constructing a
DAL. The first strategy entails a molecule consisting of two different active compounds separated by
a spacer, one example is shown in Figure 14.1

17. 12
Figure 14. Example of the “classical” DAL where two unaltered pharmacophores; ZM 241385 and ropinirole are connected
by an amide containing linker.
In this example, ZM 241385 was chosen as the A2A receptor antagonist and ropinirole, the well-
established antiparkinsonian drug, as the D2 receptor agonist (Figures 14 and 15).
Figure 15.The A2A receptor antagonist ZM241385 1 and D2 receptor agonist ropinirole 2 along with their respective
carboxylic acid analogues 3, 4 and 5.
The reference or parent molecule 1 has a PSA of 127 Å2
and MW 337.34 g/mol and 2 has a PSA of 32
Å2
and MW 260.38 g/mol (Figure 15).1
The DAL 6 (Figure 16) is a compound that is the result of the combination between the ropinirole
congener 5 and the antagonist 1. The parent compounds (1 and 2) exhibited IC50(A2A) and EC50(D2)
values of 33 nM and 304 nM, respectively, and compound 6 showed comparable activities with
IC50(A2A) 35 nM and EC50(D2) 484 nM.1
This means a slight loss in potency of roughly 1.6-fold for the D2 receptor as well as a decrease
regarding the pharmacokinetic properties as the new compound has a PSA of 187 Å2
, MW 694.33
g/mol and cLogP 3.88.

18. 13
Figure 16. 2The heterobivalent compound 6.
The heterobivalent compounds of 7a and 7b (Figure 17) have similar activities as 6, however contrary
to the loss of potency observed for 6, a 1.5-fold increase in potency towards the D2 receptor was
observed for 7b. In comparison to the DALs 6, 7a and 7b, compounds 8a-c (Figure 17) showed a
reduction in affinity towards both receptors with the exception of 8b displaying an IC50 value of 41
nM (110 nM and 1072 nM for 8a and 8b, respectively).1
From these DALs, there is no pattern indicating if the linker length has any significance for the
potency of these DALs, though as 8c had the longest linker it suffered from the biggest drop in
potency towards both receptors along with an efficacy (Emax) of only 38±5%. This poor efficacy in
dopamine response was suggested by Jörg et al. to be attributed to the long linker creating a steric
conflict with the receptor.1
Figure 17. ”Classical” heterobivalent compounds.
The triazine-linked analogue 9 (Figure 17) showed a potency on par with the parent compound 2 for
the D2 receptor (EC50: 311 nM vs. 304 nM) but at the same time 9 exhibited a decreased affinity for
the A2A receptor witch IC50 values of 120 nM vs. 33 nM).1
As expected, the monovalent compound 10 (Figure 18) exhibited no activity towards the D2 receptor

19. 14
with an EC50 value of >100000 nM. Although 10 exhibited some A2A affinity (IC50 437 nM), it was
relatively poor compared to 1 (IC50 33 nM).1
This confirms that the oxindole moiety is responsible for the D2 agonistic effect.
Figure 18.The monovalent compound 10.
The second strategy to produce DALs is by merging them together to create integrated DALs, so
called iDALs, one example is shown in Figure 19.
Figure 19. An example of the integrated DAL strategy where the tyramine moiety of ZM 241385 has been discarded in order
to integrate the pharmacophores of ropinirole and ZM241385 into a low molecular weight compound. The aim is to achieve
subtype selectivity with higher affinity together with an enhanced physiological response by the simultaneous binding to the
orthosteric binding sites of the A2A-D2 heterodimer.
The iDAL analogues were constructed by fusing the parent compounds 1 and 2 directly to one
another. However, this came with the price of the iDALs showing a decrease in potency. The same
was true for compounds 11 and 12a-c (Figure 20), 11 showed a decrease in potency at the D2
receptor but was equipotent with 1 towards the A2A receptor (IC50 of 32 nM vs. 33 nM). This low
activity at the D2 receptor can be attributed to the lack of an ionizable amino function that is
important for the binding to the D2 receptor. Conversely, as 11 lack the ionizable amino function it is
able to retain its high potency for the A2A receptor. This can be confirmed by comparing the results of
11 to the analogues 12a-c (which has an ionizable amino function).

20. 15
Figure 20. Potential iDALs and cyclic iDALs.
The cyclic monovalent analogues 15, 16a and 16b (Figure 21) as well as the cyclic iDALs 13, 14a
(Figure 20) and 14b did not exhibit any affinity towards the D2 receptor, which was suggested by Jörg
et al. to be due to the constrained ring system of their structures that does not allow the compound
to achieve the right conformation needed for optimal binding.1
Their antagonistic activity towards
the A2A receptor was either not determined or very poor with IC50 values showing a 46-fold decrease.
Yet analogue 14a stands out in the sense that it shows a decent affinity for the A2A receptor (IC50 59
nM) as well as a comparable topological PSA to the parent compound 1 (131 Å2
vs. 127 Å2
).
Figure 21. Cyclic monovalent compounds.

21. 16
In vitro studies were conducted using blood and brain tissue from rats (Wistar Han rat blood and
brain tissues), the A2A-D2 targeting heterobivalent compounds showed that although having
unfavorable physicochemical properties compound 9 (MW: 774.87 g/mol; cLogP: 4.87; PSA: 212 Å2
),
still exhibited a brain:blood ratio (kbb) of 3.66.1
The kbb value is used as an estimate to predict the
probability of a compound to penetrate the BBB where the general rule of thumb is that a kbb value
>1 means a greater likelihood of penetration into the CNS.1
In order to put compound 9’s ability to
enter the CNS into perspective, the parent compound 1 has a kbb value of 0.76.1
This ratio alone does
however not tell the whole story of how successfully the drug will be reaching its destination. One
also need to consider the amount of unbound drug that is actually able to reach the binding site
which in this case is determined by the percent fraction unbound in blood and brain tissue.
Compounds with <1% fraction unbound were considered to have a significant plasma protein
binding. Alas, compound 9 was considered to have a high plasma protein binding (2.22±0.086 %
unbound in blood and 0.61±0.034 % unbound in brain tissue), suggesting that only a limited amount
of the free drug will make it through to the CNS.1
The noncyclic iDAL 12b displayed a Kbb value of 1.29 along with a relatively low plasma protein
binding (19.76±3.16 % unbound in blood; 15.33±1.01 % unbound in brain tissue) that in contrast to
compound 9, implies that a reasonably high amount of the free drug is able to pass the BBB and
reach its target(s) within the CNS.1
Compound 14a was thought to have the potential to be the most promising cyclic iDAL of the
presented analogues, however although it exerted a fairly good Kbb of 1.07 it was not enough to
convert it into a desirable compound for further investigation as it along with 13 and 14b had next to
no affinity towards the D2 receptor.1
Of the compounds described above, particularly 9 and 12b can be regarded as highly promising
compounds for future exploration in useful PD treatment with DALs targeting the A2A-D2
heterodimer.
DALs targeting the A2A receptor and MAO-B
Caffeine (17) (Figure 22) is present in nourishing sources like chocolate, cocoa-beverages, coffee, tea
and soft drinks, thus making it one of the most consumed CNS active compounds in the world.57, 58
17
causes several different pharmacological effects both in the CNS and peripheral nervous system. The
CNS effects are related to learning, memory, cognition and sleep, however at typical dietary doses,
the pharmacological effects of 17 in the CNS involve antagonism of the A1 and A2A receptors.
Accordingly, caffeine is a beneficial lead compound for the treatment of PD, in fact, most A2A
antagonistic compounds has the xanthine ring system as their scaffold. Recently, it has been
documented that caffeine also has the ability to act as an inhibitor of MAO-A and MAO-B with Ki-
values of 0.70 nM and 3.83 nM, respectively.57, 58, 59
Interestingly, caffeine binds reversibly and
competitively to both MAO enzymes and does also have the capability to antagonize the GABAergic
receptors. While this only occurs at µM to mM levels, the inhibition of the inhibitory GABA
neurotransmission may lead to the release of the activating NT glutamate. Consequently, this makes
17 relevant for the symptomatic treatment of PD as it has the ability to inhibit the A2A receptor along
with the MAO-B enzyme.

22. 17
The MAOs are responsible for the oxidative deamination of xenobiotic amines as well as endogenous
monoamines (Figure 4). While both MAO enzymes catalyze the oxidation of epinephrine,
norepinephrine (NE), DA, 5-HT and dietary amines, they possess some substrate selectivities. The
MAO-A isoform is mainly responsible for the deamination of 5-HT and NE whereas MAO-B mainly
deaminates exogenous amines such as β-phenethylamine and benzylamine.57, 60
The MAOs are highly
expressed in the brain tissue, particularly MAO-B which makes it relevant for treatment of PD.57, 60, 61
The fact that there is a much higher concentration of MAO-B than MAO-A in the basal ganglia (the
affected part in a PD brain) makes it the target isoform. The concentration of MAO-B is also thought
to be related to age in the sense that as the brain ages, the amount and activity of the enzyme is
increased and since the B isoform is found in glial cells it means that the increase in population of
glial cells in the basal ganglia is also age related.57
In contrast, the levels of MAO-A are not affected by
age.57
Due to this, MAO-B is of more interest in the symptomatic treatment of PD.
By inhibiting MAO B, the intended effect is to impede the central metabolism of DA which creates
the net effect of a prolonged action of DA within the CNS. The positive side effect to this is a
decrease in the motor oscillations as the amount of L-DOPA required for each administration dose is
decreased.
Figure 22. Xanthine based compounds that display antagonistic traits towards the A2A receptor.
The scaffold of caffeine 17 is a good starting point for the design of MAO-B inhibitors as the
introduction of subtle changes to this scaffold has the ability to significantly modulate the potency of
the compound. (E)-8-(3-chlorostyryl)caffeine (CSC) 18 is a good example of this where the addition
of the 2-(3-chlorophenylethen-1-yl) moiety in the 8-position amplifies the potency 47000-fold
compared to 17 (Figure 22). Both compounds have the ability to inhibit the activity of adenosine
receptors as well as MAO, further adding to the effectiveness of the xanthine scaffold.
Figure 23. Xanthine-based compounds exhibiting inhibiting effects on both the monoamine oxidase B and the adenosinergic
receptors A1 and A2A.

23. 18
As previously stated, CSC (18) is a bivalent compound which possesses a high and comparable affinity
towards both the adenosine A2A receptor and the enzyme MAO-B (Ki: 26-54 nM and 80.6 nM).57
Replacement of the m-Cl-atom with another halogen like Br, leads to an IC50 value of 0.112 µM.57
While the antagonistic potency for the A1 receptor is significantly improved (Ki of >1 µM vs. 28.2 µM),
the potency for the A2A receptor was more or less retained (Ki of 0.026-0.054 µM vs. 0.0284 µM). The
trend amongst the different compounds is such that when structural modifications are made to the
scaffold at the C1, 3- and 7-positions, it often results in an increase of the potency at the A2A receptor
but with a reduced inhibition of MAO-B.57, 62
Indeed, the assessment of compounds 21, 22 and 23
(Figure 23) reveals an increase in A2A potency along with a decrease in MAO-B inhibition. The 1, 3-
diethyl and 1, 3, 7-triethyl substituted analogues 21 and 22 exhibited IC50 values of >100 µM for
MAO-B and Ki values for A2A of 0.0119 µM and 0.083 µM, respectively.57
This indicates that the
lipophilic pockets that might exist around these areas are quite narrow. However, this theory does
not necessarily apply to the lipophilic pocket located around the C-7 position, as 23 reveals that
moderately MAO-B IC50 values can be obtained (2.05 µM) while the compound is still being a strong
potent A2A receptor antagonist (Ki 32.9 µM).57
There have also been attempts to create DALs for the A2A receptor and MAO-B from non-xanthine
based scaffolds.62
One such example is the benzothiazinone analogues 24-28 (4H-3,1-benzothiazine-
4-one compounds) (Figure 24). Structural diversity was created by varying and extending the spacer
between the heterobicyclic moiety and a phenyl group (R) that possessed different functional groups.
The analogues 25 and 26 were investigated in order to test the potential role of the amide proton
acting as a HBD and to implement diversity at the fused benzene ring. Additionally, the impact on the
interaction with the target proteins was also verified by using a non- or dimethyl substituted
thiophenothiazine moiety (compounds 27 and 28).
Figure 24. Non-xanthine based compounds.
From the SAR studies on 22 analogues, it became clear that the majority of the compounds favoured
either MAO-B inhibition or A2A antagonism.62
Nonetheless, there were a handful of compounds
showing promise as DALs. Among these were compounds 29, 30, 31 and 32 (Figure 25).
Figure 25. Non-xanthine based compounds showing dual agonistic features towards both monoamine oxidase isoforms A
and B as well as for the A2A receptor.

24. 19
The phenylpropionyl derivative 29 was considered to be the reference point the different analogues
were measured against. It showed DAL-character with a relatively good potency at the A2A receptor
(Ki= 80.9 nM) along with an IC50 value for MAO-B (17.6 nM).62
By introducing a methoxy group at the
C-3 position on the phenyl ring led to 30. Compared to 29, compound 30 showed an increase in
affinity to the A2A receptor (Ki: 64.9 nM vs. 80.9 nM) but to the cost of a substantial drop in potency
for MAO-B (IC50 95.3 nM vs. 17.6 nM). By just extending the spacer on 29 increased the potency at
A2A substantially (IC50 39.5 nM) while exhibiting a comparable potency for MAO-B (IC50 34.9 nM).62
Thus, compound 31 is a potentially interesting compound for treatment of PD as it exhibits equally
high potency for both targets while exerting promising pharmacokinetic properties: cLogP 2.96, tPSA
58.5 Å2
and a MW of 324 g/mol that are appropriate for BBB transport. Analogues with fused
thiophenes as in 32 did not show as much promise as the benzene fused compounds. The potency at
the A2A receptor was on par with that of 29 (Ki: 82.5 nM vs. 80.9 nM), while exhibiting a moderate
potency for MAO-B with an IC50 value of 69.7 nM.62
Unsubstituted benzothiazinone derivatives showed the best DAL properties having the ability to
target both the receptor and the isoenzyme, where 31 in particular may be a promising lead
compound for further studies as a potential symptomatic treatment for PD.
DALs targeting the serotoninergic-dopaminergic 5-HT1A-D2 receptors
In addition to the DAergic system also the serotonergic system has been suggested as an additional
targets for new therapeutic strategies for PD. 5-HT is not able to pass the BBB due to its lipophilicity,
instead it is biosynthesized from the amino acid tryptophan in the cell body of the neuron and gets
transported to the axon terminal (Figure 2 and 26).
Figure 26. Biosynthetic route for the monoamine 5-HT.Abbreviations: TPH: L-tryptophan hydroxylase; AADC: aromatic amino
acid decarboxylase.
This particular system is related to psychoemotional, cognitive and motor functions in the CNS. The
5-HT1A receptor subtype has received a fair amount of interest as it correlates to disorders such as
depression, schizophrenia and PD. Henceforth, drugs targeting both 5-HT1A as well as D2 receptors
are of interest as they may provide new compounds useful for treatment of PD with higher efficacy
and better safety profiles.64
The importance of agonists and/or partial agonists for the 5-HT1A receptor in PD is due to its action at
the striatal serotoninergic nerve terminals where it can modify the levels of DA produced from L-
DOPA.64, 65
5-HT1A agonists also possess the ability to attenuate L-DOPA induced dyskinesia (LID)
without altering the anti-parkinsonian efficacy. Thus a dual acting compound for both the D2 and
5-HT1A could be of interest.64

25. 20
Figure 27. Dual active compounds used in clinical trials today.
Sarizotan, bifeprunox and paradoprunox (Figure 27) are examples of the most studied 5-HT1A - D2
dual receptor agonists. Sarizotan is a full agonist at the 5-HT1A receptor and only a weak partial
agonist at the D2 receptor that has shown to reduce the LID without diminishing the therapeutic
effect.33
Bideprunox, a partial agonist for both 5-HT1A and D2 receptors has been found to improve
the cognitive and negative symptoms of schizophrenia as well as providing a long lasting anti-PD
effect.64
Pardoprunox (Ki D2:7.9 nM; 5-HT1A: 9.3 nM) is presently going through phase III clinical trials
for PD treatment as it has proved to significantly ameliorate the motor symptoms related to PD. The
latter compound has in fact been in phase III trials since 2005 (by Solvay’s pharmaceutical
company)64
but there is still not enough evidence to reveal whether or not it actually can be used as
an effective monotherapy for the symptomatic treatment of PD.64
One scaffold for DA receptor agonists is the tetracyclic skeleton of apomorphine (APO) 34, an
analogue to morphine 33 (Figure 28).
Figure 28. The parent compound morphine and the tetracyclic scaffold apomorphine.
R-APO is a well-documented agonist with the ability to act on both D1 and D2 receptors and was first
synthesized in 1869.64, 65
The main drawback of the clinical use of APO is that it suffers from a short
duration of action and poor oral bioavailability. The only way to administer APO today is by
subcutaneous injections. By the introduction of small changes in the substitution pattern of 34, a
significant increase in potency towards the 5-HT1A receptor can be observed.64, 65
While the replacement of the 10-OH with a methyl group as in 35 (Figure 29) results in a noteworthy
increase in potency for 5-HT1A (Ki 296±15 for 34 vs. 0.45±0.13 nM for 35), a near complete loss in
potency for D2 (41.9±4.7 vs. 1070±54 nM) is observed. Analogue 36 (Figure 29) where the methyl
group is replaced by a propyl group, did indeed show an increase in potency for D2 receptors
compared to 35 while retaining high potency for 5-HT1A receptors. This indicates that there is a
lipophilic pocket near the 6-N position and that this is an important interaction for the activation of
D2 receptors.

26. 21
Figure 29. Apomorphine analogues.
Furthermore, 37 vs. 42 also shows an increase in affinity for D2 receptors (58.5±9.5 vs. 12.7±1.6 nM),
further solidifying the importance of the interaction to the hydrophobic pocket by the N-propyl
group. The affinity towards both receptors were well-matched for the analogues based on APO 39-
41, which lead to a retained high affinity towards 5-HT1A along with a decrease in affinity for D2
receptors. 42 and 43 showed weak agonistic effects for D2 and weak antagonistic effects for 5-HT1A
receptors (Figure 29).64
By introducing larger and more lipophilic ester moieties as in the prodrugs 44-48 (Figure 29), the
pharmacokinetics of the compounds were greatly improved as well as their potency.64
Having esters
that are harder to hydrolyze, makes it possible for a higher concentration of the active compound
(42) to make it to the site of action.
Compared to 42, compound 44 showed slightly lower potency for D2 (12.7±1.6 vs. 92±18).64
Nonetheless, the bioavailability as well as the duration of action for compound 44 was improved.64
The extension of the esters as in 45-48 as well as the diesters 49-50 revealed that the length of the
ester had no significant correlation to the activity.64
Although, for the monoesters, 46 exhibited the
highest binding potency towards both receptors with Ki values of 56±13 nM (D2) and 12±3 nM (5-
HT1A), respectively.64
While it possesses a significant anti-Parkinson effect, it also suffers from a short
half-life as well as a relatively moderate effect on LID (in rats). Both compounds 49 and 50 are
inactive at the D2 receptor (showing Ki values of >10000 nM for both compound) (Figure 29).64

27. 22
All previous analogues show good physicochemical properties such as cLogP, MW and PSA in the
range of 4.5, 350 g/mol and 29 Å2
.64
The esters obviously exerted higher cLogP values, although they
are pro-drugs and will be hydrolyzed into 42. The larger lipoic acid ester compound 51 stood out as it
showed the optimal potency. Additionally, the lipoic acid is able to readily cross the BBB.64, 65
While it
is the scaffold of 42 that exerts the best binding, the larger lipoic ester chain on 51 is ideal for optimal
pharmacokinetic properties. Since this lipoic ester is not as easily hydrolyzed as a normal ester, more
of the actual compound is able to make it to the BBB. 51 acts as a full agonist for both 5-HT1A and D2
receptors and displays the ability reduce LID in rats without attenuating its relatively high anti-
parkinsonian effect.64, 65
The EC50 values for D2 and 5-HT1A were 320 and 190 nM, respectively. All
these traits make 51 into a promising lead compound for future bioassays, which is why it as of today
undergoes early preclinical studies (Figure 29).
Figure 30. Aminotetralin analogues.
Alternatives to the apomorphine analogues are the tricyclic 52-55 (Figure 30) which have proved to
be valuable tools for investigating new compounds for treatment of PD. All compounds in Figure 26
act as D2 receptor agonists where 52 shows high D2 – 5-HT1A selectivity. However, only the R-
enantiomer of 52 showed DAergic activity, which was completely absent for S-52. For 55a and 55b,
the imidazole ring was replaced with a phenyl moiety which resulted in 55a being a potent D2
receptor agonist, however dual potency was achieved with 55b. These are good candidates for
treatment of PD, although it is worth noting that these aminotetralin analogues have generally low
oral bioavailability and show poor absorption together with rapid excretion.64

28. 23
Discussion
It is well established that the currently used treatments for PD are limited and are in need of
improvement.DALs have shown to have the potential to improve the quality of life for PD-patients as
they are designed to show higher efficacy and fewer side effects. They allow a later start with L-DOPA
treatment and will thereby decrease the risk for the development of LID. Even though it is not, as of
yet, possible to completely cure the disease, the patients should have the right to live a normal life
for as long as possible. The mono-active drugs such as the anticholinergic biperidene and
trihexyfenidyl, the DA agonist ropinirole and L-DOPA are used today, however there are a few DALs
in the pipeline, some in clinical trials. Pardoprunox is one such example.
The potential of the multiple target strategies has led to the launch of several new dual acting
compounds. The dopamine receptors have always been thought to be the most important target for
alleviating symptoms related to PD. But with the discovery that PD patients consuming coffee at a
regular basis experienced pharmacological effects such as a lowered occurrence of motor
fluctuations and neuroprotective benefits, steered the focus from the dopaminergic receptors over
to the adenosinergic receptors.66
While the dopaminergic receptors are still considered more
important and the main target, a simultaneous stimulation of both the adenosinergic and
dopaminergic receptors has a positive effect. As it is also known that the GPCRs exist as oligomers,
DALs can also be used as pharmacological tools to investigate the properties of these oligomers (such
as A2A-D2 receptor heterodimer). The problem that seems to be occurring for DALs is that the
merging of two active compounds held together by a spacer leads to undesired physicochemical
properties. The MW can in some cases become high, and with polar groups, several rotatable bonds,
HBA’s and BHD’s the compounds can be expected to show lower CNS activities. The proposed way of
solving this is by utilizing iDALs, which are DALs that has been merged instead of linking them
together. By doing this, the linker or spacer is excluded which ultimately makes the compounds
better for passing the BBB as their MW, cLogP and tPSA are reduced (eg. Figure 14 and Figure 19).
The lower the MW and the more lipophilic the compound the easier it becomes for the compound to
pass the BBB. However, the “optimal” properties of the compound do not always dictate whether it
will successfully penetrate the BBB or not.
While it is possible to target two different active sites by a drug combination (two separate drugs), it
becomes difficult to predict the pharmacokinetics and whether the drugs affect each other
negatively or not. By utilizing one compound that is able to target multiple targets, a more
predictable pharmacokinetic and pharmacodynamic relationship is obtained. Besides, the compliance
of the patients is also improved as they need only to take one drug rather than having to take
multiple drugs.
There is a wide range of different combinations of receptor targets that has proved to show promise
for treatment of PD, such as the serotonergic-dopaminergic receptors, the monoamine oxidase-B
adenosine receptor and the above mentioned adenosinergic-dopaminergic receptors. While these
targets have all produced lead compounds worthy of further exploration, DALs targeting the
adenosinergic-dopaminergic neurons seems to be the most effective. A lot of research has been
done on the A2A-D2 receptor heterodimer in particular and compounds exhibiting high potencies for
the respective receptors have been identified (like compound 6 and 9).

29. 24
Among the compounds worthy of future studies are the 5-HT1A-D2 targeting compound 51, the
xanthine based A2A-MAO-B targeting compounds 18, 20 and 23 and the non-xanthine based 29, 30,
31 and 32, and the A2A-D2 targeting 9 and 12b.
Conclusion
The construction of future DALs to further slowdown the progress of neurodegeneration in PD, is of
much need, as the compounds examined in this review points towards a bright future. Indeed, they
can be considered to be good lead compounds for future studies but they are undeniably in need of
further optimization. The compounds must become more efficient and smaller while still having a
high affinity towards their respective receptors.
Utilizing DALs to explore and study the different oligomeric receptors and their properties, might
lead to a better understanding of the direction of the design of the future dual acting compounds.
The current drugs and/or the proposed new DALs are all symptomatic for the treatment of PD. In
other words, it is not possible to actually cure the disease, it is only possible to slow it down.
Nevertheless, there are signs that points towards an improved way of treating PD that is more
controlled and more efficient, and that entails more studies around the use of DALs.

30. 25
Acknowledgments
Prima facea, I would like to express my sincere and utmost gratitude to my supervisor and Professor
Kristina Luthman for showing me her continuous support, motivation and most of all, her patience. It
is unfathomable to me how one person could endure someone asking questions and knocking on her
door every other day. This is indeed a thesis I know I would not be able to complete without her
guidance and I am eternally thankful for that.
I would further like to express my gratitude to my peer Kamil, who has written his thesis alongside
me every day, seven days a week since the start of the course. He really has been a huge help in
providing me with honest and useful feedback regarding the language and structure of my report. So
I tip my hat to you Kamil and I wish you good look in your future endeavors.
I must also place on record, my sincere thank you to the student counselors Hannah Ahlborg and
Barbara Casari. I really have been like a hen without a head and they truly have gone above and
beyond to guide me back to the right path.
Lastly, with deepest affection, I wish to give the biggest thank you to my mother, then my mother,
and then my mother and finally my father and siblings for their unconditional love and support.
Thank you all for making this possible for me.

31. 26
References
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