APJ ( IC50 = 1.75 μM )
Apelin (APJ) receptor: Functionally antagonizes APJ, with >37-fold selectivity over the angiotensin II type 1 (AT1) receptor; shows no significant binding activity against 29 other GPCRs, except for <50% inhibition (I) at 10 μM against κ-opioid receptor and <70%I at 10 μM against benzodiazepinone receptor [1]
- Apelin receptor (APLNR): Inhibits the apeliPLNR axis to suppress cholangiocarcinoma (CCA) growth [2]

In vitro activity: ML221 shows moderate permeability in a PAMPA permeability assay. Given that it is quickly metabolized in both human and mouse liver homogenates (4.2% and 4.9% remaining at 60 minutes), ML221 exhibits moderate plasma and poor microsomal stability. Over 50 μM does not demonstrate any toxicity to human hepatocytes. Limited cross reactivity is shown by ML221 against a variety of GPCRs. By preventing apelin-APJ signaling, ML221 prevents the proliferation of endothelial cells while leaving VEGF and VEGFR2 expression unaltered.

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APJ Antagonism Assays: ML221 antagonized Ap13-mediated activation of APJ in a concentration-dependent manner in both cAMP assay and β-arrestin recruitment assay, as indicated by dose-response curves fitted with a four-parameter logistic model (mean ± SEM% inhibition of Ap13) [1]
- GPCR Selectivity Profiling: ML221 (10 μM) exhibited no significant binding activity to 29 tested GPCRs; only weak activity was observed against κ-opioid receptor (<50%I) and benzodiazepinone receptor (<70%I) [1]
- CCA Cell Proliferation Inhibition: ML221 treatment decreased the gene expression of proliferative markers (Ki-67, PCNA) in Mz-ChA-1 CCA cells in a dose-dependent manner (measured by rtPCR, n=5). At 10 μM, ML221 significantly reduced Mz-ChA-1 cell proliferation and migration at 6, 12, 24, and 48 h in a wound-healing assay, though it had no significant effect on cell invasiveness after 24 h of treatment [2]
- CCA Angiogenic Factor Suppression: ML221 (dose-dependent) decreased the gene expression of angiogenic factors (VEGF-A, Ang-1, Ang-2) in Mz-ChA-1 cells (rtPCR, n=5). In HuH-28 and SG231 CCA cell lines, 10 μM ML221 significantly downregulated Ki-67 and angiogenic factors (VEGF-A, VEGF-C, Ang-1, Ang-2) (rtPCR) [2]
- Benign Cholangiocyte Effects: In benign H69 cholangiocytes, 10 μM ML221 (24 h treatment) increased Ki-67 gene expression but significantly decreased angiogenic factor expression (VEGF-A, VEGF-C, Ang-1, Ang-2) (rtPCR) [2]

In the retina of OIR model mice, intraperitoneal injection of ML221 suppresses pathological angiogenesis while promoting the recovery of normal vessels into the ischemic areas. The effects of ML221 on mechanical allodynia and heat hyperalgesia are dose-dependent and go away after 7 days of chronic constriction injury (CCI). The apelin-APJ system is implicated in both initiating and maintaining pain, as evidenced by the persistent attenuation of CCI-induced pain hypersensitivity following intraspinal delivery of ML221 both at the onset and in fully-established neuropathic pain. Apelian's effect on neuropathic pain may be mediated through ERK signaling, as evidenced by intrathecal ML221's downregulation of phosphorylated extracellular signal-related kinase (ERK) in the rat spinal cord dorsal horn.

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Xenograft Tumor Growth Inhibition: Nude (nu/nu) mice were implanted with Mz-ChA-1 cells, then treated with ML221 at 150 μg/kg via tail vein injection. ML221 significantly decreased Mz-ChA-1 tumor volume compared to untreated controls (n=12 for treated group, n=9 for control group). H&E staining of tumors showed differences between treated and control groups. Immunoblotting revealed reduced p-ERK and t-ERK expression in ML221-treated tumors, and rtPCR showed downregulated gene expression of proliferative markers (PCNA, Ki-67), angiogenic factors (VEGF-A, VEGF-C, Ang-1, Ang-2), and tumor progression markers (Vimentin, MMP-9, MMP-3) [2]

ML221's antagonistic effect on apelin-13-mediated APJ activation was evaluated through two complementary APJ function assays: β-arrestin recruitment and cAMP inhibition. Increasing concentrations of ML221 antagonized a fixed concentration of Ap13 (EC80 = 10 nM) in both assays, with a calculated IC50equal to 0.70 μM in the cAMP assay, and 1.75 μM in the β-arrestin assay.

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cAMP Assay for APJ Antagonism: Cells expressing APJ were treated with Ap13 (to activate APJ) and various concentrations of ML221. The cAMP level in cells was measured to assess the inhibition of Ap13-mediated APJ activation. The assay data were plotted as mean ± SEM% inhibition of Ap13, and a four-parameter logistic curve was fitted to determine the concentration-dependent antagonism [1]
- β-Arrestin Recruitment Assay for APJ Antagonism: APJ-expressing cells were used to detect β-arrestin recruitment induced by Ap13. Different concentrations of ML221 were added, and the extent of β-arrestin recruitment was measured. Similar to the cAMP assay, results were presented as mean ± SEM% inhibition of Ap13, with a four-parameter logistic curve fitted [1]
- GPCR Binding Profiling Assay: ML221 (10 μM) was tested for binding activity against a panel of 31 GPCRs (including APJ, AT1, κ-opioid, benzodiazepinone, and 29 other GPCRs). Binding activity was quantified as percentage inhibition (%I), and only <50%I (κ-opioid) and <70%I (benzodiazepinone) were observed besides no significant activity on others [1]

The expansion of bEnd.After being incubated with ML221 (0-30 μM) for 24 hours, 3 cells are evaluated using the BrdU incorporation assay and the MTT assay. bEnd, a mouse endothelial cell line Dulbecco's modified Eagle's medium, enhanced with 10% heat-inactivated fetal bovine serum, is used to sustain three cells. For the MTT and BrdU incorporation assays, the cells are plated in 24-well culture plates at a density of 2.5 × 10⁴/well. To perform the BrdU incorporation assay, 10 μM BrdU is added to the culture medium. The cells are fixed with 4% paraformaldehyde for 10 minutes after 2 hours. Streptavidin fluorescein isothiocyanate combined with biotinylated goat anti-mouse IgG antibodies allows for the visualization of the primary antibody. To detect nuclei, use Hoechst 33342. The number of BrdU positive cells per Hoechst positive cell is used to calculate the BrdU incorporation rate.

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APJ-Mediated Activation Inhibition in Cells: Cells expressing APJ were seeded and cultured under appropriate conditions. Ap13 was used as the APJ agonist, and ML221 was added at different concentrations. For the cAMP assay, intracellular cAMP levels were measured after incubation to evaluate ML221’s ability to inhibit Ap13-induced APJ activation. For the β-arrestin recruitment assay, a detection system specific for β-arrestin binding to activated GPCRs was used to quantify the effect of ML221 on Ap13-mediated β-arrestin recruitment. Each assay was repeated to obtain mean ± SEM% inhibition values, and dose-response curves were generated [1]
- CCA Cell Proliferation and Marker Analysis: Mz-ChA-1, HuH-28, and SG231 CCA cells, as well as H69 benign cholangiocytes, were cultured. Cells were treated with ML221 at various concentrations (including 10 μM for key experiments) for specified durations (24 h for gene expression, 6–48 h for wound-healing). rtPCR was performed to detect the expression of proliferative markers (Ki-67, PCNA) and angiogenic factors (VEGF-A, VEGF-C, Ang-1, Ang-2). A wound-healing assay was conducted by creating a scratch in the cell monolayer, and cell migration was monitored at multiple time points to assess proliferation and migration. Cell invasiveness was tested using an invasion assay kit, with 24 h of ML221 treatment [2]
- Immunofluorescence and Flow Cytometry for APLNR Expression: H69 and Mz-ChA-1 cells were fixed and stained with APLNR-specific antibodies for immunofluorescence to visualize APLNR localization. For flow cytometry, cells were harvested, incubated with APLNR antibodies, and analyzed to quantify APLNR expression levels, comparing CCA cells and benign cholangiocytes [2]
- ELISA for Apelin Secretion: Culture supernatants from CCA cells (Mz-ChA-1, HuH-28, SG231) and H69 cells were collected. ELISA kits specific for apelin were used to measure apelin concentrations, comparing secretion levels between malignant and benign cells [2]

150 μg/kg; 3× weekly via tail vein injection for 4 weeks
Male BALB/c eight week old nude (nu/nu) mice

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Xenograft Tumor Model in Nude Mice: Nude (nu/nu) mice were used. Mz-ChA-1 CCA cells were injected into the flanks of the mice to establish tumor xenografts. Once tumors formed, the treatment group (n=12) received ML221 at a dose of 150 μg/kg via tail vein injection, while the control group (n=9) received no treatment. Tumor size was monitored regularly, and tumor volume was calculated. At the end of the experiment, mice were euthanized, and tumors were harvested. Harvested tumors were subjected to H&E staining (for histopathological analysis), immunoblotting (to detect CK-19, p-ERK, t-ERK expression), and rtPCR (to measure gene expression of proliferative, angiogenic, and tumor progression markers) [2]

[1]. Bioorg Med Chem Lett . 2012 Nov 1;22(21):6656-60.

[2]. Cancer Lett . 2017 Feb 1:386:179-188.

4-Nitrobenzoic acid [4-oxo-6-[(2-pyrimidinylthio)methyl]-3-pyranyl] ester is a nitrobenic acid. ML221 (chemical name: 4-oxo-6-((pyrimidinyl-2-ylthio)methyl)-4H-pyran-3-yl-4-nitrobenzoic acid ester) was discovered through high-throughput screening (HTS) of a MLSMR library of approximately 330,600 compounds. Its synthesis and structure-activity relationship (SAR) (based on the kojic acid skeleton) have also been reported [1]. The apeliPJ system is a key regulator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular diseases; it also plays a role in energy metabolism and gastrointestinal function. ML221, as a potent APJ functional antagonist, provides a tool for studying this system [1]. The five-year survival rate for cholangiocarcinoma (CCA) is low. The apeliPLNR axis is overexpressed in CCA tissues and cells (confirmed by IHC, rtPCR, immunofluorescence, flow cytometry and ELISA). ML221 inhibits CCA growth by targeting this axis, suggesting its potential as a novel targeted therapy for CCA tumors [2].

C17H11N3O6S

385.04

385.037

C, 52.99; H, 2.88; N, 10.90; O, 24.91; S, 8.32

877636-42-5

7217941

Off-white to yellow solid powder

3.372

0

9

6

27

646

0

O=C(C1C=CC([N+](=O)[O-])=CC=1)OC1C(=O)C=C(CSC2N=CC=CN=2)OC=1

UASIRTUMPRQVFY-UHFFFAOYSA-N

InChI=1S/C17H11N3O6S/c21-14-8-13(10-27-17-18-6-1-7-19-17)25-9-15(14)26-16(22)11-2-4-12(5-3-11)20(23)24/h1-9H,10H2

[4-oxo-6-(pyrimidin-2-ylsulfanylmethyl)pyran-3-yl] 4-nitrobenzoate

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)

Solubility in Formulation 1: ≥ 2 mg/mL (5.19 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.)