ML224, also known as NCGC00242364 and ANTAG3, is a selective and inverse agonist of Thyroid Stimulating Hormone Receptor(TSHR). It suppresses the synthesis of cAMP stimulated by TSH with an IC50 of 2.3 μM. With a half-maximum inhibitory dose of 2.1 μM for TSHR and more than 30 μM for LH and FSH receptors, ANTAG3 was selectively inhibiting TSHR. ANTAG3 reduced sodium-iodide cotransporter and thyroperoxidase mRNAs by 75% and 83%, respectively, and serum free T4 by 44% in mice receiving TRH treatment. ANTAG3 reduced serum free T4 by 38% and thyroperoxidase and sodium-iodide cotransporter mRNAs by 73% and 40%, respectively, in mice administered M22. We have, in summary, created a selective TSHR antagonist that works well in vivo in mice. This could result in the development of a medication to treat Graves' disease, as it is the first report of a small-molecule TSHR antagonist that is active in vivo. Additionally, ML224 may provide a path for the creation of medications to treat TSHR-mediated hyperthyroidism, which is brought on by constitutively activating mutations or inducing auto-antibodies linked to Graves' disease.

Physicochemical Properties

Molecular Formula	C31H31N3O5
Molecular Weight	525.59
Exact Mass	525.226
Elemental Analysis	C, 70.84; H, 5.95; N, 7.99; O, 15.22
CAS #	1338824-21-7
Related CAS #	1338824-21-7
PubChem CID	50897809
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	752.3±60.0 °C at 760 mmHg
Flash Point	408.8±32.9 °C
Vapour Pressure	0.0±2.5 mmHg at 25°C
Index of Refraction	1.626
LogP	2.92
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	8
Heavy Atom Count	39
Complexity	829
Defined Atom Stereocenter Count	0
SMILES	CC(NC1=CC(C)=C(OCC2=CC(C(N3CC4=CC=CO4)NC5=C(C=CC=C5)C3=O)=CC=C2OC)C(C)=C1)=O
InChi Key	BFTSWGYWHRJVNI-UHFFFAOYSA-N
InChi Code	InChI=1S/C31H31N3O5/c1-19-14-24(32-21(3)35)15-20(2)29(19)39-18-23-16-22(11-12-28(23)37-4)30-33-27-10-6-5-9-26(27)31(36)34(30)17-25-8-7-13-38-25/h5-16,30,33H,17-18H2,1-4H3,(H,32,35)
Chemical Name	N-[4-[[5-[3-(furan-2-ylmethyl)-4-oxo-1,2-dihydroquinazolin-2-yl]-2-methoxyphenyl]methoxy]-3,5-dimethylphenyl]acetamide
Synonyms	NCGC00242364; ANTAG3; NCGC 00242364; ANTAG 3; NCGC-00242364; ANTAG-3; ML224; ML-224; ML 224
HS Tariff Code	2934.99.03.00
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	TSHR ( IC50 = 2.1 μM ); LHR ( IC50 ＞ 30 μM ); FSHR ( IC50 ＞ 30 μM )
ln Vitro	In vitro activity: ML224 is an inverse agonist and selective selective Thyroid Stimulating Hormone Receptor (TSHR) agonist. It is also referred to as NCGC00242364 and ANTAG3. With an IC50 of 2.3 μM, it suppresses the production of cAMP stimulated by TSH. TSHR inhibition was the only pathway that ANTAG3 was selective for, with half-maximum inhibitory doses for TSHR being 2.1 μM and for LH and FSH receptors being greater than 30 μM. ANTAG3 reduced serum free T4 by 44% and sodium-iodide cotransporter and thyroperoxidase mRNAs by 75% and 83%, respectively, in mice receiving TRH treatment. ANTAG3 reduced sodium-iodide cotransporter and thyroperoxidase mRNAs by 73% and 40%, respectively, and serum free T4 by 38% in mice given M22. To sum up, we created a selective TSHR antagonist that works well in mice in vivo. This could result in a medication to treat Graves disease as it is the first report of a small-molecule TSHR antagonist that is active in vivo.
ln Vivo	ANTAG3 reduced serum free T4 by 44% and sodium-iodide cotransporter and thyroperoxidase mRNAs by 75% and 83%, respectively, in mice receiving TRH treatment. ANTAG3 reduced serum free T4 by 38% in mice given M22, and thyroperoxidase and sodium-iodide cotransporter mRNAs by 40% and 73%, respectively.
Enzyme Assay	ML224, also known as NCGC00242364 and ANTAG3, is a selective and inverse agonist of Thyroid Stimulating Hormone Receptor(TSH)R). With an IC50 of 2.3 μM, it suppresses the production of cAMP stimulated by TSH. TSHR inhibition was the only pathway that ANTAG3 was selective for, with half-maximum inhibitory doses for TSHR being 2.1 μM and for LH and FSH receptors being greater than 30 μM.
Cell Assay	Cell Line: Human embryonic kidney 293 cells (stably expressing TSHRs, LHRs, or FSHRs) Concentration: 0.001-100 µM Incubation Time: 20 min Result: Showed the IC50 for stimulation by bovine TSH (1.8 nM) was 2.1 µM. Showed inhibition of LH and FSH stimulation was less than 15% for LH (1 nM) and less than 30% for FSH (1 nM) at 30 µM.
Animal Protocol	Female BALB/c mice (8 to 13-week-old; ~18.7 g) Dosage: 2 mg/mice Intraperitoneal injection via osmotic pump; single daily for 3 days
References	[1]. A selective TSH receptor antagonist inhibits stimulation of thyroid function in female mice. Endocrinology. 2014 Jan;155(1):310-4.
Additional Infomation	N-[4-[[5-[3-(2-furanylmethyl)-4-oxo-1,2-dihydroquinazolin-2-yl]-2-methoxyphenyl]methoxy]-3,5-dimethylphenyl]acetamide is a member of quinazolines.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (190.3 mM) Water: <1 mg/mL Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.76 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.76 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9026 mL	9.5131 mL	19.0262 mL
	5 mM	0.3805 mL	1.9026 mL	3.8052 mL
	10 mM	0.1903 mL	0.9513 mL	1.9026 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.