FAK-IN-2 is a potent and orally bioactive focal adhesion kinase (FAK) inhibitor (antagonist) with anti-tumor activity (FAK IC50= 35 nM). FAK-IN-2 covalently inhibits the autophosphorylation of FAK in a dose-dependent manner, and can also inhibit the formation and migration of tumor cells and induce their apoptosis.

Physicochemical Properties

Molecular Formula	C28H31CLN8O3
Molecular Weight	563.050544023514
Exact Mass	562.22
CAS #	2872588-02-6
PubChem CID	163322241
Appearance	White to off-white solid powder
LogP	4.1
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	9
Heavy Atom Count	40
Complexity	878
Defined Atom Stereocenter Count	1
SMILES	C[C@H](C(=O)N1CCN(CC1)C2=CC=C(C=C2)NC3=NC=C(C(=N3)NC4=CC=CC=C4C(=O)NC)Cl)NC(=O)C=C
InChi Key	SQMGCXJZSMCVHC-GOSISDBHSA-N
InChi Code	InChI=1S/C28H31ClN8O3/c1-4-24(38)32-18(2)27(40)37-15-13-36(14-16-37)20-11-9-19(10-12-20)33-28-31-17-22(29)25(35-28)34-23-8-6-5-7-21(23)26(39)30-3/h4-12,17-18H,1,13-16H2,2-3H3,(H,30,39)(H,32,38)(H2,31,33,34,35)/t18-/m1/s1
Chemical Name	2-[[5-chloro-2-[4-[4-[(2R)-2-(prop-2-enoylamino)propanoyl]piperazin-1-yl]anilino]pyrimidin-4-yl]amino]-N-methylbenzamide
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	IC50: 35 nM (FAK)[1]
ln Vitro	FAK-IN-2 (compound 11w) exhibits significant anti-proliferation actions on cancer cell lines (0–5 μM; 72 hours) and a considerable degree of toxicity on normal cell lines [1]. In a dose-dependent manner, FAK-IN-2 (0–30 nM; 14 days) can significantly impact the clone generation of HCT-116 cells[1]. The migration of HCT116 cells is greatly inhibited by FAK-IN-2 (10-500 nM; 24 and 48 hours) in a dose-dependent manner[1]. The phosphorylation of FAK and its downstream proteins is inhibited by FAK-IN-2 (0.001-10 μM; 4 and 24 hours) through various pathways[1]. Apoptosis and a significant cell cycle arrest at the G2/M phase are caused by -2 (0.01-1 μM; 24 or 48 hours)[1]. .
ln Vivo	FAK-IN-2 (once daily for 16 days at 5 and 15 mg/kg) possesses strong anticancer activity in dose-dependent manner in model mice without causing appreciable harm.[1] Sprague-Dawley male rat FAK-IN-2 pharmacokinetic parameters[1]. PO (5 milligrams per kilogram) IV (5 mg/kg) (μg/L) 239.87 2965.27 Cmax T1/2 (h) 4.70 7.57 Clz (L/h/kg) Tmax (h) 1.44 0.08 9.92 2.19 (μg*h/L) AUC0-t 2439.06 F% 21.02% 512.75
Cell Assay	Cell Proliferation Assay Cell Types: Hela, HCT116, MDA-MB-231, H9C2, L929, LO2, HEK293[1] Tested Tested Concentrations: 0-5 μM Incubation Duration: 72 hrs (hours) Experimental Results: demonstrated high anti-proliferation activities on cancer cell lines, as well as certain toxicity on normal cell lines. Western Blot Analysis Cell Types: HCT116 cells[1] Tested Tested Concentrations: 0.001, 0.01, 0.1, 1 and 10 μM Incubation Duration: 4 and 24 hrs (hours) Experimental Results: Inhibited the phosphorylation of FAK and its downstream proteins from multiple pathways. Cell Cycle Analysis Cell Types: HCT116 cells[1] Tested Tested Concentrations: 0.01, 0.05, 0.1 and 0.5 μM for 24 hrs (hours); 0.01, 0.05, 0.1, 0.3 and 1 μM for 48 hrs (hours) Incubation Duration: 24 and 48 hrs (hours) Experimental Results: Induced strong cell cycle arrest at the G2/M phase and apoptosis.
Animal Protocol	Animal/Disease Models: Female Balb/C nu/nu (nude) mice (HCT116-injected)[1] Doses: 5 and 15 mg/kg Route of Administration: 16 days; one time/day Experimental Results: Displayed potent antitumor effects in a dose-dependent manner without significant toxicity.
References	[1]. Design, synthesis, and biological evaluation of novel covalent inhibitors targeting focal adhesion kinase. Bioorg Med Chem Lett. 2021;54:128433.

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	1.7760 mL	8.8802 mL	17.7604 mL
	5 mM	0.3552 mL	1.7760 mL	3.5521 mL
	10 mM	0.1776 mL	0.8880 mL	1.7760 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.