Design and synthesis of selective and potent orally active S1P5 agonists.

The immunomodulatory drug fingolimod (FTY720, 2-amino-2[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal metabolite (ISP-1, myriocin), is phosphorylated in vivo by sphingosine kinases to produce (R)-FTY720-phosphate (FTY720-P). 2] FTY720-P activates sphingosine-1-phosphate (S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar concentrations and is inactive toward the S1P2 receptor. The FTY720-P-mediated activation of the S1P1 receptor on lymphocytes induces receptor internalization, which attenuates T-cell response to S1P gradients, preventing their egress from secondary lymphoid tissues. In addition to playing a role in the immune system, all S1P receptors except S1P4 are also found differentially expressed in the central nervous system and on various tumor cell types. 6] Although the precise regulation of these receptors by locally released S1P remains unclear, S1P receptors are thought to play a role in such events as astrocyte migration, oligodendrocyte differentiation, and cell survival 9] and neurogenesis. 11] To assess the relevance of individual S1P receptor subtypes for the activity of FTY720-P, selective agonists are required. Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC50 = 270 nm), but has modest selectivity against S1P1 (EC50 = 3140 nm) and S1P4 (EC50 = 100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists. To guide the optimization process, homology models of all S1P receptors were built from a crystal structure of bovine rhodopsin (PDB ID: 1F88). Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring, ~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl. In the S1P5 receptor complex, the amide group forms a hydrogen bond with OG1-Thr120. The benzamide phenyl ring lies in a large hydrophobic pocket surrounded by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and Leu271. The aniline ring undergoes a T-shaped interaction with Phe116 and hydrophobic contacts with Leu271 and Leu292. The ortho-methyl substituents fill a small pocket formed by Tyr89, Val115, and Leu292 on one side, and sit at the face of Phe196 on the other side. Inspection of sequence alignments (Figure 2) revealed two positions, one in transmembrane (TM) helix TM3 (115, S1P5 sequence) and one in TM5 (192), where S1P5 has smaller residues lining the binding site, thus creating putative pockets. We hypothesized that filling these pockets with atoms from our ligands should lead to high selectivity for the S1P5 receptor. Position 2 on the benzamide core, which was closest to the hypothesized pocket around Val115, was therefore extensively modified. Syntheses of derivative 1 A–L (Scheme 1) began with 3-fluorobromobenzene 2, which was converted into acid 3 by reaction with lithium diisopropylamide (LDA) and carbon dioxide. Nucleophilic substitution of the fluorine atom with trimethylaniline at 78 8C yielded 4. This intermediate was then used in various ways. Copper-catalyzed nucleophilic substitution of the bromine atom with various alcohols yielded ethers 5 D–N, which were amidated with ammonia using chlorodimethoxytriazine for activation to yield 1 D–J. Palladium-catalyzed substitution of the bromine atom in acid 4 with various alkylstannanes yielded 6 A–C, which were amidated as described above to yield 1 A–C. Alternatively, palladium-catalyzed substitution of the bromine atom with tributyl-(1-ethoxyvinyl)stannane yielded 9, which was cyclized to 1 L by reaction with hydrazine. Acid 4 was also amidated with allylamine, using chlorodimethoxytriazine for activation, to yield allylamide 10. Palladiumcatalyzed cyclization of this intermediate led to 1 K. All compounds were assayed for S1P5 activation in GTPgS assays, which gave more reliable structure–activity results than the FLIPR assays, at concentrations up to 10 mm. EC50 values were determined for all compounds (Table 1). Disrupting the intramolecular hydrogen bond by introducing small alkyl substituents at position 2 (compounds 1 A–C), led to a [a] Dr. H. Mattes, Prof. Dr. K. K. Dev, Dr. R. Bouhelal, Dr. C. Barske, Dr. F. Gasparini, Dr. D. Guerini, Dr. A. K. Mir, Dr. D. Orain, M. Osinde, A. Picard, C. Dubois, E. Tasdelen, S. Haessig Novartis Institute for Biomedical Research WKL-122 4002 Basel (Switzerland) Fax: (+ 41) 61 696 2455 E-mail : henri.mattes@novartis.com [b] Prof. Dr. K. K. Dev Molecular Neuropharmacology, Department of Physiology Trinity College Institute of Neuroscience (TCIN) Medical School Trinity College Dublin, Dublin 2 (Ireland) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000253.