ML264 is a novel and selective inhibitor of Krüppel-like factor 5 (KLF5) that potently inhibits the proliferation of colorectal cancer cells in vitro through modifications of the cell-cycle profile. Additionally, ML264 effectively stops the tumor's growth in 5 days in a well-established mouse xenograft model of colon cancer. Since ML264 potently inhibits the expression of KLF5 and EGR1, a transcriptional activator of KLF5, this effect is brought on by a marked decrease in proliferation. ML264 is highly active (IC50 = 29 nM is a cell-based assay for proliferation of DLD-1 cells, IC50 = 81 nM in a cell-based luciferase assay). The IEC-6 control cell line does not exhibit any cytotoxicity (IC50 >50 μM,<50% inhibition was observed at 100 μM). A number of other KLF5-expressing cell types (such as HCT116, IC50 = 560 nM; HT29, IC50 = 130 nM; and SW620, IC50 = 430 nM) also showed robust activity. An analysis of a panel of 47 selected kinases revealed that ML264 does not inhibit the kinases connected to the KLF5 pathway. By a large margin, ML264 lowers KLF5 expression, according to a Western blot analysis. These outcomes illustrate the target specificity of KLF5. According to a NCI60 panel study using ML264, the majority of colon cancer cell lines are killed off, and a number of other tumor cell lines are also affected by its cytotoxicity. Chemical stability, lack of reaction with glutathione, suitability for aqueous solubility, high stability to mouse, rat, and human hepatic microsomes, favorable characteristics related to drug-likeness, and lack of inhibition of CYP enzymes are all characteristics of ML264. It also has suitable aqueous solubility. These characteristics, combined with ML264's high cellular potency and selectivity, make it an excellent candidate for in vivo anticancer studies. It also has great potential for use in long-term in situ studies aimed at elucidating the function of KLF5 as a regulator of cellular proliferation and tumor formation in the intestinal epithelium. The potential of ML264 and its analogues as a novel therapeutic agent to stop the growth and spread of colorectal cancer is worth exploring.

Physicochemical Properties

Molecular Formula	C17H21CLN2O4S
Molecular Weight	384.88
Exact Mass	384.091
Elemental Analysis	C, 53.05; H, 5.50; Cl, 9.21; N, 7.28; O, 16.63; S, 8.33
CAS #	1550008-55-3
Related CAS #	1550008-55-3
PubChem CID	51003603
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	701.0±60.0 °C at 760 mmHg
Flash Point	377.7±32.9 °C
Vapour Pressure	0.0±2.2 mmHg at 25°C
Index of Refraction	1.599
LogP	0.46
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	5
Heavy Atom Count	25
Complexity	601
Defined Atom Stereocenter Count	0
SMILES	ClC1=CC=CC(=C1)/C=C/C(NCC(N(C)C1CCS(CC1)(=O)=O)=O)=O
InChi Key	AJCDZIDKYKCOMZ-AATRIKPKSA-N
InChi Code	InChI=1S/C17H21ClN2O4S/c1-20(15-7-9-25(23,24)10-8-15)17(22)12-19-16(21)6-5-13-3-2-4-14(18)11-13/h2-6,11,15H,7-10,12H2,1H3,(H,19,21)/b6-5+
Chemical Name	(E)-3-(3-chlorophenyl)-N-[2-[(1,1-dioxothian-4-yl)-methylamino]-2-oxoethyl]prop-2-enamide
Synonyms	CID-51003603; SID 117686865, and SR-03000002171; ML-264; ML264; ML 264
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	KLF5 (IC50 = 29 nM)
ln Vitro	ML264 potently halts DLD-1 viability (IC50 = 29 nM) with high maximal effect (>90%). Human colorectal adenocarcinoma cells are DLD-1 cells. Other cell types, such as HCT116 (human colorectal carcinoma), HT29 (human colorectal adenocarcinoma), and SW620 (human colorectal adenocarcinoma), are also significantly affected by ML264 at submicromolar doses. With inhibition below 50% at the highest dose, the IEC-6 anti-target (a nontransformed rat intestinal epithelial cell line) is largely unaffected[1]. Through changes to the cell cycle profile, this substance effectively prevents the proliferation of CRC cells in vitro[2].
ln Vivo	In a well-established mouse xenograft model of colon cancer, ML264 effectively inhibits tumor growth five days after administration. Since ML264 potently inhibits the expression of KLF5 and EGR1, a transcriptional activator of KLF5, this effect is brought on by a significant decrease in proliferation[2].
Enzyme Assay	ML264 is highly active (IC50=29 nM is a cell-based assay for proliferation of DLD-1 cells, IC50=81 nM in a cell-based luciferase assay). ML264 lacks cytotoxicity in the IEC-6 control cell line (IC50>50 μM,<50% inhibition is observed at 100 μM). Robust activity is also seen in several other KLF5-expressing cell types as well (e.g., HCT116, IC50=560 nM; HT29, IC50=130 nM; SW620, IC50=430 nM). Western blot analysis shows that ML264 significantly reduces KLF5 expression.
Cell Assay	DLD-1 and HCT116 cells are treated with 10 μM ML264 or DMSO in experiments testing cell proliferation. Live cells are collected 24, 48, and 72 hours after treatment, and the number of each are counted using a Coulter counter. DLD-1 and HCT116 cells are treated with 10 μM ML264 or with the control (DMSO) in the MTS assay. Each well receives 20 μL of MTS solution following incubations of 24, 48, and 72 hours. An analysis is then carried out in accordance with the manufacturer's instructions.
Animal Protocol	Mice: In ventilated, filtered cages with positive pressure, naked mice are kept in a pathogen-free environment. The right flank of 6-7 week old male nude mice is injected subcutaneously with 5×106 DLD-1 human colorectal cells to produce xenograft tumors. By measuring with a caliper and using established formulas, tumor volume is calculated. Mice are treated intraperitoneally (i.p.) with ML264 at doses of 10 mg/kg daily, 10 mg/kg twice daily, and 25 mg/kg twice daily for a total of 10 days when tumor volumes reach approximately 100 mm6. The control treatment is the vehicle solution. Each and every two days, mice are observed and weighed. When the tumor's largest measurement reaches 2 cm, the experiment is over. Tumors are removed and saved for additional research[2].
References	[1]. ML264: An Antitumor Agent that Potently and Selectively Inhibits Krüppel-like Factor Five (KLF5) Expression: A Probe for Studying Colon Cancer Development and Progression. [2]. ML264, A Novel Small-Molecule Compound That Potently Inhibits Growth of Colorectal Cancer. Mol Cancer Ther. 2016 Jan;15(1):72-83.

Solubility Data

Solubility (In Vitro)

DMSO: ~10mM Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3.25 mg/mL (8.44 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 32.5 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	2.5982 mL	12.9911 mL	25.9821 mL
	5 mM	0.5196 mL	2.5982 mL	5.1964 mL
	10 mM	0.2598 mL	1.2991 mL	2.5982 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.