Dimethylenastron, a quinazoline-thione analog, is a potent, cell-permeable, specific, and reversible mitotic kinesin 5 (Eg5) inhibitor with IC50 of 200 nM. The mitotic motor of ATPase activity stimulated by microtubules is called Eg5. Dimethylenastron, an Eg5 Inhibitor III, has little effect on the ATPase activity of kinesin-1, -4, -7, and -10 and may have anticancer properties. Moreover, dimethylenastron (~1 μM) causes cell cycle arrest and prevents the formation of bipolar spindles in HeLa cells and Xenopus egg extracts. After treatment for 24 hours, dimethylenastron (3 and 10 μM) concentration-dependently suppresses the cancer cells' ability to migrate, but it does not stop the cancer cells from proliferating until 72 hours later. Dimethylenastron also lessens the cancer cells' capacity to invade.

Physicochemical Properties

Molecular Formula	C16H18N2O2S
Molecular Weight	302.39
Exact Mass	302.108
Elemental Analysis	C, 63.55; H, 6.00; N, 9.26; O, 10.58; S, 10.60
CAS #	863774-58-7
Related CAS #	863774-58-7
PubChem CID	11609157
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	475.6±55.0 °C at 760 mmHg
Flash Point	241.4±31.5 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.674
LogP	2.47
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	1
Heavy Atom Count	21
Complexity	512
Defined Atom Stereocenter Count	0
SMILES	O=C1CC(C)(C)CC2NC(NC(C1=2)C1C=C(O)C=CC=1)=S
InChi Key	RUOOPLOUUAYNPY-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H18N2O2S/c1-16(2)7-11-13(12(20)8-16)14(18-15(21)17-11)9-4-3-5-10(19)6-9/h3-6,14,19H,7-8H2,1-2H3,(H2,17,18,21)
Chemical Name	4-(3-hydroxyphenyl)-7,7-dimethyl-2-sulfanylidene-3,4,6,8-tetrahydro-1H-quinazolin-5-one
Synonyms	Dimethylenastron
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	Eg5 (IC50 = 200 nM)
ln Vitro	Dimethylenastron has an IC50 of 200 nM, making it a strong Eg5 inhibitor. Five other kinesin subfamilies (kinesin 1/4/7/10 and one ungrouped, originating from four different organisms) are not inhibited by dimethylenastron. In HeLa cells, dimethylenastron (0.5, 1 μM) induces cell accumulation in G2/M[1]. After treatment for 24 hours, dimethylenastron (3 and 10 μM) concentration-dependently suppresses the cancer cells' ability to migrate, but it does not stop the cancer cells from proliferating until 72 hours later. Dimethylenastron also lessens the cancer cells' capacity to invade[2].
ln Vivo	Dimethylenastron (1.0 µmol) causes a less severe scarring, but it does not considerably extend the bleb's life in comparison to the control group. In the rabbits treated with glaucoma filtration surgery, dimethylenastron (1.0 µmol) shows a significantly reduced ratio of intraocular pressure and a milder, though not significantly reduced, subconjunctival fibrotic reaction[3].
Enzyme Assay	Dimethylenastron has an IC50 of 200 nM, making it a strong Eg5 inhibitor. Five other kinesin subfamilies (kinesin 1/4/7/10 and one ungrouped, originating from four different organisms) are not inhibited by dimethylenastron. In HeLa cells, dimethylenastron (0.5, 1 μM) induces cell accumulation in the G2/M phase.
Cell Assay	Transwell assays measure the invasion of cells in response to dimethylenastron. Matrigel or fibronectin is applied to the transwell filters' upper surface. After adding cells suspended in 200 μL serum-free media to the chamber, the chamber is put in a 24-well plate with the entire medium inside. The matrigel on the upper surface of the filters is removed with cotton swabs once the filters have been gently removed from their 24-hour incubation at 37°C. The cells on the underside of transwell filters are photographed after being fixed for 30 minutes in 4% paraformaldehyde and stained for 10 minutes in 0.1% crystal violet. The number of invading cells is counted in five random fields per filter for quantitative evaluation. The number of invasive cells in the drug-treatment group divided by the number of invasive cells in the control group is used to calculate the extent of cell invasion[2].
Animal Protocol	Following the closure of the conjunctival suture, one of the following agents is injected using a metallic needle (30 G) that is placed into the subconjunctival space at the nasal border of the superior rectus muscle: Following surgery, the rabbits in the control group receive no adjuvant, the other groups receive one unilateral subconjunctival injection of Dimethylenastron (1.0 µmol, 3.0 µmol) or the vehicle (DMSO, 99.9%, 10 mg/mL) alone at baseline. This means that the injection is given immediately after surgery, and in two additional groups, it is given twice more at days 3 and 7 after (1.0 µmol, 3.0 µmol)[3].
References	[1]. Development and biological evaluation of potent and specific inhibitors of mitotic Kinesin Eg5. Chembiochem. 2005 Jul;6(7):1173-7. [2]. Dimethylenastron suppresses human pancreatic cancer cell migration and invasion in vitro via allosteric inhibition of mitotic kinesin Eg5. Acta Pharmacol Sin. 2011 Dec;32(12):1543-8. [3]. The effect of adjuvant dimethylenastron, a mitotic Kinesin Eg5 inhibitor, in experimental glaucoma filtration surgery. Curr Eye Res. 2010 Dec;35(12):1090-8.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 31 mg/mL Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.27 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 (8.27 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	3.3070 mL	16.5349 mL	33.0699 mL
	5 mM	0.6614 mL	3.3070 mL	6.6140 mL
	10 mM	0.3307 mL	1.6535 mL	3.3070 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.