ZCL278 (ZCL-278; ZCL 278) is a potent, cell-permeable and selective Cdc42 GTPase inhibitor with potential anticancer activity. With a Kd of 11.4 μM, it suppresses Cdc42 GTPase. In human metastatic prostate cancer PC-3 cells, ZCL278 inhibited the phosphorylation of Rac/Cdc42.

Physicochemical Properties

Molecular Formula	C21H19BRCLN5O4S2
Molecular Weight	584.89
Exact Mass	582.975
Elemental Analysis	C, 43.12; H, 3.27; Br, 13.66; Cl, 6.06; N, 11.97; O, 10.94; S, 10.96
CAS #	587841-73-4
Related CAS #	587841-73-4
PubChem CID	1791111
Appearance	White to light yellow solid powder
Density	1.6±0.1 g/cm3
Index of Refraction	1.687
LogP	4.45
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	7
Heavy Atom Count	34
Complexity	800
Defined Atom Stereocenter Count	0
SMILES	O=C(NC(NC1=CC=C(S(=O)(NC2=NC(C)=CC(C)=N2)=O)C=C1)=S)COC3=CC=C(Br)C=C3Cl
InChi Key	XKZDWYDHEBCGCG-UHFFFAOYSA-N
InChi Code	InChI=1S/C21H19BrClN5O4S2/c1-12-9-13(2)25-20(24-12)28-34(30,31)16-6-4-15(5-7-16)26-21(33)27-19(29)11-32-18-8-3-14(22)10-17(18)23/h3-10H,11H2,1-2H3,(H,24,25,28)(H2,26,27,29,33)
Chemical Name	2-(4-bromo-2-chlorophenoxy)-N-[[4-[(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl]carbamothioyl]acetamide
Synonyms	ZCL-278; ZCL278; ZCL 278
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	Cdc42 (Kd = 11.4 μM)
ln Vitro	ZCL278 as a potent, cell-permeable inhibitor of Cdc42 that inhibits actin-based cellular processes such as cell motility and Golgi organization.ZCL278 eliminates microspike formation and perturbs GM130-docked Golgi structures in Swiss 3T3 fibroblast cultures, which are two of the most notable Cdc42-mediated subcellular events. Unlike the selective Rac inhibitor NSC23766, ZCL278 lowers the perinuclear accumulation of active Cdc42. In a metastatic prostate cancer cell line (PC-3), ZCL278 inhibits Cdc42-mediated neuronal branching and growth cone dynamics along with actin-based motility and migration without compromising cell viability[1]. While ZCL278 does not inhibit the nonenveloped poliovirus, it does inhibit the function of Cdc42 as an entry inhibitor for the dengue virus, vesicular stomatitis virus, lymphocytic choriomeningitis virus, and Junin virus (JUNV). ZCL278 effectively suppresses chemically induced filopodium formation in cells, a process that depends on Cdc42 activity. In the first round of dose-response studies, ZCL278 is not toxic at 200 μM concentrations, but according to flow cytometry, it inhibits JUNV with an IC50 of about 14 μM[2].
ln Vivo	ZCL278 causes a more than 33-fold reduction in the amount of JUNV RNA in the spleen, with no JUNV RNA present in 5 out of 8 mice. ZCL278 can stop JUNV replication, as evidenced by these outcomes, which are comparable to those observed in mice given gabapentin[2].
Enzyme Assay	The protein Cdc42 that has been lyophilized is reconstituted to a concentration of 5 mg/mL using a buffer that comprises 50 mM Tris, 0.5 mM MgCl2, 50 mM NaCl, 3% (wt/vol) sucrose, and 0.6% dextran. We next dilute the stock solution to 1 μM in 5 mM phosphate buffer (pH 7.4). Before each fluorescent measurement, aliquots of ZCL278 are added to a quartz cuvettete containing Cdc42 solution and incubated for five minutes. After each addition, tryptophan fluorescence at 350 nm is measured using an excitation wavelength of 275 nm. GraphPad's equimolar specific binding model is used to fit the titration curve, and the Kd is computed[1].
Cell Assay	ZCL278, NSC23766, or the Cdc42 activator are added to PC-3 cells during a 24-hour incubation period in order to measure cell viability. With a Countess Automated Cell Counter, the trypan blue dye exclusion method is used to determine the number of live and dead cells. Any null hypothesis with a probability level less than 95% is rejected, and P values are assigned for every experiment[1].
Animal Protocol	Mice: C57BL/6 mice that are four weeks old are given intravenous injections of either ZCL278 (100 μg/g) or gabapentin. The mice receive an intraperitoneal injection of JUNV Candid #1 (1×106 PFU) in a maximum volume of 1 mL using a 27 1/2-gauge needle, one hour post-treatment. When the trial is over, the mice are killed, their spleens are homogenized using a Dounce homogenizer, they are centrifuged to produce a cell pellet and supernatant, and the levels of RNA expression are assessed.
References	[1]. Small molecule targeting Cdc42-intersectin interaction disrupts Golgi organization and suppresses cell motility. Proc Natl Acad Sci U S A. 2013 Jan 22;110(4):1261-6. [2]. Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor. J Virol. 2016 Apr 14;90(9):4494-510.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~170.9 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.27 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 (4.27 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	1.7097 mL	8.5486 mL	17.0972 mL
	5 mM	0.3419 mL	1.7097 mL	3.4194 mL
	10 mM	0.1710 mL	0.8549 mL	1.7097 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.