Discovery of a Potent, Selective, and Brain-Penetrant Small Molecule that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake

Article abstract of DOI:10.1021/acs.jmedchem.8b00566

The orphan G-protein-coupled receptor GPR88 is highly expressed in the striatum. Studies using GPR88 knockout mice have suggested that the receptor is implicated in alcohol seeking and drinking behaviors. To date, the biological effects of GPR88 activatio

Full text of DOI:10.1021/acs.jmedchem.8b00566

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Article
Discovery of a Potent, Selective, and Brain-Penetrant Small-Molecule
that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake
Chunyang Jin, Ann M. Decker, Viren H. Makhijani, Joyce
Besheer, Emmanuel Darcq, Brigitte L. Kieffer, and Rangan Maitra
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00566 • Publication Date (Web): 16 Jul 2018
Downloaded from http://pubs.acs.org on July 16, 2018
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Discovery of a Potent, Selective, and Brain-Penetrant Small-Molecule that Activates the Orphan
Receptor GPR88 and Reduces Alcohol Intake
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Chunyang Jin,†* Ann M. Decker,† Viren H. Makhijani,‡ Joyce Besheer,‡# Emmanuel Darcq,§ Brigitte L.
Kieffer,§ and Rangan Maitra†
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†Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, North Carolina 27709,
United States
‡Bowles Center for Alcohol Studies, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599,
United States
#Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States
§Douglas Mental Health Research Institute, Department of Psychiatry, McGill University, Montreal, Quebec
H4H 1R3, Canada
*Corresponding author:
Dr. Chunyang Jin
Research Triangle Institute
Post Office Box 12194
Research Triangle Park, NC 27709
Telephone: 919 541ꢀ6328
Fax: 919 541ꢀ8868
Eꢀmail: cjin@rti.org
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Abstract
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The orphan Gꢀproteinꢀcoupled receptor GPR88 is highly expressed in the striatum. Studies using GPR88
knockout mice have suggested that the receptor is implicated in alcohol seeking and drinking behaviors. To
date, the biological effects of GPR88 activation are still unknown due to the lack of a potent and selective
agonist appropriate for in vivo investigation. In this study, we report the discovery of the first potent,
selective, and brainꢀpenetrant GPR88 agonist RTIꢀ13951ꢀ33 (6). RTIꢀ13951ꢀ33 exhibited an EC50 of 25 nM
in an in vitro cAMP functional assay and had no significant offꢀtarget activity at 38 GPCRs, ion channels, and
neurotransmitter transporters that were tested. RTIꢀ13951ꢀ33 displayed enhanced aqueous solubility
compared to (1R,2R)ꢀ2ꢀPCCA (2) and had favorable pharmacokinetic properties for behavioral assessment.
Finally, RTIꢀ13951ꢀ33 significantly reduced alcohol selfꢀadministration and alcohol intake in a doseꢀ
dependent manner without effects on locomotion and sucrose selfꢀadministration in rats when administered
intraperitoneally.
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Keywords: GPR88, 2ꢀPCCA, SAR, Alcohol, RTIꢀ13951ꢀ33
INTRODUCTION
Alcohol use disorders (AUDs) remain a significant clinical problem and exact great emotional, social, and
economic costs.1, 2 It is well known that alcohol interacts with a number of neurotransmitters in the brain and
triggers broad alterations in gene expression and synaptic plasticity.3ꢀ6 In regard to the brain reward system,
alcohol affects both the dopamine and opioid systems.4 Accordingly, the competitive opioid antagonist
naltrexone and the functional glutamate receptor antagonist acamprosate have been approved by the U.S.
Food and Drug Administration to treat alcoholism.7 However, both drugs face significant challenges including
lack of adherence, low efficacy, and serious side effects.7ꢀ10 In addition, alcoholism is a heterogeneous
disorder and there is no single effective treatment for all alcoholic patients.10 Therefore, new therapeutic
agents based on novel targets are urgently needed.
GPR88 is an orphan Gꢀproteinꢀcoupled receptor (GPCR) and has a high expression in both dorsal and
ventral areas of the striatum.11, 12 GPR88 has also been found in other regions of the brain, including the
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cerebral cortex, amygdala, and hypothalamus.13ꢀ17 In the striatum, GPR88 is expressed at postsynaptic sites in
medium spiny neurons (MSNs) of both direct and indirect pathways.15 A number of studies using GPR88
knockout (KO) mice have suggested that genetic ablation of GPR88 induces a state of hypersensitivity to the
dopamine system and the receptor is implicated in disease states such as schizophrenia and anxiety.18ꢀ22
Interestingly, a recent GPR88 KO study demonstrated that the GPR88ꢀdeficient mice displayed enhanced
voluntary alcohol drinking in both moderate and excessive drinking paradigms compared with wildꢀtype
(WT) mice.23 No alterations in water intake, palatability, and alcohol metabolism were observed in these
mice.23 Moreover, mice lacking the GPR88 gene showed enhanced motivation for alcohol drinking and
seeking behaviors using operant selfꢀadministration.23 Altogether, these findings suggest that weak GPR88
signaling may represent a vulnerability factor for AUDs and GPR88 might serve as a novel drug target to treat
AUDs.
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We have previously reported that GPR88 is a Gαi−coupled GPCR activated by a synthetic agonist, 2ꢀ
PCCA (1, Figure 1).24 Unfortunately, 2ꢀPCCA is expected to have poor brain bioavailability due to its high
lipophilicity (clogP 6.19) and the substrate of Pꢀglycoprotein.25 Thus, an improved agonist probe is required
to elucidate the in vivo functions of GPR88. Structureꢀactivity relationship (SAR) studies from our laboratory,
as well as others, have focused on sites AꢀC of 2ꢀPCCA and determined a preliminary understanding of
receptor tolerances at each site (Figure 1).24ꢀ27 For example, the aromatic ring on site A is essential for activity
but has limited substitution tolerance, suggesting the binding space around this region is constrained. The
(1R,2R)ꢀtrans configuration of the cyclopropane linkage is more potent than the (1S,2S)ꢀtrans and cisꢀ
isomers. On site B, the 4’ꢀposition of the biphenyl ring is well tolerated with smallꢀ to largeꢀsized alkyl or
alkoxy groups, which likely exits through a hydrophobic binding pocket to the extracellular loop consistent
with a binding position in the GPR88 homology model. On site C, the (S)ꢀamino group is important for
potency. We previously demonstrated that the ethylamine moiety of site C had tolerance for modification.24 In
this study, we continued to investigate SAR at site C and tested a series of analogues (4a−h, Figure 2) bearing
smallꢀ to largeꢀsized amino alkyl side chains with the goal of improving potency and drugꢀlike properties for
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brain penetration. We then combined the positive SAR results obtained from sites AꢀC and designed
analogues 5a and 5b, which led to the discovery of the first potent, selective, and brainꢀpenetrant smallꢀ
molecule 6 (designated as RTIꢀ13951ꢀ33) that activated GPR88 and significantly reduced alcohol
reinforcement and intake behaviors in rats.
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RESULTS AND DISCUSSION
Synthesis. The overall synthetic approach followed methods detailed in our earlier publications.24, 27
Synthesis of compounds 4a−g is shown in Scheme 1. Reductive amination of an appropriate aldehyde 7a-g
with 4ꢀbromoaniline (8a) or 4ꢀ(4’ꢀpropylphenyl)aniline (8b) using sodium triacetoxyborohydride in 1,2ꢀ
dichloroethane afforded amine 9a-g in 50−86% yields. Reaction of 9a-g with the acid chloride of racemic (±)ꢀ
transꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropanecarboxylic acid gave amide 10a-g in 49−60% yields. Suzuki coupling of
10a, 10b, and 10g with 4ꢀpropylphenylboronic acid under microwave conditions yielded 53−80% of 11a,
11b, and 11g. Removal of the Boc group with 4 M HCl in dioxane furnished 4a-g in 92−98% yields.
Synthesis of the (R)ꢀ1ꢀmethoxyethyl derivatives 4h, 5a,b, and 6 is outlined in Scheme 2. HBTUꢀassisted
amide coupling between 4ꢀbromoaniline (8a) and NꢀBocꢀOꢀmethylꢀLꢀthreonine (12) led to 13, which was
subsequently reduced with borane in THF to give amine 14 in 31% yield over two steps. Amide formation
afforded 15 in 95% yield. Suzuki coupling of 15 with arylboronic acid, followed by Boc deprotection
provided 4h and 5a,b in the range of 62−77% yields. Target compounds 4b, 4d−h, and 5a,b were 1:1
mixtures of (1R,2R)ꢀ and (1S,2S)ꢀenantiomers, differentiating at the configuration of the transꢀsubstituted
cyclopropane, determined by 1H NMR and HPLC analyses. The pure diastereomer 6 was synthesized using
the procedure analogous to that used to prepare (1R,2R)ꢀ2ꢀPCCA.24 The 1:1 diastereomeric mixture 15 was
separated by a chiral HPLC, affording (1R,2R)ꢀ15 and (1S,2S)ꢀ15 (structure not shown) in 48% and 47%
yield, respectively. The configuration of (1R,2R)ꢀ15 was assigned based on the comparison of its 1H NMR
spectrum and HPLC retention time with those of a sample synthesized from pure (1R,2R)ꢀ2ꢀ(pyridinꢀ2ꢀ
yl)cyclopropanecarboxylic acid.24 Finally, Suzuki coupling with 4ꢀ(methoxymethyl)phenylboronic acid,
followed by Boc deprotection provided RTIꢀ13951ꢀ33 (6) in 64% yield.
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Pharmacological Evaluations. We previously examined a brief SAR on site C of 2ꢀPCCA in a GloSensor
cAMP assay using stable HEK293ꢀGPR88ꢀpGloSensor22F cells.24 Later, we observed that, over time, the
HEK293 cells were physically unhealthy after transfection with GPR88 and subsequently led to deteriorated
responses to 2ꢀPCCA.26 In order to facilitate the SAR study, we recently created a stable CHOꢀPPLSꢀHAꢀ
GPR88 cell line and measured cAMP levels using the PerkinElmer Lance assay kit.27 The new assay system
is reliable and sensitive, and therefore was used in subsequent SAR studies.
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Our efforts on the optimization of 2ꢀPCCA have focused on improving its brain permeability by
lowering the lipophilicity while retaining or enhancing potency. Early SAR studies suggest that the
substituted ethylamine moiety on site C is suitable for modification. In the present study, we further explored
the SAR on this site and evaluated a series of analogues 4a−h in the GPR88 Lance cAMP assays. The results
are presented in Table 1. The unsubstituted analogue 4a was approximately 4ꢀfold less potent than 2ꢀPCCA
(501 nM vs. 126 nM). Addition of a methyl group (4b) or 1,1’ꢀdimethyl groups (4c) slightly improved
potency with an EC50 value of 398 nM and 316 nM, respectively. The potency was further improved by
adding a mediumꢀsized alkyl side chain with the isobutyl analogue 4e (EC50 = 126 nM) being equipotent to 2ꢀ
PCCA. Interestingly, substitution with a phenyl group (4f) led to a significant loss of potency (EC50 = 1260
nM), whereas the phenylmethyl analogue 4g retained a moderate activity. Further optimization of the amino
alkyl side chain of 2ꢀPCCA by inserting an oxygen to lower lipophilicity gave 4h with an EC50 value of 63
nM. In general, a mediumꢀsized alkyl side chain is well tolerated on site C, indicating a hydrophobic binding
pocket for this region. An alkoxy group can also be tolerated on this site to further enhance potency and lower
lipophilicity.
With the positive SAR results obtained on site C, we next combined our optimized biphenyl substitution
(site B) with the amino alkyl side chain (site C) in an effort to further improve the drugꢀlike properties for
CNS penetration (in general, CNS drugs have clogP = 2−428) while retaining the potency. Because compound
3 (Figure 1) showed comparable potency but has lower lipophilicity than (1R,2R)ꢀ2ꢀPCCA (2) (clogP 4.63 vs.
6.19),27 we designed and synthesized analogues 5a,b with a methoxy or a methoxymethyl group on the
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biphenyl ring, respectively. Both compounds had good agonist activity with 5b (clogP = 3.34, EC50 = 100
nM) being slightly more potent than 5a. Finally, we synthesized 6, a pure (1R,2R)ꢀdiastereomer of 5b, which
had an EC50 of 25 nM.
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Physicochemical Properties and In Vitro ADME Studies. Physiochemical properties, such as lipophilicity
(clogP), topological polar surface area (TPSA), and logBB, of select compounds (2, 3, 4h, and 6) were
calculated to predict the compound’s potential for brain penetration. In general, CNS drugs have clogP of
2−4,28 TPSA < 76 Å2,29 and logBB > −1.30 As shown in Table 2, (1R,2R)ꢀ2ꢀPCCA (2) has the highest clogP
value of 6.19, whereas RTIꢀ13951ꢀ33 (6) has the lowest clogP = 3.34. All compounds, except 6, have TPSA <
76 Å2. Compound 6 has a TPSA value of 77.68 Å2 that is just above the recommended threshold. A TPSA
cutoff of 90 Å2 has also been suggested for CNS drugs.31 All compounds have logBB > −1, suggesting that
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these compounds have the potential to penetrate the brain.
The brain permeability of select compounds were further tested in vitro using the MDCK cell line
expressing the human Pꢀglycoprotein efflux transporter. In general, compounds that have >10% permeability
(expressed as % transported from apical A to basal B) in the MDCK assay are considered desirable. Only
compound 6 had >10% permeability, whereas 2, 3, and 4h had 1%, 8%, and 3%, respectively, with 2 being
the least permeable in this series. The permeability correlated well with clogP. The solubility of select
compounds was determined using a thermodynamic solubility assay. RTIꢀ13951ꢀ33 (6) was highly soluble in
aqueous solutions with a thermodynamic solubility of 984 ± 9.4 ꢀg/mL (mean ± %CV) in the phosphateꢀ
buffered saline (PBS) solution. On the other hand, (1R,2R)ꢀ2ꢀPCCA (2) had a much lower solubility of 46.1 ±
1.8 ꢁg/mL, which is expected due to its high lipophilicity.
Receptor Selectivity. The onꢀtarget activity of (1R,2R)ꢀ2ꢀPCCA (2) and RTIꢀ13951ꢀ33 (6) was determined in
the [35S]ꢀGTPγS binding assay using striatal membrane preparations from WT mice and GPR88 KO mice.
RTIꢀ13951ꢀ33 increased [35S]ꢀGTPγS binding (EC50 = 535 nM) in mouse striatal membranes, but none in
membranes from GPR88 KO mice even at a concentration of 100 ꢀM, indicating the compound has a GPR88ꢀ
specific agonist signaling activity in the striatum (Figure 3A). By contrast, (1R,2R)ꢀ2ꢀPCCA had an EC50 of
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1140 nM in the WT mouse striatal membranes but also had a significant [35S]ꢀGTPγS binding at 10 ꢀM in
GPR88 KO mouse striatal membranes (Figure 3B). The offꢀtarget profile of RTIꢀ13951ꢀ33 was further
assessed in radioligand binding assays against a panel of 38 GPCRs, ion channels, and transporters at a single
concentration of 10 ꢀM, each in duplicate, by Eurofins PanLabs (Taipei, Taiwan). RTIꢀ13951ꢀ33 at 10 ꢁM
showed binding activity only at the kappa opioid receptor (KOR), vesicular monoamine transporter (VMAT)
and serotonin transporter (SERT) with inhibition of 64%, 54% and 55%, respectively (see Supporting
Information for the full offꢀtarget profile report). The followꢀup concentrationꢀresponse assays determined
that the compound had weak affinities at KOR (Ki = 2.29 ꢀM) and VMAT (Ki = 4.23 ꢀM), and a moderate
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affinity at SERT (Ki = 0.75 ꢀM). RTIꢀ13951ꢀ33 was then submitted to the NIMH Psychoactive Drug
Screening Program (NIMH PDSP) to evaluate its functional activity at SERT. This compound had little
inhibitory activity at SERT with an IC50 = 25.1 ± 2.7 ꢀM in the neurotransmitter transporter reuptake
inhibition assay. Taken together, RTIꢀ13951ꢀ33 is GPR88 selective and has no significant offꢀtarget liability
based on our selectivity evaluations.
Pharmacokinetic Study. On the basis of potency, receptor selectivity, and in vitro ADME properties, RTIꢀ
13951ꢀ33 was further evaluated in a snapshot pharmacokinetic (PK) testing to assess whether this compound
has sufficient brain exposure. Following an intraperitoneal (ip) dose of 10 mg/kg in rats, RTIꢀ13951ꢀ33 was
rapidly absorbed into systemic circulation, with peak plasma concentration (Cmax = 874 ng/mL, Table 3)
observed at 15 min postdose (the first sampling time point). The brain concentration peaked at 60 min with a
Cmax of 287 ng/mL, then was eliminated from the brain with an apparent halfꢀlife of 87 min. The overall brain
to plasma AUC ratio, as determined by AUC0 inf ratio, was 0.5. At 30 min, brain concentration was 242
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ng/mL (527 nM), indicating that RTIꢀ13951ꢀ33 had sufficient brain penetration for GPR88 modulation
considering it has an EC50 of 25 nM in the cAMP functional assay.
In Vivo Activities of RTI-13951-33. Because GPR88 KO mice showed enhanced motivation for alcohol
drinking and seeking behaviors,23 we sought to determine whether RTIꢀ13951ꢀ33 would be effective in vivo to
alter operant alcohol selfꢀadministration. Female Long Evans rats were trained to selfꢀadminister alcohol, as
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described in our recent publications (15% alcohol [v/v] + 2% [w/v] sucrose vs. inactive lever).32ꢀ34 This
sweetened alcohol concentration is used in all our studies because we find that it results in stable operant
responding over time and allows animals to achieve physiologically relevant and moderate alcohol intake
(e.g., 0.7–1.0 g/kg). A significant decrease in alcohol selfꢀadministration was observed at the two highest
RTIꢀ13951ꢀ33 doses tested (10 and 20 mg/kg, Figure 4A). This decrease in alcohol lever responses
corresponded to decreased alcohol intake (g/kg) at the highest dose (20 mg/kg) and a trend for a decrease at
the 10 mg/kg dose (p = 0.05, Figure 4A). Notably, there was no effect on inactive lever responses (mean ±
S.E.M.; vehicle: 0.8 ± 0.3; 5 mg/kg dose: 1.1 ± 0.4; 10 mg/kg dose: 0.5 ± 0.3; 20 mg/kg dose: 0.0 ± 0.0
responses) or locomotor rate (Figure 4B) measured during the selfꢀadministration sessions,35 suggesting that
the decrease in alcohol selfꢀadministration was not related to a general suppression of activity.
For a comparative assessment of actions on intake of a natural reward, a separate cohort of rats was
trained to selfꢀadminister sucrose (0.8% [w/v] sucrose vs. inactive lever; FR2). This sucrose concentration
was chosen as it resulted in lever responding similar to the alcohol selfꢀadministration group. There was no
difference in sucrose lever responses, or sucrose intake at any RTIꢀ13951ꢀ33 dose vs. vehicle (Figure 4C).
Interestingly, there was a trend for a reduction in locomotor rate [F(3,18) = 3.16, p = 0.05; mean ± S.E.M.;
vehicle: 27.0 ± 1.8; 5 mg/kg dose: 24.0 ± 2.0; 10 mg/kg dose: 23.6 ± 1.2; 20 mg/kg dose: 17.9 ± 3.6 beam
breaks/min], but this did not affect sucrose lever responses. Given the lack of modulatory effect on sucrose
selfꢀadministration at doses that reduced alcohol selfꢀadministration, this suggests that the compound had a
selective effect for alcohol reinforcement.
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CONCLUSIONS
In summary, we have developed a potent, selective, and brainꢀpenetrant GPR88 agonist RTIꢀ13951ꢀ33
(6) based on the 2ꢀPCCA scaffold. The in vitro pharmacological evaluations revealed that RTIꢀ13951ꢀ33 was
highly potent in the GPR88 cAMP functional assay and had no significant offꢀtarget activity based upon our
screening data of 38 GPCRs, ion channels, and transporters and had no agonist signaling activity in the
GTPγS binding assay using GPR88 KO mouse striatal membranes. RTIꢀ13951ꢀ33 displayed a good aqueous
solubility and had favorable pharmacokinetic properties for brainꢀpenetration. RTIꢀ13951ꢀ33 showed
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significant efficacy in reducing alcohol intake in a rat model of alcohol selfꢀadministration. Importantly, there
was no effect on locomotion measured during the selfꢀadministration sessions, indicating that the decrease in
alcohol selfꢀadministration was not related to a general suppression of activity. Moreover, we demonstrated a
specificity between alcohol and natural rewards, with GPR88 agonism reducing alcohol selfꢀadministration,
but not sucrose selfꢀadministration at equivalent doses, supporting a role of GPR88 signaling in the
modulation of alcohol reinforcement. Further studies of RTIꢀ13951ꢀ33 in the mouse model of alcoholꢀ
drinking and ꢀseeking behaviors using both WT and GPR88 KO mice to access the in vivo onꢀtarget
specificity are in progress. Taken together, GPR88 represents a novel drug target and should be further
studied in the development of new treatments for alcohol use disorders.
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EXPERIMENTAL SECTION
Chemistry. General Methods. Melting points were determined using a MELꢀTEMP II capillary melting point
apparatus and are uncorrected. Melting point of the diastereomeric mixture was not measured. Nuclear
magnetic resonance (1H NMR and 13C NMR) spectra were obtained on a Bruker Avance DPXꢀ300 MHz
NMR spectrometer. Chemical shifts are reported in parts per million (ppm) with reference to internal solvent.
13C NMR data of diastereomeric mixtures were not reported due to the complicity of the spectra. Mass spectra
(MS) were run on a PerkinꢀElmer Sciex API 150 EX mass spectrometer. HRMS spectra were run on a Waters
Synapt G2 HDMS QꢀTOF mass spectrometer, using electrospray ionization in positive ion mode. Analytical
thinꢀlayer chromatography (TLC) was carried out using EMD silica gel 60 F254 TLC plates. TLC visualization
was achieved with a UV lamp or in an iodine chamber. Flash column chromatography was done on a
CombiFlash Companion system using Isco prepacked silica gel columns. Unless otherwise stated, reagentꢀ
grade chemicals were obtained from commercial sources and were used without further purification. All
moistureꢀ and airꢀsensitive reactions and reagent transfers were carried out under dry nitrogen. Synthesis and
characterization of compounds 1−3, 4a,b, 4d,e, and 4g have been previously reported.24 All synthesized
compounds were ≥95% pure as determined by HPLC analyses (see Supporting Information).
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(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid (2-Amino-2-methylpropyl)-(4’-
propylbiphenyl-4-yl)amide (4c). A solution of 10c (50 mg, 0.09 mmol) and 4 M HCl in dioxane (2 mL) in
CH2Cl2 (5 mL) was stirred at room temperature for 6 h. The solvent was removed under reduced pressure.
The resulting residue was triturated with hexanes to give 4c dihydrochloride (45 mg, 95%) as an offꢀwhite
solid: mp 158−160 ºC; 1H NMR (300 MHz; CD3OD) δ 8.59 (d, J = 3.0 Hz, 1H), 8.35−8.45 (m, 1H),
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7.80−7.45 (m, 8H), 7.26 (d, J = 6.0 Hz, 2H), 4.13 (d, J = 15.0 Hz, 1H), 3.98 (d, J = 15.0 Hz, 1H), 3.78−3.58
(m, 1H), 3.10−2.95 (m, 1H), 2.62 (t, J = 7.5 Hz, 2H), 2.25−2.15 (m, 1H), 2.00−1.90 (m, 1H), 1.75−1.55 (m,
2H), 1.34 (s, 6H), 0.96 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CD3OD) δ 174.1, 157.6, 146.5, 143.9, 143.4,
143.3, 142.5, 138.2, 130.2, 129.5, 129.2, 127.8, 125.5, 125.1, 59.6, 57.7, 38.6, 27.0, 25.6, 25.5, 24.8, 17.8,
14.0; HRMS (ESI) calcd. for C28H33N3O [M + H]+: 428.2696. Found: 428.2700.
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S)-2-Amino-2-phenylethyl]-(4’-
propylbiphenyl-4-yl)amide (4f). The procedure for 4c was followed using 50 mg (0.09 mmol) of 10f to give
46 mg (97%) of 4f dihydrochloride (1:1 diastereomeric mixture) as an offꢀwhite solid: 1H NMR (300 MHz;
CD3OD) δ 8.50−8.38 (m, 1H), 8.15−8.05 (m, 1H), 7.65−7.30 (m, 9H), 7.25−6.98 (m, 6H), 4.65−4.45 (m,
1.5H), 4.05−3.85 (m, 1H), 3.68−3.25 (m, 0.5H), 2.90−2.82 (m, 0.5H), 2.82−2.70 (m, 0.5H), 2.52 (t, J = 7.5
Hz, 2H), 2.05−1.95 (m, 1H), 1.88−1.70 (m, 1H), 1.68−1.45 (m, 3H), 0.86 (t, J = 7.5 Hz, 3H); HRMS (ESI)
calcd. for C32H33N3O [M + H]+: 476.2696. Found: 476.2705.
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3R)-2-Amino-3-methoxybutyl]-(4’-
propylbiphenyl-4-yl)amide (4h). A mixture of 15 (52 mg, 0.1 mmol), 4ꢀpropylphenylboronic acid (24.6 mg,
0.15 mmol), Pd(dppf)Cl2•CH2Cl2 (8.7 mg, 0.01 mmol) and K3PO4 (76 mg, 5.4 mmol) in dimethoxyethane (1
mL) and water (0.3 mL) was heated in a sealed vessel by microwave irradiation at 160 °C for 6 min. The
resulting mixture was poured into 1 N NaOH solution (5 mL) and extracted with CH2Cl2 (3 x 10 mL). The
combined organic layers were dried (Na2SO4) and concentrated under reduced pressure. Flash column
chromatography of the crude product on silica gel using 0−20% EtOAc in hexanes afforded intermediate tertꢀ
butyl [(2S,3R)ꢀ1ꢀ{(4’ꢀpropylbiphenylꢀ4ꢀyl)ꢀ[(1R*,2R*)ꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropanecarbonyl]amino}ꢀ3ꢀ
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methoxybutanꢀ2ꢀyl]carbamate (44 mg, 79%, 1:1 diastereomeric mixture) as an oil: 1H NMR (300 MHz;
CDCl3) δ 8.30−8.25 (m, 1H), 7.55−7.40 (m, 5H), 7.32−7.15 (m, 5H), 7.02−6.95 (m, 1H), 5.12−5.00 (m, 1H),
4.42−4.28 (m, 1H), 4.00−3.80 (m, 2H), 3.58−3.48 (m, 1H), 3.45−3.36 (m, 1H), 3.26 and 3.25 (s, 3H),
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2.73−2.55 (m, 3H), 2.08−1.98 (m, 1H), 1.75−1.60 (m, 3H), 1.43 (s, 9H), 1.13 and 1.12 (2d, J = 6.0 Hz, 3H),
0.97 (t, J = 7.5 Hz, 3H); MS (ESI) m/z 558.5 [M + H]+. The coupled product was then dissolved in CH2Cl2 (5
mL) and treated with 4 M HCl in dioxane (3 mL) at room temperature for 6 h. The solvent was removed
under reduced pressure. The resulting residue was triturated with hexanes to give 4 h dihydrochloride (40 mg,
96%, 1:1 diastereomeric mixture) as an offꢀwhite solid: 1H NMR (300 MHz; CD3OD) δ 8.40−8.28 (m, 1H),
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8.00−7.88 (m, 1H), 7.65−7.25 (m, 8H), 7.20−7.05 (m, 2H), 4.30−4.18 (m, 0.5H), 4.18−4.06 (m, 0.5H),
3.98−3.86 (m, 0.5H), 3.85−3.75 (m, 0.5H), 3.68−3.52 (m, 1H), 3.52−3.38 (m, 1H), 3.23 and 3.21 (2s, 3H),
2.85−2.68 (m, 1H), 2.60−2.46 (m, 2H), 2.08−1.90 (m, 1H), 1.80−1.68 (m, 1H), 1.68−1.40 (m, 3H), 1.12−1.00
(m, 3H), 0.98−0.75 (m, 3H); HRMS (ESI) calcd. for C29H35N3O2 [M + H]+: 458.2802. Found: 458.2790.
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3R)-2-Amino-3-methoxybutyl]-(4’-
methoxybiphenyl-4-yl)amide (5a). The procedure for 4h was followed using 52 mg (0.1 mmol) of 15 and
22.8 mg (0.15 mmol) of 4ꢀmethoxyphenylboronic acid to give 32 mg (62% over two steps) of 5a
dihydrochloride (1:1 diastereomeric mixture) as an offꢀwhite solid: 1H NMR (300 MHz; CD3OD) δ 8.50−8.38
(m, 1H), 8.15−8.00 (m, 1H), 7.60−7.25 (m, 8H), 6.95−6.85 (m, 2H), 4.30−4.18 (m, 0.5H), 4.18−4.05 (m,
0.5H), 3.98−3.85 (m, 0.5H), 3.85−3.72 (m, 0.5H), 3.75 (s, 3H), 3.70−3.52 (m, 1H), 3.52−3.38 (m, 1H),
3.30−3.15 (m, 3H), 2.90−2.78 (m, 1H), 2.10−1.98 (m, 1H), 1.85−1.72 (m, 1H), 1.60−1.45 (m, 1H), 1.12−0.98
(m, 3H); HRMS (ESI) calcd. for C27H31N3O3 [M + H]+: 446.2438. Found: 446.2431.
(1R*,2R*)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3R)-2-Amino-3-methoxybutyl]-[4’-
(methoxymethyl)biphenyl-4-yl]amide (5b). The procedure for 4h was followed using 52 mg (0.1 mmol) of
15 and 24.9 mg (0.15 mmol) of 4ꢀ(methoxymethyl)phenylboronic acid to give 41 mg (77% over two steps) of
5b dihydrochloride (1:1 diastereomeric mixture) as an offꢀwhite solid: 1H NMR (300 MHz; CD3OD) δ
8.55−8.45 (m, 1H), 8.20−8.15 (m, 1H), 7.78−7.38 (m, 10H), 4.49 (s, 2H), 4.45−4.30 (m, 0.5H), 4.30−4.15 (m,
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0.5H), 4.12−3.95 (m, 0.5H), 3.95−3.80 (m, 0.5H), 3.78−3.62 (m, 1H), 3.62−3.50 (m, 1H), 3.40 (s, 3H),
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3.35−3.25 (m, 3H), 3.00−2.82 (m, 1H), 2.20−2.08 (m, 1H), 1.95−1.80 (m, 1H), 1.70−1.55 (m, 1H), 1.22−1.08
(m, 3H); HRMS (ESI) calcd. for C28H33N3O3 [M + H]+: 460.2595. Found: 460.2579.
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(1R,2R)-2-(Pyridin-2-yl)cyclopropanecarboxylic Acid [(2S,3R)-2-Amino-3-methoxybutyl]-[4’-
(methoxymethyl)biphenyl-4-yl]amide (6). The procedure for 4h was followed using 1.6 g (3.1 mmol) of
(1R,2R)ꢀ15 and 0.77 g (4.65 mmol) of 4ꢀ(methoxymethyl)phenylboronic acid to give 1.05 g (64% over two
steps) of 6 dihydrochloride as a white solid: mp 130−132 °C; 1H NMR (300 MHz; CD3OD) δ 8.40 (br s, 1H),
7.93 (t, J = 7.5 Hz, 1H), 7.67 (d, J = 6.0 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.42 (d, J
= 6.0 Hz, 4H), 4.49 (s, 2H), 4.21 (dd, J = 15.0, 9.0 Hz, 1H), 4.04 (dd, J = 15.0, 6.0 Hz, 1H), 3.60−3.50 (m,
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1H), 3.40 (s, 3H), 3.34 (s, 3H), 2.84−2.74 (m, 1H), 2.10−2.02 (m, 1H), 1.85−1.76 (m, 1H), 1.60−1.50 (m,
1H), 1.07 (d, J = 6.0 Hz, 3H) (Note: one proton singal overlaps with the CD3OD solvent peak at δ 3.32−3.30);
13C NMR (75 MHz; CDCl3) δ 174.6, 159.1, 147.0, 142.4, 142.3, 141.8, 140.2, 139.4, 129.6 (two peaks
overlap), 129.5, 128.0, 124.2, 124.1, 75.2, 75.0, 58.4, 57.4, 56.9, 51.0, 27.3, 26.6, 17.9, 15.6; HRMS (ESI)
calcd. for C28H33N3O3 [M + H]+: 460.2595. Found: 460.2583.
tert-Butyl {1-[(4’-Propylbiphenyl-4-yl)amino]-2-methyl-propan-2-yl}carbamate (9c). To a solution
of NꢀBocꢀ2ꢀaminoꢀ2ꢀmethylꢀ1ꢀpropanol (162 mg, 0.85 mmol) in waterꢀsaturated CH2Cl2 (5 mL) at room
temperature was added DessꢀMartin reagent (760 mg, 1.79 mmol) and the reaction was stirred for 1 h.
Additional waterꢀsaturated CH2Cl2 (2 mL) was added every 15 min during the reaction time. The mixture was
diluted with Et2O (30 mL) and poured into a solution of Na2S2O3 (1.4 g) in 80% saturated NaHCO3 (30 mL).
After stirring for 10 min, the layers were separated, and the aqueous layer was extracted with Et2O (30 mL).
The combined organic layers were washed with iceꢀcold saturated NaHCO3 (10 mL) and water (10 mL). The
solution was dried (Na2SO4) and concentrated under reduced pressure to give the crude aldehyde 7c. To a
solution of 4ꢀ(4’ꢀpropylphenyl)aniline (8b) (150 mg, 0.71 mmol) in 1,2ꢀdichloroethane (10 mL) was added
the above crude aldehyde, followed by NaBH(OAc)3 (301 mg, 1.42 mmol). The mixture was stirred at room
temperature overnight. Saturated NaHCO3 (10 mL) was added and the layers were separated. The aqueous
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layer was extracted with CH2Cl2 (2 x 20 mL). The combined organic layers were washed with brine (3 x 20
mL), dried (Na2SO4) and concentrated under reduced pressure. Flash column chromatography of the crude
product on silica gel using 0−30% EtOAc in hexanes afforded 9c (204 mg, 75%) as a white solid: mp
120−122 ºC; 1H NMR (300 MHz; CDCl3) δ 7.42−7.38 (m, 4H), 7.20 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 9.0 Hz,
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2H), 4.66 (s, 1H), 4.15 (br s, 1H), 3.29 (s, 2H), 2.61 (t, J = 6.0 Hz, 2H), 1.75−1.64 (m, 2H), 1.43 (s, 9H), 1.34
(s, 6H), 0.96 (d, J = 6.0 Hz, 3H); 13C NMR (75 MHz; CDCl3) δ 155.1, 148.0, 140.5, 138.8, 130.3, 128.8,
127.8, 126.2, 113.2, 79.4, 53.2, 52.8, 37.8, 28.5, 28.3, 25.9, 24.7, 14.0; MS (ESI) m/z 383.5 [M + H]+.
tert-Butyl {(1S)-2-[(4’-Propylbiphenyl-4-yl)amino]-1-phenylethyl}carbamate (9f). The procedure
for 9c was followed using 150 mg (0.71 mmol) of 8b and aldehyde 7f, prepared by oxidation of NꢀBocꢀLꢀ
phenylglycinol (203 mg, 0.85 mmol), to give 175 mg (57%) of 9f as a white solid: mp 118−120 ºC; 1H NMR
(300 MHz; CDCl3) δ 7.48−7.30 (m, 9H), 7.20 (d, J = 9.0 Hz, 2H), 6.68 (d, J = 9.0 Hz, 2H), 5.08 (br s, 1H),
4.96 (br s, 1H), 3.94 (s, 1H), 3.50 (d, J = 3.0 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 1.72−1.60 (m, 2H), 1.44 (s,
9H), 0.96 (d, J = 6.0 Hz, 3H); 13C NMR (75 MHz; CDCl3) δ 155.8, 147.1, 140.7, 138.6, 130.9, 128.9, 128.8,
127.9, 126.5, 126.2, 123.4, 80.0, 54.4, 49.8, 37.7, 28.4, 24.6, 14.0; MS (ESI) m/z 431.3 [M + H]+.
tert-Butyl (1-{(4’-Propylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-
2-methyl-propan-2-yl)carbamate (10c). To a solution of (±)ꢀtransꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropanecarboxylic
acid (78 mg, 0.39 mmol) in CH2Cl2 (10 mL) at room temperature was added oxalyl chloride (0.07 mL, 0.78
mmol) and DMF (5 µL). The mixture was stirred at 40 °C for 2 h, then cooled to room temperature and
concentrated under reduced pressure. The resulting acid chloride was dissolved in CH2Cl2 (10 mL) and treated
with 9c (125 mg, 0.33 mmol) and Et3N (0.18 mL, 1.32 mmol). The mixture was stirred at room temperature
overnight. Saturated NaHCO3 (5 mL) was added and the layers were separated. The aqueous layer was
extracted with CH2Cl2 (3 x 10 mL). The combined organic layers were washed with brine (3 x 10 mL), dried
(Na2SO4) and concentrated under reduced pressure. Flash column chromatography of the crude product on
silica gel using 0−25% EtOAc in hexanes afforded 10c (85 mg, 49%) as an oil: 1H NMR (300 MHz; CDCl3) δ
8.28 (d, J = 6.0 Hz, 1H), 7.56−7.38 (m, 5H), 7.30−7.15 (m, 5H), 7.05−6.98 (m, 1H), 5.05 (s, 1H), 4.18−3.98
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(m, 2H), 2.66−2.54 (m, 3H), 2.10−1.98 (m, 1H), 1.76−1.60 (m, 3H), 1.50−1.40 (m, 1H), 1.30 (s, 6H), 1.22 (s,
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9H), 0.97 (t, J = 6.0 Hz, 3H); 13C NMR (75 MHz; CDCl3) δ 173.5, 159.5, 154.2, 149.2, 142.4, 142.2, 139.9,
137.5, 135.9, 128.9, 128.2, 127.7, 126.8, 122.3, 121.0, 56.6, 54.9, 37.7, 28.3, 28.1, 25.8, 25.6, 25.0, 24.5,
17.6, 13.9; MS (ESI) m/z 529.2 [M + H]+.
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tert-Butyl [(1S)-2-{(4’-Propylbiphenyl-4-yl)-[(1R*,2R*)-2-(pyridin-2-
yl)cyclopropanecarbonyl]amino}-1-phenylethyl]carbamate (10f). The procedure for 10c was followed
using 115 mg (0.27 mmol) of 9f to give 75 mg (49%) of 10f (1:1 diastereomeric mixture) as an oil: 1H NMR
(300 MHz; CDCl3) δ 8.27 (d, J = 6.0 Hz, 1H), 7.60−7.40 (m, 5H), 7.38−7.12 (m, 10H), 7.05−6.95 (m, 1H),
6.05−5.90 (m, 1H), 4.98−4.80 (m, 1H), 4.70−4.50 (m, 1H), 3.50−3.38 (m, 1H), 2.78−2.55 (m, 3H), 2.10−1.98
(m, 1H), 1.80−1.60 (m, 3H), 1.46 and 1.44 (2s, 9H), 1.35−1.20 (m, 1H), 0.97 (t, J = 7.5 Hz, 3H); MS (ESI)
m/z 576.7 [M + H]+.
(2S,3R)-2-(Boc-amino)-3-methoxybutyric Acid 4-Bromophenylamide (13). To a solution of 4ꢀ
bromoaniline (8a) (11.09 g, 64.5 mmol) and NꢀBocꢀOꢀmethylꢀLꢀthreonine (12) (15 g, 64.5 mmol) in
acetonitrile (300 mL) at room temperature was added DIPEA (16.8 mL, 96 mmol) followed by HBTU (29.3
g, 77.4 mmol). The mixture was stirred overnight, then saturated NaHCO3 (100 mL) was added. The layers
were separated. The aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layers
were washed with brine (3 x 50 mL), dried (Na2SO4) and concentrated under reduced pressure. Flash column
chromatography of the crude product on silica gel using 0−25% EtOAc in hexanes afforded 13 (22.5 g, 90%)
as a white solid: mp 48−50 ºC; 1H NMR (300 MHz; CDCl3) δ 8.48 (s, 1H), 7.50−7.40 (m, 4H), 5.55 (s, 1H),
4.40−4.32 (m, 1H), 4.05−3.95 (m, 1H), 3.45 (s, 3H), 1.50 (s, 9H), 1.16 (d, J = 6.0 Hz, 3H); 13C NMR (75
MHz; CDCl3) δ 168.2, 155.9, 136.5, 132.0, 121.6, 117.0, 80.5, 76.2, 57.9, 57.2, 30.9, 28.3, 14.6; MS (ESI)
m/z 387.3 [M + H]+ (79Br), 389.5 [M + H]+ (81Br).
tert-Butyl {(2S,3R)-1-[(4-Bromophenyl)amino]-3-methoxybutan-2-yl}carbamate (14). To a solution
of 13 (21 g, 54 mmol) in THF (150 mL) at 0 °C was added 1 M borane THF complex solution (162 mL, 162
mmol) over 30 min. The mixture was then stirred at 65 °C for 2 days. After cooling to room temperature, the
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reaction was quenched with 0.1 N HCl (200 mL) and stirred for 5 min. The solution was basified with
saturated NaHCO3. The layers were separated. The aqueous layer was extracted with EtOAc (3 x 150 mL).
The combined organic layers were washed with brine (200 mL), dried (Na2SO4) and concentrated under
reduced pressure. Flash column chromatography of the crude product on silica gel using 0−10% EtOAc in
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hexanes afforded 14 (6.82 g, 34%) as an oil: 1H NMR (300 MHz; CDCl3) δ 7.23 (d, J = 9.0 Hz, 2H), 6.50 (d,
J = 9.0 Hz, 2H), 4.92 (br d, J = 9.0 Hz, 1H), 4.23 (br s, 1H), 3.81−3.71 (m, 1H), 3.58−3.48 (m, 1H), 3.33 (s,
3H), 3.27−3.18 (m, 2H), 1.46 (s, 9H), 1.18 (d, J = 6.0 Hz, 3H); 13C NMR (75 MHz; CDCl3) δ 156.6, 147.4,
131.9, 114.1, 108.5, 79.5, 75.8, 56.4, 54.3, 46.7, 28.4, 15.6; MS (ESI) m/z 373.2 [M + H]+ (79Br), 375.0 [M +
H]+ (81Br).
tert-Butyl [(2S,3R)-1-{(4-Bromophenyl)-[(1R*,2R*)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-
3-methoxybutan-2-yl]carbamate (15). The procedure for 10c was followed using 2.8 g (7.5 mmol) of 14 to
give 3.7 g (95%) of 15 (1:1 diastereomeric mixture) as a yellow foam: 1H NMR (300 MHz; CDCl3) δ
8.35−8.27 (m, 1H), 7.58−7.50 (m, 1H), 7.50−7.38 (m, 2H), 7.25−6.98 (m, 4H), 4.96 (d, J = 9.0 Hz, 1H),
4.35−4.20 (m, 1H), 3.92−3.78 (m, 2H), 3.55−3.45 (m, 1H), 3.45−3.32 (m, 1H), 3.26 and 3.25 (2s, 3H),
2.70−2.62 (m, 0.5H), 2.62−2.55 (m, 0.5H), 2.00−1.88 (m, 1H), 1.72−1.58 (m, 1H), 1.41 (s, 9H), 1.11 and 1.10
(2d, J = 6.0 Hz, 3H); MS (ESI) m/z 518.6 [M + H]+ (79Br), 520.5 [M + H]+ (81Br).
tert-Butyl [(2S,3R)-1-{(4-Bromophenyl)-[(1R,2R)-2-(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-
methoxybutan-2-yl]carbamate ((1R,2R)-15) and tert-Butyl [(2S,3R)-1-{(4-Bromophenyl)-[(1S,2S)-2-
(pyridin-2-yl)cyclopropanecarbonyl]amino}-3-methoxybutan-2-yl]carbamate ((1S,2S)-15). The
diastereomeric mixture 15 (3.4 g) was separated to (1R,2R)ꢀ15 (1.62 g, 48%) and (1S,2S)ꢀ15 (1.61 g, 47%) by
preparative HPLC using ChiralPak IA column: mobile phase, 10% isopropanol/hexanes; flow rate, 10
mL/min; detection 220 nm. The diastereomeric excess (de) of both of separated compounds was determined
to be >98% by HPLC (ChiralPak IA column; 10% isopropanol/hexanes; flow rate 1 mL/min; detection 220
nm; retention time, (1R,2R)ꢀ15: 7.95 min, (1S,2S)ꢀ15: 12.02 min). (1R,2R)ꢀ15: White foam; 1H NMR (300
MHz; CDCl3) δ 8.28 (d, J = 6.0 Hz, 1H), 7.58−7.40 (m, 3H), 7.25−6.98 (m, 4H), 4.98 (d, J = 9.0 Hz, 1H),
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4.24 (dd, J = 13.5, 9.0 Hz, 1H), 3.92−3.78 (m, 2H), 3.50 (dd, J = 12.0, 3.0 Hz, 1H), 3.45−3.32 (m, 1H), 3.26
(s, 3H), 2.72−2.62 (m, 1H), 2.00−1.90 (m, 1H), 1.70−1.58 (m, 1H), 1.42 (s, 9H), 1.11 (d, J = 6.0 Hz, 3H); 13C
NMR (75 MHz; CDCl3) δ 172.4, 159.1, 156.0, 149.1, 141.4, 135.7, 132.6, 129.7, 122.5, 121.3, 120.9, 78.8,
76.3, 56.4, 53.1, 50.7, 28.2, 27.4, 24.6, 17.7, 15.0; MS (ESI) m/z 518.2 [M + H]+ (79Br), 520.4 [M + H]+
(81Br). (1S,2S)ꢀ15: White foam; 1H NMR (300 MHz; CDCl3) δ 8.29 (d, J = 6.0 Hz, 1H), 7.52 (dd, J = 9.0, 6.0
Hz, 1H), 7.41 (d, J = 9.0 Hz, 2H), 7.20−6.98 (m, 4H), 4.99 (d, J = 9.0 Hz, 1H), 4.28 (dd, J = 13.5, 9.0 Hz,
1H), 3.85−3.75 (m, 2H), 3.49 (dd, J = 13.5, 4.5 Hz, 1H), 3.45−3.32 (m, 1H), 3.25 (s, 3H), 2.65−2.55 (m, 1H),
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1.98−1.90 (m, 1H), 1.72−1.62 (m, 1H), 1.41 (s, 9H), 1.10 (d, J = 6.0 Hz, 3H); 13C NMR (75 MHz; CDCl3) δ
172.4, 159.1, 156.0, 149.3, 141.4, 135.8, 132.7, 129.8, 122.4, 121.4, 121.0, 78.9, 76.4, 56.5, 53.2, 50.5, 28.4,
27.7, 24.7, 17.6, 15.0; MS (ESI) m/z 518.4 [M + H]+ (79Br), 520.5 [M + H]+ (81Br).
Pharmacology. Materials. Cell culture materials were purchased from Fisher SSI. Forskolin was purchased
from SigmaꢀAldrich. The Lance Ultra kit (TRF0262) was purchased from PerkinElmer.
LanceUltraTM cAMP Assay Using Stable PPLS-HA-GPR88 CHO Cells. Stimulation buffer containing
1X Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES, 0.1% BSA stabilizer, and 0.5 mM final IBMX
was prepared and titrated to pH 7.4 at room temperature. Serial dilutions of the test compounds (5 µL) and
300 nM forskolin (5 µL), both prepared at 4x the desired final concentration in 2% DMSO/stimulation buffer,
were added to a 96ꢀwell white ½ area microplate (PerkinElmer). A cAMP standard curve was prepared at 4x
the desired final concentration in stimulation buffer and 5 µL was added to the assay plate. Stable PPLSꢀHAꢀ
GPR88 CHO cells were lifted with versene and spun at 270 g for 10 min. The cell pellet was resuspended in
stimulation buffer and 4,000 cells (10 µL) were added to each well except wells containing the cAMP
standard curve. After incubating for 30 min at room temperature, EuꢀcAMP tracer and uLIGHTꢀantiꢀcAMP
working solutions were added per the manufacturer’s instructions. After incubation at room temperature for 1
h, the TRꢀFRET signal (ex 337 nm) was read on a CLARIOstar multimode plate reader (BMG Biotech, Cary,
NC).
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Data Analysis. The TRꢀFRET signal (665 nm) was converted to femtomolar cAMP by interpolating
from the standard cAMP curve. Fmol cAMP was plotted against the log of compound concentration and data
were fit to a threeꢀparameter logistic curve to generate EC50 values (Prism, version 7.0, GraphPad Software,
Inc., San Diego, CA).
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MDCK Permeability Assay. MDCKꢀmdr1 cells obtained from the Netherlands Cancer Institute were grown
on Transwell type filters (Corning) for 4 d to confluence in DMEM/F12 media containing 10% fetal bovine
serum and antibiotics as has been described previously.36, 37 Compounds were added to the apical side at a
concentration of 10 ꢀM in a transport buffer comprising of 1X Hank’s balanced salt solution, 25 mM Dꢀ
glucose and buffered with HEPES to pH 7.4. Samples were incubated for 1 h at 37 °C and carefully collected
from both the apical and basal side of the filters. Compounds selected for MDCKꢀmdr1 cell assays were
infused on an Applied Biosystems APIꢀ4000 mass spectrometer to optimize for analysis using multiple
reaction monitoring (MRM). Flow injection analysis was also conducted to optimize for mass spectrometer
parameters. Samples from the apical and basolateral side of the MDCK cell assay were dried under nitrogen
on a Turbovap LV. The chromatography was conducted with an Agilent 1100 binary pump with a flow rate of
0.5 mL/min. Mobile phase solvents were A, 0.1% formic acid in water, and B, 0.1% formic acid in methanol.
The initial solvent conditions were 10% B for 1 min, then a gradient was used by increasing to 95% B over 5
min, then returning to initial conditions. Data reported are average values from 2 to 3 measurements.
Solubility Determination. Thermodynamic solubility was measured in PBS pH 7.4 (Gibco). For each
compound, 1.0 mg of powder was combined with 1 mL of buffer to reach a targeted final concentration of 1
mg/mL. The solutions were mixed by repetitive inversion for 24 h at room temperature. Following agitation,
the samples were filtrated on Millipore multiscreen polycarbonate membrane (0.4 µm). The eluates were
diluted 1000ꢀfold with a mixture of acetonitrile/water (1:1). All samples were assessed in triplicate and
analyzed by LCꢀMS/MS using electrospray ionization against standards prepared in the same matrix.
Calibration curve ranged from 1 ng/mL to 2500 ng/mL.
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[35S]-GTPγS Binding Assay. [35S]ꢀGTPγS binding assays were performed on membrane preparations from
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WT mice or GPR88 KO mice, following our previously published methods.21, 22 To assess [35S]ꢀGTPγS
binding in the whole striatal region, brains were quickly removed after cervical dislocation and the whole
striatal region was dissected out, frozen, and stored at ꢀ80 °C until use. Membranes were prepared by
homogenizing brain samples in iceꢀcold 0.25 M sucrose solution 10 vol (mL/g wet weight of tissue). The
obtained suspensions were then centrifuged at 2500 g for 10 min. Supernatants were collected and diluted ten
times in buffer containing 50 mM TrisHCl (pH 7.4), 3 mM MgCl2, 100 mM NaCl, 0.2 mM EGTA, and then
centrifuged at 23,000 g for 30 min. The pellets were homogenized in 800 µL iceꢀcold sucrose solution (0.32
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M), aliquoted and kept at ꢀ80 °C. For [35S]ꢀGTPγS binding assays, 2 ꢀg of protein was used per well. Samples
were incubated with and without the test compound for 1 h at 25 °C in an assay buffer containing 30 mM
GDP and 0.1 nM [35S]ꢀGTPγS. Bound radioactivity was quantified using a liquid scintillation counter. Nonꢀ
specific binding was defined as binding in the presence of 10 ꢁM GTPγS; basal binding refers to binding in
the absence of the agonist. Data were expressed as a mean percentage of activation above the basal binding.
EC50 values were calculated using GraphPad Prism software.
Off-Target Selectivity Evaluation. The offꢀtarget profile of RTIꢀ13951ꢀ33 was assessed in radioligand
binding assays against a panel of 38 GPCRs, ion channels, and transporters at a single concentration of 10 ꢀM
by Eurofins PanLabs (Taipei, Taiwan) according to their standard protocols. The full methods and references
can be found at: http://www.eurofinsdiscoveryservices.com. The percentage of inhibition was given as the
average of two determinations. When significant displacement of radioligand was observed (>50% inhibition
at 10 ꢀM), complete concentrationꢀdependent displacement curves (in duplicate) were constructed to generate
IC50 values. IC50 values were determined by a nonlinear regression analysis using MathIQTM (ID Business
Solutions Ltd., Guildford, Surrey, UK). The equilibrium dissociation constant (Ki) was calculated with the
ChengꢀPrusoff equation using the observed IC50 of the tested compound, the concentration of radioligand, and
the historical values of Kd of the ligand.
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Neurotransmitter transporter assays were conducted by NIMH PDSP using Molecular Devices’
Neurotransmitter Transporter Uptake Assay Kit (R8174) with HEK293 cells stably expressing human SERT.
The full protocol can be found at: http://pdspdb.unc.edu/pdspWeb. In brief, cells were plated in PolyꢀLꢀLys
(PLL) coated 384ꢀwell black clear bottom cell culture plates in DMEM + 1% dialyzed FBS, at a density of
15,000 cells per well in a total volume of 40 ꢀL. The cells were incubated for a minimum of 6 h before being
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used for assays. Medium was removed, and 20 ꢀL of assay buffer (20 mM HEPES, 1X HBSS, pH 7.4) was
added, followed by 5 ꢀL of 5x drug solutions. The plate was incubated at 37 °C for 30 min. After incubation,
25 ꢀL of dye solution were added, and the fluorescence intensity was measured after 30 min at 37 °C, using
the FlexStation II (bottom read mode, Excitation at 440 nm, Emission at 520 nm with 510 nm cutꢀoff).
Results in relative fluorescence units (RLU) were exported and plotted against drug concentrations in Prism
7.0 for nonlinear regression to obtain IC50 values.
Pharmacokinetic Analysis. Snapshot PK study of RTIꢀ13951ꢀ33 was performed using male LongꢀEvans rats
(Paraza Pharma Inc., Montreal, Canada). Doses were formulated in 10% DMSO in saline. On the morning of
the PK study, animals were weighed, and dosing formulation volumes were calculated accordingly. The
compound was injected intraperitoneally to all animals. At selected time points (0.25, 0.5, 1, 2, 4, 8, and 24 h
postdose), animals were anesthetized to perform a cardiac puncture to collect blood for pooled plasma
analysis, followed by whole body perfusion with phosphate saline buffer (pH 7.4) to wash out any remaining
blood from the organs. Brains were harvested and homogenized by polytron 1:4 (w/v) in 25% isopropanol in
water. Brain homogenates were further pooled per corresponding time point and extracted for drug
quantification of LCꢀMS/MS. Samples were prepared and analyzed as follows: Plasma (10 µL) was mixed
with 10 µL of 0.5% formic acid in water, 100 ꢀL internal standard working solution (0.1 ꢀM
Glyburide/Labetalol in acetonitrile), vortexed, and centrifuged at 10000 g for 25 min at 4 °C. Supernatant
(100 ꢀL) was transferred to a 2 mL deepwell plate and diluted with 200 ꢀL water. Brain homogenate (50 µL)
was mixed with 10 µL of 0.5% formic acid in water, 100 ꢀL internal standard working solution (0.1 mM
Glyburide/Labetalol in acetonitrile), vortexed, and centrifuged at 10000 g for 25 min at 4 °C. Supernatant
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(200 ꢀL) was transferred to a 2 mL deepwell plate and diluted with 200 ꢀL water. LCꢀMS/MS was conducted
using an Applied Biosystems API 4000 HPLC system. Chromatography was performed with an Xbridge BEH
C18 (2.1 × 30 mm, 2.5 µm) column. Mobile phases were 0.1% formic acid in water (A), and 0.1% formic
acid in 25% isopropanol/acetonitrile (B). Initial conditions were 5% B and held for 0.1 min, followed by a
linear gradient to 95% B over 1.4 min. 95% B was held for 2.5 min, followed by a linear gradient to 98% B
over 2.55 min. 98% B was held for 3.15 min before returning to initial conditions.
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In Vivo Pharmacology. Self-administration training and testing. Female Long Evans rats (Harlan Sprague–
Dawley, Indianapolis, IN) were double housed, in ventilated cages with water and food available ad libitum in
the home cage. The colony room was maintained on a 12ꢀh light/dark cycle, with lights on at 07:00. All
experiments were conducted during the light cycle. Animals were under continuous care and monitoring by
veterinary staff from the Division of Comparative Medicine (DCM) at UNCꢀChapel Hill. All procedures were
conducted in accordance with the NIH Guide to Care and Use of Laboratory Animals and UNCꢀChapel Hill
institutional guidelines. Rats (n = 8 for the alcohol selfꢀadministration experiments and n = 7 for the sucrose
selfꢀadministration experiments) were trained using the same selfꢀadministration and training procedures
previously described in our publications.32ꢀ34 Selfꢀadministration sessions (30 min) took place 5 days/week
(M−F) with active lever responses on a fixed ratio 2 (FR2) schedule of reinforcement such that every second
response on the lever resulted in delivery of alcohol (0.1 mL) into a liquid receptacle. Responses on the
inactive lever were recorded but produced no programmed consequences. Locomotor activity was measured
during the selfꢀadministration sessions by infrared photobeams that divided the behavioral chamber into 4
parallel zones. Testing was only conducted following stable selfꢀadministration behavior, (i.e., defined as no
change greater than 15% in the total number of responses during the session prior to testing) and rats had 5
months of alcohol selfꢀadministration history prior to testing. Rats received RTIꢀ13951ꢀ33 via ip injection 30
min prior to a selfꢀadministration session. For each experiment, a repeated measures design was used such
that each rat received each dose in a randomized order, with at least one intervening selfꢀadministration
session between testing days.
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Drugs. The 15% alcohol [v/v] + 2% [w/v] sucrose solution was prepared by dissolving 95% alcohol
[v/v] and sucrose in tap water. RTIꢀ13951ꢀ33 was dissolved in sterile 0.9% saline to be administered ip at 1
mL/kg.
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Data analysis. All data were analyzed by oneꢀway repeatedꢀmeasures analysis of variance (ANOVA)
with RTIꢀ13951ꢀ33 dose as the withinꢀsubjects factor, followed by Tukey post-hoc test. Data are represented
as means ± S.E.M., and significance was declared at p < 0.05.
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SUPPORTING INFORMATION
The Supporting Information is available free of change on the ACS Publication website.
HPLC analysis of 4c, 4f, 4h, 5a, 5b and 6, and the full report for offꢀtarget radioligand binding profile (PDF)
Molecular formula strings with biological data (CSV)
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final
version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported in part by the National Institute of Mental Health (NIMH, Grant MH103708 to C.J.)
and National Institute on Alcohol Abuse and Alcoholism (NIAAA, Grant No. 16658 to B.K.; Grant
AA019682 to J.B.), National Institutes of Health, U.S.. V.H.M. was supported by T32NS007431 while
working on this project. R.M. was supported by AA023256 (NIAAA) while working on this project. We
thank Tiffany Langston and Vineetha Vasukuttan for their valuable technical assistance. The functional data
at SERT were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening
Program, Contract HHSNꢀ271ꢀ2013ꢀ00017ꢀC (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth
MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH,
Bethesda MD, USA.
ABBREVIATIONS USED
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GPCR, G proteinꢀcoupled receptor; 2ꢀPCCA, (1R,2R)ꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropane carboxylic acid ((2S,3S)ꢀ
2ꢀaminoꢀ3ꢀmethylpentyl)ꢀ(4′ꢀpropylbiphenylꢀ4ꢀyl)amide; cAMP, cyclic adenosine monophosphate; AUDs,
alcohol use disorders; KO, knockout; MSNs, medium spiny neurons; WT, wideꢀtype; CNS, central nervous
system; SAR, structure activity relationship; HBTU, 2ꢀ(1Hꢀbenzotriazolꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium
hexafluorophosphate; THF, tetrahydrofuran; NMR, nuclear magnetic resonance; MS, mass spectrometry;
HPLC, high performance liquid chromatography; TLC, thinꢀlayer chromatography; HEK, human embryonic
kidney; CHO, Chinese hamster ovary; PPLS, preꢀprolactin leader sequence; HA, human influenza
hemagglutinin; TPSA, topological polar surface area; MDCK, MadinꢀDarby canine kidney; PBS, phosphateꢀ
buffered saline; KOR, kappa opioid receptor; VMAT, vesicular monoamine transporter; SERT, serotonin
transporter; NIMH, National Institute of Mental Health, ADME, absorption, distribution, metabolism, and
excretion; PK, pharmacokinetic; DCM, dichloromethane; DME, 1,2ꢀdimethoxyethane; DIPEA, N,Nꢀ
diisopropylethylamine; RLU, relative fluorescence units; FR2, fixed ratio 2.
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Figure 1. Structures of 2ꢀPCCA (1), (1R,2R)ꢀ2ꢀPCCA (2) and 3, and SAR of sites AꢀC.
Figure 2. Structures of 2ꢀPCCA analogues 4a−h, 5a, 5b, and 6.
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Scheme 1a
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a Reagents: (a) NaBH(OAc)3, 1,2ꢀdichloroethane, rt, overnight; (b) (±)ꢀtransꢀ2ꢀ(pyridinꢀ2ꢀyl)cyclopropaneꢀ
carboxylic acid/oxalyl chloride/DCM/40 °C/2 h, concentrated, then 9a−g/Et3N/DCM, rt, overnight; (c) 4ꢀ
propylphenylboronic acid, Pd(dppf)Cl2·DCM, K3PO4, DME/H2O (3:1), microwave, 160 °C, 6 min; (d) 4 M
HCl/dioxane, DCM, rt, 6 h.
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Scheme 2a
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a Reagents: (a) HBTU, DIPEA, MeCN, rt, overnight; (b) BH3·THF, THF, 65 °C, 2 d; (c) (±)ꢀtransꢀ2ꢀ(pyridinꢀ
2ꢀyl)cyclopropanecarboxylic acid/oxalyl chloride/DCM/40 °C/2 h, concentrated, then 14/Et3N/DCM, rt,
overnight; (d) arylboronic acid, Pd(dppf)Cl2·DCM, K3PO4, DME/H2O (3:1), microwave, 160 °C, 6 min; (e) 4
M HCl/dioxane, DCM, rt, 6 h; (f) chiral HPLC separation.
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