Synthesis and pharmacological evaluation of 5-pyrrolidinylquinoxalines as a novel class of peripherally restricted κ-opioid receptor agonists.

5-Pyrrolidinyl substituted perhydroquinoxalines were designed as conformationally restricted κ-opioid receptor agonists restricted to the periphery. The additional N atom of the quinoxaline system located outside the ethylenediamine κ pharmacophore allows the fine-tuning of the pharmacodynamic and pharmacokinetic properties. The perhydroquinoxalines were synthesized stereoselectively using the concept of late stage diversification of the central building blocks 14. In addition to high κ-opioid receptor affinity they demonstrate high selectivity over µ, δ, σ1, σ2, and NMDA receptors. In the [35S]GTPγS assay full agonism was observed. Because of their high polarity, the secondary amines 14a (log D7.4=0.26) and 14b (log D7.4=0.21) did not penetrate an artificial blood-brain barrier. 14b was able to inhibit the spontaneous pain reaction after rectal mustard oil application to mice (ED50=2.35 mg/kg). This analgesic effect is attributed to activation of peripherally located κ receptors, since 14b did not affect centrally mediated referred allodynia and hyperalgesia.

Combination of cyclohexane and piperazine based κ-opioid receptor agonists: Synthesis and pharmacological evaluation of trans,trans-configured perhydroquinoxalines.

Desymmetrization of the pseudochiral (2r)-configured cyclohexane-1,2,3-triamines 8 with dimethyl oxalate led to racemic aminoquinoxaline-2,3-diones 9. Selective introduction of the κ pharmacophoric structural elements pyrrolidine and 3,4-dichlorophenylacetamide with a two-carbon distance afforded conformationally restricted κ agonists 13-15 based on the quinoxaline ring system. In competitive radioligand receptor binding studies the benzylamine 13b, the secondary amine 14b, and the carbamate 15 displayed high κ receptor affinity. The Ki value of the lead compound derived methoxycarbonyl derivative 15 is 9.7nM. However, the κ affinity of 15 is exceeded by 13b and 14b with a basic functional group instead of the methoxycarbonyl group in 1-position of the quinoxaline system. The chlorine atoms of the dichlorophenylacetyl residue are essential, since the corresponding phenylacetyl analogs show considerably reduced κ affinity. The potent κ ligands 13b, 14b and 15 are selective over the related µ- and δ-opioid receptors, σ1, σ2 and NMDA receptors. In the [(35)S]GTPγS-binding assay 13b behaved as partial agonist with lower activity than U-69,593.

Probing leukocyte traffic in lymph from oro-nasal mucosae by cervical catheterization in a sheep model.

Lymph nodes are instructed via the lymph about ongoing events in tissues both during the steady state and under provoked inflammation. In order to probe for tissue-to-node transduction mechanisms, we have developed a novel in vivo technique of pseudo-afferent lymph collection from the oro-nasal mucosae which represent the main portals of entry of micro-organisms and efficient routes for vaccination. After lateral lymph node resection of the head, a network of lymph ducts was reconstructed as checked by lymphography. Subsequent catheterization of the cervical lymph duct allowed the collection of cells that were shown to originate from the oro-nasal mucosae. These cells included dendritic cells, monocytes, granulocytes, memory CD45RAneg CD2pos integrin beta7lo CD4 T cells, CD25pos CD4, CD8, gamma/delta T cells, and B lymphocytes. This approach, which permits lymph collection over several weeks, opens a valuable and unique way to study leukocyte and particulate (micro-organisms, vaccines) trafficking from head tissue to nodes under homeostastic and immuno-stimulatory conditions in a highly physiological setting.