CCR4-351 is a potent and selective CCR4 clamp antagonist. CCR4-351 has a novel carboxyl-azetidine motif with IC50 of 22 nM and 50 nM in calcium flux and CTX assays. CCR4-351 has anti-tumor activity.

Physicochemical Properties

Molecular Formula	C24H27CL2N7O
Molecular Weight	500.42
Exact Mass	499.165
CAS #	2174938-70-4
Related CAS #	CCR4-351 hydrochloride;2174938-71-5
PubChem CID	134225255
Appearance	Light yellow to yellow solid powder
LogP	3.7
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	6
Heavy Atom Count	34
Complexity	746
Defined Atom Stereocenter Count	2
SMILES	C12N([C@@H](C3=CC=C(Cl)C=C3Cl)C)N=C(C#N)C1=NC=C(N1CC([C@H]3CCCN(CCO)C3)C1)N=2
InChi Key	PNCGVFJZWKHMCL-CVEARBPZSA-N
InChi Code	InChI=1S/C24H27Cl2N7O/c1-15(19-5-4-18(25)9-20(19)26)33-24-23(21(10-27)30-33)28-11-22(29-24)32-13-17(14-32)16-3-2-6-31(12-16)7-8-34/h4-5,9,11,15-17,34H,2-3,6-8,12-14H2,1H3/t15-,16+/m1/s1
Chemical Name	1-[(1R)-1-(2,4-dichlorophenyl)ethyl]-6-[3-[(3R)-1-(2-hydroxyethyl)piperidin-3-yl]azetidin-1-yl]pyrazolo[3,4-b]pyrazine-3-carbonitrile
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, avoid exposure to moisture.
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	CCR4-351 (compound 38) had little activity in CYP450 induction tests [1]. The IC50 of CCR4-351 is 39 nM for mouse iTreg cells and 33 nM for human iTreg cells, respectively, to inhibit their migration [1].
ln Vivo	CCR4-351 (Compound 38; 50 mg/kg; oral; daily; for 40 days) effectively suppresses tumor development [1]. CCR4-351 (0.5 mg/kg; IV) had low clearance (CL=10.2 mL/min/kg), medium volume of distribution (Vss=5.2 L/kg), T1/2 of 6.9 h, and good bioavailability (%F = 29) Orally given to mice [1]. CCR4-351 has a low clearance (CL=7.3 mL/min/kg), a half-life of 12.7 hours, and a bioavailability of 44% in dogs. CCR4-351 has low clearance (CL=3.7 mL/min/kg), extended terminal half-life (10.7 hours), and good bioavailability (%F = 41) in cynomolgus monkeys [1].
Animal Protocol	Animal/Disease Models: 6 to 8 weeks old female C57BL/6 mice with Pan02-OVA tumors [1] Doses: 50 mg/kg Route of Administration: Oral; daily; continued for 40 days Experimental Results: Significant reduction in tumor growth . Animal/Disease Models: Rat and Mouse[1] Doses: 0.5 mg/kg IV; 2 mg/kg PO Route of Administration: IV or PO Experimental Results: Rats had moderate clearance after oral administration (CL=47.6 mL/min /kg) and the bioavailability is 49%. Oral administration to mice demonstrated low clearance (CL=10.2 mL/min/kg), medium volume of distribution (Vss=5.2 L/kg), T1/2 of 6.9 hrs (hrs (hours)), and good bioavailability (%F = 29) .
References	[1]. Novel Piperidinyl-Azetidines as Potent and Selective CCR4 Antagonists Elicit Antitumor Response as Single Agent and in Combination with Checkpoint Inhibitors. J Med Chem. 2020 Jul 15.

Solubility Data

Solubility (In Vitro)

DMSO : ~190 mg/mL (~379.68 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	1.9983 mL	9.9916 mL	19.9832 mL
	5 mM	0.3997 mL	1.9983 mL	3.9966 mL
	10 mM	0.1998 mL	0.9992 mL	1.9983 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.