Dopamine D3/D2 Receptor Antagonist PF-4363467 Attenuates Opioid Drug-Seeking Behavior without Concomitant D2 Side Effects

Article abstract of DOI:10.1021/acschemneuro.6b00297

Dopamine receptor antagonism is a compelling molecular target for the treatment of a range of psychiatric disorders, including substance use disorders. From our corporate compound file, we identified a structurally unique D3 receptor (D3R) antagonist scaffold, 1. Through a hybrid approach, we merged key pharmacophore elements from 1 and D3 agonist 2 to yield the novel D3R/D2R antagonist PF-4363467 (3). Compound 3 was designed to possess CNS drug-like properties as defined by its CNS MPO desirability score (≥4/6). In addition to good physicochemical properties, 3 exhibited low nanomolar affinity for the D3R (D3 K_i = 3.1 nM), good subtype selectivity over D2R (D2 K_i = 692 nM), and high selectivity for D3R versus other biogenic amine receptors. In vivo, 3 dose-dependently attenuated opioid self-administration and opioid drug-seeking behavior in a rat operant reinstatement model using animals trained to self-administer fentanyl. Further, traditional extrapyramidal symptoms (EPS), adverse side effects arising from D2R antagonism, were not observed despite high D2 receptor occupancy (RO) in rodents, suggesting that compound 3 has a unique in vivo profile. Collectively, our data support further investigation of dual D3R and D2R antagonists for the treatment of drug addiction.

Full text of DOI:10.1021/acschemneuro.6b00297

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Article
Dopamine D3/D2 Receptor Antagonist PF-4363467 Attenuates
Opioid Drug-Seeking Behavior without Concomitant D2 Side Effects
Travis T Wager, Thomas A. Chappie, David Horton, Ramalakshmi Y. Chandrasekaran, Brian
M. Samas, Elizabeth Dunn-Sims, Cathleen Hsu, Nawshaba Nawreen, Michelle A Vanase-
Frawley, Rebecca E O'Connor, Christopher Schmidt, Keith Dlugolenski, Nancy C. Stratman,
Mark J. Majchrzak, Bethany L. Kormos, David Nguyen, Aarti Sawant-Basak, and Andy N. Mead
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00297 • Publication Date (Web): 07 Oct 2016
Downloaded from http://pubs.acs.org on October 12, 2016
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Dopamine D3/D2 Receptor Antagonist PFꢀ4363467
Attenuates Opioid DrugꢀSeeking Behavior without
Concomitant D2 Side Effects
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Travis T. Wager, Thomas Chappie, David Horton, Ramalakshmi Y. Chandrasekaran, Brian
Samas, Elizabeth R. Dunn-Sims, Cathleen Hsu, Nawshaba Nawreen, Michelle A. Vanase-
Frawley, Rebecca E. O'Connor, Christopher J. Schmidt, Keith Dlugolenski, Nancy C. Stratman,
Mark J. Majchrzak, Bethany L. Kormos, David Nguyen, Aarti Sawant-Basak, Andy N. Mead
Pfizer Worldwide Research and Development; Neuroscience Medicinal Chemistry and
Neuroscience Research Unit, 610 Main Street, Cambridge, MA 02139 (TTW, TC, CJS, KD,
NCS, BLK); Pharmacokinetics, Dynamics, and Metabolism, 610 Main Street, Cambridge, MA
02139 (DN, ASꢀB); Chemistry and Biology, Eastern Point Road, Groton, CT 06340 (RYC, BS
DH, ERDꢀS, CH, NN, MJM ANM, MAVꢀF, REO).
ABSTRACT: Dopamine receptor antagonism is a compelling molecular target for the
treatment of a range of psychiatric disorders, including substance use disorders. From our
corporate compound file we identified a structurally unique D3 receptor (D3R) antagonist
scaffold, 1. Through a hybrid approach, we merged key pharmacophore elements from 1 and D3
agonist 2 to yield the novel D3R/D2R antagonist PFꢀ4363467 (3). Compound 3 was designed to
possess CNS drugꢀlike properties as defined by its CNS MPO desirability score (≥4/6). In
addition to good physicochemical properties, 3 exhibited low nanomolar affinity for the D3R
(D3 K = 3.1 nM), good subtype selectivity over D2R (D2 K = 692 nM), and high selectivity for
i
i
D3R versus other biogenic amine receptors. In vivo, 3 doseꢀdependently attenuated opioid selfꢀ
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administration and opioid drugꢀseeking behavior in a rat operant reinstatement model using
animals trained to selfꢀadminister fentanyl. Further, traditional extrapyramidal symptoms (EPS),
adverse side effects arising from D2R antagonism, were not observed despite high D2 receptor
occupancy (RO) in rodents, suggesting that compound 3 has a unique in vivo profile.
Collectively, our data support further investigation of dual D3R and D2R antagonists for the
treatment of drug addiction.
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KEYWORDS: Substance use dependence, drug addiction, CNS MPO, dopamine D3 antagonist,
dopamine D2 antagonist, opioid, cessation, drugꢀseeking behavior, reinstatement, extrapyramidal
symptoms, locomotor.
a
Abbreviations: ADME, absorption, distribution, metabolism and excretion; CK1, casein kinase
1
; AUC, area under the curve; C , average concentration; C , efficacious concentration; Cmax,
ave eff
maximum concentration; CNS, central nervous system; CPP, conditioned place preference; DA,
dopamine; D2R, dopamine 2 receptor; D3R, dopamine 3 receptor; EPS, extrapyramidal
symptoms; HBD, hydrogen bond donor; HLM, human liver microsomes; ICSS, intracranial selfꢀ
stimulation; MDR, multiꢀdrug resistance; MPO, multiꢀparameter optimization; Pꢀgp, Pꢀ
glycoprotein; SAR, structureꢀactivity relationship; SBDD, structureꢀbased drug design.
Introduction
Drug addiction is characterized as compulsive, outꢀofꢀcontrol drug use despite negative
consequences including significant morbidity and mortality. Substance abuse is a global health
concern with major socioꢀeconomic ramifications, which has reached epidemic proportions. For
example, opioid dependency increased 74% from 1990 through 2010 and the economic cost
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attributable to illicit drug use in the United States alone is estimated to be over $500 billion
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, 2
annually.
Opioid dependency is particularly troubling in that it accounted for nearly half the
estimated 20 million disabilityꢀadjusted life years worldwide for illicit drug use in 2010 and
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more than half of the 78,000 deaths in 2010 due to illicit drug use. Furthermore, recent trends
suggest that opioid addiction afflicts young people (ages 15–29), a population that will likely
carry this burden of drug dependency throughout life. Given the continued rise in opioid
dependency, there is an urgent need to develop new medicines that can be used both to treat
addiction and to prevent relapse.
We recently disclosed that casein kinase 1 inhibitors (CK1i) attenuate opioidꢀseeking
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behavior in a rodent reinstatement model of fentanyl selfꢀadministration. While CK1 inhibition
is an attractive target for the treatment of drug addiction, dopamine (DA) receptor antagonism is
also a compelling molecular target, given the rich preclinical and clinical literature data on this
approach. In particular, the dopamine D3 receptor (D3R) has been pursued for more than a
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, 5
decade as a potential target for the treatment of substance use disorders, with numerous groups
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developing selective ligands targeting D3R.
Several lines of evidence provide a rationale to
target D3R and D2 receptors (D2R) for the treatment of substance abuse disorders. D3R and
D2R are distributed in key brain regions that regulate the rewarding, motoric, cognitive, and
emotional effects of opioids. D2R expression is widespread in the brain, with the highest
concentration found in the striatum, followed by the nucleus accumbens, globus pallidus,
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subtantia nigra and ventral tegmental area. Conversely, D3R is most abundant in the globus
pallidus, ventral pallidum/substantia innominata, and substantia nigra, which have all been
associated with motivational processing and are likely part of the neurocircuitry of addiction and
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relapse.
In contrast to D2R, D3R is virtually absent in the ventral tegmental area, which is
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associated with a rewarding response to lowꢀdose opioid injections. Blockade of D2R in the
ventral tegmental area may help reduce opioid use. Further, there is evidence that abnormal D2R
expression and function can provide a preꢀexisting vulnerability, in addition to arising as a
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consequence of substance abuse disorder. Coꢀexpression of D2R and D3R in many brain
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regions suggests a functional convergence of the signals mediated by these receptors. The brain
regions that express D2R and D3R have been shown to be important for motivation and reward,
as well as drugꢀmediated and cueꢀinduced relapse. Thus, the localization of these receptors in
these brain regions suggests a potential role for modulating addictive behaviors.
Repeated exposure to illicit drugs produces longꢀterm molecular and neurochemical
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changes in the brain in both humans and animals. Postmortem evaluation of the expression of
D3R in the brains of chronic cocaine users revealed an increased receptor density when
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compared with ageꢀmatched and drugꢀfree control subjects.
This observation of increased
receptor density has been extended to other drugs of abuse, including methamphetamine and
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alcohol.
The upꢀregulation of D3R in the addicted state suggests a role for D3R in the
neuroadaptive responses to addictive drugs, and implies that blocking D3R may reduce craving
and relapse rates.
Recent literature suggests that D3R/D2R ligands have potential efficacy in blocking
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drugꢀseeking behavior in both preclinical and clinical models of addiction.
Early work
examining the effect of D3R preferential agonists on cocaine selfꢀadministration in rats revealed
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the importance of D3Rs in the reinforcing effects of cocaine. Further research has shown that
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BP 897, a D3R partial agonist, doseꢀdependently reduced cocaine seeking; additional studies,
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however, have shown that this compound exhibits antagonism at D3R. More recently, Román
et al. reported that cariprazine, a 10ꢀfold selective D3R/D2R partial agonist, was able to reduce
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the rewarding effect of cocaine and to attenuate relapse to cocaineꢀseeking in rats. Further work
with antagonists has shown that SBꢀ277011ꢀA, a D3R/D2R antagonist, inhibits both cocaineꢀ
conditioned place preference (CPP) and reinstatement of cocaineꢀseeking behavior, as well as
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cocaineꢀinduced increases in brain stimulation reward. NGB 2904, a selective D3R antagonist,
was reported to inhibit cocaine selfꢀadministration under a progressive ratio schedule,
reinstatement of cocaineꢀseeking behavior, and enhancement of brain stimulation reward induced
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by cocaine, methamphetamine, heroin, and nicotine. Others have disclosed the discovery of
novel selective D3R antagonists and reported the potential utility of D3R antagonist in treating
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opioid addiction.
Finally, the selective D3R antagonist GSK598809 doseꢀdependently
attenuated nicotineꢀinduced CPP in rats; this effect translated into the clinic, where GSK598809
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transiently alleviated craving in smokers at subꢀmaximal receptor occupancy (72–89%). This
investigation provided the first clinical evidence supporting the hypothesis that D3R antagonism
would be effective in treating substance use disorders.
Given the growing preclinical and clinical data arising from subtypeꢀpreferring D3R/D2R
antagonists and selective D3R antagonists, antagonism of these receptors might be a promising
approach to treat substance abuse disorders and addiction. High DA receptor subtype selective
D3R antagonists are ideal for preclinical study of the specific role of D3R in addiction; however,
they may fall short of desired outcomes in the clinic due to the complex nature of addiction and
dopamine signaling. For this reason we pursued compounds that had the potential to engage
D3R at high occupancy levels, while simultaneously interacting with D2R at a wide range of
occupancies, given the potentially significant roles that both D3R and D2R play in addiction.
Based on the receptor occupancy achieved in the GSK598809 clinical study, and our knowledge
of D2Rꢀrelated EPS effects, our initial target was a compound that could achieve high D3
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receptor occupancy (>90%) with low D2 receptor occupancy (<10%). A second objective was to
establish whether it was possible to titrate in higher D2R occupancy and thereby achieve
improved efficacy in rodent models of substance use and relapse disorders. We hypothesized
that this dualꢀpharmacology strategy would improve efficacy by broadening the modulatory
influence over cueꢀreward processing pathways.
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Results and Discussion
Our reꢀentry into identifying subtypeꢀselective D3R antagonists began with a review of
our corporate file, which at the time included compounds from legacy Pfizer and Pharmacia &
Upjohn D3R and D2R antagonist and agonist programs; these analogues had been assayed for
binding and functional activity. Of the compounds that were in the file, PNUꢀ177864 (1)
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2
represented a structurally unique D3R antagonist scaffold (Table 1). Structurally, 1 had an aryl
sulphonamide, distinguishing it from many of the published selective D3R antagonists, which
3
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often contained an arylꢀpiperidine moiety. Compound 1 exhibited moderate affinity for D3R
and good selectivity over D2R (Table 1) and was shown to be a functional antagonist, as
measured by a human D3 SHꢀSY5Y GTPꢀγꢀS functional assay. The known in vivo
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2
phospholipidosis safety liability of 1 prompted us to identify an amine replacement.
Specifically, we wanted to reduce the pK of the basic amine and lower the lipophilicity of the
a
compound. This approach has been successfully applied in a histamine H3 receptor program to
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reduce phospholipidosis liability, and resulted in the identification of two clinical candidates.
At this point we revisited our corporate file, seeking other D3R ligands with predicted pK < 9
a
for the most basic center. PFꢀ592379 (2) was identified as a potential lead that retained affinity
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and selectivity for D3R over D2R, although functionally it was a full agonist (Table 1). The
physicochemical properties of 2 were significantly better than those of 1 (Table 2), thus allowing
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for considerable structural modification opportunities with which to switch the in vitro functional
activity from agonist to antagonist.
Hybridization of 1 and 2, along with application of traditional structure activity
relationships (SAR), led to the identification of PFꢀ4363467 (3) and its enantiomer PFꢀ4363476
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(not shown) (Table 1). Compound 3 exhibited single digit nM affinity for D3R (K = 3.1 nM)
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and greater than 100ꢀfold selectivity over D2R (K = 692 nM; Table 1). Further, 3 was an
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antagonist in the human D3 SHꢀSY5Y GTPꢀγꢀS functional assay and in the human D2 SHꢀSY5Y
cAMP stimulation assay (Table 1). Functional D2/D3 selectivity was in line with binding
selectivity, thus achieving our initial goal of a novel D3ꢀselective antagonist.
Table 1. Corporate file mining leads and hybrid compound 3.
D3 Ki
D2 Ki
(nM)
D2/
D3
D3 Functional
D2 Functional Functional
ID
1
a
a
b
b
(nM)
(nM)
(nM)
Activity
c
38.2
4480
117
>12
223
K = 22
K
i
> 33
Antagonist
i
c
(23.6 – 61.9) (3290 – 6090)
(10.1 ꢀ 48.9)
d
d
c
2
3
322
57.9 – 1790)
>4160
EC50 = 21
EC50 > 9,000
Agonist
(
3.1
2.47 – 3.99)
692
(539 – 890)
K = 1.1
K
i
= 464
Antagonist
i
c
(
(0.61 ꢀ 2.1)
(188 – 1147)
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Human dopamine D3 and D2 binding data reported as the geometric mean of at least 3
b
determinations. Human dopamine D3 and D2 functional data reported as the geometric mean of
c
d
at least 3 determinations. Data reported as mean antagonist activity with (95% CI). Data
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reported by Attkins et al. as agonist activity.
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The overall physicochemical properties of 3 are in line with marketed CNS drugs, with a
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CNS multiꢀparameter optimization (CNS MPO) desirability score of 4.0/6.0 (Table 2).
Improvements in ClogP, HBD, and pK were achieved relative to compound 1; most notable is
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the change in pK , which was lowered from 10.6 to 7.5. ClogD7.4 is still relatively high for 3
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(ClogD7.4 = 3.96, which may be a contributing factor in its high human liver microsome (HLM)
clearance (Table 3). Future work around this chemotype should focus on further lowering ClogP
and ClogD7.4, with a goal of improving CNS MPO desirability and aligning all ADME
attributes to desirable values.
Table 2. Summary of physicochemical properties and CNS MPO desirability
Compound 1
Component
Compound 2
Component
Compound 3
Component
Physicochemical
Property
Value
Score
Value
Score
Value
Score
MW
ClogP
TPSA
ClogD7.4
HBD
402
4.46
67.4
1.06
2
0.70
0.27
1.00
1.00
0.50
0.00
235
1.48
51.4
0.76
2
1.00
1.00
1.00
1.00
0.50
1.00
403
4.06
58.6
3.96
1
0.70
0.47
1.00
0.02
0.83
1.00
pKa
10.6
7.5
7.5
CNS MPO
3.5/ 6.0
5.5/ 6.0
4.0/ 6.0
a
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Calculated CNS MPO desirability scores were obtained using the published algorithm. Color
code for component score (x): red, x ≤ 0.33; yellow, 0.33 < x ≤ 0.66; green, x > 0.66.
In addition to good physicochemical properties and potency, compound 3 exhibited good
ꢀ6
passive permeability, with a Papp value of 8.4 × 10 cm/sec and predicted lowꢀtoꢀmoderate Pꢀgp
liability (Table 3). Brain availability (C /C ) in rats was 1.0 and free brain exposure of 3
b,u p,u
(
Cmax,b,u) at a 10 mg/kg s.c. dose was determined to be 23.7 nM. At a 10 mg/kg s.c. dose, 3 had
sufficient free drug exposure to cover the D3R K (∼7 fold). Cmax,b,u was further used to estimate
i
target occupancy (%) as:
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(
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where the dissociation constant K represents in vitro target binding potency. Using the binding
d
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K as a surrogate for K compound 3 was initially estimated to have maximum CNS target
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d,
occupancy of 88% for D3R and 5% for D2, after a 10 mg/kg s.c. dose.
Table 3. Summary of ADME properties for 3.
a
b
c
Rat Cmax,b,u Rat AUC0ꢀ∞ Rat AUC0ꢀ∞
Compound HLM
Papp
Pꢀgp
d
[nM]
C /C
b
C /C
b,u p,u
p
3
126
8.4
2.3
23.7
2.4
1.01
a
c
b
ꢀ6
Human liver microsomal clearance (ꢀL/min/mg). Passive permeability (Papp AB x 10 cm/sec).
d
MDR1 Efflux Ratio (BA/AB). Rat Cmax,b,u values are reported as maximum free drug
concentration in brain at a 10 mg/kg dose, s.c.
Compound 3 was further evaluated in a broad selectivity panel, where it demonstrated
high selectivity for D3R over receptors for other biogenic amines, ion channels, and enzymes
(Figure 1, see supplementary material, Table 1s for details). In this 129ꢀtarget selectivity panel,
3 inhibited 22 targets at >50% inhibition at 10 µM drug concentration. Of the offꢀtarget
activities tested in IC format, 3 had the highest affinity for CYP2C19 (110 nM, 34ꢀfold higher
5
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than the D3R binding IC ), followed by 5HT6 (990 nM, 309ꢀfold higher than the D3R IC ) and
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HT2A (1.4 µM, 437ꢀfold higher than the D3R IC ). Overall, compound 3 possessed an
50
excellent selectivity profile and was therefore progressed into ex vivo brain target occupancy
studies.
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Figure 1. A) Percent inhibition of a 129ꢀtarget promiscuity panel at 10 µM of compound 3.
Heat map colors for this continuum: green, 0% inhibition; yellow, 50% inhibition; red, 100%
inhibition. B) IC determination for 26 targets hitting in the promiscuity panel with inhibition
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50% for compound 3. Heat map colors continuum: green, 7,600 nM; yellow, 100 nM and red,
nM. IC determinations for the 10 highest affinity targets for compound 3 were: D3R, 4 nM;
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CYP2C19, 110 nM; D2R, 420 nM; D2S, 540 nM; 5HT6, 990 nM; 5HT2A, 1400 nM; CYP3A4b,
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300 nM; H3, 2400 nM; D1R, 2500 nM.
Using an ex vivo autoradiography technique with rat brain slices, compound 3 was
evaluated for both D3R and D2R occupancy (Table 4, Figure 2A, 2B). The 0.1 mg/kg dose
resulted in moderate D3R occupancy (33.6%) and low D2R occupancy (10.8%). High D3R
occupancy was achieved at the 3.2 mg/kg dose with near maximal occupancy (>95%) and
moderate D2R occupancy (34.7%). It was not until 32 mg/kg that D2R occupancy was over
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5%. The ex vivo receptor occupancy EC was determined to be 1.5 nM for D3R and 46.6 nM
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for D2R. The D3R occupancy EC50 is within 3ꢀfold of the binding K ; however, the D2R
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occupancy EC is 10ꢀfold leftꢀshifted when compared to the in vitro binding K . This was the
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first piece of data that suggested this compound does not behave like a traditional D3/D2
antagonist (i.e. eticlopride and sulpiride). The ex vivo D3R and D2R occupancy characteristics
of 3 represented a reasonable profile to test two hypotheses with one molecule, simply by
varying the dose. First, can a selective D3R antagonist (low dose) attenuate opioid drugꢀseeking
behavior or, second, is dual pharmacology required (high dose) to yield a robust in vivo
biological response?
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Table 4. D3R and D2R brain receptor occupancy from autoradiography techniques in rats
Treatment Cmax,b,u
D3 EVRO % RO
D2 EVRO %
Compound
a
b
(mg/kg)
(nM)
207.1
43.9
13.6
0.7
Observed
RO Observed
85.9 ± 1.7
50.6 ± 3.5
34.7 ± 1.5
10.8 ± 4.1
ꢀꢀꢀ
3
2
0
ꢀꢀꢀ
1
98.8 ± 2.4
99.9 ± 0.5
33.6 ± 4.5
6.3 ± 10.1
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0
.2
.1
3
0.032
0.2
EC50
1.5 ± 0.4 nM
46.6 ± 9.1 nM
a
b
Brain region evaluated: rat cerebellum (mean ± SEM, n = 3–4). Brain region evaluated: rat
striatum (mean ± SEM, n = 4). Ex vivo Receptor Occupancy (EVRO).
A)
B) 1
00
1
00
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Dose = 0.01
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3.2 10 mg/kg
Dose = 0.1
3.2 10
32 mg/kg
0.032
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-9
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-7
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10
10
10
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10
10
Free Brain Concentration (nM)
Free Brain Concentration (nM)
Figure 2. A) D2 (striatum) and B) D3 (cerebellum) brain receptor occupancy curves from the ex
vivo autoradiography protocol in rat. The gray and black data points represent actual measured
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RO and brain PK from individual animals. The solid line is the dataꢀfitted RO model. The D3R
binding was measured in cerebellum, while the D2R binding was measured in striatum.
Given the higherꢀthanꢀexpected D2R occupancy in the ex vivo receptor occupancy assay,
we were concerned that significant D2R side effects would be observed at low doses (≤10
mg/kg). Haloperidol and other D2R antagonists provoke prolactin secretion and elicit catalepsy
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in preclinical and clinical studies.
The induction of catalepsy in rat preclinical studies is
believed to be a predictor of extrapyramidal syndrome (EPS) in man. Further, it has been
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suggested that D2R antagonism may decrease locomotor activity. Prolactin serves as a marker
for D2R antagonism and the magnitude of response is roughly correlated with the occupancy of
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D2 receptors in the pituitary.
As expected, 3 (0.1–10 mg/kg) doseꢀdependently increases
prolactin release in rats (Figure 3A). At the highest dose tested in rats (10 mg/kg), 3 increases
plasma prolactin 9.3ꢀfold (p < 0.05). Unexpectedly, compound 3 had no significant effects on
spontaneous locomotor activity (Figure 3B) at the highest dose tested (32 mg/kg), where D2R
occupancy is 85.9%. Further, 3 did not produce a statistically significant cataleptic effect in rats
at any dose tested up to a dose of 32 mg/kg (Figure 3C). While this was surprising, there have
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been reports of selective D3R antagonists attenuating catalepsy induced by haloperidol. The
lack of significant locomotor and catalepsy side effects after administration of 3 make this a
unique compound, and we undertook further evaluations to determine whether there are
advantages of dosing 3 to both high D3R and D2R occupancy in models of addiction.
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Vehicle
Haldol, 1 mg/kg
Vehicle
A)
9.3 fold ↑ B)
C)
*
*
Haldol, 1 mg/kg
3, 1.0 mg/kg
3, 3.2 mg/kg
3, 10 mg/kg
3
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3
, 1.0 mg/kg
, 3.2 mg/kg
, 10 mg/kg
**
**
**
3, 32 mg/kg
3, 32 mg/kg
1.8 fold ↑
1
.5 fold ↑
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3
, Dose (mg/kg, SC)
Figure 3. A) Effect of 3 on prolactin. Data represent mean ± SEM, n = 5/group. B) Effects of 3
on total activity in a rat model of locomotor activity. Data represent mean ± SEM locomotor
activity over 30 minute time bins, n = 8/group. C) Effects of 3 in a rat model of catalepsy. Data
represents mean ± SEM seconds on bar demonstrating the effect of 3 (1.0–32.0 mg/kg, s.c.), n =
8
/group. ** indicates p < 0.01 compared to vehicle group.
To evaluate the utility of D3R/D2R antagonism in substance use disorder treatment, the
effect of 3 was assessed in both a cessation model of drugꢀtaking (Figure 4A) and a relapse
model of drugꢀseeking behavior (Figure 4B). With respect to cessation, administration of 3
reduced selfꢀadministration of fentanyl (Figure 4A), as indicated by a significant main effect of
dose (F(4, 312) = 23.93, p < 0.0001), a significant main effect of session (F(6,312) = 8.14, p < 0.0001)
and a significant dose by session interaction (F(24,312) = 2.64, p < 0.0001). Followꢀup analysis of
infusions for each test session by oneꢀway ANOVA revealed a significant main effect of dose
across sessions 1, 2, and 3 (F(4,37) = 7.47, p<0.001; F(4,37) = 6.89, p < 0.001; F(4,37) = 11.2, p <
0
.0001, respectively). Post hoc comparisons at each test session revealed a significant decrease
in infusions following pretreatment with 32 mg/kg of 3 for all three sessions (p < 0.001) and
when fentanyl was replaced with saline under the saline extinction condition for sessions 1 and 3
(
p < 0.05 and p < 0.01, respectively). Analysis of the average infusions obtained over three
consecutive test days in the fentanyl cessation model following treatment with 3 revealed a
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significant main effect of dose (F4,37 = 10.8, p < 0.0001) and post hoc comparisons indicated a
significant decrease in average infusions following pretreatment of 32 mg/kg and when fentanyl
was replaced with saline (saline extinction) compared to when fentanyl was available for selfꢀ
administration (vehicle; Figure 1s, supporting information). A trend for a reduction in fentanyl
selfꢀadministration was observed following 10 mg/kg of 3, but the effect did not reach statistical
significance (p = 0.08).
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To further determine the effect of 3 on fentanylꢀseeking behavior, the ability of 3 to
attenuate reinstatement of fentanyl seeking was assessed, following exposure to both a fentanyl
drug prime and fentanylꢀassociated cues. Analysis of results revealed a main effect of dose
(F(3,21) = 3.28; p < 0.05), a main effect of test (F(1,7) = 7.66; p < 0.05) and a significant dose by
test interaction (F(3,21) = 3.32; p < 0.05; Figure 4B). Post hoc comparisons revealed a significant
reinstatement effect following administration of 3.2 (t = −2.52; p < 0.05) and 10 mg/kg (t =
(7)
(7)
−2.94; p < 0.05) of 3 compared to baseline, with a strong trend for an increase following vehicle
pretreatment (p = 0.058). While the effect of vehicle on reinstatement was just outside
significance, normalization of the active lever responses to fentanyl intake obtained during the
last two sessions of fentanyl selfꢀadministration training (Figure 2s, supporting information),
revealed a main effect of dose (F(3,21) = 4.30; p < 0.05), a main effect of test (F(1,7) = 24.4; p <
0.01) and a significant dose by test interaction (F(3,21) = 4.70; p < 0.05; Figure 2s). Post hoc
analysis revealed a significant reinstatement effect following pretreatment with vehicle, 10
mg/kg of 3, or 3.2 mg/kg of 3 (p < 0.05). With respect to the effect of 3 on reinstatement of
fentanyl seeking, post hoc analysis revealed that 3 decreased active lever responses under
reinstatement test conditions at 32 mg/kg compared to vehicle. Analysis of inactive lever
responses failed to reveal a main effect of test, main effect of dose, or dose by interaction effect.
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At this dose it is expected that both the D3R and D2R receptors will be highly occupied, so dual
antagonism seems to be responsible for the observed activity. These results make a compelling
case for D3R/D2R antagonism in both the cessation and relapse phase of opioid addiction
treatment.
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A)
B)
**
Extinction
Vehicle
3
3
3
, 3.2 mg/kg
, 10 mg/kg
, 32 mg/kg
3
, Dose (mg/kg, SC)
Treatment
Figure 4. A) Effects of vehicle or 3 pretreatment on fentanyl selfꢀadministration over 3 test days
in the rat model of cessation. Data represent the average infusions of fentanyl across baseline
responding (fentanyl) before and after test sessions as well as three consecutive test sessions with
3
(Days 1–3). Saline extinction tests (Extinction) are also shown, in which saline was substituted
for fentanyl during test sessions. n = 9–11; *** p < 0.001. B) Effects of 3 on combined cue and
fentanyl primeꢀinduced reinstatement of fentanylꢀseeking behavior. Data represent mean ± SEM
active lever responses, with white bars representing baseline active lever responses and blue bars
representing active lever response on reinstatement test conditions. n = 8; ** p < 0.01
Conclusion
Via examination of compounds with D3R and D2R binding and functional data in our
corporate compound file, we identified the unique structural scaffold 1. Through a hybrid
approach, we merged key pharmacophore elements from D3 antagonist 1 and D3 agonist 2 to
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yield the novel D3R/D2R antagonist 3. Compound 3 possessed CNS drugꢀlike properties as
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defined by ADME attributes and CNS MPO desirability score (≥4/6).
In addition to good
physicochemical properties, 3 exhibited low nanomolar affinity for the D3R (D3 K = 3.1 nM)
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and good subtype selectivity over D2R (D2 K = 692 nM). The D3R functional potency of 3 was
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in line with its binding affinity, establishing 3 as an antagonist with a D3R functional K of 1.1
i
nM. Compound 3 exhibited excellent brain penetration in rats, with equivalent free drug levels
in plasma and brain compartments (C /Cp,u = 1). Given the higherꢀthanꢀexpected D2R
b,u
occupancy in the ex vivo receptor occupancy assay, we were concerned that significant D2R side
effects would be observed at low doses. When we examined 3 in models that evaluate D2Rꢀ
associated liabilities, we were surprised to discover that high D2R occupancy (86% RO) did not
result in either the locomotor or catalepsy side effects characteristic of traditional D2R
antagonists such as haloperidol and eticlopride which induce catalepsy in rodents at D2R
occupancies of >50%. While we do not fully understand why 3 was devoid of these side effects,
one potential explanation is that very high D3R occupancy counters these responses to moderate
D2 receptor blockade in specific neural pathways. An alternative explanation could be that 3 has
a unique binding mode in either D3R or D2R that differentially modulates in vivo functional
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signaling, compared to traditional D2R antagonists. Recent reports
suggest that D3/D2
dimerization is critical to the functional effects of D2R, allowing for the hypothesis that 3 is
binding in a unique way to this heteromer complex and thus changing the downstream signaling
events. Lastly, moderate affinity of 3 to 5HT2a could help explain the lack of locomotor or
catalepsy side effects, however, other compounds from this series devoid of 5HT2a activity have
similar in vivo profiles to 3. Ongoing studies, including the generation of a crystal structure of 3
interacting with either D3R or D2R, may provide insight into this potential unique binding pose,
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and single molecule fluorescence experiments could be used to determine if binding of 3 alters or
prevents dimerization of dopamine receptors, thereby differentially modulating in vivo
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functional effects. Inducedꢀfit docking of 3 into the published D3R Xꢀray crystal structure
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(
PDB ID: 3PBL) suggests the possibility of two binding modes. Neither of these binding
modes is fully predictive of the SAR and selectivity in this series, however, nor is it clear what
could potentially disrupt D3/D2 dimerization from either of these binding poses; the true binding
mode of 3 to D3R or D2R therefore remains unclear.
Compound 3 is well tolerated in animals at high D3R and D2R occupancy, allowing for
the full examination of a range of both D3R and D2R occupancy of 3 on cessation of drugꢀtaking
and relapse to drugꢀseeking. The current study demonstrated a connection between D3R/D2R
antagonism and attenuation of drugꢀseeking behavior, with compound 3 decreasing fentanyl selfꢀ
administration (cessation) and robustly attenuating the reinstatement of fentanylꢀseeking
behavior under combined cue and prime conditions. Drugꢀseeking behavior can be characterized
by the acquisition and maintenance of drugꢀtaking behavior and the relapse to drugꢀseeking
behavior, consisting of forced abstinence from drugꢀtaking and a reinstatement/relapse event
triggered by a drug prime or drug cue presentation (Figure 5). In the current model, the proposed
efficacy of a D3/D2 receptor antagonist is highlighted by a potential benefit in both cessation of
a drugꢀtaking (as evidenced by the decrease in fentanyl selfꢀadministration) as well as a
prevention of relapse (as evidenced by a decrease in fentanyl reinstatement) following
pretreatment with compound 3. Considering the increased efficacy observed following
administration of 32 mg/kg relative to 10 mg/kg, the current results suggest that addition of D2R
antagonism is beneficial in driving the efficacy of reducing drugꢀseeking behavior compared to
D3R antagonism alone. A potential alternative interpretation of these results is that compound 3
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is inducing a nonꢀspecific effect on motor function or general motivation, resulting in decreased
fentanyl selfꢀadministration and reinstatement, rather than specifically attenuating fentanylꢀ
seeking. However, results from the current study demonstrate that compound 3 does not alter
locomotor activity up to 32 mg/kg. Furthermore, the effect of compound 3 in an intracranial selfꢀ
stimulation (ICSS) model designed to understand potential anhedonic effects of compounds
revealed no change in ICSS behavior at efficacious doses, which suggests a specific effect on
fentanylꢀassociated motivation.. Thus, given that the rat reinstatement model of drug relapse
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possesses high predictive validity for efficacy in substance use disorders, these data suggest
that dual D3/D2 receptor antagonists warrant further investigation in clinical studies examining
both the drug cessation and relapse phases of substance use disorder treatment.
Figure 5. General depiction of the experiment. Selective D3/D2 antagonist 3 attenuates
fentanylꢀseeking behavior in models of both cessation of fentanyl selfꢀadministration and
reinstatement (as a model of relapse).
In summary, compound 3 is an excellent preclinical tool to functionally probe the
biology of D3R and D2R in vivo. While it does not meet our guidelines [C ≤ 250 nM (total
eff
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drug)] to move into preclinical in vivo safety testing, the discovery of 3 is a significant
27
advancement in the development of D3R/D2R antagonists for the treatment of drug addiction.
The unique in vivo profile of 3 makes it a promising candidate for preclinical testing of D3/D2
antagonism for multiple substance use disorders.
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METHODS
Physicochemical Properties and Data Analysis. For the work herein, calculated CNS MPO
3
7
desirability scores were obtained using the published algorithm,
and calculated
physicochemical properties were obtained using standard commercial packages: Biobyte for
53
ClogP calculations; for calculation of TPSA, see Ertl. Statistical analyses were carried out
using SigmaPlot version 11.0, from Systat Software, Inc., San Jose California USA,
www.sigmaplot.com .
ADME Data. Data on the following in vitro ADME properties were obtained from the company
database. All assays were performed via reported methods as described previously for: (a)
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passive apparent permeability, P , assay; (b) Pꢀglycoprotein (Pꢀgp) efflux liability assay; (c)
app
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metabolic stability, expressed as unbound intrinsic clearance (CLint,u);
and (d) plasma protein
binding, F mouse and human.
u
Chemistry
The synthesis of 3 begins with commercially available starting materials, as illustrated in Scheme
1. Conversion of 3ꢀmethylꢀ4ꢀnitrobenzoic acid (4) to the corresponding acid chloride with
thionyl chloride and catalytic DMF gave 3ꢀmethylꢀ4ꢀnitrobenzoyl chloride (5) in 91% crude
yield (500 g). Treatment of crude 5 in acetonitrile/ THF with (trimethylsilyl)diazomethane
generated the αꢀdiazoketone in situ, which by addition of 40% aq. HBr was converted to 2ꢀ
bromoꢀ1ꢀ(3ꢀmethylꢀ4ꢀnitrophenyl)ethanone (6) in 93% isolated yield (600 g). Morpholinꢀ2ꢀol 7
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5
5
5
5
5
6
was prepared in 52% isolated yield (320 g) via bromide displacement of αꢀbromoketone 6 with
2ꢀ(ethylamino)ethanol and subsequent cyclization to the lactol. Hydrodehydroxylation of 7 with
TFA/triethylsilane in CH Cl yielded 4ꢀethylꢀ2ꢀ(3ꢀmethylꢀ4ꢀnitrophenyl)morpholine (8) (200 g)
2
2
0
1
2
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4
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8
9
0
in 66% isolated yield. Reduction of the nitro group in 8 to the corresponding amine was
accomplished via Raney nickel hydrogenation in MeOH to give 4ꢀ(4ꢀethylmorpholinꢀ2ꢀyl)ꢀ2ꢀ
methylaniline (9) in 63% isolated yield (110 g). Completing the sequence, racemic sulfonamide
10 was prepared in 99% isolated yield (20 g) by treatment of aniline 9 with commercially
available 4ꢀisopropylbenzeneꢀ1ꢀsulfonyl chloride in pyridine. Preparative chiral chromatography
of this sulfonamide, followed by HCl salt formation, afforded the enantiomerically pure title
compound
(ꢀ)ꢀNꢀ{4ꢀ[(2S)ꢀ4ꢀethylmorpholinꢀ2ꢀyl]ꢀ2ꢀmethylphenyl}ꢀ4ꢀ(propanꢀ2ꢀ
yl)benzenesulfonamide (3).
Scheme 1: Synthesis of D3R/D2R antagonist 3.
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O
O
O
a
b
Br
OH
Cl
O N
O N
O N
2
2
2
4
5
6
10
11
12
13
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N
O
N
O
N
O
HO
c
d
e
O N
O N
H N
2
2
2
7
8
9
N
O
N
O
O
O
O
O
f
g
S
S
N
N
H
H
HCl
10
3 (PF-4363467)
Reagents and conditions: a) thionyl chloride, DMF; b) (trimethylsilyl)diazomethane, ACN/THF,
then 40% HBr; c) 2ꢀ(ethylamino)ethanol, Et N, CH Cl ; d) triethylsilane, TFA, CH Cl ; e) H ,
3
2
2
2
2
2
Raney Ni, MeOH; f) 4ꢀisopropylbenzeneꢀ1ꢀsulfonyl chloride, pyridine; g) chiral
chromatographic separation, then HCl/EtOAc/Et O.
2
Chemistry Experimental Section. General Information. All solvents and reagents were
obtained from commercial sources and were used as received. All reactions were followed by
TLC (TLC plates F254, Merck) or LCMS (liquid chromatographyꢀmass spectrometry) analysis.
1
3
Proton and C NMR spectra were obtained using deuterated solvents on a Varian 400 MHz
instrument. All proton shifts are reported in δ units (ppm) downfield of TMS (tetramethylsilane)
and were measured relative to the signals for chloroform (7.27 ppm) and methanol (3.31 ppm).
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13
All C shifts are reported in δ units (ppm) relative to the signals for chloroform (77.0 ppm) and
1
methanol (49.1 ppm) with Hꢀdecoupled observation. NMR abbreviations are as follows: br,
broadened; s, singlet; d, doublet; t, triplet; q, quartet; p, pentuplet; m, multiplet; dd, doublet of
doublets; dddd, doublet of doublet of doublet of doublets; sept, septuplet; tt, triplet of triplets.
Analytical analyses by UPLC were performed on a Waters Acquity system with PDA detection
0
1
2
3
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(
UV 210 nm) at 45 ºC, flow rate 0.5 mL/min, with a 95/5 buffer/acetonitrile (0 to 7.55 min),
1
0/90 buffer/acetonitrile (7.55 to 7.85 min) using the following columns and buffers: Waters
BEH C8 column (2.1 x 100 mm, 1.7 µm) with 50 mM sodium perchlorate/0.1% phosphoric acid
or 10 mM ammonium bicarbonate as buffer; Waters BEH RP C18 column (2.1 x 100 mm, 1.7
µm) or Waters HSS T3 (2.1 x 100 mm, 1.8 µm) column with 0.1% methanesulfonic acid buffer.
All melting points are uncorrected. Elemental analyses were performed by QTI Development.
Mass spectra were recorded on a Micromass ADM atmospheric pressure chemical ionization
instrument (MS, APCI). High resolution mass spectra were obtained on an Agilent LCꢀMS TOF
equipped with a Zorbax Eclipse column (50 mm x 4.6 mm, 1.8 µm XDBꢀC18) using 0.1%
aqueous formic acid as mobile phase A1 and acetonitrile containing 0.1% formic acid as mobile
phase B1. Column chromatography was carried out on silica gel 60 (32–60 mesh, 60 Ǻ) or on
preꢀpacked Biotage™ columns. The purities of final compound 3 and 4–10 as measured by
UPLC were found to be above 95%.
Preparation of 2ꢀbromoꢀ1ꢀ(3ꢀmethylꢀ4ꢀnitrophenyl)ethanone (6). A mixture of 3ꢀmethylꢀ4ꢀ
nitrobenzoic acid (4) (500.0 g, 2.8 mol), DMF (10 mL) and thionyl chloride (2.0 L, 26.8 mol)
was refluxed for 10 h. The thionyl chloride was removed in vacuo and the residue was reꢀ
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concentrated from heptane three times to yield 3ꢀmethylꢀ4ꢀnitrobenzoyl chloride (5) (500.0 g,
91%) as a brown oil which was used without purification. For analytical purposes, a portion of
1
this material was recrystallized from heptane to give a light yellow solid: H NMR (400 MHz,
10
11
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59
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CDCl ) δ ppm 8.05 ꢀ 8.08 (m, 2H), 7.98 (d, J = 8.8 Hz, 1H), 2.63 (s, 3H). The crude acid
3
chloride (500.0 g, 2.5 mol) was dissolved in CH CN/THF (1:1, 8.0 L) and cooled to 0 ºC. A
3
solution of 2 M (trimethylsilyl)diazomethane in hexanes (1.5 L, 3.0 mol) was added dropꢀwise.
The resulting mixture was warmed to rt and stirred for 1 h. The reaction was reꢀcooled to 0 ºC
and 40% aq. HBr (1.0 kg, 5.0 mol) was added dropꢀwise. After stirring at rt for 30 min, the
reaction was concentrated in vacuo to yield 2ꢀbromoꢀ1ꢀ(3ꢀmethylꢀ4ꢀnitrophenyl)ethanone (6)
1
(600.0 g, 93.0%) as a brown solid. H NMR (400 MHz, CDCl ) δ ppm 7.89 ꢀ 8.05 (m, 3H), 4.45
3
13
(
s, 2H), 2.66 (s, 3H); C NMR (100 MHz, CDCl ) δ ppm 190.2, 152.3, 137.0, 134.2, 133.5,
3
+
127.6, 125.2, 30.5, 20.3; HRMS m/z (+TOF) [M + H] calcd. for C H NO S, 257.9760; found,
9
8
3
257.9753.
Preparation of 4ꢀethylꢀ2ꢀ(3ꢀmethylꢀ4ꢀnitrophenyl)morpholinꢀ2ꢀol (7). 2ꢀBromoꢀ1ꢀ(3ꢀmethylꢀ
ꢀnitrophenyl)ethanone (6) (600.0 g, 2.3 mol), 2ꢀ(ethylamino)ethanol (228 mL, 2.6 mol) and
4
Et N (259 g, 2.6 mol) in dichloromethane (3.5 L) were stirred at rt for 16 h. The reaction mixture
3
was washed with brine (1.5 L), and the aqueous layer was extracted with ethyl acetate (3×3 L).
The combined organic layers were dried over MgSO and concentrated. The residue was purified
4
by silica gel chromatography (pet. ether/EtOAc 10:1) to give 4ꢀethylꢀ2ꢀ(3ꢀmethylꢀ4ꢀ
1
nitrophenyl)morpholinꢀ2ꢀol (7) (320 g, 52.0%) as a brown oil. H NMR (400 MHz, CDCl ) δ
3
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ppm 7.96 (d, J = 8.4 Hz, 1H), 7.57 ꢀ 7.64 (m, 2H), 4.95 (br s, 1H), 4.18 (ddd, J = 11.9, 11.9, 2.9
Hz, 1H), 3.86 (dd, J = 11.6, 3.6 Hz, 1H), 2.76 ꢀ 2.85 (m, 2H), 2.61 (s, 3H), 2.41 ꢀ 2.53 (m, 2H),
13
2
.32 (ddd, J = 11.7, 11.7, 3.8 Hz, 1H), 2.19 (d, J = 10.9 Hz, 1H), 1.09 (t, J = 7.2 Hz, 3H); C
0
1
2
3
4
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8
9
0
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8
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0
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8
9
0
1
2
3
4
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0
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2
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8
9
0
NMR (100 MHz, CDCl ) δ ppm 149.1, 147.0, 133.6, 130.6, 124.8, 94.4, 63.4, 60.9, 52.3, 52.0,
3
+
20.8, 11.9; HRMS m/z (+TOF) [M + H] calcd. for C H N O , 267.1339; found, 267.1331.
13
18
2
4
Preparation of 4ꢀethylꢀ2ꢀ(3ꢀmethylꢀ4ꢀnitrophenyl)morpholine (8). A mixture of 4ꢀethylꢀ2ꢀ(3ꢀ
methylꢀ4ꢀnitrophenyl)morpholinꢀ2ꢀol (7) (320 g, 1.2 mol), triethylsilane (720 mL, 6.0 mol) and
TFA (3.2 L) in CH Cl (6.0 L) was stirred at rt for 72 h. Saturated aq. K CO was added until the
2
2
2
3
pH was 10. The organic layer was separated and the aqueous layer was extracted with ethyl
acetate (3x1.5 L). The combined organic layers were dried over MgSO and concentrated, and
4
the residue was purified by silica gel chromatography (petroleum ether) to yield 4ꢀethylꢀ2ꢀ(3ꢀ
methylꢀ4ꢀnitrophenyl)morpholine (8) (200 g, 66.0%) as a brown oil. This material was suitable
for use without further purification. For analytical purposes, a portion was converted to the
hydrochloride salt and recrystallized from CHCl / EtOAc, yielding a light orangeꢀtinged solid:
3
1
H NMR (400 MHz, CDCl ) δ ppm 13.54 (br s, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.36 ꢀ 7.42 (m,
3
2H), 5.44 (d, J = 9.4 Hz, 1H), 4.53 ꢀ 4.61 (m, 1H), 4.23 (dd, J = 13.1, 3.1 Hz, 1H), 3.57 (t, J =
10.9 Hz, 2H), 3.10 ꢀ 3.18 (m, 2H), 2.86 ꢀ 2.98 (m, 1H), 2.56 ꢀ 2.65 (m, 4H), 1.54 (t, J = 7.2 Hz,
13
3
H); C NMR (100 MHz, CDCl ) δ ppm 149.2, 141.8, 134.3, 130.3, 125.3, 124.3, 73.8, 63.6,
3
+
5
6.2, 52.9, 50.4, 20.4, 8.9; HRMS m/z (+TOF) [M + H] calcd. for C H N O , 251.1390;
13 18
2
3
found, 251.1387.
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Preparation of 4ꢀ(4ꢀethylmorpholinꢀ2ꢀyl)ꢀ2ꢀmethylaniline (9). A mixture of 4ꢀethylꢀ2ꢀ(3ꢀ
methylꢀ4ꢀnitrophenyl)morpholine (8) (200 g, 0.80 mol) and Raney nickel (20 g) in MeOH (5.0
L) was stirred under 1 atm of H at rt for 24 h. The mixture was filtered and washed with MeOH
2
(3x100 mL). The filtrate was concentrated and the residue was purified by chromatography on
silica gel (petroleum ether/EtOAc 5:1) to give 4ꢀ(4ꢀethylmorpholinꢀ2ꢀyl)ꢀ2ꢀmethylaniline (9)
1
(
110 g, 62%) as a brown oil: H NMR (400 MHz, CDCl ) δ ppm 7.07 (s, 1H), 7.03 (dd, J = 8.1,
3
1.9 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 4.45 (dd, J = 10.4, 2.3 Hz, 1H), 4.02 (ddd, J = 11.4, 3.4,
1.4 Hz, 1H), 3.83 (ddd, J = 11.5, 11.5, 2.3 Hz, 1H), 3.59 (br s, 2H), 2.91 (ddd, J = 11.5, 2.0, 2.0
Hz, 1H), 2.81 (dd, J = 11.3, 1.8 Hz, 1H), 2.45 (q, J = 7.1 Hz, 2H), 2.15 ꢀ 2.23 (m, 4H), 2.05 (dd,
1
3
J = 11.4, 10.4 Hz, 1H), 1.11 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ ppm 144.5,
3
130.8, 128.7, 125.3, 122.4, 114.9, 78.5, 67.4, 60.5, 52.9, 52.8, 17.6, 12.0; HRMS m/z (+TOF) [M
+
+
H] calcd for C H N O; 221.1648, found, 221.1646.
1
3
20
2
N
O
O
O
N
N
O
O
O
O
f
g
S
S
N
N
H N
2
H
H
HCl
9
1
0
3 (PF-4363467)
Preparation
of
(ꢀ)ꢀNꢀ{4ꢀ[(2S)ꢀ4ꢀethylmorpholinꢀ2ꢀyl]ꢀ2ꢀmethylphenyl}ꢀ4ꢀ(propanꢀ2ꢀ
yl)benzenesulfonamide (3). 4ꢀIsopropylbenzeneꢀ1ꢀsulfonyl chloride (8.9 mL, 49.9 mmol) was
added to an ice cooled solution of 4ꢀ(4ꢀethylmorpholinꢀ2ꢀyl)ꢀ2ꢀmethylaniline (9) (11.0 g, 49.9
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mmol) in pyridine (106 mL) and the resulting reaction mixture was slowly warmed to rt. After
2
stirring overnight, the reaction was concentrated and the residual oil was dissolved in CH Cl2
and washed with 1 N NaOH, dried over MgSO , and concentrated to yield 28 g of crude Nꢀ[4ꢀ(4ꢀ
4
0
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3
4
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ethylmorpholinꢀ2ꢀyl)ꢀ2ꢀmethylphenyl]ꢀ4ꢀ(propanꢀ2ꢀyl)benzenesulfonamide. Chromatography on
a 350 g ISCO cartridge using EtOAc and 5% EtOH / EtOAc gave 20 g (>99%) of purified
1
material as an amorphous glass: H NMR (CDCl ) δ 7.66 (d, J = 8.5 Hz, 2H), 7.28 ꢀ 7.23 (m,
3
3H), 7.12 ꢀ 7.08 (m, 2H), 4.49 (dd, J = 10.3, 1.9 Hz, 1H), 4.5 ꢀ 3.95 (m, 1H), 3.82 (ddd, J = 11.6,
11.2, 2.5 Hz, 1H), 2.94 ꢀ 2.78 (m, 3H), 2.50 ꢀ 2.42 (m, 2H), 2.24 ꢀ 2.15 (m, 1H), 2.02 ꢀ 1.96 (m,
13
1
H), 1.97 (s, 3H), 1.26 ꢀ 1.20 (m, 6H), 1.05 (t, J = 9.5 Hz, 3H); C NMR (CDCl ) δ 154.7,
3
138.4, 134.2, 131.6, 129.2, 128.7, 127.5, 127.3, 125.0, 124.4, 67.3, 60.2, 52.8, 52.7, 34.5, 34.3,
+
23.8, 17.8, 11.8; LRMS m/z calcd for C H N O S, 402.56; found, 403.3 [M + H] , (APCI); R
2
2
30
2
3
f
=
0.4 (10% EtOH / EtOAc). The enantiomers of Nꢀ[4ꢀ(4ꢀethylmorpholinꢀ2ꢀyl)ꢀ2ꢀmethylphenyl]ꢀ
4ꢀ(propanꢀ2ꢀyl)benzenesulfonamide were separated by preparative chiral chromatography (10
cm x 50 cm Chiralpak AD column using 75/25 heptane/EtOH as mobile phase with a flow rate
of 500 mL/ min) to yield 7.7 g of enantiomer #1 (3, r.t. = 8.49 min; [α] = −3.025, c = 1.20 in
D
MeOH) and 7.7 g of enantiomer #2 (PFꢀ04363476; r.t. 13.43 min). The HCl salt of enantiomer
#1 (3) was prepared in EtOAc (150 mL) by adding 1 eq. of 2 N HCl/ether followed by ether (150
mL). The resulting slurry was stirred overnight, filtered, dried under N and evacuated overnight
2
1
to yield 7.7 g of the mono HCl salt of 3 as a light yellowꢀtinted solid: mp = 218–220 ºC; H
NMR (400 MHz, CDCl ) δ ppm 13.05 (br s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.28 ꢀ 7.30 (m, 3H),
3
7.10 ꢀ 7.12 (m, 2H), 7.06 (s, 1H), 5.20 (dd, J = 10.7, 1.6 Hz, 1H), 4.45 ꢀ 4.54 (m, 1H), 4.15 (dd, J
=
13.0, 3.4 Hz, 1H), 3.48 ꢀ 3.56 (m, 2H), 3.09 ꢀ 3.17 (m, 2H), 2.83 ꢀ 2.99 (m, 2H), 2.60 ꢀ 2.68
13
(
m, 1H), 2.03 (s, 3H), 1.50 (t, J = 7.3 Hz, 3H), 1.24 (d, J = 6.9 Hz, 6H); C NMR (100 MHz,
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CDCl ) δ 154.7, 137.0, 135.3, 134.3, 132.1, 128.7, 127.2, 127.1, 124.5, 124.4, 74.4, 63.6, 56.3,
3
5
2.8, 50.5, 34.1, 23.6, 17.7, 8.8; Anal. calcd. for C H N O S•HCl: C, 60.19; H, 7.12; N, 6.38;
22 30 2 3
Found: C, 60.32; H, 7.37, N, 6.26. The absolute stereochemistry was determined using small
molecule crystallography. Pertinent crystal, data collection and refinement information is
summarized in the small molecule report, supplementary material.
In vitro Materials.
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Binding Assay.
Binding assays were performed using overꢀexpressing human D2 and D3 CHO cell lines. To
determine basic assay parameters, ligand concentrations were determined from saturation
3
binding studies, where the K using [ H]ꢀspiperone (PerkinElmer NET1187250UC) for D2
d
3
binding was determined to be 1.6 nM and a K of 1.4 nM was determined for hD3 using [ H]ꢀ7ꢀ
d
OHꢀDPAT (PerkinElmer NET1169250UC). From tissue concentration curve studies, the
optimal amount of tissue was determined to be 4 mg/mL for hD2 and 7 mg/mL for hD3 per 96ꢀ
well plate. These ligand and tissue concentrations were used in timeꢀcourse studies to determine
linearity and equilibrium conditions for binding. It was found that binding was at equilibrium
with the specified amount of tissue in 20 minutes at 37 °C for both receptors. From these
parameters, K values were determined by homogenizing the specified amount of tissue for each
i
receptor in 50 mM Tris (pH 7.4 at 4 °C) containing 2.0 mM MgCl , and spinning in a centrifuge
2
at 40,000 x g for 10 minutes. The pellet was resuspended in either the D2 assay buffer [50 mM
Tris (pH 7.4 @ 37 °C), 100 mM NaCl and 1 mM MgCl ] or the D3 assay buffer [50 mM Tris
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(pH 7.4 at 37 °C), 120 mM NaCl, 5 mM MgCl , 5 mM KCl and 2 mM CaCl ]. Incubations were
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initiated by the addition of 200 µL of tissue to 96ꢀwell plates containing test drugs (2.5 µL) and
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.6 nM [ H]ꢀspiperone for D2 or 1.5 nM of [ H]ꢀ7ꢀOHꢀDPAT for D3 (both 50 µL) for a final
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assay volume of 250 ꢀL. Nonꢀspecific binding was determined by radioligand binding in the
presence of a saturating concentration of haloperidol. After the 20 minute incubation period at
37 °C, assay samples were rapidly filtered through Unifilterꢀ96 GF/B PEIꢀcoated filter plates and
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rinsed with iceꢀcold 50 mM Tris buffer (pH 7.4 at 4 °C). Membrane radioligand levels were
determined by liquid scintillation counting of the filter plates in 50 µL EcoLume. The IC value
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(concentration at which 50% inhibition of specific binding occurs) was calculated by linear
regression of the concentrationꢀresponse data in Activity Base (IDBS). K values were calculated
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according to the ChengꢀPrusoff equation.
D2 Functional Assay. cAMP Accumulation Assay in SHꢀSY5Y hD2L Cells:
Human D2Lꢀtransfected SHꢀSY5Y cells were grown at 37 °C and 5% CO in DMEM/F12
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medium supplemented with 10% dialyzed FBS and 100 ꢁg/mL G418. The cells were frozen as
assayꢀready vials and thawed immediately prior to the assay. On the day of the experiment,
compounds dissolved in DMSO were diluted in pH 7.4 assay buffer containing PBS, 5 µM
HEPES, and 100 µM IBMX (final concentrations). The vial of frozen cells was rapidly thawed
in a 37 °C water bath. Cells were washed with PBS and centrifuged to obtain a pellet. The
pellet was resuspended in warm PBS and the cells were allowed to sit for 5 minutes, prior to
counting and dilution. The reaction was initiated by addition of the cells into 384ꢀwell plates
containing test compounds; the final number of cells used in the assay was 10000–15000 cells
per well. Cells and compounds were preꢀincubated together for 10 minutes at 37 °C. NKH477
was then added to the plates at a final concentration of 3 µM to activate adenylyl cyclase and
increase basal levels of cAMP. For testing compounds in agonist mode, assay buffer was added
to the plate; in antagonist mode, a D2 agonist was added to produce an 80% agonist effect
(quinelorane, 32 nM). Plates were then returned to the 37 °C incubator and incubated for 15
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minutes at 37 °C. To stop the reaction, Cisbio cAMP Dynamic 2 screening kit reagents (cat#
62AM4PEB) were added to the plate. Homogenous timeꢀresolved fluorescenceꢀbased cAMP
(Schering) detection was determined according to the manufacturer's instruction. A Wallac
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EnVision was used to measure HTRF (excitation 320 nm, emission 665 nm/620 nm, delay time
50 ꢁs, window time 400 ꢁs). Data was analyzed based on the ratio of fluorescence intensity of
each well at 620 nm and 665 nm followed by cAMP quantification using a cAMP standard
curve. Agonist activity of a compound was measured by its ability to decrease the cAMP
production elicited by NKH477. The agonist effect was normalized to the % effect of 1 µM
quinelorane. Antagonist activity was measured by a compound’s ability to reverse the agonistꢀ
induced decrease of cAMP (in the presence of NHK477). The antagonist effect was normalized
to the % effect of NHK477 alone.
D3 Functional Assay. GTPꢀγꢀS Binding Assay in SHꢀSY5Y hD3 Cells: SHꢀSY5Y hD3 cells
were grown at 37 °C and 5% CO2 in DMEM/F12 supplemented with 10% dialyzed FBS and 100
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g/mL G418. Cell paste was frozen in a pellet and stored at −80 °C until ready for use. The day
of the assay, cell paste was homogenized using a glassꢀTeflon homogenizer in buffer containing
50 mM HEPES, 1 mM EDTA and protease inhibitors (pH 7.4, 4 °C). The membranes were
centrifuged at 40000 x g for 10 minutes. The pellet was resuspended in pH 7.4 assay buffer (20
mM HEPES, 100 mM NaCl, 30 mM MgCl ). Saponin and guanosine diphosphate (44 µg/mL
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and 30 µM final assay concentrations, respectively) were added to the membrane solution. The
membranes were incubated at 4 °C for 1 hour to promote GDP binding. Membranes were then
added to test compounds (in DMSO) in the assay plate and incubated for 30 minutes at 30 °C.
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S] GTPꢀγꢀS was added to the assay plate (final assay concentration of 1 nM) and incubated
another 30 minutes at 30 °C. To stop the reaction, assay samples were rapidly filtered through
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