SynthesisandevaluationofpotentialinhibitorsofeIF4E capbindingto7-methylGTP Phalguni Ghosh, a Chunkyung Park, a Mark S. Peterson, b Peter B. Bitterman, b Vitaly A. Polunovsky b and Carston R. Wagner a, * a Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA b Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA Received 9 August 2004; revised 25 January 2005; accepted 27 January 2005 Available online 18 March 2005 Abstract—Cap-dependent translation is initiated by the binding of eIF4E to capped mRNA (m 7 GpppN). We have prepared a small library of 7-methyl guanosine nucleoside and nucleotide analogs and evaluated their ability to inhibit eIF4E binding to 7-methyl GTP with a competitive eIF4E binding immunoassay. 5 0 -H-Phosphonate derivatives in which the 2 0 - and 3 0 -riboside hydroxyls were tethered together by an isopropylidene group were shown to be a new class of inhibitors of eIF4E binding to capped mRNA. Ó 2005 Elsevier Ltd. All rights reserved. In eukaryotes, eukaryotic initiation factor 4E (eIF4E) plays a key role in initiation of the translation process by recognition of the 5 0 terminal mRNA cap structure, 7-methyl G(5 0 )ppp(5 0 )X (X = any nucleoside). A strong interaction between the 5 0 -cap of mRNAs and eIF4E is required for initiation of translation. 1 Formation of the eIF4E—cap mRNA complex is considered to be rate limiting for translation initiation under most circum- stances 2 and its up-regulation is associated with cell growth, tumorgenicity and inhibition of apoptosis. 3,4 Recently, inhibition of cap-dependent translation by the ectopic expression of the eIF4E repressor protein, 4E binding protein 1 (4E-BP1), was shown to reduce breast cancer tumorgenicity and resistance to apopto- sis. 4 These findings have renewed interest in the develop- ment of pharmacological tools and assays capable of modulating and monitoring translation initiation in gen- eral and eIF4E binding to capped RNA in particular. 5,6 In this report, we describe the synthesis and evaluation of the ability of a small library of 7-methyl G nucleoside and nucleotide analogs to inhibit eIF4E binding to 7- methyl GTP with an immunoassay. A library of 29 members was assembled. The triphos- phates 1–3 and nucleosides 7–9 were obtained from commercial sources. The monophosphates 10–13 were prepared from guanosine monophosphate as previously described. 7 The synthesis of the m 7 G derivatives 14–29 is summarized in Scheme 1. Guanosine was first treated with dimethoxy propane and toluene sulfonic acid (TsOH) to yield 2 0 ,3 0 -isopropylidene guanosine, 16. Next, 16 was either alkylated with the appropriate alkyl or aryl halide to yield compounds, 17–19, or treated with diphenylphosphite and pyridine to afford the pro- tected H-phosphonate of guanosine, 20. Compounds 21–24 were obtained from alkylation of 20 by a proce- dure similar to that employed to synthesize compounds 17–19. Since removal of the isopropylidene from com- pounds 21–24 resulted in significant depurination, com- pound 14 was obtained by deprotection of compound 20 Dowex 50W resin, followed by alkylation of compound 13 with benzyl bromide. The amino acid phosphorami- dates of compound 20 were prepared by oxidation with timethylsilyl chloride (TMSCl) and I 2 , followed by the addition of the carbomethoxy amino acid. The carbo- methoxy group was removed by treatment with sodium hydroxide, yielding compounds 25, 26, 28, and 29. Unlike previous assays 6–8 that have monitored indirectly eIF4E antagonism in the presence of accessory proteins, we have successfully developed a high throughput pro- cedure for the direct measurement of the inhibition of eIF4E binding capped mRNA in tissue extracts. 9 Typi- cally, eIF4E from pooled cell extracts 10 was co-incu- bated with variable concentrations of the potential inhibitors in the presence of 7-methyl-GTP agarose 0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2005.01.080 * Corresponding author. Tel.: +1 612 625 2614; fax: +1 612 624 0139; e-mail: wagne003@tc.umn.edu Bioorganic & Medicinal Chemistry Letters 15 (2005) 2177–2180