CU-CPT17e is a multi-TLR (Toll-like receptor) agonist that activates TLR3, TLR8, and TLR9 with immunomodulatory activities. Therapies based on activation of multiple Toll-like receptors (TLRs) may offer superior therapeutic profiles than that of single TLR activation. CU-CPT17e was identified from a cell-based high-throughput screening of a small-molecule library based on TLR3-mediated NF-κB activation. Biochemical studies demonstrated that CU-CPT17e could induce a strong immune response via the production of various cytokines in human monocytic THP-1 cells. Furthermore, CU-CPT17e inhibited the proliferation of HeLa cancer cells by triggering apoptosis and arresting the cell cycle at the S phase. These results showcase potential therapeutic applications of CU-CPT17e in both vaccine adjuvants and anticancer therapies based on multi-TLR activation.

Physicochemical Properties

Molecular Formula	C27H24N2O8
Molecular Weight	504.488067626953
Exact Mass	504.153
CAS #	2109805-75-4
Related CAS #	2109805-75-4
PubChem CID	132585194
Appearance	Light yellow to yellow solid powder
LogP	5
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	6
Heavy Atom Count	37
Complexity	804
Defined Atom Stereocenter Count	0
SMILES	O1C2C=C(C(=CC=2C=CC21CCOCC2)OCC1C=CC(=CC=1)[N+](=O)[O-])OCC1C=CC(=CC=1)[N+](=O)[O-]
InChi Key	LHTNFHWDTHQECR-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H24N2O8/c30-28(31)22-5-1-19(2-6-22)17-35-25-15-21-9-10-27(11-13-34-14-12-27)37-24(21)16-26(25)36-18-20-3-7-23(8-4-20)29(32)33/h1-10,15-16H,11-14,17-18H2
Chemical Name	6,7-bis[(4-nitrophenyl)methoxy]spiro[chromene-2,4'-oxane]
Synonyms	CU-CPT-17e; CU-CPT17e;CU CPT17e;CU-CPT 17e;CUCPT17e
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-CPT17e showed substantial NF-κB activation in TLR3, TLR8 and TLR9 HEK293 cells, with EC50 values of 4.80±0.73, 13.5±0.58 and 5.66±0.17 μM, respectively. CU-CPT17e dramatically boosted NF-κB activity, boosting NF-κB activation by 13.9±0.9 times, with an EC50 value of 4.8±0.7 μM. CU-CPT17e suppresses the proliferation of HeLa carcinoma cells by activating apoptosis and stopping the cell cycle in S phase. The induction of apoptosis by CU-CPT17e in HeLa cells was examined. HeLa cells were grown in increasing doses of CU-CPT17e or poly I:C or blank control (DMSO) for 24 h. Treatment with CU-CPT17e at varied concentrations (10 to 40 μM) for 24 hours resulted in a 10% to 17% rise in the apoptotic cell population, which was more effective than poly I:C at 5 μg/mL. These data imply that the antiproliferative activity of CU-CPT17e on HeLa cells may be due to its ability to directly trigger apoptosis [1].
ln Vivo
Animal Protocol
References	[1]. Discovery of Small Molecules as Multi-Toll-like Receptor Agonists with Proinflammatory and Anticancer Activities. J Med Chem. 2017 Jun 22;60(12):5029-5044.

Solubility Data

Solubility (In Vitro)

DMSO: 5 mg/mL Water:<1mg/mL Ethanol:<1 mg/mL

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	1.9822 mL	9.9110 mL	19.8220 mL
	5 mM	0.3964 mL	1.9822 mL	3.9644 mL
	10 mM	0.1982 mL	0.9911 mL	1.9822 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.