RGB-286638 2HCl, the dihydrochloride salt of RGB-286638, is a multi-targeted cyclin-dependent kinase (CDK) inhibitor. It has IC50 values of 1, 2, 3, 4, 5, and 5 nM for cyclin T1-CDK9, B1-CDK1, E-CDK2, D1-CDK4, E-CDK3, and p35-CDK5 kinase activity inhibition; IC50 values of 3, 5, 50, and 54 nM for GSK-3β, TAK1, Jak2, and MEK1 kinase activity inhibition are also observed. RGB-286638 inhibits transcriptional CDKs, which in turn initiates P53-dependent and -independent anti-multiple myeloma (MM) activity. When MM tumor growth was inhibited and in vivo survival was extended, GB-286638 treatment led to MM cytotoxicity in vitro. Both wt-p53 and mutant-p53 cells showed caspase-dependent apoptosis, which was closely linked to transcription inhibition and RNA polymerase II phosphorylation downregulation.

Physicochemical Properties

Molecular Formula	C29H37CL2N7O4
Molecular Weight	618.6
Exact Mass	617.228
CAS #	784210-87-3
Related CAS #	RGB-286638 free base;784210-88-4
PubChem CID	11285001
Appearance	Light yellow to yellow solid
LogP	4.243
Hydrogen Bond Donor Count	5
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	8
Heavy Atom Count	42
Complexity	857
Defined Atom Stereocenter Count	0
SMILES	Cl.Cl.O(C)CCN1CCN(CC2C=CC(=CC=2)C2C3C(C4C(=CC=CC=4C=3NN=2)NC(NN2CCOCC2)=O)=O)CC1
InChi Key	WJVMGQMXUBAAPL-UHFFFAOYSA-N
InChi Code	InChI=1S/C29H35N7O4.2ClH/c1-39-16-13-34-9-11-35(12-10-34)19-20-5-7-21(8-6-20)26-25-27(32-31-26)22-3-2-4-23(24(22)28(25)37)30-29(38)33-36-14-17-40-18-15-36;;/h2-8H,9-19H2,1H3,(H,31,32)(H2,30,33,38);2*1H
Chemical Name	1-[3-[4-[[4-(2-methoxyethyl)piperazin-1-yl]methyl]phenyl]-4-oxo-1H-indeno[1,2-c]pyrazol-5-yl]-3-morpholin-4-ylurea;dihydrochloride
Synonyms	RGB-286638 2HCl; RGB 286638 2HCl; RGB2866382HCl
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	T1-CDK9 (IC50 = 1 nM); cyclin B1-CDK1 (IC50 = 2 nM); cyclin E-CDK2 (IC50 = 3 nM); cyclin D1-CDK4 (IC50 = 4 nM); cyclin E-CDK3 (IC50 = 5 nM); p35-CDK5 (IC50 = 5 nM); cyclin H-CDK7 (IC50 = 44 nM); cyclin D3-CDK6 (IC50 = 55 nM); GSK-3β (IC50 = 3 nM); JAK2 (IC50 = 50 nM); MEK1 (IC50 = 54 nM); Fms (IC50 = 1 nM); TAK1 (IC50 = 5 nM); JNK1a1 (IC50 = 17 nM); JNK1a2 (IC50 = 40 nM); C-src (IC50 = 25 nM); AMPK (IC50 = 41 nM)
ln Vitro	RGB-286638 is a CDK inhibitor (CDKI) derived from indenopyrazole that exhibits Ki-nanomolar activity against transcriptional CDKs. AMPK, Jak2, MEK1, TAK1, GSK-3β, and other tyrosine and serine/threonine non-CDK enzymes are all inhibited by RGB-286638. By using the MTT assay to measure viability at 24 and 48 hours, the effects of RGB-286638 (12.5-100nM) treatment are examined on the growth of human p53-wt (MM.1S, MM.1R, and H929) and p53-mutant (U266, OPM1, and RPMI) MM cells. After 48 hours, the half-maximally effective concentrations (EC50) vary from 20 to 70 nM. Following 50nM treatment, dose-dependent growth differences between p53-wt and -mutant cells are seen, with p53-wt MM.1S, MM.1R, and H929 showing a slight increase in sensitivity to RGB-286638 treatment at 48 hours[1].
ln Vivo	The highest tolerated dosage in SCID mice, according to dose-finding studies using RGB-286638, is 40 mg/kg/day IV therapy. RGB-286638 treatment for five days intravenously significantly suppresses the growth of MM tumors; the maximum TGI (%) in the 30 mg/kg and 40 mg/kg treated cohorts is recorded at day 14 after treatment completion, at 85.06% and 86.34%, respectively. Each of the two treated groups has a log10 cell kill (LCK Td: 4.5 days) of 1.6. The first death in the control group occurred on day 24, while in the treated groups it occurred on day 43. This suggests that treatment with RGB-286638 is also linked to improved survival. This study did not result in any toxic deaths. The highest percentage of body weight (BW) loss was noted on day 5 (8.4%) at a dosage schedule of 30 mg/kg and on day 15 (9.9%) after a dosage of 40 mg/kg. The following two weeks saw weight recovery[1].
Enzyme Assay	The Nuclear Extraction Kit is used to isolate nuclear proteins from MM.1S cells that have been exposed to 50nM RGB-286638 for 1, 4, and 8 hours. A specific double-stranded DNA sequence containing the p53 response element is coated on 96-well plates, and nuclear protein aliquots are added for an overnight incubation. By adding a particular primary antibody that targets p53, p53 in the nuclear extract can be identified. A sensitive colorimetric readout at 450 nm is achieved by adding a secondary antibody conjugated to HRP. Triple-checking is done on every experiment[1].
Cell Assay	Colorimetric assays are used to measure the efficacy of drugs at progressively higher RGB-286638 concentrations (0-100nM). 100 μL of isopropanol containing 0.04 HCl is added to each well after 10μL of 5 mg/mL MTT is pulsed into each well of p53-expressing wild-type (MM.1S, MM.1R, H929) or mutant (U266, OPM1, RPMI) cells from 24- or 48-hour cultures. The cells are then incubated at 37°C for 4 hours. Using a spectrophotometer, absorbance measurements are made at 570 nm wavelength (corrected using readings at 630 nm). Three duplicates of each experiment are run[1].
Animal Protocol	40 mg/kg; i.v. Mice: RGB-286638's in vivo anti-MM activity is assessed using an MM xenograft model. Agennix AG prepares and supplies the RGB-286638 dosing solutions, which come in 2 and 3 mg/mL in 5% dextrose/water (D5W) pH5.2 as well as D5W pH5.2 for the vehicle control dosing group. The mice used are CB-17 severe combined immunodeficient (SCID). A total of forty male mice aged five to six weeks are exposed to 2 Gy [200 rad] of radiation using a cesium 137 (137Cs)-irradiator source; 24 hours post-irradiation, the mice measure 2.5 x 106 MM. Subcutaneous inoculation of the upper back is done with 1S cells. Mice are randomized into three cohorts when the tumor weight is roughly 100 mg, and they are given daily IV tail vein injections for five days straight with either RGB-286638 30 mg/kg (8 mice), 40 mg/kg (9 mice), or control vehicle alone (10 mice). Every other day, the body weight and tumor volume of the animals are measured using calipers. Volume of tumor is estimated. From the first day of treatment until death, survival is assessed. From the first day of treatment until the first sacrifice day, measurements taken with a caliper are used to assess the growth of the tumor. Tumor growth inhibition (TGI) percentage is computed.
References	[1]. Small-molecule multi-targeted kinase inhibitor RGB-286638 triggers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs. Leukemia. 2013 Dec;27(12):2366-75.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 50 mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 7.5 mg/mL (12.13 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 75.0 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: ≥ 7.5 mg/mL (12.13 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 75.0 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	1.6166 mL	8.0828 mL	16.1655 mL
	5 mM	0.3233 mL	1.6166 mL	3.2331 mL
	10 mM	0.1617 mL	0.8083 mL	1.6166 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.