CLK1-IN-3 (compound 10ad) is a potent and specific Clk1 inhibitor (antagonist) with IC50 of 5 nM and is over 300-fold selective over Dyrk1A. CLK1-IN-3 also showed relatively potent inhibitory effects on Clk2 and Clk4, with IC50s of 42 and 108 nM, respectively. CLK1-IN-3 effectively induces autophagy in vitro. CLK1-IN-3 may be utilized in acute liver injury (ALI) research.

Physicochemical Properties

Molecular Formula	C24H23FN6O
Molecular Weight	430.477427721024
Exact Mass	430.191
CAS #	2922550-28-3
PubChem CID	168355546
Appearance	Off-white to light yellow solid powder
LogP	2.7
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	32
Complexity	636
Defined Atom Stereocenter Count	0
InChi Key	LQKHTRXKHPHEPP-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H23FN6O/c1-30-6-8-31(9-7-30)20-4-2-16(3-5-20)24(32)29-19-11-21-22(15-28-23(21)27-14-19)17-10-18(25)13-26-12-17/h2-5,10-15H,6-9H2,1H3,(H,27,28)(H,29,32)
Chemical Name	N-[3-(5-fluoropyridin-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-4-(4-methylpiperazin-1-yl)benzamide
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	CLK1 5 nM (IC50) CLK2 42 nM (IC50) CLK4 108 nM (IC50) DYRK1A 1521 nM (IC50)
ln Vitro	CLK1-IN-3 (compound 10ad) has the capacity to inhibit both Clk1 and Clk2, which gives it anti-tumor potential [1]. In a dose-dependent manner, CLK1-IN-3 (10 μM-1000 μM) may bind to Clk1 protein and efficiently block its degradation [1]. In HeLa, BNLCL.2, and HCT 116 cells, CLK1-IN-3 (0–10 μM, 24 h) initiates autophagy [1]. The autolysosomal marker SQSTM1/p62 is degraded more readily when CLK1-IN-3 is present [1].
ln Vivo	In the acetaminophen (HY-66005, APAP)-induced ALI model, CLK1-IN-3 (compound 10ad) (0-40 mg/kg, intraperitoneal injection, once) can significantly inhibit ALI without obvious hepatocyte death[1]. With an acceptable pharmacokinetic profile and a relatively long half-life of 5.29 hours, CLK1-IN-3 (10 mg/kg; IV, PO, IP, once) has an oral bioavailability of 19.5%[1].
Cell Assay	Western Blot Analysis[1] Cell Types: Hela cells, BNLCL.2 and HCT 116 cells Tested Concentrations: 0.2, 1, 5, and 10 μM Incubation Duration: 24 h Experimental Results: Elevated the expression level of LC3II protein (a marker of autophagosomes) as well as the ratio of LC3II to LC3I (a sensitive index of autophagy) in a dose-dependent and time-dependent manner.
Animal Protocol	Animal/Disease Models: Male C57BL/6 mice(8 weeks, injected acetaminophen (HY-66005) (500 mg/kg, ip))[1] Doses: 10, 20, and 40 mg/kg Route of Administration: IP, once Experimental Results: diminished serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels Dramatically in dose-dependent. Animal/Disease Models: Male balb/c (Bagg ALBino) mouse: (aged 8 weeks)[1] Doses: 10 mg/kg Route of Administration: IV, PO, IP, once (pharmacokinetic/PK Analysis) Experimental Results: pharmacokinetic/PK Parameters of CLK1-IN-3 in male balb/c (Bagg ALBino) mouse:[1]. IV (10 mg/kg) PO ( 10 mg/kg) IP (10 mg/kg) Cmax (ng/mL) 13166.5±875.9 1457.4±177.3 4654.3±435.3 T1/2 (h) 2.96±1.2 5.29±2.1 3.27 ±1.1 AUC0-t (ng/mL* h) 9520.5±1011.3 1860.2±411.0 5010.4±987.2 CL (L/h/kg) 1.05±0.10 5.51±1.00 3.58±0.82 F (%) 19.5%
References	[1]. Rational design and appraisal of selective Cdc2-Like kinase 1 (Clk1) inhibitors as novel autophagy inducers for the treatment of acute liver injury (ALI). Eur J Med Chem. 2023 Mar 15;250:115168.

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	2.3230 mL	11.6149 mL	23.2299 mL
	5 mM	0.4646 mL	2.3230 mL	4.6460 mL
	10 mM	0.2323 mL	1.1615 mL	2.3230 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.