Antagonist and partial agonist modulators of the dopamine D3 receptor (D3R) have emerged as promising therapeutics for the treatment of substance abuse and neuropsychiatric disorders. However, development of druglike lead compounds with selectivity for the D3 receptor has been challenging because of the high sequence homology between the D3R and the dopamine D2 receptor (D2R). In this effort, we synthesized a series of acylaminobutylpiperazines incorporating aza-aromatic units and evaluated their binding and functional activities at the D3 and D2 receptors. Docking studies and results from evaluations against a set of chimeric and mutant receptors suggest that interactions at the extracellular end of TM7 contribute to the D3R versus D2R selectivity of these ligands. Molecular insights from this study could potentially enable rational design of potent and selective D3R ligands.
Antagonist and partial agonist modulators of the dopamine D3 receptor (n class="Chemical">D3R) have emerged as promising therapeutics for the treatment of substance abuse and neuropsychiatric disorders. However, development of druglike lead compounds with selectivity for the D3 receptor has been challenging because of the high sequence homology between the D3R and the dopamine D2 receptor (D2R). In this effort, we synthesized a series of acylaminobutylpiperazines incorporating aza-aromatic units and evaluated their binding and functional activities at the D3 and D2 receptors. Docking studies and results from evaluations against a set of chimeric and mutant receptors suggest that interactions at the extracellular end of TM7 contribute to the D3R versus D2R selectivity of these ligands. Molecular insights from this study could potentially enable rational design of potent and selective D3R ligands.
Dysregulation of the
dopaminergic system is implicated in several
pathological conditions including n class="Disease">schizophrenia, Parkinson’s
disease, depression, and addiction. Dopaminergic signaling is mediated
through two types of receptors termed D1-like (D1, D5) and D2-like
(D2, D3, D4) receptors. Among the various approaches, targeting the
dopamine D3 receptor (D3R) with antagonist or partial agonist ligands
has emerged as a promising area for the development of medications
for the treatment of substance abuse and neuropsychiatric disorders.[1,2] The D3 dopamine receptor subtype is expressed primarily in mesolimbic
regions of the brain including the nucleus accumbens and has been
implicated in the pathophysiology of drug addiction.[3] Studies in animal models have demonstrated that D3R activation
is involved in the reinforcing and motivational effects of cocaine.[4−9] Long-term exposure to cocaine results in up-regulation of D3 receptors
as demonstrated in post-mortem studies of cocaine-overdose fatalities.[10,11] Positron emission tomography (PET) studies have also shown an up-regulation
of D3R over D2R in methamphetamine polydrug abusers.[12] Preclinical studies with a number of D3R antagonist or
partial agonist ligands, such as those shown in Figure 1 (1–5), have demonstrated
that D3R ligands can effectively suppress motivation to self-administer
drugs and prevent drug-associated cue-induced craving and relapse
to drug taking.[13−20]
Figure 1
Structures
of dopamine D3 receptor selective ligands.
In addition, several lines of evidence indicate that D3 receptors
play an important role in the pathophysiology of schizophrenia.[21] Elevated levels of n class="Chemical">D3R expression in the mesolimbic
regions of the brain of schizophrenicpatients have been demonstrated.[22] Overexpression of D3R has been proposed to be
responsible for the sensitization to dopamine agonists. Inhibition
of D3R function may, therefore, attenuate positive symptoms associated
with schizophrenia without causing the extrapyramidal side effects
associated with classical D2R antagonists. Moreover, D3R antagonists
have been shown to enhance D3 receptor mediated release of acetylcholine
in the frontal cortex and, therefore, may have beneficial effects
on attention and memory loss associated with schizophrenia.[21] Indeed, studies with D3R selective or D3R preferring
antagonists have confirmed their efficacy as antipsychotic and procognitive
agents.[23−27]
Structures
of dopamine D3 receptor selective ligands.In the design and development of novel D3R ligands, a primary
challenge
is achieving a high degree of selectivity for n class="Chemical">D3R over the highly
homologous D2R for ligands with druglike characteristics. These issues,
as well as the progress made in the development of D3R selective ligands,
have been the subject of several reviews.[28−34] In the search for novel D3R selective ligands, compounds possessing
a 4-phenylpiperazine tethered to an amide via a four-carbon linker
such as that found in structures 1 and 3 have emerged as a particularly promising group of ligands. Previous
structure–activity relationship (SAR) studies on this class
of compounds, generically represented in Figure 2, have elucidated the importance of the length and composition of
the linker, the carboxamide function, the substituent group on the
piperazine (referred to as the “head group”), and the
substituent group on the amide moiety (referred to as the “tail
group”) in modulating the affinity and intrinsic activity of
this class of compounds.[35−38]
Figure 2
Schematic representation of the generic pharmacophore
for the acylaminobutylpiperazine
class of ligands.
Schematic representation of the generic pharmacophore
for the n class="Chemical">acylaminobutylpiperazine
class of ligands.
Structural comparisons
of the D3R crystal structure[39] and n class="Gene">D2R
homology model as well as docking studies
suggest that a putative orthosteric binding site near transmembrane
helices (TM) 5 and 6 and part of extracellular loop II (ELII) may
contribute to D3R selectivity of the ligands that occupy this site.[40,41] The head group of arylpiperazine class of ligands is accommodated
in this region. Hydrophobic substituents on the head groups could
potentially explore differences in the residues Val350 (D3R)/Ile365
(D2R) and Thr353 (D3R)/Ile368 (D2R) at this orthosteric binding site.
Therefore, we pursued a series of ligands possessing various aryl
or heteroaryl head groups with nonpolar aliphatic substituents that
may explore this region (Table 1). In an effort
to keep the overall lipophilicity low, in this series of ligands (6–25) we incorporated imidazo[1,2-a]pyridine as the tail group.
Table 1
Binding Affinity
of Imidazo[1,2-a]pyridinecarboxamides with
Head Group Variations
Partition
coefficients (CLogP) was
calculated using ChemBioOffice Ultra 2010.
Displacement of [125I]IABN
from HEK cell membranes stably expressing human D3R.
Displacement of [125I]IABN
from HEK cell membranes stably expressing human D2LR. Ki values are the mean ± SEM from three
or more independent experiments.
D2R Ki/D3R Ki.
The use of a heteroaryl
functionality such as 2-indolyl-[35,36,42−44] and 2-benzofuranyl[16,35,42−47] as tail groups has been shown to yield ligands with selectivity
for D3 versus D2 receptors. Recent studies have implicated interaction
of these substituents at a secondary binding pocket (SBP) near the
transmembrane helices 1, 2, and 7 and ELI and ELII as contributing
to the D3R selectivity of such ligands.[38,39] To gain insight
into such selectivity conferring tail group interactions, we pursued
another series of compounds possessing isosteric heterocyclic systems
in place of imidazo[1,2-a]pyridine (26–31). Additional analogues were designed to investigate
potential salt bridge/polar interactions (32–36) and hydrophobic interactions (37–39) of the tail group (Table 2). The
results and insights gained from the synthesis, receptor binding,
and molecular modeling studies are presented herein.
Table 2
Binding Affinity of 2-tert-Butyl-6-trifluoromethylpyrimidines
with Tail Group Variations
Partition coefficients (CLogP) was
calculated using ChemBioOffice Ultra 2010.
Displacement of [125I]IABN
from HEK cell membranes stably expressing human D3R.
Displacement of [125I]IABN
from HEK cell membranes stably expressing human D2LR. Ki values are the mean ± SEM from three
or more independent experiments.
D2R Ki/D3R Ki.
Results and Discussion
Synthesis
The target compounds 6–25 listed
in Table 1 and 26–34 and 37–39 listed in Table 2 were synthesized using
a general procedure involving the coupling of a carboxylic acid with
the appropriate n class="Chemical">aminobutylpiperazine, as depicted in Scheme 1. The coupling of the amine with the acids was performed
using either BOP-Cl or HATU as the coupling agent. The desired aminobutylpiperazine
intermediates 43 were prepared from the corresponding
piperazines 41 by alkylation with bromobutylphthalimide 40 followed by deprotection of the resulting intermediate 42 with hydrazine hydrate.
Reagents and conditions: (a)
K2CO3, CH3CN, reflux; (b) N2H4·n class="Chemical">H2O, MeOH, reflux; (c) R′CO2H, BOP-Cl, Et3N, CH2Cl2,
rt or R′CO2H, HATU, Et3N, CH3CN, rt.
The 5-hydroxymethyl and 5-(dimethylaminomethyl)
compounds 35 and 36 were synthesized as
shown in Scheme 2 starting with n class="Chemical">2-amino-6-hydroxymethylpyridine
(44).
Scheme 2
Synthesis of Target Compounds 35 and 36
Reagents and conditions: (a)
BrCH2COCO2Et, EtOH, reflux, 3 h (70%); (b) TIPSCl,
imidazole, DMF, 70 °C, 2 h (72%); (c) (i) NaOH, MeOH–THF–H2O, 2 h, (ii) AcOH, 81%; (d) amine [43, R = 2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl], HATU,
Et3N, CH3CN, rt, 16 h (22%); (e) Et4NF, THF, rt, 16h (62%); (f) (i) MeSO2Cl, Et3N, CH2Cl2, 1 h, (ii) Me2NH, THF,
rt, 6 h (23%).
Synthesis of Target Compounds 35 and 36
Reagents and conditions: (a)
BrCH2COCO2Et, EtOH, reflux, 3 h (70%); (b) TIPSCl,
imidazole, n class="Chemical">DMF, 70 °C, 2 h (72%); (c) (i) NaOH, MeOH–THF–H2O, 2 h, (ii) AcOH, 81%; (d) amine [43, R = 2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl], HATU,
Et3N, CH3CN, rt, 16 h (22%); (e) Et4NF, THF, rt, 16h (62%); (f) (i) MeSO2Cl, Et3N, CH2Cl2, 1 h, (ii) Me2NH, THF,
rt, 6 h (23%).
Binding Affinities of Head
Group Variants
The affinity
of the compounds at D3 and D2 receptors were determined using previously
established displacement binding assays using [125I]n class="Chemical">IABN
as the radioligand and membrane preparations from HEK cells stably
expressing human D3 or D2L receptors.[43,48] The data for compounds containing head group variations are shown
in Table 1. Compounds 6–10, possessing phenyl and substituted phenyl groups, displayed Ki values of <10 nM at D3R with D2R/D3R selectivity
ratio in the range of 5.4- to 56-fold. Compared to the unsubstituted
phenylpiperazine compound 6, the 2-methoxyphenylpiperazine
compound 7 displayed ∼3-fold enhancement in affinity
at the D3R. However, it also displayed nearly 10-fold higher affinity
at D2R, thus resulting in a reduction of D2R/D3R selectivity. The
highest D3R affinity (in the subnanomolar range) was displayed by
compound 8, the 2,3-dichlorophenylpiperazine analogue.
Earlier studies have shown that the effect of 2-methoxyphenyl and
2,3-dichlorophenyl substituents on binding affinity and selectivity
varies depending upon the linker and the nature of the arylamide moiety.[47,49] The improved affinity and selectivity of compound 8 compared to compound 7, however, are associated with
an increase in lipophilicity (CLogP of 3.6 for 7 vs 5.1
for 8). Interestingly, the introduction of a trifluoromethyl
group at the 3-position of the phenyl ring of compound 6 led to an increase in affinity at D3R without significantly affecting
affinity at D2R, thus improving the binding selectivity (compound 9 D2R Ki/D3R Ki = 56). In addition, the introduction of an electron
withdrawing cyano substituent at the 5-position (compound 10) led to a modest decrease in affinity at D3R and D2R as well as
a decrease in D3R selectivity. The introduction of a tert-butyl group at the 3- and 5-position gave compound 11, which displayed significant reductions in affinity at both the
D3R and the D2R. Incorporation of a bicyclic heterocyclic functionality,
such as 4-quinolinyl, 2-quinolinyl, and 4-quinazolinyl rings, led
to compounds (12–18) with lower affinities
at both D3R and D2R. Whereas the unsubstituted 4-quinolinyl compound 12 and its 7-chloro analogue 15 displayed moderate
affinity at D3R, the 2-trifluoromethyl and 7-trifluoromethyl analogues 13 and 14 had much lower affinity at D3R.
Further, a series of compounds were prepared in which various substituents
were introduced at the 2- and 6-position of the pyrimidine ring. The
isomeric n class="Chemical">methyl-trifluoromethyl pyrimidines 19 and 20 displayed low affinities at the D3R. However, the isomeric
methyl-tert-butyl compounds 21 and 22 displayed interesting differences in their binding profiles
at D3R and D2R. For example, the 2-methyl-6-tert-butyl
isomer 21 had greatly reduced affinity at both receptors,
while the 2-tert-butyl-6-methyl isomer 22 had Ki < 10 nM at D3R with nearly
a 40-fold selectivity over D2R. Incorporation of a bulky tert-butyl group at the 2- and 6-position resulted in compound 23 with lower affinity (D3R Ki = 19 nM) compared to the compound 24 (D3R Ki = 8.4 nM) possessing a tert-butyl and
cyclopropyl group at the 2- and 6-position, respectively. Among the
pyrimidinyl variants containing a tert-butyl group
at the 2-position (compounds 22–25), the most dramatic effect was observed with the 6-trifluoromethyl
compound 25, which was the most potent (D3R Ki = 4.2 nM) and D3R-selective (D2R Ki/D3R Ki ratio =122) compound among
the 4-pyrimidinylpiperazines. The improved affinity and selectivity
of the 2-tert-butyl compound 25 as compared
to the 2-methyl compound 19 highlight the importance
of the tert-butyl substituent at the 2-position of
the pyrimidine ring for D3R affinity and selectivity. To gain insight
into the role of tert-butyl substituents in influencing
binding affinity and D2R/D3R selectivity, we performed a retrospective
analysis of the binding modes of compounds 19–25.
The docked poses of these compounds display a preference
for two
opposite orientations of the pyrimidine ring. The docked poses of
compounds that exemplify these opposite orientations are shown in
Figure 3 for compounds 21 and 22 and in Figure S1 (Supporting Information) for compounds 19 and 20. In the binding
mode of compound 21, the nitrogen at the 3-position of
the pyrimidine ring is buried facing the intracellular side which
we defined as the “b” (buried) orientation as opposed
to the “e” (exposed) orientation shown for compound 22. Analogously, compound 19 has “b”
as opposed to “e” orientation in compound 20. The “e” orientation exposes a more polar surface
of the pyrimidine ring to the aqueous environment than “b”,
and it is expected to be the more favorable orientation. Binding affinity
differences between the isomers 21 and 22 or between isomers 19 and 20 support this
notion that the “e” orientation is the more favorable
of the two. The docked pose of compound 25 (Figure 4 and Figure S2 in Supporting
Information) is very similar to that obtained for compound 22 with the pyrimidine ring having the “e” orientation.
In this orientation, the trifluoromethyl group forms polar interactions
with Thr115 and Ser196 while the tert-butyl group
participates in favorable hydrophobic interactions with Ile183 (ELII),
Val189 (TM5), Val350 (TM6) in D3R corresponding to Ile184, Val190,
and Ile365 in D2R. This interaction may contribute to the binding
potency and D3R selectivity of compound 25 for the following
reasons. (1) The D2R side chain of residue Ile365 exposed in this
pocket is bulkier than the corresponding V350 residue in the D3R,
contributing to a smaller available space in D2R than in the D3R.
(2) Val190 of D2R participates in an interhelical interaction with
Ile368. Such an interaction is absent in D3R where the residue corresponding
to Ile368 is Thr353 which hydrogen-bonds to the helical backbone of
TM6. This difference likely affects the interhelical packing between
the extracellular ends of TM5 and TM6 and may contribute to D3R/D2R
subtype selectivity of ligands.
Figure 3
Docked poses of compounds 21 and 22 at
the D3 receptor are illustrated. For clarity, only the side chain
orientations of the refined D3 crystal structure used for docking
are shown. Atoms are colored by atom type (C, light yellow; O, red;
N, blue) except for ligand carbon atoms as shown. The region of the
pyrimidine ring relevant to the “e” and “b”
orientations is circled.
Figure 4
(A) Docked pose of compound 25 in the D3R binding
site. Residues forming favorable interactions with the ligand are
shown. Atoms are colored by atom type (C, light yellow; O, red; N,
blue) except ligand carbon atoms are colored orange. TM1 and TM5–7
helices are displayed as ribbons. (B) Schematic representation of
the interactions between compound 25 and D3R residues.
Polar interactions are indicated with dashed lines and nonpolar/steric
interactions with gray contour lines.
Partition
coefficients (CLogP) was
calculated using ChemBioOffice Ultra 2010.Displacement of [125I]IABN
from n class="Gene">HEK cell membranes stably expressing humanD3R.
Displacement of [125I]IABN
from n class="Gene">HEK cell membranes stably expressing human D2LR. Ki values are the mean ± SEM from three
or more independent experiments.
D2R Ki/n class="Chemical">D3R Ki.
Docked poses of compounds 21 and 22 at
the D3 receptor are illustrated. For clarity, only the side chain
orientations of the refined D3 crystal structure used for docking
are shown. Atoms are colored by atom type (C, light yellow; O, red;
N, blue) except for ligand n class="Chemical">carbon atoms as shown. The region of the
pyrimidine ring relevant to the “e” and “b”
orientations is circled.
(A) Docked pose of compound 25 in the D3R binding
site. Residues forming favorable interactions with the ligand are
shown. Atoms are colored by atom type (C, light yellow; O, red; N,
blue) except ligand n class="Chemical">carbon atoms are colored orange. TM1 and TM5–7
helices are displayed as ribbons. (B) Schematic representation of
the interactions between compound 25 and D3R residues.
Polar interactions are indicated with dashed lines and nonpolar/steric
interactions with gray contour lines.
Binding Affinities of Tail Group Variants
The binding
affinities of this series of compounds are listed in Table 2. Replacement of the imidazo[1,2-a]pyridine group in compound 25 with a n class="Chemical">2-indolyl moiety
gave compound 26 that had lower binding potency at both
D3R and D2R (2.6-fold and 1.8-fold, respectively) and was less selective
for D3R (122-fold vs 83-fold). The 2-benzimidazole compound 27 had D3R binding potency comparable to that of compound 25 but with reduced D3R selectivity. The incorporation of
a 2-pyridothienyl group gave compound 28 with reduced
binding affinity at the D3R. Introduction of an additional nitrogen
atom in the imidazopyridine system gave the imidazopyrimidine (29), imidazopyrazine (30), and imidazopyridazine
(31) analogues, all of which showed slightly reduced
D3R binding affinity and D3R selectivity. The amino, dimethylaminomethyl,
and hydroxymethyl compounds 32–36, designed to explore potential polar and salt bridge interactions
by these substituents, displayed moderate binding affinity at D3R
with Ki values in the range of 25–45
nM and diminished (<21-fold) D3R selectivity.
Partition coefficients (CLogP) was
calculated using ChemBioOffice Ultra 2010.Displacement of [125I]IABN
from n class="Gene">HEK cell membranes stably expressing humanD3R.
Displacement of [125I]IABN
from n class="Gene">HEK cell membranes stably expressing human D2LR. Ki values are the mean ± SEM from three
or more independent experiments.
D2R Ki/n class="Chemical">D3R Ki.
Previous studies have
suggested that the occupation of a secondary
binding pocket formed by transmembrane helices TM1, -2, -7 and extracellular
loops ELI and ELII by tail groups of aminobutylpiperazines contributes
to the n class="Chemical">D3R selectivity.[38] Prior to the
determination of the D3R crystal structure, Geneste and co-workers
docked a ligand series related to compound 25 into a
D3R model and predicted the positioning of the amide and tail groups
near TM7 to form interactions with Thr368.[50−52] While our docking
results predicted a single preferred orientation for the head group
region of compound 25 and its analogues, we found that
the arylamide tail group may adopt three distinct possible orientations
within the secondary pocket formed by transmembrane helices TM1, -2,
-7 and extracellular loops ELI and ELII at D2R and D3R. In one of
these possible orientations the tail group is in proximity to Glu90
(D3R)/Glu95 (D2R), while a second orientation places the tail group
near the ELII loop residues Val180 (D3R)/Glu181 (D2R) and Ser182 (D3R)/Ile183
(D2R). In the third possible orientation the tail group forms aromatic
interactions with Tyr365 (D3R)/Tyr379 (D2R) while its amide group
hydrogen-bonds with Thr369 (D3R)/Thr383 (D2R). In order to gain insight
as to which might be the most likely placement of the imidazo[1,2-a]pyridine tail group of compound 25 and its
analogues in the secondary binding pocket, we designed the two analogues 34 and 36 containing a charged amine substituent
introduced at distinct positions of the imidazo[1,2-a]pyridine, which could potentially interact with Glu90 (D3R)/Glu95
(D2R) or Glu181 (D2R). Binding affinities of these analogues and lead
compound 25 were evaluated at the wild type receptors
and a set of chimeric and single-point mutant receptors to test for
interactions predicted by these alternative binding modes.
Binding
affinities of compound 25 and the designed
analogues 34 and 36 at chimeric receptors
D3/D2 ELII (human D3 receptor with the D2 ELII loop), D2/D3 ELII (n class="Species">human
D2 receptor with the D3 ELII loop) and the single point D3 receptor
mutants Glu90Ala, Glu90Gln, Ser182Ile are listed in Table 3. All three compounds displayed only a moderate
change in affinities between the wild type D3R and D3R-D2R ELII, suggesting
the lack of significant stabilizing interactions between the positively
charged tail groups of compounds 34 and 36 with Glu181 in D3/D2 ELII and, by extension, in the D2R. This is
further corroborated by the observation that, compared to affinity
at wild type D2R, these compounds show slight improvement in affinity
at the D2/D3 ELII chimeric receptor that has a valine substitution
for Glu181. Furthermore, compounds 25, 34, and 36 did not display a marked change in their binding
affinities at Glu90Ala, Glu90Gln, Ser182Ile compared to the wild type
D3R, indicating the lack of significantly stabilizing salt bridge
or polar interactions between the positively charged amine substituent
of compounds 34 and 36 with Glu90 or Ser182
of D3R.
Table 3
Affinity of Selected Compounds at
Human Dopamine D2R, D3R, Chimeric, and Mutant Receptors
Ki ± SEM (nM)a
compd
D3R wild
type
D2R wild
type
D3R-D2R ELIIb
D2R-D3R ELIIc
D3R (S182I)d
D3R (E90A)e
D3R (E90Q)f
25
8.4 ± 0.8 (3)
702 ± 167 (3)
40 ± 9 (3)
148 ± 10 (3)
12 ± 2 (3)
13 ± 3 (3)
20 ± 4 (3)
34
9.1 ± 3.0 (4)
191 ± 5 (3)
27 ± 6 (3)
72 ± 8 (3)
16 ± 2 (3)
16 ± 5 (4)
19 ± 3 (3)
36
16 ± 5 (4)
245 ± 30 (3)
44 ± 7 (3)
176 ± 25 (3)
24 ± 5 (3)
12 ± 4 (4)
12 ± 2 (3)
Binding
affinities derived from
competition binding experiments using [125I]IABN as the
radioligand. The number of independent experiments is shown in parentheses.
Chimeric D3 receptor possessing
extracellular loop II of D2 receptor.
Chimeric D2 receptor possessing
extracellular loop II of D3 receptor.
D3 receptor with serine 182 mutated
to isoleucine.
D3 receptor
with glutamate 90 mutated
to alanine.
D3 receptor
with glutamate 90 mutated
to glutamine.
Binding
affinities derived from
competition binding experiments using [125I]IABN as the
radioligand. The number of independent experiments is shown in parentheses.Chimeric D3 receptor possessing
extracellular loop II of D2 receptor.Chimeric D2 receptor possessing
extracellular loop II of D3 receptor.D3 receptor with serine 182 mutated
to isoleucine.D3 receptor
with glutamate 90 mutated
to alanine.D3 receptor
with glutamate 90 mutated
to glutn class="Chemical">amine.
These results
suggest that the side chains of Glu90 (D3)/n class="Chemical">Glu95
(D2) or Glu181 (D2)/Val180 (D3) do not significantly contribute to
the binding of the arylamide tail groups of compounds 25, 34, and 36. In docked poses, the tail
group orientation that is consistent with these results is shown in
Figure 5. The protonated dimethylamino nitrogen
in the docked poses of compounds 34 and 36 are too distant (6.4 and 8.7 Å, respectively) to engage in
salt bridge interactions with the carboxylate group of Glu90 of the
D3R. Binding interactions of compounds 34 and 36 include hydrogen bonding between the amide and Thr369 in TM7. The
distance between the tail group’s amide NH of compounds 34 and 36 and the Thr369 side chain oxygen are
2.4 and 2.9 Å, respectively. Thr369 also hydrogen-bonds with
a ring nitrogen of the imidazo[1,2-a]pyridine in
compounds 34 and 36 (heavy atom distances
of 3.2 and 3.1 Å, respectively). These hydrogen bonds may play
a role in the positioning of the imidazopyridine ring for favorable
aromatic interactions with Tyr365 in D3R (Tyr379 in D2R) in TM7.
Figure 5
(A) Docked
poses of compounds 34 and 36 at the D3R
crystal structure. D3R residues are shown with light
yellow colored carbons and underlined residue numbers. D2R residues
with sequence differences from D3R are shown with carbons colored
gray. Amino acids included in mutation experiments and discussed in
text are also shown. Ligand carbon atoms are in cyan for 34 and orange for 36. Fluorine atoms are colored dark
green. All other atoms are colored by atom type. (B) Schematic representation
of the tail region of the poses shown in (A). Polar interactions are
illustrated with dashed lines, nonpolar/steric interactions with gray
contour lines.
(A) Docked
poses of compounds 34 and 36 at the D3R
crystal structure. n class="Chemical">D3R residues are shown with light
yellow colored carbons and underlined residue numbers. D2R residues
with sequence differences from D3R are shown with carbons colored
gray. Amino acids included in mutation experiments and discussed in
text are also shown. Ligand carbon atoms are in cyan for 34 and orange for 36. Fluorine atoms are colored dark
green. All other atoms are colored by atom type. (B) Schematic representation
of the tail region of the poses shown in (A). Polar interactions are
illustrated with dashed lines, nonpolar/steric interactions with gray
contour lines.
In order to test the
hypothesis that tail groups engage in aromatic
interactions as predicted by our docked poses, we designed and evaluated
binding affinities of compounds 37–39 (Table 2). Whereas the n class="Chemical">piperidine-4-carboxamide
analogues 37 and 38 displayed only moderate
affinity, the cyclohexanecarboxamide analogue 39 displayed single digit nanomolar affinity at D3R. Interestingly,
its affinity at D2R was markedly diminished, thus providing a ligand
with >150-fold selectivity for D3R over D2R. Docking results of
compound 39 at D3R and D2R predict aromatic–aromatic
interactions
between the phenyl ring of the 4-phenylcyclohexanecarboxamide
group of compound 39 and the Tyr365D3R side chain (Figure 6) corresponding to Tyr379 in D2R. Sequence and structural
comparison between the D3R crystal structure and D2R model suggests
that interactions with this tyrosine in TM7 may contribute to D3R
selectivity of ligands as follows. The nonconservative amino acid
difference Thr368 (D3R)/Phe382 (D2R) within the interhelical region
of TM6—TM7 likely affects interhelical packing near the extracellular
end of these helices leading to structural differences between D2R
and D3R. Another sequence difference at the extracellular end of TM7,
at its TM1 interface, is Tyr36 in D3R corresponding to Leu41 in D2R.
Furthermore, the region of the TM7—TM1 interface is rich in
sequence difference amino acids. TM1 is the least conserved helix,
only 65% identical between D2R, D3R sequences as compared to overall
78% identity within the seven transmembrane helical region. These
differences may position the extracellular end of TM7 closer to TM1
and more distant from TM6 in the case of the D2R compared to D3R.
The D3R selectivity of ligands, therefore, may arise from direct interactions
with residues near the extracellular end of TM7 such as Tyr365 (D3R)/Tyr379
(D2R). Indeed, it has been suggested that subtle differences in relative
positioning of TM1 and TM7 helices in D2R and D3R may contribute to
selectivity even if the ligands interact with the same amino acid
side chains in D2R and D3R.[39] Consistent
with this notion, docked poses of ligands with high D3R selectivity
(e.g., 25–27 and 39)
display interactions between their pendent aryl group and the tyrosine
residue at the extracellular end of TM7 in both D3R and D2R. In a
recent study, mutagenesis results coupled with docking and molecular
dynamics simulations led to the identification of Gly94 (D3R) in the
ELI loop as an important contributor to D3R/D2R selectivity of ligands
through its ability to modulate the size and shape of the secondary
binding pocket.[53] The ELI loop in D3R contains
two glycine residues (Gly93, Gly94), while this loop in D2R is one
residue shorter containing one glycine residue (Gly98). Interestingly,
in the docked poses of our ligands, Gly94 is in proximity to tail
groups, for example, in the case of compound 25 (Figure
S2 in Supporting Information), the α-carbon
of Gly94 is at 3.4 Å distance from the closest heavy atom in
the imidazopyridine ring of this ligand docked at D3R.
Figure 6
Docked pose of compound 39 in the D3R binding site.
Residues forming favorable interactions with the ligand are shown.
Atoms are colored by atom type. Fluorine atoms are colored dark green.
Ligand carbon atoms are shown in orange.
Docked pose of compound 39 in the D3R binding site.
Residues forming favorable interactions with the ligand are shown.
Atoms are colored by atom type. n class="Chemical">Fluorine atoms are colored dark green.
Ligand carbon atoms are shown in orange.
Functional Activity
In an effort to assess the intrinsic
activity profile of the compounds, functional activity evaluations
were performed using assays that measure various end points such as
the mitogenesis assay, the cyclase assay, β-arrestin recruitment
assay, and the GTPγS binding assay. In the mitogenesis assay
using CHOp cells expressing n class="Species">human D3 and D2 dopamine receptors, the
evaluated compounds did not display appreciable intrinsic efficacy
at D3 or D2 receptors (Table 4).[54] The maximum stimulation of [3H]thymidine
incorporation at a ligand concentration of 10 μM was <20%
of the full agonist quinpirole. These compounds displayed varying
antagonist potencies in inhibiting mitogenesis induced by the agonist
quinpirole at the D3R and D2R. With the exception of compound 7 which displayed D3R antagonist IC50 value (3.0
nM) similar to its D3R binding Ki value
(3.5 nM), the D3R antagonist potencies of all of the evaluated compounds
were weaker than their binding affinities at the D3R. The D2R/D3R
antagonist selectivity of the compounds in the mitogenesis assay was
also much lower than the selectivity in the binding assay. The pyrimidinyl
compound 25 displayed moderate antagonist potency with
IC50 of 157 nM at D3R. However, it displayed 15-fold D3R
antagonist selectivity due to its much weaker potency at D2R.
Table 4
Efficacy of Selected Ligands in Mitogenesis
Assay
agonist
activity
antagonist activity
compd
D3R % stimulationa
D2R % stimulationa
D3R IC50 (nM)b
D2R
IC50 (nM)b
selectivity
ratioc
7
6.4
<20
3.0 ± 1.3
26 ± 8
8.7
8
<20
<20
7.2 ± 3.3
55 ± 16
7.6
12
<20
ndd
120 ± 22
ndd
nae
17
<20
ndd
940 ± 150
ndd
nae
25
0
0
157 ± 34
2300 ± 1100
15
27
<20
<20
208 ± 74
490 ± 150
2.4
29
<20
<1
51 ± 21
205 ± 48
4.0
30
<20
<20
245 ± 33
420 ± 120
1.7
31
<1
<1
637 ± 99
1160 ± 110
1.8
Percent stimulation at 10 μM
normalized to the maximal stimulation by quinpirole. Results are from
two independent experiments.
Inhibition potency against stimulation
of mitogenesis by standard agonist quinpirole (30 nM). IC50 values are mean ± SEM from at least three independent experiments,
each conducted with duplicate determinations.
(D2R IC50)/(D3R IC50).
Not determined because of weak binding
affinity at D2R (Ki > 500 nM).
Not applicable.
Percent stimulation at 10 μM
normalized to the maximal stimulation by quinpirole. Results are from
two independent experiments.Inhibition potency against stimulation
of mitogenesis by standard agonist quinpirole (30 nM). IC50 values are mean ± SEM from at least three independent experiments,
each conducted with duplicate determinations.(D2R IC50)/(n class="Chemical">D3R IC50).
Not determined because of weak binding
affinity at D2R (Ki > 500 nM).Not applicable.Selected compounds were evaluated
in the adenylyl cyclase assay
which measures the ability of the compounds to inhibit forskolin-dependent
stimulation of adenylyl cyclase activity.[43,48] The evaluations were carried out using n class="CellLine">HEK 293 cells expressing
humanD3 and D2 dopamine receptors (Table 5).
Table 5
Efficacy of Selected Ligands in Adenylyl
Cyclase Assay
compd
D3R % inhibitiona
D2R % inhibitiona
7
44 ± 6
25 ± 5
8
66 ± 13
45 ± 1
9
70 ± 4
32 ± 4
12
50 ± 6
1.4 ± 6.7
17
70 ± 18
19 ± 6
25
28 ± 7
–1.9 ± 13
26
38 ± 4
ndb
27
12 ± 11
5.6 ± 4.1
29
51 ± 10
9.2 ± 7.3
30
38 ± 5
ndb
31
32 ± 7
ndb
34
47 ± 4
8.9 ± 5.0
39
52 ± 13
ndb
Percent inhibition
values were normalized
to the percent inhibition of the full agonist quinpirole at D3R (100
nM) and D2R (1 μM). For D3 receptors the maximum inhibition
by quinpirole ranged from 38% to 53%, and for D2 receptors the maximum
inhibition was >90%. The test compounds were used at a concentration
equal to approximately 10 times the Ki value in the radioligand binding assays. The values are the mean
± SEM from three or more independent experiments.
Not determined because of insolubility
of the compounds at 10 times their binding Ki values at D2R.
Percent inhibition
values were normalized
to the percent inhibition of the full agonist quinpirole at n class="Chemical">D3R (100
nM) and D2R (1 μM). For D3 receptors the maximum inhibition
by quinpirole ranged from 38% to 53%, and for D2 receptors the maximum
inhibition was >90%. The test compounds were used at a concentration
equal to approximately 10 times the Ki value in the radioligand binding assays. The values are the mean
± SEM from three or more independent experiments.
Not determined because of insolubility
of the compounds at 10 times their binding Ki values at D2R.In the adenylyl cyclase inhibition assay, all of the evaluated
compounds displayed a partial agonist profile at D3R with intrinsic
efficacies ranging from 12% (compound 27) to 70% (compounds 9 and 17). Interestingly, among the compounds
possessing identical n class="Chemical">arylamide groups (7–9, 12, 17, and 25),
compound 25, which has a 2-tert-butyl-6-trifluoromethylpyrimidinyl
head group, displayed lower efficacy at the D3R (28%) than compounds
possessing substituted phenyl, 4-quinolinyl, and 4-quinazolinyl head
groups. Among the compounds possessing a 2-tert-butyl-6-trifluoromethylpyrimidinyl
head group (25–27, 29–31, 34, and 39), the
benzimidazole-2-carboxamide 27 displayed the lowest efficacy
at the D3R (12%).
Binding of agonists at D3R and n class="Gene">D2R is known
to lead to the recruitment
of β-arrestin to the receptor. Selected compounds were evaluated
for their activity on β-arrestin-2 recruitment using a cell-based
receptor/β-arrestin interaction assay (DiscoveRx PathHunter).[55] In this assay, the interaction of β-arrestin
with a GPCR is monitored using β-galactosidase (β-gal)
enzyme fragment complementation. Activation of the receptor by an
agonist results in the translocation of β-arrestin to active
receptor, which leads to the formation of an active β-galactosidase
enzyme. The activity of β-galactosidase is then measured by
addition of chemiluminescent detection reagents. These assays were
performed according to manufacturer’s protocol using CHO-K1
cells expressing human D2L receptors and U2OS cells expressing
human D3 receptors. Intrinsic agonist or inverse agonist activity
of the compounds was determined by evaluating the ability of the compounds
to stimulate or inhibit basal activity. In these evaluations, all
of the tested compounds (Table 6) were found
to be devoid of agonist activity and displayed very weak inverse agonist
activity at both D3R and D2R. The maximum inhibition of the basal
activity observed was <21% (compound 33) at D3R and
<30% (compound 34) at D2R at 500 nM concentration
of the test compounds. The antagonist potency of the compounds was
assessed by determining the ability of these compounds to inhibit
the agonist activity of ligands PD128907 and pergolide at D3R and
D2R, respectively. Haloperidol was included as a standard antagonist
ligand. The results are presented in Table 6.
Table 6
Antagonist Potencies of Selected Ligands
in the β-Arrestin-2 Recruitment Assay
compd
D3R IC50 ± SEM, nMa
D2R IC50 ± SEM, nMb
selectivity
ratioc
7
0.7 ± 0.1
6.6 ± 5.1
9.4
8
0.2 ± 0.1
91 ± 7
455
9
1.8 ± 0.8
60 ± 12
33
12
52 ± 7
161 ± 78
3.1
17
409 ± 25
248 ± 63
0.6
25
0.6 ± 0.1
261 ± 54
435
26
5.6 ± 1.8
418 ± 80
75
27
8.5 ± 2.1
301 ± 50
35
29
0.4 ± 0.2
37 ± 17
93
30
0.3 ± 0.1
49 ± 12
163
31
0.3 ± 0.1
22 ± 15
73
33
14 ± 3
311 ± 53
22
34
7.2 ± 3.7
217 ± 10
30
36
29 ± 3
281 ± 43
9.7
39
6.8 ± 1.8
>500
>73
haloperidol
0.06 ± 0.02
0.16 ± 0.02
2.7
IC50 values for inhibition
of (+)-PD128907-induced arrestin translocation in U2OS cells expressing
D3R.
IC50 values
for inhibition
of pergolide-induced arrestin translocation in CHO-K1 cells expressing
D2LR.
(D2R IC50)/(D3R IC50).
IC50 values for inhibition
of (+)-PD128907-induced arrestin translocation in n class="CellLine">U2OS cells expressing
D3R.
IC50 values
for inhibition
of pergolide-induced arrestin translocation in n class="CellLine">CHO-K1 cells expressing
D2LR.
(D2R IC50)/(n class="Chemical">D3R IC50).
Most of the evaluated compounds displayed potent (<1.0
nM) to
moderately potent (>1.0 nM) antagonist activity in inhibiting agonist
stimulated recruitment of β-arrestin-2 by D3 receptors. Compounds
that displayed subnanomolar antagonist potency at n class="Chemical">D3R include 7, 8, 25, 29–31. The antagonist potency of the compounds at the D3R, in
general, correlates with their binding affinities (Pearson’s
correlation coefficient between binding pKi and antagonist pIC50, r = 0.77; P = 0.008). Similar to their binding affinity at D2R, the
antagonist potency of these ligands at D2R was weaker than at D3R.
The binding affinity and antagonist potency at D2R, however, was not
as well correlated, as they were at D3R. In this assay, compounds 8 and 25, both possessing the imidazo[1,2-a]pyridine tail group, with the 2,3-dichlorophenyl (8) or 2-tert-butyl-6-trifluoromethyl-4-pyrimidinyl
(25) head group emerged as high potency ligands with
very high (>400-fold) D3R selectivity. A comparison of the profile
of compound 25 with compound 26 indicates
that incorporation of the imidazo[1,2-a]pyridine-2-carboxamide
group (25) provides a more favorable D3R binding and
D3R antagonist functional selectivity than the indole-2-carboxamide
group (26).
On the basis of the relatively high
D3 over D2 antagonist selectivity
in the β-arrestin-2 recruitment assay, compounds 8, 25, 30, and 39 were selected
for evaluation in the [35S]GTPγS binding functional
assays using n class="Species">CHO cell lines expressing humanD3R and D2R.[56−58] These compounds did not stimulate [35S]GTPγS binding,
and up to 1 μM concentration these compounds caused less than
30% inhibition of basal binding at D2R and D3R. Compounds 25, 30, and 39 at 10 μM caused inhibition
of 40–60% of D3R basal [35S]GTPγS binding,
indicating some inverse agonist activity which did not appear pharmacologically
relevant compared with their binding affinity assessed with [125I]IABN. The potencies of these compounds as antagonists
were then determined using the “shift” experiments[59] to obtain antagonist Ke values based on their ability to reduce the apparent potency
of dopamine in stimulating [35S]GTPγS binding in
CHO-D2R and CHO-D3R cells (Table 7). For compound 39, the antagonist potency at D2R could not be determined
in shift experiments, as drug concentrations substantially higher
than 1 μM were required to shift the dopamine curve; such high
concentrations had inverse agonist activity invalidating the shift
method.
Table 7
Antagonist Potencies of Selected Compounds
in [35S]GTPγS Binding Assay
compd
CHO-hD3 Ke (nM)a
CHO-hD2 Ke (nM)a
selectivity
ratiob
8
0.0038 ± 0.0013
1.1 ± 0.2
289
25
1.3 ± 0.4
111 ± 25
85
30
0.33 ± 0.05
25 ± 6
76
39
19 ± 4
nac
nac
The antagonist Ke value was calculated
from “shift” experiments
as described in Experimental Section. Dopamine
dose–response curves were generated in the absence or presence
of compound 8 (10 nM for D2R; 0.1 nM for D3R), compound 25 or 39 (1 μM for D2R; 100 nM for D3R),
or compound 30 (50 nM for D2R; 1 nM for D3R). The fixed
concentrations of drug were chosen as being high enough to shift the
dopamine stimulation curves to the right but low enough to not appreciably
inhibit [35S]GTPγS binding below baseline based on
the inverse agonist activity. The Ke was
calculated from the increase in ED50 observed with test
compound (see Experimental Section). Results
are the mean ± SEM for three to four independent experiments
assayed in triplicate.
(CHO-hD2 Ke)/(CHO-hD3 Ke).
Measurement could not be made
with
shift protocol: Concentrations of drug that by themselves did not
appreciably inhibit binding below baseline did not substantially shift
the dopamine curve to the right.
The antagonist Ke value was calculated
from “shift” experiments
as described in Experimental Section. Dopamine
dose–response curves were generated in the absence or presence
of compound 8 (10 nM for D2R; 0.1 nM for D3R), compound 25 or 39 (1 μM for D2R; 100 nM for D3R),
or compound 30 (50 nM for D2R; 1 nM for D3R). The fixed
concentrations of drug were chosen as being high enough to shift the
dopamine stimulation curves to the right but low enough to not appreciably
inhibit [35S]GTPγS binding below baseline based on
the inverse agonist activity. The Ke was
calculated from the increase in ED50 observed with test
compound (see Experimental Section). Results
are the mean ± SEM for three to four independent experiments
assayed in triplicate.(CHO-hD2 Ke)/(n class="Gene">CHO-hD3 Ke).
Measurement could not be made
with
shift protocol: Concentrations of drug that by themselves did not
appreciably inhibit binding below baseline did not substantially shift
the n class="Chemical">dopamine curve to the right.
Among the compounds possessing the 2-tert-butyl-6-trifluoromethylpyrimidinylpiperazine
head group (compounds 25, 30, and 39), compounds 25 and 39 displayed
n class="Chemical">D3R antagonist potency somewhat similar to their binding potency.
The antagonist potency of imidazopyrazine compound 30 was moderately higher at D3R and D2R compared to its binding affinities
at these receptors. Interestingly, compound 8 which contains
the 2,3-dichlorphenylpiperazinyl head group displayed extraordinary
potency at D3R in the picomolar range.
Increasing evidence indicates
that GPCR ligands including n class="Chemical">D3R and
D2R ligands can display differing intrinsic efficacy and/or potency
for different signaling pathways linked to the same receptor.[48,60−65] These types of effects have been referred to by various terms which
include stimulus trafficking, functional selectivity, collateral efficacy,
and biased agonism. The manifestation of functional selectivity in
our series of compounds is exemplified by the functional activity
profile of compounds 8, 25, and 30. At D3R, these compounds behave as antagonists in the [35S]GTPγS binding, β-arrestin-2 recruitment, and mitogenesis
assays but function as partial agonists in the cyclase assay.
Among the compounds studied, compound 25 displayed
favorable D3R binding affinity (Ki <
5 nM) and n class="Gene">D2R/D3R binding selectivity (>100-fold) with antagonist–partial
agonist functional activity profile and was chosen for pharmacological
evaluations in vivo. This compound, coded as SR 21502, was evaluated
in rats and was found to produce significant decreases in cocaine
reward, cocaine seeking, preference for cocaine-associated environments,
and cocaine induced locomotor activity at doses that had no effect
on food reward or spontaneous locomotor activity, indicating that
it selectively inhibits cocaine’s rewarding and stimulant effects.[66,67]
Summary and Conclusions
In an effort to identify dopamine
D3 receptor ligands with selectivity
for n class="Chemical">D3R versus D2R, we designed a series of ligands based on the acylaminobutylarylpiperazine
pharmacophore incorporating, primarily, aza-aromatic systems on the
acyl and piperazine moieties. Docking of these ligands at the binding
sites of the humanD3R crystal structure and our D2R homology model
provide insights into molecular features contributing to binding affinity
and binding selectivity of this panel of ligands. Among the ligands
possessing a 4-pyrimidinylpiperazine group, the affinity differences
between isomeric compounds at the D3 receptor could be traced to a
favorable orientation of the pyrimidine ring nitrogen positioned toward
the solvent-exposed extracellular side of the receptor with simultaneous
occupation of a small pocket near helices TM5 and TM6 formed by Val189,
Ile183, and Val350 by a hydrophobic substituent from the 2-position
of the pyrimidine ring. The results from binding experiments with
chimeric and mutant receptors support the hypothesis that D3 receptor
selectivity over D2 receptor conferred by tail groups such as the
imidazo[1,2-a]pyridine arises through interaction
with the tyrosine residue (D3RTyr365, D2RTyr379) at the extracellular
end of TM7, which is a region with significant structural differences
between D2 and D3 receptors. These structural insights provide a basis
for the rational design of future ligands with improved binding affinity
and selectivity for the dopamine D3 receptors.
Results from
functional assays characterized the investigated class
of compounds as antagonists or partial agonists with varying potencies
depending upon the functional end point of the assay. From this series,
compound 25 was selected as a lead compound and evaluated
in rat models of n class="Chemical">cocaine self-administration and conditioned place
preference. Results from these studies showed that compound 25 was effective at significantly attenuating cocaine self-administration,
cocaine reward, cue-induced reinstatement of cocaine-seeking and blocking
cocaine-induced place preference.[66,67] Further studies
on the development of dopamine D3R selective ligands are in progress
and will be the subject of future publications.
Experimental
Section
General Methods
Melting points were determined in open
capillary tubes with a Mel-Temp melting point apparatus and are uncorrected. n class="Chemical">1H NMR spectra were recorded on a Nicolet 300NB spectrometer
operating at 300.635 MHz. Chemical shifts are expressed in parts per
million downfield from tetramethylsilane. Spectral assignments were
supported by proton decoupling. Mass spectra were recorded on a Varian
MAT 311A double-focusing mass spectrometer in the fast atom bombardment
(FAB) mode or on a Bruker BIOTOF II in electrospray ionization (ESI)
mode. Elemental analyses were performed by Atlantic Microlab, Inc.
(Atlanta, GA) or by the Spectroscopic and Analytical Laboratory of
Southern Research Institute. Analytical results indicated by elemental
symbols were within ±0.4% of the theoretical values. Thin layer
chromatography (TLC) was performed on Analtech silica gel GF 0.25
mm plates. Flash column chromatography was performed with E. Merck
silica gel 60 (230–400 mesh). Yields are of purified compounds
and were not optimized. On the basis of NMR and combustion analysis
data, all final compounds reported in the manuscript are >95% pure.
General Amidation Procedures
Procedure A
To a solution of the
carboxylic acid (1
equiv, 1 mmol) in anhydrous n class="Chemical">CH2Cl2 (10 mL) was
added BOP-Cl (1 equiv), and the mixture was stirred at room temperature
for 2.5 h under nitrogen atmosphere. Triethylamine (3 equiv) and the
appropriate amine (1 equiv) were added, and the mixture was stirred
at room temperature overnight. The reaction mixture was concentrated
under reduced pressure, and the residue was partitioned between CHCl3 and water. The organic layer was separated, dried over anhydrous
sodium sulfate, filtered, and the filtrate was evaporated under reduced
pressure. The residue obtained was purified by column chromatography
over silica gel to obtain the desired product.
Procedure
B
To a solution of the carboxylic acid (1
equiv, 1 mmol) in n class="Chemical">acetonitrile (12 mL) was added HATU (1 equiv), and
the mixture was stirred at room temperature for 15 min. Triethylamine
(3 equiv) and the appropriate amine (1 equiv) were added, and the
mixture was stirred at room temperature overnight. The mixture was
concentrated under reduced pressure, and the residue obtained was
partitioned between CHCl3 and saturated aqueous sodium
bicarbonate. The organic layer was separated, dried over anhydrous
sodium sulfate, filtered, and the solvent was removed under reduced
pressure. The crude product thus obtained was purified by chromatography
over a column of silica gel to obtain the desired product.
To a
solution of imidazo[1,2-a]pyridine-2-carboxylic acid
(0.162 g, 1.0 mmol) in anhydrous n class="Chemical">CH2Cl2 (10
mL) was added 0.254 g (1.0 mmol) of BOP-Cl, and the mixture was stirred
at room temperature for 2.5 h under nitrogen atmosphere. Triethylamine
(0.42 mL, 3.0 mmol) and 4-(4-phenylpiperazin-1-yl)butan-1-amine (0.233
g, 1.0 mmol) were added, and the mixture was stirred at room temperature
overnight. The reaction mixture was concentrated under reduced pressure,
and the residue was partitioned between CHCl3 and water.
The organic layer was separated, dried over anhydrous sodium sulfate,
filtered, and the filtrate was evaporated under reduced pressure.
The residue obtained was purified by column chromatography over silica
gel (CHCl3–MeOH, 95:5) to obtain 0.164 g (44%) of
the desired product 6 as a colorless solid. Mp 169–171
°C. TLC R = 0.39
(CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.47–1.63 (m, 4H), 2.34 (t,
2H) 2.44–2.52 (m, 4H), 3.11 (t, 4H), 3.22–3.35 (m, 2H),
6.76 (td, 1H), 6.89 (dd, J = 8.6, 9.0 Hz, 2H), 6.97
(td, 1H), 7.19 (td, 2H), 7.32 (td, 1H), 7.55 (dd, J = 9.0, 9.0 Hz, 1H), 8.31–8.39 (m, 2H), 8.56 (dt, 1H). ESI
MS m/z 378 (M + H)+.
Anal. (C22H27N5O) C, H, N.
To a
solution of imidazo[1,2-a]pyridine-2-carboxylic acid
(0.08 g, 0.5 mmol) inn class="Chemical">acetonitrile (6 mL) was added HATU (0. 190 g,
0.5 mmol), and the mixture was stirred at room temperature for 15
min. Triethylamine (0.21 mL, 1.5 mmol) and 4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)butan-1-amine
(0.15 g, 0.5 mmol) were then added, and the mixture was stirred at
room temperature overnight. The mixture was concentrated under reduced
pressure, and the residue obtained was partitioned between CHCl3 and saturated aqueous sodium bicarbonate. The organic layer
was separated, dried over anhydrous sodium sulfate, filtered, and
the solvent was removed under reduced pressure. The crude product
thus obtained was purified by chromatography over a column of silica
gel (CHCl3–MeOH, 96:4) to obtain 0.065 g (29%) of
the desired product 9 as a colorless solid. Mp 144–146
°C. TLC R = 0.33
(CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.45–1.62 (m, 4H), 2.35 (t,
2H), 2.47–2.51 (m, 4H), 3.22 (t, 4H), 3.27–3.34 (m,
2H), 6.96–6.99 (m, 1H), 7.04 (d, J = 7.9 Hz,
1H), 7.14 (bs, 1H), 7.19 (dd, J = 8.2, 8.6 Hz, 1H),
7.31–7.34 (m, 1H), 7.37–7.42 (m, 1H), 7.56 (dd, J = 9.0, 9.0 Hz, 1H), 8.30–8.40 (m, 2H), 8.57 (dt,
1H). ESI MS m/z 446 (M + H)+. Anal. (C23H26F3N5O·0.25H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid and n class="Chemical">3-(4-(4-aminobutyl)piperazin-1-yl)-5-(trifluoromethyl)benzonitrile
according to general procedure A. The crude product was purified by
column chromatography over silica gel (CHCl3–MeOH,
97:3) to obtain compound 10 as a colorless solid in 29%
yield. Mp 124–126 °C. TLC R = 0.40 (CHCl3–MeOH, 92.5:5). 1H NMR (DMSO-d6) δ 1.45–1.66
(m, 4H), 2.35 (t, 2H), 2.44–2.56 (m, 4H), 3.18–3.35
(m, 6H), 6.93–7.02 (m, 1H), 7.31–7.32 (m, 1H), 7.46
(s, 1H), 7.50 (s, 1H), 7.55–7.62 (m, 1H), 7.64 (s, 1H), 8.34
(s, 1H), 8.43 (t, 1H), 8.54–8.62 (m, 1H). ESI MS m/z 471 (M + H)+. Anal. (C24H25F3N6O·0.25H2O)
C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid and n class="Chemical">4-(4-(3,5-di-tert-butylphenyl)piperazin-1-yl)butan-1-amine
according to general procedure B. The crude product was purified by
column chromatography over silica gel (CHCl3–MeOH,
96:4) to obtain compound 11 as a colorless solid in 28%
yield. TLC R = 0.37
(CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 1.25 (s, 18H), 1.45–1.64 (bs,
4H), 2.32–2.72 (bs, 6H), 3.10–3.22 (bs, 4H), 3.28–3.38
(m, 2H), 6.72 (d, J = 1.5 Hz, 2H), 6.86 (t, 1H),
6.97 (td, 1H), 7.32 (td, 1H), 7.55 (dt, 1H), 8.34 (d, J = 0.8 Hz, 1H), 8.39 (t, 1H), 8.52 (dt, 1H). ESI MS m/z 490 (M + H)+. Anal. (C30H43N5O·H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid (0.65 g, 4.0 mmol) and 4-(4-(quinazolin-4-yl)piperazin-1-yl)n class="Chemical">butan-1-amine
(1.136 g, 4.0 mmol) in the presence of BOP-Cl (1.38 g, 5.42 mmol)
and triethylamine (1.68 mL, 12 mmol) in CH2Cl2 (20 mL) according to general procedure A. The crude product was
purified by column chromatography over silica gel (EtOAc–MeOH,
8:1) to obtain 0.12 g (7%) of compound 17 as a pale yellow
solid. Mp 130–134 °C. TLC R = 0.10 (CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 1.66–1.78 (m, 4H), 2.45–2.54
(m, 2H), 2.64–2.74 (m, 4H), 3.42–3.55 (m, 2H), 3.82–3.94
(m, 4H), 6.91–7.00 (m, 1H), 7.32–7.40 (m, 1H), 7.50–7.61
(m, 2H), 7.76–7.86 (m, 2H), 7.97–8.05 (m, 2H), 8.27
(d, J = 0.76 Hz, 1H), 8.43–8.50 (m, 1H), 8.56
(s, 1H). ESI MS m/z 430 (M + H)+. Anal. (C24H27N7O) C, H,
N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid and n class="Chemical">4-(4-(2-(tert-butyl)quinazolin-4-yl)piperazin-1-yl)butan-1-amine
according to general procedure A. The crude product was purified by
chromatography over a column of silica gel using CHCl3–MeOH,
92.5:7.5, as the eluent to obtain compound 18 as a pale
yellow solid in 18% yield. Mp 59–61 °C. TLC R = 0.44 (CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.50 (s,
9H), 1.52–1.68 (m, 2H), 1.77–1.88 (m, 2H), 3.17 (bs,
2H), 3.24–3.41 (m, 4H), 3.64 (d, J = 11.1
Hz, 2H), 4.11 (bs, 2H), 4.85 (bs, 2H), 7.32 (t, 1H), 7.64–7.82
(m, 3H), 8.04 (t, 1H), 8.20 (d, J = 8.4 Hz, 1H),
8.45 (d, J = 8.2 Hz, 1H), 8.75 (s, 1H), 8.86 (d, J = 6.8 Hz, 1H), 9.10 (s, 1H). ESI MS m/z 486 (M + H)+. Anal. (C28H35N7O·0.25H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid (0.102 g, 0.63 mmol) and 4-(4-(6-methyl-2-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)n class="Chemical">butan-1-amine
(0.20 g, 0.63 mmol) in the presence of HATU (0.24 g, 0.63 mmol) and
triethylamine (0.26 mL, 0.189 mmol) in acetonitrile (7.0 mL) according
to general procedure B. The crude product was purified by column chromatography
over silica gel (CHCl3–MeOH, 95:5) to obtain 0.076
g (26%) of compound 20 as a colorless solid. Mp 118–120
°C. TLC R = 0.60 (CHCl3–MeOH, 90:10). 1H NMR (DMSO-d6) δ 1.43–1.61 (m, 4H), 2.33 (t, 2H), 2.37 (s,
3H), 2.42 (t, 4H), 3.26–3.34 (m, 2H), 3.65 (bs, 4H), 6.96 (s,
1H), 6.97–6.99 (m, 1H), 7.31–7.36 (m, 1H), 7.56 (m,
1H), 8.31 (d, J = 0.8 Hz, 1H), 8.37 (t, 1H), 8.55–8.59
(m, 1H). ESI MS m/z 462 (M + H)+. Anal. (C22H26 F3N7O·0.25H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid and n class="Chemical">4-(4-(6-(tert-butyl)-2-methylpyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(0.305 g, 1.0 mmol) according to general procedure B. The crude product
was purified by column chromatography over silica gel (CHCl3–MeOH, 96:4) to obtain compound 21 as a colorless
viscous oil in 35% yield. TLC R = 0.32
(CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 1.21 (s, 9H), 1.45–1.64 (m,
4H), 2.22–2.35 (m, 2H), 2.34 (s, 3H), 2.46 (t, 4H), 3.28–3.34
(m, 2H), 3.58 (t, 4H), 6.47 (s, 1H), 6.96 (td, 1H), 7.33 (td, 1H),
7.58 (dd, J = 8.6, 8.2 Hz, 1H), 8.37 (t, 2H), 8.56
(dt, 1H). ESI MS m/z 450 (M + H)+. Anal. (C25H35N7O·75H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid and n class="Chemical">4-(4-(2-(tert-butyl)-6-methylpyrimidin-4-yl)piperazin-1-yl)butan-1-amine
according to general procedure B. The crude product was purified by
column chromatography over silica gel (CHCl3–MeOH,
96:4) to obtain compound 22 as a light brown viscous
oil in 20% yield. TLC R = 0.17 (CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 1.25 (s, 9H), 1.44–1.60 (m, 4H), 2.23
(s, 3H), 2.23–2.46 (m, 6H), 3.24–3.44 (m, 2H), 3.58
(bs, 4H), 6.47 (s, 1H), 6.97 (td, 1H), 7.37 (td, 1H), 7.56 (dd, J = 9.4, 9.0 Hz, 1H), 8.34 (s, 1H), 8.37 (t, 1H), 8.56 (dt,
1H). ESI MS m/z 450 (M + H)+. Anal. (C25H35N7O·H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid and n class="Chemical">4-(4-(2,6-di-tert-butylpyrimidin-4-yl)piperazin-1-yl)butan-1-amine
according to general procedure B. The crude product was purified by
column chromatography over silica gel (CHCl3–MeOH,
92:8) to obtain compound 23 as an off-white solid in
30% yield. Mp 90–94 °C. TLC R = 0.54 (CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 1.24 (s, 9H), 1.27 (s, 9H), 1.45–1.60
(m, 4H), 2.33 (t, 2H), 2.41 (t, 4H), 3.28–3.34 (m, 2H), 3.60
(t, 4H), 6.45 (s, 1H), 6.96 (td, 1H), 7.33 (td, 1H), 7.58 (dd, J = 9.4, 9.0 Hz, 1H), 8.34 (d, J = 0.8
Hz, 1H), 8.38 (t, 1H) 8.57 (dt, 1H). ESI MS m/z 492 (M + H)+. Anal. (C28H41N7O·0.75H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyridine-2-carboxylic
acid and n class="Chemical">4-(4-(2-(tert-butyl)-6-cyclopropylpyrimidin-4-yl)piperazin-1-yl)butan-1-amine
according to general procedure B. The crude product was purified by
column chromatography over silica gel (CHCl3–MeOH,
92:8) to obtain compound 24 as a colorless solid in 36%
yield. Mp 34–37 °C. TLC R = 0.19 (CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 0.81–0.96 (2m, 4H), 1.28 (s,
9H), 1.45–1.60 (m, 4H), 1.84–1.90 (m, 1H), 2.32 (t,
2H), 2.40 (t, 4H), 3.27–3.33 (m, 2H), 3.58 (t, 4H), 6.49 (s,
1H), 6.97 (td, 1H), 7.32 (td, 1H), 7.56 (dd, J =
9.4, 9.0 Hz, 1H), 8.37 (t, 2H), 8.56 (dt, 1H). ESI MS m/z 476 (M + H)+. Anal. (C27H37N7O·H2O) C, H, N.
Imidazo[1,2-a]pyridine-2-carboxylic acid (5.89 g, 36.6 mmol) was reacted
with n class="Chemical">4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(13.14 g, 36.6 mmol) in the presence of BOP-Cl (9.3 g, 36.6 mmol)
and triethylamine (15.0 mL) in CH2Cl2 (350 mL)
as described in general procedure A. The crude product was purified
by column chromatography over silica gel (EtOAc–hexane, 2:1)
to yield 6.0 g (32%) of the desired product. A solution of the free
base in ether was treated with 1.0 M solution of HCl in Et2O to obtain the dihydrochloride salt as a light brown solid. Mp 253–255
°C. TLC R = 0.29 (CHCl3–MeOH, 90:10). 1H NMR (DMSO-d6) δ 1.31 (s, 9H), 1.62–1.69 (m, 2H), 1.80–1.88
(m, 2H), 2.88–3.19 (broad hump, 1H), 3.10–3.17 (m, 2H),
3.37–3.33 (q, 2H), 3.52–3.60 (m, 4H), 4.32–4.86
(b, 2H), 7.11 (s, 1H), 7.13–7.18 (m, 1H), 7.50–7.57
(t, 1H), 7.65 (dd, J = 9.2, 9.0 Hz, 1H), 8.53 (s,
1H), 8.57 (bs, 1H), 8.68 (d, J = 6.9 Hz, 1H), 11.0–11.8
(b, 1H). ESI MS m/z 504 (M + H)+. Anal. (C25H32F3N7O·2HCl·0.25H2O) Calcd: C, 51.68; H, 5.98; N,
16.87.Cl, 12.20. Found: C, 51.64; H, 6.00; N, 16.89, Cl, 11.97.
This compound
was prepared from indole-2-carboxylic acid and n class="Chemical">4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
according to general procedure B. The crude product was purified by
column chromatography over silica gel (CHCl3–MeOH,
92:8) to obtain compound 26 as a colorless solid in 35%
yield. Mp 70–72 °C. TLC R = 0.54 (CHCl3–MeOH, 92.5:7.5). 1H NMR
(DMSO-d6) δ 1.28 (s, 9H), 1.43–1.63
(m, 4H), 2.35 (t, 2H), 2.42 (t, 4H), 3.26–3.34 (m, 2H), 3.71
(bs, 4H), 7.00–7.02 (m, 2H), 7.16–7.19 (m, 2H), 7.41
(d, J = 8.2 Hz, 1H), 7.59 (d, J =
8.3 Hz, 1H), 8.44 (t, 1H), 7.78 (t, 1H). ESI MS m/z 503 (M + H)+. Anal. (C26H33 F3N6O·0.25H2O) C, H, N.
This compound was prepared from benzimidazole-2-carboxylic
acid and n class="Chemical">4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
according to general procedure A. The crude product was purified by
column chromatography over silica gel (CHCl3–MeOH,
92:8) to obtain compound 27 as a colorless solid in 18%
yield. Mp 80–72 °C. TLC R = 0.56 (CHCl3–MeOH, 90:10). 1H NMR
(DMSO-d6) δ 1.28 (s, 9H), 1.44–1.66
(m, 4H), 2.36 (t, 2H), 2.43 (t, 4H), 3.28–3.48 (m, 2H), 3.64–3.68
(m, 4H), 7.03 (s, 1H), 7.22–7.36 (m, 2H), 7.52 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 7.5 Hz, 1H),
8.97 (t, 1H), 13.12 (s, 1H). ESI MS m/z 504 (M + H)+. Anal. (C25H32F3N7O) C, H, N.
This
compound was prepared from thieno[2,3-b]pyridine-2-carboxylic
acid (0.10 g, 0.56 mmol) and 4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(0.20 g, 0.56 mmol) in the presence of n class="Chemical">HATU (0.211 g, 0.56 mmol) and
triethylamine (0.23 mL, 1.68 mmol) in acetonitrile (7.0 mL) according
to general procedure B. The crude product was purified by column chromatography
over silica gel (CHCl3–MeOH, 92:8) to yield 0.125
g (43%) of compound 28 as a colorless solid. Mp 122–124
°C. TLC R = 0.49 (CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.28 (s, 9H), 1.44–1.62 (m, 4H), 2.34
(t, 2H), 2.45 (t, 4H), 3.33 (q, 2H), 3.71 (bs, 4H), 7.03 (s, 1H),
7.49 (q, 1H), 8.08 (s, 1H), 8.40 (dd, J = 1.6, 1.6
Hz, 1H), 8.65 (dd, J = 1.5, 1.5 Hz, 1H), 8.84 (t,
1H). ESI MS m/z 521 (M + H)+. Anal. (C25H31F3N6OS·H2O) C, H, N.
This
compound was prepared from imidazo[1,2-a]pyrazine-2-carboxylic
acid (0.50 g, 3.07 mmol) and 4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(0.75 g, 2.09 mmol) in the presence of n class="Gene">BOP-Cl (1.1 g, 4.32 mmol) and
triethylamine (3.0 mL, 21.52 mmol) in CH2Cl2 (20 mL) according to general procedure A. The crude product was
purified by column chromatography over silica gel (CHCl3–MeOH, 92:8) to obtain 0.412 g (39%) of compound 30 as an off-white solid. Mp 200–202 °C. TLC R = 0.25 (CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 1.28 (s,
9H), 1.47–1.67 (m, 4H), 2.32 (t, J = 5.96
Hz, 2H), 2.44–2.48 (m, 4H), 3.21–3.36 (m, 2H), 3.71
(bs, 4H), 7.04 (s, 1H), 7.95 (d, J = 4.66 Hz, 1H),
8.50 (s, 1H), 8.61–8.66 (m, 2H), 9.12 (d, J = 0.77 Hz, 1H). ESI MS m/z 505
(M + H)+. Anal. (C24H31F3N8O·2HCl·0.75H2O) C, H, N.
This
compound was prepared from 3-aminothieno[2,3-b]pyridine-2-carboxylic
acid (0.109 g, 0.56 mmol) and 4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(0.20 g, 0.56 mmol) in the presence of n class="Chemical">HATU (0.213 g, 0.56 mmol) and
triethylamine (0.23 mL, 1.68 mmol) in acetonitrile (10 mL) according
to general procedure B. The crude product was purified by column chromatography
over silica gel (CHCl3–MeOH, 96:4) to obtain 0.09
g (30%) of compound 32 as an off-white solid. Mp 138–140
°C. TLC R = 0.46 (CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.28 (s, 9H), 1.42–1.62 (m, 4H), 2.33
(t, 2H), 2.43 (t, 4H), 3.24 (q, 2H), 3.71 (s, 4H), 7.03 (s, 1H), 7.14
(s, 2H), 7.44 (q, 1H), 7.71 (t, 1H), 8.4 (dd, J =
1.6, 1.6 Hz, 1H), 8.61 (dd, J = 1.5, 1.5 Hz, 1H).
ESI MS m/z 536 (M + H)+. Anal. (C25H32F3N7OS·0.25H2O) C, H, N.
To an ice cold solution of dimethylamine (15
mL, 2 M solution in n class="Chemical">THF, 30.0 mmol) was added acetic acid (3.65 mL)
followed by 2.20 mL of 37% aqueous formaldehyde solution (30.0 mmol).
Ethyl indole-2-carboxylate (3.0 g, 15.8 mmol) in methanol (150 mL)
was then added, and the resulting solution was heated under reflux
for 4 h. The mixture was concentrated to 20% of its volume in vacuo
and diluted with water (50 mL). The aqueous solution was washed with
CHCl3 (2 × 50 mL). The aqueous layer was separated,
chilled, and basified to pH 12 by addition of 20% aqueous NaOH. The
mixture was extracted with CH2Cl2 (3 ×
50 mL). The organic extract was dried with sodium sulfate and the
solvent was removed under reduced pressure to obtain ethyl 3-((dimethylamino)methyl)-1H-indole-2-carboxylate. Yield 2.8 g (72%). 1H
NMR (DMSO-d6) δ 1.34–1.38
(m, 3H), 2.16 (s, 6H), 3.89 (s, 2H), 4.31–4.37 (m, 2H), 7.05–7.42
(m, 4H), 11.59 (s, 1H). ESI MS m/z 247 (M + H)+.
Step 2
The above
ester (1.0 g, 4.06 mmol) was dissolved
in 20 mL of n class="Chemical">dioxane–water (90:10), treated with NaOH (0.8 g,
20.0 mmol), and the mixture was stirred at room temperature for 4
h. The reaction mixture was diluted with EtOAc (75 mL), and the organic
layer was separated, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to obtain 3-((dimethylamino)methyl)-1H-indole-2-carboxylic acid. Yield (0.58 g, 65%). ESI MS m/z 219 (M + H)+ The acid thus
obtained was used in the next step without further purification.
Step 3
The acid (0.091 g, 0.42 mmol) obtained above
was coupled with 4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(0.151 g, 0.42 mmol) in the presence of n class="Chemical">HATU (0.16 g, 0.42 mmol) and
triethylamine (0.18 mL, 1.26 mmol) in acetonitrile (10 mL) according
to general procedure B. The crude product was purified by column chromatography
over silica gel (CHCl3–MeOH, 97:3) to obtain 0.10
g (43%) of compound 33 as an off-white solid. Mp 174–176
°C. TLC R = 0.43 (CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.28 (s, 9H), 1.56–1.66 (m, 4H), 2.23
(s, 6H), 2.37 (t, 2H), 2.44 (t, 4H), 3.28–3.38 (m, 2H), 3.70
(bs, 4H), 7.03 (s, 1H), 7.04–7.08 (m, 2H), 7.17 (td, 1H), 7.40
(d, 1H, J = 8.2 Hz), 7.65 (d, J = 8.2 Hz, 2H), 10.40 (s, 1H), 11.58 (s, 1H). ESI MS m/z 560 (M + H)+. Anal. (C29H40F3N7O) C, H, N.
This
compound was prepared from 3-((dimethylamino)methyl)imidazo[1,2-a]pyridine-2-carboxylic acid dihydrochloride (0.164 g, 0.56
mmol) and n class="Chemical">4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(0.20 g, 0.56 mmol) in the presence of HATU (0.213 g, 0.56 mmol) and
triethylamine (0.235 mL, 1.68 mmol) in acetonitrile (7.0 mL) according
to general procedure B. The crude product was purified by column chromatography
over silica gel (CHCl3–MeOH, 98:2) to obtain 0.119
g (38%) of compound 34 as a colorless oil. TLC R = 0.38 (CHCl3–MeOH, 95:5). 1H NMR (DMSO-d6) δ 1.28 (s,
9H), 1.41–1.64 (m, 4H), 2.16 (s, 6H), 2.34 (t, 2H), 2.43 (t,
4H), 3.22–3.36 (m, 2H), 3.72 (bs, 4H), 4.17 (s, 2H), 6.92–7.05
(m, 2H), 7.38 (t, 1H), 7.57 (d, J = 9.0 Hz, 1H),
8.38 (t, 1H), 8.44 (d, J = 6.7 Hz, 1H). ESI MS m/z 561 (M + H)+. Anal. (C28H39F3N8O·0.5H2O) C, H, N.
A solution of 2-amino-6-hydroxymethylpyridine
(5.0 g, 40.28 mmol) and n class="Chemical">ethyl 3-bromopyruvate (1.57 g, 80.56 mmol)
in ethanol (150 mL) was stirred with heating under reflux for 3 h.
The reaction mixture was cooled to room temperature and concentrated
under reduced pressure. The residue obtained was purified by chromatography
over a column of silica gel using hexane–EtOAc (75:25) to yield
6.2 g (70%) of ethyl 5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-carboxylate (45). ESI MS m/z 221 (M + H)+.
To a stirred solution of the above compound
(3.89 g, 17.66 mmol) and imidazole (3.5 g, 51.4 mmol) in n class="Chemical">DMF (12 mL)
was added chlorotriisopropylsilane (6.82 g, 35.4 mmol) dropwise, and
the reaction mixture was heated at 70 °C for 2 h. After cooling
to room temperature, the reaction mixture was diluted with EtOAc (200
mL) and washed with saturated aqueous sodium carbonate (150 mL). The
organic phase was washed with brine, dried over anhydrous sodium sulfate,
and concentrated under reduced pressure. The residue thus obtained
was purified by silica gel column using CH2Cl2–EtOAc (1:1) as the eluent to obtain 4.8 g (72%) of ethyl
5-(((triisopropylsilyl)oxy)methyl)imidazo[1,2-a]pyridine-2-carboxylate (46). ESI MS m/z 377 (M + H)+.
To a solution of the above silyl ether 4.8 g
(12.7 mmol) in 50 mL of n class="Chemical">methanol/THF/water (2:2:1) was added NaOH
(2.1 g). The mixture was stirred at room temperature for 2 h. The
mixture was neturalized by the addition of acetic acid, and the solvents
were removed under reduced pressure. The residue obtained was purified
over a column of silica using CH2Cl2–MeOH
(7:1) to give 3.6 g (81%) of 5-(((triisopropylsilyl)oxy)methyl)imidazo[1,2-a]pyridine-2-carboxylic acid (47). ESI MS m/z 349 (M + H)+.
Step 4
To a solution of the above acid (1.02 g, 2.92
mmol) in acetonitrile (15 mL) was added n class="Chemical">HATU (1.11 g, 2.92 mmol) and
Et3N (0.6 mL, 4.31 mmol). The mixture was stirred for 10
min, and 4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(1.05 g, 2.92 mmol) was added. The stirring was continued for 16 h
at room temperature. The reaction mixture was concentrated under reduced
pressure, and the residue was dissolved in CHCl3 and washed
with aqueous sodium bicarbonate. The CHCl3 extract was
dried over anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The crude product thus obtained was purified by
chromatography over a column of silica gel (CHCl3–MeOH
97:3) to obtain 0.45 g (22%) of N-(4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butyl)-5-(((triisopropylsilyl)oxy)methyl)imidazo[1,2-a]pyridine-2-carboxamide (48).
Step 5
To a solution of the above compound 0.45 g (0.65
mmol) in THF (20 mL) was added n class="Chemical">tetraethylammonium fluoride (0.146
g, 0.98 mmol), and the mixture was stirred at room temperature for
16 h. The mixture was then concentrated and partitioned between CHCl3 and water. The organic layer was separated, dried over anhydrous
sodium sulfate, filtered, and the solvent was removed under reduced
pressure. The residue thus obtained was purified by chromatography
over a column of silica gel (CHCl3–MeOH, 92:8) to
obtain 0.216 g (62%) of the desired product as a colorless oil. TLC R = 0.46 (CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.28 (s,
9H), 1.41–1.60 (m, 4H), 2.34 (t, 2H), 2.44 (t, 4H), 3.01–3.28
(m, 2H), 3.70 (bs, 4H), 4.78 (d, J = 7.8 Hz, 2H),
5.71–5.78 (m, 1H), 6.94 (dd, J = 6.6, 6.6
Hz, 1H), 7.03 (s, 1H), 7.30–7.40 (m, 1H), 7.53 (d, J = 9.0 Hz, 1H), 8.28 (s, 1H), 8.40 (t, 1H). ESI MS m/z 534 (M + H)+. Anal. (C26H34F3N7O2·75H2O) C, H, N.
A solution
of hydroxymethyl compound 35 (0.125 g, 0.235 mmol) and
triethylamine (0.1 mL, 0.702 mmol) inn class="Chemical">dichloromethane (5 mL) was cooled
in ice bath and treated dropwise with methanesulfonyl chloride (0.15
g, 1.3 mmol). The mixture was allowed to warm to room temperature
and stirred at room temperature for 1 h. To the mixture was then added
dimethylamine (0.25 mL, 1 M solution in THF), and the mixture was
stirred at room temperature for 6 h. Volatiles were removed under
reduced pressure, and the residue was partitioned between CHCl3 and saturated aqueous sodium bicarbonate. The organic layers
were separated and dried over anhydrous sodium sulfate. Filtration,
removal of the solvent under reduced pressure, and purification by
silica gel column chromatography (CHCl3–MeOH 97:3)
afforded 0.03 g (23%) of the desired product 36 as a
colorless solid. Mp 62–64 °C. TLC R = 0.64 (CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.28 (s, 9H),
1.44–1.61 (m, 4H), 2.21 (s, 6H), 2.34 (t, 2H), 2.43 (t, 4H),
3.09–3.35 (m, 2H), 3.72 (bs, 4H), 3.76 (s, 2H), 6.93 (d, J = 6.7 Hz, 1H), 7.03 (s, 1H), 7.26–7.34 (m, 1H),
7.55 (d, J = 9.0 Hz, 1H), 8.31 (s, 1H), 8.39 (t,
1H). ESI MS m/z 561 (M + H)+. Anal. (C28H39F3N8O·H2O) C, H, N.
This compound was prepared from 1-phenylpiperidine-4-carboxylic
acid and 4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
according to general procedure A. The crude product was purified by
column chron class="Disease">matography over silica gel (EtOAc–MeOH, 10:1) to
obtain compound 37 as a colorless solid in 26% yield. Mp 178–180 °C. TLC R = 0.30 (CHCl3–MeOH, 92.5:7.5). 1H NMR (DMSO-d6) δ 1.28 (s,
9H), 1.42–1.46 (m, 4H), 1.58–1.77 (m, 4H), 2.22–2.35
(m, 3H), 2.42 (t, 4H), 2.61–2.69 (m, 2H), 3.06 (dd, J = 11.7, 11.7 Hz, 2H), 3.66–3.72 (m, 6H), 6.71–6.77
(m, 1H), 6.91–6.94 (m, 2H), 7.04 (s, 1H), 7.16–7.22
(m, 2H), 7.79 (t, 1H). ESI MS m/z 547 (M + H)+. Anal. (C29H41F3N6O·25H2O) C, H, N.
This compound was prepared from trans-4-phenylcyclohexanecarboxylic acid and n class="Chemical">4-(4-(2-(tert-butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine
(0.359 g, 1.0 mmol) according to general procedure A. The crude product
was purified by column chromatography over silica gel (EtOAc–MeOH,
80:20) to obtain compound 39 as a colorless solid in
30% yield. Mp 137–139 °C. TLC R = 0.69 (CHCl3–MeOH, 92.5:7.5). 1H NMR
(DMSO-d6) δ 1.28 (s, 9H), 1.37–1.55
(m, 8H), 1.78–1.86 (m, 4H), 2.11–2.19 (m, 1H), 2.31
(t, 2H), 2.42 (t, 4H), 2.49–2.51 (m, 1H), 3.06 (dd, J = 11.9, 11.7 Hz, 2H), 3.71 (bs, 4H), 7.04 (s, 1H), 7.13–7.31
(m, 5H), 7.71 (t, 1H). ESI MS m/z 546 (M + H)+. Anal. (C30H42F3N5O·25H2O) C, H, N.
A solution of 4-(tert-butyl)-6-chloro-2-methylpyrimidine
(7.98 g, 43.2 mmol) in absolute n class="Chemical">ethanol (100 mL) was added dropwise
to a boiling solution of piperazine (18.46 g, 215 mmol) in ethanol
(175 mL) over a period of 2 h. The reaction mixture was refluxed for
12 h, cooled to room temperature, and the solvent was removed under
reduced pressure. The residue was treated with ice-cold water (1 L)
and stirred for 15 min. After the mixture was allowed to stand for
an hour, the solid crystalline product obtained was collected by filtration
and dried under reduced pressure over P2O5 to
obtain 9.09 g (90%) of 4-(tert-butyl)-2-methyl-6-(piperazin-1-yl)pyrimidine.
ESI MS m/z 235 (M + H)+.
A mixture of the above piperazine (7.5 g, 32.0
mmol), n class="Chemical">N-(4-bromobutyl)phthalimide (9.03 g, 32.0
mmol), and potassium carbonate (5.30 g, 38.35 mmol) in acetonitrile
(200 mL) was stirred at room temperature overnight. The reaction mixture
was filtered, and the filtrate was concentrated under reduced pressure.
The residue thus obtained was purified by flash chromatography over
a column of silica gel using CHCl3–MeOH 98:2 as
the eluent to obtain 11.5 g (82%) of 2-(4-(4-(6-(tert-butyl)-2-methylpyrimidin-4-yl)piperazin-1-yl)butyl)isoindoline-1,3-dione.
ESI MS m/z 436 (M + H)+.
A mixture of the above (11.5 g, 32.05 mmol)
and hydrazine hydrate (64%, 5.01 g, 64.10 mmol) in n class="Chemical">methanol (150 mL)
was heated under reflux for 2 h. The reaction mixture was cooled to
room temperature, and the volatiles were removed under reduced pressure.
The residue was dissolved in ether (150 mL), filtered, and the filtrate
was concentrated under reduced pressure. The residue obtained was
purified by chromatography over a column of silica using CHCl3–MeOH–NH4OH (85:13:2) as the eluent
to yield 7.6 g (94%) of the desired product. 1H NMR (DMSO-d6) δ 1.22 (s, 9H), 1.37–1.38 (m,
2H), 1.41–1.50 (m, 2H), 2.28 (t, J = 7.0 Hz,
2H), 2.39 (t, J = 5.1 Hz, 2H), 2.54 (t, J = 6.8 Hz, 2H), 3.57 (s, 4H), 6.47 (s, 1H). ESI MS m/z 306 (M + H)+.
This compound was prepared from 2-(tert-butyl)-4-chloroquinazoline using procedures analogous
to those described for compound 43a. Overall yield: 66%.
ESI MS m/z 342 (M + H)+.
This compound was prepared from 2-(tert-butyl)-4-chloroquinazoline using procedures analogous to those described
for compound 43a. Overall yield: 66%. ESI MS m/z 319 (M + H)+.
Binding Affinity
A filtration binding assay was used
to characterize the binding properties of the D2, D3, chimeric D2/D3,
and mutant n class="Chemical">dopamine receptors. Direct binding and competition curves
were performed using [125I]IABN with dopamine receptors
stably expressed in HEK 293 cells. Stably transfected cells were harvested
by centrifugation. The cell pellet was resuspended in cold (4 °C)
homogenization buffer (50 mM Tris-HCl, pH 7.4, with 10 mM EDTA, 150
mM NaCl) by vortexing and then homogenizing with a Polytron (Brinkmann
Instruments, Westbury, NY). The homogenate was centrifuged at 12 000g at 4 °C and the membrane pellet resuspended in buffer
and kept at −80 °C. Tissue homogenates (50 μL) were
suspended in 50 mM Tris-HCl/150 mM NaCl/10 mM EDTA buffer, pH 7.5,
and incubated with 50 μL of [125I]IABN and 50 μL
of test ligand at 37 °C for 60 min. Nonspecific binding was defined
using 2 μM (+)-butaclamol. For competition experiments, the
radioligand concentration was generally equal to the Kd value and the concentration of the competitive inhibitor
ranged over 5 orders of magnitude. Binding was terminated by addition
of cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH 7.5) and filtration
over a glass-fiber filter (Whatman no. 32, Piscataway, NJ). Filters
were washed, and the radioactivity was measured using a Packard γ
counter with an efficiency of 75%. The protein concentration of the
membranes was determined using a BCA reagent (Pierce, Rockford, IL)
and BSA as the protein standard.
Estimates of the Kd and maxin class="Gene">mum binding sites (Bmax)50 were obtained using unweighted linear regression analysis
of data. Data from competitive inhibition experiments were modeled
using nonlinear regression analysis to determine the concentration
of inhibitor that inhibits 50% of the specific binding of the radioligand
(IC50). Since transfected cells expressing receptor were
used for this study, competition curves were modeled for a single
site usingwhere B is the amount of
ligand bound to tissue, B0 is the amount
of ligand bound in the absence of competitive inhibitor, L is the concentration of the competitive inhibitor, Bns is the nonspecific binding of the radioligand (defined
using a high concentration of a structurally dissimilar competitive
inhibitor), and IC50 is the concentration of competitive
inhibitor that inhibits 50% of the total specific binding. Data from
competition dose–response curves were analyzed using Tablecurve
program (Jandel/Systat Software Inc., San Jose, CA). IC50 values were converted to equilibrium dissociation constants (Ki values).
Generation of Chimeric
and Mutant Receptors
Four methods
were utilized to construct the D2/D3 receptor chimeras used in these
studies. The methods for the preparation of these chimeric receptor
genes have been previously described.[37,68,69] The first method exploits a unique n class="Gene">Pst1 restriction site in the first intracellular loop (I1) that is common
to both the human D2 and human D3 receptor cDNAs. The second method
involved making chimeric D2/D3 receptors. A human D3 receptor cDNA
and a humanD2 receptor cDNA were cloned in tandem into the pIRESneo2
vector with a unique restriction site (Hpa1) located
between the two receptor cDNAs specifically within the junctions at
the helical TMS regions TMS IV and TMS V, using site directed mutagenesis
techniques (Quick-Change site-directed mutagenesis kit, Stratagene/Agilent
Technologies, Santa Clara, CA). Finally, the D2/D3E2 and D3/D2E2 receptor
loop chimeras were prepared using the Quick Change kit strategy with
synthetic oligonucleotides encoding the E2 loop with the appropriate
5′ and 3′ flanking region. The authenticity of the chimeric
receptor was verified by DNA sequencing, and the expression of the
receptor construct in HEK 293 cells was verified by radioligand binding
using [125I]IABN.
Mitogenesis Assays
Agonist stimulation of D2 or D3
dopamine receptors leads to an increase in mitogenic activity.[54] These assays are based onn class="Chemical">[3H]thymidine
uptake by cells that are proliferating. CHO cells expressing humanD2 or D3 receptors (CHOp-D2 or CHOp-D3) were maintained in α-MEM
with 10% FBS, 0.05% pen–strep, and 200 μg/mL of G418.
To measure stimulation of mitogenesis (agonist assay) or inhibition
of quinpirole stimulation of mitogenesis (antagonist assay), CHOp-D2
or CHOp-D3 cells were seeded in a 96-well plate at a concentration
of 5000 cells/well. The cells were incubated at 37 °C in α-MEM
with 10% FBS. After 48–72 h, the cells were rinsed twice with
serum-free α-MEM and incubated for 24 h at 37 °C. Serial
dilutions of test compounds were made by the Biomek robotics system
in serum-free α-MEM. In the functional assay for agonists, the
medium was removed and replaced with 100 μL of test compound
in serum-free α-MEM. In the antagonist assay, the serial dilution
of the putative antagonist test compound was added in 90 μL
(1.1× of final concentration) and 300 nM quinpirole (30 nM final)
was added in 10 μL. After another 24 h incubation at 37 °C
of CHOp-D2 cells or 16 h incubation of CHOp-D3 cells, 0.25 μCi
[3H]thymidine in α-MEM supplemented with 10% FBS
was added to each well and the plates were further incubated for 2
h at 37 °C. The cells were trypsinized by addition of 10×
trypsin solution (1% trypsin in calcium–magnesium-free phosphate-buffered
saline), and the plates were filtered and counted as usual. Quinpirole
was run as an internal control, and dopamine was included for comparative
purposes. Butaclamol was run as standard antagonist.
Data Analysis
Data were analyzed using GraphPad Prism,
and agonist potency was expressed as EC50 values or % stimulation.
Antagonist potency was expressed as IC50 values. In these
assays, based on the % maximum stimulation as compared to the standard
agonist n class="Chemical">quinpirole (100%), the compounds were classified into following
categories: ≥90% full agonist; 70–90% partial to full
agonist; <70% partial agonist; 0–20% potential antagonist.
Whole Cell Adenylyl Cyclase Assay
Whole cell cyclic
AMP accumulation was measured by an adaptation of the method of Shimizu
and co-workers.[70] Transfected HEK 293 cells
were treated with serum-free medium containing n class="Chemical">[2,8-3H]adenine,
and cells were incubated at 37 °C for 75 min. The medium was
replaced with serum-free medium containing 0.1 mM 3-isobutyl-1-methylxanthine
(Sigma-Aldrich, St. Louis, MO). Forskolin (100 μM final) was
added in the presence or absence of test drug to a total volume of
500 μL and incubated at 37 °C for 20 min. The reaction
was stopped by addition of 500 μL of 10% trichloroacetic acid
and 1 mM cyclic AMP. After centrifugation, the supernatants were fractionated
using Dowex AG1-X8 and neutral alumina to separate the [3H]ATP and the [3H]cAMP. Individual samples were corrected
for column recovery by monitoring the recovery of the cyclic AMP using
spectrophotometric analysis at OD 259 nm. The percent conversion of
[3H]ATP into [3H]cAMP was then calculated as
the percent of inhibition of the [3H]cAMP accumulation
relative to the assay performed in the absence of a test compound,
minus basal activity. The classic D2-like dopamine receptor antagonist
and agonist (haloperidol and quinpirole, respectively) were included
in the assay design as reference compounds. The percent maximum response
is the value for the inhibition normalized to the value obtained for
the full agonist quinpirole. Values are reported as the mean values
± SEM. The concentrations for all test compounds was ≥10×
the Ki value of the compound at human
D2 or D3 dopamine receptors, obtained from competitive radioligand
binding experiments.[69]
β-Arrestin
Recruitment Assays
Effect of compounds
on agonist induced recruitment of β-arrestin by D3R and D2LR was measured using DiscoverRx PathHunter eXpress kits. Briefly,
n class="CellLine">U2OS DRD3 (DiscoveRx no. 93-0591E3) and CHO-K1 DRD2L (DiscoveRx no.
93-0579E2) cells were seeded at a density of 2500 cells/well in 384-well
white, clear-bottom plates (Corning no. 3707) with PathHunter Cell
Plating Reagents, CP0 (DiscoveRx no. 93-0563R0) and CP2 (DiscoveRx
no. 93-0563R2), respectively, and incubated at 37 °C for 48 h.
For the determination of EC80 values of standard agonists,
the cells were treated with multiple concentrations (starting from
190 nM, 1:3 dilution, 12 dose points) of (+)-PD128907 (DiscoveRx no.
92-1163) for D3R and Pergolide (DiscoveRx no. 92-1162) for D2LR. After incubation at 37 °C for 90 min, the DiscoveRx
detection reagent was added, the plates were incubated at room temperature
for 60 min, and the luminescence was measured on a Synergy 4 plate
reader (BioTek). The data were plotted, and the agonist EC80 values were calculated using GraphPad Prism software. For antagonist
activity, the cells were plated as described above and treated with
multiple concentrations (starting from 500 nM, half log dilution,
10 dose points) of the test compounds in plating reagent with 0.1%
DMSO. Following a 30 min preincubation at 37 °C, the cells were
treated with an EC80 dose (8 nM PD128907 for D3R and 6
nM pergolide for D2R) of the agonist and incubated at 37 °C for
an additional 90 min. The DiscoveRx detection reagent was then added,
and the plates were incubated at room temperature for 60 min. Luminescence
was measured, and the IC50 values were calculated. For
determination of agonist or inverse agonist activity, the cells were
plated and incubated for 48 h as described above and treated with
multiple concentrations (starting from 5 μM, half log dilution,
11 dose points) of the test compounds, incubated at 37 °C for
120 min, treated with detection reagents, and the luminescence was
measured. The activity was calculated as percent stimulation or inhibition
of the basal luminescence in the absence of the inhibitors, as the
EC50 or IC50 values of the compounds were greater
than the highest tested concentration
of 5 μM. All of the compounds were tested in the counterscreen
for inhibition of β-galactosidase enzyme using the EFC BlockDetect
kit (DiscoveRx no. 92-0004) in dose response mode and were found to
be devoid of any significant inhibitory activity at the highest tested
concentration of 5 μM.
[35S]GTPγS
Binding Assays
CHO cells
expressing n class="Species">human D2L and D3 receptors served as the source
for membrane fractions which were washed and resuspended with assay
buffer containing Mg2+, Na+, EGTA, and bovine
serum albumin as in our previous work.[55−57] The GTPγS binding
assays were performed in a final volume of 0.5 mL for CHO D2 and 1
mL for CHO D3 containing test compound or dopamine (1 mM for D2 cells,
and 100 μM for D3 cells) as indicator of binding plateau, [35S]GTPγS (0.11 nM for CHO D2 and 0.07 nM for CHO D3,
1250 Ci/mmol, PerkinElmer, Boston, MA 02118), and cell membrane suspension
(in assay buffer and GDP for final concentration of 3 μM for
CHO D2 and 6 μM for CHO D3). After preincubation with test compounds,
cell membranes and GDP were placed for 15 min at 30 °C in a shaking
water bath. [35S]GTPγS was added, and incubation
proceeded for an additional 45 min. Cell membranes were harvested
on Brandel GF/B filtermats with a 24-pin Brandel harvester (Biomedical
Research & Development Laboratories, Inc., Gaithersburg, MD).
Nonspecific binding of [35S]GTPγS measured in the
presence of 10 μM GTPγS was a very small fraction (5%
or less) of basal binding in the absence of test compounds and does
not impact the EC50 (concentration producing half-maximal
stimulation) of the test compound estimated by nonlinear logarithmic
fitting (logistics model) with OriginPro 7.0. The plateau binding
(maximal binding stimulation) with test compound was expressed as
percent of maximal binding observed with the full agonist dopamine
(% Emax). The test compounds 8, 25, 30, and 39 did
not stimulate GTPγS binding in three independent experiments.
The last three compounds at high micromolar concentrations showed
weak inverse agonist activity at D3R resulting in [35S]GTPγS
binding below baseline, but this was not further studied in the present
work. Antagonist activity was assessed by testing a fixed concentration
that by itself had little or no inverse agonist effect for its ability
to shift the concentration curve of an agonist stimulating [35S]GTPγS binding as described for opioid receptors by Sally
and co-workers.[59] The fixed concentrations
of test compound were as listed in the Results and
Discussion, and the agonist used was dopamine (0.001 μM
to 100 uM for CHO D2 and 0.1 nM to 10 μM for CHO D3). Ke is the functional Ki (equilibrium dissociation constant) of an antagonist and is calculated
according to the following equation: [test compound]/(EC50-2/EC50-1 – 1), where EC50-2 is the EC50 value in the presence of the test compound
and EC50-1 is the value in the absence of the test
compound.
Molecular Modeling and Ligand Docking
Refinement
of the Dopamine D3 Receptor Crystal Structure
The humann class="Chemical">D3R
crystal structure in complex with the antagonist eticlopride
at 2.89 Å resolution[39] (PDB code 3PBL) was refined using
Prime preparation and refinement tools of the Protein Preparation
Wizard implemented in the Schrödinger software package. After
the addition of hydrogens and detection of disulfide bonds the structure
was optimized by applying default parameters of the Impref utility
using the OPLS2001 force field. Ligand structures were prepared using
the LigPrep utility at neutral pH.
D2R Homology Model Development
A model of the D2 receptor
was developed based on the n class="Species">human D2 receptor sequence (UniProtKB accession
code P14416) and the D3 receptor crystal structure (PDB code 3PBL) as the homology
modeling template. The generated model includes transmembrane and
loop regions except for intracellular loop 3, which is missing in
the D3 crystal structure where it is replaced by T4-lysozyme. InsightII/Homology
modeling tools were used for initial homology model building. D2 loop
conformations in loop regions containing gap(s) within the D3/D2 sequence
alignment were generated in sets of 10. From these, D2 loop structures
that closely overlap with the corresponding D3 loops in the crystal
structure were selected. Loop side chains were adjusted using a built-in
rotamer library to avoid steric clashes with the rest of the structure.
Disulfide bridges involving any cysteine residues in loops were reproduced
in the D2 model structure except for a disulfide bond that is within
extracellular loop III, D3:Cys355-Cys358, which could not be reproduced
in D2 because the loop is shorter in the D2R than in the D3R. The
sequence identity between our D2 model and the D3 crystal structure
is 71.5% (including loops). In order to relax the obtained D2 homology
model structure, refinement tools of the Protein Preparation Wizard
were applied. The structure was optimized in the OPLS2001 force field
using default parameters of the Impref utility, analogous to the refinement
of the D3 receptor crystal structure. The root-mean-square deviation
of α-carbon atoms between the refined D2R model and D3R crystal
structure was 0.7.
InducedFit Ligand Docking
InducedFit
docking combines
Glide docking with Prime structural refinement tools to account for
side chain flexibility in the ligand binding site.[71] The center of the docking grid was defined as the centroid
of the cocrystallized eticlopride; the enclosing box size was determined
automatically. InducedFit settings were at default values except for
removing the side chain of a Tyr (D3:Tyr373, D2:387) in the initial
step of the protein preparation constrained refinement in order to
facilitate the initial docking of ligands longer than eticlopride.
In the InducedFit workflow this step was followed by an initial Glide
docking run using softened potentials (van der Waals scaling of 0.70
for the receptor and 0.50 for ligands). The obtained structures were
then refined using Prime side chain optimization of residues within
5 Å from docked ligand poses. The derived “induced-fit”
receptor structures were then utilized for the final step of Glide
re-docking with default parameters, applied to structures within 30
kcal/mol of the lowest energy structures. The same InducedFit protocols
were applied for ligand docking at both D3 and D2 receptor structures.
Authors: Ellen Y T Chien; Wei Liu; Qiang Zhao; Vsevolod Katritch; Gye Won Han; Michael A Hanson; Lei Shi; Amy Hauck Newman; Jonathan A Javitch; Vadim Cherezov; Raymond C Stevens Journal: Science Date: 2010-11-19 Impact factor: 47.728
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Authors: Carsten Hocke; Olaf Prante; Stefan Löber; Harald Hübner; Peter Gmeiner; Torsten Kuwert Journal: Bioorg Med Chem Lett Date: 2004-08-02 Impact factor: 2.823
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Authors: Sarah E Williford; Catherine J Libby; Adetokunbo Ayokanmbi; Arphaxad Otamias; Juan J Gordillo; Emily R Gordon; Sara J Cooper; Matthew Redmann; Yanjie Li; Corinne Griguer; Jianhua Zhang; Marek Napierala; Subramaniam Ananthan; Anita B Hjelmeland Journal: PLoS One Date: 2021-05-04 Impact factor: 3.240
Figure 1
Figure 2
Scheme 1
Scheme 2
Figure 3
Figure 4
Figure 5
Figure 6