FRAX1036 (FRAX-1036; FRAX 1036) is a selective p21-activated kinase 1 (PAK1) inhibitor with potential anticancer activity. With Ki values of 23.3 nM and 72.4 nM, respectively, it inhibits PAK1 and PAK2.

Physicochemical Properties

Molecular Formula	C28H32CLN7O
Molecular Weight	518.062
Exact Mass	517.235
Elemental Analysis	C, 64.92; H, 6.23; Cl, 6.84; N, 18.93; O, 3.09
CAS #	1432908-05-8
Related CAS #	1432908-05-8
PubChem CID	71557891
Appearance	White to light yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	669.8±65.0 °C at 760 mmHg
Flash Point	358.9±34.3 °C
Vapour Pressure	0.0±2.0 mmHg at 25°C
Index of Refraction	1.624
LogP	3.73
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	7
Heavy Atom Count	37
Complexity	803
Defined Atom Stereocenter Count	0
SMILES	O=C1C(C2=CC=C(C3=NC(C)=CN=C3)C=C2Cl)=CC4=CN=C(NCCC5CCN(C)CC5)N=C4N1CC
InChi Key	RYCBSFIKWACFBY-UHFFFAOYSA-N
InChi Code	InChI=1S/C28H32ClN7O/c1-4-36-26-21(16-32-28(34-26)31-10-7-19-8-11-35(3)12-9-19)13-23(27(36)37)22-6-5-20(14-24(22)29)25-17-30-15-18(2)33-25/h5-6,13-17,19H,4,7-12H2,1-3H3,(H,31,32,34)
Chemical Name	6-[2-chloro-4-(6-methylpyrazin-2-yl)phenyl]-8-ethyl-2-[2-(1-methylpiperidin-4-yl)ethylamino]pyrido[2,3-d]pyrimidin-7-one
Synonyms	FRAX 1036; FRAX-1036; FRAX1036
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	PAK1 (Ki = 23.3 nM); PAK2 (Ki = 72.4 nM); PAK4 (Ki = 2.4 μM)
ln Vitro	FRAX1036 is a PAK inhibitor that has corresponding Kis values of 23.3 nM, 72.4 nM, and 2.4 μM for PAK1, PAK2, and PAK4. When FRAX1036 (2.5 μM) and docetaxel are combined, it changes the phosphorylation of stathmin, causes the apoptotic marker cleaved PARP, and accelerates the apoptotic process in MDA-MB-175 and HCC2911 cells.It also modifies the organization of microtubules, mitosis, and cell fate in U2OS cells. Additionally, FRAX1036 exhibits notably efficient inhibition on U2OS cells [1]. When combined with KRAS prenylation inhibitors, FRAX1036 (10 μM) inhibits the growth of non-small cell lung cancer (NSCLC) cells[2].
ln Vivo	Frax1036 treatment slows the growth of KT21 tumors and is unlikely to cause significant blood brain barrier permeability in mice.
Enzyme Assay	The phosphorylation of a FRET peptide substrate (Ser/Thr19) labeled with coumarin and fluorescein is measured using the Z'-LYTETM assay to determine the activity/inhibition of human recombinant PAK1 (kinase domain), PAK2 (full length), or PAK4 (kinase domain). The samples used in the 10 μL assay mixtures include 20 pM PAK1; 50 pM PAK2; 90 pM PAK4), 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA, and 50 mM HEPES (pH 7.5). In black polypropylene 384-well plates, incubations are conducted at 22°C. The assay is started by adding 2.5 μL of assay buffer containing 4× ATP (160 μM PAK1; 480 μM PAK2; 16 μM PAK4). The enzyme, FRET peptide substrate, and serially diluted test compounds (FRAX1036, etc.) are preincubated together in assay buffer (7.5 μL) for 10 minutes prior to the assay. After the assay mixtures are incubated for 60 minutes, 5 μL of Z'-LYTETM development reagent is added to quench them, and an hour later, the emissions of fluorescein (520 nm) and coumarin (445 nm) are measured following excitation at 400 nm. A measurement is made of the emission ratio (445 nm/520 nm) to determine the extent of substrate phosphorylation[1].
Cell Assay	Prior to being treated with DMSO, FRAX1036, and/or docetaxel, cells are plated at 10,000 cells/well in 96-well plates for 24 hours in order to perform caspase 3/7 activation apoptosis assays. The reagent caspase 3/7 is added at a dilution of 1:1000. Using an IncuCyte Zoom Live-content imaging system, cells are imaged at 10× magnification at 37°C and 5% CO2. During the course of 36 to 72 hours, two images are acquired per well, every two or four hours. IncuCyte analysis software is used to identify and measure green (apoptotic) cells/images in data. Every condition is carried out three times. Excel is used to plot averages with SEM at each time point. When comparing the combination of FRAX1036 and docetaxel with each individual agent in Prism at the final time point, a t-test is run. The final apoptotic cell count is divided by the total cell count to determine the apoptotic index based on the apoptosis assays. Excel is used to plot averages with SEM, and a t-test is used to compare the efficacy of FRAX1036 and docetaxel in combination with each agent used alone in Prism[1].
Animal Protocol	Pak2-deficient mice 30 mg/kg by oral gavage
References	[2]. Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis. BMC Cancer. 2015 May 9;15:381.

Solubility Data

Solubility (In Vitro)	DMSO: ~5.3 mg/mL (~10.2 mM)
Solubility (In Vivo)	Solubility in Formulation 1: 10 mg/mL (19.30 mM) in 50% PEG300 +50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O 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	1.9303 mL	9.6514 mL	19.3028 mL
	5 mM	0.3861 mL	1.9303 mL	3.8606 mL
	10 mM	0.1930 mL	0.9651 mL	1.9303 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.