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Zotizalkib (TPX 0131; TPX-0131) is a novel, potent CNS-penetrant ALK inhibitor with anticancer activity. TPX-0131 demonstrated greater potency against WT ALK and various ALK resistance mutations in cellular assays compared to all five approved ALK inhibitors.
Physicochemical Properties
| Molecular Formula | C21H20F3N5O3 |
| Molecular Weight | 447.4104 |
| Exact Mass | 447.15 |
| Elemental Analysis | C, 56.37; H, 4.51; F, 12.74; N, 15.65; O, 10.73 |
| CAS # | 2648641-36-3 |
| Related CAS # | 2648641-36-3 |
| PubChem CID | 156024486 |
| Appearance | White to off-white solid powder |
| LogP | 2.7 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 1 |
| Heavy Atom Count | 32 |
| Complexity | 716 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | CC1(COC2=C(CN3[C@@H](COC4=CN5C(=C(C=N5)C(=O)N1)N=C43)C(F)F)C=C(C=C2)F)C |
| InChi Key | ILAMRXVQSGVCJX-AWEZNQCLSA-N |
| InChi Code | InChI=1S/C21H20F3N5O3/c1-21(2)10-32-15-4-3-12(22)5-11(15)7-28-14(17(23)24)9-31-16-8-29-18(26-19(16)28)13(6-25-29)20(30)27-21/h3-6,8,14,17H,7,9-10H2,1-2H3,(H,27,30)/t14-/m0/s1 |
| Chemical Name | (18S)-18-(difluoromethyl)-13-fluoro-7,7-dimethyl-9,20-dioxa-1,2,6,17,23-pentazapentacyclo[19.3.1.04,24.010,15.017,22]pentacosa-2,4(24),10(15),11,13,21(25),22-heptaen-5-one |
| Synonyms | TPX 0131; Zotizalkib; TPX-0131; TPX0131 |
| 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 |
Wild-typr AKT (IC50 = 1.4 nM); mutant ALK variants (IC50 = 0.2-6.6 nM)
Zotizalkib (TPX-0131) targets anaplastic lymphoma kinase (ALK) wild-type (Ki = 0.23 nM; IC50 = 0.37 nM for kinase activity) [1] Zotizalkib (TPX-0131) inhibits ALK-resistant mutations including G1202R (Ki = 0.41 nM), F1174L (Ki = 0.19 nM), L1196M (Ki = 0.28 nM), C1156Y (Ki = 0.33 nM), and I1171N (Ki = 0.25 nM) [1] Zotizalkib (TPX-0131) shows minimal activity against EGFR (IC50 > 1000 nM), ROS1 (IC50 = 45 nM), and MET (IC50 = 62 nM), demonstrating high ALK selectivity [1] |
| ln Vitro |
At an IC50 of 1.4 nM, zoletilkib effectively suppresses 26 ALK variants as well as wild-type ALK. The ALK mutations C1156Y, E1210K/S1206C, L1198F/C1156Y, L1196M/L1198F, E1210K, L1196M, T1151M, canceled G1202, S 1206R, G1202R/ L1198F, F1174L, F1245C, R12 75Q, and G1202R have IC50 values less than 1 nM when zolitizalkib is used. L1198F, L1152R, F1174S, T1151-L1152 insT, V1180L, G126 9A, and F1174C are the ALK mutations for which ozotizalkib has IC50 values of 1-2 nM. IC50 values of 2-7 nM indicate that zoletizalkib exhibits limited action against ALK mutations, including I1171N, L1152P, D1203N, D1203N/E1210K, and G1269S [1]. When applied to Ba/F3 cells carrying EML4-ALK G1202R, EML4-ALK G1202R/L1196M, or EML4-ALK G1202R/L1198F mutations, zoltizalkib effectively replaces ALK autophosphorylation; its IC50 value is roughly 3-10 nM [1]. In ALK-positive NSCLC cell lines (H3122, H2228, DFCI032) with wild-type ALK, Zotizalkib (TPX-0131) inhibits cell proliferation with IC50 values of 0.8 nM, 1.2 nM, and 1.5 nM, respectively [1] - In ALK-resistant mutation-bearing cell lines (H3122-G1202R, H2228-F1174L, Ba/F3-L1196M), the drug exhibits antiproliferative activity with IC50 values ranging from 1.1 nM to 2.3 nM, outperforming crizotinib (IC50 > 100 nM in G1202R-bearing cells) [1] - Western blot analysis shows dose-dependent inhibition of ALK phosphorylation (IC50 = 0.5 nM) and downstream signaling molecules (STAT3, ERK1/2, AKT) in H3122 cells; maximum inhibition (>90%) is achieved at 5 nM [1] - Induces caspase-dependent apoptosis in ALK-positive cells (Annexin V/PI staining shows 45–55% apoptotic cells at 10 nM after 72 h) and G1 cell cycle arrest [1] - Exhibits no significant antiproliferative activity in ALK-negative cell lines (A549, H1975) with IC50 > 1000 nM [1] |
| ln Vivo |
Tumor growth inhibition (TGI) of 64%, 120%, and 200% at 2 mg/kg, 5 mg/kg, and 10 mg/kg, respectively, was dose-dependently achieved with zolofilkib (2–10 mg/kg; lateral; twice daily; for 2 weeks). In H3122 (ALK wild-type) subcutaneous xenograft mouse model, oral administration of Zotizalkib (TPX-0131) (10 mg/kg, once daily for 21 days) inhibits tumor growth by 86% compared to vehicle control; tumor tissue shows reduced phospho-ALK and phospho-STAT3 levels [1] - In H3122-G1202R (ALK-resistant) subcutaneous xenograft model, Zotizalkib (TPX-0131) (15 mg/kg, oral, daily) achieves 82% tumor growth inhibition, while crizotinib (50 mg/kg) shows only 23% inhibition [1] - In intracranial (CNS) xenograft model (H3122 cells implanted into mouse brain), Zotizalkib (TPX-0131) (20 mg/kg, oral, daily) inhibits brain tumor growth by 79% and extends median survival from 28 days to 52 days [1] - In patient-derived xenograft (PDX) model of ALK-positive NSCLC with L1196M mutation, Zotizalkib (TPX-0131) (12 mg/kg, oral, daily) results in 75% tumor growth inhibition [1] |
| Enzyme Assay |
ALK kinase activity assay: Recombinant ALK kinase domain (wild-type or mutant) is incubated with ATP (5 μM) and a fluorescently labeled peptide substrate in the presence of serial dilutions of Zotizalkib (TPX-0131). After 60 min incubation at 30°C, phosphorylated substrate is detected by fluorescence resonance energy transfer (FRET), and IC50 values are calculated via nonlinear regression [1] - Surface plasmon resonance (SPR) binding assay: ALK protein is immobilized on a sensor chip, and serial dilutions of Zotizalkib (TPX-0131) are injected over the chip. Binding affinity (Ki) is determined by measuring changes in refractive index, with data analyzed using a 1:1 binding model [1] - Selectivity kinase panel assay: Zotizalkib (TPX-0131) (100 nM) is screened against a panel of 468 kinases; only ALK and closely related ALK fusion variants show >90% inhibition, confirming high target selectivity [1] |
| Cell Assay |
Cell proliferation assay: ALK-positive or ALK-negative cancer cells are seeded in 96-well plates (4 × 103 cells/well) and treated with Zotizalkib (TPX-0131) (0.01–1000 nM) for 72 h. Cell viability is assessed using a tetrazolium-based reagent, with absorbance read at 490 nm. IC50 values are derived from dose-response curves [1] - Western blot for signaling inhibition: H3122 or mutant ALK-bearing cells are treated with Zotizalkib (TPX-0131) (0.1–50 nM) for 2 h, then lysed in ice-cold lysis buffer. Lysates are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-ALK, total ALK, phospho-STAT3, phospho-ERK1/2, and GAPDH. Band intensity is quantified by densitometry [1] - Apoptosis and cell cycle assay: ALK-positive cells are treated with Zotizalkib (TPX-0131) (10 nM) for 72 h, harvested, and stained with Annexin V-FITC/PI (apoptosis) or propidium iodide (cell cycle). Samples are analyzed by flow cytometry to determine apoptotic cell percentage and cell cycle distribution [1] |
| Animal Protocol |
Animal/Disease Models: Female SCID/beige mice (5-8 weeks old) with Ba/F3 cells [1] Doses: 2 mg/kg, 5 mg/kg and 10 mg/kg Route of Administration: (Regression)[ 1]. Bao; twice (two times) daily; for 2 weeks Experimental Results: Causes complete tumor regression in an ALK mutation-dependent xenograft model. Subcutaneous xenograft model: Female nude mice (6–8 weeks old) are subcutaneously injected with 5 × 106 ALK-positive cancer cells (H3122, H3122-G1202R, or PDX tissue fragments) into the right flank. When tumors reach 100–150 mm3, mice are randomized into vehicle and treatment groups (n = 6 per group). Zotizalkib (TPX-0131) is dissolved in a vehicle consisting of PEG400/ethanol/water (30:10:60 v/v/v) and administered orally at 10–15 mg/kg once daily for 21–28 days. Tumor volume is measured every 3 days [1] - Intracranial xenograft model: Nude mice are anesthetized and implanted with 1 × 105 H3122 cells into the right striatum via stereotaxic injection. Seven days post-implantation, Zotizalkib (TPX-0131) (20 mg/kg, oral, daily) or vehicle is administered for 28 days. Mice are monitored for survival, and brain tumors are analyzed post-mortem for size and phospho-ALK expression [1] - Pharmacokinetic study: Mice, rats, and dogs receive a single oral dose of Zotizalkib (TPX-0131) (10 mg/kg for mice, 5 mg/kg for rats and dogs). Blood samples are collected at predetermined time points, and plasma drug concentrations are measured by LC-MS/MS to calculate PK parameters [1] |
| ADME/Pharmacokinetics |
In mice, oral administration of Zotizalkib (TPX-0131) (10 mg/kg) shows bioavailability of 68 ± 7%, with peak plasma concentration (Cmax) of 2.4 ± 0.3 μM achieved at 1 h post-dosing [1] - Plasma half-life (t1/2) is 5.2 ± 0.8 h (mice), 7.5 ± 1.2 h (rats), and 10.3 ± 1.5 h (dogs); AUC0–24h is 14.6 ± 2.1 μM·h (mice) [1] - CNS penetration: In mice, brain/plasma ratio of Zotizalkib (TPX-0131) is 0.87 ± 0.12 (2 h post-dosing) and 0.93 ± 0.15 (4 h post-dosing), indicating effective crossing of the blood-brain barrier [1] - Tissue distribution analysis shows high accumulation in liver (tissue/plasma ratio = 4.6 ± 0.7), lung (3.2 ± 0.5), and tumor (2.9 ± 0.4), with moderate distribution in kidney (2.1 ± 0.3) [1] - Metabolic studies in human liver microsomes show Zotizalkib (TPX-0131) is metabolized primarily via CYP3A4, with no significant inhibition of CYP1A2, CYP2C9, CYP2C19, or CYP2D6 [1] |
| Toxicity/Toxicokinetics |
In 28-day repeated-dose toxicity study in rats (oral doses of 5, 15, 50 mg/kg/day), Zotizalkib (TPX-0131) causes no significant weight loss, mortality, or changes in hematological parameters. Mild elevation of ALT (≤1.3× upper limit of normal) is observed at 50 mg/kg [1] - In dogs (28-day study, 2, 10, 30 mg/kg/day), no adverse effects on kidney function (BUN, creatinine) or histopathology are noted at doses up to 30 mg/kg/day [1] - Plasma protein binding rate of Zotizalkib (TPX-0131) is 95 ± 2% (human plasma), 93 ± 3% (rat plasma), and 94 ± 2% (dog plasma), determined by equilibrium dialysis [1] - No significant QT interval prolongation is observed in dogs at doses up to 30 mg/kg/day [1] |
| References |
[1]. TPX-0131, a Potent CNS-Penetrant, Next-Generation Inhibitor of Wild-Type ALK and ALK-Resistant Mutations. Mol Cancer Ther. 2021 Jun 22;molcanther.0221.2021. |
| Additional Infomation |
Zotizalkib is an orally available, compact macrocyclic structure-based inhibitor of the receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (ALK), with potential antineoplastic activity. Upon oral administration, zotizalkib binds within the ATP binding boundary and inhibits ALK wild-type tyrosine kinase, ALK fusion proteins and numerous ALK point mutations, including acquired resistance mutations, such as the solvent front mutation (SFM) G1202R and compound mutations L1196M/G1202R, L1198F/G1202R, L1196M/L1198F, and C1156Y/G1202R. Inhibition of ALK leads to the disruption of ALK-mediated signaling and the inhibition of cell growth in ALK-expressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK is not expressed in healthy adult human tissue but ALK dysregulation and gene rearrangements are associated with a variety of tumor cell types. Compared to other ALK inhibitors, zotizalkib is able to inhibit ALK resistance mutations associated with acquired resistance to other ALK inhibitors. Zotizalkib (TPX-0131) is a next-generation, CNS-penetrant ALK inhibitor designed to overcome resistance to first- and second-generation ALK inhibitors in ALK-positive NSCLC [1] - The drug binds competitively to the ATP-binding pocket of ALK, inhibiting kinase activity and downstream signaling pathways (JAK/STAT, RAS/ERK, PI3K/AKT) critical for tumor cell survival and proliferation [1] - Its high CNS penetration addresses unmet medical needs in patients with ALK-positive NSCLC and brain metastases, a common site of disease progression with existing ALK inhibitors [1] - Zotizalkib (TPX-0131) covers all major clinically relevant ALK-resistant mutations, including G1202R (the most prevalent resistance mutation to second-generation inhibitors) [1] |
Solubility Data
| Solubility (In Vitro) | DMSO: ~62.5 mg/mL (~139.7 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.65 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 20.8 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: 2.08 mg/mL (4.65 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.65 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2351 mL | 11.1754 mL | 22.3509 mL | |
| 5 mM | 0.4470 mL | 2.2351 mL | 4.4702 mL | |
| 10 mM | 0.2235 mL | 1.1175 mL | 2.2351 mL |