ZMF-10 is a potent PAK1 inhibitor (antagonist) with IC50s of 174 nM, 1.038 μM and 1.372 μM for PAK1, PAK2 and PAK3 respectively. ZMF-10 can inhibit PAK1 activity and affect PAK1-regulated apoptosis, endoplasmic reticulum stress and migration in MDA-MB-231 cells. ZMF-10 could be used in anti-cancer research.

Physicochemical Properties

Molecular Formula	C19H17F6N7O
Molecular Weight	473.375003576279
Exact Mass	473.139
CAS #	2415295-37-1
PubChem CID	162663870
Appearance	Typically exists as solid at room temperature
LogP	3.6
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	3
Heavy Atom Count	33
Complexity	663
Defined Atom Stereocenter Count	0
InChi Key	KGUGNCJQOQZVJW-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H17F6N7O/c20-18(21,22)10-5-11(19(23,24)25)7-13(6-10)30-17(33)29-12-1-3-32(4-2-12)16-14-8-28-31-15(14)26-9-27-16/h5-9,12H,1-4H2,(H2,29,30,33)(H,26,27,28,31)
Chemical Name	1-[3,5-bis(trifluoromethyl)phenyl]-3-[1-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperidin-4-yl]urea
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

ln Vitro	In a dose-dependent manner, ZMF-10 (0-10 μM; 48 hours) demonstrates strong antiproliferative action [1]. ZMF-10 (20 μM; 24 hours) prevents PAK1 Ser199 and Thr212 from being phosphorylated [1]. With early and late apoptotic cells, ZMF-10 (10–40 μM; 24 hours) down-regulates ERK expression and inhibits the phosphorylation of c-Raf, MEK, and ERK; it also up-regulates Bax expression and down-regulates Bcl-2 expression. The increase in number induces apoptosis in a dose-dependent manner [1]. ZMF-10 (10–40 μM; 24 hours) suppresses JNK1/2 and cell motility in MDA-MB-231 cells, as well as PI3K-AKT-mTOR signaling and mTOR phosphorylation [1].
Cell Assay	Cell Proliferation Assay Cell Types: MDA-MB-231[1] Tested Concentrations: 0-10 μM Incubation Duration: 48 hrs (hours) Experimental Results: demonstrated potent anti-proliferative activity in a dose-dependent manner with an IC50 value of 3.48 μM. Immunofluorescence Cell Types: MDA-MB-231[1] Tested Concentrations: 20 μM Incubation Duration: 24 hrs (hours) Experimental Results: Inhibition of PAK1 phosphorylation at Ser199 and Thr212. Western Blot Analysis Cell Types: MDA-MB-231[1] Tested Concentrations: 10, 20 and 40 μM Incubation Duration: 24 hrs (hours) Experimental Results: Down-regulate ERK expression, inhibit c-Raf, MEK and ERK phosphorylation; up-regulate Bax expression, down-regulate Bcl -2 expression. Apoptosis analysis Cell Types: MDA-MB-231[1] Tested Concentrations: 10, 20 and 40 μM Incubation Duration: 24 hrs (hours) Experimental Results: Induction of apoptosis in a dose-dependent manner with increase in early and late apoptotic cell populations Death.
References	[1]. Design, synthesis and biological evaluation of 1H-pyrazolo [3,4-d]pyrimidine derivatives as PAK1 inhibitors that trigger apoptosis, ER stress and anti-migration effect in MDA-MB-231 cells. Eur J Med Chem. 2020;194:112220.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1125 mL	10.5623 mL	21.1247 mL
	5 mM	0.4225 mL	2.1125 mL	4.2249 mL
	10 mM	0.2112 mL	1.0562 mL	2.1125 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.