TH257 (TH-257; TH 257) is a novel and selective allosteric inhibitor of LIM kinases-LIMK1 and LIMK2 with important biological activity. It can be used as a chemical probe for LIMK1 and LIMK2, as it inhibits them with IC50 values of 39 nM and 84 nM, respectively. An αC and DFG-out conformation-induced binding pocket is the target of the allosteric inhibitor TH-257. TH257 exhibits remarkable selectivity, as demonstrated by the KINOMEscan assay at 1 μM, which shows no discernible activity against the broader kinome.

Physicochemical Properties

Molecular Formula	C24H26N2O3S
Molecular Weight	422.5398
Exact Mass	422.166
Elemental Analysis	C, 68.22; H, 6.20; N, 6.63; O, 11.36; S, 7.59
CAS #	2244678-29-1
Related CAS #	2244678-29-1
PubChem CID	137796294
Appearance	Light yellow to yellow solid powder
LogP	4.7
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	9
Heavy Atom Count	30
Complexity	611
Defined Atom Stereocenter Count	0
InChi Key	VNCIWNGCMAKKEO-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H26N2O3S/c1-2-3-18-26(19-20-10-6-4-7-11-20)24(27)21-14-16-23(17-15-21)30(28,29)25-22-12-8-5-9-13-22/h4-17,25H,2-3,18-19H2,1H3
Chemical Name	N-benzyl-N-butyl-4-(phenylsulfamoyl)benzamide
Synonyms	TH 257; TH257; TH-257
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	LIMK1 (IC50 = 84 nM); LIMK2 (IC50 = 39 nM)
ln Vitro	Promising biochemical and cellular effectiveness are demonstrated by TH-257. However, it is not a feasible candidate for use as an in vivo instrument due to its exceedingly fast exterior clearance [2].
References	[1]. Manetti F. Recent advances in the rational design and development of LIM kinase inhibitors are not enough to enter clinical trials. Eur J Med Chem. 2018 Jul 15;155:445-458. [2]. Comparative Analysis of Small-Molecule LIMK1/2 Inhibitors: Chemical Synthesis, Biochemistry, and Cellular Activity. J Med Chem. 2022 Oct 27;65(20):13705-13713.

Solubility Data

Solubility (In Vitro)

DMSO: 85~250 mg/mL (201.2~591.7 mM) Ethanol: ~85 mg/mL (~201.2 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.92 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 (4.92 mM) (saturation unknown) 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. 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 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: ≥ 2.08 mg/mL (4.92 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	2.3666 mL	11.8332 mL	23.6664 mL
	5 mM	0.4733 mL	2.3666 mL	4.7333 mL
	10 mM	0.2367 mL	1.1833 mL	2.3666 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.