Bafetinib (INNO-406; NS187)

Alias: INNO-406; INNO 406; NS187; NS187; INNO406;NS-187; NS 187

Cat No.:V0679 Purity: ≥98%

COA Product Data Instructions for use SDS

Bafetinib (INNO-406; NS187) Chemical Structure CAS No.: 859212-16-1
Product category: Bcr-Abl

This product is for research use only, not for human use. We do not sell to patients.

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Other Forms of Bafetinib (INNO-406; NS187):

Lyn-IN-1

Official Supplier of:

Top Publications Citing lnvivochem Products

Nature 2021, 597(7874):119-125.

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Cancer Cell 2021,39(4):529-547.e7.

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Immunity 2023,56(3):620-634.e11.

J Am Chem Soc 2022,144:21831-21836.

Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

PNAS Nexus 2022,1(3):pgac063.

ACS Nano 2023,17(10):9110-9125.

Biomaterial 2023 Sep16:302:122333.

Purity & Quality Control Documentation

Current batch：

Purity: ≥98%

COA

MSDS

Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Clinical Trial Information
Biological Data

Product Description

Bafetinib (formerly INNO406; NS-187), an investigational anticancer drug originally developed by Nippon Shinyaku and later licensed to CytRx, is an orally bioavailable dual Bcr-Abl/Lyn inhibitor with potential antineoplastic activity. It inhibits Bcr-Abl/Lyn with IC50s of 5.8 nM/19 nM in cell-free assays. In Bcr-Abl–positive KU812 mouse model, Bafetinib significantly inhibited tumor growth, and completely inhibited tumor growth without causing adverse effects at a dose of 20 mg/kg/day.

Biological Activity I Assay Protocols (From Reference)

ln Vitro

In vitro activity: Bafetinib blocks WT Bcr-Abl autophosphorylation and its downstream kinase activity with IC50 of 11 nM and 22 nM in K562 and 293T cells, respectively. Bafetinib suppresses the growth of the Bcr-Abl-positive cell lines including K562, KU812, and BaF3/wt cells potently without effects on the proliferation of the Bcr-Abl-negative U937 cell line. Moreover, Bafetinib exhibits a dose-dependent antiproliferative effect against Bcr-Abl point mutant cell lines, such as BaF3/E255K cells. In Bcr-Abl+ leukemia cell lines, Bafetinib induces both caspase-mediated and caspase-independent cell death by blocking the phosphorylation of Bcr-Abl.

Kinase Assay: Bcr-Abl kinase assays are performed in 25 μL of reaction mixture containing 250 μM peptide substrate, 740 Bq/μL [γ-33P]ATP, and 20 μM cold adenosine triphosphate (ATP) by using the SignaTECT protein tyrosine kinase assay system. Each Bcr-Abl kinase is used at a concentration of 10 nM. Kinase assays for Abl, Src, and Lyn are carried out with an enzyme-linked immunosorbent assay (ELISA) kit. The inhibitory effects of NS-187 against 79 tyrosine kinases are tested with KinaseProfiler.

Cell Assay: K562, BaF3/wt, BaF3/E255K, and BaF3/T315I cells are plated at 1 × 103 in 96-well plates, whereas KU812 and U937 cells are plated at 5 × 103in 96-well plates. Cells are incubated with serial dilutions of Bafetinib for 3 days. Cell proliferation is measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nacalai Tesque) assay, and the 50% inhibitory concentration (IC50) values are calculated by fitting the data to a logistic curve.

ln Vivo

In Bcr-Abl–positive KU812 mouse model, Bafetinib (0.2 mg/kg/day) significantly inhibits tumor growth, and completely inhibits tumor growth without adverse effects at 20 mg/kg/day. For Balb/c mice, Bafetinib shows maximal tolerated dose of 200 mg/kg/d and bioavailability value (BA) of 32%. In a Central nervous system (CNS) leukemia model bearing Ba/F3/wt bcr-ablGFP, Ba/F3/Q252H, or Ba/F3/M351T cells, combination treatment of Bafetinib (60 mg/kg) and cyclosporine A (CsA) (50 mg/kg) leads to more significant inhibition of leukemia growth in the brain than either Bafetinib or CsA alone.

Animal Protocol

Bafetinib is dissolved in 0.5% methylcellulose; ≤20 mg/kg/day; p.o.

KU812 xenograft is established by subcutaneous injection of KU812 cells into the right flank of Balb/c-nu/nu female mice.

References

Blood.2005 Dec 1;106(12):3948-54;Blood.2007 Jan 1;109(1):306-14.

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula	C30H31F3N8O
Molecular Weight	576.62
CAS #	859212-16-1
Related CAS #	859212-16-1;887650-05-7;
SMILES	O=C(NC1=CC=C(C)C(NC2=NC=CC(C3=CN=CN=C3)=N2)=C1)C4=CC=C(CN5C[C@@H](N(C)C)CC5)C(C(F)(F)F)=C4
InChi Key	ZGBAJMQHJDFTQJ-DEOSSOPVSA-N
InChi Code	InChI=1S/C30H31F3N8O/c1-19-4-7-23(13-27(19)39-29-36-10-8-26(38-29)22-14-34-18-35-15-22)37-28(42)20-5-6-21(25(12-20)30(31,32)33)16-41-11-9-24(17-41)40(2)3/h4-8,10,12-15,18,24H,9,11,16-17H2,1-3H3,(H,37,42)(H,36,38,39)/t24-/m0/s1
Chemical Name	(S)-N-(3-([4,5'-bipyrimidin]-2-ylamino)-4-methylphenyl)-4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)benzamide
Synonyms	INNO-406; INNO 406; NS187; NS187; INNO406;NS-187; NS 187
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)

Solubility Data

Solubility (In Vitro)

DMSO: 100 mg/mL (173.4 mM)

Water:<1 mg/mL

Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.34 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 25.0 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: ≥ 2.5 mg/mL (4.34 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 0.5% methylcellulose+0.2% Tween 80: 30 mg/mL

(Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7342 mL	8.6712 mL	17.3424 mL
	5 mM	0.3468 mL	1.7342 mL	3.4685 mL
	10 mM	0.1734 mL	0.8671 mL	1.7342 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.

Calculator

Mass

Concentration

Volume

Molecular Weight*

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Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

Calculate the Mass of a compound required to prepare a solution of known volume and concentration
Calculate the Volume of solution required to dissolve a compound of known mass to a desired concentration
Calculate the Concentration of a solution resulting from a known mass of compound in a specific volume

See Example

An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

Enter 350.26 in the Molecular Weight (MW) box
Enter 10 in the Concentration box and choose the correct unit (mM)
Enter 5 in the Volume box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

Concentration (Start)

Volume (Start)

Concentration (End)

Volume (End)

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Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
Enter 25 into the Concentration (End) box and select the correct unit (mM)
Enter 25 into the Volume (End) box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

Molecular formula

Total molecular weight

g/mol

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Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

Volume (to add to vial)

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Desired Reconstitution Concentration

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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
Click the “Calculate” button
The answer appears in the Volume (to add to vial) box

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

Dosage mg/kg

Average weight of animals g

Dosing volume per animal μL

Number of animals

Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

% DMSO

% Tween 80

% ddH₂O

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Calculation results

Working concentration： mg/mL；

Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O，mix and clarify.

Note： (1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.

Clinical Trial Information

NCT Number	Recruitment	interventions	Conditions	Sponsor/Collaborators	Start Date	Phases
NCT01215799	Completed	Drug: Bafetinib	Hormone Refractory Prostate Cancer	CytRx	August 2010	Phase 2
NCT01144260	Completed	Drug: bafetinib	B-Cell Chronic Lymphocytic Leukemia	CytRx	June 2010	Phase 2
NCT01234740	Completed	Drug: bafetinib Procedure: microdialysis	Adult Anaplastic Astrocytoma Adult Anaplastic Ependymoma	City of Hope Medical Center	December 2010	Phase 1
NCT00352677	Completed	Drug: INNO-406	Chronic Myeloid Leukemia Acute Lymphocytic Leukemia	CytRx	July 2006	Phase 1

Biological Data

The tyrosine kinase inhibitor bafetinib blocks PAR2-TRPV4 coupling. (A) Bafetinib (1–10 μM) concentration dependently inhibited the sustained [Ca2+]i response to SLIGRL (30 μM), without affecting the peak response to SLIGRL or GSK1016790A (GSK, 30 nM). (B) Analysis showing concentration-dependent inhibition of the SLIGRL-induced coupling response with bafetinib in the TRPV4-transfected HEK293 cells. (C) 10 μM bafetinib inhibited the sustained [Ca2+]i response to trypsin, but did not affect the peak response to trypsin or the response to GSK1016790A. (D) 10 μM bafetinib inhibited trypsin-induced coupling in TRPV4 HEK cells compared with vehicle-treated (Veh) controls. Data are presented as mean ± SEM of n = 6–7 experiments.*P < 0.05, significantly different from NT HEK control. #P < 0.05, significantly different from vehicle-treated TRPV4 HEK control.

Bafetinib inhibits the expression of PD-L1 in vivo. (A) Tumor image of Balb/c mice treated with or without Bafetinib (30 mg/kg daily). (B) Tumor volume of Balb/c mice treated with or without Bafetinib (30 mg/kg daily). (C) The body weights of Balb/c mice were measured every other day. (D) Expression of PD-L1 in tumors of Balb/c mice. The relative protein level of PD-L1 in CT26 was quantitatively analyzed below. (E) Tumor volume of immunodeficient nude mice treated with or without Bafetinib (30 mg/kg daily). (F) The body weights of immunodeficient nude mice. (G) Expression of PD-L1 in tumors of immunodeficient nude mice. The relative protein level of PD-L1 in CT26 was quantitatively analyzed below. Bars, mean ± SEM (n = 6). *, p < 0.05. n. s: not significant. (H) H292 cells were treated with control Bafetinib (2.5 μM) or anti-PD-L1. T cells were isolated from peripheral blood and stimulated via anti-CD3/CD28/CD2. Co-incubation was carried out with these treated H292 cells for 8–12 h (T cells: Tumor cells = 5:1). After incubation, surviving cells were then fixed and stained with crystal violet. Sacle bar: 300 μm.

Bafetinib inhibits the expression of PD-L1 in lung cancer. (A) Screening on H292 cells treated with different small molecule drugs (10 μM) for 24 h. (B) The expression of PD-L1 protein was measured by Western blot in H292 cells, which were treated with Bafetinib (0.625, 1.25, and 2.5 μM) for 24 h. The relative protein level of PD-L1 in H292 was quantitatively analyzed on the right. (C) Expression of B7-H3, Galectin-9, PD-L1, and CD47 was measured by Western blot in H292 cells when treated with Bafetinib (2.5 μM) for 24 h (D, E) The expression of PD-L1 protein was measured by Western blot in H460, H358, PC9 cells, and primary lung cancer when treated with Bafetinib (0.625, 1.25, and 2.5 μM) for 24 h. (F) Surface PD-L1 expression on H292 treated with Bafetinib (0.625, 1.25, and 2.5 μM) was determined by flow cytometry. Cells were estimated for PD-L1 or mouse IgG control antibodies. Data were the mean ± SEM of quadruplicate experiments. The data were analyzed by one-way ANOVA with Dunnett’s post hoc test. ***, p < 0.001; *, p < 0.05.