Positive modulators of N-methyl-d-aspartate receptor: structure-activity relationship study on steroidal C-17 and...

N-methyl-d-aspartate receptors (NMDARs) are crucial therapeutic targets, modulated by endogenous neurosteroids like pregnenolone sulfate (PES). This study investigates a novel structure-activity relationship approach focusing on the steroidal D-ring, employing the bioisosteric replacement of C-17 or C-20 keto groups with oximes and oxime ethers. We synthesized a series of pregn-5-ene and androst-5-ene derivatives (11-23) and evaluated their positive allosteric modulator (PAM) activity on recombinant GluN1/GluN2B receptors via a patch-clamp technique in HEK293 cells. Our study revealed that pregnenolone-derived C-20 oxime ethers are potent and efficacious PAMs of NMDAR. Several analogues have been demonstrated as more potent than PES (E _max = 116%; EC₅₀ = 21.7 µM). Compound 12 (C-20 ethyl oxime ether, C-3 hemiglutarate) displayed the highest efficacy, potentiating NMDAR currents over 6-fold greater than PES (E _max = 673 ± 121%; EC₅₀ = 8.7 ± 1.1 µM). Compound 17 (C-20 methyl oxime ether analogue) exhibited the highest potency, being over 3.5-fold more potent than PES (E _max = 503 ± 68%; EC₅₀ = 6.1 ± 0.4 µM). In contrast, some C-17 analogues and derivatives with bulkier C-20 oxime substituents showed complex modulatory behavior. Promisingly, key compounds demonstrated favorable in vitro ADME profiles, including high metabolic stability and, for 12, excellent thermodynamic solubility. These results validate C-20 oxime ether modification of the pregnenolone scaffold as an effective strategy for generating potent NMDAR PAMs with potentially superior efficacy and drug-like properties compared to endogenous modulators.