Design and characterization of optimized adenosine A2A/A 1 receptor antagonists for the treatment of Parkinson's disease

Article abstract of DOI:10.1021/jm201640m

The design and characterization of two, dual adenosine A _2A/A₁ receptor antagonists in several animal models of Parkinson's disease is described. Compound 1 was previously reported as a potential treatment for Parkinson's disease. Fu

Full text of DOI:10.1021/jm201640m

Article
pubs.acs.org/jmc
Design and Characterization of Optimized Adenosine A2A/A1
Receptor Antagonists for the Treatment of Parkinson's Disease
Brian C. Shook,* Stefanie Rassnick, Nathaniel Wallace, Jeffrey Crooke, Mark Ault, Devraj Chakravarty,
J. Kent Barbay, Aihua Wang, Mark T. Powell, Kristi Leonard, Vernon Alford, Robert H. Scannevin,
Karen Carroll, Lisa Lampron, Lori Westover, Heng-Keang Lim, Ronald Russell, Shawn Branum,
Kenneth M. Wells, Sandra Damon, Scott Youells, Xun Li, Derek A. Beauchamp, Kenneth Rhodes,
and Paul F. Jackson
Janssen Research and Development, L.L.C., Welsh and McKean Roads, P.O. Box 776, Spring House, Pennsylvania 19477, United
States
ABSTRACT: The design and characterization of two, dual
adenosine A2A/A1 receptor antagonists in several animal models
of Parkinson's disease is described. Compound 1 was previously
reported as a potential treatment for Parkinson's disease. Further
characterization of 1 revealed that it was metabolized to reactive
intermediates that caused the genotoxicity of 1 in the Ames and
mouse lymphoma L51784 assays. The identification of the metabolites enabled the preparation of two optimized compounds 13
and 14 that were devoid of the metabolic liabilities associated with 1. Compounds 13 and 14 are potent dual A2A/A1 receptor
antagonists that have excellent activity, after oral administration, across a number of animal models of Parkinson's disease
including mouse and rat models of haloperidol-induced catalepsy, mouse and rat models of reserpine-induced akinesia, and the
rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation.
targeted therapies work well to address the PD-related motor
disturbances, they all produce undesirable side effects
(dyskinesia, hallucinations, and on−off effects) that become
more severe and problematic with continued treatment. The
aforementioned therapies typically show reduced efficacy as
motor functions deteriorate and the disease progresses.
Moreover, these treatments do not alter disease progression
and do not address the comorbidities associated with PD
including mood, postural instability, or cognitive disturbances.
These comorbidities stem from progressive neurodeneration in
non-DA brain systems and therefore are not amenable to
treatment with agents targeting DA signaling.
The limitations of DA replacement agents initiated non-DA-
based approaches for the treatment of PD. One attractive
nondopaminergic strategy that has been widely targeted is the
modulation of adenosine receptors.8 Adenosine is a purine
nucleotide produced by all metabolically active cells. Unlike
classical neurotransmitters in the brain, adenosine is not
packaged into vesicles or released from axon terminals in a
Ca2+-dependent manner. Rather, extracellular adenosine in
brain is derived from degradation of other purine nucleotides
and from intracellular adenosine released via transmembrane
transport.9 Adenosine acts as a neuromodulator that
coordinates responses to DA and other neurotransmitters in
areas of the brain that are responsible for motor function,
learning, and memory.9 Adenosine exerts a tonic inhibitory
INTRODUCTION
■
Parkinson's disease (PD) is a chronic, progressive neuro-
degenerative disease that is characterized by progressive
impairment in motor function that is often accompanied by
anxiety, depression, and cognitive impairment.1 The majority of
motor impairments of PD are caused by a gradual loss of
dopamine-producing neurons in the ventral midbrain and
concomitant loss of dopamine (DA) input to forebrain
(striatal) motor structures.2,3 The cellular mechanisms under-
lying the relatively selective loss of midbrain DA neurons
remains poorly understood. The loss of DA input to the
neostriatum leads to dysregulation of striatal function and the
classic motor symptoms of PD, such as resting tremor,
muscular rigidity, akinesia, and bradykinesia. Although the
loss of striatal DA precipitates many of the classical motor
symptoms of PD, chronic neurodegeneration and inflammatory
processes also affect other brain regions. Degenerative
processes in these regions are likely to underlie the depression,
cognitive impairment, and postural/gait instability in PD
patients.
Understanding the loss of dopaminergic cells led to the
majority of treatments that restore DA signaling and thereby
reduce the severity of the motor symptoms. DA replacement
therapy using levodopamine (L-DOPA), the precursor to DA,
remains the gold-standard treatment for PD. Other approaches
include inhibition of DA turnover using monoamine oxidase
type B (MAO-B) inhibitors,4 catechol O-methyl-transferase
(COMT) inhibitors,5 and inhibition of DA reuptake6 or direct
agonists7 of postsynaptic DA receptors. Although the DA-
Received: December 2, 2011
Published: January 12, 2012
© 2012 American Chemical Society
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Scheme 1. Metabolism of Compound 1
effect of DA receptor signaling in forebrain motor and limbic
structures. Adenosine is comprised of four distinct receptor
subtypes designated A1, A2A, A2B, and A3 belonging to the G
protein-coupled receptor superfamily.10 A1 and A3 receptors are
coupled to inhibitory G proteins, while A2A and A2B receptors
are coupled to stimulatory G proteins. On the basis of in situ
hybridization histochemistry and quantitative receptor auto-
radiography, the greatest densities of A2A receptor expression in
rodent brain are found in the neostriatum with lower levels in
olfactory, neocortical, and limbic system structures.11 On the
basis of [11C]-TMSX positron emission tomography (PET)
imaging in human subjects and on autoradiographic studies of
postmortem brain sections, the distribution of A2A receptors in
human brain closely matches the distribution in rodent brain,
with a high concentration in the neostriatum and much lower
levels in other brain regions.12
The loss of DA input into the neostriatum is a hallmark of
PD and underlies many of the cardinal motor symptoms of this
disorder. Also, it has been reported that overexpression of A2A
receptors correlates with the motor symptoms in PD.13
Adenosine A2A receptors colocalize and physically associate
with D2 receptors in the striatum.2,3 A2A and D2 receptors have
opposing effects on adenylate cyclase and cyclic adenosine
monophosphate (cAMP) production in cells, such that
activation of A2A receptors inhibits D2 receptor signaling.
Conversely, A2A receptor antagonists enhance D2-dependent
signaling as shown by induction of immediate early gene c-fos
expression in the striatopallidal pathway14 and facilitate other
D2-mediated responses. Of importance to PD, pharmacological
blockade of A2A receptors has shown dramatic beneficial effects
in preclinical animal models of PD,15 reversing haloperidol-
induced catalepsy, showing potentiation of DA-mediated
responses in DA-depleted [6-hydroxydopamine (6-OHDA)-
treated] animals and dramatic relief of parkinsonian symptoms
in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-trea-
ted nonhuman primates.16 These lines of evidence suggested
that A2A and D2 receptors play unique and integrative roles in
striatal function. A2A antagonists facilitate DA receptor signaling
and thereby normalize motor function in animal models of DA
dysregulation. As a result of these findings, the adenosine A2A
receptor has become a sought after target for treating PD.
Blockade of A2A signaling by selective A2A receptor antagonists
(e.g., KW-6002,17 istradefylline; SCH-420814, preladenant18)
was shown to be beneficial for not only enhancing the
therapeutic effects of L-DOPA but also reducing dyskinesia
from long-term L-DOPA treatment.19 Preladenant completed
phase II clinical trials for PD, and Merck (Schering-Plough) is
currently recruiting for several phase III trials.20
antagonists facilitate DA release in the striatum and, like A2A
receptors, potentiate DA-mediated responses. Antagonism of
both the A2A and the A1 would be synergisticinhibition of the
A1 receptor will facilitate DA release, while inhibition of the A2A
receptor will enhance postsynaptic responses to DA. Interest-
ingly, the A1 receptor is also concentrated in neocortical and
limbic system structures that are important for cognitive
function and has been implicated in antidepressant action.
Pharmacological inhibition of A1 receptors enhances neuro-
transmitter release in the hippocampus23 and enhances
performance in animal models of learning and memory.24
ASP-5854 is a dual A2A/A1 antagonist having binding affinities
of 1.8 and 9.0 nM for A2A and A1, respectively.25 ASP-5854 has
shown very good efficacy in a number of animal models of
PD,26 and it has shown positive effects in two models of
cognition, the scopolamine-induced memory deficits in the
mouse Y-maze and the rat passive avoidance test.27 In contrast,
the selective A2A antagonist KW-6002 had minimal or no effect
in the same models, suggesting that the A1 component could
provide added benefit to PD patients. These data suggest that a
dual A2A/A1 adenosine receptor antagonist may offer a unique
and exciting approach to treating both the motor and the
nonmotor disturbances of PD.
RESULTS AND DISCUSSION
■
We recently published the in vivo characterization of an
arylindenopyrimidine compound 1 as a dual A2A/A1 receptor
antagonist.28 Compound 1 was identified as an ideal dual A2A/
A1 receptor antagonist having excellent functional in vitro
activity (A2A Ki = 4.1 nM; A1 Ki = 17.0 nM) with good
pharmacokinetics (PK) and desirable brain levels. Compound 1
also showed excellent beneficial effects in several preclinical
animal models of PD across a number of different species
including mouse29 and rat30 models of neuroleptic-induced
catalepsy, mouse model of reserpine-induced akinesia,31 rat 6-
OHDA lesion model of drug-induced rotation,32 and MPTP-
treated nonhuman primate model.33
Further evaluation of 1 revealed that it was genotoxic in both
the Ames and the mouse lymphoma L5178Y assays following
metabolic activation.34 Incubation of 1 with Aroclor 1254-
induced rat liver S9 and human liver S9 produced at least two
reactive metabolites, the endocyclic iminium ion 2 and the aryl
aldehyde 3 (Scheme 1). It was concluded that one of these
reactive metabolites was responsible for the genotoxicity based
on several mechanistic studies. Compound 1 also showed some
adverse events in the 28 day GLP toxicity study in nonhuman
primates, which may be related to the reactive metabolites, but
that has not been confirmed. Knowing the metabolic soft spots
of the molecule allowed us to design analogues that might slow
down or eliminate the oxidative metabolism that occurred with
compound 1.
Quantitative autoradiographic analyses in rodent, postmor-
tem human brain sections,12 and [11C-MPDX] PET imaging in
human subjects21 show that adenosine A1 receptors are
concentrated in neocortex, hippocampus, and striatum. On
the basis of anatomical and in vivo microdialysis studies, A1
receptors appear to be localized presynaptically of DA axon
terminals where they inhibit DA release.22 A1 receptor
The primary site of metabolism was occurring on the
methylenes adjacent to the nitrogen inside the pyrrolidine ring.
The addition of methyl groups on one, not shown, or both
sides (4) of the nitrogen atom gave compounds that were
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devoid of the metabolic liability seen with 1. It appeared that
the metabolism was more influenced by the steric bulk of the
methyl groups rather than the electronics as one might expect
the methine carbons to be more susceptible to oxidation. Also,
the methyl groups were able to block metabolism at the
benzylic position. The dimethylpyrrolidine 4 maintained good
functional (human cell lines) in vitro activity for both A2A and
A1, but 4 had decreased in vivo activity in the haloperidol-
induced catalepsy model in mouse as compared to 1 (Table 1).
Next, we replaced the benzylic pyrrolidine with various
substituted pyridine compounds exemplified by 6−12. The
thought was to remove the electron-rich ring with the pyridine,
a system that would still increase solubility. Compounds 6−10
had very potent activity for A2A and A1, while maintaining
excellent activity in the mouse catalepsy model. A dose
response was run using 6 in the mouse catalepsy model and
showed that it had an ED50 of <1 mg/kg after oral
administration. Not surprisingly, the benzylic pyridines were
extensively metabolized at the benzylic position, which resulted
in poor plasma exposures and very short half-lives (<0.5 h).
The behavioral testing was generally conducted 1 h after dosing
the A2A/A1 antagonist, but a duration of action study showed
that 6 and 7 were active in the mouse catalepsy model at 4 and
2 h, respectively, after a 10 mg/kg po dose despite the very
short half-life. The suboptimal PK profiles and benzylic
metabolism prompted us to prepare the ketone 11 and the
gem-difluoro 12 to try and block metabolism, improve stability,
and increase in vivo exposure. The metabolism was eliminated,
and the compounds maintained good in vitro activity, but
neither 11 or 12 was active in the mouse catalepsy model after
a 10 mg/kg oral dose. Although no PK or brain levels were
determined for 11 or 12, the poor physical properties, that is,
solubility, may be responsible for low absorption and lack of
activity. Moving the pyridine ring out by one or two
methylenes results in decreased in vitro activity.
Table 1. Human Functional A2A and A1 in Vitro Activity and
Reversal of Neuroleptic-Induced Catalepsy in Mice
Converting the benzyl amines to their corresponding amides
gave compounds with excellent in vitro and in vivo activity.28c
Compound 13 is a representative compound from the amide
series that had optimal potency (ED50 = 0.4 mg/kg) in the
mouse catalepsy model as compared to other amides that were
prepared (Figure 1). Cyclic amines generally gave amide
compounds that were slightly more potent than acyclic amines.
The idea behind preparing the amides was 2-fold: first,
oxidizing the benzylic position would eliminate the oxidative
metabolism at that position; second, the electron-withdrawing
nature of the carbonyl would pull electron density out of the
nitrogen heterocycle, making the positions α to the nitrogen
less susceptible to oxidation. Introduction of a basic nitrogen,
like the one found in the piperazine, was critical to maintain
good solubility. Fortunately, this approach was successful as we
observed no metabolism on the methylenes adjacent to the
nitrogen of the amide nor did we see any metabolism on the
methylenes adjacent to the nitrogen in the 4-position.
Another strategy to eliminate the benzylic metabolism was to
prepare a variety of phenyl ethers from this series.28c
Converting the benzylic methylene to an oxygen atom
eliminated the metabolic potential at that position, similarly
to the carbonyl from the amides. Moving the solubilizing group,
nitrogen heterocycle, out a few atoms also eliminated the
oxidative metabolism from occurring inside the ring as seen
with compound 1, while maintaining good activity. Compound
14 represents the optimized compound out of a variety of
ethers that were synthesized. The morpholine ring gave optimal
in vitro and in vivo activity and had an ED50 of <0.1 mg/kg in
the mouse catalepsy model (Figure 1).
a
The in vivo activity of reversing haloperidol-induced catalepsy is
reported as an ED50, % reversal of at a single dose, or not active at a
single dose. Not active corresponds to <50% reversal of catalepsy.
Behavioral testing was conducted 1 h after dosing of A2A/A1
antagonist.
Haloperidol, a neuroleptic medication that inhibits D2
receptors,35 was used to induce catalepsy. In the rodent,
catalepsy is characterized as a loss of voluntary motion where
limbs uncharacteristically remain in placed positions, which
mimics the muscular stiffness seen in PD patients. Efficacy in
this model is defined as reversal of catalepsy, that is, moving
from the placed positions (refer to the Experimental Section for
a more detailed description of the mouse catalepsy model).
This was the primary in vivo model that was used to evaluate
compounds quickly by assessing oral and central nervous
system activity. Also, the model typically required very little
compound (5−10 mg), making it a robust tool with very fast
turnaround. The bicyclic pyrrolidine 5 was also devoid of the
metabolic liabilities and showed improved in vivo activity with
an ED50 of 1 mg/kg in mouse catalepsy, but this was still 5-fold
less potent than 1.
Chemistry. The synthesis of compounds 4−13 started with
the commercially available 6-methylindanone 15, which was
deprotonated with NaHMDS and reacted with CS2 followed by
MeI to afford the dithioketenacetal 16 (Scheme 2).28,36
Compound 16 reacts with guanidine to form an intermediate
amino pyrimidine, not shown, that is then oxidized to the
corresponding ketone by passing air through the solution to
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Figure 1. Reversal of neuroleptic-induced catalepsy in mice by compounds 13 and 14. In this behavioral model, catalepsy was induced in male Balb/
c mice by subcutaneous administration of haloperidol, a neuroleptic D2 receptor antagonist. Compound 1 was used as the positive control for this
model at 10 mg/kg, po. Compounds 13 (left panel) and 14 (right panel) were administered orally 30 min after haloperidol (1 mg/kg, sc). Behavioral
testing was conducted 1 h after dosing of 13 and 14. Each value represents average ( SEM) time in cataleptic position of n = 8−12 mice per
treatment group during a 60 s test session. Both compounds produced a dose-dependent reversal of haloperidol-induced catalepsy. The ED50 of 13
for inhibiting haloperidol-induced catalepsy in mice was 0.4 mg/kg, po, and the minimum effective dose is shown to be 1 mg/kg, po. The ED50 of 14
for inhibiting haloperidol-induced catalepsy in mice was <0.1 mg/kg, po, and the minimum effective dose is shown to be 0.1 mg/kg, po. Asterisks
indicate significant differences as compared with the haloperidol + vehicle control treatment group (***P < 0.001, Dunnett's test of multiple
comparisons).
give 17.37 The amino pyrimidine was protected using excess
(Boc)2O and 4-(dimethylamino)pyridine (DMAP) to give 18.
Efforts to mono-Boc compound 17 were unsuccessful as the di-
Boc compound was forming while starting material was present.
Also, the reaction was very sluggish without the addition of
DMAP. The phenyl substituent was installed via a modified
Suzuki reaction under Liebeskind type conditions using the
methylthioether as the coupling partner to afford 19.38 The
Suzuki reaction proceeded smoothly with the di-Boc protected
amino pyrimidine but could not be accomplished on the
unprotected amino pyrimidine 17 under the same conditions.
Compound 19 underwent benzyl bromination to afford the
corresponding bromide 20, which was a key intermediate that
was carried through paths 1−3 to prepare compounds 4−13.
Following path 1, the Boc groups were removed with TFA
followed by alkylation with 2,5-dimethylpyrrolidine or 7-
azabicyclo[2.2.1]heptane to give the desired target compounds
4 and 5, respectively. Following path 2, the benzyl bromide 20
was reacted, under Suzuki conditions, with several pyridylbor-
onic acids to provide their corresponding compounds 6−10.
The pyridyl compound 6 was oxidized using air, under basic
conditions in NMP, to give the diketone 11 (see insert Scheme
2). Reaction of 11 with DAST gave the gem-difluoro compound
12. Following path 3, the bromide 20 was converted to the
corresponding aldehyde, not shown, using NMO in the
presence of molecular sieves followed by further oxidation to
the acid 21 with KMnO4. The acid 21 was coupled with 1-
methylpiperazine using HATU followed by removal of the Boc
groups with TFA afforded the amide 13.
give the corresponding phenol 25. Attempts to deprotect using
BBr3 were completely unsuccessful. However, EtSNa in DMF
was also effective in removing the methoxy group but was not
as efficient as the LiCl. The phenol was then reacted with 4-(2-
chloroethyl)morpholine in the presence of t-BuOK to give the
ether 14.
PK. Compounds 13 and 14 were of high interest because of
their in vivo potency in the mouse catalepsy model, shown
above, and because of their different structural features. The PK
data of 13 and 14 were determined in mouse, rat, and monkey
and compared to the PK data of 1 (Table 2). Compounds 1,
13, and 14 had moderate to high clearances in all species. In
general, the compounds had good oral bioavailability and low
to moderate half-lives in all species. Amide 13 had half-lives 2−
4-fold higher in rodents as compared to compounds 1 and 14.
Plasma levels of 13 and 14 were good in all species and were
comparable to 1 in mouse and rat but were 2−4-fold higher in
monkeys as compared to 1. The concentration of 14 found in
rat brain (4161 nM) was comparable to the brain concentration
(3552 nM) found using compound 1. Interestingly, 13 had a
much lower exposure in the brain (459 nM) but showed similar
efficacy in the mouse catalepsy model as compared to 1 (Table
1). The different brain exposures of 13 and 14 were
compelling, and we thought that further characterization of
both compounds would complement one another. Our goal
was to profile both compounds to get a better understanding of
what may be the ideal brain exposure and PK profile for a dual
adenosine A2A/A1 antagonist for the treatment of PD.
Mouse Catalepsy. The mouse catalepsy data,29 Figure 1,
were collected at a single time point, 1 h postdose of 13 or 14.
Another study was performed using the same model of
neuroleptic-induced catalepsy in mice to examine the duration
of action of 13 and 14.35 Catalepsy time was measured in
haloperidol (1 mg/kg, sc)-treated mice after oral administration
of 13 (10 mg/kg), 14 (10 mg/kg), vehicle, or positive control 1
(10 mg/kg). Catalepsy was measured at 30 min and 1, 2, and 4
The synthesis of 14 started with the commercially available
6-methoxyindanone 22, which was condensed with benzalde-
hyde to afford the benzylidene 23 (Scheme 3).28 The
benzylidene 23 was reacted with guanidine and, upon
aromatization, was oxidized to the corresponding ketone 24
using air in a one-pot reaction as described above.37 The
methoxy group was deprotected by LiCl in NMP at 180 °C to
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Scheme 2. Synthesis of Compounds 4−13
h after oral dosing of the respective treatment groups. The
results in Figures 2 and 3 show that haloperidol induced
catalepsy at each measurement period and that 13 and 14 were
effective in reversing haloperidol-induced catalepsy at all
measurement periods. These data suggest that in mice, the
duration of action for in vivo efficacy of 13 and 14 is at least 4 h
following oral administration at 10 mg/kg. Extended measure-
ment periods past 4 h were not examined nor were lower doses
of compounds 13 or 14.
Rat Catalepsy. In vivo efficacy of 13 and 14 was examined
in a rat model of neuroleptic-induced catalepsy.30 This model
was carried out analogously to that described for mice except
that the maximum duration for rat was 180 s as compared to 60
s for mice. Results in Figure 4 show that both 13 and 14 were
effective in reversing the haloperidol-induced catalepsy having
ED50 values of 0.6 and 0.3 mg/kg, po, respectively, and each
having minimum effective doses of 1 mg/kg, po. As shown in
mice, the reversal in rat was also dose-dependent.
A study was performed using the rat model of neuroleptic-
induced catalepsy to examine the duration of action of 14. The
results in Figure 5 shows that haloperidol induced catalepsy at
each measurement period and that 14 was effective in reversing
haloperidol-induced catalepsy at 1, 2, and 4 h after a 1 mg/kg
oral dose. Positive results were also observed for 1 (positive
control) at 10 mg/kg, po. These data suggest that in rats, the
duration of action for in vivo efficacy of 14 is at least 4 h
following oral administration.
Mouse and Rat Models of Reserpine-Induced Akine-
sia. To evaluate the potential antiparkinsonian properties of 13
and 14, locomotor activity was studied in a mouse model of
reserpine-induced akinesia.31,39 Reserpine is an alkaloid that
depletes monoamine by inhibiting its vesicular uptake, resulting
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Scheme 3. Synthesis of Compound 14
Table 2. Pharmacokinetic Data for Compounds 1, 13, and 14
compd
IV/oral dose (mg/kg)
CL (mL/min/kg)
Vss, iv (L/kg)
mouse
oral t1/2 (h)
oral plasma Cmax (nM)
F (%)
brain Cmax (nM)
1
13
14
2/10
2/10
2/10
440
37
13
11
5
0.8
3.8
1.1
655
1695
1081
100
41
97
61
rat
1
13
14
2/10
2/10
2/10
85
52
19
7
6
1
1.6
3.6
1.2
1263
968
64
45
18
3552
459
1652
4161
monkey
1
13
14
1/10
2/10
2/10
56
15
7
7
4
2
3.2
4.3
5.5
626
1459
2769
39
47
55
in a dramatic reduction of spontaneous locomotor activity
(akinesia). Efficacy was defined as reversal of reserpine-induced
akinesia and was quantified as total distance traveled (cm)
(refer to the Experimental Section for a more detailed
description of the mouse and rat reserpine models).
Locomotion was studied 60 min after oral administration of
13 or 14 (0.10, 1.0, 3.0, 6.0, or 10.0 mg/kg), 1 (10 mg/kg,
positive control), or vehicle in mice that were pretreated with
reserpine (0.6 mg/kg, sc) 18 h earlier (Figure 6). The total
distance traveled was recorded during a 30 min measurement
period. Reserpine produced a marked decrease in horizontal
and vertical locomotor activity. As shown in Figure 6, reserpine-
induced akinesia was reversed by 13 and 14 at 1, 3, 6, and 10
mg/kg, po, and the minimum effective dose was 1 mg/kg, po,
for each compound. An analogous experiment was also
performed in rats using 13 and 14 except the distance traveled
was recorded during a 60 min measurement period instead of
30 min for the mouse model. Reserpine again produced a
marked decrease in locomotor activity. Both 13 and 14
produced a dose-dependent reversal of akinesia with both
compounds having a minimum effective dose of 1 mg/kg, po.
6-OHDA Lesion Model in Rats. Compounds 13 and 14
were studied in the 6-OHDA-lesion model of drug-induced
rotation in rats.32 In this model, the neurotoxin 6-OHDA (12
μg) is microinjected unilaterally in the medial forebrain bundle
to produce a targeted degeneration of midbrain DA neurons in
the pars compacta of the substantia nigra. 6-OHDA-induced
neurotoxicity causes a dramatic deficit in DA neurons that is
accompanied by denervation-induced supersensitivity of
postsynaptic DA receptors in the striatum of the lesioned
side. An imbalance in DA activity between the two sides of the
brain causes asymmetry in motor behavior that can be
enhanced by drug treatment. For example, drugs that stimulate
postsynaptic DA receptors produce an imbalance in DA
signaling that favors the lesion side and induces rotation
behavior (turning) toward the side opposite (contralateral to)
the lesion side (refer to the Experimental Section for a more
detailed description of the 6-OHDA-lesion model).40
One rotation count was defined as one 360° turn. Figure 8
(left panel) shows that prior to L-DOPA administration, 14
alone was able to produce a modest, dose-dependent increase
in the number of contralateral rotations observed in the 6-
OHDA lesioned rats, implying that 14 was able to provide a
modest enhancement of DA neurotransmission, especially via
the supersensitive D2 receptors at the lesioned side. Similar
results were seen with compound 13 (not shown). In contrast,
L-DOPA (10 mg/kg, po) was able to produce a more
pronounced enhancement in the number of contralateral
rotations, consistent with the known increase in DA trans-
mission that this drug produces (Figure 8, right panel). More
importantly, a dose-dependent, synergistic effect is realized
when L-DOPA is administered to animals that were dosed
previously with the adenosine receptor antagonists 13 (not
shown) or 14 with the minimum effective dose being 1 mg/kg,
po, for both compounds. These results suggest that DA must be
present in the brain for an adenosine antagonist to be optimally
effective in treating PD as the modest effect of 13 or 14 alone
on the behavior of animals would probably not suffice. It is
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Figure 2. Duration of action of 13 in the mouse model of neuroleptic-induced catalepsy. Catalepsy was induced by subcutaneous administration of
haloperidol. Compound 13 was administered orally 30 min after haloperidol (1 mg/kg, sc). Behavioral testing was conducted 30 min and 1, 2, and 4
h after administration of vehicle, 13, or 1 (positive control) in male Balb/c mice. Each value represents average ( SEM) time in cataleptic position
of n = 8 mice per treatment group during a 60 s test session. Haloperidol-induced catalepsy was evident at all measurement periods. Compound 13
was effective in reversing haloperidol-induced catalepsy 30 min and 1, 2, and 4 h after oral dosing. Asterisks indicate significant differences as
compared with the haloperidol + vehicle control treatment group (***P < 0.001, Dunnett's test of multiple comparisons).
known that approximately 80% of DA neurons are depleted
before any PD symptoms, that is, motor dysfunction, is
apparent.41 Therefore, it is critical to identify PD patients at an
earlier stage to maximize the benefit of adenosine receptor
antagonists in treating PD. While this remains the ultimate
challenge, significant efforts are being put forth to evaluate and
treat PD patients to enhance quality of life.
In summary, we have shown that the dual A2A/A1 receptor
antagonist 1 was metabolized into reactive intermediates that
caused genotoxicity and possibly the adverse events seen in the
28 day GLP toxicity study in nonhuman primates. Optimization
of the scaffold led to the identification of 13 and 14 as dual
A2A/A1 receptor antagonists having complementary brain PK
profiles. Both 13 and 14 showed excellent efficacy across a
number of animal models of PD including mouse and rat
catalepsy, mouse and rat reserpine-induced akinesia, and 6-
OHDA lesion model in rats. Despite the fact that 14 had ∼8-
fold higher brain levels in rats as compared to 13, both 13 and
14 exhibit very similar efficacy profiles across the various PD
models. Further evaluation will be needed to differentiate or
distinguish compounds 13 and 14.
EXPERIMENTAL SECTION
■
General Information. All proton and carbon nuclear magnetic
resonance spectra were determined using a 400 MHz Bruker NMR
with the appropriate internal standards. High-resolution MS was
performed on a JEOL Accutof JMS-T100 LC with a DART CE
ionization source operating in the positive mode. Reagent grade
chemicals and solvents were purchased from Aldrich, Oakwood, or
TCI. All chromatographies were carried out on a combi-flash system
equipped with an automated fraction collector. All final compounds
were purified to ≥95% purity as determined by Agilent 1100 series
high-performance liquid chromatography (HPLC) with UV detection
at 254 nm using the following method: Supelcosil ABZ+PLUS, 3.3 cm
× 2.1 cm, 11 min; 1.2 mL/min flow rate; and 5−95% 0.1% TFA in
CH3CN/0.1% TFA in H2O. Experimental procedures and spectral
listings for compounds 1 and 16−20 were previously reported.28a
2-Amino-8-((2,5-dimethylpyrrolidin-1-yl)methyl)-4-phenyl-5H-
indeno[1,2-d]pyrimidin-5-one (4). Neat TFA (8 mL, 102.8 mmol)
was added to a CH2Cl2 solution (24 mL) of 20 (1.94 g, 3.4 mmol),
and the mixture was stirred at room temperature. After 2 h, the
solution was concentrated in vacuo, and saturated aqueous NaHCO3
was added to the material. The resulting suspension was sonicated, and
the precipitate was collected by filtration and washed with water. The
collected solid was dried under high vacuum to afford 1.1 g (90%) of
2-amino-8-(bromomethyl)-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-
one as a yellow solid that was used without further purification. Rt =
1
4.75 min. H NMR (400 MHz, chloroform-d): δ 8.03−8.12 (m, 2H),
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Figure 3. Duration of action of 14 in the mouse model of neuroleptic-induced catalepsy. Catalepsy was induced by subcutaneous administration of
haloperidol. Compound 14 was administered orally 30 min after haloperidol (1 mg/kg, sc). Behavioral testing was conducted 30 min and 1, 2, and 4
h after administration of vehicle, 14, or 1 (positive control) in male Balb/c mice. Each value represents average ( SEM) time in cataleptic position
of n = 8 mice per treatment group during a 60 s test session. Haloperidol-induced catalepsy was evident at all measurement periods. Compound 14
was effective in reversing haloperidol-induced catalepsy 30 min and 1, 2, and 4 h after oral dosing. Asterisks indicate significant differences as
compared with the haloperidol + vehicle control treatment group (***P < 0.001, Dunnett's test of multiple comparisons).
Figure 4. Reversal of neuroleptic-induced catalepsy in rats by compounds 13 and 14. In this behavioral model, catalepsy was induced in Sprague−
Dawley rats by subcutaneous administration of haloperidol, a neuroleptic D2 receptor antagonist. Compound 1 was used as the positive control for
this model at 10 mg/kg, po. Compounds 13 (left panel) and 14 (right panel) were administered orally 60 min after haloperidol (1 mg/kg, sc).
Behavioral testing was conducted 1 h after dosing of 13 and 14. Each value represents average ( SEM) time in cataleptic position of n = 10 rats per
treatment group during a 180 s test session. Both compounds produced a dose-dependent reversal of haloperidol-induced catalepsy. The ED50 values
of 13 and 14 for inhibiting haloperidol-induced catalepsy in rats were 0.6 and 0.3 mg/kg, po, respectively, and the minimum effective dose was
shown to be 1 mg/kg, po, for both 13 and 14. Asterisks indicate significant differences as compared with the haloperidol + vehicle control treatment
group (***P < 0.001, Dunnett's test of multiple comparisons).
7.89 (d, J = 0.98 Hz, 1H), 7.72 (d, J = 7.58 Hz, 1H), 7.46−7.62 (m,
4H), 5.88 (br. s., 2H), 4.56 (s, 2H) ppm. 13C NMR (101 MHz,
chloroform-d): δ 187.6, 176.1, 165.9, 164.5, 144.2, 140.6, 136.4, 135.2,
133.4, 131.4, 129.7, 128.1, 124.0, 122.0, 108.0, 32.1 ppm. HRMS, m/z
calcd for C18H13BrN3O [(M + H)+], 366.0237; found, 366.0254.
Neat 2,5-dimethylpyrrolidine-mix of cis- and trans-isomers (1.1 mL,
9.3 mmol) was added to a THF solution (20 mL) of 2-amino-8-
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Figure 5. Duration of action of 14 in the rat model of neuroleptic-induced catalepsy. Catalepsy was induced by subcutaneous administration of
haloperidol. Compound 14 (1 mg/kg) was administered orally 60 min after haloperidol (1 mg/kg, sc). Behavioral testing was conducted 15 min and
1, 2, and 4 h after administration of vehicle, 14, or 1 (positive control) in Sprague−Dawley rats. Each value represents average ( SEM) time in
cataleptic position of n = 10−12 rats per treatment group during a 180 s test session. Haloperidol-induced catalepsy was evident at all measurement
periods. Compound 14 was effective in reversing haloperidol-induced catalepsy 1, 2, and 4 h after oral dosing of 1 mg/kg. Asterisks indicate
significant differences as compared with the haloperidol + vehicle control treatment group (***P < 0.001, Dunnett's test of multiple comparisons).
Figure 6. Reversal of reserpine-induced akinesia by 13 (left) and 14 (right) in mice. In this behavioral model, akinesia was induced by subcutaneous
administration reserpine, a monoamine-depleting drug in male CF-1 mice. Compounds 13, 14, 1 (10 mg/kg, positive control), or vehicle were
administered orally 60 min before the behavioral test in the locomotor activity boxes. Each value shows the mean ( SEM) of the total distance
traveled during the 30 min measurement period in the behavioral test session. Reserpine produced a marked decrease in locomotor activity as
compared with vehicle + vehicle-treated controls. Compounds 13 and 14 reversed reserpine-induced akinesia with similar effectiveness to the control
1, N = 14−16 mice per treatment group. Asterisks indicate significant differences as compared with the reserpine + vehicle control group (**P <
0.01, ***P < 0.001, Hochberg test of multiple comparisons).
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Figure 7. Reversal of reserpine-induced akinesia by 14 (left) and 13 (right) in rats. In this behavioral model, akinesia was induced by subcutaneous
administration reserpine, a monoamine-depleting drug in male Wistar rats. Compounds 13, 14, 1 (10 mg/kg, positive control), or vehicle was
administered orally 60 min before the behavioral test in the locomotor activity boxes. Each value shows the mean ( SEM) of the total distance
traveled during a 60 min measurement period in the behavioral test session. Reserpine produced a marked decrease in locomotor activity as
compared with vehicle + vehicle-treated controls. Compounds 13 and 14 reversed reserpine-induced akinesia with similar effectiveness to the control
1, N = 16−18 rats per treatment group. Asterisks indicate significant differences as compared with the reserpine + vehicle control group (*P < 0.05,
**P < 0.01, and ***P < 0.001, Hochberg test of multiple comparisons).
Figure 8. Compound 14 potentiates the effects of L-DOPA in the 6-OHDA-lesioned rat model of PD. Six weeks after administration of 6-OHDA,
the behavioral test was started by dosing rats with L-DOPA [10 mg/kg, po coadministered with carbidopa (2.5 mg/k, sc)] alone or with each dose of
14 (0.1, 1.0, 3.0, 6.0, and 10 mg/kg, po). The effects of 14 alone at 10 mg/kg, po (indicated in both the left and the right panels as 10), were also
studied in this model. For clarity, two separate groups of animals were dosed at 10 mg/kg, po. The rotational behavior was determined as each
animal was tethered to a high-resolution optical sensor connected to an automated computerized system that quantifies circular motion. One
rotation count was defined as one 360° turn. Each value represents the mean ( SEM) of total contralateral rotations of n = 16 rats per treatment
group during the 2 h before and after L-DOPA administration. Asterisks in the left panel indicate significant differences as compared with vehicle
control treatment group, and asterisks in the right panel indicate significant differences as compared with the L-DOPA + vehicle control treatment
group (*P < 0.05, **P < 0.01, and ***P < 0.001, Tukey's test of multiple comparisons).
(bromomethyl)-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one (1.7 g, 4.6
mmol) and triethyl amine (Et3N) (1.3 mL, 9.3 mmol), and the
resulting mixture was heated to 75 °C. After 3 h, the mixture was
concentrated in vacuo, dissolved into CH2Cl2. The mixture was
washed with saturated aqueous NaHCO3 and brine (2×), dried
(Na2SO4), concentrated, and purified via column chromatography to
give 1.0 g (57%) of 4. Compound 4 was dissolved in CH2Cl2 and
added dropwise to an excess of HCl in ether. The resulting precipitate
was filtered off to give 1.1 g of 2-amino-8-((2,5-dimethylpyrrolidin-1-
yl)methyl)-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one (4) as the di-
HCl salt. Rt = 5.57 min. 1H NMR (400 MHz, DMSO-d6): δ 8.12 (d, J
= 9.60 Hz, 1H), 8.01 (d, J = 8.59 Hz, 4H), 7.87 (d, J = 7.58 Hz, 1H),
7.72−7.80 (m, 1H), 7.45−7.62 (m, 4H), 4.58 (d, J = 4.55 Hz, 2H),
3.45−3.55 (m, 2H), 2.07−2.24 (m, 2H), 1.59−1.75 (m, 2H), 1.34 (s,
3H), 1.33 (s, 3H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 186.6,
175.2, 165.1, 164.8, 139.7, 136.9, 136.2, 136.1, 135.5, 130.9, 129.4,
127.6, 123.8, 123.2, 110.9, 63.1, 52.6, 29.3, 16.4 ppm. HRMS, m/z
calcd for C24H25N4O [(M + H)+], 385.2023; found, 385.2036.
8-(7-Azabicyclo[2.2.1]heptan-7-ylmethyl)-2-amino-4-phenyl-5H-
indeno[1,2-d]pyrimidin-5-one (5). The same procedure described to
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prepare 4 was followed except that 7-azabicyclo[2.2.1]heptane was
ppm. HRMS, m/z calcd for C23H16ClN4O [(M + H)+], 399.1007;
found, 399.1013.
used in place of 2,5-dimethylpyrrolidine to give the title compound in
1
2-Amino-8-nicotinoyl-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one
(11). Powdered NaOH (420 mg, 10.5 mmol) was added to an NMP
solution (1.5 mL) of 2-amino-4-phenyl-8-(pyridin-3-ylmethyl)-5H-
indeno[1,2-d]pyrimidin-5-one (6) (400 mg, 1.1 mmol). Air was
bubbled into the solution at a steady rate using a steel needle, and the
mixture was heated to 80 °C. After 18 h, the air needle was removed,
and water was added. The resulting solid was filtered off, washed with
water, and dried in vacuo. The crude solid was dissolved in THF and
dry packed onto silica gel. Column chromatography gave 110 mg
(26%) of 2-amino-8-nicotinoyl-4-phenyl-5H-indeno[1,2-d]pyrimidin-
5-one (11). Rt = 3.56 min. 1H NMR (400 MHz, DMSO-d6): δ 8.97 (s,
1H), 8.89 (d, J = 4.55 Hz, 1H), 8.13−8.25 (m, 3H), 8.00−8.10 (m,
4H), 7.86 (d, J = 7.58 Hz, 1H), 7.66 (dd, J = 5.31, 7.83 Hz, 1H), 7.48−
7.61 (m, 3H) ppm. 13C NMR (151 MHz, DMSO-d6): δ 194.1, 186.9,
175.4, 165.9, 165.5, 153.7, 150.6, 141.4, 140.1, 140.0, 137.7, 136.0,
135.2, 132.9, 131.6, 130.0, 128.3, 124.3, 123.9, 122.0, 111.7 ppm.
HRMS, m/z calcd for C23H15N4O2 [(M + H)+], 379.1190; found,
379.1221.
40% yield. Rt = 3.04 min. H NMR (400 MHz, DMSO-d6): δ 8.05−
8.16 (m, 3H), 8.01 (d, J = 7.07 Hz, 2H), 7.97 (d, J = 7.58 Hz, 1H),
7.75 (d, J = 7.58 Hz, 1H), 7.48−7.61 (m, 3H), 4.38 (d, J = 6.06 Hz,
2H), 3.95 (br. s., 2H), 2.24 (d, J = 8.59 Hz, 2H), 2.04 (d, J = 8.59 Hz,
2H), 1.77 (d, J = 8.59 Hz, 2H), 1.67 (d, J = 8.08 Hz, 2H) ppm. 13C
NMR (101 MHz, DMSO-d6): δ 186.8, 175.4, 164.8, 164.7, 139.9,
137.7, 136.8, 135.4, 135.3, 131.0, 129.5, 127.7, 123.3, 123.2, 111.0,
62.7, 48.3, 26.9, 25.1 ppm. HRMS, m/z calcd for C24H23N4O [(M +
H)+], 383.1866; found, 383.1848.
2-Amino-4-phenyl-8-(pyridin-3-ylmethyl)-5H-indeno[1,2-d]-
pyrimidin-5-one (6). A dioxane/water (4:1) solution (5 mL) of 2-
amino-8-(bromomethyl)-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one
(175 mg, 0.48 mmol), 3-pyridylboronic acid (94 mg, 0.77 mmol),
K2CO3 (133 mg, 0.96 mmol), and Pd(dppf)Cl2 (39 mg, 0.05 mmol)
was heated in the microwave at 120 °C for 10 min. The resulting
mixture was diluted with THF and EtOAc, washed with water and
brine, dried (Na2SO4), concentrated, and purified to give 112 mg
(64%) of 6. Compound 6 was dissolved in THF added dropwise to an
excess of HCl in ether. The resulting precipitate was filtered off to give
115 mg of 2-amino-4-phenyl-8-(pyridin-3-ylmethyl)-5H-indeno[1,2-
2-Amino-8-(difluoro(pyridin-3-yl)methyl)-4-phenyl-5H-indeno-
[1,2-d]pyrimidin-5-one (12). 2-Amino-8-nicotinoyl-4-phenyl-5H-
indeno[1,2-d]pyrimidin-5-one (110 mg, 0.29 mmol) was dissolved in
neat DAST (1 mL), and the mixture was heated to 70 °C. After 4 h,
the mixture was concentrated in vacuo and partitioned between DCM
and water. The organic layer was dried (Na2SO4), concentrated, and
purified via column chromatography to give 27 mg (23%) of 2-amino-
8-(difluoro(pyridin-3-yl)methyl)-4-phenyl-5H-indeno[1,2-d]-
1
d]pyrimidin-5-one (6) as the di-HCl salt. Rt = 3.01 min. H NMR
(400 MHz, DMSO-d6): δ 9.01 (s, 1H), 8.84 (d, J = 5.05 Hz, 1H), 8.54
(d, J = 8.08 Hz, 1H), 7.91−8.12 (m, 5H), 7.73 (s, 1H), 7.65 (s, 2H),
7.46−7.59 (m, 3H), 4.39 (s, 2H) ppm. 13C NMR (101 MHz, DMSO-
d6): δ 186.9, 175.4, 164.6, 164.3, 145.9, 145.7, 141.7, 140.3, 140.1,
135.3, 135.2, 134.9, 133.5, 130.8, 129.4, 127.6, 127.0, 123.5, 121.4,
111.0, 37.1 ppm. HRMS, m/z calcd for C23H17N4O [(M + H)+],
365.1379; found, 365.1395.
5-((2-Amino-5-oxo-4-phenyl-5H-indeno[1,2-d]pyrimidin-8-yl)-
methyl)nicotinonitrile (7). The same procedure used to prepare 6 was
followed except that (5-cyanopyridin-3-yl)boronic acid was used in
place of 3-pyridylboronic acid to give the title compound in 64% yield.
Rt = 4.33 min. 1H NMR (400 MHz, DMSO-d6): δ 8.86−8.97 (m, 2H),
8.34 (s, 1H), 7.99 (d, J = 7.07 Hz, 3H), 7.45−7.70 (m, 7H), 4.25 (s,
2H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 186.9, 175.4, 164.5,
164.2, 153.6, 150.4, 146.4, 140.0, 139.9, 136.5, 135.2, 134.7, 133.4,
130.8, 129.4, 127.6, 123.4, 121.2, 116.8, 111.0, 109.0, 37.3 ppm.
HRMS, m/z calcd for C24H16N5O [(M + H)+], 390.1349; found,
390.1395.
1
pyrimidin-5-one (12). Rt = 4.10 min. H NMR (400 MHz, DMSO-
d6): δ 8.89 (s, 1H), 8.78 (d, J = 3.54 Hz, 1H), 8.15 (br. s., 1H), 8.09
(d, J = 7.58 Hz, 2H), 8.01 (d, J = 7.07 Hz, 2H), 7.92 (d, J = 7.58 Hz,
1H), 7.78−7.84 (m, 2H), 7.47−7.63 (m, 4H) ppm. 13C NMR (151
MHz, DMSO-d6): δ 186.8, 175.3, 165.8, 165.5, 152.3, 146.8, 140.7,
138.5, 136.0, 134.1, 132.5, 132.4, 131.5, 130.7, 130.0, 128.2, 124.5,
124.3, 120.1, 117.8, 111.5 ppm. HRMS, m/z calcd for C23H15F2N4O
[(M + H)+], 401.1208; found, 401.1196.
2-Amino-8-(4-methylpiperazine-1-carbonyl)-4-phenyl-5H-
indeno[1,2-d]pyrimidin-5-one (13). Neat piperazine (0.4 mL, 3.6
mmol) was added to a THF solution (60 mL) of acid 21 (1.7 g, 3.3
mmol), diisopropylethylamine (1.7 mL, 9.9 mmol), and HATU (1.3 g,
3.3 mmol). The resulting mixture was heated to 40 °C. After 18 h, the
mixture was concentrated and purified via column chromatography to
give 1.8 g (91%) of bis-tert-butyl (8-(4-methylpiperazine-1-carbonyl)-
5-oxo-4-phenyl-5H-indeno[1,2-d]pyrimidin-2-yl)carbamate that was
used immediately. The bis-tert-butyl (8-(4-methylpiperazine-1-carbon-
yl)-5-oxo-4-phenyl-5H-indeno[1,2-d]pyrimidin-2-yl)carbamate (1.8 g,
3.0 mmol) was then stirred in 25 mL of CH2Cl2/TFA (4:1). After 3 h,
the mixture was concentrated, neutralized with saturated aqueous
NaHCO3, and filtered to give 1 g of crude 2-amino-8-(4-
methylpiperazine-1-carbonyl)-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-
one (13). The solid was purified via column chromatography to give
893 mg (75%) of 13 as the free base, which was dissolved in THF and
added to 10 mL of 1 N HCl in ether, concentrated, and dried in vacuo
to give 2-amino-8-(4-methylpiperazine-1-carbonyl)-4-phenyl-5H-
indeno[1,2-d]pyrimidin-5-one (13) as the di-HCl salt. Rt = 2.63
2-Amino-8-((5-fluoropyridin-3-yl)methyl)-4-phenyl-5H-indeno-
[1,2-d]pyrimidin-5-one (8). The same procedure used to prepare 6
was followed except that (5-fluoropyridin-3-yl)boronic acid was used
in place of 3-pyridylboronic acid to give the title compound in 74%
1
yield. Rt = 3.92 min. H NMR (400 MHz, DMSO-d6): δ 8.62 (d, J =
6.06 Hz, 2H), 8.20 (br. s., 2H), 7.91−8.03 (m, 3H), 7.69 (s, 1H),
7.62−7.68 (m, 2H), 7.45−7.61 (m, 3H), 4.27 (s, 2H) ppm. HRMS,
m/z calcd for C23H16FN4O [(M + H)+], 383.1303; found, 383.1229.
2-Amino-8-((3-fluoropyridin-4-yl)methyl)-4-phenyl-5H-indeno-
[1,2-d]pyrimidin-5-one (9). The same procedure used to prepare 6
was followed except that (3-fluoropyridin-4-yl)boronic acid was used
in place of 3-pyridylboronic acid to give the title compound in 69%
yield. Rt = 3.63 min. 1H NMR (400 MHz, DMSO-d6): δ 8.66 (s, 1H),
8.48 (d, J = 4.04 Hz, 1H), 8.02−8.15 (br. s., 2H), 7.99 (d, J = 7.07 Hz,
2H), 7.44−7.71 (m, 7H), 4.28 (s, 2H) ppm. 13C NMR (101 MHz,
DMSO-d6): δ 186.9, 175.4, 164.6, 164.3, 145.2, 144.8, 142.5, 140.0,
136.6, 135.3, 135.0, 133.4, 130.9, 129.5, 127.7, 126.4, 123.5, 121.0,
119.7, 111.0, 33.7 ppm. HRMS, m/z calcd for C23H16FN4O [(M +
H)+], 383.1303; found, 383.1322.
2-Amino-8-((4-chloropyridin-3-yl)methyl)-4-phenyl-5H-indeno-
[1,2-d]pyrimidin-5-one (10). The same procedure used to prepare 6
was followed except that (4-chloropyridin-3-yl)boronic acid was used
in place of 3-pyridylboronic acid to give the title compound in 56%
yield. Rt = 3.60 min. 1H NMR (400 MHz, DMSO-d6): δ 8.99 (s, 1H),
8.73 (d, J = 5.56 Hz, 1H), 8.15 (br. s., 2H), 7.90−8.03 (m, 3H), 7.45−
7.68 (m, 6H), 4.40 (s, 2H) ppm. 13C NMR (101 MHz, DMSO-d6): δ
186.7, 175.4, 164.2, 164.1, 147.5, 145.3, 144.6, 139.8, 134.9, 135.7,
134.7, 134.7, 133.6, 131.0, 129.5, 127.7, 126.5, 123.4, 121.0, 111.0, 35.7
1
min. H NMR (400 MHz, chloroform-d): δ ppm 2.34 (s, 3 H), 2.39
(br. s., 2 H), 2.52 (d, J = 2.20 Hz, 2 H), 3.46 (br. s., 2 H), 3.84 (br. s., 2
H), 5.86 (br. s., 2 H), 7.46−7.64 (m, 4 H), 7.78 (d, J = 7.58 Hz, 1 H),
7.85 (s, 1 H), 8.07 (dd, J = 7.83, 1.71 Hz, 2 H) ppm. 13C NMR (101
MHz, DMSO-d6): δ 186.5, 175.1, 167.8, 165.0, 164.9, 140.2, 139.6,
137.0, 135.5, 131.5, 130.9, 129.4, 127.6, 123.2, 119.3, 110.9, 57.1, 51.3,
42.2 ppm. HRMS, m/z calcd for C23H22N5O2 [(M + H)+], 400.1768;
found, 400.1780.
2-Amino-8-(2-morpholinoethoxy)-4-phenyl-5H-indeno[1,2-d]-
pyrimidin-5-one (14). Solid potassium tert-butoxide (t-BuOK) (9.7 g,
86.5 mmol) was added to a DMF solution (200 mL) of 25 (10.0 g,
34.6 mmol) and 4-(2-chloroethyl)morpholine hydrochloride (7.1 g,
38.1 mmol), and the resulting solution was heated to 75 °C. After 4 h
at 75 °C, the mixture was cooled to room temperature diluted with
tetrahydrofuran (THF) and EtOAc and washed with brine, water, and
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brine. The organics were dried over Na2SO4 and dry packed onto silica
gel. Column chromatography gave 7.0 g (50%) of the desired ether 14
as the free base, which was dissolved in THF and added to 100 mL of
2 N HCl in ether, concentrated, and dried in vacuo to give 2-amino-8-
(2-morpholinoethoxy)-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one
(14) as the di-HCl salt. Rt = 2.85 min. 1H NMR (300 MHz,
chloroform-d): δ ppm 2.53−2.67 (m, 4 H), 2.86 (t, J = 5.65 Hz, 2 H),
3.66−3.83 (m, 4 H), 4.25 (t, J = 5.65 Hz, 2 H), 5.76 (br. s., 2 H), 7.01
(dd, J = 8.10, 2.45 Hz, 1 H), 7.35 (d, J = 2.26 Hz, 1 H), 7.43−7.57 (m,
3 H), 7.66 (d, J = 8.29 Hz, 1 H), 7.98−8.10 (m, 2 H) ppm. 13C NMR
(101 MHz, DMSO-d6): δ 186.2, 174.9, 164.1, 163.1, 162.4, 141.8,
134.6, 131.0, 129.5, 129.5, 127.7, 125.0, 118.7, 111.5, 107.2, 63.0, 62.7,
54.5, 51.5 ppm. HRMS, m/z calcd for C23H23N4O3 [(M + H)+],
403.1765; found, 403.1754.
was heated to 80 °C and air was bubbled through the solution using a
steel needle. After 16 h, the mixture was cooled to room temperature,
water was added, and the resulting precipitate was filtered and washed
with water and cold EtOH. The red solid was dried in vacuo to give
5.0 g (56%) of 2-amino-8-methoxy-4-phenyl-5H-indeno[1,2-d]-
1
pyrimidin-5-one (24). Rt = 4.04 min. H NMR (400 MHz, DMSO-
d6): δ 8.00 (d, J = 6.57 Hz, 2H), 7.92 (br. s., 2H), 7.62 (d, J = 8.59 Hz,
1H), 7.45−7.58 (m, 3H), 7.27 (d, J = 2.53 Hz, 1H), 7.15 (dd, J = 2.53,
8.08 Hz, 1H), 3.93 (s, 3H) ppm. 13C NMR (101 MHz, DMSO-d6): δ
186.5, 174.8, 164.8, 164.2, 164.1, 142.1, 135.6, 130.6, 129.5, 128.7,
127.5, 124.9, 117.7, 111.5, 106.1, 55.9 ppm. HRMS, m/z calcd for
C18H14N3O2 [(M + H)+], 304.1081; found, 304.1065.
2-Amino-8-hydroxy-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one
(25). An NMP solution (25 mL) of 24 (4.0 g, 13.2 mmol), LiCl (5.6 g,
132.0 mmol), and water (0.5 mL) were heated to 180 °C. After 4 h,
the mixture was diluted with THF and EtOAc, washed with water and
brine, dried (Na2SO4), filtered, and dry packed onto silica gel. Column
chromatography gave 2.4 g (63%) of 2-amino-8-hydroxy-4-phenyl-5H-
2-((Bis-tert-butoxycarbonyl)amino)-5-oxo-4-phenyl-5H-indeno-
[1,2-d]pyrimidine-8-carboxylic Acid (21). Solid N-methyl morpholine
N-oxide (2.5 g, 21.2 mmol) was added to a CH3CN solution (300
mL) of 7 (6.0 g, 10.6 mmol) and 4 Å ms (10.5 g). After 18 h at room
temperature, the mixture was filtered, and the filtrate was diluted with
EtOAc and washed with water and brine, dried (Na2SO4), and purified
via column chromatography to give 3.6 g (68%) of bis-tert-butyl (8-
formyl-5-oxo-4-phenyl-5H-indeno[1,2-d]pyrimidin-2-yl)carbamate. Rt
1
indeno[1,2-d]pyrimidin-5-one (25). Rt = 3.58 min. H NMR (400
MHz, DMSO-d6): δ 10.76 (s, 1H), 7.99 (d, J = 6.57 Hz, 2H), 7.87 (br.
s., 2H), 7.42−7.59 (m, 4H), 7.15 (d, J = 2.02 Hz, 1H), 6.95 (dd, J =
2.02, 8.08 Hz, 1H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 186.7,
175.0, 164.9, 163.9, 163.2, 142.4, 135.7, 130.6, 129.5, 127.6, 127.4,
125.3, 118.8, 111.6, 107.9 ppm. HRMS, m/z calcd for C17H12N3O2
[(M + H)+], 290.0924; found, 290.0923.
1
= 6.81 min. H NMR (400 MHz, chloroform-d): δ 10.17 (s, 1H),
8.43 (s, 1H), 8.24 (d, J = 7.07 Hz, 2H), 8.13 (d, J = 7.58 Hz, 1H), 7.96
(d, J = 7.58 Hz, 1H), 7.49−7.63 (m, 3H), 1.55 (s, 18H) ppm. 13C
NMR (101 MHz, chloroform-d): δ 190.6, 188.0, 175.3, 150.3, 141.2,
139.3, 134.8, 134.3, 132.3, 130.3, 128.3, 124.7, 122.6, 84.2, 77.3, 77.2,
77.0, 76.7, 27.9 ppm. HRMS, m/z calcd for C28H28N3O6 [(M + H)+],
502.1973; found, 502.1995.
Pharmacokinetic Studies. Compound 1, 13, or 14 was
administered to fasted Sprague−Dawley rats (male), Balb/c mice
(male), and Cynomolus monkeys (male and female) by a single iv or
oral administration. Compound 1 was formulated in 10% solutol for
the iv injection. Compound 1 was formulated in 0.5% methylcellulose
and dosed orally as a solution. The iv concentrations of 1 were 2 mg/
kg for mice and rats and were 1 mg/kg for monkeys. The iv
concentrations of 13 and 14 were 2 mg/kg for mice, rats, and
monkeys. The oral concentrations of 1 were 10 mg/kg for all species.
Discrete blood samples collected from mice and rats were withdrawn
from 3 to 4 animals per time point at selected intervals postdose via
orbital sinus (rats) or cardiac (mice) puncture. Serial blood collections
from monkeys were withdrawn by venipuncture. Plasma was obtained
by centrifugation, processed by acetonitrile precipitation, and then
analyzed by LC-MS/MS. The limit of quantitiation (LOQ) was 0.7−2
ng/mL. Noncompartmental analysis was performed on individual
plasma concentrations using WinNonlin (version 4.0.4). For the tissue
distribution studies, a 10 mg/kg solution of 1, 13, or 14 in 0.5%
methylcellulose was administered orally to fasted Sprague−Dawley rats
(male). At each time point, four animals were euthanized for collection
of blood, plasma, and brain. Each tissue was weighed and
homogenized in methanol. The methanolic extracts were evaporated
and then reconstituted in mobile phase for analysis by LC-MS/MS.
Blood and plasma were treated with acetonitrile, and the supernatants
were analyzed for drug concentration by LC-MS/MS.
Mouse Catalepsy Study. Haloperidol, a neuroleptic medication
that inhibits D2 receptors, was used to induce catalepsy. In the rodent,
catalepsy is characterized as a loss of voluntary motion where limbs
uncharacteristically remain in placed positions. Catalepsy was
measured in haloperidol (1 mg/kg, sc)-treated mice (fasted, male
Balb/c mice) after oral administration of 1 (0.01, 0.10, 1.0, or 10.0
mg/kg, po), L-DOPA [300 mg/kg; coadministered with carbidopa (75
mg/kg)], or vehicle. Compounds 4−14 were given orally at 10 mg/kg
to the haloperidol-treated mice using 1 (10 mg/kg, po) as the positive
control, and the data were reported as the percent reversal of catalepsy.
Dose responses of catalepsy were measured in haloperidol (1 mg/kg,
sc)-treated mice (fasted, male Balb/c mice) after oral administration 4,
5, 6, 13, or 14 (0.1, 1.0, 3.0, or 10.0 mg/kg, po), 1 (10 mg/kg, po), or
vehicle. Animals were randomly assigned to treatment groups, and
behavioral testing was performed blind to treatment. Control mice
received the respective sc and po vehicles. Haloperidol was dissolved
in 0.3% tartaric acid in 0.9% saline. L-DOPA was diluted in 0.5%
methylcellulose and dosed as a suspension. Compounds 1, 13, and 14
were diluted in 0.5% methylcellulose and dosed orally. Compounds 1,
13, and 14 were administered orally 30 min after haloperidol.
Solid KMnO4 was added to an acetone/water solution (100 mL/25
mL) of bis-tert-butyl (8-formyl-5-oxo-4-phenyl-5H-indeno[1,2-d]-
pyrimidin-2-yl)carbamate (3.6 g, 7.2 mmol), and the resulting mixture
was heated to 55 °C. After 14 h, the mixture was cooled to room
temperature and filtered. The filtrate was diluted with EtOAc and
washed with water and brine, dried (Na2SO4), concentrated, and
purified by column chromatography to give 2.1 g (57%) of 2-((bis-tert-
butoxycarbonyl)amino)-5-oxo-4-phenyl-5H-indeno[1,2-d]pyrimidine-
8-carboxylic acid (21). Rt = 6.00 min. 1H NMR (400 MHz,
chloroform-d): δ 8.54−8.65 (m, 1H), 8.26−8.36 (m, 1 H), 8.17−
8.24 (m, 2H), 7.78−7.89 (m, 1H), 7.48−7.63 (m, 3H), 1.53 (s, 18H)
ppm. 13C NMR (101 MHz, DMSO-d6): δ 187.5, 175.0, 163.7, 163.3,
160.4, 159.9, 150.0, 134.9, 134.0, 132.0, 131.6, 130.0, 129.9, 128.2,
127.9, 124.3, 118.7, 83.9, 27.3 ppm. HRMS, m/z calcd for C28H28N3O7
[(M + H)+], 518.1922; found, 518.1887.
(E)-2-Benzylidene-6-methoxy-2,3-dihydro-1H-inden-1-one (23).
An aqueous solution (5 mL) of NaOH (1.5 g, 38.5 mmol) was
added dropwise to an ethanol (EtOH) solution (30 mL) of 6-
methoxy-1-indanone (5.0 g, 30.8 mmol) and benzaldehyde (3.4 g, 32.4
mmol). A precipitate formed immediately. An additional 30 mL of
EtOH was added, and the resulting slurry was stirred vigorously for 0.5
h. The slurry was cooled in an ice bath, filtered, and washed with cold
EtOH. The collected solid was dried in vacuo to give 7.3 g (95%) of
(E)-2-benzylidene-6-methoxy-2,3-dihydro-1H-inden-1-one (23). Rt =
1
5.39 min. H NMR (400 MHz, DMSO-d6): δ 7.79 (d, J = 7.07 Hz,
2H), 7.59 (d, J = 8.08 Hz, 1H), 7.43−7.56 (m, 4H), 7.31 (dd, J = 2.53,
8.08 Hz, 1H), 7.26 (d, J = 2.53 Hz, 1H), 4.06 (s, 2H), 3.84 (s, 3H)
ppm. 13C NMR (101 MHz, DMSO-d6): δ 193.1, 159.1, 142.6, 138.4,
135.8, 134.8, 132.6, 130.7, 129.7, 128.9, 127.5, 123.4, 105.5, 55.4, 31.1
ppm. HRMS, m/z calcd for C17H15O2 [(M + H)+], 251.1067; found,
251.1056.
2-Amino-8-methoxy-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one
(24). Powdered NaOH (5.8 g, 146.0 mmol) was added to an EtOH
solution (130 mL) of guanidine acetate (17.4 g, 146.0 mmol). After 30
min, the sodium acetate (NaOAc) was filtered off, and the filtrate was
added to an EtOH suspension (80 mL) of 23 (7.3 g, 29.2 mmol). The
resulting mixture was heated to reflux overnight. The homogeneous
solution was cooled in ice for 30 min and filtered to give 6.0 g of crude
material (des-ketone), which was used without further purification.
The crude material (6.0 g) was dissolved in NMP (80 mL), and
powdered NaOH (1.2 g, 31.1 mmol) was added. The resulting mixture
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Behavioral testing was conducted 1 h after dosing of 1, 13, and 14.
The behavioral test trial (maximum duration of 60 s) began by placing
the forepaws of fasted, male Balb/c mice on a horizontal bar elevated
3.5 cm above the bench. The cataleptic state was regarded as over, and
the trial ended when the animal came off the bar by either placing its
forepaws on the bench or climbing onto the bar with all of its limbs.
Each value represents average ( SEM) time in cataleptic position of n
= 8−12 mice per treatment group during a 60 s test session. Asterisks
indicate significant differences as compared with the haloperidol +
vehicle control group (***P < 0.001, Dunnett's test of multiple
comparisons).
two infrared beams placed 2.5 cm apart and 2 cm above the cage floor,
and the total distance traveled was quantified in cm. Locomotion was
studied 60 min after oral administration of compound 13 or 14 (0.1,
1.0, 3.0, 6.0, and 30 mg/kg, po), 1 (10 mg/kg), or vehicle in mice that
were pretreated with reserpine (0.6 mg/kg, sc) 18 h earlier. Mice used
were fasted male CF-1 mice. Animals were randomly assigned to
treatment groups. Control mice received the respective sc and po
vehicles. Reserpine was diluted in 0.5% acetic acid in distilled water
and dosed sc. Compounds 13, 14, and 1 were diluted in 0.5%
methylcellulose and dosed as a solution. Reserpine produced a marked
decrease in horizontal and vertical locomotor activity. As shown in
Figure 6, reserpine-induced akinesia was reversed by 13 and 14 at 1.0,
3.0, 6.0, and 10 mg/kg, po. Akinesia was also reversed by 1 at 10 mg/
kg as the positive control. In Figure 6, each value represents the mean
( SEM) of the total distance traveled of n = 14−16 mice per
treatment group during the 30 min measurement period in the
behavioral test session. Asterisks indicate significant differences as
compared with the reserpine + vehicle control group (**P < 0.01,
***P < 0.001, Hochberg test of multiple comparisons).
Reserpine-Induced Akinesia Study in Rats. Reserpine is an
alkaloid that depletes monoamines by inhibiting their vesicular uptake,
resulting in a dramatic reduction of spontaneous locomotor activity
(akinesia). Efficacy is defined as reversal of reserpine-induced akinesia.
Locomotion was studied in open field activity boxes (L, 17.5 in; W,
17.5 in; and H, 12 in), each containing 20 equally spaced pairs of
horizontal infrared photocell beams along one axis. A PC running the
activity analysis software measured activity automatically. One activity
count corresponded to the consecutive interruption of two infrared
beams placed 2.5 cm apart and 2 cm above the cage floor, and the total
distance traveled was quantified in cm. Locomotion was studied 60
min after oral administration of 13 (0.1, 1.0, 3.0, 6.0, and 10 mg/kg,
po), 14 (0.1, 1.0, 3.0, and 10 mg/kg, po), 1 (10 mg/kg), or vehicle in
rats that were pretreated with reserpine (0.6 mg/kg, sc) 18 h earlier.
Animals were randomly assigned to treatment groups. Control rats
received the respective sc and po vehicles. Rats used were fasted, male
Wistar rats. Reserpine was diluted in 1% acetic acid in distilled water,
13 and 14 were diluted in 20% HPbCD, and 1 was diluted in 0.5%
methylcellulose. Reserpine produced a marked decrease in motor
activity. As shown in Figure 6, reserpine-induced akinesia was reversed
by 13 at 1.0, 3.0, 6.0, and 10 mg/kg, po, and by 14 at 1.0, 3.0, and 10
mg/kg, po. Akinesia was also reversed by 1 at 10 mg/kg as the positive
control. In Figure 7, each value represents the mean ( SEM) of the
total distance traveled of n = 16−18 rats per treatment group during
the 60 min measurement period in the behavioral test session.
Asterisks indicate significant differences as compared with the
reserpine + vehicle control group (*P < 0.05, **P < 0.01, and ***P
< 0.001, Hochberg test of multiple comparisons).
Rat 6-OHDA Lesion Model of Drug-Induced Rotation. The
neurotoxin 6-OHDA (12 μg) is microinjected unilaterally in the
medial forebrain bundle to produce a targeted degeneration of
midbrain DA neurons in the pars compacta of the substantia nigra.
Fasted, male Sprague−Dawley rats weighing 227−260 g at the time of
surgery were used in this study. 6-OHDA-induced neurotoxicity is
relatively selective for monoaminergic neurons because DA and
noradrenergic transporters preferentially take it up. 6-OHDA
accumulates in the cytosol of neurons and generates reactive oxygen
species, which then inhibit mitochondrial respiratory enzymes leading
to metabolic deficits and DA cell death. Degeneration of dopaminergic
neurons in the injected side of the brain is accompanied by
denervation-induced supersensitivity of postsynaptic DA receptors in
the striatum of the lesioned side. An imbalance in DA activity between
the two sides of the brain causes asymmetry in motor behavior that
can be enhanced by drug treatment. For example, drugs that stimulate
postsynaptic DA receptors produce and imbalance in DA signaling that
favors the lesion side and induces rotation behavior (turning) toward
the side opposite (contralateral to) the lesion side. Three weeks after
administration of 6-OHDA, rats were challenged with drugs acting on
the dopaminergic system and studied in a behavioral test chamber.
Each animal was tethered to high-resolution optical sensor connected
to an automated computerized system that quantifies circular motion.
A separate study was performed using the same model of
neuroleptic-induced catalepsy to examine the duration of action of
13 and 14 in mice. Catalepsy time was measured in haloperidol (1
mg/kg, sc)-treated mice (fasted, male Balb/c mice) after oral
administration of 13 (0 or 10 mg/kg), 14 (0 or 10 mg/kg), 1 (10
mg/kg), or vehicle. Compounds 1, 13, and 14 were diluted in 0.5%
methylcellulose. Catalepsy was measured repeatedly 30 min and 1, 2,
and 4 h after oral dosing of the respective groups. Each value
represents average ( SEM) time in cataleptic position of n = 8 mice
per treatment group during a 60 s test session. Asterisks indicate
significant differences as compared with the haloperidol + vehicle
control group (***P < 0.001, Dunnett's test of multiple comparisons).
Rat Catalepsy Study. Haloperidol, a neuroleptic medication that
inhibits D2 receptors, was used to induce catalepsy. In the rodent,
catalepsy is characterized as a loss of voluntary motion where limbs
remain in placed positions. The time to descend from the bar was
measured in haloperidol (1 mg/kg, sc)-treated male Sprague−Dawley
rats (fasted) after oral administration of 13 or 14 (0.10, 1.0, 3.0, or
10.0 mg/kg), 1 (10 mg/kg), or vehicle. Animals were randomly
assigned to treatment groups, and behavioral testing was performed
blind to treatment. Control rats received the respective sc and po
vehicles. Haloperidol was dissolved in 0.3% tartaric acid in 0.9% saline.
Compound 1 was diluted in 0.5% methylcellulose and dosed as a
solution. Compounds 13 and 14 were diluted in 20% HPβCD and
dosed as a solution. Compounds 1, 13, and 14 were administered
orally 30 min after haloperidol. Behavioral testing was conducted 1 h
after dosing of 1, 13, and 14. The behavioral test trial (maximum
duration of 180 s) began by placing the forepaws of male Sprague−
Dawley rats on a horizontal bar elevated 3.5 cm above the bench. The
cataleptic state was regarded as over, and the trial ended when the
animal came off the bar by either placing its forepaws on the bench or
climbing onto the bar with all of its limbs. Each value represents
average ( SEM) time in cataleptic position of n = 10−16 rats per
treatment group during a 180 s test session. Asterisks indicate
significant differences as compared with the haloperidol + vehicle
control group (***P < 0.001, Dunnett's test of multiple comparisons).
A separate study was performed using the same model of
neuroleptic-induced catalepsy to examine the duration of action of
14 in rats. Catalepsy time was measured in haloperidol (1 mg/kg, sc)-
treated rats (fasted, male Sprague−Dawley rats) after oral admin-
istration of 14 (0 or 1.0 mg/kg), 1 (10 mg/kg), or vehicle. Compound
1 was diluted in 0.5% methylcellulose and dosed as a solution.
Compound 14 was diluted in 20% HPβCD and dosed as a solution.
Compounds 1 and 14 were administered orally 30 min after
haloperidol. Catalepsy was measured repeatedly 15 min and 1, 2,
and 4 h after oral dosing of the respective groups. Each value
represents average ( SEM) time in cataleptic position of n = 10 rats
per treatment group during a 180 s test session. Asterisks indicate
significant differences as compared with the haloperidol + vehicle
control group (***P < 0.001, Dunnett's test of multiple comparisons).
Reserpine-Induced Akinesia Study in Mice. Reserpine is an
alkaloid that depletes monoamines by inhibiting their vesicular uptake,
resulting in a dramatic reduction of spontaneous locomotor activity
(akinesia). Efficacy is defined as reversal of reserpine-induced akinesia.
Locomotion was studied in open field activity boxes (L, 17.5 in; W,
17.5 in; and H, 12 in), each containing 20 equally spaced pairs of
horizontal infrared photocell beams along one axis. The activity was
measured automatically by a PC running the activity analysis software.
One activity count corresponded to the consecutive interruption of
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Article
One rotation count was defined as one 360° turn. To test the effects of
neuronal loss on drug responsiveness, motor asymmetry was examined
in a screening test with subcutaneous administration of a postsynaptic
DA agonist, apomorphine (0.05 mg/kg, sc). This test was repeated 1
week later and then followed 2 weeks later with a behavioral test to L-
DOPA (10 mg/kg, po) coadministered with carbidopa (2.5 mg/kg,
po). Animals showing an average response of at least 125 contralateral
rotations in 1 h after apomorphine were included in further studies.
Sixteen animals selected equidistant from the median L-DOPA
response were used to test the effects of compound 1 on rotational
behavior. To exclude a priming effect of L-DOPA, L-DOPA [10 mg/
kg, po, coadministered with carbidopa (2.5 mg/kg, po)] was
administered alone or with each dose of 13 or 14 (0.1, 1.0, 3.0, 6.0,
and 10.0 mg/kg, po) to all animals on separate testing days in a
randomized order, with each rat serving as its own control. The effects
of 13 and 14 alone were also studied in the model. This within-
subjects repeated measure design was carried out with at least two
nondrug days elapsing between drug-testing sessions. Control rats
received the respective po vehicles. Compounds 13 and 14 were
diluted in 20% HPβCD and dosed as solutions. L-DOPA and
carbidopa were diluted in 0.5% methylcellulose and coadministered as
suspensions. Each behavioral test session began with a 30 min interval
of acclimation in the test chamber, followed by administration of 13,
14, or vehicle and measurement of behavior for 60 min. Thereafter, the
behavioral effects of L-DOPA (coadministered with carbidopa) or
vehicle were measured for 120 min. Each value represents the mean
( SEM) of total contralateral rotations of n = 16 rats per treatment
group during the 2 h before and after L-DOPA administration.
Asterisks indicate significant differences as compared with the L-
DOPA + vehicle control treatment group (*P < 0.05, **P < 0.01, and
***P < 0.001) Tukey's test of multiple comparisons).
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: 215-628-7047. Fax: 215-540-4612. E-mail: bshook@its.
jnj.com.
ABBREVIATIONS USED
■
6-OHDA, 6-hydroxydopamine; MPTP, 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine; PD, Parkinson's disease; DA,
dopamine; L-DOPA, levodopamine; MAO-B, monoamine
oxidase type B; COMT, catechol O-methyl-transferase;
cAMP, cyclic adenosine monophosphate; PK, pharmacoki-
netics; PET, positron emission tomography; DMAP, 4-
(dimethylamino)pyridine
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