ETI41 targets endosomal Toll-like receptors (TLRs), specifically binding to the nucleoside-binding Site I of TLR7 (IC₅0 = 0.63 microM) and TLR9 (IC₅0 = 0.16 microM), while sparing surface TLRs including TLR1/TLR2, TLR2/TLR6, TLR4, and TLR5. Endosomal TLRs (TLR3, TLR7, TLR8, TLR9) are localized within endosomal compartments of immune cells and recognize pathogen-associated molecular patterns; their aberrant activation is implicated in inflammatory and autoimmune diseases. By binding to TLR7 and TLR9, ETI41 blocks the recruitment of adaptor proteins such as MyD88, thereby inhibiting downstream pro-inflammatory signaling pathways. The compound also affects p38 MAPK, TNF Receptor, ERK, JNK, NF-kappaB, and Interleukin Related pathways, as confirmed by immunoblotting and biophysical analyses.

ETI41 (0-200 μM, 4-24 h) effectively inhibited the production of TNF-α in mouse macrophages (RAW 264.7) and human B lymphoblast (Daudi) cell lines induced by TLR agonists (e.g., Imiquimod (IMQ) for TLR7 and ODN2395 for TLR9) in a dose-dependent manner without inducing cytotoxic effects [1]. ETI41 (24 h) showed inhibitory activity against endosome TLRs with IC50 values ranging from approximately 100 to 1,000 nM, and inhibited the activity of TLR3, TLR7 and TLR8 in a concentration-dependent manner within a concentration range of 31.2 nM to 10 μM [1]. ETI41 (5-10 μM, 20 min-8 h) inhibited IMQ or ODN2395-induced phosphorylation of MAPKs (p-ERK, p-JNK, and p-p38), nuclear translocation of the NF-κB p65 subunit, Iκ-Bα degradation, and IRF7 expression in RAW 264.7 cells [1]. ETI41 (0.2-1 μM, 30 min) significantly inhibited the production of IL-12p40, IFN-β, and CD40 in primary bone marrow-derived dendritic cells (BMDCs) stimulated with ODN2395 [1]. ETI41 (10 μM, 2-4 h) can attenuate the upregulation of multiple inflammation-related genes induced by IMQ, including IL1-β, CXCL2, IL18RAP, TNF, PDCD1, NLRP3, NFKBIZ, CCL3, CCL4, KDM6B, ZC3H12A and PTGS2[1].

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In vitro, ETI41 (0‑200 microM, 4‑24 h) dose-dependently inhibits TNF-alpha production in mouse RAW 264.7 macrophages and human Daudi B lymphoblasts stimulated with TLR7 agonist IMQ or TLR9 agonist ODN2395, without inducing cytotoxicity. ETI41 (24 h) inhibits endosomal TLRs with IC₅0 values ranging from approximately 100 to 1,000 nM, and within 31.2 nM to 10 microM concentration-dependently inhibits TLR3, TLR7, and TLR8 activity. ETI41 (5‑10 microM, 20 min‑8 h) blocks IMQ or ODN2395‑induced phosphorylation of MAPKs (p‑ERK, p‑JNK, p‑p38), nuclear translocation of NF‑kappaB p65, Ikappa‑Balpha degradation, and IRF7 expression. ETI41 (0.2‑1 microM, 30 min) significantly inhibits IL‑12p40, IFN‑beta, and CD40 production in ODN2395‑stimulated primary bone marrow‑derived dendritic cells. At 10 microM (2‑4 h), it attenuates IMQ‑induced upregulation of multiple inflammation‑related genes including IL1‑beta, CXCL2, TNF, NLRP3, CCL3, and CCL4.

ETI41 (60 mg/kg, orally, once daily from day 2 to day 5) improved IMQ-induced electrode psoriasis[1]. ETI41 (60 mg/kg, orally, once daily from day 2 to day 9) improved IL-23-induced electrode psoriasis[1]. ETI41 (30 mg/kg, orally, once daily for 39 days) effectively improved symptoms in a systemic lupus erythematosus (SLE) model[1].

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In vivo, ETI41 demonstrates therapeutic efficacy in mouse autoimmune models following oral administration. In an IMQ‑induced psoriasis mouse model, ETI41 (60 mg/kg, orally, once daily from day 2 to day 5) improves psoriasis‑like symptoms. In an IL‑23‑induced psoriasis model, ETI41 (60 mg/kg, orally, once daily from day 2 to day 9) also reduces disease severity. In an MRL/MpJ‑Fasˡᵖʳ/ᴶ SLE mouse model, ETI41 (30 mg/kg, orally, once daily for 39 days) effectively ameliorates SLE symptoms, including reduced alopecia and skin rash, decreased relative lymph node weight, and modulation of serum complement C3 levels. RNA sequencing revealed that ETI41 modulates expression of inflammation‑associated genes. All compounds were dissolved in a vehicle comprising ethanol, PEG400, and distilled water at a 1:4:5 ratio. These findings highlight the therapeutic potential of ETI41 for treating autoimmune diseases.

Non‑cellular biochemical assays for ETI41 typically involve biophysical analyses to confirm selective binding to endosomal TLRs without affecting surface TLRs. Immunoblotting is performed to assess TLR inhibition and downstream signaling blockade. Surface plasmon resonance (SPR) or similar binding assays are used to determine the binding affinity of ETI41 to purified TLR7 and TLR9 proteins, calculating IC₅0 values from concentration‑response curves (TLR7 IC₅0 = 0.63 microM, TLR9 IC₅0 = 0.16 microM). Additionally, a pH assay using RAW 264.7 cells treated with ETI41 and pHrodo Red Dextran or LysoSensor Yellow/Blue DND‑160 may be conducted to assess effects on lysosomal pH, which is relevant to endosomal TLR function. These cell‑free binding and biophysical protocols are standard for characterizing selective TLR inhibitors.

Cell Viability Assay[1]
Cell Types: murine RAW 264.7 and human Daudi cells
Tested Concentrations: 1.6-200 μM
Incubation Duration: 24 h
Experimental Results: Exhibited no cytotoxicity in both murine RAW 264.7 and human Daudi cells at concentrations up to 10 μM.

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Western Blot Analysis[1]
Cell Types: RAW 264.7 cells
Tested Concentrations: 5 and 10 μM
Incubation Duration: 20, 30, 40, and 50 min, 6 and 8 h
Experimental Results: Reduced p-ERK, p-JNK and p-p38 levels. Suppressed the nuclear translocation of the NF-κB p65 subunit. Downregulated IRF7 expression and prevented the degradation of Iκ-Bα.

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In vitro cell‑based assays are performed using murine RAW 264.7 macrophages, human Daudi B lymphoblasts, and primary bone marrow‑derived dendritic cells (BMDCs). Cells are seeded in multi‑well plates and pre‑treated with a dose range of ETI41 (0.2 microM to 200 microM) for 30 min to 24 h. Cells are then stimulated with TLR7 agonist IMQ (2 microM) or TLR9 agonist ODN2395 (1 microM). TNF‑alpha production in supernatant is quantified by ELISA. MAPK phosphorylation (p‑ERK, p‑JNK, p‑p38), NF‑kappaB p65 nuclear translocation, Ikappa‑Balpha degradation, and IRF7 expression are assessed by Western blotting. IL‑12p40, IFN‑beta, and CD40 production in BMDCs are measured by ELISA. Total mRNA is extracted using TRIzol, reverse transcribed, and inflammation‑related gene expression is analyzed by qRT‑PCR. Cell viability is assessed using CCK‑8 or MTT assays to confirm no cytotoxicity.

Animal/Disease Models: Female C57BL/6J mice (6 weeks old) induced with psoriasis via intradermal injection of recombinant murine IL-23 (500 ng) around the ear daily for 8 days[1]
Doses: 60 mg/kg
Route of Administration: p.o., daily from day 2 to day 9
Experimental Results: Improved disease symptoms comparable to or superior to the positive control (anti-IL-17A antibody, 30 mg/kg, i.p., dose on day 2, 5, and 8), without significant differences in body weight. Significantly decreased ear and epidermal thicknesses, CD68 expression and Ki-67 keratinocyte proliferation.

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Animal/Disease Models: Female C57BL/6J mice (6 weeks old) induced with psoriasis via daily topical application of Aldara cream (IMQ, 62.5 mg/cm²) for 4 days post-shaving[1]
Doses: 60 mg/kg
Route of Administration: p.o., daily from day 2 to day 5
Experimental Results: Significantly reduced the Psoriasis Area and Severity Index (PASI) scores, epidermal acanthosis, dermal thickness and keratinocyte proliferation. Inhibited IL-17A and IL-23 expression and dermal inflammatory cell infiltration.

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Animal/Disease Models: Female MRL/MpJ-Faslpr/J lupus-prone mice (14 weeks old)[1]
Doses: 30 mg/kg
Route of Administration: p.o., daily for 39 days
Experimental Results: Prevented weight gain or loss and decreased lymphnode weights. Reduced alopecia and skin rashes compared with the vehicle and HCQ (60 mg/kg, P.O., daily for 39 days). Reduced serological markers associated with SLE, such as antinuclear antibody (ANA), anti-dsDNA antibodies and IgG in the kidney, compared to HCQ group. Demonstrated significant reduction in SLE symptoms at half the dose of HCQ, suggesting high efficacy.

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In vivo animal studies are conducted using female C57BL/6J mice (6‑weeks‑old) for psoriasis models and MRL/MpJ‑Fasˡᵖʳ/ᴶ mice for SLE models. For IMQ‑induced psoriasis, Aldara cream (62.5 mg/cm2) is applied topically daily from day 1 to day 4. ETI41 is administered orally at 60 mg/kg daily from day 2 to day 5. For IL‑23‑induced psoriasis, recombinant mouse IL‑23 (0.25 mg/kg) is injected intradermally around the ear daily. ETI41 is administered orally at 60 mg/kg daily from day 2 to day 9. Psoriasis Area and Severity Index (PASI) scores (erythema, scaling, thickness) are recorded daily. For SLE models, ETI41 is administered orally at 30 mg/kg daily for 39 days, with HCQ (60 mg/kg) as positive control. Body weight is measured every 3 days. At endpoint, skin, blood, and lymph node samples are collected for histology, ELISA, and RNA‑seq analysis. All compounds are dissolved in a vehicle comprising ethanol, PEG400, and distilled water at a 1:4:5 ratio.

Detailed pharmacokinetic parameters (half‑life, Cₘₐₓ, AUC, bioavailability, volume of distribution, clearance, protein binding) for ETI41 have not been fully disclosed in the public literature. However, as an orally active compound with demonstrated in vivo efficacy in mouse models at doses of 30‑60 mg/kg (oral), ETI41 exhibits sufficient oral absorption and systemic exposure to achieve therapeutic effects. The compound is formulated in a vehicle comprising ethanol, PEG400, and distilled water at a 1:4:5 ratio for oral administration in animal studies. Storage recommendations: powder at ‑20 degC for up to 3 years; in solvent at ‑20 degC for up to 6 months. The compound is stable at ambient temperature for up to one month as a powder during shipping.

Safety Data Sheet (SDS) classifications for ETI41 vary by supplier. One SDS reports no hazardous classification, stating the substance is "not a hazardous substance or mixture" under GHS criteria, with no GHS hazard statements and no other hazards identified. However, another SDS reports an acute toxicity oral Category 4 (H302: Harmful if swallowed) and aquatic toxicity Category 1 (H400/H410: Very toxic to aquatic life with long‑lasting effects). Precautionary statements include P264 (wash skin thoroughly after handling), P270 (do not eat, drink or smoke when using), P273 (avoid release to the environment), P301+P312 (if swallowed and feeling unwell, call a poison center/physician), P330 (rinse mouth), and P391 (collect spillage). Recommended personal protective equipment includes gloves, lab coat, and safety goggles. The compound is for research use only and not for human or veterinary use.

[1]. Discovery of ETI41 and ETI41: novel selective endosomal Toll-like receptor inhibitors for the treatment of autoimmune diseases. Exp Mol Med. 2025 Sep;57(9):1951-1962.

ETI41 is a research‑grade small molecule that has not entered clinical trials and is not approved by any regulatory authority (FDA, EMA, PMDA) for human therapeutic use. It was developed and reported by Kim et al., with the primary reference being Experimental & Molecular Medicine (2025, DOI: 10.1038/s12276‑025‑01526‑w). The mechanism of action involves selective binding to endosomal TLR7 and TLR9 at their nucleoside‑binding Site I, blocking MyD88‑dependent pro‑inflammatory signaling pathways including MAPK phosphorylation, NF‑kappaB activation, and IRF expression, thereby inhibiting production of inflammatory cytokines (TNF‑alpha, IL‑1beta, IL‑6, IL‑12p40) and type I interferons (IFN‑beta). The compound is labeled exclusively as “for research use only” and is available for preclinical autoimmune disease research focusing on psoriasis, systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. Its molecular weight is 314.47, purity >98%, and CAS number is 2773474‑99‑8.

C19H30N4

314.47

2773474-99-8

Typically exists as solids at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)