ML346 (CID767276) is a novel and potent activator of heat shock protein 70 (Hsp70) (EC50 = 4600 nM in HeLa cell toxicity assay). ML346 is a member of the barbituric acid class of substances that, in conformational disease models, induce HSF-1-dependent chaperone expression and restore folding of proteins.

Physicochemical Properties

Molecular Formula	C14H12N2O4
Molecular Weight	272.26
Exact Mass	272.079
Elemental Analysis	C, 61.76; H, 4.44; N, 10.29; O, 23.51
CAS #	100872-83-1
Related CAS #	100872-83-1
PubChem CID	767276
Appearance	Orange to red solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.669
LogP	0.88
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	20
Complexity	452
Defined Atom Stereocenter Count	0
SMILES	O(C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])/C(/[H])=C(\[H])/C(/[H])=C1\C(N([H])C(N([H])C\1=O)=O)=O
InChi Key	IXYLVJHFJKDHRM-NSCUHMNNSA-N
InChi Code	InChI=1S/C14H12N2O4/c1-20-10-7-5-9(6-8-10)3-2-4-11-12(17)15-14(19)16-13(11)18/h2-8H,1H3,(H2,15,16,17,18,19)/b3-2+
Chemical Name	5-[(E)-3-(4-methoxyphenyl)prop-2-enylidene]-1,3-diazinane-2,4,6-trione
Synonyms	ML-346; ML 346; ML346; CID-767276; CID767276; CID 767276
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	HSP70 ( EC50 = 4.6 μM )
ln Vitro	ML346 is a newly discovered Hsp70 activator, exhibiting an EC50 of 4600 nM in HeLa cells. Proteostasis, CFTR-mediated iodide conductance, and proper folding of proteins expressed in two distinct cellular compartments are all restored by ML346 at 10 μM. In WT MEF cells, ML346 (Compound F1) strongly induces Hsp70, the oxidative stress response genes HO1 and GCLM, as well as a 2.5-fold upregulation of BiP. It also induces multiple responses. ML346 (0.5-25 μM) doubles the protection against H2O2-induced apoptosis and shows cytoprotective effects in cells following a 35-minute severe heat shock.
ln Vivo
Enzyme Assay	The procedure involves incubating HeLa cells for three or six hours with either DMSO (negative control), the PRs A1, A3, and ML346 (F1), or the positive controls MG132 (10 μM) and lactacystin (6 μM). After lysing the cells for five minutes on ice in homogenization buffer (50 mM Tris-HCl, pH7.5, 250 mM sucrose, 5 mM MgCl2, 2 mM ATP, 1 mM DTT, 0.5 mM EDTA, and 0.025% digitonin), the total protein content of the whole cell extract is calculated. The assay buffer (50 mM Tris-HCl, pH 7.5, 40 mM KCl, 5 mM MgCl2, 0.5 mM ATP, 1 mM DTT, 0.05 mg/mL BSA) is mixed with 3 μg of whole cell extracts in a black 96-well plate. The reaction is started by adding a 2× (200 μM) fluorogenic peptide substrate Suc-LLVY-AMC. Fluorescence is measured with a Synergy H4 multi-mode microplate reader every ten minutes[2].
Cell Assay	HeLa cells are plated in 100 μL of DMEM supplemented with 10% FBS and 1% Pen/Strep/Neo at a density of 10,000 cells per well in black 96-well plates. Before adding the compound, the plates are incubated for 16 hours at 37°C, 5% CO2, and 95% relative humidity. To the sample or control wells, 1 μL of hit compounds (ML346) in DMSO or DMSO alone is added, respectively. After that, the plates are kept in the incubator for a full day. Following incubation, 200 μL of PBS is used to wash the cells twice, and 200 μL of calcein AM (1 μg/mL) solution is added to each well. Following a 45-minute incubation period at 37°C and 5% CO2, the cells are fluoresced using an Analyst GT multimode reader. The expressed percentage of cytotoxicity is in relation to wells containing cells that were treated with DMSO alone (100%)[2].
Animal Protocol
References	[1]. ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases.Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. 2012 Dec 17. [2]. Small-molecule proteostasis regulators for protein conformational diseases. Nat Chem Biol. 2011 Dec 25;8(2):185-96.

Solubility Data

Solubility (In Vitro)

DMSO: > 10mM Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.25 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.6730 mL	18.3648 mL	36.7296 mL
	5 mM	0.7346 mL	3.6730 mL	7.3459 mL
	10 mM	0.3673 mL	1.8365 mL	3.6730 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.