Ribociclib (formerly NVP-LEE011; LEE011; LEE-011; trade name: Kisqali) is a potent, orally bioavailable and highly specific inhibitor of CDK4/6 (cyclin-dependent kinase) with potential antineoplastic activity. Its IC50s for CDK4/6 inhibition are 10 nM and 39 nM, respectively. In March 2017, the FDA approved Ribociclib as a treatment for postmenopausal women who had an advanced form of breast cancer. Ribociclib works by reducing the levels of phosphorylated FOXM1 and RB. Out of 17 human neuroblastoma cell lines tested, 12 showed sensitivity to Ribociclib treatment (mean IC50=306±68 NM). By stopping the G0-G1 cell cycle, ribociclib treatment may significantly reduce the rate of cell proliferation. Treatment with LEE011 could markedly inhibit cell proliferation in 12 out of 17 human neuroblastoma-derived cell lines.

Physicochemical Properties

Molecular Formula	C23H30N8O
Molecular Weight	434.54
Exact Mass	434.254
Elemental Analysis	C, 63.57; H, 6.96; N, 25.79; O, 3.68
CAS #	1211441-98-3
Related CAS #	Ribociclib hydrochloride;1211443-80-9;Ribociclib-d6 hydrochloride;Ribociclib succinate;1374639-75-4;Ribociclib succinate hydrate;1374639-79-8;Ribociclib-d6;1328934-40-2;Ribociclib-d8;2167898-24-8
PubChem CID	44631912
Appearance	Yellow solid powder
Density	1.4±0.1 g/cm3
Boiling Point	730.8±70.0 °C at 760 mmHg
Flash Point	395.8±35.7 °C
Vapour Pressure	0.0±2.4 mmHg at 25°C
Index of Refraction	1.723
LogP	-0.74
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	5
Heavy Atom Count	32
Complexity	636
Defined Atom Stereocenter Count	0
SMILES	N1(CCNCC1)C1C=NC(NC2N=C3N(C(C(N(C)C)=O)=CC3=CN=2)C2CCCC2)=CC=1
InChi Key	RHXHGRAEPCAFML-UHFFFAOYSA-N
InChi Code	InChI=1S/C23H30N8O/c1-29(2)22(32)19-13-16-14-26-23(28-21(16)31(19)17-5-3-4-6-17)27-20-8-7-18(15-25-20)30-11-9-24-10-12-30/h7-8,13-15,17,24H,3-6,9-12H2,1-2H3,(H,25,26,27,28)
Chemical Name	7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide
Synonyms	LEE 011; Ribociclib; LEE011; LEE-011; trade name: Kisqali; Ribociclib (LEE011); LEE 011; 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide;
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	CDK4 (IC50 = 10 nM); CDK6 (IC50 = 39 nM)
ln Vitro	Treating a panel of 17 neuroblastoma cell lines with Ribociclib (LEE011) across a four-log dose range (10 to 10,000 nM). In 12 of the 17 neuroblastoma cell lines that were studied, treatment with ribociclib dramatically reduces substrate adherent growth in comparison to the control (mean IC50=306±68 nM, taking only sensitive lines into consideration; sensitivity is defined as an IC50 of less than 1 μM). After being treated with ribofloxacilb, two neuroblastoma cell lines (IMR5 and BE2C) that have been shown to be sensitive to CDK4/6 inhibition accumulate cells in the G0/G1 phase of the cell cycle in a dose-dependent manner. At concentrations of 100 nM (p=0.007) and 250 nM (p=0.01), respectively, of Ribociclib, this G0/G1 arrest becomes significant[2].
ln Vivo	Ribociclib (LEE011; 200 mg/kg) or a vehicle control is administered once daily for 21 days to CB17 immunodeficient mice carrying BE2C, NB-1643 (MYCN amplified, sensitive in vitro), or EBC1 (non-amplified, resistant in vitro) xenografts. Since none of the xenograft models exhibit weight loss or other toxicity indicators, this dosage strategy is well tolerated. During the course of the 21-day treatment period, mice carrying either the BE2C or 1643 xenografts (both, p<0.0001) showed a significant delay in tumor growth, which did not resume after treatment[2]. CDK4/6 inhibition by Ribociclib (LEE011) causes tumor growth delay in vivo [2] Given the observed differential sensitivity of neuroblastoma cell lines to CDK4/6 inhibition, we assayed for in vivo efficacy using neuroblastoma cell-line derived xenografts representing the extremes of in vitro sensitivity. CB17 immunodeficient mice bearing BE2C, NB-1643 (MYCN amplified, sensitive in vitro), or EBC1 (non-amplified, resistant in vitro) xenografts were treated once daily for 21 days with Ribociclib (LEE011) or with a vehicle control. This dosing strategy was well tolerated, as no weight loss or other signs of toxicity were observed in any of the xenograft models. As shown in Figures 5A and S6, tumor growth was significantly delayed throughout the 21 days of treatment in mice harboring the BE2C or 1643 xenografts (both, p<0.0001), although growth resumed post-treatment (data not shown). By contrast, as anticipated by the in vitro data, tumor growth suppression was less robust in the EBC1 xenograft model (p=0.51). Assessment of the Ki67 proliferation marker by immunohistochemistry confirmed that proliferation was impaired only in the BE2C and 1643 xenograft models, as tumors resected from separate cohorts of BE2C or 1643 xenografted mice demonstrated comparatively weaker staining following 7 days of treatment with Ribociclib (LEE011) than with the vehicle control, while no Ki67 staining differences were observed in the EBC1 xenografts (Figure 5B). Phosphorylation of RB was also substantially diminished in the BE2C and 1643 xenografts, while only a minimal decrease was detected in the EBC1 model (Figures 5B and 5C) [2].
Enzyme Assay	Ribociclib, a powerful, oral, and highly selective inhibitor of CDK4/6 (cyclin-dependent kinase), with IC50s of 10 nM and 39 nM, respectively, was previously known as LEE011, NVP-LEE011; trade name: Kisqali. In March 2017, the FDA approved Ribociclib as a treatment for postmenopausal women who had an advanced form of breast cancer. Ribociclib works by reducing the levels of phosphorylated FOXM1 and RB. Out of 17 human neuroblastoma cell lines tested, 12 showed sensitivity to ribofacilb treatment (mean IC50=306±68 NM). By stopping the G0-G1 cell cycle, ribociclib treatment may significantly reduce the rate of cell proliferation. Treatment with LEE011 could markedly inhibit cell proliferation in 12 out of 17 human neuroblastoma-derived cell lines.
Cell Assay	In 35 mm plates, cells are grown for 24 hours, then treated with 500 nM Ribociclib for 6 days. The cells are then fixed, and overnight staining is done. Then, using an Axio Observer D.1 phase contrast microscope, cells are imaged for SA-β-gal. By counting the number of positive cells in three different microscope frames and normalizing to the control, one can calculate the percentage of SA-β-gal positive cells. In order to evaluate apoptotic activity, cells are treated with Ribociclib, plated in triplicate in 96-well plates, and then 16 hours later, caspase 3/7 activation is measured 16 hours after Caspase-Glo 3/7 treatment. As a positive control, SN-38-treated cells are employed[2].
Animal Protocol	Mice: The xenografts derived from BE2C, NB-1643, or EBC1 cell lines are subcutaneously implanted into the right flank of CB17 SCID-/-mice. Then, for a total of 21 days, animals with engrafted tumors measuring 200–600 mm3 are randomly assigned to receive oral treatment with 200 mg/kg Ribociclib in 0.5% methylcellulose (n = 10) or vehicle (n = 10). Throughout the course of treatment, the tumor burden is calculated on a regular basis using the formula (π/6)×d2, where d is the mean tumor diameter measured with a caliper.
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Ribociclib is orally bioavailable, highly selective inhibitor of CDK4/6 kinases with inhibitory IC50 concentrations in the low nanomolar range. Following oral dosing, ribociclib was rapidly absorbed with median Tmax ranging from 1 to 5 hours. Plasma concentrations increased approximately 2- to 3-fold from Cycle 1 Day 1 to Cycle 1 Day 18/21 due to accumulation, with steady state reached by approximately Day 8 on the basis of trough concentrations after repeated daily dosing. Dose-proportionality analyses demonstrated that exposure to ribociclib increased with dose, with both Cmax and area under the curve (AUC) increasing slightly more than proportional to dose, over the dose range 50–1,200 mg/day Biological Half-Life 32.6 hours
Toxicity/Toxicokinetics	Hepatotoxicity In the large clinical trials, adverse events were common and led to dose reductions in 45% of patients and discontinuation in 7%. In preregistration clinical trials, ALT elevations occurred in 46% of ribociclib vs 36% of control subjects and elevations above 5 times the ULN in 10% vs 1%. In one study, 1% of recipients developed clinically apparent liver injury with jaundice, but all recovered. The liver injury arose after 3 to 5 cycles and presented with asymptomatic elevations in serum ALT followed by symptoms and jaundice. Immunoallergic and autoimmune features were not present, although liver histology sometimes showed autoimmune hepatitis-like features. Recovery was slow (3 to 5 months), but ultimately complete. Restarting ribociclib resulted in more rapid and severe recurrence. Thus, experience with ribociclib is limited, but it appears to be capable of causing significant liver injury. Likelihood score: C (probable cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of ribociclib during breastfeeding. Because protein binding of ribociclib is 70%, clinically important amounts of the drug might pass into breastmilk. The manufacturer recommends that breastfeeding be discontinued during ribociclib therapy and for at least 3 weeks after the final dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date.
References	[1]. Molecular Pathways: Targeting the Cyclin D-CDK4/6 Axis for Cancer Treatment. Clin Cancer Res. 2015 Jul 1;21(13):2905-10. [2]. Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma. Clin Cancer Res. 2013 Nov 15;19(22):6173-82.
Additional Infomation	Ribociclib is a member of piperazines and a member of pyridines. Ribociclib is a selective cyclin-dependent kinase inhibitor, a class of drugs that help slow the progression of cancer by inhibiting two proteins called cyclin-dependent kinase 4 and 6 (CDK4/6). These proteins, when over-activated, can enable cancer cells to grow and divide too quickly. Targeting CDK4/6 with enhanced precision may play a role in ensuring that cancer cells do not continue to replicate uncontrollably. Ribociclib was approved by the U.S. FDA in March, 2017 as Kisqali. Ribociclib is a Kinase Inhibitor. The mechanism of action of ribociclib is as a Kinase Inhibitor, and Cytochrome P450 3A Inhibitor. Ribociclib is a unique cyclin-dependent kinase inhibitor that is used in combination with aromatase inhibitors in the treatment of postmenopausal women with metastatic breast cancer. Ribociclib is associated with a moderate rate of serum aminotransferase elevations during therapy, and to clinically apparent liver injury in a proportion of these. Ribociclib is an orally available cyclin-dependent kinase (CDK) inhibitor targets at cyclin D1/CDK4 and cyclin D3/CDK6 cell cycle pathway, with potential antineoplastic activity. Ribociclib specifically inhibits CDK4 and 6, thereby inhibiting retinoblastoma (Rb) protein phosphorylation. Inhibition of Rb phosphorylation prevents CDK-mediated G1-S phase transition, thereby arresting the cell cycle in the G1 phase, suppressing DNA synthesis and inhibiting cancer cell growth. Overexpression of CDK4/6, as seen in certain types of cancer, causes cell cycle deregulation. See also: Ribociclib Succinate (active moiety of). Drug Indication Kisqali (ribociclib) is a selective cyclin-dependent kinase inhibitor, a class of drugs that help slow the progression of cancer by inhibiting two proteins called cyclin-dependent kinase 4 and 6 (CDK4/6). These proteins, when over-activated, can enable cancer cells to grow and divide too quickly. Targeting CDK4/6 with enhanced precision may play a role in ensuring that cancer cells do not continue to replicate uncontrollably. Kisqali is indicated for the treatment of women with hormone receptor (HR)‑positive, human epidermal growth factor receptor 2 (HER2)‑negative locally advanced or metastatic breast cancer in combination with an aromatase inhibitor or fulvestrant as initial endocrine-based therapy, or in women who have received prior endocrine therapy. In pre‑ or perimenopausal women, the endocrine therapy should be combined with a luteinising hormone‑releasing hormone (LHRH) agonist. Treatment of neuroblastoma Mechanism of Action Inhibition of cyclin-dependent kinase 4 and 6 (CDK4/6) may provide protection against oncogenic processes in specific tissue types. For example, CDK4 is not required for normal mammary tissue development based on knockout mouse studies, but it is needed for growth of Ras-induced mammary tumors, suggesting a potential therapeutic window for treatment with lower toxicity. Ribociclib was reported to be a most selective CDK4/6 inhibitor and to have dose dependent antitumor activity in a number of preclinical models. It inhibited growth of tumor cells by arresting the cells at the G1 checkpoint, which prevents the tumor cells from proliferating.

Solubility Data

Solubility (In Vitro)

DMSO: ~7 mg/mL (~16.1 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1 mg/mL (2.30 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1 mg/mL (2.30 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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: ≥ 0.89 mg/mL (2.05 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 8.9 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 4: ≥ 0.89 mg/mL (2.05 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 8.9 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 5: ≥ 0.89 mg/mL (2.05 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 8.9 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 6: 5% DMSO+40% PEG 300+5%Tween80+ 50%ddH2O: 1.1mg/ml  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3013 mL	11.5064 mL	23.0128 mL
	5 mM	0.4603 mL	2.3013 mL	4.6026 mL
	10 mM	0.2301 mL	1.1506 mL	2.3013 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.