human vasopressin 1a (hV1a); mV1a
RO5028442 is a highly potent and selective antagonist of the human vasopressin 1a (hV1a) receptor. Its binding affinity (Ki) for the hV1a receptor is 1 nM. It exhibits excellent selectivity with a Ki >30000 nM for the human V2 receptor (>30000-fold selective) and a Ki of 9891 nM for the human oxytocin receptor (OTR) (>9891-fold selective) [1].

RG7713 (RO5028442) (Compound 8) exhibits good selectivity for human oxytocin (hOT) and human V2 (hV2) receptors, moderate affinity for mice, and great binding and functional affinity for hV1a. The solubility of RG7713 (RO5028442) is high. For a set of 89 targets, RG7713 (RO5028442) was determined to be extremely selective. Ultimately, it was concluded that RG7713 (RO5028442) was a chemical appropriate for clinical research [1].

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Receptor Binding Profile: In radioligand competition binding assays using membranes from HEK293 cells expressing human receptors, RO5028442 demonstrates high affinity for the hV1a receptor (Ki = 1 nM). It shows no significant binding to a panel of 89 other targets, confirming its high selectivity [1].
Functional Antagonist Activity: In a functional antagonist calcium-flux assay using CHO cells stably expressing the human V1a receptor, RO5028442 inhibits vasopressin-induced calcium signaling. Its apparent functional affinity (Kb) is 2.6 nM [1].

Central AVP administration stimulates V1a, V1b, and oxytocin receptors and induces scratching behavior in mice. In V1a knockout mice, AVP does not induce scratching. In order to probe the potential of our new class of V1a receptor antagonists to antagonize brain V1a receptors, 25 was administered to mice i.c.v. prior to the treatment with AVP. Gratifyingly, 25 was found to dose-dependently suppress scratching. On the basis of its superior pharmacokinetic profile due to the lower clearance and higher volume of distribution compared to those of 24 and 25, we consequently selected 27 for peripheral i.p. administration. In spite of its poor brain penetration, 27 also showed a dose-dependent suppression of AVP induced scratching (Figure 8).[1]

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Central Target Engagement (Scratching Model): Although the primary efficacy data for RO5028442 is not presented, tool compounds from the same series (25 and 27) demonstrated dose-dependent inhibition of central AVP-induced scratching in mice after i.c.v. and i.p. administration, respectively. This model validates that antagonism of brain V1a receptors can produce a quantifiable behavioral effect [1].
Pharmacokinetics in Mice: In mice, after a 2 mg/kg i.v. dose, RO5028442 has a plasma clearance (Cl) of 91 mL/min/kg and a volume of distribution (Vss) of 5.8 L/kg. After a 10 mg/kg oral dose, the brain-to-plasma concentration ratio is 1.4, indicating excellent brain penetration [1].

Human V1a, Human V2, Human OTR, and Mouse V1a Binding Affinity Measurement[1] The human and mouse receptors were cloned by RT-PCR from total human liver RNA (V1a), kidney RNA (V2), mammary gland RNA (OTR), or mouse liver RNA (mouse V1a). Cell membranes were prepared from HEK293 cells transiently transfected with expression vector coding for human V1a, human V2, or mouse V1a. For human OTR membrane preparation, a stable HEK clone expressing the receptor was selected. The transient or stable cells were grown in 20 L fermenters.[1] For each receptor, 50 g of cell pellet was resuspended in 30 mL of ice cold lysis buffer (50 mM HEPES, 1 mM EDTA, and 10 mM MgCl2 adjusted to pH 7.4 + complete cocktail of protease inhibitor (Roche Diagnostics) and homogenized with Polytron for 1 min. The preparation was centrifuged 20 min at 500g at 4 °C, the pellet discarded, and the supernatant centrifuged for 1 h at 43,000g at 4 °C (19’000 rpm). The pellet was resuspended in lysis buffer + sucrose 10%. The protein concentration was determined by the Bradford method and aliquots stored at −80 °C until use.[1] For vasopressin receptor binding studies, 60 mg of yttrium silicate SPA beads were mixed with an aliquot of membrane in binding buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM MgCl2) for 15 min with mixing. 50 μL of bead/membrane mixture was then added to each well of a 96 well plate, followed by 50 μL of 4 nM 3H-vasopressin (American Radiolabeled Chemicals). For total binding measurements, 100 μL of binding buffer was added to the respective wells; for nonspecific binding, 100 μL of 8.4 mM cold vasopressin or cold oxytocin for hOTR was added; and for compound testing, 100 μL of a serial dilution of each compound in 2% DMSO was added. The plate was incubated for 1 h at room temperature, centrifuged 1 min at 1000g, and counted on a Packard Top-Count.[1] Binding to human OTR was measured by filtration binding using 1 nM 3H-oxytocin final concentration in 50 mM Tris, 5 mM MgCl2, and 0.1% BSA (pH 7.4) buffer containing membranes. After compound addition as described above and 1 h of incubation at room temperature, the binding was terminated by rapid filtration under vacuum through GF/C filters, presoaked for 5 min with assay buffer, and washed 5 times with ice-cold assay buffer before counting. Nonspecific binding counts were subtracted from each well and data normalized to the maximum specific binding set at 100%. To calculate the IC50, the curve was fitted using a nonlinear regression model (XLfit) and the Ki calculated using the Cheng–Prussoff equation. Saturation binding experiments performed for each assay indicated that a single homogeneous population of binding sites was being labeled. For receptor binding affinity (Ki) determination, compounds were tested at least 2 times in duplicate, important compounds were tested between 3 and 5 times in duplicate[1].

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Stable Cell Culture and the Human V1a Calcium Flux Assay Using Fluorescent Imaging[1] CHO cells were stably transfected with expression plasmids encoding human V1a and grown in F-12 K, containing 10% fetal bovine serum, 1% penicillin–streptomycin, 1% l-glutamate, and 200 μg/mL geneticin at 37 °C in a 10% CO2 incubator at 95% humidity. Cells were plated for 24 h at 50,000 cells/well in clear bottomed 96 well plates and were dye loaded for 60 min with 2 μM Fluo-4-AM in assay buffer. After cell washing, the plate was loaded on a fluorometric imaging plate reader (FLIPR), compound dilution series added to the cells, and agonist activity measured. None of the compounds tested had agonistic activity. After 20 min of incubation, a concentration of vasopressin (V1a agonist) giving 80% of the maximum signal was added to the plate and the calcium signal recorded for 5 min. The calcium signal reduction due to the antagonist activity of the compounds was fitted to a single site competition equation with formula y = A + ((B – A)/(1 + ((x/C)D))), where y is the % normalized fluorescence, A is the minimum y, B is the maximum y, C is the IC50 (concentration inhibiting 50% of the agonist induced fluorescence), x is the log10 of the concentration of the competing compound, and D the Hill coefficient. IC50 values were transformed in apparent Kb values using the formula: Kb = IC50/1 + (agonist EC80/agonist EC50). Agonist EC80 and EC50 were determined on the same day and the same cells in an independent experiment. For functional antagonism (Kb) determination, all compounds were tested at least 2 times in duplicate, and important compounds were tested between 3 and 5 times in duplicate[1].

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Human V1a Calcium Flux Assay: CHO cells stably expressing the human V1a receptor were plated in 96-well plates and loaded with the calcium-sensitive dye Fluo-4-AM. After washing, various concentrations of RO5028442 were added to the cells and incubated for 20 minutes to check for any agonist activity (none was observed). Subsequently, a concentration of vasopressin (the V1a agonist) that produces 80% of the maximal signal (EC80) was added to each well. The calcium signal was recorded for 5 minutes using a Fluorometric Imaging Plate Reader (FLIPR). The reduction in calcium signal due to antagonist activity was measured. IC50 values were determined by fitting the data to a single-site competition equation. Apparent Kb values were calculated from the IC50 using the formula: Kb = IC50 / (1 + (Agonist EC80 / Agonist EC50)) [1].

Inhibition of AVP Induced Scratching in Mice[1] NMRI male mice (19–21 g) were used with n = 8 per dose. Animals received an injection of either a potential V1a antagonist (different doses i.c.v. or i.p.- 5 or 30 min prior to AVP treatment, respectively) or vehicle. AVP was then administered (i.c.v.) under a short isoflurane anesthesia at a dose of 3 ng/5 μL 2 min prior to behavioral testing. Animals were then observed for 5 min, and the time spent on scratching was recorded. AVP was dissolved in artificial CSF (cerebrospinal fluid); V1a antagonists were administered i.p. in a 0.3% tween 80 in NaCl 0.9% solution or i.c.v. in artificial CSF.

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Mouse Brain Penetration Study:** Male mice were administered a 10 mg/kg oral dose of RO5028442. The compound was formulated in a vehicle (details not specified for this compound but for related compounds, a 0.3% Tween 80 in 0.9% NaCl solution was used for i.p. administration). At a specified time point post-dose, brain and plasma samples were collected. The compound concentrations in both matrices were measured, and the brain-to-plasma ratio was calculated, which was 1.4 [1].
* **Mouse IV Pharmacokinetic Study:** Male mice were administered a 2 mg/kg intravenous dose of RO5028442. Blood samples were collected at various time points, and plasma concentrations were determined. Pharmacokinetic parameters, including clearance (Cl) and volume of distribution (Vss), were calculated using non-compartmental analysis [1].

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Mouse Brain Penetration Study: Male mice were administered a 10 mg/kg oral dose of RO5028442. The compound was formulated in a vehicle (details not specified for this compound but for related compounds, a 0.3% Tween 80 in 0.9% NaCl solution was used for i.p. administration). At a specified time point post-dose, brain and plasma samples were collected. The compound concentrations in both matrices were measured, and the brain-to-plasma ratio was calculated, which was 1.4 [1].
Mouse IV Pharmacokinetic Study: Male mice were administered a 2 mg/kg intravenous dose of RO5028442. Blood samples were collected at various time points, and plasma concentrations were determined. Pharmacokinetic parameters, including clearance (Cl) and volume of distribution (Vss), were calculated using non-compartmental analysis [1].

Solubility: RO5028442 exhibits high aqueous solubility, measured at 20 mg/L (or 5 μg/mL in another unit given) at pH 6.5 [1].
Lipophilicity and P-gp Efflux: The compound is designed to have a balanced lipophilicity to avoid P-glycoprotein (P-gp) mediated efflux. It shows weak P-gp transport, which contributes to its excellent brain penetration [1].
Pharmacokinetic Parameters in Mouse: Plasma clearance (Cl) = 91 mL/min/kg; Volume of distribution at steady state (Vss) = 5.8 L/kg (after 2 mg/kg i.v.). Brain-to-plasma concentration ratio = 1.4 (after 10 mg/kg p.o.) [1].

It is stated that the compound was identified as suitable for human clinical studies, implying a favorable preclinical safety profile. A CEREP selectivity screen against 89 targets showed no significant off-target activities, suggesting a low risk of off-target-related toxicity [1].

[1]. Discovery of highly selective brain-penetrant vasopressin 1a antagonists for the potential treatment of autism via a chemogenomic and scaffold hopping approach. J Med Chem. 2015 Mar 12;58(5):2275-89.

RO5028442 has been used in autism research trials.

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RO5028442 is a highly potent, selective, and brain-penetrant vasopressin 1a (V1a) receptor antagonist discovered through a combination of high-throughput screening, chemogenomics, and scaffold hopping. It was developed for the potential treatment of autism and other psychiatric disorders with social emotional dysfunction, such as anxiety disorders and schizophrenia. The rationale is that a brain-penetrant V1a antagonist may have antidepressant, anxiolytic, and pro-social effects by modulating the social brain. Key to its design is high selectivity over the closely related V2 and oxytocin receptors to avoid peripheral side effects and preserve the pro-social effects of oxytocin. The compound has an excellent pharmacokinetic profile with high solubility, weak P-gp efflux, and good brain penetration (brain/plasma ratio of 1.4 in mice). It was found to be suitable for and has progressed into human clinical studies for autism (NCT01474278) [1].

437.187

C, 68.56; H, 6.44; Cl, 8.09; N, 9.59; O, 7.31

920022-47-5

1228088-30-9 (RO5285119; Balovaptan)

59657596

Typically exists as white to off-white solids at room temperature

1.3±0.1 g/cm3

621.3±55.0 °C at 760 mmHg

329.6±31.5 °C

0.0±1.8 mmHg at 25°C

1.646

3.68

0

3

4

31

651

0

QZXVLRCMAHJVIP-UHFFFAOYSA-N

InChI=1S/C25H28ClN3O2/c1-27(2)13-14-29-16-21(20-8-7-19(26)15-23(20)29)24(30)28-11-9-25(10-12-28)22-6-4-3-5-18(22)17-31-25/h3-8,15-16H,9-14,17H2,1-2H3

[6-chloro-1-[2-(dimethylamino)ethyl]indol-3-yl]-spiro[1H-2-benzofuran-3,4'-piperidine]-1'-ylmethanone

RO-5028442; RO5028442; Ro-5028442; LZ0EU1YHCK; UNII-LZ0EU1YHCK; (6-Chloro-1-(2-(dimethylamino)ethyl)indol-3-yl)-spiro(1H-isobenzofuran-3,4'-piperidine)-1'-yl-methanone; CHEMBL3416885; RO 5028442

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~20 mg/mL (~45.67 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (4.57 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 20.0 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 mg/mL (4.57 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 20.0 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 mg/mL (4.57 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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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