Rigid Benzene Ring Substitution of Capsaicin's Double Bond: Synthesis of TRPV1 Agonist with Anti-fatigue Efficacy.

To search for safe and efficient anti-fatigue active molecules, 16 capsaicin (CAP) derivatives were synthesized by replacing the unsaturated carbon-carbon double bond in capsaicin with a rigid benzene ring via condensation, chlorination, and amidation reactions using vanillylamine hydrochloride as the starting material, with yields ranging from 44.1 to 79.1%. Their structures were confirmed by 1H-NMR, 13C-NMR, and MS (electrospray ionization [ESI]). In vitro assays demonstrated that N8 exerted superior transient receptor potential vanilloid 1 (TRPV1) agonistic activity compared to CAP at a concentration of 10 μM, upregulated peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) expression in a concentration-dependent manner (1.25-10 μM), and showed no significant toxicity to C2C12 myotube cells at 0.78-100 μM. In vivo evaluations in mice (15 mg/kg, 31-d gavage) demonstrated that N8 had no adverse effect on body weight but significantly prolonged rotarod duration (143.5%, p < 0.001) and forced swimming time (75.6%, p < 0.001), increased serum lactate dehydrogenase (LDH) levels (p < 0.01), decreased serum urea nitrogen (SUN) levels (p < 0.001) and lactic acid (LA) accumulation (p < 0.001), and elevated hepatic and muscle glycogen contents (p < 0.001) compared with the fatigue control group. Mechanistic studies via Western blot, mitochondrial fluorescence staining, cellular thermal shift assay, and molecular docking revealed that N8 had better binding stability to TRPV1 than CAP (relative binding rate at 65°C: 86.7 vs. 21.5%), activated the TRPV1 channel, synergistically upregulated the expression of cluster of differentiation 36 (CD36), carnitine palmitoyltransferase 1M (CPT1M), SURF1, and cytochrome c1 (CYC1), promoted mitochondrial biogenesis, and optimized muscle energy metabolism. These results indicate that N8 demonstrates superior anti-fatigue activity both in vitro and in vivo compared to CAP, making it a potential candidate for anti-fatigue drug development.