E: i) bile acid derivatives, for example lithocholic acid (LCA, compound 1)20,21 and cholanic acid,22 two competitive Eph receptor antagonists getting a moderate preference for the EphA receptor subfamily; ii) salicylic-acid derivatives,23, 24 exemplified by 4-(two,5dimethyl-1H-pyrrol-1-yl)-2-hydroxybenzoic acid, which inhibit the EphA2 and EphA4 receptors;23,24 iii) doxazosin,25 the marketed 1-adrenoreceptor antagonist that has been lately shown to bind and activate EphA2 and EphA4 receptor subtypes; iv) some polyphenols and polyphenol metabolites.26-28 Among these classes of Eph-ephrin system modulators, we recently focused our attention on LCA, a compound characterized by a (five)-cholan-24-oic acid scaffold, which competitively displaces ephrin-A1 in the ligand-binding domain of EphA2.21 Inside the present operate, we report the synthesis and structure-activity connection (SAR) profile of an extended series of -amino acid conjugates of LCA, developed beginning from a theoretical binding mode of LCA in to the EphA2 binding website. The synthesized compounds had been examined for their ability to disrupt EphA2-ephrin-A1 binding and to stop EphA2 phosphorylation within a prostate cancer cell line.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCHEMISTRYLithocholic acid (LCA, compound 1) was bought from Sigma when compounds 2, 4-7 and 12-21 had been synthesized based on a procedure equivalent to that described in references.29,30 Methyl ester hydrochlorides of -amino acids have been purchased from industrial suppliers (3a, 4b-7b, 12b, 14b, 16b-18b, 20b) or synthesized following step i of Scheme 1 (i.e. methyl ester hydrochloride derivatives 13b, 15b, 19b and 21b). The methylJ Med Chem. Author manuscript; readily available in PMC 2014 April 11.Incerti et al.Pageester hydrochloride of the proper -amino acid was reacted with 1 (LCA), working with N-(3dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) as coupling agent. The resulting amides three, 4a-7a, 12a-21a had been hydrolyzed with NaOH to provide compounds two, 4-7, and 12-21. Compounds 8 and 9 had been synthesized as outlined by the procedure reported in Scheme two. Methyl ester hydrochlorides 8c and 9c had been ready starting from O-benzyl L- or D-serine. Then compounds 8c and 9c have been coupled to 1 (as described above), PRMT4 Inhibitor Biological Activity providing the corresponding amide conjugates 8b and 9b. Reductive deprotection of intermediates 8b and 9b afforded 8a and 9a. These compounds have been hydrolyzed giving the final NPY Y1 receptor Antagonist Source solutions 8 and 9. Compounds ten and 11 were synthesized based on the procedure reported in Scheme 3. The amino group of L- or D-asparagine was protected with di tert-butyl dicarbonate (Boc2O). This reaction gave compounds 10d and 11d, which have been transformed in the corresponding benzyl esters 10c and 11c. The Boc protection was then removed giving 10b and 11b, which in turn were coupled to 1 to acquire compounds 10a and 11a.31 The final solutions ten and 11 were obtained by removing the benzyl ester protection by way of hydrogenation.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptRESULTS AND DISCUSSIONMolecular modeling and discovery of glycolithocholic acid (two) as an EphA2 antagonist Molecular modeling investigations previously performed by our group22 suggested that LCA (1) can mimic the binding mode of ephrin-A1 towards the EphA2 receptor32 by inserting its cyclopenta[a]perhydrophenanthrene scaffold in to the hydrophobic EphA2 receptor ligandbinding channel and forming a salt bridge with Arg103 (Figure 2A),.