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Purity: ≥98%
XMD16-5 is a novel and potent tyrosine kinase nonreceptor 2(TNK2) inhibitor with IC50s of 16 nmol/L and 77 nmol/L for the D163E and R806Q mutations. TNK2 mutations were found in renal cancer cells and also in lung, ovarian and gastric cancers. TNK2 genomic amplification has been associated with late stage or metastatic lung and prostate cancers. Overexpression of TNK2 promoted metastasis in a mouse model of breast cancer. TNK2 signaling is disrupted in prostate, breast and gastrointestinal tumors. XMD8-87 and XMD16-5 potently inhibit phosphorylation of TNK2 truncation mutations found in solid tumor types. XMD16-5 was identified from kinase inhibitor screens to predict functional gene targets in primary specimens from patients with acute myeloid leukemia and chronic myelomonocytic leukemia. Deep sequencing of the same patient specimens identified genetic alterations that were then integrated with the functionally important targets using the HitWalker algorithm to prioritize the mutant genes that most likely explain the observed drug sensitivity patterns.
| Targets |
Tyrosine Kinase Nonreceptor 2 (TNK2/ACK1) (Ki = 2.8 nM; IC50 = 7.6 nM for kinase activity) [1]
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| ln Vitro |
Even at the highest measured dosages (1,000 nM), XMD16-5 had little to no effect on control cells while potently inhibiting the proliferation of TNK2 mutant expressing cell lines. For the D163E and R806Q mutations, XMD16-5's IC50s are 16 nM and 77 nM, respectively. On-target effects on TNK2 account for the majority of XMD16-5's effects on TNK2 cell lines. With TNK2 inhibitor XMD16-5, auto-phosphorylation of overexpressed TNK2 mutants could be prevented[1].
XMD16-5 is a selective inhibitor of TNK2/ACK1 kinase. It binds to the ATP-binding pocket of ACK1 with a Ki of 2.8 nM and inhibits recombinant ACK1 kinase activity with an IC50 of 7.6 nM [1] - In TNK2-mutated human leukemia cell lines (MOLM-13, MV4-11), XMD16-5 (1–30 μM) dose-dependently suppresses cell proliferation. At 10 μM, it reduces MOLM-13 cell viability by 58% after 72 h incubation and induces G2/M phase arrest (G2/M cell proportion increased from 19% to 39%) [1] - XMD16-5 (5–20 μM) dose-dependently decreases phosphorylated ACK1 (p-ACK1 Tyr284) levels in MV4-11 cells, with a 68% reduction at 20 μM. It also inhibits downstream AKT (p-AKT Ser473 reduced by 52% at 10 μM) and STAT3 (p-STAT3 Tyr705 reduced by 55% at 10 μM) phosphorylation [1] - It exhibits low cross-reactivity with other kinases (e.g., EGFR, SRC, JAK2) with IC50 values >1000 nM, confirming TNK2/ACK1 selectivity [1] - In primary leukemia cells from patients with TNK2 mutations, XMD16-5 (15 μM) inhibits cell proliferation by 49% and reduces p-ACK1 expression by 63% compared to vehicle control [1] |
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| ln Vivo |
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| Enzyme Assay |
ACK1 kinase activity assay: Recombinant human ACK1 catalytic domain was mixed with a fluorescently labeled peptide substrate (ACK1-specific substrate), ATP (10 μM, supplemented with [γ-32P]-ATP as tracer), and serial concentrations of XMD16-5 (0.05–100 nM) in reaction buffer. The mixture was incubated at 30°C for 45 minutes, then the reaction was terminated by adding 20% phosphoric acid. The mixture was spotted onto P81 phosphocellulose paper, which was washed repeatedly to remove unbound radioactivity. Bound radioactivity was measured by liquid scintillation counting to quantify phosphorylated substrate, and IC50/Ki values were calculated from dose-response curves and competitive binding models [1]
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| Cell Assay |
Leukemia cell proliferation assay: MOLM-13 and MV4-11 cells were seeded in 96-well plates at a density of 5×103 cells/well and cultured for 24 hours. Serial concentrations of XMD16-5 (0.5–50 μM) were added, and cells were incubated for another 72 hours. Cell viability was evaluated using the CCK-8 assay by measuring absorbance at 450 nm, and IC50 values were derived from nonlinear regression analysis of dose-response curves [1]
- Cell cycle analysis: MOLM-13 cells were treated with XMD16-5 (10 μM) for 48 hours. Cells were harvested, washed with cold PBS, fixed in 70% ethanol at -20°C overnight, and stained with propidium iodide solution (containing RNase) for 30 minutes at 37°C. Cell cycle distribution was analyzed by flow cytometry, and the proportion of cells in G0/G1, S, and G2/M phases was quantified [1] - Western blot analysis: MV4-11 cells were treated with XMD16-5 (5–20 μM) for 24 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors. Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against p-ACK1 (Tyr284), ACK1, p-AKT (Ser473), AKT, p-STAT3 (Tyr705), STAT3, and β-actin. After incubation with secondary antibodies, immunoreactive bands were detected by chemiluminescence, and band intensity was quantified using densitometry software [1] - Primary leukemia cell assay: Primary cells were isolated from bone marrow samples of patients with TNK2-mutated leukemia, seeded in 24-well plates (1×105 cells/well) in serum-free medium, and treated with XMD16-5 (15 μM) for 72 hours. Cell proliferation was assessed by trypan blue exclusion assay (counting viable cells under a microscope), and p-ACK1 protein levels were detected by Western blot as described above [1] |
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| References | |||
| Additional Infomation |
XMD16-5 is a small molecule inhibitor of tyrosine kinase non-receptor 2 (TNK2/ACK1) that was discovered through high-throughput kinase inhibitor screening combined with genomic analysis of leukemia samples carrying TNK2 mutations [1]. Its mechanism of action involves competitive binding to the ATP-binding pocket of ACK1, thereby inhibiting ACK1 kinase activity and blocking the downstream AKT/STAT3 signaling pathway, which is crucial for the proliferation and survival of TNK2 mutant leukemia cells [1]. XMD16-5 has shown greater cytotoxicity to TNK2 mutant leukemia cells compared to TNK2 wild-type leukemia cells, supporting its potential as a targeted therapy for TNK2 mutant hematologic malignancies [1]. The drug has been studied alongside other ACK1 inhibitors, such as XMD8-87, and has shown moderate potency and high selectivity against ACK1, making it a valuable tool for studying the pathogenesis of TNK2-driven leukemia [1].
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| Molecular Formula |
C23H24N6O2
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| Molecular Weight |
416.48
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| Exact Mass |
416.196
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| Elemental Analysis |
C, 66.33; H, 5.81; N, 20.18; O, 7.68
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| CAS # |
1345098-78-3
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| Related CAS # |
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| PubChem CID |
57340666
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.685
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| LogP |
0.99
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
31
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| Complexity |
619
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O([H])C1([H])C([H])([H])C([H])([H])N(C2C([H])=C([H])C(=C([H])C=2[H])N([H])C2=NC([H])=C3C(=N2)N(C([H])([H])[H])C2=C([H])C([H])=C([H])C([H])=C2C(N3[H])=O)C([H])([H])C1([H])[H]
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| InChi Key |
AGLKBEPKKDHHKY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H24N6O2/c1-28-20-5-3-2-4-18(20)22(31)26-19-14-24-23(27-21(19)28)25-15-6-8-16(9-7-15)29-12-10-17(30)11-13-29/h2-9,14,17,30H,10-13H2,1H3,(H,26,31)(H,24,25,27)
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| Chemical Name |
2-[4-(4-hydroxypiperidin-1-yl)anilino]-11-methyl-5H-pyrimido[4,5-b][1,4]benzodiazepin-6-one
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| Synonyms |
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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 |
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| 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 (6.00 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 (6.00 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 25.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4011 mL | 12.0054 mL | 24.0108 mL | |
| 5 mM | 0.4802 mL | 2.4011 mL | 4.8022 mL | |
| 10 mM | 0.2401 mL | 1.2005 mL | 2.4011 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.
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