Positive Modulators of N-Methyl-D-Aspartate Receptor: Structure-Activity Relationship Study on Steroidal C-17 and C-20 Oxime Ethers

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 rat GluN1/GluN2B receptors via patch-clamp 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 (Emax = 116%; EC50 = 21.7 {micro}M). Compound 12 (C-20 ethyl oxime ether, C-3 hemiglutarate) displayed the highest efficacy, potentiating NMDAR currents over 6-fold more than PES (Emax = 673 {+/-} 121%; EC50 = 8.7 {+/-} 1.1 {micro}M). Compound 17 (C-20 methyl oxime ether analogue) exhibited the highest potency, being over 3.5-fold more potent than PES (Emax = 503 {+/-} 68%; EC50 = 6.1 {+/-} 0.4 {micro}M). 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.