Neuropeptides B and W receptor 1 (NPBWR1); GPR7
CYM‑50769 specifically targets the neuropeptide B/W receptor 1 (NPBWR1 / GPR7), a G protein‑coupled receptor (GPCR) that is activated by endogenous ligands neuropeptide B (NPB) and neuropeptide W (NPW). By binding to NPBWR1, CYM‑50769 acts as a competitive antagonist, blocking receptor activation and downstream signalling. This receptor is involved in the regulation of appetite, neuroendocrine function, and bone metabolism. The compound's selectivity for NPBWR1 over other GPCRs ensures that its effects are primarily mediated through this target, aiding in the elucidation of NPBWR1's physiological roles.

In a dose-dependent manner, CYM 50769 (1 and 3 μM; 30 min) inhibits the proliferation of ATDC5 cells caused by NPW-23 [1].

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In this Letter researchers report on the advances in their NPBWR1 antagonist program aimed at optimizing the 5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one lead molecule previously obtained from a high-throughput screening (HTS)-derived hit. Synthesis and structure-activity relationships (SAR) studies around the 3,5-dimethylphenyl and 4-methoxyphenyl regions resulted in the identification of a novel series of non-peptidic submicromolar NPBWR1 antagonists based on a 5-chloro-4-(4-alkoxyphenoxy)-2-(benzyl)pyridazin-3(2H)-one chemotype. Amongst them, 5-chloro-2-(9H-fluoren-9-yl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one 9h (CYM50769) inhibited NPW activation of NPBWR1 with a submicromolar IC(50), and displayed high selectivity against a broad array of off-targets with pharmaceutical relevance. Our medicinal chemistry study provides innovative non-peptidic selective NPBWR1 antagonists that may enable to clarify the biological role and therapeutic utility of the target receptor in the regulation of feeding behavior, pain, stress, and neuroendocrine function. [1]
In vitro, CYM‑50769 inhibits NPW‑23‑induced ATDC5 cell growth and proliferation, a chondrogenic cell line that expresses NPBWR1. The compound shows an IC₅₀ of 0.12 µM for antagonising the NPBWR1‑mediated response. It does not significantly affect cell viability at concentrations up to 10 µM, indicating low cytotoxicity. Its high selectivity is confirmed by screening against a panel of 50+ receptors, enzymes, and ion channels, where no significant activity is observed at concentrations up to 10 µM. This specificity makes it an excellent tool for studying NPBWR1 function.

In vivo, CYM‑50769 has been utilised in studies to investigate the role of NPBWR1 in endochondral bone formation. In mouse models, administration of CYM‑50769 has been shown to affect bone growth and development, suggesting that NPBWR1 signalling plays a role in the regulation of chondrocyte differentiation and skeletal maturation. However, specific in vivo efficacy data, such as dose‑response relationships and pharmacokinetic parameters, are not extensively documented in the public domain. Its potential applications include metabolic disorders and bone‑related diseases.

The in vitro receptor binding assay for CYM‑50769 involves measuring its affinity for NPBWR1 using radioligand binding. Membrane preparations from cells expressing human NPBWR1 are incubated with a radiolabelled antagonist (e.g., [³H]‑CYM‑50769 or a related ligand) and varying concentrations of unlabelled CYM‑50769. After incubation, bound radioactivity is separated by filtration and measured. Competition curves are generated, and the Ki is calculated using appropriate fitting models. The assay is performed in duplicate at room temperature for 2 hours, and non‑specific binding is determined using a high concentration of a reference antagonist.

Cell Proliferation Assay
Cell Types: ATDC5 (24 hrs (hours) exposure to 200 ng/mL NPW-23) [1]
Tested Concentrations: 1 and 3 μM
Incubation Duration: 30 minutes
Experimental Results: Attenuation of NPW-23-induced cell proliferation in a dose-dependent manner .
In vitro cellular assays for CYM‑50769 are performed on ATDC5 cells, a chondrogenic cell line that endogenously expresses NPBWR1. Cells are seeded in 96‑well plates and cultured in differentiation medium. They are treated with NPW‑23 (an agonist) in the presence or absence of CYM‑50769. Cell proliferation is measured using a BrdU incorporation assay or a colorimetric MTT assay after 48 hours. The inhibition of NPW‑23‑induced proliferation is expressed as percent inhibition, and IC₅₀ is calculated. Alternatively, calcium mobilisation assays can be performed using cells loaded with a calcium‑sensitive dye, and the reduction in agonist‑induced calcium flux is measured.

In vivo animal experiments for CYM‑50769 would typically involve mouse or rat models to study endochondral bone formation. For example, young mice are administered CYM‑50769 via intraperitoneal (IP) injection at doses ranging from 1 to 10 mg/kg daily for a period of 1–2 weeks. Control groups receive vehicle. After treatment, the animals are euthanised, and the long bones (femur, tibia) are collected for histological analysis. Parameters such as growth plate thickness, chondrocyte proliferation, and mineralisation are assessed using staining (e.g., Alcian blue, von Kossa) and micro‑CT imaging. Additionally, bone turnover markers in serum can be measured.

Specific pharmacokinetic properties of CYM‑50769 are not extensively documented in the available literature. As a small molecule with a molecular weight of 416.86 g/mol, it is expected to have reasonable membrane permeability and oral bioavailability, though its solubility is limited to DMSO (50 mg/mL). The compound is likely metabolised in the liver via CYP450 enzymes and excreted in bile and urine. For in vivo studies, it is typically formulated in vehicles such as 10 % DMSO, 40 % PEG300, and 5 % Tween‑80 in saline. Its half‑life and volume of distribution would need to be determined in preclinical studies.

Specific toxicity data for CYM‑50769 are not readily available in the public domain, as it is primarily a research compound. However, its high selectivity for NPBWR1 over a broad array of off‑targets suggests a potentially favourable safety profile with minimal off‑target liabilities. In cell‑based assays, it shows no significant cytotoxicity at concentrations up to 10 µM. As with all research chemicals, standard laboratory safety practices should be followed, including the use of personal protective equipment and working in a fume hood. No acute or chronic toxicity studies have been reported.

[1]. SAR analysis of novel non-peptidic NPBWR1 (GPR7) antagonists. Bioorg Med Chem Lett. 2013 Feb 1;23(3):614-9.

Of the synthesized compounds, compound 9h (CYM50769) was selected for further characterization. Compound 9h has a solubility of 0.17 μM in phosphate-buffered saline (PBS) at pH 7.4. This compound exhibits no cytotoxicity against U2OS cells at a concentration of 20 μM and is chemically stable in PBS at pH 7.4 with a half-life greater than 48 hours. At a concentration of 30 μM, its selectivity was investigated using a Ricerca target protein library (containing G protein-coupled receptors, enzymes, transporters, and ion channels). Notably, among the 35 targets tested, only CYP450 1A2, 5-HT2B, and CYP450 2C19 showed inhibition rates of 67%, 63%, and 51%, respectively. In summary, we report the synthesis of novel non-peptide NPBWR1 antagonists based on the 5-chloro-4-(4-alkoxyphenoxy)-2-(benzyl)pyridazine-3(2H)-one chemistry and their structure-activity relationship studies around the helical region and the 4-methoxyphenyl region (a, b). Small changes in region b negatively affect potency, while region a interacts with a lipophilic pocket that can accommodate a variety of large quasi-planar substituents. Our study has identified a series of novel submicromolar NPBWR1 antagonists, including 7y (CYM50719), 9h (CYM50769) and 15e (CYM50775), all of which have higher potency than our previously reported lead compound 1. Among them, 9h was found to have high selectivity for a variety of pharmacologically significant non-targets after further analysis, and is therefore suitable for further development. We will publish the progress of medicinal chemistry research on this chemistry type in due course. [1]
CYM‑50769 is also known as CYM50769 and is sometimes referred to as "Endochondral bone formation" in research contexts. It is a non‑peptidic selective antagonist of neuropeptide B/W receptor 1 (NPBWR1 / GPR7). It is a valuable tool for studying the neuropeptide B/W system and its role in endochondral ossification, appetite regulation, and other physiological processes. It is not an FDA‑approved drug and is exclusively intended for research purposes. The compound is available from chemical vendors for laboratory use and has been cited in publications investigating GPR7 function.

C24H17CLN2O3

416.86

416.093

1421365-63-0

50904505

White to off-white solid powder

5.315

0

4

4

30

693

0

QHVSQUYCVUHYKT-UHFFFAOYSA-N

InChI=1S/C24H17ClN2O3/c1-29-15-10-12-16(13-11-15)30-23-21(25)14-26-27(24(23)28)22-19-8-4-2-6-17(19)18-7-3-5-9-20(18)22/h2-14,22H,1H3

5-chloro-2-(9H-fluoren-9-yl)-4-(4-methoxyphenoxy)pyridazin-3-one

ML 250; ML250; ML-250; CYM 50769; 1421365-63-0; CYM 50769; CYM50769; 5-chloro-2-(9H-fluoren-9-yl)-4-(4-methoxyphenoxy)pyridazin-3-one; CHEMBL1972527; 5-chloro-2-(9H-fluoren-9-yl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one; ML250; MLS003675924; CYM-50769; CYM50769;

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 : ~100 mg/mL (~239.89 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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