hV1a ( Ki = 1 nM ); mV1a ( Ki = 39 nM )

Balovaptan (RG7314) exhibits selectivity for hV1a over hV2 receptors that is >30000-fold, and selectivity over the human oxytocin receptor (hOTR) that is 9891-fold[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]

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].

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].

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.

[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.

Balovaptan is under investigation in clinical trial NCT01793441 (A Study of RG7314 to Investigate Efficacy and Safety in Individuals With Autism Spectrum Disorders (ASD)).

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Antidiuretic Hormone Receptor Antagonists: Endogenous compounds and drugs that inhibit or block the activity of ANTIDUIRETIC HORMONE RECEPTORS.

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From a micromolar high throughput screening hit 7, the successful complementary application of a chemogenomic approach and of a scaffold hopping exercise rapidly led to a low single digit nanomolar human vasopressin 1a (hV1a) receptor antagonist 38. Initial optimization of the mouse V1a activities delivered suitable tool compounds which demonstrated a V1a mediated central in vivo effect. This novel series was further optimized through parallel synthesis with a focus on balancing lipophilicity to achieve robust aqueous solubility while avoiding P-gp mediated efflux. These efforts led to the discovery of the highly potent and selective brain-penetrant hV1a antagonist RO5028442 (8) suitable for human clinical studies in people with autism.[1]

C22H24CLN5O

409.918

409.166

C, 64.46; H, 5.90; Cl, 8.65; N, 17.09; O, 3.90

1228088-30-9

46200932

White to off-white solid powder

1.4±0.1 g/cm3

597.0±60.0 °C at 760 mmHg

314.9±32.9 °C

0.0±1.7 mmHg at 25°C

1.702

3.16

ClC1C=CC2=C(C=1)CN(C)CC1=NN=C(C3CCC(CC3)OC3C=CC=CN=3)N12

GMPZPHGHNDMRKL-UHFFFAOYSA-N

InChI=1S/C22H24ClN5O/c1-27-13-16-12-17(23)7-10-19(16)28-20(14-27)25-26-22(28)15-5-8-18(9-6-15)29-21-4-2-3-11-24-21/h2-4,7,10-12,15,18H,5-6,8-9,13-14H2,1H3

8-chloro-5-methyl-1-(4-pyridin-2-yloxycyclohexyl)-4,6-dihydro-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine

RG7314; RG-7314; RO-5285119; RG 7314; RG7314; RO5285119; Balovaptan [INN]; Balovaptan [USAN]; RAX5D5AGV6; RG-7314; RO5285119

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: ~50 mg/mL (~122.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.10 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 25.0 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: ≥ 2.5 mg/mL (6.10 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 25.0 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: ≥ 2.5 mg/mL (6.10 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 25.0 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.)