BACKGROUND AND PURPOSE The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine-based compound with structural similarity to previously described CCR8-specific agonists, but containing a unique phenyl-tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1. EXPERIMENTAL APPROACH Single point mutations were introduced in CCR1 and CCR8, and their effect on small molecule ligand-induced receptor activation was examined through inositol trisphosphate (IP3) accumulation. The molecular interaction profile of the agonist was verified by molecular modeling. KEY RESULTS The chemokine receptor conserved glutamic acid in TM-VII served as a common anchor for the positively charged amine in the piperidine ring. However, whereas the phenyl-tetrazol group interacted with TyrIV: 24 (Tyr172) and TyrIII: 09 (Tyr114) in the major binding pocket (delimited by TM-III to VII) of CCR8, it also interacted with TrpII: 20 (Trp90) and LysII: 24 (Lys94) in the minor counterpart (delimited TM-I to III, plus TM-VII) in CCR1. A straightening of TM-II by Ala-substitution of ProII: 18 confirmed its unique role in CCR1. The extracellular loop 2 (ECL-2) contributed directly to the small molecule binding site in CCR1, whereas it contributed to efficacy, but not potency in CCR8. CONCLUSION AND IMPLICATIONS Despite high ligand potency and efficacy and receptor similarity, this dual-active and bitopic compound binds oppositely in CCR1 and CCR8 with different roles of ECL-2, thereby expanding and diversifying the influence of extracellular receptor regions in drug action.

Activation of CCR8 by its ligand CCL1 may play an important role in diseases such as asthma, multiple sclerosis, and cancer. The study of small molecule CCR8 antagonists will help establish the validation of these hypotheses. We report the design, synthesis, and progress toward optimization of potent small molecule CCR8 antagonists identified from a high-throughput screen. These analogues exhibit good potency in binding and chemotaxis assays, show good selectivity versus the hERG channel, and have good eADME (early absorption, distribution, metabolism, and excretion) profiles.

[reaction: see text] Reaction of o-azidobenzenesulfonamides with polymer-supported triphenylphosphine affords the corresponding iminophosphoranes. Subsequent reaction with isocyanates gives 3-amino-1,2,4-benzothiadiazine 1,1-dioxides in high yields and purities. The reaction has been successfully applied to the synthesis of derivatives with various substituents at the 2- and 3-positions and in the benzenoid ring.