Enbezotinib, also known as TPX-0046, is a novel and potent RET/SRC inhibitor for treating RET-driven cancers. In enzymatic assays, TPX-0046 exhibited low nanomolar potency against WT and many mutant RET and SRC and did not retain VEGFR2. TPX-0046 potently inhibits RET phosphorylation and cell proliferation/growth in internally engineered Ba/F3 KIF5B-RET, TT and LC2/ad cells with IC50 of approximately 1 nM. TPX-0046 was effective against SFM G810R in the Ba/F3 cell proliferation/growth assay with an average IC50 of 17 nM, while similar agents BLU-667 and LOXO-292 had IC50 > 500 nM. TPX-0046 demonstrated significant in vivo anti-tumor efficacy in multiple RET-driven cancer cell-derived and patient-derived xenograft tumor models.

Physicochemical Properties

Molecular Formula	C21H21FN6O3
Molecular Weight	424.43
Exact Mass	424.17
Elemental Analysis	C, 59.43; H, 4.99; F, 4.48; N, 19.80; O, 11.31
CAS #	2359649-81-1
Related CAS #	Enbezotinib (enantiomer);2359650-19-2
PubChem CID	146662764
Appearance	White to off-white solid powder
LogP	1.8
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	0
Heavy Atom Count	31
Complexity	702
Defined Atom Stereocenter Count	3
SMILES	O1C2=NC=C(F)C=C2CN2[C@]3([H])CCC[C@]3([H])OC3=CN4C(N=C23)=C(C=N4)C(=O)NC[C@@H]1C
InChi Key	BYYQDEOVMILBQT-XZJROXQQSA-N
InChi Code	InChI=1S/C21H21FN6O3/c1-11-6-23-20(29)14-8-25-28-10-17-19(26-18(14)28)27(15-3-2-4-16(15)31-17)9-12-5-13(22)7-24-21(12)30-11/h5,7-8,10-11,15-16H,2-4,6,9H2,1H3,(H,23,29)/t11-,15+,16-/m0/s1
Chemical Name	(13E,14E,15aR,18aS,5S)-35-Fluoro-5-methyl-15,15a,16,17,18,18a-hexahydro-4-oxa-7-aza-1(5,3)-cyclopenta[b]pyrazolo[1',5':1,2]pyrimido[4,5-e][1,4]oxazina-3(3,2)-pyridinacyclooctaphan-8-one
Synonyms	TPX-0046; TPX 0046; TPX0046; Enbezotinib;
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

ln Vitro	Compound 5 (enbezotinib) (0.3-300 nM) inhibits p-RET (y905) in cells triggered by RET [1].
ln Vivo	Embetinib (Compound 5) decreases the growth of tumors in mice when given twice daily for 27 days at a dose of 2–5 mg/kg [1].
References	[1]. Macrocyclic compounds for treating disease. WO2019126121A1.
Additional Infomation	Enbezotinib is an orally bioavailable selective dual inhibitor of fusions and mutations involving the proto-oncogene receptor tyrosine kinase rearranged during transfection (ret) and the src family tyrosine kinases, with potential antineoplastic activity. Upon oral administration, enbezotinib specifically targets and binds to ret mutants and ret-containing fusion products. This results in an inhibition of cell growth of tumor cells that exhibit increased ret activity. By inhibiting src kinase-mediated signaling and reducing the src-initiated recruitment of multiple receptor tyrosine kinases involved in bypass resistance, enbezotinib may be able to overcome tumor resistance which may increase its therapeutic effect. Ret overexpression, activating mutations, and fusions result in the upregulation and/or overactivation of ret tyrosine kinase activity in various cancer cell types; dysregulation of ret activity plays a key role in the development and progression of these cancers. Src tyrosine kinases are upregulated in many tumor cells and play important roles in tumor cell proliferation, survival, migration, invasion and angiogenesis. Src upregulation is seen in tumors with acquired resistance to RET inhibitors.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~235.61 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.90 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 (4.90 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	2.3561 mL	11.7805 mL	23.5610 mL
	5 mM	0.4712 mL	2.3561 mL	4.7122 mL
	10 mM	0.2356 mL	1.1781 mL	2.3561 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.