**Background:** Obesity is a major health problem with increasing prevalence, predisposing to insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular diseases. Current pharmacotherapies primarily reduce energy intake but are limited by compensatory metabolic adaptations and side effects. Mitochondrial uncouplers, such as 2,4-dinitrophenol, increase energy expenditure by dissipating the proton gradient across the inner mitochondrial membrane, but their systemic use is limited by a narrow therapeutic window and toxicities including hyperthermia, arrhythmia, and cataracts. Recent studies have shown that targeting uncouplers to specific tissues, such as the liver or adipose tissue, can improve safety. This study aimed to achieve adipose tissue-selective accumulation of uncouplers by conjugating them with hydrophobic hydrocarbon chains via an ether bond.
**Methods:** The authors synthesized FCCP analogs with alkyl ether groups of varying chain lengths (C1, C4, C8, C12) and selected the C8-conjugated analog (NMY1009) for further study. They also synthesized 2,6-dinitro-4-(octyloxy)phenol, a C8-conjugated analog of 2,6-dinitrophenol. Uncoupling activity was measured in isolated mouse liver mitochondria using a Clark-type oxygen electrode and in differentiated 3T3 L1 adipocytes using a Seahorse flux analyzer. In vivo toxicity was assessed by daily intraperitoneal (i.p.) injections of NMY1009 at doses from 1 to 100 mg/kg for seven days. Pharmacokinetics were evaluated by measuring plasma and tissue concentrations after a single i.p. dose of 5 mg/kg NMY1009 or 2,6-dinitro-4-(octyloxy)phenol. Functional effects were studied in mice fed a high-fat diet (45% fat) for three weeks with daily i.p. administration of 3 mg/kg NMY1009, measuring body composition, blood markers, glucose tolerance, and energy expenditure via indirect calorimetry. Metabolic stability was assessed by incubating NMY1009 and FCCP with rat liver microsomes and in aqueous buffer, with drug concentrations measured by LC-MS/MS. Glutathione (GSH) adduct formation was analyzed by ESI mass spectrometry.
**Key Results:** NMY1009 and 2,6-dinitro-4-(octyloxy)phenol exhibited potent uncoupling activity. In isolated mitochondria, NMY1009 had an EC50 of approximately 100 nM, slightly less potent than FCCP. 2,6-dinitro-4-(octyloxy)phenol had an EC50 of 3.7 μM, compared to 136 μM for 2,6-dinitrophenol and 9.6 μM for 2,4-dinitrophenol. In vivo, NMY1009 showed selective accumulation in adipose tissue, with highest drug concentrations in different adipose depots and very low levels in other tissues. However, no significant functional effects were observed after three weeks of treatment: there were no changes in body weight, fat mass, blood glucose, insulin, triglycerides, free fatty acids, 3-hydroxybutyrate, leptin, or glucose tolerance (AUC 2109 ± 179 vs 2090 ± 288 mmol/l × min for vehicle vs NMY1009). Energy expenditure and respiratory exchange ratio were also unchanged. NMY1009 was found to be metabolically unstable, with a half-life of approximately 30 min in rat liver microsomes and rapid degradation in aqueous buffer (about 18% remained after 24 h). Mass spectrometry revealed O-dealkylation (loss of the octyl group) as the primary degradation pathway. Both FCCP and NMY1009 formed adducts with GSH, but GSH reactivity did not significantly contribute to NMY1009 metabolism. Similarly, 2,6-dinitro-4-(octyloxy)phenol showed very low tissue concentrations and rapid plasma clearance, indicating instability.
**Clinical Implications:** This study demonstrates that conjugation of mitochondrial uncouplers with a C8-hydrocarbon chain via an ether bond can achieve potent uncoupling activity and selective adipose tissue accumulation. However, the rapid metabolic instability of these compounds, likely due to O-dealkylation, prevented any therapeutic effect in vivo. The findings suggest that alternative conjugation strategies, such as direct C–C linkage of the hydrocarbon chain to the phenolic ring, may be necessary to achieve stable, adipose-selective uncouplers. The approach remains promising, as previous work with ester-linked conjugates (e.g., tetradecanoic acid-2,4-dinitrophenol) has shown efficacy and improved safety. Future studies should focus on developing metabolically stable lipophilic uncouplers to harness the potential of adipose tissue-targeted mitochondrial uncoupling for obesity treatment.