BTB-1 is a novel, potent and the first small molecule inhibitor of the mitotic motor protein Kif18A. It exhibits selectivity within the Kif4, Eg5, MKLP-1, MKLP-2, MPP1, CENP-E, and MCAK kinesin subgroups. Antimitotic medications target kinesin motors because they have a variety of functions during mitosis. Kif18A is frequently overexpressed in solid tumors, so drugs such as BTB-1 are not only very interesting for basic research but may also lead to new approaches in the treatment of human diseases. BTB-1 was added during in vitro motility tests, and this reversibly inhibited Kif18A's ability to glide through MT. Surprisingly, BTB-1 traps Kif18A on the microtubule, while its binding site is similar to that of well-studied Kif11 inhibitors that prevent tight microtubule binding.

Physicochemical Properties

Molecular Formula	C12H8CLNO4S
Molecular Weight	297.71
Exact Mass	296.986
Elemental Analysis	C, 48.41; H, 2.71; Cl, 11.91; N, 4.70; O, 21.50; S, 10.77
CAS #	86030-08-2
Related CAS #	86030-08-2
PubChem CID	291461
Appearance	White to off-white solid powder
Density	1.476g/cm3
Boiling Point	471.2ºC at 760mmHg
Melting Point	115ºC
Flash Point	238.8ºC
Index of Refraction	1.623
LogP	4.685
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	2
Heavy Atom Count	19
Complexity	422
Defined Atom Stereocenter Count	0
SMILES	[O-][N+](C1C(S(C2C=CC=CC=2)(=O)=O)=CC=C(Cl)C=1)=O
InChi Key	VZDUQPHKUBZMLW-UHFFFAOYSA-N
InChi Code	InChI=1S/C12H8ClNO4S/c13-9-6-7-12(11(8-9)14(15)16)19(17,18)10-4-2-1-3-5-10/h1-8H
Chemical Name	1-(benzenesulfonyl)-4-chloro-2-nitrobenzene
Synonyms	BTB-1; BTB 1; BTB1
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	Kif18A (IC50 = 1.69 μM)
ln Vitro	BTB-1 blocks the motility of Kif18A in a reversible manner. BTB-1 slows down the progress of cells through mitosis by inhibiting Kif18A in a manner that is competitive with adenosine triphosphate (ATP) but uncompetitive with microtubules. No other tested mitotic kinesin is significantly inhibited by 100 μM BTB-1. Only when the motor protein is connected to its pseudosubstrate microtubules does BTB-1 compete with ATP for Kif18A binding. BTB-1 treatment causes dose-dependent accumulation of HeLa cells during mitosis[1]. BTB-1 demonstrates cytotoxicity, exhibiting an EC50 value of 35.8 δM. When 50 μM BTB-1 is applied to HeLa cells, significant abnormalities in chromosome alignment and spindle morphology are observed. Treatment with high BTB-1 concentrations does not cause spindles to elongate[2].
ln Vivo
Enzyme Assay	In DMSO, BTB-1 is prepared. The His-Kif18Amotor sactivityis observed as ATP concentration increases when 3 μM Mts and increasing BTB-1 concentrations (0.21 μM, 0.42 μM, 0.85 μM, 1.7 μM) or DMSO as a control are present[1].
Cell Assay	In a reversible way, BTB-1 inhibits Kif18A motility. Cell division is slowed down by BTB-1, which inhibits Kif18A in a way that is competitive for adenosine triphosphate (ATP) but uncompetitive for microtubules. It was found that none of the other tested mitotic kinesins are significantly inhibited by 100 μM BTB-1. BTB-1 and ATP are only in competition for Kif18A binding when the motor protein is attached to its pseudosubstrate microtubules. BTB-1 treatment causes dose-dependent accumulation of HeLa cells during mitosis. With an EC50 value of 35.8 μM, BTB-1 exhibits cell toxicity. Severe defects in spindle morphology and chromosome alignment are observed in HeLa cells treated with 50 μM BTB-1. Longitudinal spindles are not produced by treatment with high BTB-1 concentrations.
Animal Protocol
References	[1]. BTB-1: a small molecule inhibitor of the mitotic motor protein Kif18A. Angew Chem Int Ed Engl. 2009;48(48):9072-6. [2]. Synthesis and biological evaluation of optimized inhibitors of the mitotic kinesin Kif18A. ACS Chem Biol. 2015 Feb 20;10(2):554-60.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 100 mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.40 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 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 (8.40 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 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 (8.40 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 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	3.3590 mL	16.7949 mL	33.5897 mL
	5 mM	0.6718 mL	3.3590 mL	6.7179 mL
	10 mM	0.3359 mL	1.6795 mL	3.3590 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.