Total Synthesis and Biological Evaluation of the Protein Phosphatase 2A Inhibitor Cytostatin and Analogues

Abstract

The total synthesis of the natural product cytostatin is described which inhibits protein phosphatase 2A. Cytostatin has anti-metastatic properties and induces apoptosis. On the basis of this synthesis the relative and absolute configuration of cytostatin could be assigned. Key structural elements of cytostatin are an alpha,beta-unsaturated lactone group and a side chain embodying a phosphate and a rather unstable (Z,Z,E)-triene subunit. In addition, the natural product carries six stereocenters. For the construction of the stereocenters reagent-controlled transformations were used in order to ensure maximum stereochemical flexibility. The Evans syn-aldol reaction was chosen to establish the stereochemistry at C-4, C-5, C-9 and C-10; C-6 was introduced by means of the Evans asymmetric alkylation. In all cases the same chiral auxiliary was employed as stereodirecting group. The stereocenter at C-11 was established by an asymmetric reduction using CBS-oxazaborolidine. Temporary protection of the phosphate group was achieved best by using the base-labile 9-fluorenylmethyl group, which could be cleanly cleaved by an excess of triethylamine; this reaction yielded analytically pure phosphates after a simple aqueous work-up. The (Z,Z,E)-triene embodied in cytostatin was synthesized by means of a Stille coupling as key transformation. The synthesis sequence established in this way readily gave access to a series of analogues with simplified structure. Initial biological testing of these analogues proved that the alpha,beta-unsaturated lactone, the C-11-hydroxy group and a fully deprotected phosphate moiety at C-9 are essential for the PP2A-inhibitory activity of cytostatin. The rather unstable triene moiety in the side chain can be replaced by other lipophilic residues with only moderate decrease of biological activity. Other phosphatases, that is, PP1, VHR, PTP1B, CD45, were not inhibited by cytostatin or any of the analogues, demonstrating the high selectivity of this compound. These findings will be useful for the design and synthesis of cytostatin-derived chemical tools for the study of biological processes influenced by PP2A.

Principles, implementation, and application of biology-oriented synthesis (BIOS)

“…Screening of a BIOS-derived a,b-unsaturated d-lactone compound collection in two cell based screens yielded small molecule modulators of cell cycle progression and of viral entry via the secretory pathway (Leßmann et al, 2007). Extensive screening and synthesis efforts also resulted in highly selective phosphatase inhibitors with enhanced properties (Bialy and Waldmann, 2004;Umarye et al, 2007).…”

Structure-Activity Relationship Studies of Fostriecin, Cytostatin, and Key Analogs, with PP1, PP2A, PP5, and (β12–β13)-Chimeras (PP1/PP2A and PP5/PP2A), Provide Further Insight into the Inhibitory Actions of Fostriecin Family Inhibitors

“…Subsequently, fostriecin (Walsh et al, 1997;Buck et al, 2003), cytostatin (Bialy and Waldmann, 2004;Lawhorn et al, 2006), and structurally related natural products [phospholine, leustroducsin, and phoslactomycins (Usui et al, 1999;Kawada et al, 2003); Fig. 1] have all been shown to inhibit a subset of PPP-family serine/threonine protein phosphatases.…”

“…Fostriecin acts as a potent inhibitor of PP2A/PP4 (IC 50 0.2-4 nM) and a weak inhibitor of PP1 and PP5 (PP2A/PP4 versus PP1/PP5 selectivity Ͼ10 4 ) (Walsh et al, 1997;Buck et al, 2003). Cytostatin is also a potent and selective inhibitor of PP2A (PP2A IC 50 ϭ 20 -400 nM; PP2A versus PP1/PP5 Ͼ 10 3 ) (Bialy and Waldmann, 2004;Lawhorn et al, 2006). Phospholine, leustroducsin H, and phoslactomycins are weaker inhibitors of PP2A (Usui et al, 1999;Kawada et al, 2003) and have not been examined using other phosphatases.…”

“…Following the first total synthesis of fostriecin (Boger et al, 2001), at least nine total or formal syntheses have been reported (Chavez and Jacobsen, 2001;Esumi et al, 2002;Reddy and Falck, 2002;Miyashita et al, 2003;Maki et al, 2005;Trost et al, 2005). The total synthesis of cytostatin (Bialy and Waldmann, 2004;Lawhorn et al, 2006;Jung et al, 2008) and cytostatin analogs (Lawhorn et al, 2006;Jung et al, 2008) have also been reported. These studies have sparked renewed interest in the potential for development of more stable derivatives that may have utility as novel antitumor drugs.…”

Synthesis and Immobilization of erythro‐C14‐ω‐Aminosphingosine‐1‐phosphate as a Potential Tool for Affinity Chromatography

“…Conversion to Fm-protected phosphate 8 was achieved by a onepot treatment with Fm-substituted diisopropylphosphoramidate followed by oxidation of the resulting trivalent phosphorus to pentavalent phosphorus. [16] The high stability of the Fm groups towards acidic hydrolysis allowed for concomitant cleavage of the TBDPS group and both N-Boc groups with a mixture of HF and HCl to give 3. The terminal amino group of 3 was expected to react regioselectively with the reactive groups of the selected affinity matrix, [9] that is, Affi-Gel 10 loaded with 15 mmol mL À1 of NHS esters linked to the gel beads by decapeptide spacers.…”