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Purity: ≥98%
ML221 is a potent functional antagonist of the apelin (APJ) receptor. It originated from an HTS that gathered about 330,600 compounds using the MLSMR library. ML221 suppresses apelin-13-induced APJ activation, exhibiting IC50 values of 0.70 μM in the cAMP assay, 1.75 μM in the β-arrestin assay, and EC 80 of 10 nM in both assays. ML221 exhibits >37-fold selectivity for APJ in comparison to the closely related angiotensin II type 1 (AT1) receptor in assays conducted on cells. Apart from the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM), this antagonist did not exhibit any noteworthy binding activity against 29 other GPCRs.
| Targets |
APJ ( IC50 = 1.75 μM )
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| Enzyme Assay |
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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| Cell Assay |
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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| References |
| Molecular Formula |
C17H11N3O6S
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| Molecular Weight |
385.04
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| Exact Mass |
385.04
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| Elemental Analysis |
C, 52.99; H, 2.88; N, 10.90; O, 24.91; S, 8.32
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| CAS # |
877636-42-5
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| Appearance |
Solid powder
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| SMILES |
C1=CN=C(N=C1)SCC2=CC(=O)C(=CO2)OC(=O)C3=CC=C(C=C3)[N+](=O)[O-]
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| InChi Key |
UASIRTUMPRQVFY-UHFFFAOYSA-N
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| InChi Code |
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
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| Chemical Name |
[4-oxo-6-(pyrimidin-2-ylsulfanylmethyl)pyran-3-yl] 4-nitrobenzoate
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5971 mL | 12.9857 mL | 25.9713 mL | |
| 5 mM | 0.5194 mL | 2.5971 mL | 5.1943 mL | |
| 10 mM | 0.2597 mL | 1.2986 mL | 2.5971 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Representative dose response curve for ML221.Bioorg Med Chem Lett. 2012 Nov 1; 22(21): 6656–6660. |
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GPCR profiling panel forML221.Bioorg Med Chem Lett. 2012 Nov 1; 22(21): 6656–6660. |
A: Positive APLNR staining by IHC in four human CCA tissues (bottom row) compared to adjacent non-malignant liver tissues (top row).Cancer Lett.2017Feb 1;386:179-188. |
A: Treatment of benign human cholangiocytes (H69) with 10 μM of ML221 over 24 h increased Ki-67 gene expression, but significantly decreased expression of angiogenic factors (VEGF-A, VEGF-C, Ang-1, and Ang-2) via rtPCR. B. Treatment of HuH-28 cholangiocarcinoma cells with 10 μM of ML221 over 24 h significantly decreased Ki-67 gene expression, as well as angiogenic factors (VEGF-A, VEGF-C, Ang-1, and Ang-2) via rtPCR. C. Treatment of SG231 cholangiocarcinoma cells with 10 μM of ML221 over 24 h significantly decreased Ki-67 gene expression, as well as angiogenic factors (VEGF-A, VEGF-C, Ang-1, and Ang-2) via rtPCR. Cancer Lett.2017Feb 1;386:179-188. |
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A: Apelin treatment promotes Mz-ChA-1 gene expression of Ki-67 and PCNA (left), whereas, ML221 treatment decreases Mz-ChA-1 gene expression of Ki-67 and PCNA in a dose dependent manner (n = 5) via rtPCR. B: Gene expression of angiogenic factors (VEGF-A, Ang-1, and Ang-2) is increased when Mz-ChA-1 cells are treated with increasing concentrations of apelin (left), whereas, gene expression is decreased with increasing concentrations of ML221, an APLNR antagonist (right) (n = 5) via rtPCR. C: 10 μM of ML221 treatment significantly decreases cell proliferation and migration at 6, 12, 24, and 48 h during wound-healing assay (* = P < 0.05). D: Mz-ChA-1 cell invasiveness did not significantly change following treatment with 10 μM of ML221 for 24 h compared to untreated control cells.Cancer Lett.2017Feb 1;386:179-188. |
A: Parenteral administration of ML221 in nu/nu mice decreases Mz-ChA-1 tumor size (bottom row) compared to untreated control tumors (top row). B: Mz-ChA-1 tumor volume significantly increases in untreated control tumors (n = 9) compared to ML221 treated tumors (n = 12). C: H&E staining of control (left) and ML221 treated (right) Mz-ChA-1 tumors isolated from nu/nu mice.
A: Immunoblots of protein isolated from control and ML221 treated Mz-ChA-1 tumors shows expression of CK-19. p-ERK and t-ERK expression is decreased in ML221 treated Mz-ChA-1 tumors compared to untreated Mz-ChA-1 tumors. B: Control and ML221 treated Mz-ChA-1 tumors demonstrate positive staining for CK-19, a cholangiocyte specific marker, shown by IHC. C: IHC shows positive staining for APLNR in control and ML221 treated Mz-ChA-1 tumors. D. Mz-ChA-1 tumors treated with ML221 demonstrated decreased gene expression of proliferative markers (PCNA, Ki-67), angiogenic factors (VEGF-A, VEGF-C, Ang-1, and Ang-2), and markers of tumor progression (Vimentin, MMP-9, MMP-3) via rtPCR. |
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