Synthesis of inhibitors targeting the downstream enzymes in the isoprenoid biosynthetic pathways

Abstract

The nitrogenous bisphosphonates pamidronate, alendronate, risedronate, and zoledronate are used clinically in the treatment of bone disease. All of these drugs inhibit the enzyme farnesyl diphosphate synthase (FDPS), which mediates production of farnesyl diphosphate (FPP). However, because it is a branch point in isoprenoid biosynthesis, FPP is involved in the biosynthesis of several different substrates at the same time. One key enzyme downstream of FDPS in isoprenoid biosynthesis is geranylgeranyl diphosphate synthase (GGDPS) which affords the geranylgeranyl diphosphate (GGPP) necessary for prenylation of the small GTPases such as Ras, Rab, Rho and Rac, that are important signaling proteins. Non-nitrogenous analogues of the clinical drugs, including mono-and bisisoprenoid bisphosphonates, have been developed more recently. These new analogues have been found to inhibit GGDPS selectively. Because it is important to inhibit the generation of GGPP, selective inhibition of GGDPS is highly desirable. In previous research, digeranyl bisphosphonate (DGBP) was discovered to show good inhibition of GGDPS. In order to obtain more potent analogues of the compound DGBP, and to study the biological effect of an α-alkoxy group on bisphosphonate compounds, a series of ether bisphosphonates has been prepared and studied. A second important enzyme in isoprenoid biosynthesis is geranylgeranyl transferase II (GGTase II). This enzyme transfers GGPP to Rab proteins, and thus converts the parent proteins to lipoproteins which are essential for their proper cellular vii localization. One known inhibitor of this enzyme is the chemical 3-PEHPC, but a high concentration of this compound is necessary to generate any cellular effects. In an effort to study the cellular effects that result from inhibition of this enzyme, and to develop more potent inhibitors, my research has focused on modification of 3-PEHPC to obtain derivatives that may have improved biological activity. Both the known compounds 3-PEHPC and 3-PEPC, and new structures, including the first generation PEHPC N-oxides and the second-generation compounds prepared through click chemistry, have been prepared and tested for activity in this system. viii

Synthesis of geminal bisphosphonates as potential inhibitors of GGDPS

“…To confirm that this allylic azide rearrangement was the cause for the mixture of isomers in triazole bisphosphonates 25-27, geraniol, a C10 E-allylic alcohol, and nerol, the parallel C10 Z-allylic alcohol, were converted first to the corresponding bromides and then to the azides, which then were subjected to click reaction conditions. 29 A 2:1 ratio of E:Z olefin isomers was observed in the NMR spectrum for each click product, regardless of whether geraniol or nerol was the starting material, 31 thus confirming that the mixture of isomers was indeed from the allylic azide rearrangement.…”

“…38 Lastly, the tetraethyl ester of triazole bisphosphonate 42 was hydrolyzed under standard conditions to afford the tetra sodium salt 43. 31,33 The sodium salt of bisphosphonate 41 was also seen as a potential source of biological activity if the epoxide could survive the ester hydrolysis. The time allowed for reaction with NaOH was found to be crucial, as the epoxide itself is susceptible to hydrolysis, and diol formation was observed even with reaction times as short as 3.5 minutes.…”

“…Bisphosphonate 41 was successfully hydrolyzed with TMSBr and collidine, followed by treatment with NaOH for just two minutes to give the tetra sodium salt 44. 33 1) TMSBr, collidine 0 °C to rt 2) NaOH, 2 min The geranyl 33 and farnesyl 31 analogues of compound 43 were both synthesized in a similar fashion as the neryl triazole bisphosphonate salt (43), starting from commercially available geraniol and trans, trans-farnesol, respectively. The corresponding geranyl 33 and farnesyl epoxide sodium salts also were made in order to evaluate their biological activity.…”

“…However, it was difficult to determine if formaldehyde was formed because it is volatile. It also was challenging to conclude whether diethyl methyl phosphate (97) or diethyl phosphate (98) was produced, given that both compounds have similar 31 P NMR shifts (between 0 and -1 ppm), and a peak at -1 ppm was observed in the 31 P NMR spectrum. Basic hydrolysis of diethyl (benzyloxymethyl)phosphonate ( 99) only provided ethyl hydrogen phosphonate 100, just as stated in the literature.…”

“…60 To establish if the benzyl group would be removed faster than the phosphonate esters would be dealkylated, a model reaction was done by allowing diethyl (benzyloxymethyl)phosphonate (99) to react with BBr3 (Figure 13). After 30 minutes, the 31 The next step in this approach to a homoneryl compound was removal of the benzyl protecting group in order obtain an alkylated α-hydroxy phosphonate. The benzylprotected (hydroxymethyl)phosphonate 103a was allowed to react with BBr3 (Figure 40)…”