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.

Physicochemical Properties

Molecular Formula	C17H11N3O6S
Molecular Weight	385.04
Exact Mass	385.037
Elemental Analysis	C, 52.99; H, 2.88; N, 10.90; O, 24.91; S, 8.32
CAS #	877636-42-5
Related CAS #	877636-42-5
PubChem CID	7217941
Appearance	Off-white to yellow solid powder
LogP	3.372
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	6
Heavy Atom Count	27
Complexity	646
Defined Atom Stereocenter Count	0
SMILES	O=C(C1C=CC([N+](=O)[O-])=CC=1)OC1C(=O)C=C(CSC2N=CC=CN=2)OC=1
InChi Key	UASIRTUMPRQVFY-UHFFFAOYSA-N
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
Chemical Name	[4-oxo-6-(pyrimidin-2-ylsulfanylmethyl)pyran-3-yl] 4-nitrobenzoate
Synonyms	ML 221; ML221; ML-221
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	APJ ( IC50 = 1.75 μM )
ln Vitro	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.
ln Vivo	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.
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.
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.
Animal Protocol	150 μg/kg; 3× weekly via tail vein injection for 4 weeks Male BALB/c eight week old nude (nu/nu) mice
References	[1]. Bioorg Med Chem Lett . 2012 Nov 1;22(21):6656-60. [2]. Cancer Lett . 2017 Feb 1:386:179-188.
Additional Infomation	4-nitrobenzoic acid [4-oxo-6-[(2-pyrimidinylthio)methyl]-3-pyranyl] ester is a nitrobenzoic acid.

Solubility Data

Solubility (In Vitro)

DMSO: ~9 mg/mL (~23.4 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

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.