C34

C34 is an orally active, selective Toll-like receptor 4 (TLR4) antagonist. C34 exerts anti-inflammatory effects by inhibiting the TLR4/MyD88/NF-κB signaling pathway, thereby reducing the production of pro-inflammatory cytokines^[1-2]. C34 alleviates systemic inflammation in various disease models, including endotoxemia, necrotizing enterocolitis, ulcerative colitis, and acute kidney injury. TLR4-IN-C34 is applicable for research in immunology, inflammation, and related disorders^[3-4].

In vitro, BV2 microglial cells were pretreated with C34 (10-100μM) for 2 hours, followed by LPS (200ng/mL) stimulation for 24 hours. C34 significantly inhibited LPS-induced activation of the TLR4/MyD88/NF-κB signaling pathway, downregulated the release of pro-inflammatory factors (TNF-α, IL-1β, IL-6, MCP-1, and NO), and reduced ROS generation and NLRP3 inflammasome activation^[5]. Rat enteric glial cells were pretreated with C34 (50μM) for 1 hour, then exposed to Clostridioides difficile toxins TcdA (50ng/mL) or TcdB (1ng/mL) for 18 hours. C34 significantly suppressed toxin-induced TLR4/NF-κB p65 nuclear translocation and TNF-α protein expression, and decreased apoptosis^[6].

In vivo, in an isoprenaline (ISO; 100mg/kg)-induced Wistar rat model, C34 (1mg/kg or 3mg/kg; administered intraperitoneally 1 hour before each of two ISO injections), C34 significantly reduced serum creatinine levels and ameliorated renal tissue inflammation, fibrosis, and apoptosis^[7]. In a Pilocarpine (40mg/kg; i.p.)-induced status epilepticus Wistar rat model, C34 (1μg/rat; single intracerebroventricular injection) was administered 1 hour or 24 hours after pilocarpine injection. Administration at 1 hour post-induction, C34 significantly reduced hippocampal neuronal death, decreased levels of the apoptotic factor caspase-3 and inflammatory mediators TNF-α and NF-κB, whereas treatment at 24 hours, C34 showed a markedly diminished therapeutic effect^[8].

References:
[1] Wipf P, Eyer BR, Yamaguchi Y, et al. Synthesis of anti-inflammatory α-and β-linked acetamidopyranosides as inhibitors of toll-like receptor 4 (TLR4). Tetrahedron Lett. 2015 Jun 3;56(23):3097-3100.
[2] Neal MD, Jia H, Eyer B, et al. Discovery and validation of a new class of small molecule Toll-like receptor 4 (TLR4) inhibitors. PLoS One. 2013 Jun 12;8(6):e65779.
[3] Wei K, Zou Y, Xu H, et al. TLR4-IN-C34 attenuates the progression of osteoarthritis through inhibiting inflammation, angiogenesis and pain. Cell Signal. 2025 Dec;136:112084.
[4] Filippova LV, Fedorova AV, Nozdrachev AD. Mechanism of Activation of Enteric Nociceptive Neurons via Interaction of TLR4 and TRPV1 Receptors. Dokl Biol Sci. 2018 Mar;479(1):44-46.
[5] Zhang SS, Liu M, Liu DN, et al. TLR4-IN-C34 Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses via Downregulating TLR4/MyD88/NF-κB/NLRP3 Signaling Pathway and Reducing ROS Generation in BV2 Cells. Inflammation. 2022 Apr;45(2):838-850.
[6] Barbosa MLL, Costa DVDS, de Pacífico DM, et al. Role of TLR4 in Enteric Glia Response to Clostridioides Difficile Toxins: Insights From In Vivo and In Vitro Studies. J Cell Mol Med. 2025 Nov;29(22):e70943.
[7] Abdelsalam HM, Helal MG, Abu-Elsaad NM. TLR4-IN-C34 protects against acute kidney injury via modulating TLR4/MyD88/NF-κb axis, MAPK, and apoptosis. Iran J Basic Med Sci. 2022 Nov;25(11):1334-1340.
[8] Varmazyar R, Naderi N, Javid H, et al. Neuroprotective Effects of Early TLR4 Blockade with Compound C34 in Temporal Lobe Epilepsy: Alleviation of Neuroinflammation and Apoptosis. Iran J Pharm Res. 2025 Mar 8;24(1):e159165.