Original articleSynthesis and characterization of novel phosphonocarboxylate inhibitors of RGGT
Graphical abstract
Introduction
Compounds bearing a phosphonocarboxylate (PC) scaffold, in which phosphonate and carboxylate groups are linked via one carbon atom have received considerable attention as potential antiviral [1], [2], [3] and anticancer agents [4], [5]. They are therefore structurally similar to pyrophosphate, an important endogenous regulator of mineralization, but unlike pyrophosphate they are resistant to hydrolysis.
The interest in nitrogen-containing PCs was initiated during structure–activity relationship (SAR) studies of the well established nitrogen-containing bisphosphonates (BP), which inhibit osteoclast-mediated bone resorption by targeting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS) (Fig. 1) [6].
The first PC (1c, 3-PEHPC, NE-10790), derived from bisphosphonate 2, in which one phosphonic group was exchanged for a carboxylic group, has no effect on FPPS, but was found to be the first selective inhibitor of RGGT [7], inhibiting osteoclast activity in vitro [7], in vivo [8] and exhibiting anti-cancer properties both in vitro and in an animal model [9], [10]. Comparable potency was observed for analogs 1a–b [4]. Much higher potency against RGGT was achieved with the subsequent generation of PCs, compounds 3a–c, derived from BP 4 (Fig. 2) [11], [12].
RGGT is responsible for geranylgeranylation of Rab proteins (Fig. 1), the largest family of small GTPases. Both RGGT and Rab proteins have recently been implicated in numerous diseases including cancer, neurological disorders, bacterial and viral infections [13], [14], [15]. To date a few classes of RGGT inhibitors have been identified, including PC derivatives of BPs [4], [7], [11], [12], tripeptide analogues [16], [17], compounds derived from GGTase 1 inhibitors, with pentasubstituted pyrrolidine analogs [18], compounds derived from FTase inhibitor, based on a tetrahydrobenzodiazepine scaffold [19], [20] and triazole-based BPs [21].
The lack of a crystal structure of the RGGT–PC complex limits the possibilities for the rational design of more potent PC inhibitors and therefore further elaboration of the pharmacophore model by synthesizing and evaluating the biological activity of new analogs is required.2 It has been proposed that PCs interact with the TAG tunnel of RGGT, a tunnel adjacent to the GGPP binding site that is not present in the other prenyl transferases, FTase and GGT-1, making it a promising site for selective targeting of RGGT [17]. The TAG tunnel is thought to accommodate the first GG-cysteine prior to addition of the second geranylgeranyl group, explaining why the PCs inhibit only the second geranylgeranylation step [22].
We based our studies on the trend observed for the PC–BP pairs that have already been investigated (1–2 and 3–4), which implied a possible correlation between structure–activity relationships for inhibition of FPPS by BPs and RGGT by PCs with respect to the nitrogen-containing group.
Therefore, we focused on the synthesis of 5, the PC analog of the potent 3rd generation heterocyclic BP, zoledronic acid 9, and PC analogs (6–8) of the 2nd generation aminoalkyl BPs, pamidronic 10, alendronic 11 and ibandronic 12 acids (Fig. 3), which are all potent, clinically used BPs. Until now, only analogs bearing a heterocyclic amine side chain have been tested (Fig. 2: 1a–c, 3a–c) [4], [7], [11], [12].
The second group of compounds that we generated were analogs of PCs with reported potency against RGGT (1c, 3c), in which the hydroxyl group was exchanged for an alkyl substituent (Fig. 4). The influence of a hydrophobic substituent that mimics the geranylgeranyl pyrophosphate (GGPP) substrate of RGGT on PC activity has not been studied before. We synthesized five analogs of 3-PEHPC (1d–h), modified with alkyl chains of different lengths and one analog of 3-IPEHPC (3g). We expected that an increase in the hydrophobicity of the PCs might result in increased activity due to the inhibitor fitting into the GGPP binding pocket and/or improved cell permeability [23].
Section snippets
Chemistry
A variety of approaches to the synthesis of PCs have been described. They include formation of C–P bond, via introduction of phosphonic group either in the Arbuzov–Michaelis reaction of trialkyl phosphite with α-bromoesters [24], or in a reaction of enolate and chlorodialkyl phosphite [25], or reaction of diethyl phosphite with α-ketoester [11]. In other methods the carboxylic moiety is introduced into the carbon adjacent to the phosphonate group using lithium alkylphosphonate and diethyl
Discussion
The cellular assays identified two PC analogs as potent and specific inhibitors of RGGT (5b > 5a) (Table 1). 5b was as potent as 3-IPEHPC (3c), which is the most potent PC inhibitor of RGGT yet identified. However, 5b is more selective, since at high concentrations 3c also inhibits Rap1a prenylation, most likely due to inhibition of GGPPS [11].
The cell-based FRET-assays clearly showed that 5a and 5b both significantly decreased the membrane localization of dual-geranylgeranylated Rabs, but not
Conclusions
In summary, we have developed simple routes for the synthesis of novel PC analogs of anti-resorptive bisphosphonates, which were predicted to extend the library of PC-type inhibitors of RGGT. Indeed, biological evaluation of the compounds identified two analogs of zoledronic acid, 5a and 5b, to be highly effective RGGT inhibitors in intact cells, with 5b more potent than 5a and of comparable potency and higher selectivity to the most potent and selective PC inhibitor of RGGT reported thus far.
Experimental section
NMR spectra were measured at 250.13 or 700 MHz for 1H NMR, 62.90 or 170 MHz for 13C NMR, 101.30 MHz for 31P NMR, 235.31 MHz for 19F NMR. Chemical shifts (δ) are reported in parts per million (ppm) relative to: in 1H NMR: internal residual CHCl3 in CDCl3 (δ 7.26), or internal residual HDO in D2O (pH ∼ 12, δ 4.76); in 31P NMR: external 85% H3PO4 (0 ppm); in 13C NMR: CDCl3 (77.00 ppm); in case of solutions in D2O signals from residual solvents were used as reference (EtOH (15.17 and 58.13) or
Acknowledgement
KMB is grateful for financial support provided by Ministry of Science and Higher Education in Poland (N N204 519839 and IP2010 003070). FPC is grateful for financial support from the Alliance for Better Bone Health. AKN is grateful for support by the graduate school, National Doctoral Programme in Informational and Structural Biology (ISB). This work was supported by the Academy of Finland (252381) fellowship grant, the Sigrid Juselius Foundation, the Cancer Society of Finland and the
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These authors contributed equally.