# Recent Advances in the Development of Mammalian Geranylgeranyl Diphosphate Synthase Inhibitors
## 3 GGDPS Inhibitors  
In 1992, [PERSON] et al., reported 3-aza-2,3-dihydrogenarylgeranyl diphosphate (3-azaGGPP) (3, Figure 2) as an inhibitor of GGDPS [27]. Since it was modelled upon GGPP, 3-azaGGPP contains a longer carbon chain than the previously developed bisphosphonates and specifically inhibited GGDPS,but not FDPS, isolated from rat liver. In 1999, [PERSON] et al. reported the cloning, expression and partial characterization of human GGDPS [28]. While FDPS and GGDPS share limited sequence homology, the five amino acid motif characteristic of trans-prenyl transferases is present in both enzymes. This fueled investigation into bisphosphonates and other compounds resembling FDPS inhibitors as potential inhibitors of GGDPS. Overall, the differences in the structures between the two synthases have allowed for the development of distinct sets of specific inhibitors. While the most potent of the FDPS inhibitors are small molecules containing a nitrogen heterocycle and a bisphosphonate head group, the majority of the GGDPS inhibitors prepared to date (described below) incorporate one or more longer alkyl chains into the bisphosphonate-based structure. In 2002, a set of bisphosphonates and azaprenyl diphosphates was reported with IC\({}_{50}\) values against purified GGDPS enzyme ranging from 0.14-690 uM [29]. Such results suggested that bisphosphonate compounds containing isopenoid chains might also function as inhibitors of GGDPS. These prior studies prompted the design of a library of isoprenoid bisphosphonates, including digeranyl bisphosphonate (DGBP, **4**, Figure 2), which inhibited geranylgeranylation of small GTPases, but not farnesylation [30]. Shortly thereafter, it was discovered that DGBP specifically inhibits GGDPS, resulting in depletion of intracellular GGPP [31]. DGBP was reported to have an IC\({}_{50}\) value of \(\sim\)200 nM against purified enzyme and inhibited protein geranylgeranylation, but not farnesylation, in cultured cells [31]. Structure-function work suggested that with regard to the alkyl substituents on the carbon central to the phosphonates, at least one geranyl chain (or a longer isoprenoid) was required for significant inhibition of GGDPS. Crystallographic studies showed that DGBP's V-shaped structure occupied the enzyme's active site, with the bisphosphonate group complexing with magnesium ions and the two prenyl side chains occupying the substrate-binding site of FPP, as well as the product-binding site of GGDPS [32]. Preparation of the isoprenoid bisphosphonates proved to be relatively straightforward (Figure 3). When treated with base and an alkyl halide, commercial tetraethyl methylene bisphosphonate (**14**) can be alkylated to obtain the monoalkyl derivative **15**, and the process can be repeated to introduce a second alkyl substituent (**16**). Final hydrolysis readily affords the corresponding salts **17**. Evaluation of a series of mono- and dialkylated compounds prepared in this way revealed that the di-substituted compounds are generally more potent than the mono-substituted compounds [33]. Assessment of a series of aromatic bisphosphonates (e.g., **5**, Figure 2) demonstrated selective inhibition of GGDPS over FDPS, albeit at lower potency than DGBP [34]. Through a similar strategy, a series of geminal bisphosphonate ethers was prepared based upon the hypothesis that incorporation of the ether moiety would lower the pKa3 and improve activity. For this series, the commercial phosphonate **18** was first converted to an ether through a classical Williamson synthesis. After introduction of the ether, treatment of phosphonate **19** with base and diethyl chlorophosphate resulted in C-P bond formation and gave the bisphosphonate **20**. Subsequent C-alkylation (to **21**) and hydrolysis of the phosphonate esters give the desired ethers **22**. The most potent reported example of this set, the \(O\),\(C\)-digeranyl geminal bisphosphonate **6** had an IC\({}_{50}\) of 82 nM against GGDPS in an vitro enzyme assay [35].