Design and synthesis of non-hydrolyzable homoisoprenoid \xce\xb1-monofluorophosphonate inhibitors of PPAPDC family integral membrane lipid phosphatases
An efficient, diversity oriented synthesis of homoisoprenoid \xce\xb1-monofluorophosphonates utilizing electrophilic fluorination is presented along with their activity as inhibitors of PPAPDC2 family integral membrane lipid phosphatases. These novel phosphatase-resistant analogues of isoprenoid monophosphates are a platform for further structure-activity relationship studies and provide access to other isoprenoid family members where the phosphate ester oxygen is replaced by a \xce\xb1-monofluoromethylene moiety. 2009 Elsevier Ltd. All rights reserved.
<p>Farnesyl diphosphate (FPP, 1) is a key intermediate in the mevalonate pathway for the biosynthesis of cholesterol and is the precursor for the biosynthesis of geranylgeranyl diphosphate (GGPP, 2), ubiquinone, heme and dolichol.1, 2 FPP and GGPP also function as prenyl donors for the isoprenylation of many proteins involved in cell growth and proliferation.3–5 The mevalonate pathway makes these isoprenoid diphosphates without synthesizing the corresponding isoprenols.6 However, isoprenols such as farnesol 3 and geranylgeraniol 4 are produced in cells and tissues by sequential enzymatic dephosphorylation of their diphosphates (Scheme 1).7, 8</p><p>PDP1/PAPDC2 in mammalian cells decreases protein isoprenylation and results in defects in cell growth and Cultured cells can incorporate exogenously provided isoprenols into sterols and proteins which requires their conversion to the corresponding diphosphates.7, 9–13 Together, these observations identify a route for interconversion of isoprenols and isoprenoid diphosphates that is independent of the mevalonate pathway. This process may enable recycling of "scavenged" isoprenols or could allow independent regulation of flux or substrate channeling to produce individual mevalonate pathway endproducts which have diverse functions.</p><p>Two members of the phosphatidic acid phosphatase domain containing (PPAPDC) class of integral membrane phosphatases are broad specificity lipid phosphomonoesterases that exhibit a preference for lipid diphosphate substrates including isoprenoid diphosphates.7, 14, 15 In vitro, one of these enzymes, PDP1/PPAPDC2 can catalyze the sequential hydrolysis of isoprenoid diphosphates first to the corresponding monophosphate and then to the isoprenol. Overexpression of PDP1/PAPDC2 in mammalian cells decreases protein isoprenylation and results in defects in cell growth and cytoskeletal organization that are associated with dysregulation of isoprenylated Rho family of GTPases suggesting that this enzyme may be a functionally involved in interconversion of isoprenols and isoprenoid diphosphates. Two related proteins PPAPDC1A and PPAPDC1B are also broad specificity enzymes. While their activities against isoprenoid diphosphates have not been reported, in vitro both enzymes actively hydrolyze the plant lipid diacylglycerol pyrophosphate (DGPP). Studying the physiological functions of these PPAPDC2 family proteins is hampered by the lack of small molecule probes and selective inhibitors. Thus, we have prepared a series of metabolically stabilized isoprenoid monophosphate mimics that we show are inhibitors of a representative member of the PPAPDC family of integral membrane lipid phosphatases.</p><p>Phosphonates are commonly used as hydrolytically stabilized analogues of phosphate monoesters.16–19 Metabolically stabilized inhibitors of protein farnesyl transferase based on farnesyl-α-difluoromethlenephosphonate have been prepared.20 Recent experimental studies indicate that the α-monofluoromethylene phosphonate is a better mimic of phosphate monoesters than either the methylene or difluoromethylene derivatives and the utility of this moiety as a probe of biochemical function has been demonstrated in recent years.21–24 We had previously shown that analogues of FPP where the isoprene units were replaced by substituted aniline moieties, (Scheme 1, AGPP 5), can serve as substrates for a variety of isoprenoid diphosphate utilizing enzymes, including the PPAPDC family integral membrane phosphatases and the protein prenyl transferases FTase and GGTase.25–27 In addition, an unidentified cellular pathway, possibility involving a kinase, converts isoprenols 3, 4, and AGOH 6 to their corresponding diphosphates 1, 2, and 5.7 We took advantage of this substrate promiscuity to design potential inhibitors based on both natural and unnatural aniline substituted isoprenoids.</p><p>The synthetic strategy for the target α-monofluorophosphonates is outlined in scheme 2 and was based on installing the fluorine prior to incorporation of the aniline group, followed by revealing the charged phosphonic acid in the last step by deprotecting the phosphonate esters with trimethylsilyl bromide (TMSBr). This strategy enables the introduction of structural diversity into the isoprenoid moiety after establishing the common α-monofluoromethylene replacement of the bridging phosphate ester oxygen.</p><p>Our initial approach was to incorporate the α-fluoromethlenephosphonate diester en bloc. Attempts to alkylate (EtO)2P(O)CFHZnBr or (EtO)2P(O)CFHCu or the α-lithiated α-fluoro-trimethylsilylphosphonate carbainion made from diethyldibromofluoromethylphosphonate with geranyl bromide proved unsuccessful in our hands.28, 29 The failure of these approaches led us to abandon the α-flurophosphonate moiety as a synthon and incorporate the fluorine via electrophilic fluorination with N-fluorobenzenesulfonamide (NFBS). Of the available electrophilic fluorinating agents, NFBS was chosen for its selective reactivity under mild conditions and ease of handling and storage.30,31,32 Accordingly, lithiation of either commercially available dimethyl or diethylmethylphosphonate, followed by alkylation with either geranyl or farnesyl bromide gave 7a-b and 8a-b in quantitative yield.19 Treatment of phosphonate 7a-b with n-butyllithium at -98°C in THF/HMPA generated the lithiated carbanion which was fluorinated using NFBS to give α-fluorophosphonate 9a-b in 71% and 68% yield respectively. The yield of α-monofluorophosphonate was reduced or absent when other bases such as LiHDMS, KHDMS or t-BuLi as well as when different solvent mixtures were employed (Table 1). The yield of α-monofluorophosphonate was also reduced to 38% when the diethyl ester 8a-b was used, suggesting that steric bulk around the phosphonate interferes with anion access to NFBS. Optimum deprotection of α-monofluorophosphonates 9a and 9b was achieved using a 15:30:1 molar ratio of TMSBr, pyridine and the phosphonate diester at 0°C for 45 min. followed by quenching with ice-cold water and stirring for another 4 hr at 0°C. TMSBr alone, other ratios of TMSBr and pyridine or stirring with aq. alkali for several days resulted in difficult to separate mixtures of partially hydrolyzed esters and other byproducts.19, 33 Attempt to optimize deprotection of ethyl esters 10a-b only gave partially hydrolyzed compounds along with starting material.</p><p>We then adapted our previously reported strategy for the synthesis of anilinogeraniol derivatives to prepare α-monofluorophosphonates 16a-f as outlined in scheme 2.34 Oxidation of phosphonates 9a with SeO2 provided the corresponding mixture of allylic alcohol 12 and aldehyde 13 respectively.10, 34 The alcohol 12 was recovered and oxidized to the desired aldehyde 13 with manganese dioxide. Reductive amination of the appropriate anilines 14a-f with aldehyde 13 using NaBH(OAc)3 gave α-monofluorophosphonate esters 15a-f. As expected from previous work, reductive amination of the anilines with the α,β-unsaturated aldehyde gives a mixture of cis/trans isomers about the 7,8 double bond in an approximately 1:10 ratio.34 Attempts to separate the isomers by column chromatography, silica-HPLC, or reverse-phase HPLC were unsuccessful. Deprotection of the phosphonate esters with the optimized TMSBr/pyridine conditions gave the desired α-monofluorophosphonic acids 16a-f which were stored at −20°C immediately after purification.</p><p>We used membranes from insect cells expressing PPAPDC1b as a source of activity to investigate the ability of the α-monofluorophosphonates to inhibit dephosphorylation of lipid phosphate substrates by this enzyme. Like PPAPDC2, PPAPDC1B hydrolyzes the representative substrate diacylglycerol pyrophosphate presented in mixed phospholipid and detergent micells with an apparent KM of 130 μM (Figure S1, supplementary data). A more detailed characterization of the PPAPDC1B enzyme will be published elsewhere. Phosphatase activity was determined in assays containing a fixed 10 μM concentration of the indicated α-monofluorophosphonates and the data are presented as a % inhibition observed in reactions containing 400 μM 1,2-dioctanoyl-sn-glycerol 3-phosphate (DGPP) substrate. Assuming a purely competitive mode of inhibition, the most potent of these compounds 16d, 16e and 16f inhibit PPAPDC1B activity with Ki values of ~10 μM. (Table 2). Phosphonate analogues of phosphatidic acid are competitive inhibitors of the related enzyme PPAPDC2 with comparable inhibition constants (Ki = 0.4 μM).35 These new inhibitors are therefore promising chemical tools to investigate the biological function and substrate-activity relationship of PPAPDC family of integral membrane lipid phosphatases and may prove to be of value for further investigations of the interconversion of isoprenoid diphosphates and their corresponding isoprenols in regulation of the mevalonate pathway.</p>