Journal of Medicinal Chemistry · 18 authors, 7 centres
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This study reports the design and synthesis of 19 2,7-diazaspiro[4.4]nonane derivatives as sigma receptor ligands. Compound AD258 (9d) showed high affinity for both sigma-1 and sigma-2 receptors (Ki S1R = 3.5 nM, Ki S2R = 2.6 nM) and exerted potent antiallodynic effects in a capsaicin-induced pain model at very low doses (0.6–1.25 mg/kg) without motor impairment. These findings identify AD258 as a promising lead for developing novel analgesics targeting sigma receptors.
**Background:** Pain is a major health issue with limited effective treatments. Sigma receptors (S1R and S2R) are emerging targets for analgesia. S1R antagonists have shown clinical promise, and S2R modulators may also contribute to pain relief. This study aimed to develop novel sigma receptor ligands based on a 2,7-diazaspiro[4.4]nonane scaffold and evaluate their analgesic potential.
**Methods:** Nineteen compounds were synthesized and tested for S1R and S2R binding affinity using radioligand assays ([3H]-(+)-pentazocine for S1R, [3H]DTG for S2R) in rat liver homogenates. Molecular docking studies were performed using crystal structures (PDB IDs 5HK1 and 7M95). The most promising compounds (8a, 9a, 9d, 14a) were evaluated for in vitro cytotoxicity in human corneal epithelial (HCE) cells using LDH and MTT assays. In vivo antiallodynic effects were assessed in a capsaicin-induced mechanical hypersensitivity model in mice. Motor coordination was tested using the rotarod test. Functional activity was determined by a phenytoin shift assay and by co-administration with the S1R agonist PRE-084. Selectivity profiling was performed against opioid, serotonin, cannabinoid, and NMDA receptors. ADMET properties (solubility, chemical/plasma stability, metabolic stability in liver microsomes, hERG inhibition) were also evaluated.
**Key Results:** Compound 9d (AD258) exhibited high binding affinity for both S1R (Ki = 3.5 nM) and S2R (Ki = 2.6 nM) with a Ki S2R/S1R ratio of 0.7. It showed negligible cytotoxicity in HCE cells up to 50 μM. In the capsaicin-induced allodynia model, 9d produced dose-dependent and full reversal of mechanical hypersensitivity, reaching maximum effect at very low doses (0.6–1.25 mg/kg s.c.), outperforming the standard S1R antagonist BD-1063 (effective at 10–40 mg/kg). The antiallodynic effect of 9d was fully reversed by the S1R agonist PRE-084, confirming S1R antagonism as the mechanism. In the rotarod test, 9d at 1.25 mg/kg did not impair motor coordination, whereas pregabalin (60 mg/kg) significantly reduced latency to fall. The phenytoin shift assay confirmed 9d acts as an S1R antagonist (Ki ratio without/with phenytoin = 0.6). Selectivity profiling showed no significant affinity (<50% inhibition at 1 μM) for opioid (MOR, DOR, KOR), serotonin (5HT2A, SERT), cannabinoid (CB1, CB2), or NMDA receptors. ADMET evaluation: aqueous solubility 2.14 mM (0.71 mg/mL), chemical stability T1/2 >24 h at pH 7.4, plasma stability T1/2 >24 h, metabolic half-life ~55–56 min in mouse and human liver microsomes (CLint 123.7 and 126.7 μL/min/mg protein, respectively). However, hERG inhibition was observed (IC50 = 0.085 μM), indicating a potential liability.
**Clinical Implications:** Compound AD258 (9d) is a potent, mixed S1R/S2R antagonist with robust antiallodynic effects at very low doses and no motor impairment, making it a promising lead for developing novel analgesics for chronic pain conditions. The high hERG inhibition requires further optimization to improve cardiac safety. The study validates the 2,7-diazaspiro[4.4]nonane scaffold for sigma receptor targeting and provides a strong foundation for future medicinal chemistry efforts.