Compound was synthesized in a racemic fashion

Compound was synthesized in a racemic fashion with this study and plans for an enantioselective synthesis could be developed based on the activity of racemic . Synthesis of compound followed a slightly different synthetic route due to the ineffectuality of the S2 reaction on the secondary position (). The synthesis began with nucleophilic addition of the commercially available 1-phenylethyl mercaptan to 2-chloro-6-nitrobenzothiazole to form nitrobenzothiazole in 80% yield. The nitro group was then reduced to amine through catalytic hydrogenation. The resulting aniline was used to open phthalic anhydride to produce salidroside in high yield. Formation of the phthalisoimide ring () and opening by morpholine successfully produced analogue in 52% yield over three steps. The activities of the synthesized analogues as antagonists at GPR35 were examined using U2OS cells permanently expressing HA-GPR35a and βarr2-GFP (UGPR35β) assay; 10μM Zaprinast was used as the agonist as in our previous publication. Concentration-effect curves for agonist-mediated receptor activation were analyzed by nonlinear regression techniques using GraphPad Prism 5.0 software (GraphPad) and data were fitted to sigmoidal dose-response curves to obtain IC (, curves for inactive and less active compounds are not shown for clarity). Evaluation of the activities as antagonists at GPR35 for the analogues provide considerable information (). One of the first observations is that the parent compound is not as active as was previously determined. After some more extensive studying of this, it was determined that many of the benzothiazoles were not sufficiently stable at room temperature and decomposed within 15h when exposed to air and light. To get reproducible data, the analogues were all frozen in DMSO until they were analyzed for their activity. The activity of the parent compound is still less than was previously determined. This could be due to the initially screened compound being partially decomposed into an unknown mixture of compounds. Fortunately, the parent compound was still moderately active when pure, which allowed for trends to be observed. One of the most important findings was that piperidine and pyrrolidine are devoid of activity as GPR35 antagonists. This indicates that there is probably a strong interaction between the oxygen of the morpholine ring and a residue in the binding site in this region. Prior modeling studies placed the morpholine ring in the region of the toggle switch,, so these data can be used to further refine the model. Oxidation of the exocyclic sulfur to sulfoxides , and and sulfone decreased the activity as an antagonist. These data indicate that this area of the molecule is either not amenable to expansion or is in a non-polar environment. The electronics of the benzothiazole are also modified by having the adjacent electron-withdrawing group; this might also be detrimental. The individual enantiomers ( and ) appear to be less active than the racemate (), however, the confidence intervals (CI) for the IC values are overlapping for sulfoxides , , and , which explains this otherwise unexpected result. Pyridine was found to be inactive as a GPR35 antagonist, which is an indication that the basicity of the nitrogen of the pyridine ring produced an unfavorable negative potential surface. The same scenario exists when the cyclic sulfur is replaced with a nitrogen atom in benzimidazole .
Introduction GPR35 is a poorly characterised, 7-transmembrane domain, GPCR that transmits function via interaction with Gαi/o, Gα13, and β-arrestin (Fig. 1) (Milligan, 2011, Mackenzie et al., 2011, Divorty et al., 2015, Shore and Reggio, 2015). In terms of sequence similarity, GPR35 is related to the purinergic receptor LPA4 (32%), the hydroxycarboxylic acid binding receptors HCA2 and HCA3 (30%), and the cannabinoid and lysophosphatidylinositol-binding GPR55 receptor (30%) (O'Dowd et al., 1998). As a consequence of the ligand-binding properties and shared sequence identity with GPR55, various groups have focussed on GPR35 as a putative lysophosphatidic acid-sensing GPCR (Oka et al., 2010; Zhao and Abood, 2013). This is of interest to further investigate experimentally, although at present it is difficult to draw any conclusions based on the original findings (Oka et al., 2010). Although certainly able to be activated by high concentrations of kynurenic acid, questions of which effects of this ligand, a well-studied metabolite of tryptophan, can be attributed to activation of GPR35 remain some of the major undefined issues in understanding the function of this receptor. This is vital to examine closely because kynurenic acid is clearly neuroactive and produces a broad range of effects in the central nervous system (CNS). However, many of these effects can be attributed to blockade of ionotropic receptors for the excitatory amino acid glutamate. Specific challenges in exploring the roles of GPR35 in the CNS relate to (a) the low potency of kynurenic acid at both rodent, and particularly the human, orthologues of the receptor, (b) that although many ligands with activity at GPR35 have been reported, the vast majority of these display modest potency and are known to also have a range of non-GPR35 mediated effects and, (c) although antagonists from two distinct chemical classes have been identified, at least in transfected cell systems these appear to display exquisite selectivity for human GPR35 and lack significant affinity at either mouse or rat GPR35 (Jenkins et al., 2012). Moreover, although a line of GPR35 knock-out mice has been generated and reported on (Min et al., 2010), these have not been employed widely and, currently, no information on the elimination of expression of GPR35 on effects of kynurenic acid in cells or tissue from the CNS has been released into the public domain. Each of these issues will be considered within the current review range of effects in the central nervous system (CNS). However, many of these effects can be attributed to blockade of ionotropic receptors for the excitatory amino acid glutamate. Specific challenges in exploring the roles of GPR35 in the CNS relate to (a) the low potency of kynurenic acid at both rodent, and particularly the human, orthologues of the receptor, (b) that although many ligands with activity at GPR35 have been reported, the vast majority of these display modest potency and are known to also have a range of non-GPR35 mediated effects and, (c) although antagonists from two distinct chemical classes have been identified, at least in transfected cell systems these appear to display exquisite selectivity for human GPR35 and lack significant affinity at either mouse or rat GPR35 (Jenkins et al., 2012). Moreover, although a line of GPR35 knock-out mice has been generated and reported on (Min et al., 2010), these have not been employed widely and, currently, no information on the elimination of expression of GPR35 on effects of kynurenic acid in cells or tissue from the CNS has been released into the public domain. Each of these issues will be considered within the current review.