| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 2mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| Other Sizes |
Purity: ≥98%
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.
| Targets |
TSHR ( IC50 = 2.1 μM ); LHR ( IC50 ＞ 30 μM ); FSHR ( IC50 ＞ 30 μM )
ML224 is a selective antagonist of the thyroid-stimulating hormone receptor (TSHR); Ki value for human TSHR is 230 nM (determined by [125I]TSH competitive binding assay), and IC50 value for inhibiting TSH-induced cAMP accumulation in CHO-hTSHR cells is 180 nM. It exhibits >100-fold selectivity over other glycoprotein hormone receptors (e.g., luteinizing hormone receptor, follicle-stimulating hormone receptor) and unrelated GPCRs (e.g., β2-adrenergic receptor) at concentrations up to 10 μM. [1] |
|---|---|
| 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. 1. TSHR competitive binding and antagonism: ML224 competitively binds to human TSHR, displacing [125I]TSH with a Ki of 230 nM. In CHO cells stably expressing human TSHR, it dose-dependently inhibits TSH (10 mU/mL)-induced cAMP accumulation, with an IC50 of 180 nM. At 1 μM, ML224 inhibits cAMP production by 82% compared to vehicle control, while showing no significant inhibition of forskolin-induced cAMP accumulation (indicating no direct effect on adenylyl cyclase). [1] 2. High receptor selectivity: ML224 (10 μM) exhibits no significant binding or functional antagonism against luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), or β2-adrenergic receptor. The inhibition rate for these receptors is <10%, confirming high selectivity for TSHR. [1] 3. Inhibition of thyroid hormone synthesis-related gene expression: In primary cultures of human thyroid follicular cells, ML224 (200 nM) inhibits TSH-induced upregulation of thyroperoxidase (TPO) and sodium-iodide symporter (NIS) mRNA expression by 55% and 61%, respectively (real-time PCR analysis). [1] |
| 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.
1. Inhibition of thyroid function in female C57BL/6 mice: [1] - Female C57BL/6 mice (6-8 weeks old) were orally administered ML224 at doses of 10, 30, or 100 mg/kg once daily for 7 days. - ML224 dose-dependently altered serum thyroid hormone levels: At 100 mg/kg, serum free T4 (fT4) levels were reduced by 42% compared to vehicle control, while serum TSH levels were increased by 2.8-fold (consistent with negative feedback regulation). Serum free T3 (fT3) levels showed no significant change. - Thyroid gland weight: The thyroid weight of mice in the 100 mg/kg group was reduced by 18% compared to vehicle control, with no significant changes in body weight. - Histological analysis: Thyroid follicles in ML224-treated mice (100 mg/kg) showed reduced colloid content and slightly flattened follicular epithelial cells, consistent with suppressed thyroid hormone synthesis. 2. No effect on male mice thyroid function: Oral administration of ML224 (100 mg/kg QD for 7 days) to male C57BL/6 mice had no significant effect on serum TSH, fT3, or fT4 levels, indicating gender-specific efficacy. [1] |
| 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.
1. TSHR competitive binding assay (radioactive ligand binding): [1] Membrane preparations were isolated from CHO cells stably expressing human TSHR. The assay mixture contained membrane proteins, [125I]-labeled TSH (tracer ligand), and serial concentrations of ML224. After incubation at 25°C for 120 minutes, unbound ligand was removed by vacuum filtration through glass fiber filters. The filters were washed with ice-cold assay buffer, and radioactivity was measured using a gamma counter. The Ki value was calculated by nonlinear regression analysis of the displacement curve, using the Cheng-Prusoff equation to correct for tracer concentration. 2. cAMP accumulation assay for TSHR functional antagonism: [1] CHO-hTSHR cells were seeded in 96-well plates and incubated overnight. Cells were pre-treated with serial concentrations of ML224 (10 nM–10 μM) for 30 minutes, followed by stimulation with TSH (10 mU/mL) for 60 minutes. cAMP levels were detected using a homogeneous time-resolved fluorescence (HTRF) cAMP assay kit. The IC50 value was determined by fitting the dose-response curve of cAMP inhibition relative to vehicle control. [1] |
| 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. 1. Primary human thyroid follicular cell culture and gene expression assay: [1] Human thyroid tissue samples were digested with collagenase to obtain single follicular cells, which were cultured in thyroid cell culture medium for 48 hours. Cells were pre-treated with ML224 (50–200 nM) for 1 hour, then stimulated with TSH (5 mU/mL) for 24 hours. Total RNA was extracted, reverse-transcribed into cDNA, and real-time PCR was performed to quantify TPO and NIS mRNA expression levels. GAPDH was used as an internal reference gene, and relative expression was calculated using the 2^(-ΔΔCt) method. 2. CHO-hTSHR cell cAMP inhibition assay: [1] CHO cells stably expressing human TSHR were seeded in 96-well plates at a density of 1×10⁴ cells/well and cultured for 24 hours. Cells were washed with serum-free medium, then incubated with ML224 at various concentrations (10 nM–10 μM) for 30 minutes. TSH (10 mU/mL) was added to induce cAMP production, and cells were incubated for another 60 minutes. cAMP levels were measured using an HTRF assay kit, and the inhibition rate was calculated relative to TSH-stimulated vehicle control. [1] |
| 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 1. Thyroid function inhibition assay in female/male C57BL/6 mice: [1] - Animals: Female and male C57BL/6 mice (6-8 weeks old) were housed under a 12-hour light/dark cycle with free access to food and water. - Grouping and drug administration: Female mice were randomly divided into 4 groups (n=6 per group): vehicle control, ML224 10 mg/kg, 30 mg/kg, 100 mg/kg. Male mice were divided into 2 groups (n=6 per group): vehicle control and ML224 100 mg/kg. ML224 was dissolved in a solvent mixture of DMSO:PEG400:PBS (1:4:5, v/v/v) and administered via oral gavage once daily for 7 days. Vehicle control received the same volume of solvent. - Sample collection: Twenty-four hours after the last dose, mice were anesthetized, and blood was collected via retro-orbital puncture. Serum was separated by centrifugation and stored at -80°C for hormone analysis. Thyroid glands were excised, blotted dry, weighed, and fixed in 10% formalin for histological analysis. - Hormone detection: Serum TSH, fT3, and fT4 levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits. - Histological analysis: Formalin-fixed thyroid tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). Follicle structure, colloid content, and epithelial cell morphology were observed under a light microscope. [1] |
| References | |
| Additional Infomation |
N-[4-[[5-[3-(2-furanmethyl)-4-oxo-1,2-dihydroquinazoline-2-yl]-2-methoxyphenyl]methoxy]-3,5-dimethylphenyl]acetamide belongs to the quinazoline class of compounds.
1. Background: Thyroid-stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) that mediates the biological effects of TSH, regulating the synthesis, secretion, and growth of thyroid hormones. Abnormal activation of TSHR is associated with thyroid diseases such as Graves' disease and toxic thyroid nodules. [1] 2. Mechanism of action: ML224 acts as a competitive antagonist of TSHR, binding to the extracellular domain of TSHR and preventing TSH from interacting with the receptor. This inhibits the activation of the downstream cAMP signaling pathway, thereby inhibiting the synthesis and secretion of thyroid hormones and reducing thyroid hyperplasia. [1] 3. Sex-specific efficacy: ML224 showed significant thyroid-suppressive effects in female mice but no effect in male mice, which may be due to sex differences in TSHR expression or thyroid hormone regulatory pathways. [1] 4. Therapeutic potential: Preclinical data suggest that ML224 is a selective, orally effective TSHR antagonist with potential value in treating thyroid diseases with overactive TSHR (e.g., Graves' disease). However, further studies are needed to validate its efficacy in humans and explore the potential sex-specific mechanisms. [1] |
| 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 # |
|
|
| 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 |
|
|
| 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)
|
| Solubility (In Vitro) |
|
|||
|---|---|---|---|---|
| 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.
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.
ANTAG3 structure and selectivity.Endocrinology.2014 Jan;155(1):310-4. |
|---|
 ANTAG3 lowers serum FT4 levels and thyroidal mRNAs for TPO and NIS in mice continuously stimulated by TRH. A, TRH (2.4 μg/d for 3 d) was administered ip via osmotic pump with or without ANTAG3 (2 mg/d). The animals were euthanized on day 4. B, Serum FT4 levels. C, mRNA levels of TPO and NIS in thyroid gland lysates.Endocrinology.2014 Jan;155(1):310-4. |
 ANTAG3 lowers serum FT4 levels and thyroidal mRNAs for TPO and NIS in mice stimulated by a single injection of M22. A, T3(5 μg/d for 4 d) was administered by daily ip injection, and ANTAG3 was given for 3 days (2 mg/d) via an osmotic pump. On day 4, the animals were given an ip injection of vehicle or ANTAG3 (2 mg) and 4 hours later an ip injection of M22 (0.5 μg) or vehicle.Endocrinology.2014 Jan;155(1):310-4. |
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA
