CU-CPT-8m is a novel, potent and specific antagonist of Toll-like receptor 8 (TLR8) with immunomodulatory effects. It inhibits TLR8 with an IC50 of 67 nM. Endosomal Toll-like receptors (TLR3, TLR7, TLR8, and TLR9) are highly analogous sensors for various viral or bacterial RNA and DNA molecular patterns. Nonetheless, few small molecules can selectively modulate these TLRs. CU-CPT-8m is the first human TLR8-specific small-molecule antagonists via a novel inhibition mechanism. Crystal structures of two distinct TLR8-ligand complexes validated a unique binding site on the protein-protein interface of the TLR8 homodimer. Upon binding to this new site, the small-molecule ligands stabilize the preformed TLR8 dimer in its resting state, preventing activation. CU-CPT-8m is able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens. These results not only suggest a novel strategy for TLR inhibitor design, but also shed critical mechanistic insight into these clinically important immune receptors.

Physicochemical Properties

Molecular Formula	C14H12N4O
Molecular Weight	252.271
Exact Mass	252.101
CAS #	125079-83-6
Related CAS #	125079-83-6(CU-CPT-8m);
PubChem CID	15042086
Appearance	White to off-white solid powder
LogP	1.2
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	2
Heavy Atom Count	19
Complexity	348
Defined Atom Stereocenter Count	0
SMILES	O=C(C1=C2N=CC=C(C3=CC=CC(C)=C3)N2N=C1)N
InChi Key	HNKGGVGQAVODNJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C14H12N4O/c1-9-3-2-4-10(7-9)12-5-6-16-14-11(13(15)19)8-17-18(12)14/h2-8H,1H3,(H2,15,19)
Chemical Name	7-(m-tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Synonyms	CU-CPT-8m; CU-CPT 8m; CU-CPT8m;CU CPT 8m
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	CU-CPT-8m is a selective TLR8 antagonist, with an IC50 of 67±10 nM and low cytotoxicity. The Kd value of CU-CPT-8m is determined to be 220 nM. CU-CPT-8m solely suppresses the proinflammatory response in the TLR8-overexpressing cells strongly confirms that CU-CPT-8m directly recognizes TLR8 in cells. It is particularly remarkable that TLR7 signaling is not impacted at doses up to 75 μM. TLR7 and TLR8 are closely related and have several similar ligands. Treatment of 1 μM CU-CPT-8m totally abolishes the rise of TNF-α and IL-8 mRNA levels produced by R848. CU-CPT-8m suppresses R848-induced TNF-α production in the differentiated THP-1 monocytes cells in a dose- dependent manner with an IC50 of 90±10 nM, which is in good agreement with its IC50 value found in HEK-Blue TLR8 cells[1].
ln Vivo
Animal Protocol
References	[1]. Small-molecule inhibition of TLR8 through stabilization of its resting state. Nat Chem Biol. 2018 Jan;14(1):58-64.

Solubility Data

Solubility (In Vitro)

DMSO: >30 mg/mL Water:<1mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (9.91 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 1.25 mg/mL (4.96 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 1.25 mg/mL (4.96 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.9640 mL	19.8200 mL	39.6401 mL
	5 mM	0.7928 mL	3.9640 mL	7.9280 mL
	10 mM	0.3964 mL	1.9820 mL	3.9640 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.