New Substituted Piperazines as Ligands for Melanocortin Receptors. Correlation to the X-ray Structure of “THIQ”

Abstract

A series of piperazine analogues of the melanocortin 4 receptor (MC4R) specific small-molecule agonist "THIQ" was synthesized and characterized structurally and pharmacologically. First, several THIQ imitations lacking the triazole moiety were prepared. Syntheses included acylation of 4-phenylpiperazine or 4-cyclohexylpiperazine. In two cases the tertiary amine function was replaced by the corresponding N-oxide. To obtain more complex structures, a 4-substituted piperazine ring was formed by alkylation of the primary amino group of cyclohexane-derived amino alcohols with N,N-bis(2-chloroethyl)benzylamine. The hydroxylic group of the intermediate was first activated with methanesulfonyl chloride, and the sulfonic ester formed in situ was introduced into the reaction with the sodium salt of 1,2,4-triazole. In one case (i.e., preparation of 23c) introduction of the 1,2,4-triazole moiety was performed at a carbon of the cyclohexane ring. In addition, this intermediate contained a piperazine moiety connected via its nitrogen atom to a cyclohexane ring carbon neighboring the reaction center. As established in NMR and X-ray investigations herein, this substitution proceeded with retention of the initial trans configuration of 1,2-disubstituted cyclohexane. To obtain pure enantiomers of 23c, its precursor 21c was subjected to chiral chromatography on a Chirobiotic V column. The derivatives (R,R)-21cand (S,S)-21c obtained were introduced into further syntheses steps, giving (R,R)-23c and (S,S)-23c, respectively. Melanocortin MC(1,3-5) receptor binding studies showed that all tested piperazine derivatives were active. Several compounds showed clear selectivity for MC4R, with submicromolar affinities being obtained. Among them, one substance, (R,R)-23c, displayed a biphasic curve in displacement of [125I]NDP-MSH on MC4R [K(i)high = 1 nM and K(i)low = 260 nM]. This biphasic competition curve was similarly biphasic to the competition curve obtained herein using THIQ. An X-ray study performed on crystals of the THIQ sulfate salt revealed two closely related conformations, which resemble the shape of the letter "Y", where piperidine and 4-chlorophenyl groups are situated close to each other, but the 1,2,3,4-tetrahydroisoquinoline residue is remote, the triazole function being highly exposed to the environment. The crystals of the dinitrate salt of (R,R)-23c showed a different conformation, where parts of the molecule are spread out almost symmetrically around the central section. Molecular modeling, based on the THIQ crystal structure and the functional similarity of THIQ and (R,R)-23c, allowed us to suggest a possible "bioactive" conformation of (R,R)-23c that is similar to the crystal conformation of THIQ.

Interactions of Human Melanocortin 4 Receptor with Nonpeptide and Peptide Agonists,

“…Our models are consistent with receptor-bound conformations of agonists that have been proposed previously (18,48,(56)(57)(58)(59). The best-fitting conformation of small agonist, THIQ was similar to the crystal structure of this molecule (see Results).…”

“…L265A mutation demonstrates a large effect only on the binding affinity and the potency of small-molecule agonists (Table 1) probably because the aliphatic side-chain of Leu 265 is more tightly packed with the cyclohexane ring of the peptidomimetics than with the corresponding planar ring of Trp 9 of the peptide. It can not be excluded, however, that D126A and H264A mutations may also impair plasma membrane targeting and/or assembly, as radiolabel binding and functional activation is dramatically reduced for these mutants.Our models are consistent with receptor-bound conformations of agonists that have been proposed previously (18,48,(56)(57)(58)(59). The best-fitting conformation of small agonist, THIQ was similar to the crystal structure of this molecule (see Results).…”

Dynamics of ligand binding to GPCR: Residence time of melanocortins and its modulation

“…A non-peptide radioligand [ 125 I]THIQ (also referred to as I-Mex2) [36] has also been used as a radioligand for MC4 receptors but is not commercially available. This ligand is derivative of 4-cyclopiperidine and has at least 250-fold selectivity for MC4 receptors over other MC receptor subtypes [37]. Kinetic studies of [ 125 I]NDP binding to MC4 receptors revealed heterogeneity in association, where only some binding sites followed the regularities of simple bimolecular reactions, depending linearly on radioligand concentration [38].…”

Design of cyclic peptides with agonist activity at melanocortin receptor-4

“…A lot of nonpeptide agonists and antagonists, such as piperidine-, 6,7 piperazine-, 8,9 or guanidine 10 -based ligands, were developed and evaluated. On the other hand, peptidic MC-4R ligands were also investigated based on the sequence of endogenous MC-R agonists, for example, ACTH and a-, b-, and c-MSH.…”