RRD-251 is an inhibitor (blocker/antagonist) of the retinoblastoma tumor suppressor protein (Rb)-Raf-1 interaction (Rb-Raf-1 interaction) and has potent antiproliferation, anti-angiogenic and anti-tumor activities.

Physicochemical Properties

Molecular Formula	C8H9CL3N2S
Molecular Weight	271.59
Exact Mass	269.955
CAS #	72214-67-6
PubChem CID	12526767
Appearance	White to off-white solid powder
Density	1.47g/cm3
Boiling Point	345.3ºC at 760 mmHg
Flash Point	162.6ºC
Index of Refraction	1.643
LogP	4.722
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	3
Heavy Atom Count	14
Complexity	189
Defined Atom Stereocenter Count	0
InChi Key	COMNQRICZGJVLE-UHFFFAOYSA-N
InChi Code	InChI=1S/C8H8Cl2N2S.ClH/c9-6-2-1-5(7(10)3-6)4-13-8(11)12;/h1-3H,4H2,(H3,11,12);1H
Chemical Name	(2,4-dichlorophenyl)methyl carbamimidothioate;hydrochloride
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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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	Rb-Raf-1 interaction, apoptosis[1]
ln Vitro	In vitro, RRD-251 (10-50 μM; 24 hours) suppresses the proliferation of melanoma[1]. In non-small cell lung cancer cells, RRD-251 (50 μM; 2 hours) suppresses Rb-Raf-1 interaction and Rb phosphorylation [1]. RRD-251 causes cell cycle arrest (20–50 μM; 4 hours) and apoptosis (50 μM; 18 hours)[1]. Apoptosis regulating protein and cell cycle expression are modified by RRD-251[1].
ln Vivo	Melanomas treated with RRD-251 (50 mg/kg; ip; qod; for 14 days) have anti-cancer properties in vivo[1].
Cell Assay	Cell Proliferation Assay[1] Cell Types: SK-MEL-28 cells, SK-MEL-5 cells, SK-MEL- 2 cells Tested Concentrations: 10 μM, 20 μM, 50 μM Incubation Duration: 24 hrs (hours) Experimental Results: Inhibited melanoma growth in-vitro. Western Blot Analysis[1] Cell Types: SK-MEL-28 cells, SK-MEL-5 cells, SK -MEL-2 cells Tested Concentrations: 50 μM Incubation Duration: 2 hrs (hours) Experimental Results: demonstrated the depletion of phosphorylated-Rb in RRD-251 treated cells. Apoptosis Analysis[1] Cell Types: SK-MEL-28 cells, SK-MEL-5 cells, SK-MEL-2 cells Tested Concentrations: 50 μM Incubation Duration: 18 hrs (hours) Experimental Results: Induced apoptosis. Cell Cycle Analysis[1] Cell Types: SK-MEL-28 cells, SK-MEL-5 cells Tested Concentrations: 20 μM, 50 μM Incubation Duration: 4 hrs (hours) Experimental Results: Resulted in a dose dependent inhibition of cell cycle progression in SK-MEL-28 and SK-MEL-5 cells, respectively.
Animal Protocol	Animal/Disease Models: 8-wk-old female athymic nude mice, with SK-ME-28 xenograft[1] Doses: 50 mg/kg Route of Administration: intraperitoneal (ip)administration, qod, for 14 days Experimental Results: Inhibits the growth of SK-ME-28 xenograft in nude mice.
References	[1]. Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazine. Mol Cancer Ther. 2010 Dec; 9(12): 3330–3341.

Solubility Data

Solubility (In Vitro)

DMSO : 125 mg/mL (460.25 mM)

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.6820 mL	18.4101 mL	36.8202 mL
	5 mM	0.7364 mL	3.6820 mL	7.3640 mL
	10 mM	0.3682 mL	1.8410 mL	3.6820 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.