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Theliatinib (formerly HMPL-309; HMPL309; HMPL 309; xiliertinibum; xiliertinib) is a novel and highly potent EGFR-TKI (epidermal growth factor receptor-tyrosine kinase inhibitor) with potential antitumor and anti-angiogenesis activities. It inhibits EGFR with IC50 values of 3 nM and 22 nM against EGFR and EGFR T790M/L858R mutant, and a Ki value of 0.05 nM against EGFR of the wild typ
Physicochemical Properties
| Molecular Formula | C25H26N6O2 |
| Molecular Weight | 442.512944698334 |
| Exact Mass | 442.21 |
| Elemental Analysis | C, 67.86; H, 5.92; N, 18.99; O, 7.23 |
| CAS # | 1353644-70-8 |
| Related CAS # | Theliatinib tartrate;2413487-72-4 |
| PubChem CID | 54759275 |
| Appearance | White to off-white solid powder |
| LogP | 3.1 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 33 |
| Complexity | 754 |
| Defined Atom Stereocenter Count | 2 |
| SMILES | CN1CC[C@H]2[C@@H]1CN(C2)C(=O)NC3=C(C=C4C(=C3)C(=NC=N4)NC5=CC=CC(=C5)C#C)OC |
| InChi Key | FSXCKIBROURMFT-VGSWGCGISA-N |
| InChi Code | InChI=1S/C25H26N6O2/c1-4-16-6-5-7-18(10-16)28-24-19-11-21(23(33-3)12-20(19)26-15-27-24)29-25(32)31-13-17-8-9-30(2)22(17)14-31/h1,5-7,10-12,15,17,22H,8-9,13-14H2,2-3H3,(H,29,32)(H,26,27,28)/t17-,22+/m1/s1 |
| Chemical Name | (3aR,6aR)-N-[4-(3-ethynylanilino)-7-methoxyquinazolin-6-yl]-1-methyl-2,3,3a,4,6,6a-hexahydropyrrolo[2,3-c]pyrrole-5-carboxamide |
| Synonyms | Theliatinib; HMPL309; HMPL 309; HMP-L309; xiliertinibum; xiliertinib |
| 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 |
EGFR (IC50 = 3 nM); EGFR (Ki = 0.05 nM); EGFR (L858R/T790M) (IC50 = 22 nM) Theliatinib exhibits dose-dependent anti-tumor activity in a panel of PDECX (patient-derived esophageal cancer xenograft) models with an overall strong correlation between EGFR H score and tumor growth inhibition. Additionally, the anti-tumor activity of EGFR TKIs, particularly theliatinib, is reduced by aberrant activation or gene mutations of other targets, including PI3K and FGFR[1]. |
| ln Vitro |
Theliatinib exhibits dose-dependent anti-tumor activity in a panel of PDECX (patient-derived esophageal cancer xenograft) models with an overall strong correlation between EGFR H score and tumor growth inhibition. Additionally, the anti-tumor activity of EGFR TKIs, particularly theliatinib, is reduced by aberrant activation or gene mutations of other targets, including PI3K and FGFR[1]. Theliatinib inhibits EGF-stimulated EGFR phosphorylation in A431 cells with an IC₅₀ of 0.007 ± 0.002 μM. It inhibits the proliferation/survival of A431 epidermoid carcinoma cells (IC₅₀ = 0.8 μM), H292 lung cancer cells (IC₅₀ = 0.058 μM), and FaDu pharynx cancer cells (IC₅₀ = 0.354 μM). [1] Theliatinib exhibits 3–7 fold greater potency than erlotinib or gefitinib at both the enzyme and cellular levels. [1] |
| ln Vivo |
At the conclusion of the trial, theliatinib (2–15 mg/kg; PO; daily; for 21 days; NOD-SCID mice; PDECX 1T0950 model) treatment showed 75% tumor shrinkage with a dosage response [1]. Theliatinib demonstrated potent dose-dependent antitumor activity in patient-derived esophageal cancer xenograft (PDECX) models with high EGFR expression. In two PDECX models with both EGFR gene amplification and high protein expression (PDECX1T0326, H score=290; PDECX1T0950, H score=280), theliatinib at 15 mg/kg/day induced tumor regression (31.6% and 75.3%, respectively). In PDECX models with high EGFR protein expression but without gene amplification (e.g., PDECX1T0781, H score=270; PDECX1T1315, H score=295), theliatinib at 15 mg/kg/day achieved 91.8%-95.9% tumor growth inhibition (TGI). Efficacy was attenuated in models with concurrent high EGFR expression and PIK3CA mutation (e.g., PDECX1T0472) or FGFR1 overexpression (e.g., PDECX1T0327). Moderate efficacy was observed in a model with medium EGFR expression (PDECX1T0474, H score=180; 63.6% TGI), and minimal effect was seen in a model with low EGFR expression (PDECX1T0773, H score=15). In comparative studies, 15 mg/kg/day theliatinib was significantly more effective than 20 mg/kg/day gefitinib in most models. [1] |
| Enzyme Assay |
EGFR kinase inhibition was determined using a fluorescence-based kinase assay kit. Recombinant EGFR kinase was dissolved in a reaction buffer containing HEPES, MgCl₂, and EGTA. The final reaction mixture contained the kinase, a tyrosine-containing substrate peptide, ATP at various concentrations (10–1000 μM), and the test compound. The reaction was incubated at 25°C for 60 minutes in a 384-well plate. A development reagent was then added, and after another incubation, fluorescence was measured at excitation 400 nm and emission 445/520 nm. The Ki values for theliatinib, gefitinib, and erlotinib were calculated using the Michaelis-Menten equation in GraphPad Prism software. [1] |
| Cell Assay |
One overnight incubation at 37°C and 5% CO2 is followed by the seeding of duplicate A431 cells (1 × 104 cells/well) in exponential phase in DMEM containing 10% FBS. Following that, 10 μL of test compounds (erlotinib, gefitinib, and theliatinib) at tested concentrations (10~0.005 μM, threefold gradient dilution) are added to each well, with 0.5% DMSO as the final concentration. Add 10 μL/well CCK-8 solution and incubate the cells for an additional hour after 48 hours. We measure the optical density at 450 nm to determine cell survival. For EGFR phosphorylation inhibition, A431 cells were seeded, serum-starved for 24 hours, and then treated with serially diluted compounds for 60 minutes. Cells were stimulated with recombinant human EGF (20 ng/mL) for 45 minutes, then lysed. Cell lysates were transferred to assay plates pre-coated with an anti-EGFR capture antibody. Phosphorylated EGFR was detected using a europium-labeled anti-phospho-tyrosine antibody and a fluorescence enhancement solution, with fluorescence measured at 620 nm emission (excitation 340 nm). For the cell survival assay, A431, H292, or FaDu cells were seeded, treated with compounds for 48 hours, and then cell viability was assessed using a tetrazolium-based reagent (CCK-8). Optical density was measured at 450 nm. [1] |
| Animal Protocol |
Animal/Disease Models: NOD-SCID (severe combined immunodeficient) mouse injected with esophageal cancer cells (PDECX 1T0950 model) [1] Doses: 2 mg/kg, 5 mg/kg, 15 mg/kg Route of Administration: po (po (oral gavage)) daily; one time/day; Results lasting 21 days: Tumor growth was attenuated in a dose-dependent manner in the PDECX 1T0950 model. The antitumor efficacy of theliatinib was evaluated in female or male BALB/c nude or NOD-SCID mice bearing patient-derived esophageal cancer xenografts (PDECX). When tumor volumes reached 250–500 mm³, mice were randomized into groups. Theliatinib was suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) and administered orally (p.o.) once daily (qd) at doses of 2, 5, or 15 mg/kg. Treatment duration varied by model (up to 24 days). Tumor volumes were measured regularly, and tumor growth inhibition (TGI) was calculated. [1] |
| References |
[1]. Anti-tumor efficacy of theliatinib in esophageal cancer patient-derived xenografts models with epidermal growth factor receptor (EGFR) overexpression and gene amplification. Oncotarget. 2017 Apr 19;8(31):50832-50844. |
| Additional Infomation |
Xiliertinib is an orally available, ATP-competitive inhibitor of the epidermal growth factor receptor (EGFR), with potential antineoplastic activity. Upon oral administration, xiliertinib binds to and inhibits the activity of EGFR. This prevents EGFR-mediated signaling, and may lead to both induction of cell death and inhibition of tumor growth in EGFR-overexpressing cells. EGFR, a receptor tyrosine kinase mutated in many tumor cell types, plays a key role in tumor cell proliferation and tumor vascularization. Theliatinib is a novel EGFR tyrosine kinase inhibitor under investigation in a Phase I clinical trial (NCT02601248). Its chemical name is (3aR,6aR)-N-(4-(3-ethylnylphenylamino)-7-methoxyquinazolin-6-yl)-1-methyl-hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxamide. The study proposes that esophageal cancer patients with high EGFR protein expression (IHC H score ≥ 270), with or without EGFR gene amplification, may benefit from theliatinib treatment. [1] |
Solubility Data
| Solubility (In Vitro) | DMSO: 1~5 mg/mL (2.3~11.3 mM) |
| 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 | 2.2598 mL | 11.2992 mL | 22.5984 mL | |
| 5 mM | 0.4520 mL | 2.2598 mL | 4.5197 mL | |
| 10 mM | 0.2260 mL | 1.1299 mL | 2.2598 mL |