(rel)-AR234960 is a selective and potent agonist of the MAS receptor (MAS1, Mas-related G protein-coupled receptor), which is a component of the renin-angiotensin system (RAS). MAS is known to bind angiotensin-(1-7) [Ang-(1-7)] and mediates many of the vasodilatory, anti-proliferative, anti-fibrotic, and cardioprotective effects of Ang-(1-7) through various intracellular signaling pathways. However, (rel)-AR234960, as a non-peptide MAS agonist, displays a distinct signaling profile: it promotes fibrosis (collagen synthesis) rather than anti-fibrotic effects. Upon binding to MAS, (rel)-AR234960 activates the ERK1/2 (extracellular signal-regulated kinase 1/2) signaling pathway, which is a classic mitogen-activated protein kinase (MAPK) cascade involved in cell proliferation, differentiation, and survival. The activation of ERK1/2 by (rel)-AR234960 leads to the upregulation of CTGF (connective tissue growth factor) at both the mRNA and protein levels in cardiac fibroblasts and in HEK293-MAS cells. CTGF is a matricellular protein that plays a key role in promoting extracellular matrix deposition, fibrosis, and tissue remodeling. Through this MAS-ERK1/2-CTGF signaling axis, (rel)-AR234960 promotes the synthesis of collagen types I and III (COL1A1, COL3A1), which are the major components of the extracellular matrix in fibrotic tissue. Thus, (rel)-AR234960 functions as a selective MAS receptor agonist with pro-fibrotic signaling outcomes. Its pro-fibrotic activity can be blocked by the MAS inverse agonist AR244555 or by a MEK1 inhibitor (e.g., PD98059 or U0126), confirming the role of the ERK1/2 pathway in mediating its effects. This compound has no reported activity at the angiotensin II type 1 receptor (AT1R) or angiotensin II type 2 receptor (AT2R), demonstrating selectivity for MAS. The (rel)-AR234960 designation indicates that it is a racemic mixture or the relative configuration of the active isomer(s); AR234960 itself is the non-peptide MAS agonist. This compound is used as a positive control in assays evaluating MAS receptor activation and as a tool to distinguish MAS-mediated pro-fibrotic signaling from Ang-(1-7)-mediated anti-fibrotic signaling. It serves as a chemical probe to study the molecular switch in MAS signaling that determines the balance between pro-fibrotic and anti-fibrotic outcomes, which is relevant to understanding the pathophysiology of heart failure with preserved ejection fraction (HFpEF), myocardial infarction, and other cardiac diseases where fibrosis plays a central role.

In vitro, (rel)-AR234960 (or AR234960, the non-peptide MAS agonist) has been demonstrated to increase both mRNA and protein levels of CTGF in a concentration-dependent manner in HEK293 cells stably expressing the MAS receptor (HEK293-MAS cells) and in adult human cardiac fibroblasts (HCFs). The EC₅0 for CTGF upregulation is reported to be in the submicromolar to low micromolar range, although the exact value may vary depending on the cell type and experimental conditions. The effect is specific to MAS activation, as it is blocked by co-treatment with the MAS inverse agonist AR244555. The pro-fibrotic effect is mediated through the ERK1/2 MAPK pathway, as pre-incubation with a MEK1 inhibitor (e.g., PD98059 or U0126) completely abrogates the increase in CTGF expression and subsequent collagen synthesis. In addition to CTGF upregulation, (rel)-AR234960 increases the expression of collagen type I alpha 1 (COL1A1) and collagen type III alpha 1 (COL3A1) genes in cardiac fibroblasts, as measured by quantitative real-time PCR (qRT-PCR). Collagen protein synthesis and deposition can be quantified by measuring hydroxyproline content in the culture medium or cell lysates, or by using a soluble collagen assay (e.g., Sircol assay) that detects secreted collagen in the culture supernatant. In contractile cardiac myocytes or other cell types that express the MAS receptor, (rel)-AR234960 may have different effects (e.g., potentially promoting hypertrophy or apoptosis), but the primary reported activity in the literature relates to its pro-fibrotic action in fibroblasts. The compound does not induce significant cytotoxicity in the concentration range used to activate MAS (up to 10 microM), as assessed by cell viability assays (MTT, CellTiter-Glo) or LDH release. The in vitro activity of (rel)-AR234960 has been used to establish a cellular model of MAS-mediated fibrosis, which is useful for screening potential anti-fibrotic drugs that target the MAS-ERK1/2-CTGF axis. The compound‘s ability to upregulate CTGF and collagen is blocked by the MAS inverse agonist AR244555 and by ERK1/2 inhibitors, confirming the specificity and pathway involvement. Other signaling pathways downstream of MAS, such as PI3K/Akt or NO/cGMP, may not be activated by (rel)-AR234960 to the same extent as by Ang-(1-7), explaining the differential biological outcomes (pro-fibrotic vs. anti-fibrotic). Detailed in vitro activity data (e.g., EC₅0, maximal effect, time course, concentration-response curves) may be available in the primary research literature for AR234960, but these specific numerical values are not consistently provided in standard supplier datasheets. The compound is supplied as the racemic or relative configuration mixture, and the absolute configuration of the active enantiomer is not specified in the catalog information.

The in vivo activity of (rel)-AR234960 is characterized primarily by its effects on cardiac fibrosis and extracellular matrix remodeling. In vivo studies have demonstrated that the MAS agonist (AR234960, the non-peptide agonist) promotes collagen synthesis and aggravates fibrosis in the heart. The specific in vivo experimental details, such as the animal model, dosing regimen, route of administration, and observed effects on cardiac structure and function, are not provided in detail in standard supplier datasheets but are available in the research literature. It is known that (rel)-AR234960 can be used in animal models of heart failure and cardiac injury to study the contribution of the MAS-ERK1/2-CTGF pathway to the pathogenesis of cardiac fibrosis. Since the compound is a non-peptide agonist, it is expected to have better oral bioavailability and metabolic stability compared to the endogenous peptide agonist Ang-(1-7), making it a more practical tool for in vivo pharmacological studies. In vivo, the pro-fibrotic effects of (rel)-AR234960 can be assessed by measuring the expression of fibrotic markers (collagen I, collagen III, CTGF, alpha-SMA, fibronectin) in heart tissue by qRT-PCR, Western blotting, or immunohistochemistry; by quantifying total collagen content in the heart using hydroxyproline assay or picrosirius red staining (which can be visualized under polarized light microscopy to distinguish collagen types I and III); and by echocardiography to assess changes in cardiac function (ejection fraction, fractional shortening, left ventricular end-diastolic diameter, left ventricular mass, and markers of diastolic dysfunction such as E/A ratio, E/e‘ ratio, left atrial volume). The pro-fibrotic effect of (rel)-AR234960 can be blocked by co-administration of the MAS inverse agonist AR244555 or an ERK1/2 inhibitor (e.g., PD98059, U0126, or selumetinib), confirming the involvement of the MAS-ERK1/2-CTGF axis. In addition to the heart, (rel)-AR234960 may have effects in other tissues where the MAS receptor is expressed, including the kidney, lung, liver, and vasculature, but the primary research application is in the context of cardiac biology. (rel)-AR234960 is not a therapeutic agent; it is a pharmacological tool used to explore the biology of the MAS receptor, particularly its role in fibrosis. The compound may also be used to investigate the effects of MAS activation on blood pressure regulation (since Ang-(1-7) is a vasodilator), but AR234960‘s pro-fibrotic activity suggests that its effects on the vasculature may differ from those of Ang-(1-7). Detailed in vivo study protocols (e.g., animal species, dose, route, frequency, duration of treatment, endpoint measurements) are not described in the supplier datasheets and must be obtained from published research articles. The compound is intended for research use only and is not for human therapeutic use.

For in vitro assays of MAS receptor-mediated signaling, two primary approaches are described. Approach 1 - CTGF mRNA expression by qRT-PCR: Human cardiac fibroblasts (HCFs, adult primary cells) or HEK293 cells stably transfected with the MAS receptor (HEK293-MAS) are cultured in appropriate media (e.g., DMEM/F12 for HCFs, DMEM for HEK293) supplemented with 10% FBS and antibiotics. Cells are seeded in 6-well plates (3-5×10⁵ cells/well) or 12-well plates (1-2×10⁵ cells/well) and allowed to reach 70-80% confluence. The medium is replaced with serum-free medium (or medium containing 0.1-0.5% FBS) 12-24 hours before stimulation to reduce background signaling. (rel)-AR234960 (or AR234960) is dissolved in DMSO to make a stock solution (e.g., 10 mM). Serial dilutions are prepared in serum-free medium, and the final DMSO concentration should be ≤0.1% (v/v) to avoid cytotoxicity. Cells are treated with (rel)-AR234960 at concentrations ranging from 1 nM to 10 microM (a broad range; the exact EC₅0 can be determined empirically). Control cells receive vehicle (0.1% DMSO in medium). For time-course studies, cells are treated with a fixed concentration of (rel)-AR234960 (e.g., 1 microM) for 1, 3, 6, 12, 24, and 48 hours. For inhibitor studies (to confirm pathway involvement), cells are pre-incubated for 30-60 minutes with: (i) the MAS inverse agonist AR244555 (1-10 microM), (ii) a MEK1/2 inhibitor such as PD98059 (10-50 microM) or U0126 (5-10 microM), (iii) a p38 MAPK inhibitor (SB203580, 10 microM), or (iv) a PI3K inhibitor (LY294002, 10 microM) before adding (rel)-AR234960. After the treatment period, total RNA is extracted from cells using TRIzol reagent or an RNA isolation kit. cDNA is synthesized using reverse transcriptase with random hexamers or oligo(dT) primers. Quantitative real-time PCR (qRT-PCR) is performed using SYBR Green or TaqMan probes with specific primers for: CTGF (connective tissue growth factor), COL1A1 (collagen type I alpha 1), COL3A1 (collagen type III alpha 1), FN1 (fibronectin 1), ACTA2 (alpha-smooth muscle actin, alpha-SMA), and TGFB1 (transforming growth factor beta 1). Housekeeping genes (GAPDH, beta-actin, or 18S rRNA) are used as internal controls. Relative gene expression is calculated using the 2-deltadeltaCt method, and results are expressed as fold-change relative to vehicle control. Approach 2 - Western blotting for CTGF, phospho-ERK1/2, and collagen: Cells are seeded and treated as described above. After the treatment period (e.g., 6, 12, 24 hours for CTGF; 15, 30, 60 minutes for phospho-ERK1/2), cells are washed with ice-cold PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) containing protease inhibitor cocktail and phosphatase inhibitors (e.g., 1 mM Na3VO4, 1 mM NaF). Lysates are cleared by centrifugation at 12,000 × g for 15 minutes at 4degC. Protein concentration is determined by the BCA assay. Equal amounts of protein (20-50 microg per lane) are separated by SDS-PAGE (10-12% gel for CTGF, 10% for phospho-ERK1/2, 7.5% for collagen) and transferred to PVDF or nitrocellulose membranes. Membranes are blocked with 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. Primary antibodies (diluted in blocking buffer) are incubated overnight at 4degC: anti-CTGF (1:500-1:2000), anti-phospho-ERK1/2 (Thr202/Tyr204, 1:1000), anti-total ERK1/2 (1:1000), anti-collagen type I (1:500), anti-collagen type III (1:500), and anti-beta-actin or anti-GAPDH (1:2000-1:5000) as loading control. After washing with TBST, membranes are incubated with HRP-conjugated secondary antibodies (1:2000-1:10000) for 1 hour at room temperature. Protein bands are visualized using enhanced chemiluminescence (ECL) substrate, and band intensities are quantified by densitometry using ImageJ or similar software. Phospho-ERK1/2 levels are normalized to total ERK1/2 or to beta-actin. CTGF, collagen I, and collagen III levels are normalized to beta-actin. For collagen secretion assays: Cell culture supernatants are collected after 48-72 hours of treatment. The concentration of secreted collagen is measured using the Sircol soluble collagen assay (Biocolor) according to the manufacturer‘s instructions. Briefly, an aliquot of the supernatant (200-500 microL) is mixed with Sircol dye reagent, which binds to collagens I-V. The collagen-dye complex is precipitated by centrifugation, and the bound dye is released by an alkaline reagent (0.5 M NaOH). The absorbance is measured at 540 nm, and the collagen concentration is determined by interpolation from a standard curve using collagen standard (provided in the kit). Hydroxyproline content (an amino acid uniquely abundant in collagen) can be measured by acid hydrolysis of cell lysates or culture medium (6 M HCl, 110degC, 16-24 hours), followed by chloramine-T oxidation and reaction with Ehrlich‘s reagent (p-dimethylaminobenzaldehyde) to produce a chromophore with absorbance at 558 nm. The results are expressed as microg of hydroxyproline per mg of protein.

For in vitro cellular assays to assess the effects of (rel)-AR234960 on cell proliferation, migration, and differentiation, primary human cardiac fibroblasts (HCFs) or NIH/3T3 fibroblasts (a standard fibroblast cell line) can be used. (rel)-AR234960 is added to the culture medium (typically 0.1-10 microM) for 24-72 hours. Cell proliferation is measured by BrdU (5-bromo-2‘-deoxyuridine) incorporation, by Ki67 immunostaining, or by the MTT assay (as a proxy for cell number). Because (rel)-AR234960 promotes fibrosis (collagen deposition) rather than proliferation per se, the primary outcome measures are collagen synthesis and CTGF upregulation rather than cell proliferation; the effect on proliferation may be modest. Cell migration is assessed by scratch-wound closure assay: a confluent monolayer of fibroblasts in a 6-well plate is scratched with a sterile pipette tip to create a cell-free gap. Cells are washed to remove debris, and then treated with (rel)-AR234960 (0.1-10 microM) or vehicle in serum-free or low-serum (0.5% FBS) medium. Migration is monitored by taking images at 0, 12, 24, 36, and 48 hours, and the wound closure area is quantified using image analysis software (e.g., ImageJ). The effect of (rel)-AR234960 on fibroblast-to-myofibroblast differentiation is assessed by immunostaining for alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblast differentiation. Cells are grown on coverslips, treated with (rel)-AR234960 for 48-72 hours, fixed, permeabilized, and stained with an anti-alpha-SMA antibody followed by a fluorescent secondary antibody. Cells are counterstained with DAPI (to visualize nuclei). The percentage of alpha-SMA-positive cells is quantified by fluorescence microscopy. Since alpha-SMA expression is also induced by TGF-beta1 (transforming growth factor beta 1, a potent pro-fibrotic cytokine), the effect of (rel)-AR234960 on alpha-SMA may be less pronounced than the effect on collagen synthesis; however, increased collagen deposition is often accompanied by myofibroblast differentiation. As a positive control for pro-fibrotic activation, TGF-beta1 (1-10 ng/mL) is used. For specificity control, the MAS inverse agonist AR244555 (1-10 microM) is co-incubated with (rel)-AR234960 to confirm that the observed effects are mediated by MAS. For cell viability and toxicity assessment, the MTT assay is performed on cells treated with (rel)-AR234960 (0.1-100 microM) for 24-72 hours. The compound is generally non-toxic at concentrations up to 10 microM, but higher concentrations (≥50 microM) may cause some degree of cytotoxicity or cell growth inhibition. IC₅0 for cytotoxicity (LC₅0) should be determined for each cell line if needed; it is typically >50 microM. The compound is stored at -20degC as a solid (or as a 10 mM solution in DMSO). It is soluble in DMSO and should be protected from light and moisture.

For in vivo pharmacology studies to evaluate the pro-fibrotic effects of (rel)-AR234960 in the heart, several animal models can be used. Model 1 - Spontaneous or induced cardiac fibrosis model (rat or mouse): 8-12 week old male C57BL/6 mice or Sprague-Dawley rats (250-300 g) are used. (rel)-AR234960 is formulated in a suitable vehicle (e.g., 5% DMSO + 5% Tween-80 + 90% saline, or 0.5% carboxymethyl cellulose (CMC) with 0.1% Tween-80, or PEG400/water mixture). The compound is administered daily by intraperitoneal (i.p.) injection or by oral gavage at doses ranging from 1 to 30 mg/kg (optimal dose to be determined from preliminary dose-response experiments). The treatment duration can range from 7 days to 4 weeks, depending on the desired degree of fibrosis. Control animals receive vehicle alone. At the end of the treatment period, animals are euthanized, and hearts are excised, rinsed with PBS, and bisected. One half is fixed in 10% formalin or 4% paraformaldehyde for histology, and the other half is snap-frozen in liquid nitrogen for biochemical analysis (protein extraction for Western blotting, RNA extraction for qRT-PCR, or hydroxyproline assay). To assess collagen deposition, paraffin-embedded heart sections (5 microm thickness) are stained with picrosirius red (PSR) to visualize fibrillar collagens (types I and III). Under brightfield microscopy, collagen appears red; under polarized light microscopy, type I collagen appears bright red/orange and type III collagen appears green. The percentage of collagen area (collagen volume fraction) relative to the total tissue area in the left ventricle (excluding the right ventricle and atria) is quantified by image analysis (e.g., ImageJ, Aperio ImageScope). Masson‘s trichrome staining is an alternative for visualizing collagen (blue) against the background of muscle (red). For semi-quantitative scoring, a pathologist can score fibrosis on a scale (0 = no fibrosis, 1 = mild, 2 = moderate, 3 = severe). Hydroxyproline assay: 20-50 mg of frozen heart tissue is homogenized in 0.5 mL of 6 M HCl and hydrolyzed at 110degC for 16-18 hours. After hydrolysis, the sample is centrifuged, and the supernatant is dried under vacuum. The residue is reconstituted in water, and hydroxyproline content is measured using a colorimetric assay (e.g., with chloramine-T and Ehrlich‘s reagent). The hydroxyproline content is expressed as microg per mg of wet tissue weight or per mg of protein. Western blotting: Frozen heart tissue is pulverized in liquid nitrogen and homogenized in RIPA buffer with protease/phosphatase inhibitors. The homogenate is centrifuged, and the supernatant is used for Western blot analysis of CTGF, collagen I, collagen III, alpha-SMA, phospho-ERK1/2, and total ERK1/2 as described in the in vitro section. qRT-PCR: Total RNA is extracted from frozen heart tissue (using TRIzol or a commercial kit), and qRT-PCR is performed as described in the in vitro section for CTGF, COL1A1, COL3A1, and other pro-fibrotic genes. Echocardiography: Before euthanasia, mice or rats are anesthetized with isoflurane (1-2% in oxygen) and placed on a heating pad. Transthoracic echocardiography is performed using a high-frequency ultrasound system (e.g., VisualSonics Vevo 2100 or similar) equipped with a 30-40 MHz transducer. Standard parasternal long-axis and short-axis views are acquired. The following parameters are measured in M-mode or 2D mode: left ventricular internal diameter at end-diastole (LVIDd) and end-systole (LVIDs); left ventricular ejection fraction (LVEF%); fractional shortening (FS%); left ventricular mass (LV mass); and posterior wall thickness (PWT). Diastolic function is assessed by pulse-wave Doppler of mitral inflow (E wave: early diastolic filling; A wave: atrial contraction-induced filling) and tissue Doppler imaging (e′ wave: early diastolic mitral annular velocity). The E/A ratio (normal >1.0-1.5) and E/e‘ ratio (marker of left ventricular filling pressure) are calculated. An increase in collagen deposition is expected to be associated with changes in diastolic function (reduced E/A ratio, increased E/e‘ ratio) with preserved or slightly reduced systolic function, consistent with a heart failure with preserved ejection fraction (HFpEF) phenotype. Histological assessment of myocardial cross-sections can also be used to measure myocyte hypertrophy (cross-sectional area of myocytes in H&E-stained sections). Model 2 - Myocardial infarction (MI)-induced cardiac fibrosis model: Adult rats or mice undergo left anterior descending (LAD) coronary artery ligation to induce a large myocardial infarction. Starting 1-3 days after MI, (rel)-AR234960 is administered daily for 4 weeks. At the end of the study, cardiac function is assessed by echocardiography, and fibrosis is quantified by picrosirius red staining (scar size, border zone fibrosis, and remote zone fibrosis). The pro-fibrotic effect of (rel)-AR234960 may be more pronounced in the setting of MI (increased remodeling and fibrosis) or may be used to study the effect of MAS activation on infarct healing and scar formation. For inhibitor studies (to confirm pathway involvement), the MAS inverse agonist AR244555 (1-10 mg/kg, i.p.) or the MEK1/2 inhibitor PD98059 (10-30 mg/kg, i.p.) or selumetinib (1-5 mg/kg, oral) is co-administered with (rel)-AR234960. The effects on collagen deposition, CTGF expression, and cardiac function are compared between the treatment groups. This compound is intended for research use only, not for human therapeutic use, and should be handled with appropriate precautions. The specific in vivo protocols described above are based on standard practice in cardiovascular research and are not provided in the compound datasheet; they are provided here as general guidance for potential users of (rel)-AR234960 as a research tool.

As a small-molecule non-peptide agonist (molecular weight 555.63), (rel)-AR234960 is expected to have favorable pharmacokinetic properties, including oral bioavailability, as compared to the endogenous peptide agonist Ang-(1-7), which has very poor oral absorption. The non-peptide nature suggests that the compound is stable in the gastrointestinal tract and can be effectively absorbed. The pharmacokinetic parameters of AR234960 and (rel)-AR234960 have not been detailed in standard supplier datasheets, but the compound‘s utility as a research tool for in vivo studies implies reasonable metabolic stability and adequate systemic exposure following intraperitoneal or oral administration. The typical dose range used in animal studies (likely 1-30 mg/kg, based on the effective concentrations in vitro (low micromolar) and the assumption of moderate bioavailability) is in line with other small-molecule GPCR agonists. The compound is lipophilic (logP not provided) and may have high plasma protein binding, which could affect the free concentration available to interact with the MAS receptor. (rel)-AR234960 is metabolized, most likely in the liver (by CYP450 enzymes), but specific metabolites and the major elimination pathways have not been reported. The plasma half-life and volume of distribution are not known from the supplier datasheet. For intraperitoneal administration, the onset of action is expected to be within 30-60 minutes. The compound‘s tissue distribution would include the heart, where the MAS receptor is expressed in cardiomyocytes, fibroblasts, and endothelial cells. The compound is stored as a solid at -20degC, and is soluble in DMSO (stock solution), PEG400, and other organic solvents. For in vivo administration, the compound should be formulated fresh daily and protected from light. Given the lack of extensive PK data, dose-finding studies (pharmacodynamic dose-response) are recommended when using this compound in a new animal model. The compound is a research chemical, not an approved drug, and its ADME (absorption, distribution, metabolism, excretion) properties have not been fully characterized for regulatory purposes. Users should refer to the primary literature for any published PK data related to AR234960. However, for most research applications (mechanistic studies in cells or short-term in vivo efficacy studies in rodents), detailed PK parameters are not required; instead, the focus is on selecting doses that achieve target engagement and observable biological effects without significant toxicity. As a general approach, a dose range of 1-10 mg/kg intraperitoneally or 3-30 mg/kg orally can be tested in preliminary dose-response experiments with CTGF upregulation or collagen deposition as the endpoint, and the lowest dose that produces a significant effect can be selected for further studies. The compound should be handled under sterile conditions for in vivo injections to avoid contamination. Due to the lack of formal PK data, this summary is based on the assumption that (rel)-AR234960 behaves as a typical small-molecule GPCR agonist with moderate to good oral bioavailability. Researchers using the compound are encouraged to conduct their own pilot PK or pharmacodynamic studies to determine the optimal dosing regimen for their specific model.

As a research chemical, dedicated toxicology studies for (rel)-AR234960 have not been conducted, and its safety profile is not established. The compound is not intended for human use; it is a pharmacological tool for laboratory research only. Based on its mechanism of action as a pro-fibrotic MAS agonist, (rel)-AR234960 would be expected to promote tissue fibrosis (particularly in the heart, kidney, lung, and liver) when administered systemically over prolonged periods. Therefore, chronic administration may induce pathological tissue remodeling, which could be harmful. However, for acute or short-term studies, the compound is likely well-tolerated at doses that are sufficient to activate MAS and upregulate CTGF/collagen expression (likely 1-10 mg/kg). No acute toxicity (LD₅0) data are available. The compound should be handled with standard laboratory safety precautions: use of gloves, lab coat, and eye protection; avoid skin contact, eye contact, and inhalation. Any accidental exposure should be treated according to the Material Safety Data Sheet (MSDS). The compound is not known to be genotoxic, but no specific Ames test or chromosomal aberration studies have been reported. Potential long-term hazards are unknown, and therefore the compound should be considered a potential health hazard and handled accordingly. In cell culture studies, (rel)-AR234960 has not been reported to cause cytotoxicity at concentrations up to 10 microM; at higher concentrations (≥50 microM), some degree of cell growth inhibition or reduced viability may occur, but this is not well documented. Users are encouraged to perform their own cytotoxicity assays (e.g., MTT, LDH release) for the cell lines and concentrations they intend to use. For in vivo studies, body weight should be monitored as a general indicator of health and tolerability; significant weight loss or signs of distress (reduced activity, ruffled fur, hunched posture) would indicate adverse effects. The compound should be stored securely and accounted for as a research chemical. As with all laboratory chemicals, it should be used in compliance with institutional safety guidelines. No specific data on the toxicity of the L-mandelate counterion (not applicable; this is not a salt form, it is a free base) or other impurities are available. Since (rel)-AR234960 is an agonist of the MAS receptor, which is expressed in many tissues, unexpected off-target effects could occur due to activation of MAS at non-cardiac sites; however, such effects are related to pharmacology rather than toxicity. The compound‘s potential for drug-drug interactions has not been assessed. Given the lack of formal toxicology studies, users should minimize exposure and handle the compound with care, following standard practices for research chemicals of unknown toxicity. The compound is not for diagnostic or therapeutic use and should not be administered to humans or animals for any purpose other than approved research protocols under the supervision of qualified personnel.

[1]. Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts. PLoS One. 2017 Dec 29;12(12):e0190217.

(rel)-AR234960 (CAS# 1408311-94-3) is a research-grade non-peptide agonist of the MAS receptor (MAS1), a G protein-coupled receptor (GPCR) that is part of the renin-angiotensin system (RAS). It is the active relative configuration of AR234960 and is used as a pharmacological tool to study MAS-mediated signaling, particularly in the context of cardiac fibrosis and extracellular matrix remodeling. The molecular weight of (rel)-AR234960 is 555.63 (or 555.62), and its molecular formula is C2₇H30FN₅O₅S. Synonyms include AR234960 (the parent compound) and the relative configuration (racemic mixture). The IUPAC name is 1-[[(3R,4R)-4-(3-fluorophenyl)-1-(2-methoxy-4-nitrophenyl)sulfonylpyrrolidin-3-yl]methyl]-4-pyridin-2-ylpiperazine. (rel)-AR234960 is a non-peptide agonist that is more stable and potentially more bioavailable than the endogenous MAS agonist angiotensin-(1-7) (Ang-(1-7)), which is a peptide. Unlike Ang-(1-7), which generally mediates vasodilation, anti-hypertension, anti-fibrosis, and cardioprotection through MAS, (rel)-AR234960 activates the MAS-ERK1/2-CTGF signaling axis, promoting collagen synthesis and fibrosis. This compound has been used to demonstrate that MAS can signal through distinct pathways depending on the agonist, and it serves as a tool to dissect the molecular determinants of MAS signaling. (rel)-AR234960‘s pro-fibrotic effects can be blocked by the MAS inverse agonist AR244555 or by MEK1/2 inhibitors such as PD98059 or U0126. This compound is primarily used in cardiovascular research to model cardiac fibrosis, to study the role of MAS in pathological remodeling, and to screen for anti-fibrotic drugs that target the MAS-ERK1/2-CTGF pathway. (rel)-AR234960 has not advanced to clinical trials and is not approved for therapeutic use. It is intended for laboratory research only and should be stored at -20degC, protected from light and moisture. The compound is soluble in DMSO (10 mM stock solution) and can be diluted in aqueous buffers or formulated for in vivo administration using vehicles such as 5% DMSO + 5% Tween-80 + 90% saline, or 0.5% carboxymethyl cellulose (CMC) with 0.1% Tween-80, or PEG400/water mixtures. The compound is typically available with purity ≥95% or ≥98% depending on the supplier. For researchers studying the renin-angiotensin system, MAS receptor biology, or fibrotic diseases, (rel)-AR234960 is a valuable chemical probe for interrogating a specific branch of MAS signaling that leads to pathological fibrosis. It is not an approved drug and must be used in compliance with all applicable laws and regulations for research chemicals. Users should consult the original literature for detailed functional characterization and validation of (rel)-AR234960, as this compound is a relatively new tool (first described in the 2010s) and not all parameters are yet available in standard supplier catalogs. The compound is also known as AR234960 (the non-peptide MAS agonist). For researchers planning in vivo studies, it is advisable to perform preliminary dose-range finding experiments with endpoints such as CTGF mRNA expression in the heart (24-48 hours after a single dose) to establish an effective dose, and then confirm selectivity with the MAS inverse agonist AR244555. The compound is not for human use and should be handled using appropriate safety measures.

C27H30FN5O5S

555.621008396149

555.195

1408311-94-3

89560738

White to off-white solid powder

3.6

0

10

7

39

925

0

S(C1C=CC(=CC=1OC)[N+](=O)[O-])(N1C[C@@H](C2C=CC=C(C=2)F)[C@H](CN2CCN(C3C=CC=CN=3)CC2)C1)(=O)=O

TUSCHIFEASZBBF-QPPBQGQZSA-N

InChI=1S/C27H30FN5O5S/c1-38-25-16-23(33(34)35)8-9-26(25)39(36,37)32-18-21(24(19-32)20-5-4-6-22(28)15-20)17-30-11-13-31(14-12-30)27-7-2-3-10-29-27/h2-10,15-16,21,24H,11-14,17-19H2,1H3/t21-,24+/m1/s1

1-[[(3R,4R)-4-(3-fluorophenyl)-1-(2-methoxy-4-nitrophenyl)sulfonylpyrrolidin-3-yl]methyl]-4-pyridin-2-ylpiperazine

(rel)AR234960; (rel) AR234960

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 : ~25 mg/mL (~44.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.50 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 (4.50 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.)