Phosphorylation by Sphingosine Kinase 2 is Essential for in vivo Potency of FTY720 Analogues

active principle (S)-FTY720-phosphate which binds to four of the five known sphingosine-1-phosphate receptors (S1P1–5). It has been convincingly demonstrated that the immunomodulatory effect is primarily mediated by S1P1. Sphingosine kinases (SPHKs) have been identified as the enzymes that catalyze the mono-O-phosphorylation of the amino alcohol head group of FTY720. We and others have found that SPHK2 is much more efficient than SPHK1 in phosphorylation of FTY720 (Table 1). Indeed, in vivo experiments have demonstrated that SPHK2 is essential for the lymphopenia induced by FTY720. Although apoptosis induced by FTY720 at higher concentrations has been shown to be receptor independent, a recent publication suggests that SPHK2 is also essential for this pathway. The finding that only one of the prochiral hydroxy groups in FTY720 is phosphorylated in vivo suggests that the enzymatic phosphorylation reaction can be tuned by offering various stereochemical features in the substrate. For the chiral analogues 1a and 1b (Figure 2) it has already been demonstrated that induction of lymphopenia correlates with SPHK phosphorylation efficiency as well as with the potency as agonists of the corresponding phosphates toward S1P1. For potent S1P1 agonists that do not induce lymphopenia, a difference in SPHK activity has already been suspected as a possible reason for their lack of in vivo efficacy. Herein we report the synthesis and systematic investigation of FTY720-like amino alcohols featuring two stereocenters in the head group in order to establish the relative importance of SPHK phosphorylation compared with potency toward S1P1 for lymphopenia induction in vivo. Primary alcohols 1a and 1b were prepared as reported previously by using a Schcllkopf auxiliary-based approach. For the synthesis of these two compounds as well as the secondary alcohols 2a and 3a, the N-Boc-protected amino alcohol 4 served as the precursor (Scheme 1). Using the protocol described by Ley et al. , this alcohol was oxidized to the aldehyde 5. The addition of methyl magnesium bromide resulted [a] Dr. K. Hcgenauer, Dr. A. Billich, Dr. P. Nussbaumer Novartis Institutes for Biomedical Research Brunner Strasse 59, 1235 Wien (Austria) Fax: (+43)1-80166-354 E-mail : klemens.hoegenauer@novartis.com [b] C. Pally, Dr. M. Streiff, Dr. T. Wagner, Dr. K. Welzenbach Novartis Institutes for Biomedical Research 4002 Basel (Switzerland) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author: all experimental protocols and analytical data. Figure 1. FTY720 is phosphorylated by SPHKs. Table 1. In vivo and in vitro data.