CLK8 is a potent and specific CLOCK inhibitor that disrupts the interaction between CLOCK and BMAL1 and interferes with CLOCK nuclear translocation. CLK8 may be utilized to study diseases associated with weakened circadian rhythms.

Physicochemical Properties

Molecular Formula	C29H26N2O6
Molecular Weight	498.526547908783
Exact Mass	498.179
CAS #	898920-65-5
PubChem CID	4869040
Appearance	White to off-white solid powder
LogP	4.4
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	6
Heavy Atom Count	37
Complexity	910
Defined Atom Stereocenter Count	0
SMILES	C(N)(=O)C1=CC=CC=C1NC(COC1=C2C(C3=CC=CC=C3)=CC(=O)OC2=C2CCC(C)(C)OC2=C1)=O
InChi Key	SVELPNHVEDZZRQ-UHFFFAOYSA-N
InChi Code	InChI=1S/C29H26N2O6/c1-29(2)13-12-19-22(37-29)15-23(35-16-24(32)31-21-11-7-6-10-18(21)28(30)34)26-20(14-25(33)36-27(19)26)17-8-4-3-5-9-17/h3-11,14-15H,12-13,16H2,1-2H3,(H2,30,34)(H,31,32)
Chemical Name	2-[[2-[(8,8-dimethyl-2-oxo-4-phenyl-9,10-dihydropyrano[2,3-h]chromen-5-yl)oxy]acetyl]amino]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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
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	CLOCK[1]
ln Vitro	In U2OS and NIH 3T3 cells, CLK8 (10-40 μM; 4-6 d) increases the amplitude of the Bmal1-dLuc signal in a dose-dependent manner without causing periodic variations [1]. In HEK293T cells, CLK8 (10–40 μM) diminishes the BMAL1–CLOCK connection while having no effect on the interactions between CLOCK–F80A, K220A, and BMAL1 [1]. In U2OS cells, CLOCK's nuclear localization is decreased by CLK8 (20 μM; 2 d) [1].
ln Vivo	In mouse liver whole cell lysates, CLK8 (25 mg/kg; a single ip) lowers CLOCK levels while leaving BMAL1 and CRY1 levels unaltered[1]. At dosages of 5 and 25 mg/kg, CLK8 (5-1000 mg/kg; ip) did not exhibit any mortality or clinical signs (dyspnea, hyporeflexia, decreased motor activity, piloerection, stooped posture, and corneal opacification)[1].
Animal Protocol	Animal/Disease Models: Male C57BL/6J mice (8 weeks, 18-24 g)[1] Doses: 25 mg/kg Route of Administration: A single ip Experimental Results: A decrease in CLOCK levels was detected in whole cell lysates of the mouse livers, whereas the levels of BMAL1 and CRY1 were unaltered. diminished the abundance of CLOCK in the nucleus. The abundances of cytosolic and nuclear BMAL1 and CRY1 were unaltered. diminished Cry1 transcriptional level.
References	[1]. A CLOCK-binding small molecule disrupts the interaction between CLOCK and BMAL1 and enhances circadian rhythm amplitude. J Biol Chem. 2020 Mar 13;295(11):3518-3531.

Solubility Data

Solubility (In Vitro)

DMSO: 50 mg/mL (100.29 mM)

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	2.0059 mL	10.0295 mL	20.0590 mL
	5 mM	0.4012 mL	2.0059 mL	4.0118 mL
	10 mM	0.2006 mL	1.0029 mL	2.0059 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.