Synthesis and structure-activity relationships of N,N'-di-o-tolylguanidine analogues, high-affinity ligands for the haloperidol-sensitive sigma receptor.

With an eye toward the development of novel atypical antipsychotic agents, we have studied the structure-affinity relationships of N,N'-di-o-tolylguanidine (DTG, 3) and its congeners at the haloperidol-sensitive sigma receptor. A number of DTG analogues were synthesized and evaluated in in vitro radioligand displacement experiments with guinea pig brain membrane homogenates, using the highly sigma-specific radioligands [3H]-3 and [3H]-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and the phencyclidine (PCP) receptor specific compounds [3H]-N-[1-(2-thienyl)-cyclohexyl]piperidine and [3H]-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine. The affinity of N,N'-diarylguanidines for the sigma receptor decreases with increasing steric bulk of ortho substituents larger than C2H5. Hydrophobic substituents are generally preferred over similarly positioned hydrophilic ones. Furthermore, electroneutral substituents are preferred over strongly electron donating or withdrawing groups. Significant binding to the sigma receptor is usually retained as long as at least one side of the guanidine bears a preferred group (e.g. 2-CH3C6H5). Replacement of one or both aryl rings with certain saturated carbocycles (e.g. cyclohexyl, norbornyl, or adamantyl) leads to a significant increase in affinity. By combining the best aromatic and best saturated carbocyclic substituents in the same molecule, we arrived at some of the most potent sigma ligands described to date (e.g. N-exo-2-norbornyl-N'-(2-iodophenyl)guanidine, IC50 = 3 nM vs [3H]-3). All of the compounds tested were several orders of magnitude more potent at the sigma receptor than at the PCP receptor, with a few notable exceptions. This series of disubstituted guanidines may be of value in the development of potential antipsychotics and in the further pharmacological and biochemical characterization of the sigma receptor.

Identification of the binding subunit of the sigma-type opiate receptor by photoaffinity labeling with 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2-methyl[4,6-3H]phenyl)guanidine.

The sigma-type opiate receptor is a distinct binding site in the brain that may mediate some of the psychotomimetic effects caused by benzomorphan opiates and phencyclidine in humans. We have developed a synthetic, highly selective ligand for this receptor, 1,3-di-o-tolylguanidine (DTG). To identify the binding protein(s) of the sigma receptor, we have now synthesized a radiolabeled azide derivative of DTG, 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2-methyl[4,6-3H]phenyl)-guanidine ([3H]N3DTG). In guinea pig brain membrane binding assays conducted in the dark, [3H]N3DTG bound reversibly, selectively, and with high affinity (Kd = 10 nM) to sigma receptors. The drug specificity profile of reversible [3H]-N3DTG binding was identical to that of [3H]DTG and 3H-labeled (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding indicating that [3H]N3DTG is a selective sigma receptor ligand. Guinea pig brain membranes were photoaffinity-labeled with [3H]N3DTG. NaDodSO4/PAGE of detergent-solubilized membrane extract identified a single 29-kDa radioactive band. Sepharose Cl-6B gel chromatography of photolabeled brain membranes solubilized with the nondenaturing detergent sodium cholate showed a radioactive complex with a Stoke's radius of 4.6 nm (Mr, 150,000) that may represent the intact sigma receptor complex. NaDodSO4/PAGE of this complex showed that the radiolabeled material was a 29-kDa polypeptide that may be the binding subunit of the sigma receptor. The specific sigma receptor photoaffinity ligand described here should be a useful tool for purifying and characterizing the sigma receptor.