ONO-4059 analogue, an analogue of ONO-4059, is a novel, covalent, selective and orally bioavailable BTK inhibitor with potential anticancer and anti-inflammatory activity. It inhibits BTK with IC50 of 23.9 nM. ONO-4059 has demonstrated high antitumor activity in several pre-clinical models. It potently and dose-dependently reverse clinical arthritis and prevented bone damage in the CIA model. ONO-4059 inhibited the TMD-8 cell growth and Btk phosphorylation in a concentration-dependent manner. Furthermore, the decrease in Btk phosphorylation subsequently down-regulated Erk phosphorylation. After the combination of ONO-4059 with doxorubicin, etoposide, vincristine and dexamethasone, increased apoptosis ratio was observed, 25, 20, 17 and 29%, respectively.

Physicochemical Properties

Molecular Formula	C25H24N6O3
Molecular Weight	456.50
Exact Mass	456.19
Elemental Analysis	C, 65.78; H, 5.30; N, 18.41; O, 10.51
CAS #	1351635-67-0
Related CAS #	Tirabrutinib;1351636-18-4;Tirabrutinib hydrochloride;1439901-97-9
PubChem CID	89455219
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	714.0±70.0 °C at 760 mmHg
Flash Point	385.6±35.7 °C
Vapour Pressure	0.0±2.3 mmHg at 25°C
Index of Refraction	1.677
LogP	1.82
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	34
Complexity	751
Defined Atom Stereocenter Count	1
SMILES	C=CC(=O)N1CCC[C@@H](C1)N2C3=NC=NC(=C3N(C2=O)C4=CC=C(C=C4)OC5=CC=CC=C5)N
InChi Key	KSUDUUBCXJUFRL-SFHVURJKSA-N
InChi Code	InChI=1S/C25H24N6O3/c1-2-21(32)29-14-6-7-18(15-29)31-24-22(23(26)27-16-28-24)30(25(31)33)17-10-12-20(13-11-17)34-19-8-4-3-5-9-19/h2-5,8-13,16,18H,1,6-7,14-15H2,(H2,26,27,28)/t18-/m0/s1
Chemical Name	(S)-9-(1-acryloylpiperidin-3-yl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one
Synonyms	ONO 4059-Analog; ONO-4059-Analog;ONO-WG-307;ONO4059-Analog; 6-amino-7-(4-phenoxyphenyl)-9-[(3S)-1-prop-2-enoylpiperidin-3-yl]purin-8-one; (S)-9-(1-acryloylpiperidin-3-yl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one; (S)-9-(1-acryloylpiperidin-3-yl)-6-amino-7-(4-phenoxyphenyl)-7H-purin-8(9H)-one; ONO4059 analog?; CHEMBL3991931; SCHEMBL14798147; (R)-9-(1-acryloylpiperidin-3-yl)-6-amino-7-(4-phenoxyphenyl)-7h-purin-8(9h)-one; GS 4059-Analog; GS-4059-Analog; GS4059-Analog.
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	BTK
ln Vitro	In vitro activity: In TMD-8 cells, ONO-4059 inhibits cell growth with IC50 of 3.59 nM, and induces cell apoptosis. The combination of of ONO-4059 with doxorubicin, etoposide, vincristine or dexamethasone causes the increased apoptosis ratio. Kinase Assay: ONO-4059 analog is the analog of ONO-4059, ONO-4059 is a highly potent and selective Btk inhibitor. IC50 value: sub-nM range. Cell Assay: ONO-4059 inhibited the TMD-8 cell growth and Btk phosphorylation in a concentration-dependent manner. Furthermore, the decrease in Btk phosphorylation subsequently down-regulated Erk phosphorylation. After the combination of ONO-4059 with doxorubicin, etoposide, vincristine and dexamethasone, increased apoptosis ratio was observed, 25, 20, 17 and 29%, respectively.
ln Vivo	For the 100-200 mm3 tumour groups, tumour growth inhibition at the final treatment day was 23% in QD, 72.9% in BD and 100% in dose mixed in food, groups respectively. For the 400-450 mm3 tumour groups, no growth inhibition was observed in the QD group and, growth inhibitions of 27.5% in BD and 100% in dose mixed in food were observed. The PK concentration and phosphorylated Btk inhibition levels of those animals whose dose was mixed in with food were higher than that of other treatment groups.
Enzyme Assay	Determination of covalent binding. [Biochim Biophys Acta Gen Subj. 2020 Apr;1864(4):129531.] Protein labeling experiments were performed using BTK at a final concentration of 2 μM in a buffer solution containing 10 mM HEPES, pH 7.5, 150 mM sodium chloride, 10 mM magnesium chloride, 2 mM Tris(2-carboxyethyl)phosphine (TCEP), and 1% glycerol. Inhibitors were added to a final concentration of 10 μM, with a final concentration of 1% DMSO in all samples. Four conditions were tested, each run in triplicate: BTK + tirabrutinib, BTK + staurosporine, BTK + ibrutinib, and BTK + DMSO control. After compound addition, samples were incubated overnight at 4 °C in a rotating shaker (1200 rpm). After an 18-h incubation, aliquots were collected from each condition for analysis and this time point was termed t = pre-chase. A chase step was then performed with the remaining sample by addition of ibrutinib into the BTK + tirabrutinib and BTK + staurosporine samples to a final concentration of 100 μM. An equivalent amount of DMSO was added to the BTK + ibrutinib and BTK + DMSO control samples to maintain the same volume. After incubating for 6 h at 4 °C, the remaining sample was collected at the final time point, termed t = post-chase. Aliquots taken at both time points were analyzed at the time of collection using mass spectrometry and enzyme activity assays. Mass spectrometry analysis was performed on an Agilent 6210 Time of Flight Mass Spectrometer with an Agilent 1200 Rapid Resolution HPLC using Masshunter B.05 Acquisition software. Samples were run on an Agilent Zorbax 300 Extend C18 rapid resolution column at 70 °C, using reverse phase chromatography with a gradient from 20% to 90% acetonitrile containing 0.1% formic acid. Data were processed using Agilent MassHunter Qualitative Analysis B.06, with a BioConfirm workflow allowing for protein deconvolution to obtain neutral mass values.
Animal Protocol	ONO-4059 has demonstrated anti-tumour activity in several pre-clinical models. ONO-4059 potently and dose-dependently reverse clinical arthritis and prevented bone damage in the CIA model Mouse tumor and CIA models
References	Blood.2016Jan 28;127(4):411-9.
Additional Infomation	See also: Tirabrutinib (annotation moved to).

Solubility Data

Solubility (In Vitro)

DMSO: 20 mg/mL (43.8 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (5.48 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. 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 (5.48 mM) 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; with ultrasonication. 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. Solubility in Formulation 3: 2.5 mg/mL (5.48 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1906 mL	10.9529 mL	21.9058 mL
	5 mM	0.4381 mL	2.1906 mL	4.3812 mL
	10 mM	0.2191 mL	1.0953 mL	2.1906 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.