Volasertib (BI 6727) tri HCl is an orally bioactive, potent, ATP-competitive inhibitor of Polo-like kinase 1 (PLK1) with IC50 of 0.87 nM. Volasertib tri HCl inhibits PLK2 and PLK3 with IC50 of 5 and 56 nM, respectively. Volasertib tri HCl induces mitotic arrest and apoptosis. Volasertib tri HCl is a dihydropterinone analogue that displays significant anticancer effect in multiple cancer models.

Physicochemical Properties

Molecular Formula	C34H53CL3N8O3
Molecular Weight	728.20
Exact Mass	726.331
CAS #	946161-17-7
Related CAS #	Volasertib;755038-65-4
PubChem CID	16718566
Appearance	Typically exists as solid at room temperature
LogP	7.326
Hydrogen Bond Donor Count	5
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	10
Heavy Atom Count	48
Complexity	996
Defined Atom Stereocenter Count	1
SMILES	Cl.Cl.Cl.O=C(C1C=CC(=C(C=1)OC)NC1=NC=C2C(=N1)N(C(C)C)[C@@H](C(N2C)=O)CC)NC1CCC(CC1)N1CCN(CC1)CC1CC1
InChi Key	JFEPFDDQDQBWIL-NGDVEHRBSA-N
InChi Code	InChI=1S/C34H50N8O3.3ClH/c1-6-28-33(44)39(4)29-20-35-34(38-31(29)42(28)22(2)3)37-27-14-9-24(19-30(27)45-5)32(43)36-25-10-12-26(13-11-25)41-17-15-40(16-18-41)21-23-7-8-23;;;/h9,14,19-20,22-23,25-26,28H,6-8,10-13,15-18,21H2,1-5H3,(H,36,43)(H,35,37,38);3*1H/t25?,26?,28-;;;/m1.../s1
Chemical Name	N-[4-[4-(cyclopropylmethyl)piperazin-1-yl]cyclohexyl]-4-[[(7R)-7-ethyl-5-methyl-6-oxo-8-propan-2-yl-7H-pteridin-2-yl]amino]-3-methoxybenzamide;trihydrochloride
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	PLK1 0.87 nM (IC50) PLK2 5 nM (IC50) PLK3 56 nM (IC50)
ln Vitro	In numerous cell lines, volasertib trihydrochloride (BI 6727 trihydrochloride; 0.01-10000 nM; 72 hours) shows EC50 values ranging from 11 to 37 nmol/L[1]. Cells with a 4N DNA content accumulate when exposed to volasertib trihydrochloride (10–1000 nM) for 24 hours, which is suggestive of a cell cycle block in the G2-M phase[1]. At 48 hours, volasertib trihydrochloride (100 nM; 24-72 hours) causes cell death[1].
ln Vivo	In human colon cancer xenograft models, volasertib trihydrochloride (BI 6727 trihydrochloride; a total weekly dose of 50 mg/kg; Oral; once a week, twice a week, or daily; for 40 days) exhibits comparable efficacy[1]. In human colon cancer xenograft models, volasertib trihydrochloride (15, 20, or 25 mg/kg/day; iv; 2 consecutive days per week; for 40 days) causes a considerable delay in tumor growth and even tumor regression [1]. kg administered once a week or 10 mg/kg orally;1] markedly inhibits the formation of tumors in a non-small cell lung cancer xenograft model derived from NCI-H460 cells. In HCT 116 tumor-bearing nude mice, volasertib (a single dose of 40 mg/kg; iv) significantly (13-fold) increases the number of mitotic cells[1]. Volasertib exhibits a large terminal half-life (Vss = 22 L/kg, t1/2 = 54 h) and a high volume of distribution in mice (Vss = 7.6 L/kg, t1/2 = 46 h)[1].
Cell Assay	Cell Proliferation Assay[1] Cell Types: Multiple cell lines Tested Concentrations: 0.01-10000 nM Incubation Duration: 72 hrs (hours) Experimental Results: Inhibited proliferation of multiple cell lines derived from various cancer tissues, including carcinomas of the colon (HCT 116, EC50=23 nmol/L) and lung (NCI-H460, EC50=21 nmol/L), melanoma (BRO, EC50=11 nmol/L), and hematopoietic cancers (GRANTA-519, EC50 =15 nmol/L; HL-60, EC50=32 nmol/L; THP-1, E50=36 nmol/L and Raji, EC50=37 nmol/L) with EC50 values of 11 to 37 nmol/L. Apoptosis Analysis [1] Cell Types: NCI-H460 cells Tested Concentrations: 100 nM Incubation Duration: 24, 48, 72 hrs (hours) Experimental Results: G2-M arrest at 24 hrs (hours) was followed by induction of apoptosis at 48 hrs (hours). Cell Cycle Analysis[1] Cell Types: NCI-H460 cells Tested Concentrations: 10, 30, 100, 300, 1000 nM Incubation Duration: 24 hrs (hours) Experimental Results: Resulted in accumulation of cells with 4N DNA content, indicative of a cell cycle block in G2-M phase.
Animal Protocol	Animal/Disease Models: Female BomTac:NMRI- Foxn1nu (nude) mice (Taconic) were grafted sc with HCT 116 human colon carcinoma cells (ATCC CCL-247)[1] Doses: A total weekly dose of 50 mg/kg Route of Administration: Oral; once a week, twice a week, or daily; for 40 days Experimental Results: demonstrated comparable efficacy and were well tolerated. Animal/Disease Models: Female BomTac:NMRI-Foxn1nu (nude) mice and male Wistar rats of the strain Crl:WI[1] Doses: 35 mg/kg (mice) or 10 mg/kg (rat) (pharmacokinetic/PK Analysis) Route of Administration: IV 5-minute infusion; a single dose 5-minute infusion Experimental Results: Had high volume of distribution and a long terminal half-life in mice (Vss=7.6 L/kg, t1/2= 46 h) and rats (Vss=22 L/kg, t1/2=54 h).
References	[1]. Volasertib suppresses tumor growth in cervical cancer. Am J Cancer Res. 2015 Nov 15;5(12):3548-59. [2]. BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity.Clin Cancer Res. 2009 May 1;15(9):3094-102. Epub.

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	1.3732 mL	6.8662 mL	13.7325 mL
	5 mM	0.2746 mL	1.3732 mL	2.7465 mL
	10 mM	0.1373 mL	0.6866 mL	1.3732 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.