Sometime ago we have described diamino

Sometime ago we have described diamino-1,3,5-triazines as histamine H4R ligands,12, 13 and aryl derivatives of the 1,3,5-triazine, which attenuated inflammatory and nociceptive response in vivo in the rodent models of inflammation induced by Cyclic di-GMP and zymosan. In this work we describe new derivatives of 1,3,5-triazines as histamine H4R ligands. These compounds were designed based on the structure of compound KB8 and the structure of JNJ39758979 (2; Fig. 1) as shown in Scheme 1. Our previous published KB8 had hH4R affinity in a low micromolar concentration range, whereas JNJ39758979 and other alkyl derivatives of 2,4-diaminopyrimidines described by Saval et al. demonstrated high affinities for hH4R with Ki values below 100 nM. Moreover, taking account of a general construction pattern of hH4R ligands suggested by Schreeb et al. a 4-methylpiperazine moiety was chosen as basic group. This group is often present in hH4R ligands and increases affinity in most cases. As a central core the triazine ring was chosen. The modifications were made in the lipophilic part of the molecule and different (un)branched alkyl (alkenyl) substituents were introduced into the 6 position of the triazine ring. To check the tolerability of H4R for polar substituents, one ether analogue was synthesized and tested within this small series. Moreover, 4-tert-butyl-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (tBMTA; Table 1) the compound previously described by Altenbach et al., was resynthesized and pharmacological characterized. This compound was oryginally synthesized by Abbott researchers as an analogue of the pyrimidine derivative (which had been found in the screening of chemical library in the FLIPR calcium assay). tBMTA was found to be a moderate hH4R antagonist with a pKb of 7.04 and not further modified by Abbott researchers. Furthemore, in order to determine possible protein-ligand interactions, docking studies for a set of herein described ligands were performed. Additionally, the antinociceptive activity in vivo was examined for the most potent compound in this series, 4-(cyclohexylmethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (14).
Results and discussion
Conclusions To sum up, basing on available literature data, a small series of alkyl-1,3,5-triazinyl piperazines were designed and synthesized. Some of the compounds proved to have submicromolar affinities for hH4R (e.g. 14, Ki = 160 nM), whereas two compounds (9 and 16) were deprived of an activity. Antagonist behavior of selected compounds was confirmed in the cellular aequorin-based functional assay (9 and 14) and in [S]GTPγS binding assay (14). Furthermore, in in vivo studies, compound 14 was able to attenuate inflammatory pain in tested models of pain (formalin test, carrageenan-induced inflammation). To sum up, alkyl-1,3,5-triazine derivatives appear as promising structures for further development of potent hH4R ligands. However, future perspectives should include evaluation of longer (un)branched linkers (butyl, pentyl) as well as (un)branched alkenyl substituent to be profitable for increased hH4R affinity.
Experimental part All reagents were purchased from commercial suppliers. JNJ7777120 was synthesized in Department of Technology and Biotechnology of Drugs (Jagiellonian University Medical College). Melting points (Mp.) were determined on a MEL-TEMP II (LD Inc., USA) melting point apparatus and are uncorrected. IR spectra were measured as KBr pellets on FT Jasco IR spectrometer. Mass spectra (LC/MS) were performed on Waters TQ Detector (Water Corporation., Milford, USA) mass spectrometer. Retention times (tR) are given in minutes. The UPLC/MS purity of all final compounds was determined (%). 1H NMR spectra were recorded on a Mercury 300 MHz PFG spectrometer (Varian, Palo Alto, California, USA) in DMSO‑d6. Chemical shifts were expressed in parts per million (ppm) using the solvent signal as an internal standard. Data are reported in the following order: multiplicity (br, broad; cyclohex, cyclohexyl; m, multiplet; Pp, piperazine; q, quartet; s, singlet; sxt, sextet; t, triplet;), approximate coupling constants J expressed in Hertz (Hz), number of protons. TLC data were obtained with Merck silica gel 60F254 aluminum sheets with the following detection with UV light and evaluation with Dragendorff’s reagent (solvent system: methylene chloride: methanol 1:1).