TLR7 agonist 2 is a novel, oral, potent and selective agonist of Toll-like Receptor 7 (TLR7) with antiviral activity. It activates TLR7 with a LEC of 0.4 μM. It has the potential for the immunotherapy of viral hepatitis. Oral administration of the TLR7 agonist 2 effectively induced a transient interferon stimulated gene (ISG) response in mice and cynomolgus monkeys.

Physicochemical Properties

Molecular Formula	C17H16N6O2
Molecular Weight	336.347942352295
Exact Mass	336.133
CAS #	1642857-69-9
PubChem CID	86346192
Appearance	White to off-white solid powder
LogP	1.3
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	5
Heavy Atom Count	25
Complexity	441
Defined Atom Stereocenter Count	0
SMILES	O(CC1C=C(C)ON=1)C1=C2C(C=CN2CC2C=CC=CN=2)=NC(N)=N1
InChi Key	FVKOYFLAKSGBBV-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H16N6O2/c1-11-8-13(22-25-11)10-24-16-15-14(20-17(18)21-16)5-7-23(15)9-12-4-2-3-6-19-12/h2-8H,9-10H2,1H3,(H2,18,20,21)
Chemical Name	4-[(5-methyl-1,2-oxazol-3-yl)methoxy]-5-(pyridin-2-ylmethyl)pyrrolo[3,2-d]pyrimidin-2-amine
Synonyms	TLR7 agonist 2; TLR7 agonist-2
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	LEC: 0.4 μM (TLR7)[1]
ln Vitro	TLR7 agonist 2: In HEK293 cells, it has a lowest effective concentration (LEC) of 0.4 μM and is a strong and specific Toll-like Receptor 7 (TLR7) agonist. When applied to human TLR8, TLR7 agonist 2 has a lethal dose of >100 μM and is proven to be selective for TLR7 over TLR8. In addition to not being a time-dependent inhibitor of CYP450 3A4, TLR7 agonist 2 exhibits low inhibition across five CYP450 isozymes (IC50 >10 μM). A limited suppression of 3H-dofetilide binding to the hERG potassium ion channel (IC50 >50 μM) is seen by TLR7 agonist 2[1].
ln Vivo	It is discovered that TLR7 agonist 2 is quickly eliminated in tandem with our target profile. Between 0.3 and 3 mg/kg, both Cmax and AUC increase more than dose-proportionally; between 3 and 10 mg/kg, they increase less than dose-proportionally. At modest doses, TLR7 agonist 2 can elicit an antiviral interferon stimulated gene (ISG) response without eliciting an IFNα response. Additionally, starting at 0.3 mg/kg, TLR7 agonist 2 reduces the serum HBV viral load by 2.7 logs, with a maximal reduction of 3.1 logs reported for doses between 1 and 5 mg/kg[1].
Cell Assay	The ability of TLR7-agonist-1 to activate human TLR7 and/or TLR8 is assessed by using HEK293 cells. Briefly, HEK293 cells are grown in culture medium (DMEM supplemented with 10% FCS and 2 mM Glutamine). Transfected cells are then detached with Trypsin-EDTA, washed in PBS and resuspended in medium to a density of 1.67×105 cells/mL. Thirty microliters of cells are then dispensed into each well in 384-well plates, where 10 μL of TLR7-agonist-1 in 4% DMSO is already present. Following 6 hours incubation at 37°C, 5% CO2, the luciferase activity is determined by adding 15 μL of Steady Lite Plus substrate to each well and readout performed on a microplate imager. Lowest effective concentrations (LEC) values are determined for TLR7-agonist-1[1].
References	[1]. Identification and Optimization of Pyrrolo[3,2-d]pyrimidine Toll-like Receptor 7 (TLR7) Selective Agonists for the Treatment of Hepatitis B. J Med Chem. 2017 Jul 27;60(14):6137-6151.

Solubility Data

Solubility (In Vitro)

DMSO : ~160 mg/mL (~475.69 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	2.9731 mL	14.8655 mL	29.7309 mL
	5 mM	0.5946 mL	2.9731 mL	5.9462 mL
	10 mM	0.2973 mL	1.4865 mL	2.9731 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.