Structure-Functional-Selectivity Relationship Studies of Novel Apomorphine Analogs to Develop D1R/D2R Biased Ligands

Article abstract of DOI:10.1021/acsmedchemlett.9b00575

Loss of dopamine neurons is central to the manifestation of Parkinson's disease motor symptoms. The dopamine precursor L-DOPA, the most commonly used therapeutic agent for Parkinson's disease, can restore normal movement yet cause side-effects such as dyskinesias upon prolonged administration. Dopamine D1 and D2 receptors activate G-protein- A nd arrestin-dependent signaling pathways that regulate various dopamine-dependent functions including locomotion. Studies have shown that shifting the balance of dopamine receptor signaling toward the arrestin pathway can be beneficial for inducing normal movement, while reducing dyskinesias. However, simultaneous activation of both D1 and D2Rs is required for robust locomotor activity. Thus, it is desirable to develop ligands targeting both D1 and D2Rs and their functional selectivity. Here, we report structure-functional-selectivity relationship (SFSR) studies of novel apomorphine analogs to identify structural motifs responsible for biased activity at both D1 and D2Rs.

Full text of DOI:10.1021/acsmedchemlett.9b00575

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Letter
Structure-Functional-Selectivity Relationship Studies of Novel
Apomorphine Analogs to Develop D1R/D2R Biased Ligands
Hyejin Park, Aarti N. Urs, Joseph Zimmerman, Chuan Liu, Qiu Wang, and Nikhil M Urs
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00575 • Publication Date (Web): 06 Jan 2020
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ACS Medicinal Chemistry Letters
Structure-Functional-Selectivity Relationship Studies of Novel
Apomorphine Analogs to Develop D1R/D2R Biased Ligands
4
Hyejin Park, +§ Aarti N. Urs, ‡§ Joseph Zimmerman,‡ Chuan Liu,+ Qiu Wang+* and Nikhil M. Urs‡*
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+Department of Chemistry, Duke University, Durham, NC 27708
‡Department of Pharmacology and Therapeutics, University of Florida, Gainesville FL 32610
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KEYWORDS: apomorphine analog, D1R/D2R, biased ligands, structure-functional-selectivity relationships (SFSR)
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ABSTRACT: Loss of dopamine neurons is central to the manifestation of Parkinson’s disease motor symptoms. The dopamine
precursor L-DOPA, the most commonly used therapeutic agent
for Parkinson’s disease, can restore normal movement yet cause
side-effects such as dyskinesias upon prolonged administration.
HO
G-protein
Dyskinesia
Dopamine D1 and D2 receptors activate G-protein- and arrestin-
dependent signaling pathways that regulate various dopamine-
dependent functions including locomotion. Studies have shown
that shifting the balance of dopamine receptor signaling toward
the arrestin pathway can be beneficial for inducing normal
movement, while reducing dyskinesias. However, simultaneous
activation of both D1 and D2Rs is required for robust locomotor
activity. Thus, it is desirable to develop ligands targeting both D1
and D2Rs and their functional selectivity. Here, we report
structure-functional-selectivity relationships (SFSR) studies of
HO
Me
N
SFSR studies of novel
apomorphine analogs toward
biased D1R/D2R ligands
Locomotor
improvement
β-arrestin
novel apomorphine analogs to identify structural motifs responsible for biased activity at both D1 and D2Rs.
Parkinson’s disease (PD) is a neurodegenerative disorder
caused by degeneration of dopamine neurons and loss of
dopamine signaling in the striatum.1 The loss of dopaminergic
neurons in PD causes motor dysregulation, resulting in tremor,
rigidity and slowed movements.2 Dopamine signaling is
mediated by two main G-protein-coupled receptors (GPCRs),
D1-like (D1R and D5R) and D2-like (D2R, D3R, D4R)
receptors, both of which have been therapeutic targets for
developing antiparkinsonian and antipsychotic drugs.3
Although D1 and D2 receptors are involved in similar
biological activities, they couple and activate different G-
protein complexes, either increasing (D1-like) or decreasing
(D2-like) the production of 3`, 5`- cyclic Adenosine Mono
Phosphate (cAMP). These receptors are found in similar or
unique anatomical regions in the central nervous system, and
respond to dopamine with different sensitivity. Thus, targeting
different dopamine receptors have distinct therapeutic outcome
and clinical efficacy.3-5
Figure 1. Structures of L-DOPA and (R)-(–)-Apomorphine
12, 13
induce beneficial locomotor effects.3,
These studies
suggest that the side effects of dopamine receptor agonist
drugs can be repressed by reducing activation of the G-protein
pathway.
Selective activation of a signaling pathway in GPCRs can be
achieved using functionally selective, or so called biased
ligands.14 These biased ligands can selectively activate a single
or subset of signaling cascades responsible for therapeutic
effects, in contrast to traditional agonists that stimulate all
possible responses upon binding to a receptor.15-17 Since L-
DOPA, apomorphine or other dopamine receptor agonists
induce dyskinesias and activate both signaling pathways,
development of -arrestin biased agonists provides a strategic
therapeutic approach to treat motor symptoms of PD without
inducing dyskinesias.18 There are a number of reported D1R
G-protein pathway selective ligands,19-22 which show
therapeutic efficacy but their dyskinesia activity has not been
reported. However, there are no known -arrestin biased D1R
agonists to date. Conversely, β-arrestin biased D2R agonists
have been reported.23 Herein, our approach is to target both D1
and D2Rs, since the activation of both D1 and D2Rs is
required for a robust locomotor response. We therefore
focused our efforts on parent drug scaffolds that are dual
D1/D2R agonists.
PD is commonly treated with the dopamine precursor L-3,4-
dihydroxyphenylalanine (L-DOPA) or dopamine receptor
agonists such as apomorphine (Figure 1). However, chronic
treatment with L-DOPA, (R)-apomorphine or other dopamine
receptor agonists often leads to abnormal involuntary
movements called dyskinesias (L-DOPA induced dyskinesia,
LIDs).6 In addition to the canonical G-protein coupled
pathway, dopamine also activates
a
distinct β-arrestin
8
mediated pathway.7,
Previous studies have shown that
overactivation of the G-protein mediated pathway is associated
with the manifestation of dyskinesias.9 On the other hand, β-
arrestin 2 signaling may act as a major contributor in
alleviating locomotor abnormalities,10, 11 as the recruitment of
-arrestin 2 not only desensitizes G-protein signaling, but also
activates a G-protein-independent signaling pathway that can
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Page 2 of 10
chemiluminescent sensor (GloSensor assay, Promega) and β-
Our search for novel dual D1/D2R -arrestin biased agonists
started from (R)-(–)-apomorphine as a core scaffold (Figure
2). Apomorphine, a non-selective unbiased D1R and D2R
agonist, has been clinically used to treat PD motor
symptoms.24 We decided to start from (R)-(–)-apomorphine in
search for novel dual D1/D2R -arrestin biased agonists for
two main reasons. First, with apomorphine as a known PD
drug, its structurally close analogs are expected to retain
dopamine receptor affinity and therapeutic properties. Second,
there have been some structure-activity relationship (SAR)
studies in the literature for apomorphine analogs,25, 26 although
there are no structure-functional-selectivity relationship
(SFSR) studies.
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arrestin 2 recruitment (Bioluminiscence Resonance Energy
Transfer or BRET assay) at D1 and D2 receptors using
standard in vitro dose-response assays (see SI). The results in
the tables show the potency (EC50, pEC50) and efficacy (Emax
)
values calculated using the sigmoidal dose-response function
in GraphPad Prism 7.0. The percent response values were
normalized to dopamine. Transduction coefficients i.e. log
(/KA) were calculated for each compound at both pathways
(G-protein and -arrestin 2) based on the Black and Leff
operational model, where KA is the equilibrium dissociation
constant and  is the agonist efficacy. Bias factors were
calculated based on the method of Kenakin et al,33 where log
(/KA) for each compound was calculated in relation to the
reference agonist dopamine, and the log (/KA) i.e bias
factor was calculated by subtracting the relative transduction
coefficients log (/KA) of the two pathways for each
compound.
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Herein, we report the synthesis of novel apomorphine
analogs and the SFSR examination on how various structural
modifications affect biased signaling at D1 and D2Rs. To
determine whether modifying various structural elements of
apomorphine could result in biased signaling, we divided its
scaffold into three primary regions (Figure 2). These studies
will provide insights toward discovery of -arrestin biased
analogs of apomorphine that can potentially activate
locomotion without inducing dyskinesias.
Scheme 1. Synthesis of novel apomorphine analogs 3–6.
Figure 2. Three regions of apomorphine (2) for SFSR studies.
Our studies began with the phenyl ring, which was the least
explored structural moiety in previous studies. We chose to
create an array of analogs with substituents at either the C-1 or
C-3 position, including bromine and alkyl groups. The
synthesis of these compounds was achieved by a regioselective
[4+2] cyclization of aryne precursor and isoquinoline
derivative as a key step to afford the core aporphine ring as
described in Scheme 1. The aryne precursor I and the
isoquinoline derivatives II-A and II-B were synthesized
following
a
previously reported procedure.27 The CsF-
promoted [4+2]-cyclization of isoquinoline II-A and II-B with
2-trimethylsilylaryl triflate I delivered the core intermediates
III-A and III-B, which were hydrolyzed to free amines IV-A
and IV-B under microwave heating. Finally, reductive
methylation yielded analogs 3 and 4 which bear a bromine at
C-1 and at C-3, respectively. To examine the effects of C-
substituents on the phenyl ring, we next performed a cross-
coupling reaction of racemic 4 for the synthesis of ethyl-
substituted apomorphine derivative 5, as well as a coupling
reaction of (R)-4 for the synthesis of (R)-6. This synthetic
route gave a racemic mixture of apomorphine analogs. Yet the
racemic mixture has shown to be less than half as potent as the
(R)-(–) isomer,28 since the (S)-(+) enantiomer is known to be a
DA receptor antagonist that blocks the (R)-(–) isomer
actions.29-31 Thus, we separated two enantiomers via either
chiral resolution with (+)-DBTA32 or chiral HPLC to generate
enantiopure novel apomorphine analogs (R)-3 and (S)-3, (R)-4
and (S)-4, as well as (R)-5 and (S)-5, for comparison in the
SFSR studies.
(a) HMDS, 80 C, 2 h (quant.); (b) LDA, TMSCl, THF, 20 h
(96%); (c) TBAF, THF, 0 C, 15 min (70%); (d) Tf2O, pyridine,
DCM, 0 C, 18 h (46%); (e) Ac2O, pyridine, 70 C, 2 h (97–99%);
(f) (ClOC)2, FeCl3, H2SO4, DCM, MeOH; (g) Ac2O, pyridine, 60
C, 3 h (11–33%); (h) CsF, MeCN, 80 C, 24 h (26–51%); (i)
LiOH, EtOH/H2O, MW, 180 C, 40 min (59–83%); (j) CH2O,
NaBH4, MeOH, 1.5 h (43–55%); (k) (+)-DBTA, EtOAc/iPrOH;
(l) Chiral HPLC (Lux 5µm Celluclose-1, 250 × 4.6 mm); (m)
Pd(dppf)Cl2, Zn(Et)2, 1,4-dioxane, 70 ºC, 23 h (39%, one step);
(n) Pd(dppf)Cl2, Zn(Me)2, 1,4-dioxane, 70 ºC, 17 h (29%).
All compounds were evaluated for their activities for
stimulating G-protein signaling using a cAMP level-dependent
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As shown in Table 1, both enantiomers of bromine and
alkyl substituted apomorphine analogs showed a general
reduction in activity in all assays for both receptors. However,
compounds (R)-3 and (R)-4 showed weak bias toward G-
1
2
3
4
5
Table 1. Agonist activities of apomorphine analogs with substituents on C-1 and C-3 of phenyl ring on D1R and D2R.
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cAMP
-arrestin
(S)/
(R)
Cmpd
R1
R3
EC50 (nM) pEC50 ± SEM Emax (%) ± SEM EC50 (nM) pEC50 ± SEM Emax (%) ± SEM Bias G
D1R
Apo
(R)-3
(S)-3
(R)-4
(S)-4
(R)-5
(S)-5
(R)-6
(R)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
H
Br
Br
H
H
H
520.8
>10,000
>10,000
>10,000
>10,000
36.86
6.3 ± 0.87
<5.00
13.02 ± 4.9
N.D.
3.77
239.2
8.4 ± 0.15
6.62 ± 0.45
<5.00
94.92 ± 3.17
26.61 ± 3.67
N.D.
41.50
N.C.
N.C.
N.C.
N.C.
N.C
0
H
<5.00
N.D.
>10,000
150.2
Br
Br
Et
Et
Me
<5.00
N.D.
6.82 ± 0.89
6.41 ± 1.56
<5.00
19.98 ± 4.96
0.6325 ± 1.48
N.D.
H
<5.00
N.D.
385.9
H
7.43 ± 2.1
6.60 ± 0.79
9.10 ± 0.45
13.67 ± 1.88
15.04 ± 1.65
14.17 ± 0.9
>10,000
505.1
H
246.8
6.30 ± 0.65
7.06 ± 0.84
9.204 ± 2.02
5.099 ± 1.03
H
0.8
87.44
N.C
D2R
Apo
(R)-3
(S)-3
(R)-4
(S)-4
(R)-5
(S)-5
(R)-6
(R)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
H
Br
Br
H
H
H
10.1
8.0 ± 0.08
7.26 ± 3.11
<5.00
75.0 ± 2.43
7.28 ± 2.25
N.D.
1.61
8.8 ± 0.56
<5.00
<5.00
<5.00
<5.00
<5.00
<5.00
<5.00
60 ± 7.7
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
2.16
N.C
N.C
N.C
N.C
N.C
N.C
N.C
55.37
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
H
>10,000
>10,000
>10,000
>10,000
613.5
Br
Br
Et
Et
Me
<5.00
N.D.
H
<5.00
N.D.
H
<5.00
N.D.
H
6.21 ± 2.86
5.59 ± 2.48
7.33 ± 2.86
9.51 ± 3.6
H
2561
EC50 and Emax values are from three independent experiments performed in duplicate or triplicate. Emax values are calculated as % response
normalized to dopamine. N.C. not calculable, N.D. not determined.
substituted Apo analogs (S)-3 and (S)-4 in HEK-293 cells. D1R
antagonist SCH23390 (SCH) and D1R antagonist raclopride
(RAC) were used as reference antagonists. Data are presented as
percent of the total SCH or RAC response (mean ± SEM).
protein signaling at D1Rs while having no recruitment of -
arrestin 2. Thus, we decided to test antagonist activities of
both enantiomers of compounds 3–6 at both D1R and D2Rs
(Fig 3, Table 2). Interestingly, compounds (S)-3 and (S)-4
showed biased antagonism (comparing IC50’s only) for -
arrestin 2 at both D1 and D2 receptors. The (S)-enantiomer of
apomorphine is known to be an antagonist at dopamine
receptors,29-31 but this is the first study that shows biased
antagonism of the (S)-enantiomer.
It is known that apomorphine’s activity as a dopamine
receptor agonist originates from the catechol ring and
phenethylamine moiety.34 Thus, substitutions on the catechol
Figure 3. Dose response curves of antagonist activities of
bromine substituted apomorphine analogs. In vitro GloSensor
and BRET antagonist assays at D1Rs and D2Rs for bromine
ring may influence the binding of apomorphine at D1/D2R
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toward biased signaling. To probe this hypothesis, a series of followed by O-acetyl protection readily gave the brominated
1
2
3
4
C-9 substituted apomorphine analogs were prepared by direct
and chlorinated apomorphine analogs 7 and 8, respectively
(Scheme 2). Subsequent coupling reactions of bromoarene 7
afforded apomorphine derivatives 9–12 bearing an alkyl or
aryl substituent at C-9 position on the catechol ring (See SI).
functionalization of apomorphine (Scheme 2).
First, the treatment of (R)-(–)-apomorphine (2) with N-
bromosuccinimide (NBS) and N-chlorosuccinimide (NCS)
Table 2. Antagonist activities of apomorphine analogs with substituents on C-1 and C-3 of phenyl ring on D1R and D2R.
5
6
7
8
9
cAMP
-arrestin
(S)/
(R)
IC50 (nM)
pIC50 ± SEM
Emax (%) ± SEM
IC50 (nM)
D1R
pIC50 ± SEM
Emax (%) ± SEM
Cmpd
R1
R2
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SCH
(R)-3
(S)-3
(R)-4
(S)-4
(R)-5
(S)-5
(R)-6
4.34
4080
110.7
815.7
232.4
1603
1522
1427
8.36 ± 0.14
5.39 ± 0.44
7.0 ± 0.26
5.83 ± 0.51
6.6 ± 0.32
5.8 ± 0.58
5.82 ± 0.62
5.85 ± 0.4
93.7 ± 5.23
105.46 ± 9.87
90.0 ± 10
6.96
>10,000
3887
8.16 ± 0.16
4.83 ± 0.24
5.41 ± 0.25
5.29 ± 0.34
5.13 ± 0.24
5.07 ± 0.3
<5.00
87.8 ± 6.1
113.67 ± 5.95
101.94 ± 6.04
94.27 ± 7.44
112.5 ± 6.9
106.47 ± 8.17
N.D
(R)
(S)
(R)
(S)
(R)
(S)
(R)
Br
Br
H
H
H
Br
Br
Et
69.97 ± 11.1
83.73 ± 11.1
71.73 ± 12.5
80.48 ± 11.8
78.36 ± 9.78
5115
H
7461
H
8590
H
Et
>10,000
>10,000
H
Me
<5.00
N.D
D2R
RAC
(R)-3
(S)-3
(R)-4
(S)-4
(R)-5
(S)-5
(R)-6
1.83
30.35
337.5
45.07
1957
28.2
8.74 ± 0.07
7.52 ± 0.83
6.5 ± 0.32
7.35 ± 0.76
5.71 ± 0.3
7.55 ± 0.98
7.39 ± 0.56
5.44 ± 0.51
95 ± 2.76
67.4 ± 12.3
82.33± 9
104.2
0.124
365.2
3222
7 ± 0.14
9.91 ± 3.5
6.44±0.94
5.49 ± 0.65
5.05±0.74
N.C.
89.28 ± 4.9
13.83 ± 7.7
17.26±6.9
53.88 ± 5.55
28.6 ± 12.92
N.C.
(R)
(S)
(R)
(S)
(R)
(S)
(R)
Br
Br
H
H
H
Br
Br
Et
71.5 ± 11.1
76.53 ± 9.9
60.82 ± 13.4
83.47 ± 10.2
96.73 ± 7.75
H
8900
H
N.C.
H
Et
40.77
3629
>10,000
N.C.
<5.00
N.D
H
Me
N.C.
N.C.
IC50 and Emax values are from three independent experiments performed in duplicate or triplicate. Emax values are normalized to SCH23390
(SCH) as an antagonist reference for D1 receptor or raclopride (RAC) as an antagonist reference for D2 receptor. N.C. not calculatable.
N.D. Not determined.
Scheme 2. C-9 Functionalization of apomorphine.
methyl protecting groups, starting from either commercially
available (R)-(–)-apomorphine (2) or apomorphine analog 4.
Scheme 3. Catechol protection of apomorphine.
(a) NBS, TFA, 0 ºC, then Ac2O, pyridine (51%); (b) NCS, TFA,
0 ºC, then Ac2O, pyridine (49%); (c) 7, Pd(dppf)Cl2, Zn(Me)2,
1,4-dioxane, 70 ºC, 24 h (57%); (d) 7, Pd(dppf)Cl2, Zn(Et)2, 1,4-
dioxane, 70 ºC, 24 h (35%); (e) 7, Pd(dppf)Cl2, Zn(iPr)2, 1,4-
dioxane, 70 ºC, 20 h (19%); (f) 7, 3-methoxyphenylboronic
acid, Pd(OAc)2, RuPhos, K3PO4, THF, H2O, rt, 17 h (17%).
Meanwhile, we tested the effects of various catechol
protecting groups, as several protecting groups have been
used in our design and synthesis of novel apomorphine
analogs. It is also known that apomorphine, containing the
10, 11-dihydroxy group, is prone to auto-oxidation and has
limited bioavailability,24, 35 both of which may be improved
upon the presence of a protecting group. As outlined in
Scheme 3, we prepared apomorphine derivatives 13–16
which bear acetyl, methylene, methoxy methyl (MOM), or
(a) Ac2O, pyridine, rt, 5 h (quant); (b) KOH, CH2Br2, DMSO,
80 ºC, 4.5 h (61%); (c) NaOH, MOMCl, CH2Cl2, 0 C, 16 h
(5%); (d) RuCl2(p-cymene)2, K2CO3, i-PrOH, 100 ºC, 24 h
(92%); (e) Chiral HPLC (Lux 5µm Celluclose-1, 250 × 4.6
mm).
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Overall, increasing steric bulkiness of the substituents on
1
C-9 led to pronounced loss of activity of -arrestin 2
recruitment at both D1R (no activity) and D2R (Emax< 36%)
(Table 3). Some analogs such as 9 exhibited low bias for G-
protein activity (7.2 for D1R and 1.78 for D2R), which is the
opposite selectivity we desire. Taken together, these results
indicate that halogen and alkyl substituents on the catechol
ring largely do not contribute to functional selectivity, but
rather reduce agonism for both G-protein and -arrestin 2 at
dopamine receptors.
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57
58
59
60
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Table 3. Agonist activities of apomorphine analogs with C-9 modifications on the catechol ring on D1R and D2R.
1
2
3
4
5
6
7
8
9
cAMP
-arrestin
Cmpd
PG
R9
EC50 (nM) pEC50 ± SEM Emax (%) ± SEM EC50 (nM) pEC50 ± SEM Emax (%) ± SEM Bias G
D1R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Apo
7
H
H
Br
520.8
>10000
>10000
14.45
6.3 ± 0.87
<5
13.02 ± 4.9
N.D
3.77
333.9
194.3
1555
1548
951.4
669.1
20.4
8.4 ± 0.15
6.45 ± 0.54
6.71 ± 0.48
5.81 ± 0.1
94.92 ± 3.17
37.94 ± 8.29
43.25 ± 8.5
67.04 ± 3.19
17.49 ± 4.71
17.09 ± 7.84
24.9 ± 4
41.50
N.C
N.C
7.2
Ac
Ac
Ac
Ac
Ac
Ac
Ac
8
Cl
<5
N.D
9
Me
7.84 ± 1.32
9.42 ± 2.1
<5
3.69 ± 0.33
3.77 ± 0.37
N.D
10
11
12
13
Et
0.38
5.81 ± 0.57
6.02 ± 0.99
6.18 ± 0.36
7.69 ± 0.23
0
i-Pr
3-OMePh
H
>10000
41.36
N.C
N.C
N.C
7.38 ± 2.64
<5
3.5 ± 2.6
N.D
>10000
84.07 ± 8.04
D2R
Apo
7
H
10.1
1412
604
8.0 ± 0.08
5.9 ± 0.43
6.22 ± 0.68
5.74 ± 0.1
5.79 ± 0.24
<5
75.0 ±2 .43
6.47 ± 1.32
6.7 ± 2.0
36.32 ± 1.32
16.04 ± 1.77
N.D
1.61
>10000
>10000
579.7
8.8 ± 0.56
<5
60 ± 7.7
N.D
2.16
N.C
N.C
1.78
0.66
N.C
N.C
1.2
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Br
Cl
8
<5
N.D
9
Me
1812
1636
>10000
N.A
6.24 ± 0.16
5.45 ± 0.25
<5
94.84 ± 6.46
94.75 ± 11.44
N.D
10
11
12
13
Et
3586
i-Pr
>10000
>10000
12.86
3-OMePh
H
N.A
N.A
<5
N.D
111.2
7.0 ± 0.18
66.9 ± 5.5
7.9 ± 0.27
71.2 ± 8.1
EC50 and Emax values are from three independent experiments performed in duplicate or triplicate. Emax values are calculated as % response
normalized to dopamine. N.A. no activity at maximum concentration tested (10–4 M), N.C. not calculable, N.D. not determined.
Table 4. Agonist activities of apomorphine analogs with modifications on the catechol ring on D1R and D2R.
cAMP
-arrestin
EC50 (nM)
pEC50 ± SEM Emax (%) ± SEM EC50 (nM) pEC50 ± SEM Emax (%) ± SEM
D1R
Bias G
Cmpd
PG
14
15
–CH2–
MOM
Me
>10000
8.45
<5
8.1 ± 1.26
N.C
N.D
5.74 ± 3.1
N.C
112.2
>10000
N.C
7.0 ± 1.8
<5
8.3 ± 6.2
N.D
N.C
N.C
N.C
N.C
(R)-16
(S)-16
N.C
N.C
N.C
Me
N.C
N.C
N.C
N.C
N.C
N.C
D2R
14
15
–CH2–
MOM
Me
102
0.3
7.0 ± 1.84
9.5 ± 2.0
N.C
3.6 ± 2.8
2.0 ± 1.9
N.C
>10000
8236
<5
N.D
140
N.C
35.81
N.C
5.08 ± 0.19
5.7 ± 0.6
5.8 ± 0.2
(R)-16
(S)-16
N.C
N.C
1913
75.1 ± 19.4
107.6 ± 10.0
Me
N.C
N.C
1496
N.C
EC50 and Emax values are from three independent experiments performed in duplicate or triplicate. Emax values are calculated as % response
normalized to dopamine. N.C. not calculable, N.D. not determined.
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greater efficacy and potency at both D1 and D2Rs for -
1
2
3
4
arrestin 2 recruitment and G-protein signaling (Table 5).
Methylene-dioxy protection of the N-propyl substituted
apomorphine (19, MDO-NPA) led to reduced activity at all
pathways but to a lesser degree (Table 5, Figure 5) than
apomorphine analogs 14–16.
5
In this study, we employed (R)-apomorphine, a known
agonist of dopamine receptors, as our parent scaffold,
synthesized a series of novel analogs, and evaluated their
SFSR toward identifying -arrestin biased analogs for
D1R/D2R. We systematically examined substitutions on
different regions of the apomorphine scaffold and performed
cell-based screening assays for G-protein versus β-arrestin
recruitment activity at D1 and D2Rs.
6
7
8
9
10
11
12
13
14
15
16
The SFSR studies of the substitution on the catechol group
indicate that among various protecting groups (13–16), O-
acetylation resulted in the least change in activities of
apomorphine, compared to methylene, methoxy methyl, or
methyl groups. Modifications at C-9 position of the catechol
ring (7–12) largely inhibited arrestin activity although
compounds such as 9 were slightly biased for the Gαs-cAMP
pathway.
Figure 4. Dose response curves comparing apomorphine
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(Apo) and catechol protected analogs. In vitro GloSensor
and BRET agonist assays at D1Rs and D2Rs for catechol
protected Apo analogs (13–15) in HEK-293 cells. Data are
presented as percent of the total DA response (mean ± SEM).
The SFSR studies of the lower phenyl ring revealed that
substitutions at either the C-1 or C-3 position generally
abolished activity at both pathways for both receptors. Yet
these findings are confounded by the fact that these analogs
bear methyl protected catechol ring, while the methyl
protection group itself reduces activity at all pathways except
for G-protein at D2R. Interestingly, (S)-3 and (S)-4 showed -
arrestin biased antagonism (IC50 only) at both D1 and D2
receptors. Although these results do not align with our main
interest of discovery of -arrestin biased agonists, such
compounds might be useful for conditions of
hyperdopaminergia that are associated with psychiatric
disorders such as schizophrenia or ADHD, which will be
investigated in our future work.
Among the three protecting groups, the O-acetyl-protected
13 (Table 4) but not the methylene protected (14), methoxy
methyl protected (15) or methyl protected (16) apomorphine,
had the least change in activities in all assays (Figure 4, green
line and Table 4) compared to apomorphine (Figure 4, blue
line and Table 4). Thus, 13 could be a desirable derivative for
further modifications, as it preserves activity relatively well
compared to other protected analogs, and the diester
derivatives have shown to prevent oxidation and prolong the
half-life of apomorphine.36, 37
Lastly, we decided to study the N-alkyl substitution effects
on functional selectivity. Previous studies on N-alkyl
substitutions of apomorphine showed that (R)-N-
propylnorapomorphine (NPA, 17) has a higher affinity at D2
receptor, with selectivity over D1 receptor.35, 38, 39 However,
there were no functional selectivity profiles available. Here we
chose to evaluate a set of derivatives of NPA 17–19 for their
functional selectivity on D1 and D2 receptors (Scheme 4 and
Table 5).
The SFSR studies of the N-alkyl chain were performed on
the N-propyl analogs of apomorphine, based on previous
findings that show NPA is a selective D2 agonist.38 We show
that NPA has picomolar potency for the D2R G-protein
pathway but nanomolar potency for D1R G-protein and D2R
β-arrestin 2 pathways. At low doses NPA produces catalepsy
and inhibition of locomotor activity, whereas higher doses it
increases locomotor activity. This phenomenon can be
explained by the higher potency observed at the D2R G-
protein and arrestin pathways compared to D1R, especially at
lower picomolar doses. Such low doses might preferentially
activate presynaptic D2 autoreceptors causing catalepsy.
Although NPA showed potent activation of the D2R -arrestin
2 pathway, it did show higher efficacy than apomorphine at
the D1R -arrestin 2 pathway, suggesting that the N-alkyl
chain may play an important role in functional selectivity for
the arrestin pathway, which will be examined in the future
studies.
Scheme 4. N-propylnorapomorphine (NPA) and analogs.
In our study, N-propyl substitution (NPA) showed almost
100-fold higher potency at D2R (pEC50: 10.4 ± 0.09) over
D1R (pEC50: 8.96 ± 0.15) at the G-protein pathway, which is
consistent with previous findings.38 Interestingly, NPA (17)
also showed increased efficacy and potency for -arrestin 2
recruitment at D2R (pEC50: 8.93 ± 0.04 Emax: 93.83 ± 1.74)
over D1R (pEC50: 5.73 ± 0.11, Emax: 72.23 ± 4.91) (Table 5
and Figure 5). However, NPA (17) showed increased efficacy
for -arrestin 2 recruitment at D1R when compared to
In summary, these novel apomorphine-based analogs
provide valuable chemical tools and SFSR information in
deciphering receptor pharmacology implicated in Parkinson’s
disease and other dopamine-related disorders.
apomorphine (Figure 5 blue line and Table 5, D1R Emax
:
13.02 ± 4.9). Compared to the acetyl protected N-methyl
analog 13, the acetyl protected N-propyl analog 18 exhibited
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1
2
3
4
Table 5. Agonist activities of N-propylnorapomorphine analogs on D1R and D2R
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cAMP
-arrestin
EC50 (nM) pEC50 ± SEM Emax (%) ± SEM EC50 (nM) pEC50 ± SEM Emax (%) ± SEM
D1R
Bias G
Cmpd
R
PG
Apo
13
Methyl
Methyl
Propyl
Propyl
H
Ac
H
520.8
>10000
1884
6.3 ± 0.87
<5
13.02 ± 4.9
N.D
3.77
20.4
1.1
8.4 ± 0.15
7.69 ± 0.23
8.96 ± 0.15
7.5 ± 0.12
6.14±0.16
94.92 ± 3.17
84.07 ± 8.04
84.87 ± 5.7
88.8 ± 4
41.50
N.C
19.32
0.79
0
17
5.73 ± 0.11
5.3 ± 0.3
5.71 ± 0.91
72.23 ± 4.91
52.5 ± 13.74
7.44 ± 3.1
18
Ac
5496
31.7
717.5
D2R
1.61
12.86
0.04
0.373
7.7
19
Propyl –CH2–
1949
57.8 ± 4.1
Apo
13
Methyl
Methyl
Propyl
Propyl
H
Ac
H
10.1
111.2
1.18
6.34
520
8.0 ± 0.08
7.0 ± 0.18
8.93 ± 0.04
8.2 ± 0.04
6.3 ± 0.05
75.0 ± 2.43
66.9 ± 5.5
93.83 ± 1.74
92.12 ± 1.6
79.41
8.8 ± 0.56
7.9 ± 0.27
10.4 ± 0.09
9.43 ± 0.13
8.11 ± 0.13
60 ± 7.7
71.2 ± 8.1
2.16
1.2
17
94.38 ± 3.42
86.52 ± 4.3
85.43 ± 5.17
0.85
0.6
18
Ac
19
Propyl –CH2–
2.7
EC50 and Emax values were calculated with % response normalized to dopamine. N.C. not calculable. N.D. not determined.
Experimental and characterization data for all compounds and
biological experiment procedures
AUTHOR INFORMATION
Corresponding Author
Nikhil M. Urs: nikhilurs@ufl.edu
Qiu Wang: qiu.wang@duke.edu
§H. Park and A. N. Urs contributed equally to this work.
ACKNOWLEDGMENT
We thank the financial support to this project from the Michael J.
Fox Foundation (MJFF #11764 to N.U.) and the Alfred P. Sloan
Foundation (to Q.W.).
ABBREVIATIONS
PD, Parkinson’s disease; GPCR, G protein-coupled receptor;
Figure 5. Dose response curves for N-propyl substituted
apomorphine analogs. In vitro GloSensor and BRET agonist
assays at D1Rs and D2Rs for N-propyl Apo analogs 17
(NPA), 18 and 19 in HEK-293 cells. Data are presented as
percent of the total DA response (mean ± SEM).
SFSR,
structure-functional-selectivity
relationship;
D1R,
dopamine D1 receptor; D2R, dopamine D2 receptor; BRET, β-
arrestin bioluminiscence resonance energy transfer; L-DOPA, L-
3,4-dihydroxyphenylalanine; DCM, dichloromethane; LDA,
lithium diisopropylamide; DBTA, dibenzoyl tartaric acid; APO,
apomorphine; SCH, SCH23390; RAC, raclopride; NPA, N-
propylnorapomorphine.
ASSOCIATED CONTENT
Supporting Information
REFERENCES
The Supporting Information is available free of charge on the
ACS Publications website.
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