DBeQ (JRF-12; JRF 12; JRF12) is a reversible and ATP-competitive p97 inhibitor with potential antineoplastic activity. Its IC50 value for p97 inhibition is 1.5 μM. By interfering with the ubiquitin-dependent and autophagic protein clearance pathways, DBeQ can be used to treat cancer.

Physicochemical Properties

Molecular Formula	C22H20N4
Molecular Weight	340.42
Exact Mass	340.168
Elemental Analysis	C, 77.62; H, 5.92; N, 16.46
CAS #	177355-84-9
Related CAS #	177355-84-9
PubChem CID	676352
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	573.1±52.0 °C at 760 mmHg
Melting Point	149 °C
Flash Point	300.4±30.7 °C
Vapour Pressure	0.0±1.6 mmHg at 25°C
Index of Refraction	1.735
LogP	4.34
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	26
Complexity	403
Defined Atom Stereocenter Count	0
SMILES	N([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])C1C2=C([H])C([H])=C([H])C([H])=C2N=C(N=1)N([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]
InChi Key	QAIMUUJJAJBPCL-UHFFFAOYSA-N
InChi Code	InChI=1S/C22H20N4/c1-3-9-17(10-4-1)15-23-21-19-13-7-8-14-20(19)25-22(26-21)24-16-18-11-5-2-6-12-18/h1-14H,15-16H2,(H2,23,24,25,26)
Chemical Name	2-N,4-N-dibenzylquinazoline-2,4-diamine
Synonyms	DBeQ; DBEQ; JRF12; JRF-12; JRF 12.
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	p97( IC50 = 1.5 μM ); Vps4（ IC50 = 11.5 μM ）
ln Vitro	DBeQ inhibits the degradation of UbG76V-GFP, ODD-Luc, and Luc-ODC at IC50 values of 2.6 μM, 56 μM, and 45 μM in HeLa cells. DBeQ exhibits a minimum of 50-fold reduction in its potency towards NSF and 26S proteasome. DBeQ exhibits competitive inhibition of p97 in relation to ATP, exhibiting a Ki of 3.2 μM, indicating its binding to the D2 domain's active site. TCRα-GFP degradation in HEK293 cells is potently blocked by DBeQ (10 μM). In HEK293 cells, DBeQ does not raise the level of p21, but it does induce CHOP in a concentration-dependent manner within 3 hours. In Hela cells, DBeQ (15 μM) causes a significant build-up of LC3-II in the nucleus along with membrane-enriched and cytosolic fractions. Rather than stimulating autophagy in HeLa cells, DBeQ functions by preventing the autophagic degradation of LC3-II. In HeLa cells, activation of the "executioner" caspases-3 and -7 is rapidly promoted by DBeQ (10 μM). While STS activates both pathways to a similar degree, DBeQ activates the intrinsic caspase-9 apoptotic pathway more than the extrinsic caspase-8 pathway. HeLa and Hek293 cells exhibit intermediate sensitivity to DBeQ, which is five times more active against multiple myeloma (RPMI8226) cells than normal human fetal lung fibroblasts (MRC5).[1] In HeLa cells, p97-dependent versus independent UPS reporter substrates can be stabilized with a 20-fold selectivity demonstrated by DBeQ. In the autophagy and ERAD pathways, DBeQ hinders substrate degradation.[2] In HeLa cells, DBeQ (12 μM) shows a dose-dependent inhibition of intracellular neutralization. The virus and antibody degradation in the fate-of-capsid experiment is completely inhibited by DBeQ (10 μM), but the degradation of IgG Fc is not prevented. As an antibody concentration increases, DBeQ (9 μM) diminishes the initial neutralization gradient. According to [3], DBeQ has similar effects to rapamycin in U20S cells by reducing the phosphorylation of MTOR targets both basally and in response to stimuli.[4]
ln Vivo
Enzyme Assay	Assay Buffer is put into each well of a 96-well plate. It contains 20 μL of 2.5× concentration, where 1× = 50 mM Tris (pH 7.4), 20 mM MgCl22, 1 mM EDTA, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP). 10 μL of purified p97 (25 μL of 50 μM) is added to each well after being diluted in 975 μL of 1× Assay Buffer. Following the addition of 10 μL of either DBeQ or 5% DMSO to each well, the plate is incubated for 10 minutes at room temperature. The ATPase assay involves filling each well with 10 μL of 500 μM ATP (pH 7.5), letting it sit at room temperature for 60 minutes, and then adding 50 μL of Kinase Glo Plus reagent. Finally, it is left to sit at room temperature in the dark for 10 minutes. An Analyst AD reads luminosity. In triplicate, DBeQ is assayed at the following concentrations: 0, 0.048, 0.24, 1.2, 6, and 30 μM.
Cell Assay	A solid white 384-well plate is used for cell seeding, with 5,000 cells per well. The indicated duration of DBeQ treatment or 48 hours of luciferase or p97 siRNA (10 nM) transfection are applied to the cells. One minute of 500 rpm shaking is used to mix the caspase-3/7 Glo, caspase-6 Glo, caspase-8 Glo, or caspase-9 Glo. After incubation for an hour at room temperature, the luminosity signal is measured. Through the use of CellTiter-Glo reagen, cellular viability is assessed. For a duration of 48 hours, cells are subjected to seven concentrations of MG132 or DBeQ (threefold serial dilutions beginning at 33 μM) in order to ascertain the half-life of the cells. Finding the percentage of luminescence signal normalized to cells treated with DMSO is how IC50 values are computed.
Animal Protocol
References	[1]. Proc Natl Acad Sci U S A . 2011 Mar 22;108(12):4834-9. [2]. Autophagy . 2011 Sep;7(9):1091-2. [3]. Proc Natl Acad Sci U S A . 2012 Nov 27;109(48):19733-8. [4]. Autophagy . 2013 May;9(5):799-800.

Solubility Data

Solubility (In Vitro)

DMSO : 35.71~68 mg/mL ( 104.9~199.8 mM ) Ethanol : ~5 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.34 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 (7.34 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	2.9375 mL	14.6877 mL	29.3755 mL
	5 mM	0.5875 mL	2.9375 mL	5.8751 mL
	10 mM	0.2938 mL	1.4688 mL	2.9375 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.