Novel bivalent ligands for D2/D3 dopamine receptors: Significant cooperative gain in D2 affinity and potency

Article abstract of DOI:10.1021/ml3002117

This report describes development of a series of novel bivalent molecules with a pharmacophore derived from the D2/D3 agonist 5-OH-DPAT. The spacer length in the bivalent compounds had a pronounced influence on affinity for D2 receptors. A 23-fold increase of D2 affinity was observed at a spacer length of 9 or 10 (compounds 11d and 14b) as compared to monovalent 5-OH-DPAT (K _i; 2.5 and 2.0 vs 59 nM for 11d and 14b vs 5-OH-DPAT, respectively). The functional potency of 11d and 14b indicated a 24- and 94-fold increase in potency at the D2 receptor as compared to 5-OH-DPAT (EC₅₀; 1.7 and 0.44 vs 41 nM for 11d and 14b vs 5-OH-DPAT, respectively). These are the most potent bivalent agonists for the D2 receptor known to date. This synergism is consonant with cooperative interaction at the two orthosteric binding sites in the homodimeric receptor.

Full text of DOI:10.1021/ml3002117

Letter
pubs.acs.org/acsmedchemlett
Novel Bivalent Ligands for D2/D3 Dopamine Receptors: Significant
Cooperative Gain in D2 Affinity and Potency
Sanjib Gogoi,†,∥ Swati Biswas,† Gyan Modi,† Tamara Antonio,‡ Maarten E. A. Reith,‡,§
and Aloke K. Dutta*,†
†Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48202, United States
§
‡Department of Psychiatry and Department of Pharmaclogy, New York University, New York, New York 10016, United States
S
* Supporting Information
ABSTRACT: This report describes development of a series of
novel bivalent molecules with a pharmacophore derived from
the D2/D3 agonist 5-OH-DPAT. The spacer length in the
bivalent compounds had a pronounced influence on affinity for
D2 receptors. A 23-fold increase of D2 affinity was observed at
a spacer length of 9 or 10 (compounds 11d and 14b) as
compared to monovalent 5-OH-DPAT (Ki; 2.5 and 2.0 vs 59
nM for 11d and 14b vs 5-OH-DPAT, respectively). The
functional potency of 11d and 14b indicated a 24- and 94-fold
increase in potency at the D2 receptor as compared to 5-OH-
DPAT (EC50; 1.7 and 0.44 vs 41 nM for 11d and 14b vs 5-
OH-DPAT, respectively). These are the most potent bivalent
agonists for the D2 receptor known to date. This synergism is consonant with cooperative interaction at the two orthosteric
binding sites in the homodimeric receptor.
KEYWORDS: bivalent ligands, D2/D3 dopamine receptors, cooperative gain, affinity, potency, synergism
protein-coupled receptors (GPCRs) make up one of the
largest and diverse family of mammalian proteins.
action results in increased affinity theoretically equivalent to the
product of the binding contribution of both putative sites.14,15
This can also give rise to higher functional potency, greater
selectivity, and better therapeutic agents.16 Bivalent ligands with
varying linker lengths for dopamine receptors and other GPCR
have been reported.17−21
G
Consequently, GPCRs are targeted most frequently in
therapeutics development for numerous diseases.1,2 The
prototypical GPCR structure consists of seven trans membrane
domains as the central core, connected by extracellular and
intracellular loops. The traditional concept of participation of a
monomeric receptor in the signal transduction process has been
increasingly challenged by evidence for the existence of homo-
and heterodimeric receptors.3 A number of GPCRs have been
shown to form dimers, which include opioid receptors,4
adrenergic receptors,5 and muscarinic receptors.6 Dopamine
receptors are also G protein-coupled and act via modulation of
adenylase cyclase activity. Both dopamine D2 and D3 receptors
can exist as homodimers in cell lines expressing these
receptors.7,8 Furthermore, the dopamine D2 receptor has
been shown to form a dimer in human and rat brain tissues.9
Recently, dopamine D1 and D2 receptors also have been
shown to form heterodimers.10
Bivalent ligands for GPCRs have been reported going back
more than a decade.4,11,12 Bivalent ligands typically consist of
two pharmacophoric moieties connected by a spacer with an
appropriate length, which then allows the two “heads” to
interact either with two orthosteric binding sites of receptors on
adjacent protomers of a dimer or with another relevant,
allosteric site at the same receptor. Bivalents are classified as
either homo- or heterobivalent ligands depending on the nature
of pharmacophores.13 Such simultaneous cooperative inter-
In our quest to probe dimeric forms of either D2 or D3
receptors in cells expressing these cloned receptors, we have
designed a prototypical structure of bivalent molecules by
linking the well-known dopamine D2/D3 agonist molecule 5-
hydroxy-2-(dipropylamino)tetralin (5-OH-DPAT) via a meth-
ylene spacer of varying length (Figure 1). Site-directed
mutagenesis has demonstrated that the amino tetralin moiety
interacts with an agonist binding domain involving TM-3 and
TM-5 for activation of either D2 or D3 receptors.22,23 In our
own study, we have demonstrated the critical importance of
basic nitrogen and hydroxyl functionalities in TM3 and TM5
for binding activity of 5-OH-DPAT.24 Suitable potent bivalent
agonists or antagonists for dopamine receptors might find
potential therapeutic use for treatment of neuropsychiatric and
neurodegenerative diseases like Parkinson's disease.
Scheme 1 describes synthesis of target compound 5.
Optically active (−)-5-methoxy aminotetralin25 was acylated
Received: July 23, 2012
Accepted: October 26, 2012
Published: October 26, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
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increase, consonant with a cooperative interaction resulting in
synergistic enhancement of affinity at D2. The synthesis of 7-,
9- and 10-linkers produced 14a, 11d, and 14b, which exhibited
further improvement in affinity, with 9- and 10-linkers
producing the highest affinity for D2 receptors. A further
increase of the spacer length to 12 methylene units (14c)
decreased the affinity to a level that was comparable to
compound 5 with a linker length of 2. Upon further increase of
the spacer length to 14 (14d), the affinity dropped to that of 5-
OH-DPAT (see also Figure 2). At the D3 receptor, less
dramatic changes occurred upon varying the linker length, and
the 4-linker compound 11a started the bivalent compounds of
this spacer length at an affinity lower (higher Ki) than the
parent compound 5-OHDPAT (Table 1 and Figure 2).
Increasing the length from four linkers up gradually (but
modestly) increased D3 affinity, with the 6-linker compound
displaying the first significant affinity enhancement, just as
observed for D2 affinity. The highest D3 affinity was seen with
a linker length of nine methylene units [11d, Ki (D3); 0.91
nM], whereas further elongation (14b, 14c, and 14d) resulted
in a lowering of affinity. In comparison, in the case of the D2
receptor, 11d along with 14b with a 10-linker spacer, produced
the highest affinity [Ki (D2); 2.0−2.5 nM; statistically the two
compounds were indistinguishable at D2]. Compounds 11d
and 14b were selected for further functional analysis (see
below).
Figure 1. Molecular structures of known dopamine D2 and D3
agonists.
with chloroacetyl chloride under basic conditions to produce
intermediate 2. Further N-alkylation of 1 with intermediate 2 in
presence of a base provided 3, which upon reduction with
lithium aluminum hydride (LAH) produced 4. Finally,
demethylation in presence of boron tribromide at −40 °C
produced the final bivalent target compound 5.
Similarly, Scheme 2 describes synthesis of compounds 11a−
d. Compound 1 was treated with either appropriate acid
chloride or acid to yield intermediates 7a−d. Hydrolysis of
ester by lithium hydroxide produced acid intermediate 8a−d.
Acid coupling with amine 1 under standard 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide-hydrochloride
(EDCI) coupling conditions in the presence of hydroxybenzo-
triazole (HOBt) produced amide intermediate 9a−d. Reduc-
tion of amide followed by demethylation yielded the final
compounds 11a−d.
The gain in affinity for 11d and 14b at D2 (and to a lesser
extent at D3) is consonant with a cooperative effect of
simultaneous interaction of the two heads with two distal
binding sites. As pointed out by Kuhhorn et al.,17 binding of
bivalent ligands to two sites will induce cooperativity with
binding of the first head facilitating binding of the second head
due to being placed in the vicinity of the second binding site.
This binding cooperativity can be reflected in increased Hill
numbers (as compared with unity for binding of monovalent
neutral antagonists). Thus, neutral antagonist binding to D2
receptors displays Hill numbers up to 2.0 in the case of the
bivalent compounds in the study of Kuhhorn et al.17 However,
McRobb et al.19 report no increases in Hill numbers over unity
for clozapine propylamine bivalent antagonists at D2 receptors,
indicating that this is a ligand-dependent phenomenon. Full
monovalent agonists, recognizing high-affinity D2 states,
generally display Hill numbers below unity (0.5−0.7).17 Our
compounds are full agonists (Table 2) and display increases in
Scheme 3 describes synthesis of compounds 14a−d. Two
molecules of amine 1 were coupled with the appropriate diacid
to produce intermediate diamides 12a−d, which on reduction
with LAH produced amines 13a−d. The final targets 14a−d
were synthesized after demethylation with boron tribromide.
Scheme 4 describes the synthesis of compounds 16 and 19. In
both cases, straightforward synthesis approaches were adopted.
In our design of this series of bivalent ligands, we
incorporated methylene units to build the spacer separating
the two pharmacophoric head groups, with a spacer length
varying from 2 to 14 methylene units (Table 1). We included
the agonist 5-OH-DPAT as the parent compound for
comparison purposes. The four-linker 11a exhibited similar
affinity at the D2 receptor as 5-OH-DPAT (Ki; 70 vs 59 nM for
11a vs 5-OH-DPAT, respectively) (Table 1). Moving from a 4-
linker to a 5-linker did not affect the affinity of 11b significantly
as compared to 11a. Interestingly, switching from the 5-linker
(11b) to the 6-linker (11c) induced a significant, 12-fold
a
Scheme 1
a
Reagents and conditions: (i) Chloroacetyl chloride, Et3N, CH2Cl2, 0 °C to RT, 2 h. (ii) (S)-5-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-
amine (1), K2CO3, acetonitrile, reflux, 3 h. (iii) LiAlH4, THF, 80 °C, 3 h. (iv) BBr3, CH2Cl2, −40 °C to RT, 12 h.
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ACS Medicinal Chemistry Letters
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a
Scheme 2
a
Reagents and conditions: (i) Et3N, CH2Cl2, 0 °C to RT, 3 h. (ii) EDCl, HOBT, Et3N, CH2Cl2, RT, 12 h. (iii) LiOH, MeOH:H2O (3:2), RT, 12 h.
(iv) LiAlH4, THF, 80 °C, 3 h. (v) BBr3, CH2Cl2, −40 °C to RT, 12 h.
a
Scheme 3
a
Reagents and conditions: (i) For compound 12a: (1,5)-heptanedioic acid, EDCl·HCl, HOBt, Et3N, CH2Cl2, rt, 24 h; for compound 12b: (1,8)-
decanedioic acid, EDCl·HCl, HOBt, Et3N, CH2Cl2, rt, 24 h; for compound 12c: (1,10)-dodecanedioic acid, EDCl·HCl, HOBt, Et3N, CH2Cl2, rt, 24
h; for compound 12d: (1, 12)-tetradecanedioic acid, EDCl·HCl, HOBt, Et3N, CH2Cl2, rt, 24 h. (ii) LiAlH4, THF, reflux, 3 h. (iii) BBr3, CH2Cl2, −78
°C to rt, 12 h.
Hill number as a function of bivalent binding to D2 (Table 1).
Thus, 11a, the 4-linker compound, has a Hill less than unity as
does 5-OH-DPAT, and with increasing linker length, the Hill
numbers go up in the 1.3−1.8 range. The two bivalent
compounds with the highest D2 potency, 11d and 14b, had
Hill numbers of 1.3 and 1.4, with clearly steeper binding
inhibition curves as compared with 5-OH-DPAT with an
average Hill of 0.8. Therefore, our Hill numbers strongly
indicate cooperative binding of our lead bivalent compounds.
To assess any contribution of a nine methylene spacer, by
itself, to affinity for D2/D3 receptors, monovalent 16 with a
nine methylene spacer length was synthesized and monitored
for D2/D3 receptor binding. It is apparent that 16 exhibited
similar affinity for D2 as 5-OH-DPAT (Ki; 59 vs 68.8 nM for 5-
OH-DPAT vs 16, respectively) and exhibited lower affinity for
D3 as compared to 5-OH-DPAT. In our effort to further
evaluate any contribution of hydrophobic or related effect in
production of high affinity in 11d and 14b, nine-linker 19 with
a terminal phenyl group was designed and synthesized. The
binding data indicate much weaker activity of 19 at D2 (Ki =
141) and D3 receptors (23 nM). This clearly indicates that the
methylene spacer units did not contribute toward the enhanced
affinity of 11d and 14b. It furthermore indicates that enhanced
interaction of lead bivalent compounds does not result from
enhanced hydrophobic or π-stacking interaction.
Following binding analysis, selected lead 11d and 14b were
subjected to the [35S]GTPγS (guanosine 5′-[g-thio]-
triphosphate) functional assay for D2 and D3 receptors and
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Letter
a
Scheme 4
a
Reagents and conditions: (i) Nonanal, NaCNBH3, acetic acid, 1,2-dichloroethane, 12 h, rt. (ii) 48% aqueous HBr, reflux, 6 h. (iii) NaCNBH3,
CH3OH, room temperature, overnight.
Table 1. Inhibition Constants for Competing for
[3H]Spiperone Binding to Cloned rD2L and rD3 Receptors
a
Expressed in HEK Cells
Ki (nM)
Hill slopes
HEK-rD2
D2/
D3
D-compound
HEK-rD2
59 11
HEK-rD3
1.4 0.3
parent
b
5-OH-DPAT
0.83 0.07
43
bivalent (spacer
length)
5 (2)
16
2
1.1 0.1
0.56 0.04
0.72 0.06
1.1 0.01
1.5 0.2
1.3 0.1
1.4 0.1
1.8 0.2
1.4 0.2
1.1 0.3
4.0 1.0
3.6 0.6
2.2 0.4
1.1 0.4
0.9 0.3
1.8 0.6
14
11a (4)
11b (5)
11c (6)
14a (7)
11d (9)
14b (10)
14c (12)
14d (14)
monovalent
16
70 22
75 19
5.9 0.2
3.9 0.7
2.5 0.5
2.0 0.4
17
21
2.7
3.6
2.7
1.1
1.0
1.7
11
2
10
31
3
5
55 11
Figure 2. Interaction of 11d, 14b, and 5-OH-DPAT with rD2
receptors expressed in HEK-293 cells. Isotherms were obtained in
competition experiments with [3H]spiperone as described in the
experimental section (see the Supporting Information). The Hill
slopes for the curve 11d (nH = 1.31) and 14b (nH = 1.43) were higher
than monovalent 5-OH-DPAT (nH = 0.83), indicating a possible
cooperative binding interaction with the D2 receptor dimer. Each
69 (4)
9
7.3 1.1
23
9.4
6.1
19
140 30
4
a
Results are means SEMs for 3−7 experiments each performed in
b
triplicate. Average of conglomerate of 27 separate experiments
(including data sets collected previously under the same conditions).
point is the average
SEM (vertical bar) of 4−6 independent
compared with the full agonists dopamine, ropinirole, and 5-
OH-DPAT (Table 2). The assays were carried out with the
cloned human D2 and D3 receptors expressed in Chinese
hamster ovary (CHO) cells. Dopamine and ropinirole, as
observed by previously, were more potent in activating D3 than
D2. The results also indicated that 11d and 14b potently
stimulate both D2 and D3 receptors. In this regard, 11d and
14b exhibited more than 24- and 94-fold potency as compared
to its monovalent counterpart 5-OH-DPAT in activating the
D2 receptor (EC50 values of 1.7 and 0.44 nM, respectively, vs
41 nM for 5-OH-DPAT, Table 2 and Figure 2), gains similar to
or exceeding those observed for binding affinity. The 94-fold
increase for 14b indicates remarkable synergistic enhancement
of potency for this compound for the D2 receptor. The linker
length of our lead bivalent compounds is ∼13.5 Å (between the
two pharmacophoric N-atoms) or ∼22.6 Å (between the two
pharmacophoric −OH groups), which is different from the
bivalent compounds reported by others. In this regard, there
are a number of different linker lengths for bivalent interactions
that have been reported in the literature. The antagonist/
experiments assayed in triplicate. For Ki values for each curve, see
Table 1. The averages shown for 5-OH-DPAT are from a subset of six
experiments. The x-axis concentrations are expressed as Ki taking into
account the conversion from IC50 as indicated in the methods (see the
Supporting Information).
antagonist bivalent ligands reported by Gmeiner et al. for
interaction at the D2 receptor were based on aryl piperazine
and aminoindane moieties.17,18 In a recent report by McRobb
et al. on a bivalent antagonist, clozapine-like compound at the
D2 receptor, a different, shorter linker length was reported.19 In
addition, for agonist interaction at other GPCRs, a shorter
linker length was also reported for optimal interaction.21 At this
point, it is not known what dominant interaction forces might
play a role in initiating the binding interaction of these varying
bivalent ligands with the D2 homodimer.
In the case of the D3 receptor, there was not the same gain
for bivalent interaction as observed for D2 (Table 2). The
present bivalent ligands have higher receptor affinities for D3
receptor to begin with (as compared with their corresponding
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ACS Medicinal Chemistry Letters
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a
Table 2. Stimulation of [35S]GTPgS Binding to hD2 and hD3 Receptors Expressed in CHO Cells
hCHO-D2
hCHO-D3
b
b
compd
EC50 (nM) [35S]GTPγS
% Emax
100
74 0.9
EC50 (nM) [35S]GTPγS
% Emax
D2/D3
dopamine
240 40
300 10
5.8 1.3
100
42
30
65
b
ropinirole
5-OH-DPAT
11d
10
2
67
75
92
74
8
41
6
80
100
97
4
3
4
0.63 0.08
0.53 0.04
4
1
5
1.7 0.1
3.2
0.46
14b
0.44 0.09
0.94 0.28
a
b
EC50 values (nM) are means SEMs for 3−6 experiments each performed in triplicate. Data from ref 27.
D2 affinities and potencies, Tables 1 and 2); therefore, it may
be more difficult to show additional gains in potency through
bivalent interactions. D3 receptors also couple to G protein less
effectively than D2 receptors,26 and we speculate that this
underlies the generally higher Hill numbers observed for D3
binding (data not shown).
ABBREVIATIONS
■
GTPγS, guanosine 5′-[g-thio]triphosphate; 5-OH-DPAT, 5-
hydroxy-2-(dipropylamino)tetralin; CHO, Chinese hamster
ovary; HEK, human embryonic kidney
REFERENCES
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■
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ASSOCIATED CONTENT
* Supporting Information
Elemental analysis data for all final targets. This material is
■
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available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 1-313-577-1064. Fax: 1-313-577-2033. E-mail: adutta@
wayne.edu.
■
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Present Address
∥CSIR-NEIST, Jorhat, Assam, 785006, India.
Funding
This work is supported by the National Institute of Neuro-
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We are grateful to Dr. K. Neve, Oregon Health and Science
University (Portland, OR), for D2L and D3 expressing HEK
cells. We are also grateful to Dr. J. Shine, Garvan Institute for
Medical Research (Sydney, Australia), for D2L expressing
CHO cells.
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