Ro5-3335 (Ro-53335) is a novel, potent benzodiazepine-based inhibitor of core binding factor (CBF) with the potential for treating leukemia. As a RUNX1-CBFβ interaction inhibitor, it represses RUNX1/CBFB-dependent transactivation.

Physicochemical Properties

Molecular Formula	C₁₃H₁₀CLN₃O
Molecular Weight	259.691
Exact Mass	259.051
CAS #	30195-30-3
Related CAS #	139339-45-0; 2328140-37-8 ;30195-30-3;
PubChem CID	64983
Appearance	Off-white to light yellow solid powder
Density	1.46g/cm3
Boiling Point	501.6ºC at 760mmHg
Melting Point	256 °C (分解)
Flash Point	257.1ºC
Index of Refraction	1.712
LogP	2.031
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	1
Heavy Atom Count	18
Complexity	371
Defined Atom Stereocenter Count	0
InChi Key	XWNMORIHKRROGW-UHFFFAOYSA-N
InChi Code	InChI=1S/C13H10ClN3O/c14-8-3-4-10-9(6-8)13(11-2-1-5-15-11)16-7-12(18)17-10/h1-6,15H,7H2,(H,17,18)
Chemical Name	7-Chloro-1,3-dihydro-5-(1H-pyrrol-2-yl)-2H-1,4-benzodiazepin-2-one
Synonyms	Ro5-3335 Ro 5 3335 CBFβ-Runx1 inhibitor II Ro 5-3335
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	RUNX1-CBFβ interaction[1]
ln Vitro	Ro5-3335 exhibits antiproliferative effect against human CBF leukemia cell lines, as demonstrated by its IC50 values of 1.1 μM, 21.7 μM, and 17.3 μM for ME-1, Kasumi-1, and REH, respectively[1]. In zebrafish embryos, Ro5-3335 prevents final hematopoiesis [1]. Although Ro5-3335 alters the complex's structure or widens the gap between RUNX1 and CBFβ, it does not entirely dissolve the RUNX1-CBFβ interaction[1].
ln Vivo	In zebrafish models, Ro5-3335 has been found to be an inhibitor of RUNX1–CBFβ function[1]. ?A RUNX1-ETO transgenic zebrafish's preleukemic phenotype is salvaged by Ro5-3335[1]. ?In a mouse CBFB-MYH11 leukemia model, Ro5-3335 (300 mg/kg/d; po; for 30 days) lowers the burden of leukemia[1].
Animal Protocol	Animal/Disease Models: C57BL/6 mice (leukemic model)[1] Doses: 300 mg/kg Route of Administration: Oral administration; daily; for 30 days Experimental Results: decreased the number of c-kit+ cells in the transplanted mice and leukemic cell infiltration in the livers, bone marrow and spleen.
References	[1]. Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1-CBFβ interaction. Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14592-7.
Additional Infomation	Ro 5-3335 is a 1,4-benzodiazepinone that is nordazepam in which the phenyl substituent has been replaced by a 1H-pyrrol-2-yl group. It inhibits gene expression in HIV-1 at the transcriptional level through interference with Tat-mediated transactivation. It has a role as an antineoplastic agent, an anti-HIV-1 agent, a RUNX1 inhibitor and a HIV-1 Tat inhibitor. It is a 1,4-benzodiazepinone, an organochlorine compound and a member of pyrroles.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~385.07 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.63 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 (9.63 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	3.8507 mL	19.2537 mL	38.5075 mL
	5 mM	0.7701 mL	3.8507 mL	7.7015 mL
	10 mM	0.3851 mL	1.9254 mL	3.8507 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.