GPR88 (EC50 = 25 nM)

In the GPR88 cAMP functional test, RTI-13951-33 has an EC50 of 25 nM, making it a strong, selective, brain-penetrating GPR88 agonist. RTI-13951-33 elevates [35]-GTPγS binding (EC50, 535 nM) in mouse striatal membranes, but not in GPR88 KO mice's membranes [1]. While RTI-13951-33 has a moderate affinity for the serotonin transporter (SERT; Ki = 0.75 μM), a weak affinity for the kappa opioid receptor (KOR; Ki = 2.29 μM), and a vesicular monoamine transporter (VMAT; Ki = 4.23 μM), it has a poor inhibitory impact on SERT (IC50, 25.1±2.7 μM)[1].

In a rat model of alcohol self-administration, RTI-13951-33 (10 and 20 mg/kg, i.p.) decreases alcohol lever responses in a dose-dependent manner [1]. The sufficient brain penetration of RTI-13951-33 (10 mg/kg, ip) is demonstrated by its t1/2 in rat brain and plasma, which are 48 and 87 minutes, respectively [1].

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In Vivo Activities of RTI-13951-33 . [1]
Because GPR88 KO mice showed enhanced motivation for alcohol drinking and seeking behaviors,23 we sought to determine whether RTI-13951-33 would be effective in vivo to alter operant alcohol self-administration. Female Long Evans rats were trained to self-administer alcohol, as described in our recent publications (15% alcohol [v/v] + 2% [w/v] sucrose vs inactive lever).32–34 This sweetened alcohol concentration was used in all of our studies because we found 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 self-administration was observed at the two highest RTI-13951-33 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 ± SEM; 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 self-administration sessions,35 suggesting that the decrease in alcohol self-administration was not related to a general suppression of activity.

[35S]-GTPγS Binding Assay. [1]
[35S]-GTPγS binding assays were performed on membrane preparations from WT mice or GPR88 KO mice, following our previously published methods. 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 ice-cold 0.25 M sucrose solution 10 vol (mL/g wet weight of tissue). The obtained suspensions were then centrifuged at 2500g for 10 min. Supernatants were collected and diluted 10 times in buffer containing 50 mM TrisHCl (pH 7.4), 3 mM MgCl2, 100 mM NaCl, and 0.2 mM EGTA and then centrifuged at 23000g for 30 min. The pellets were homogenized in 800 μL of ice-cold sucrose solution (0.32 M), aliquoted, and kept at −80 °C. For [35S]-GTPγS binding assays, 2 μg 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. Nonspecific binding was defined as binding in the presence of 10 μM 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.

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Off-Target Selectivity Evaluation. [1]
The off-target profile of RTI-13951-33 was assessed in radioligand binding assays against a panel of 38 GPCRs, ion channels, and transporters at a single concentration of 10 μM by Eurofins PanLabs 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 μM), complete concentration-dependent displacement curves (in duplicate) were constructed to generate IC50 values. IC50 values were determined by a nonlinear regression analysis using MathIQ. The equilibrium dissociation constant (Ki) was calculated with the Cheng–Prusoff equation using the observed IC50 of the tested compound, the concentration of radioligand, and the historical values of Kd of the ligand. [1]
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 poly-l-Lys (PLL) coated 384-well black clear bottom cell culture plates in DMEM + 1% dialyzed FBS at a density of 15000 cells per well in a total volume of 40 μL. The cells were incubated for a minimum of 6 h before being used for assays. Medium was removed, and 20 μL of assay buffer (20 mM HEPES, 1× HBSS, pH 7.4) was added, followed by 5 μL of 5× drug solutions. The plate was incubated at 37 °C for 30 min. After incubation, 25 μL 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 cutoff). Results in relative fluorescence units (RLU) were exported and plotted against drug concentrations in Prism 7.0 for nonlinear regression to obtain IC50 values.

MDCK Permeability Assay. [1]
MDCK-mdr1 cells were grown on Transwell type filters 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 μM in a transport buffer comprising 1× Hank’s balanced salt solution, 25 mM d-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 MDCK-mdr1 cell assays were infused on an Applied Biosystems API-4000 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.

Pharmacokinetic Analysis.[1]
A snapshot PK study of RTI-13951-33 was performed using male Long’Evans 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% 2-propanol in water. Brain homogenates were further pooled per corresponding time point and extracted for drug quantification of LC-MS/MS. Samples were prepared and analyzed as follows: Plasma (10 μL) was mixed with 10 μL of 0.5% formic acid in water, 100 μL internal standard working solution (0.1 μM glyburide/labetalol in acetonitrile), vortexed, and centrifuged at 10000g for 25 min at 4 °C. Supernatant (100 μL) was transferred to a 2 mL deep-well plate and diluted with 200 μL of water. Brain homogenate (50 μL) was mixed with 10 μL of 0.5% formic acid in water, 100 μL internal standard working solution (0.1 mM glyburide/labetalol in acetonitrile), vortexed, and centrifuged at 10000g for 25 min at 4 °C. Supernatant (200 μL) was transferred to a 2 mL deep-well plate and diluted with 200 μL of water. LC-MS/MS was conducted using an Applied Biosystems API 4000 HPLC system. Chromatography was performed with an Xbridge BEH C18 (2.1 mm × 30 mm, 2.5 μm) column. Mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in 25% 2-propanol/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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Self-Administration Training and Testing.[1]
Female Long–Evans rats were double housed in ventilated cages with water and food available ad libitum in the home cage. The colony room was maintained on a 12-h light/dark cycle, with lights on at 07:00. Rats (n = 8 for the alcohol self-administration experiments and n = 7 for the sucrose self-administration experiments) were trained using the same self-administration and training procedures previously described in our publications.32–34 Self-administration 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 self-administration sessions by infrared photobeams that divided the behavioral chamber into four parallel zones. Testing was only conducted following stable self-administration 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 self-administration history prior to testing. Rats received RTI-13951-33 via ip injection 30 min prior to a self-administration 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 self-administration session between testing days.

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Drugs.[1]
The 15% alcohol [v/v] + 2% [w/v] sucrose solution was prepared by dissolving 95% alcohol [v/v] and sucrose in tap water. RTI-13951-33was dissolved in sterile 0.9% saline to be administered ip at 1 mL/kg .

Pharmacokinetic Study. [1]
On the basis of potency, receptor selectivity, and in vitro ADME properties, RTI-13951-33 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, RTI-13951-33 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 half-life 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 ng/mL (527 nM), indicating that RTI-13951-33 had sufficient brain penetration for GPR88 modulation considering it has an EC50 of 25 nM in the cAMP functional assay.

[1]. Discovery of a Potent, Selective, and Brain-Penetrant Small Molecule that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake. J Med Chem. 2018 Aug 9;61(15):6748-6758.

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 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 brain-penetrant GPR88 agonist RTI-13951-33 (6). RTI-13951-33 exhibited an EC50 of 25 nM in an in vitro cAMP functional assay and had no significant off-target activity at 38 GPCRs, ion channels, and neurotransmitter transporters that were tested. RTI-13951-33 displayed enhanced aqueous solubility compared to (1 R,2 R)-2-PCCA (2) and had favorable pharmacokinetic properties for behavioral assessment. Finally, RTI-13951-33 significantly reduced alcohol self-administration and alcohol intake in a dose-dependent manner without effects on locomotion and sucrose self-administration in rats when administered intraperitoneally. [1]

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In summary, we have developed a potent, selective, and brain-penetrant GPR88 agonist RTI-13951-33 (6) based on the 2-PCCA scaffold. The in vitro pharmacological evaluations revealed that RTI-13951-33 was highly potent in the GPR88 cAMP functional assay and had no significant off-target 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. RTI-13951-33 displayed a good aqueous solubility and had favorable pharmacokinetic properties for brain-penetration. RTI-13951-33 showed significant efficacy in reducing alcohol intake in a rat model of alcohol self-administration. Importantly, there was no effect on locomotion measured during the self-administration sessions, indicating that the decrease in alcohol self-administration was not related to a general suppression of activity. Moreover, we demonstrated a specificity between alcohol and natural rewards, with GPR88 agonism reducing alcohol self-administration but not sucrose self-administration at equivalent doses, supporting a role of GPR88 signaling in the modulation of alcohol reinforcement. Further studies of RTI-13951-33 in the mouse model of alcohol drinking and seeking behaviors using both WT and GPR88 KO mice to assess the in vivo on-target 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. [1]

C28H35CL2N3O3

532.50

531.20554

RTI-13951-33;2244884-08-8

137628671

White to light yellow solid powder

3

5

10

36

630

4

C[C@H]([C@@H](CN(C1=CC=C(C=C1)C2=CC=C(C=C2)COC)C(=O)[C@@H]3C[C@H]3C4=CC=CC=N4)N)OC.Cl.Cl

YEUXFGDNARDKIP-DBJQVROASA-N

InChI=1S/C28H33N3O3.2ClH/c1-19(34-3)26(29)17-31(28(32)25-16-24(25)27-6-4-5-15-30-27)23-13-11-22(12-14-23)21-9-7-20(8-10-21)18-33-2;;/h4-15,19,24-26H,16-18,29H2,1-3H3;2*1H/t19-,24-,25-,26-;;/m1../s1

(1R,2R)-N-[(2R,3R)-2-amino-3-methoxybutyl]-N-[4-[4-(methoxymethyl)phenyl]phenyl]-2-pyridin-2-ylcyclopropane-1-carboxamide;dihydrochloride

RTI-13951-33 hydrochloride; CHEMBL4452474; RTI13951-33 hydrochloride; CHEMBL-4452474;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~50 mg/mL (~93.90 mM)
H2O :~50 mg/mL (~93.90 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (187.79 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)