Riviciclib HCl (P276-00) is a flavonoid analog which acts as a novel inhibitor of CDK1 (cyclin-dependent kinase), CDK4 and CDK9 with IC50 of 79 nM, 63 nM and 20 nM, respectively. P276-00 has the capacity to stop transcription of cells in the G1/S phase. In experiments using NSCLC cell lines, P276-00 demonstrated a strong ability to suppress cell proliferation, which in turn reduced the potential for colony formation by obstructing the cell cycle. By reducing the expression of CCND1, P276-00 treatment for 12 hours could significantly inhibit the ability of head and neck squamous carcinoma cell lines (FaDu, Detroits-562, SCC-25) to proliferate. The IC50 value for these cell lines varied between 0.8 and 1.7 uM.

Physicochemical Properties

Molecular Formula	C21H21CL2NO5
Molecular Weight	438.301
Exact Mass	437.08
Elemental Analysis	C, 57.55; H, 4.83; Cl, 16.18; N, 3.20; O, 18.25
CAS #	920113-03-7
Related CAS #	Riviciclib;920113-02-6
PubChem CID	23643975
Appearance	Off-white to light yellow solid powder
LogP	4.044
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	3
Heavy Atom Count	29
Complexity	628
Defined Atom Stereocenter Count	2
SMILES	OC1C=C(O)C2C(C=C(C3C=CC=CC=3Cl)OC=2C=1[C@@H]1CCN(C)[C@H]1CO)=O.Cl
InChi Key	OOVTUOCTLAERQD-OJMBIDBESA-N
InChi Code	InChI=1S/C21H20ClNO5.ClH/c1-23-7-6-12(14(23)10-24)19-15(25)8-16(26)20-17(27)9-18(28-21(19)20)11-4-2-3-5-13(11)22;/h2-5,8-9,12,14,24-26H,6-7,10H2,1H3;1H/t12-,14+;/m1./s1
Chemical Name	2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methylpyrrolidin-3-yl]chromen-4-one;hydrochloride
Synonyms	P-27600; P 27600; P27600; P276-00; P-276-00; P 276-00; Riviciclib HCl
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	CDK9- Cyclin T1 (IC50 = 0.020 μM); cdk4-cyclin D1 (IC50 = 0.063 μM); CDK1-Cyclin B (IC50 = 0.079 μM); cdk2-cyclin A (IC50 = 0.224 μM); cdk2-cyclin E (IC50 = 2.500 μM); cdk6-cyclin D3 (IC50 = 0.396 μM); CDK9-cyclin H (IC50 = 2.900 μM)
ln Vitro	P276-00 exhibits selectivity toward Cdk4-D1 that is 40 times greater than that of Cdk2-E[1]. It is found to be highly selective for cancer cells as compared with normal fibroblast cells[1]. It exhibits potent antiproliferative effects against a variety of human cancer cell lines, including HCT-116, U2OS, H-460, HL-60, HT-29, SiHa, MCF-7, Colo-205, SW-480, PC-3, Caco2, and T-24. The IC50 ranges from 300 to 800 nmol/L. In an ATP-competitive manner, P276-00 can down-regulate Cdk4 and cyclin D1, as well as lower phosphorylation of pRb Ser780 that is specific to Cdk4. Through the activation of cellular caspase-3 activity and the creation of DNA ladders, P276-00 also causes apoptosis[1].
ln Vivo	P276-00 can significantly inhibit the growth of murine colon cancer (CA-51) when given intraperitoneally (i.p.) at a dose of 50 mg/kg per day for 20 treatments. However, an increased dose of 60 mg/kg (30 mg/kg twice daily) given every other day intraperitoneally for seven treatments, in the murine lung carcinoma model (Lewis lung), exhibits a notable inhibition in the growth[2]. Additionally, it inhibits the growth of human non-small cell lung carcinoma H-460 and human colon carcinoma HCT-116 xenografts[2]. Its maximum tolerated dose, according to efficacy studies, is 78 mg/kg/d[2].
Enzyme Assay	The Cdk4-D1/Cdk2-E enzyme assay is conducted using Millipore Multiscreen filtration plates in a 96-well format. A single filter plate is used for each step of the assay. After prewetting the filtration wells with 100 μL of kinase buffer (which contains 50 mmol/L HEPES (pH of 7.5), 10 mmol/L MgCl2, and 1 mmol/L EGTA), the solution is vacuum-withdrawn. Vacuum is applied to the filter plate while it is on the vacuum manifold. Each well receives 50 μL of GST-Rb bound to GSH-Sepharose beads in kinase buffer (0.5 μg GST-Rb/50 μL). A reaction mix containing ATP (cold + hot) and 4× phosphatase inhibitor mix (40 μmol/L unlabeled ATP, 10 μCi/mL γ32P-ATP, 40 mmol/L h-glycerophosphate, 4 mmol/L DTT, 0.4 mmol/L NaF, 0.4 mmol/L sodium orthovanadate) diluted in kinase buffer is added to each well in approximately 25 μL. An additional 25 μL volume is then added containing either the test compound (4×final concentration in kinase buffer) or kinase buffer alone (control). To start the reaction, add 50 μL (100 ng) of human Cdk4-D1/Cdk2-E enzyme in kinase buffer to each well. The reaction is then allowed to run at 30°C for 30 minutes. The filter plate is air-dried and put in a Multiscreen adapter plate after the reaction is finished and vacuum is applied once more. The plate is then cleaned three times using the TNEN buffer, which is composed of 20 mmol/L Tris (pH, 8.0), 100 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% nonidet-P40. The plate is covered with a Top-Seal A film after adding 30 μL of Packard Microscint-O cocktail. With a Top Count scintillation counter, 32P-GST-Rb is quantified in 96-well filter plates. The initial concentration of each compound tested is 1 μmol/L. More profiling is done on compounds exhibiting 50% or more inhibition in order to determine their IC50.
Cell Assay	In a 96-well plate, the cells are seeded at a density of 3,000–5,000 per well, depending on the type of cell, in 180 μL of culture medium. The cells are then allowed to adhere over night in the incubator. Compounds in varying concentrations are added to the wells, and they are then incubated at 37°C for 48 hours. Each well receives an addition of 3H-thymidine (0.25 μCi), and the radiolabel is allowed to be incorporated for a duration of 5 to 7 hours. After the incubation period, cells are harvested onto GF/B unifilter plates using a Packard Filtermate Universal harvester, and the 96-well liquid scintillation counter Packard Top Count is used to count the plates.
Animal Protocol	H-460 xenograft 50 mg/kg once daily or 30 mg/kg twice daily i.p.
References	[1]. Mol Cancer Ther . 2007 Mar;6(3):918-25. [2]. Mol Cancer Ther . 2007 Mar;6(3):926-34.
Additional Infomation	Riviciclib Hydrochloride is the hydrochloride salt form of riviciclib, a flavone and cyclin dependent kinase (CDK) inhibitor with potential antineoplastic activity. Riviciclib selectively binds to and inhibits Cdk4/cyclin D1, Cdk1/cyclin B and Cdk9/cyclin T1, serine/threonine kinases that play key roles in the regulation of the cell cycle and cellular proliferation. Inhibition of these kinases leads to cell cycle arrest during the G1/S transition, thereby leading to an induction of apoptosis, and inhibition of tumor cell proliferation.

Solubility Data

Solubility (In Vitro)

DMSO: 50~88 mg/mL (114.1~200.8 mM) Ethanol: ~7 mg/mL (~16 mM) Water: ~88 mg/mL (~200.8 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.75 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 20.8 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: ≥ 2.08 mg/mL (4.75 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.75 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2815 mL	11.4077 mL	22.8154 mL
	5 mM	0.4563 mL	2.2815 mL	4.5631 mL
	10 mM	0.2282 mL	1.1408 mL	2.2815 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.