Lerociclib (G1T38) is a novel, potent, orally bioavailable and selective inhibitor of CDK4/6, with IC50s of 1 nM, 2 nM for CDK4/CyclinD1 and CDK6/CyclinD3, respectively. G1T38 is a competitive, nanomolar inhibitor of CDK4/6 with high selectivity for CDK4-cyclin D1 and CDK6-cyclin D3, as shown by biochemical profiling. Low EC50 (<100 in='' rb='' competent='' cell='' lines='' compared='' to=''>3 μM in Rb null cells) is seen in G1T38. In vivo, G1T38 administered orally once a day results in a notable and long-lasting inhibition of tumor growth in HER2/neu GEMM and MCF7 xenograft breast cancer models.

Physicochemical Properties

Molecular Formula	C26H34N8O
Molecular Weight	474.6012
Exact Mass	474.29
Elemental Analysis	C, 65.80; H, 7.22; N, 23.61; O, 3.37
CAS #	1628256-23-4
Related CAS #	Lerociclib dihydrochloride;2097938-59-3
PubChem CID	86269224
Appearance	Solid powder
LogP	3.4
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	4
Heavy Atom Count	35
Complexity	750
Defined Atom Stereocenter Count	0
InChi Key	YPJRHEKCFKOVRT-UHFFFAOYSA-N
InChi Code	InChI=1S/C26H34N8O/c1-18(2)32-10-12-33(13-11-32)20-6-7-22(27-16-20)30-25-28-15-19-14-21-24(35)29-17-26(8-4-3-5-9-26)34(21)23(19)31-25/h6-7,14-16,18H,3-5,8-13,17H2,1-2H3,(H,29,35)(H,27,28,30,31)
Chemical Name	4-[[5-(4-propan-2-ylpiperazin-1-yl)pyridin-2-yl]amino]spiro[1,3,5,11-tetrazatricyclo[7.4.0.02,7]trideca-2,4,6,8-tetraene-13,1'-cyclohexane]-10-one
Synonyms	Lerociclib; G1T38; G1T-38; G1T 38; G1-T38; G1 T-38; G1 T38
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	cdk2/cyclin A (IC50 = 1.5 μM); CDK2/cyclinE (IC50 = 3.6 μM); Cdk4/cyclin D1 (IC50 = 1 nM); cdk6/cyclin D3 (IC50 = 2 nM); CDK9/Cyclin T (IC50 = 28 nM); CDK5/p35 (IC50 = 0.832 μM); CDK1/cyclinB1 (IC50 = 2.4 μM); CDK7/Cyclin H/MAT1 (IC50 = 2.4 nM); Cdk5/p25 (IC50 = 1.2 nM)
ln Vitro	Lerociclib is the least selective member of the CDK family against CDK9/cyclin T, with a biochemical IC50 that is approximately thirty times lower than that of CDK4/cyclin D1. With an EC50 of less than 20 nM, lerociclib causes a strong and long-lasting G1 arrest in CDK4/6 dependent cells. When CDK4/6 dependent WM2664 cells are treated with Lerociclib for 24 hours, a dose-dependent increase of cells in the G1 phase of the cell cycle is seen. More than 300 times the biochemical IC50 is maintained through 300 nM, sustaining this arrest. When compared to vehicle controls, WM2664 cells treated with 30-1000 nM of Lerociclib for 24 hours show a complete inhibition of RB phosphorylation. Lerociclib treatment results in almost total inhibition of RB phosphorylation by 16 hours after treatment, with RB phosphorylation reduced within 1 hour after treatment. With EC50 concentrations as low as 23 nM, lerociclib effectively inhibits the proliferation of a wide range of tumor cell lines, including breast, melanoma, leukemia, and lymphoma[1].
ln Vivo	Lerociclib-treated mice in this HER2+ breast cancer model show 8% tumor regression after 21 days of treatment, whereas control animals show a 577% increase in tumor burden during the same time period. The MCF7 xenograft model exhibits tumor regression in 10 days when daily treatment with 100 mg/kg of Lerocyclib or palbociclib is administered in comparison to the mice treated with a vehicle. Tumor growth inhibition is seen in the 10, 50, and 100 mg/kg Lerociclib cohorts after 27 days of treatment (approximately 12%, 74%, and 90% inhibition, respectively). In the 10, 50, and 100 mg/kg dosage cohorts, daily oral palbociclib treatment results in an 18%, 66%, and 87% inhibition of tumor growth, respectively. It's interesting to note that lerociclib is much more effective than palbociclib at 50 mg/kg. Comparing Lerocyclib and palbociclib at the 50 mg/kg dose in the ER+ZR-75-1 breast cancer xenograft model yields similar results. With an overall tumor growth delay of 60% and 77% TGI, mice treated with lerociclib show that lerocyclib alone is highly effective in treating this NSCLC tumor model.
Cell Assay	The cells in Costar 3903 96-well plates are seeded with 1000 cells per well for SupT1, Daudi, MCF7, ZR-75-1, A2058, WM2664, and H69; plated at 4000 cells per well for MV-4-11 and BV173; plated at 8,000 cells per well for Tom-1; and plated at 20,000 cells per well for NALM-1. Plates are dosed with Lerociclib (G1T38) at a nine-point dose concentration ranging from 10 μM to 1 nM after a 24-hour period. After four or six days, cell viability is assessed. GraphPad Prism 5 statistical software is used for data analysis after plates are processed on the BioTek Synergy2 multi-mode plate reader[1].
Animal Protocol	Mice: Lerocyclib (G1T38) (100 mpk, medicated diet) is tested in female MMTV-NEU mice. Body composition is evaluated at the start of treatment, and weight measurements (in grams) are kept track of and used to calculate gross toxicity. NSCLC PDX CTG0159 tumors are implanted in female naked mice. After tumors reach a volume between 150 and 300 mm3, mice are randomly assigned to treatment groups, and dosing is started. Lerociclib (G1T38) at a dose of 100 mg/kg or the vehicle is taken orally for 28 days in a row. A lung adenocarcinoma model of H1975 NSC is implanted into female NCI Ath/nu mice. When tumors are 100–150 mm3 in size on average, mice are randomly assigned to treatment groups. For the duration of the study, mice are given oral doses of afatinib (20 mg/kg), erlotinib (70 mg/kg), or Lerociclib (50 or 100 mg/kg) either alone or in combination (Lerociclib+erlotinib or Lerociclib+afatinib). Up until the mice reach a tumor burden of 1500 mm3, all tumors are measured twice a week.
References	[1]. Preclinical development of G1T38: A novel, potent and selective inhibitor of cyclin dependent kinases 4/6 for use as an oral antineoplastic in patients with CDK4/6 sensitive tumors. Oncotarget. 2017 Jun 27;8(26):42343-42358
Additional Infomation	Lerociclib is under investigation in clinical trial NCT02983071 (G1T38, a CDK 4/6 Inhibitor, in Combination With Fulvestrant in Hormone Receptor-positive, Her2-negative Locally Advanced or Metastatic Breast Cancer). Lerociclib is an orally bioavailable inhibitor of cyclin-dependent kinase (CDK) types 4 (CDK4) and 6 (CDK6), with potential antineoplastic activity. Upon administration, lerociclib selectively inhibits CDK4 and CDK6, which inhibits the phosphorylation of retinoblastoma protein (Rb) early in the G1 phase, prevents CDK-mediated G1-S phase transition and leads to cell cycle arrest. This suppresses DNA replication and decreases tumor cell proliferation. CDK4 and 6 are serine/threonine kinases that are upregulated in many tumor cell types and play a key role in the regulation of both cell cycle progression from the G1-phase into the S-phase and tumor cell proliferation.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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	2.1070 mL	10.5352 mL	21.0704 mL
	5 mM	0.4214 mL	2.1070 mL	4.2141 mL
	10 mM	0.2107 mL	1.0535 mL	2.1070 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.