ML216 (formerly known as CID-49852229) is a novel potent inhibitor of the Bloom's syndrome protein (BLM) helicase which is responsible for DNA unwinding activity. The results indicated that the IC50s for full length BLM and BLM636–1298 were 3.0 and 0.97 μM, respectively. The conserved RecQ helicase family includes the Bloom's syndrome protein, BLM. While there are cell lines with no BLM, these show progressive genomic instability, making it difficult to separate the primary from secondary effects of BLM loss. ML216 exhibits cell-based activity and is able to promote the toxicity of aphidicolin, cause sister chromatid exchanges, and inhibit the proliferation of cells that express BLM but not those that do not. According to these findings, ML216 exhibits a high degree of BLM selectivity in cultured cells. ML216 thus has the potential to be useful as an anticancer agent because it targets BLM.

Physicochemical Properties

Molecular Formula	C15H9F4N5OS
Molecular Weight	383.32
Exact Mass	383.046
Elemental Analysis	C, 47.00; H, 2.37; F, 19.82; N, 18.27; O, 4.17; S, 8.36
CAS #	1430213-30-1
Related CAS #	1430213-30-1
PubChem CID	49852229
Appearance	Light yellow to yellow solid powder
Density	1.6±0.1 g/cm3
Index of Refraction	1.641
LogP	4.11
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	3
Heavy Atom Count	26
Complexity	491
Defined Atom Stereocenter Count	0
SMILES	O=C(NC1=CC=C(F)C(C(F)(F)F)=C1)NC2=NN=C(S2)C3=CC=NC=C3
InChi Key	WMCOYUSJXXCHFH-UHFFFAOYSA-N
InChi Code	InChI=1S/C15H9F4N5OS/c16-11-2-1-9(7-10(11)15(17,18)19)21-13(25)22-14-24-23-12(26-14)8-3-5-20-6-4-8/h1-7H,(H2,21,22,24,25)
Chemical Name	1-[4-fluoro-3-(trifluoromethyl)phenyl]-3-(5-pyridin-4-yl-1,3,4-thiadiazol-2-yl)urea
Synonyms	CID-4985229; CID 4985229; CID4985229; ML216; ML-216; ML 216
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	BLMfull-length ( IC50 = 2.98 μM ); BLM636-1298 ( IC50 = 0.97 μM )
ln Vitro	ML216 (12.5-50 μM; 24-72 hours; PSNG5 and PSNG13cells) treatmentsuppresses PSNF5 cell proliferation, but not PSNG13 cell proliferation in a concentration-dependent manner[1]. ML216 treatment increases the frequency of sister chromatid exchanges (SCEs) in a statistically significant way in PSNF5 cells, but not in PSNG13 cells[1]. ML216 enhances the aphidicolin sensitivity of PSNF5 cells, but has no sensitizing effect on isogenic PSNG13 cells lacking BLM[1]. ML216 inhibits WRN500-946 (IC50 of 12.6 μM), which is 2.5 times more sensitive to inhibition than the full length WRN (IC50 of 5 μM). Based on IC50 values, BLM is slightly more sensitive than WRN to ML216's inhibition (1.7-fold). Even though ML216 clearly inhibits WRN, this substance seems to be specific to BLM in human cells. Both WRN+ and WRN? cell proliferation is equally inhibited by ML216, and both cell types are similarly sensitized to aphidicolin[1].
ln Vivo	Though ML216 inhibits unwinding by the sequence-related BLM and WRN helicases in vitro in a manner similar to that of WRN helicases, the drug's mechanism of action in vivo appears to be specific for BLM based on ML216's apparent reliance on BLM for biological effects in human cells. Informational would be a co-crystal structure of BLM in complex with the inhibitor. A certain conformation of WRN that renders it resistant to ML216 may be induced by cellular cues in vivo[2].
Cell Assay	Cell Line: PSNG5 and PSNG13cells Concentration: 12.5 μM or 50 µM Incubation Time: 24 hours, 48 hours, 72 hours Result: Inhibited the proliferation of PSNF5 cells, but not of PSNG13 cells, and did so in a concentration-dependent manner.
References	[1]. A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells. Chem Biol. 2013 Jan 24;20(1):55-62. [2]. A new development in DNA repair modulation: discovery of a BLM helicase inhibitor. Cell Cycle. 2013 Mar 1;12(5):713-4.
Additional Infomation	1-[4-fluoro-3-(trifluoromethyl)phenyl]-3-(5-pyridin-4-yl-1,3,4-thiadiazol-2-yl)urea is a member of ureas.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 1.5 mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: 2 mg/mL (5.22 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6088 mL	13.0439 mL	26.0879 mL
	5 mM	0.5218 mL	2.6088 mL	5.2176 mL
	10 mM	0.2609 mL	1.3044 mL	2.6088 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.