1988;107:1307C1316. mixture of four diastereomers).21c Since a practical route to functionalized 2,2-dichloro acids appeared impracticable, the ATRC-FR approach to Glutaminase-IN-1 chaetomellic acid A analogues lost any appeal. Now as Glutaminase-IN-1 part of a project about the acute kidney injury (AKI) from ischaemia-reperfusion in rat, we were asked to develop a versatile way to chaetomellic acid A (1), and analogues, for the prevention of the ischemic damage, through the inhibition of the pathway Ras/ERK1/2. Here we describe the new synthetic method and the serendipitous discovery of an ACA analogue having a higher Glutaminase-IN-1 affinity for the FTase than the natural product. 2. Results and discussion 2.1. Synthesis of ACA To solve the intrinsic problems of the ATRC-FR paths to maleic anhydrides, we have recently studied the copper catalyzed radical cyclization (RC) of configured C=C bond,23b the same geometry was assigned to 10, and to the other enamides we prepared in this work. Using a reasonably pure sample of 10 (a condition that has to be maintained also with the other enamides we prepared), the radical cyclization proceeded smoothly giving, as expected, the disulfide 11 and the thioacetal 12 (Scheme 7, path and respectively). With the anhydride 18 in our hand, we were ready to test the thio-click reaction. We focused on the preparation of 19, an isosteric ACA analogue. Thus, the radical addition of butanethiol to 18 had to be realized. Since anhydride 18 carried two olefinic functions a problem of chemoselectivity could raise. The two C=C bonds are, however, quite different: one is electron-poor and tetrasubstituted, whereas the other is usually electron rich and monosubstituted. As the thiyl radical is usually electrophilic37 and the rate of radical attack controlled by steric and polar factors,38 we anticipated that attack at the apical methylene carbon should be favored.34d At the beginning we tried the initiation of the radical chain at room temperature, using organoboranes (such as triethylborane or 2-ethylbenzo[computational analysis has been carried out around the interaction of thia-analogue 27 with FTase in order to get insights into its moderate increased inhibition potency with respect to the parent Glutaminase-IN-1 compound 2. After an extensive analysis of the X-ray structures of FTase available in the PDB data bank, the X-ray structure of rat FTase complexed with farnesyl pyrophosphate (FPP) (pdb ref code 1FT2)44 and of the ternary complex in which the rat FTase interacts with the FPT-II FPP analog and the substrate peptide CVLS (pdb ref code 1TN8)45 were selected. Superposition of the two 3D structures by alignment of all enzyme C atoms shows that the structures of the enzyme in these complexes are essentially identical, and the location and conformation of the isoprenoid and nonreactive isoprenoid analogs are very comparable. In fact, only a few minor side chain rearrangements are observed in the proximity of the anionic head binding sites of the isoprenoid analogs, and of the C-terminal carboxylate residues of the CVLS peptide. The choice of the conformation of the thia-analogue 27 (among the many low-energy quasi-extended conformations it can assume) to be considered for docking experiments was based on: a) the best alignment with the isoprenoid analogs, taken as references; (Physique 5a and b) the best fit of the molecular volume of 27 and the volume of the supermolecule formed by FPP and FPT-II FPP, which can be considered to reflect the overall shape and the conformational flexibility of the enzyme binding site (Physique 5b). Open in a separate window Physique 5 a) Alignment of FPP, in the conformation assumed in the 1FT2 pdb structure (blue), FPT-II FPP, in the conformation assumed in the PDGFRA 1TN8 pdb structure (yellow), and of the thia-analogue 27 in the quasi-extended conformation chosen (atom colors: carbon atoms are in green, oxygen atoms in red, and sulfur atom in orange). b) Superposition of the molecular volume of 27 (green) and the volume of the supermolecule (white) formed by FPP and FPT-II FPP. In the physique the hydrogen atoms are omitted for clarity. The structural motif of hydrophilic head group of 27 is usually well accommodated into the highly positively charged pocket, located near the subunit interface and adjacent to the catalytic zinc ion, Glutaminase-IN-1 which constitutes the site of the diphosphate moiety of farnesyl diphosphate (FPP) in the crystal structures of the binary and ternary complexes.7c,44,46 This pocket is formed by amino acid residues K164, Y200, and H201 from the -subunit of the enzyme and Y300, K294, R291, H248 from the -subunit (Determine 6, top). Open in a separate window Physique 6 The conversation of inhibitor 27 and the FTase binding.