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Purity: ≥98%
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, andcompletely inhibited tumor growth without causing adverse effects at a dose of 20mg/kg/day.
| Targets |
Abl (IC50 5.8 nM in an ELISA-based kinase assay; IC50 72 nM in purified Abl kinase domain assay); Lyn (IC50 19 nM); Src (IC50 1700 nM); Fyn (inhibited at 0.1 μM, exact IC50 not reported); Arg (inhibited at 0.1 μM); wild-type Bcr-Abl (cellular IC50 11 nM in K562, 22 nM in 293T, 63 nM in BaF3/wt for autophosphorylation); Bcr-Abl E255K mutant (cellular IC50 98 nM in 293T, 340 nM in BaF3); Bcr-Abl mutants M244V, G250E, Q252H, Y253F, E255K, E255V, F317L, M351T, E355G, F359V, H396P, F486S (in vitro IC50 ranging from 81 nM to 1400 nM, see Table 2); no inhibition of T315I (>10,000 nM). [1]
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| 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. NS-187 blocks autophosphorylation of wild-type Bcr-Abl in K562 cells (IC50 11 nM) and in 293T cells (IC50 22 nM), 25 and 55 times more potent than imatinib respectively. [1] It inhibits phosphorylation of CrkL and ERK in K562 and BaF3/wt cells at much lower concentrations than imatinib. [1] For Bcr-Abl E255K mutant, NS-187 inhibits autophosphorylation in BaF3/E255K cells (IC50 340 nM) and blocks CrkL/ERK phosphorylation, while imatinib is ineffective. [1] No inhibition of T315I mutant autophosphorylation or CrkL/ERK in BaF3/T315I cells (IC50 >10,000 nM). [1] NS-187 suppresses proliferation of Bcr-Abl-positive cell lines K562 (more potent than imatinib, exact IC50 not given), KU812 (more potent than imatinib), BaF3/wt (more potent), and BaF3/E255K (concentration-dependent, more potent than imatinib), but has no effect on Bcr-Abl-negative U937 cells or BaF3/T315I cells (up to 10 μM). [1] It inhibits PDGFR and c-Kit phosphorylation with IC50 similar to imatinib (PDGFR IC50 56 nM, c-Kit IC50 840 nM in cell-based assays), but does not inhibit EGFR at clinically relevant concentrations (IC50 >10,000 nM). [1] |
| 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.
In a KU812 subcutaneous xenograft model in Balb/c-nu/nu mice, oral NS-187 at 0.2 mg/kg/d (bid) significantly inhibited tumor growth, and at 20 mg/kg/d completely inhibited tumor growth without adverse effects; imatinib required 200 mg/kg/d for complete inhibition. NS-187 was at least 10-fold more potent than imatinib. [1] In a BaF3/wt intravenous leukemia model, NS-187 (6-200 mg/kg/d, oral bid for 11 days) prolonged survival in a dose-dependent manner compared to vehicle; imatinib at 400 mg/kg/d had little effect (all mice died by day 25). [1] In a BaF3/E255K (imatinib-resistant mutant) intravenous model, NS-187 at 120 mg/kg/d (oral bid for 26 days) significantly prolonged survival compared to vehicle and imatinib (200 mg/kg/d). [1] |
| Enzyme Assay |
Purified Abl kinase domains (wild-type and 13 point mutants, amino acids 229-515) were generated using baculovirus expression in Sf9 cells and purified on Q-Sepharose and HisTrap columns. Kinase assays were performed in 25 μL reaction mixture containing 250 μM peptide substrate, [γ-33P]ATP (740 Bq/μL), and 20 μM cold ATP using the SignaTECT protein tyrosine kinase assay system. Each Bcr-Abl kinase was used at 10 nM. [1]
Kinase assays for Abl, Src, and Lyn were carried out with an ELISA kit. [1] The inhibitory effects of NS-187 against 79 tyrosine kinases were tested with KinaseProfiler. At 0.1 μM, NS-187 inhibited Abl, Arg, Fyn, and Lyn. [1] |
| Cell Assay |
Cell lines (K562, BaF3/wt, BaF3/E255K, BaF3/T315I, KU812, U937, NHDF, NCI-H526, A431) were cultured in RPMI 1640 or DMEM with 10% FBS, 2 mM L-glutamine, and appropriate supplements at 37°C, 5% CO2. For inhibition of intracellular tyrosine phosphorylation, cells were treated with serial dilutions of compounds for 1.5 hours. Serum-starved NHDF, NCI-H526, and A431 cells were then stimulated with PDGF (50 ng/mL), SCF (100 ng/mL), or EGF (100 ng/mL) for 10 minutes. Cells were lysed with RIPA buffer, and equal amounts of lysate protein were analyzed by Western blotting using antiphosphotyrosine antibody (PY20-HRP), and antibodies against c-Abl, c-Kit, CrkL, EGFR, ERK1, or PDGFR. [1]
For cell proliferation assays, cells were plated in triplicate in 96-well plates (1×10^3 or 5×10^3 cells/well) and incubated with serial dilutions of compounds for 3 days. Cell proliferation was measured by MTT assay, and IC50 values were calculated by fitting data to a logistic curve. [1] |
| 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.
For subcutaneous xenograft model: Balb/c-nu/nu female mice (8 weeks old) were injected subcutaneously with 2.5×10^7 KU812 cells into the right flank. Seven days after inoculation, mice were randomized into groups of 5, and NS-187, imatinib, or vehicle (0.5% methylcellulose) was administered orally twice a day by gavage for 10 consecutive days. Tumor sizes were measured at least twice a week by caliper and calculated as (d^2 × D)/2/1000 cm^3. [1] For BaF3/wt leukemia model: Balb/c-nu/nu mice were injected intravenously with 1×10^6 BaF3/wt cells via tail vein. The next day, mice were randomized into groups of 7, and compounds or vehicle were administered orally twice a day for 11 consecutive days. Survival was analyzed by Kaplan-Meier method with log-rank test. [1] For BaF3/E255K leukemia model: Balb/c mice were injected intravenously with 5×10^4 BaF3/E255K cells. The next day, mice were randomized into groups of 3, and compounds or vehicle were administered orally twice a day for 26 consecutive days. Survival analysis as above. [1] |
| ADME/Pharmacokinetics |
In preliminary pharmacokinetic studies in Balb/c mice given oral NS-187 at 30 mg/kg: Tmax = 2 hours; Cmax = 661 ng/mL; AUC0-∞ = 2294 ng·h/mL; T1/2 = 1.0 hour; bioavailability (BA) = 32%. [1]
The maximum tolerated dose (MTD) of NS-187 in Balb/c or Balb/c-nu/nu mice was 200 mg/kg/d (100 mg/kg twice a day). At this dose, estimated Cmax was 2226 ng/mL (4.0 μM). [1] |
| Toxicity/Toxicokinetics |
NS-187 was well tolerated in mice. At doses up to 200 mg/kg/d (100 mg/kg bid), body weights of treated tumor-bearing mice were not significantly different from untreated mice [1]
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| References | |
| Additional Infomation |
Bafetinib is a biaryl compound. Bafetinib has been used in clinical trials to treat various cancers, including adult glioma, adult mixed glioma, adult glioblastoma, chronic myeloid leukemia, and acute lymphoblastic leukemia. Bafetinib is an orally effective 2-phenylaminopyrimidine derivative with potential antitumor activity. INNO-406 specifically binds to and inhibits the Bcr/Abl fusion protein tyrosine kinase, an abnormal enzyme resulting from the Philadelphia chromosome translocation associated with chronic myeloid leukemia (CML). Furthermore, the drug also inhibits the Src family member Lyn tyrosine kinase, which is upregulated in imatinib-resistant CML cells and various solid tumor cells. The inhibition of these specific tyrosine kinases by INNO-406 reduces cell proliferation and induces apoptosis. A significant proportion of CML patients are resistant to imatinib, sometimes due to point mutations in the Bcr/Abl fusion protein kinase domain. INNO-406 has dual inhibitory activity, has been shown to overcome this resistance, and is a potent drug for treating imatinib-resistant CML.
NS-187 is a 2-phenylaminopyrimidine-class compound. It binds to Abl with a similar mode as imatinib, stabilized by hydrogen bonds with Met318, Thr315, Glu286, and Asp381. The compound inhibits Lyn but not Src or Yes due to the presence of Gln252 (vs Cys252 in Src/Yes), which facilitates hydrogen bonding and induced fit of the P-loop. [1] NS-187 does not inhibit the T315I Bcr-Abl mutant because the mutation eliminates a crucial hydrogen bond and sterically blocks the binding site. [1] It has potential as a novel agent for imatinib-resistant Philadelphia chromosome-positive leukemias (CML and Ph+ ALL). [1] |
| Molecular Formula |
C30H31F3N8O
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|---|---|
| Molecular Weight |
576.62
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| Exact Mass |
576.257
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| CAS # |
859212-16-1
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| Related CAS # |
859212-16-1;887650-05-7;
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| PubChem CID |
11387605
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Melting Point |
166-168°C
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| Index of Refraction |
1.640
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| LogP |
3.03
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
42
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| Complexity |
872
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC1=C(C=C(C=C1)NC(=O)C2=CC(=C(C=C2)CN3CC[C@@H](C3)N(C)C)C(F)(F)F)NC4=NC=CC(=N4)C5=CN=CN=C5
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| InChi Key |
ZGBAJMQHJDFTQJ-DEOSSOPVSA-N
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| 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
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| Chemical Name |
(S)-N-(3-([4,5'-bipyrimidin]-2-ylamino)-4-methylphenyl)-4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)benzamide
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| Synonyms |
INNO-406; INNO 406; NS187; NS187; INNO406;NS-187; NS 187
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
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| 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. View More
Solubility in Formulation 3: 0.5% methylcellulose+0.2% Tween 80: 30 mg/mL |
| 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.
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.
(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.
| 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 |
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