E: i) bile acid derivatives, which include lithocholic acid (LCA, compound 1)20,21 and cholanic acid,22

E: i) bile acid derivatives, which include 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 which 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 mTORC1 Activator Accession Eph-ephrin technique modulators, we recently focused our attention on LCA, a compound characterized by a (five)-cholan-24-oic acid scaffold, which competitively displaces ephrin-A1 from the ligand-binding domain of EphA2.21 Within the present work, we report the synthesis and structure-activity relationship (SAR) profile of an extended series of -amino acid conjugates of LCA, made beginning from a theoretical binding mode of LCA into the EphA2 binding internet site. The synthesized compounds have been examined for their ability to disrupt EphA2-ephrin-A1 binding and to stop EphA2 phosphorylation in 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 while compounds 2, 4-7 and 12-21 were synthesized according to a process comparable to that described in references.29,30 Methyl ester hydrochlorides of -amino acids were bought from commercial 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; offered in PMC 2014 April 11.Incerti et al.Pageester hydrochloride with the suitable -amino acid was reacted with 1 (LCA), working with N-(3dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) as coupling agent. The resulting amides 3, 4a-7a, 12a-21a were hydrolyzed with NaOH to provide compounds two, 4-7, and 12-21. Compounds eight and 9 were synthesized as outlined by the procedure reported in Scheme two. Methyl ester hydrochlorides 8c and 9c had been ready beginning from O-benzyl L- or D-serine. Then compounds 8c and 9c were coupled to 1 (as described above), providing the corresponding amide conjugates 8b and 9b. Reductive deprotection of intermediates 8b and 9b afforded 8a and 9a. These compounds had been hydrolyzed giving the final goods 8 and 9. Compounds 10 and 11 were synthesized as outlined by the procedure reported in Scheme three. The amino group of L- or D-asparagine was protected with di tert-butyl dicarbonate (Boc2O). This reaction gave compounds 10d and 11d, which were transformed inside the corresponding benzyl esters 10c and 11c. The Boc protection was then removed giving 10b and 11b, which in turn had been coupled to 1 to acquire compounds 10a and 11a.31 The final goods ten and 11 were obtained by removing the benzyl ester protection by means 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 to the EphA2 receptor32 by Nav1.7 Antagonist Storage & Stability inserting its cyclopenta[a]perhydrophenanthrene scaffold in to the hydrophobic EphA2 receptor ligandbinding channel and forming a salt bridge with Arg103 (Figure 2A),.