PROTAC-O4I2 is a PROTAC targeting splicing factor 3B1 (SF3B1). PROTAC-O4I2 induces FLAG-SF3B1 degradation in K562 cells with IC50 of 0.244 μM. PROTAC-O4I2 also induced apoptosis in K562 WT cells.

Physicochemical Properties

Molecular Formula	C29H29CLN6O5S
Molecular Weight	609.10
Exact Mass	608.16
CAS #	2785323-62-6
PubChem CID	162641046
Appearance	White to yellow solid powder
Density	1.453±0.06 g/cm3(Temp: 20 °C; Press: 760 Torr)(Predicted)
LogP	4.9
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	12
Heavy Atom Count	42
Complexity	1030
Defined Atom Stereocenter Count	0
SMILES	ClC1C=CC(=CC=1)NC1=NC(=CS1)C(NCCCCCCNC1=CC=CC2C(N(C(C=21)=O)C1C(NC(CC1)=O)=O)=O)=O
InChi Key	UWYLVQJEOCYCEW-UHFFFAOYSA-N
InChi Code	InChI=1S/C29H29ClN6O5S/c30-17-8-10-18(11-9-17)33-29-34-21(16-42-29)25(38)32-15-4-2-1-3-14-31-20-7-5-6-19-24(20)28(41)36(27(19)40)22-12-13-23(37)35-26(22)39/h5-11,16,22,31H,1-4,12-15H2,(H,32,38)(H,33,34)(H,35,37,39)
Chemical Name	2-(4-chloroanilino)-N-[6-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino]hexyl]-1,3-thiazole-4-carboxamide
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	Cereblon
ln Vitro	PROTAC-O4I2 preferentially degrades SF3B1 and prevents the formation of tumors in cells by introducing thalidomide into the ubiquitin E3 ligase cereblon (CRBN) [1]. STAT-O4I2 inhibits and degrades SF3B1 in K562 cells. SF3B1 WT, SF3B1 OE, and SF3B1 K700E cells are non-proliferatively affected by PROTAC-O4I2, with IC50 values of 228, 63, and 90 nM, respectively [1]. With an IC50 value of 0.244 μM in K562 cells, PROTAC-O4I2 causes FLAG-SF3B1 degradation in a concentration-dependent manner [1].
ln Vivo	PROTAC-O4I2 (10 μM) inhibits the maintenance and development of the tumor, hence considerably increasing the survival rate of Drosophila in the intestinal tumor model [1].
Cell Assay	Cell Proliferation Assay[1] Cell Types: K562 control cells (WT), cells overexpressing SF3B1 (OE), and cells expressing SF3B1K700E (K700E) Tested Concentrations: 1 pM, 0.1 nM, 10 nM, 1 μM, 100 μM Incubation Duration: 72 hrs (hours) Experimental Results: In K562 WT cells, the parental compound O4I2 demonstrated a marginal anti-proliferation effect (IC50 >10 μM). In In contrast, PROTAC-O4I2 demonstrated Dramatically higher toxicity with an IC50 of 228 nM, nearly 3-fold less potent than pladienolide B (IC50, 76 nM). Cells overexpressing SF3B1 WT was slightly resistant to pladienolide B (IC50, 134 nM), but more sensitive to PROTAC-O4I2 (an IC50 value of 63 nM). Apoptosis Analysis[1] Cell Types: K562 cell Tested Concentrations: 1 μM Incubation Duration: 48 h Experimental Results: Induced cellular apoptosis s in cells expressing SF3B1WT or SF3B1K700E.
Animal Protocol	Animal/Disease Models: Drosophila melanogaster[1] Doses: 10 μM Route of Administration: Flies were fed on a round filter paper loaded with PROTAC-O4I2 in a 5% sucrose solution , maintained at 18℃ and flipped into a freshly prepared vial every 2 days. Experimental Results: diminished stem cell activity, blocked the initiation and growth of tumor, and improved the survival of the Drosophila ISC tumor model.
References	[1]. A PROTAC targets splicing factor 3B1. Cell Chem Biol. 2021 Nov 18;28(11):1616-1627.e8.

Solubility Data

Solubility (In Vitro)

DMSO : 250 mg/mL (410.44 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.6418 mL	8.2088 mL	16.4177 mL
	5 mM	0.3284 mL	1.6418 mL	3.2835 mL
	10 mM	0.1642 mL	0.8209 mL	1.6418 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.