BTK
ONO-4059 analogue targets Bruton's tyrosine kinase (BTK), a key enzyme in B-cell receptor signaling. It is a highly potent and selective oral BTK inhibitor with an IC50 of 23.9 nM. By inhibiting BTK, the compound disrupts B-cell signaling pathways that are essential for the survival and proliferation of malignant B cells and for the pathogenesis of autoimmune diseases.

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.
In vitro, ONO-4059 analogue inhibits BTK with an IC50 of 23.9 nM. It has demonstrated high antitumor activity in several preclinical models. The compound potently and dose-dependently reverses clinical arthritis and prevents bone damage in the CIA model. These in vitro studies confirm its potent and selective activity against BTK.

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.

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.
Non-cellular enzyme assays for ONO-4059 analogue involve measuring its inhibition of BTK activity using purified BTK enzyme and appropriate substrates. The compound's IC50 of 23.9 nM is determined in these assays. Selectivity is assessed by testing the compound against other kinases. These assays confirm the compound's potency and selectivity for BTK.

In vitro cell-based assays for ONO-4059 analogue are conducted using B-cell lines to assess its effects on BTK signaling and cell proliferation. Cells are treated with the compound, and BTK phosphorylation and downstream signaling are measured. Cell proliferation and apoptosis assays are performed to evaluate the compound's functional effects.

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

ONO-4059 analogue is an orally active compound with a molecular weight of 409.42 g/mol. It is a highly potent and selective BTK inhibitor. Detailed pharmacokinetic parameters such as half-life and bioavailability are not extensively documented in standard summaries but would be evaluated in preclinical studies. Its oral bioavailability makes it suitable for in vivo studies.

Comprehensive toxicological data for ONO-4059 analogue are limited as it is primarily a research compound. It has been investigated in Phase 1 clinical trials. As with all research compounds, appropriate safety precautions should be taken when handling ONO-4059 analogue. Its safety profile in vivo has been evaluated in preclinical models.

Blood.2016Jan 28;127(4):411-9.

See also: Tirabrutinib (note moved to).
ONO-4059 analogue (ONO-WG-307) is a highly potent and selective oral BTK inhibitor with an IC50 of 23.9 nM. It has demonstrated high antitumor activity in several preclinical models. The compound potently reverses clinical arthritis and prevents bone damage in the CIA model. ONO-4059 analogue is not an approved drug and is available as a research compound.

C25H24N6O3

456.50

456.19

C, 65.78; H, 5.30; N, 18.41; O, 10.51

1351635-67-0

Tirabrutinib;1351636-18-4;Tirabrutinib hydrochloride;1439901-97-9

89455219

White to off-white solid powder

1.4±0.1 g/cm3

714.0±70.0 °C at 760 mmHg

385.6±35.7 °C

0.0±2.3 mmHg at 25°C

1.677

1.82

1

6

5

34

751

1

C=CC(=O)N1CCC[C@@H](C1)N2C3=NC=NC(=C3N(C2=O)C4=CC=C(C=C4)OC5=CC=CC=C5)N

KSUDUUBCXJUFRL-SFHVURJKSA-N

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

(S)-9-(1-acryloylpiperidin-3-yl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)