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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC 2017 August 15.
Published in final edited form as:
PMCID: PMC4955788
NIHMSID: NIHMS797332

A New Motif for Inhibitors of Geranylgeranyl Diphosphate Synthase

Abstract

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

Keywords: GGDP synthase, inhibition, isoprenoid biosynthesis, bisphosphonate monoester, prodrug

Graphical abstract

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1. Introduction

Geranylgeranyl diphosphate synthase (GGDPS) is a key enzyme in the later stages of the mevalonate pathway in humans1,2 and other organisms.3 Among other applications, the linear C20 diterpenoid that it affords (geranylgeranyl diphosphate or GGDP) is utilized to convert a variety of proteins to lipoproteins through post-translational modification, and the resulting proteins are central to important signaling and protein transport processes. Clinical drugs such as zoledronate (1) and pamidronate (2, Figure 1)4 that inhibit the upstream enzyme farnesyl diphosphate synthase (FDPS) are used for treatment of osteoporosis, Paget's disease, and malignant bone disease including myeloma.5 However, there is evidence that suggests that these drugs exert their effect through depletion of GGDP,2 which increases interest in identification of compounds that inhibit GGDPS directly.

Figure 1
Some bisphosphonates in current clinical use.

One compound that has been found to inhibit GGDPS selectively is digeranyl bisphosphonate (3, Figure 2).6 The bisphosphonate head group is important to binding with the enzyme through coordination with magnesium cations in the active site. However, early investigation into the nature of substrate binding elucidated the importance of the two nonpolar side chains as well, because they can occupy hydrophobic channels within the enzyme. This led to the hypothesis that binding would be enhanced with a ‘V-shaped’ inhibitor.1 Early inhibitors attained a V-shape through placement of two isoprenoid chains on the α-carbon of the bisphosphonate,6,7 but some linear compounds also show significant activity. For example, we recently reported that a linear triazole bisphosphonate is also a potent inhibitor of this enzyme (IC50 = 45 nM).8 This clearly supports the view that still other strategies for including isoprenoid chains on a methylene bisphosphonate may offer opportunities for potent inhibitors. In this paper, we report the synthesis and initial bioassays of a family of compounds that can achieve a shape similar to digeranyl bisphosphonate by placement of one isoprenoid chain on the α-carbon and a second isoprenoid chain as a phosphonate ester (e.g. 4). Among other attractive features, this structure could more closely mimic the degree of negative charge found in a diphosphate monoester, and this general structure still allows for incorporation of a third substituent on the α-carbon.

Figure 2
V- and U-shaped isoprenoid bisphosphonates.

2. Synthesis

At the outset of our work we decided to target a prodrug of structures like compound 4 rather than the salts themselves for a variety of reasons. Highly charged phosphonate salts may have trouble crossing the cell membrane,9,10 while more lipophilic prodrugs can penetrate rapidly by simple diffusion and liberate the charged phosphonate once inside the cell.11 Various analyses of cell lysate easily can distinguish between compounds that inhibit GGDPS and those that inhibit geranylgeranyl transferase I and/or II (GGTase I and GGTase II), thus providing information on both activity and selectivity from one assay. The specific prodrug form selected was the pivaloyloxymethyl or POM group, in part because of its long history12 and in part because it already is in clinical use in both antibiotics13 and especially in antiviral agents.14 The POM group is cleaved by nonspecific esterases within the cell, which liberates pivalic acid, formaldehyde, and the phosphonic acid, while simple alkyl esters of phosphonic acids are far more stable to metabolism.11

The synthetic sequence began with commercial dimethyl methylphosphonate (5). After treatment of this phosphonate with n-BuLi, reaction with freshly prepared geranyl bromide (6) gave the alkyl phosphonate 7. The mono methyl ester mono acid chloride 8 then could be obtained by reaction with oxalyl chloride in DMF.15 In principal, reaction of this acid chloride with any alcohol should afford a mixed diester. However, out of concern for the lability of an allylic ester like that derived from geraniol,16 S-(−)-citronellol was employed to provide the mixed ester 9. Phosphonylation of compound 9 could be accomplished by treatment with strong base and dimethyl chlorophosphate.17-19 This gave the key intermediate 10 in modest yield.

Compound 10 served as the point of divergence to obtain several products of similar structure. The direct reaction of this trimethyl ester with POMCl in the presence of sodium iodide gave the triPOM compound 11 in low yield,16 and the larger citronellyl ester appeared to be replaced more slowly than the methyl groups under these conditions. To introduce an additional substituent at the α-carbon, treatment of ester 10 with sodium hydride followed by reaction with an alkylating agent (methyl iodide, or allyl, prenyl, or geranyl bromide) gave the expected products 12–15. With each of these four compounds, reaction with POMCl and sodium iodide converted the methyl esters to POM groups while preserving the isoprenoid ester, albeit in low yields. Nevertheless, through these parallel reactions the desired products 16–19 were obtained in quantities sufficient for bioassay.

It should be noted that compounds 11 and 1619 were obtained as mixtures of stereoisomers. While the (S)-citronellol employed was a single enantiomer, no effort was expended at this stage to control the stereochemistry at the adjacent phosphorus or at the α-carbon. This is readily apparent in the 31P NMR spectra of these compounds (cf. the Supporting Information). In the phosphorus spectrum of compound 9, two resonances of equal intensity are observed reflecting two stereoisomers at phosphorus. Introduction of the second phosphorus through formation of compound 10 results in a more complex 31P NMR spectrum, with two clusters of peaks reflecting both formation of the new stereocenter at the α-carbon and phosphorus-phosphorus coupling. Similar clusters are observed in most of the subsequent products, although introduction of the second geranyl chain in compounds 15 and 19 eliminates the stereocenter at the α-carbon, diminishes the number of diastereomers, and simplifies those phosphorus spectra.

Assuming that the POM groups function as prodrugs as intended,11 the phosphorus stereocenter will be destroyed once the POM group of the mixed phosphonate ester is hydrolyzed within the cell. The stereochemistry at the α-carbon is more permanent, at least in compounds 16–18 and probably in compound 10 at physiological pH, so even after POM hydrolysis the materials tested would be a mixture of two diastereomers. In theory, these diastereomers can be separated but because such separations can prove challenging it was decided to postpone any efforts along these lines until the biological activity of the new structures were determined. While there may be some risks associated with this approach a similar strategy has proven useful with two olefin stereoisomers, where the material first demonstrated activity as a mixture20 and subsequent synthesis of the individual stereoisomers allowed identification of the more active isomer.21

After significant activity was observed in the first representatives of this U-shaped series (vide infra), we began to investigate the importance of stereochemistry through preparation of an R-(+)-citronellol ester of the most active inhibitor, compound 16. Accordingly, the acid chloride 8 was allowed to react with the R-(+)-citronellol enantiomer to afford the mixed ester 20 (Scheme 2). Phosphonylation to the bisphosphonate 21 was achieved under conditions parallel to those used to obtain compound 10, and methylation also proceeded smoothly to give compound 22. Transformation of this trimethyl ester to the corresponding POM derivative 23 completed this reaction sequence.

Scheme 2
Synthesis of an R-(+)-citronellol ester (23).

3. Biological results and discussion

To gauge the impact of these new compounds on isoprenoid metabolism, their ability to disrupt geranylgeranylation in myeloma cells was determined. As shown in Figure 3A-B, concentration-dependent effects were observed for all tested compounds within the S-(−)-citronellol ester series. Disruption of protein geranylgeranylation was demonstrated via two methods: 1) immunoblot analysis for unmodified Rap1a which is a substrate of GGTase I (Fig 3A); and, 2) ELISA for intracellular lambda light chain which is a marker for disruption of Rab GTPase geranylgeranylation22 (Figure 3B). Of the five compounds examined, the methyl derivative 16 most potently disrupted protein geranylgeranylation. When ranked from most potent to least potent, the order is 16 > 18 > 1719 > 11. The HMG-CoA reductase inhibitor lovastatin, which inhibits the synthesis of mevalonate and therefore depletes cells of all isoprenoids downstream of mevalonate, was used as a positive control in these studies.22

Figure 3
The new bisphosphonates disrupt protein geranylgeranylation in human myeloma cells

The cytotoxic activities of these compounds were tested in three human-derived myeloma cell lines. As shown in Table 1, the structure-function relationship was the same across all tested cell lines with the methyl derivative 16 the most potent and the geranyl derivative 19 the least potent. In general, the cytotoxic activity correlated with the potency of these compounds in disrupting cellular geranylgeranylation with the exception of compound 11. The limited activity of the geranyl compound 19 is somewhat disappointing, given that the three isoprenoid chains could offer multiple ways to accomplish binding in the FDP and GGDP channels. Even so, the activity associated with compound 16 shows that this template has promise for development of new GGDPS inhibitors.

Table 1
Cytotoxic activity of the tested compounds in human myeloma cell lines. The EC50's were determined from MTT assays are shown as “mean ± standard deviation”.

After identification of compound 16 as the most potent member of this family, add-back experiments were performed with this compound and isoprenoid intermediates. As shown in Figure 4A-B, while addition of either mevalonate or GGDP prevents lovastatin's effects on disrupting protein geranylgeranylation, only GGDP prevents the effects of compound 16. In aggregate the results of these studies are consistent with these agents inhibiting GGDPS because there is global disruption of geranylgeranylation of both GGTase I and GGTase II substrates and addition of GGDP can prevent these effects.

Fig 4
The novel bisphosphonate 16 disrupts protein geranylgeranylation by depletion of GGDP

Finally, bioassays were performed to determine whether the stereochemistry of the citronellyl group has an impact on activity. As shown in Figure 5, the (R)-(+)-citronellol analogue 23 was compared directly with the (S)-(−) citronellol analogue 16. In general the potency of the two compounds appeared very similar, with compound 23 displaying slightly more activity than compound 16 at lower concentrations based on levels of unmodified Rap1a and intracellular light chain.

Fig 5
Effect of citronellol stereochemistry on activity

4. Conclusions

In conclusion, we have developed a synthetic route for preparation of bisphosphonate isoprenoid esters and used this strategy to prepare a small group of new isoprenoid bisphosphonates. These compounds have been examined in cell-based assays for their impact on isoprenoid metabolism, and found to function as inhibitors of GGDPS. The most active compounds in this series, bisphosphonates 16 and 23, show activity at sub-micromolar activity in both immunoblot and ELISA analyses. Given that both materials are still mixtures of diastereomers at the α-carbon even after hydrolysis of the prodrug groups, additional efforts to obtain the individual isomers appear to be justified. Further synthetic studies along those lines, as well as assays of the corresponding salts with the isolated enzyme, will be reported in due course.

5. Experimental procedures and methods

5.1 General experimental conditions

Tetrahydrofuran was freshly distilled from sodium/benzophenone, while methylene chloride was distilled from calcium hydride prior to use and toluene was dried over molecular sieves. All other reagents and solvents were purchased from commercial sources and used without further purification. All reactions in nonaqueous solvents were conducted in flame-dried glassware under a positive pressure of argon and with magnetic stirring. All NMR spectra were obtained at 300, 400, or 500 MHz for 1H, and 75, 100, or 125 MHz for 13C, with internal standards of (CH3)4Si (1H, 0.00) or CDCl3 (1H, 7.27; 13C, 77.2 ppm) for non-aqueous samples or D2O (1H, 4.80) and 1,4-dioxane (13C, 66.7 ppm) for aqueous samples. The 31P chemical shifts were reported in ppm relative to 85% H3PO4 (external standard). High resolution mass spectra were obtained at the University of Iowa Mass Spectrometry Facility. Silica gel (60 Å, 0.040–0.063 mm) was used for flash chromatography.

5.2 (E)-Dimethyl (4,8-dimethylnona-3,7-dien-1-yl)phosphonate (7)

Dimethyl methylphosphonate (2.56 mL, 22.9 mmol) was added to an oven-dried round bottom flask with THF (100 mL). The solution was cooled in a dry ice/acetone bath for 20 minutes, and then n-butyl lithium (10.08 mL, 25.2 mmol) was added as a 2.5 M solution in hexanes. After 1.5 hours freshly prepared geranyl bromide (5.47 g, 25.2 mmol) was added dropwise and the reaction was stirred and allowed to reach room temperature overnight (15 h). The reaction was diluted with diethyl ether, quenched by addition of water, and washed with water (3 × 20 mL), and the organic layer was dried (Na2SO4). The mixture was vacuum filtered through celite, and the filtrate was concentrated to give a dark yellow oil. Final purification via flash chromatography (EtOAc, silica gel) gave the desired product (7) as a yellow oil (5.67 g, 95%): 1H NMR (300 MHz, CDCl3) δ 5.15–5.06 (m, 2H), 3.74 (d, JPH = 10.8 Hz, 6H), 2.34–2.23 (m, 2H), 2.07–1.98 (m, 4H), 1.86–1.72 (m, 2H), 1.73 (s, 3H), 1.71 (s, 3H), 1.67 (s, 3H); 31P NMR (121 MHz, CDCl3) δ +34.4.

5.3 (E)-Methyl (4,8-dimethylnona-3,7-dien-1-yl)phosphonochloridate (8)

Compound 7 (1.93 g, 7.4 mmol) was diluted with anhydrous toluene and then concentrated under reduced pressure (3 × 3 mL) to remove any residual water. The phosphonate then was dissolved in dichloromethane (25 mL) and dimethyl formamide was added (1 drop). The solution was then cooled on an ice bath for 30 minutes. Oxalyl chloride (1.91 mL, 22.3 mmol) was added, and the solution was allowed to react for 13 hours. The excess oxalyl chloride and solvent were removed in vacuo and the resulting deep red oil was utilized without further purification. The 31P NMR spectrum showed nearly complete conversion to product (approximately a 13:1 ratio of product to starting material by integration, or 92% by 31P NMR). 31P NMR (121 MHz, CDCl3) δ +46.1 (product peak), +34.8 (residual starting material).

5.4 (S)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-4,8-dimethylnona-3,7-dien-1-yl)phosphonate (9)

Compound 8 (1.963 g, 7.4 mmol) was dissolved in anhydrous toluene (25 mL). After (S)-(−)-citronellol (2.71 mL, 14.8 mmol) and freshly distilled triethylamine (2.07 mL, 14.8 mmol) were added sequentially, the solution was allowed to react overnight. Upon addition of triethylamine formation of a white precipitate and a color change from deep red to dark brown was noted. The reaction was quenched by addition of deionized water (5 mL). A cloudy emulsion formed which clarified after washing with brine (3 × 10 mL). The organic layers were combined, dried (Na2SO4), and vacuum filtered through celite, and the filtrate was concentrated in vacuo to afford a dark yellow oil. Final purification by flash chromatography (silica gel, 50% EtOAc in hexanes) gave the desired product 9 as a light yellow oil (1.695 g, 59% yield, 64% based on recovered 7): 1H NMR (300 MHz, CDCl3) δ 5.15-5.05 (m, 3H), 4.16–4.01 (m, 2H), 3.73 (d, JPH = 10.5 Hz, 3H), 2.35-2.23 (m, 2H), 2.05–1.91 (m, 6H), 1.82–1.70 (m, 2H), 1.68 (s, 6H), 1.62 (s, 3H), 1.60 (s, 6H), 1.57–1.13 (m, 5H), 0.92 (d, J = 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 135.9 (d, JCP = 1.2 Hz), 130.8, 130.7, 124.2, 123.8, 122.7 (d, JCP = 16.4 Hz), 63.5, 51.5, 39.2, 37.2 (d, JCP = 5.3 Hz), 36.6, 28.7 (d, JCP = 0.9 Hz), 26.2, 25.1 (d, JCP = 137.9 Hz), 25.3, 25.0, 20.7, 20.5, (d, JCP = 4.6 Hz), 18.9, 17.2, 15.6, 13.8; 31P NMR (121 MHz, CDCl3) δ +33.11, +33.07; HRMS (ES+, m/z) calcd for (M+H)+ C22H42O3P: 385.2872; found: 385.2875.

5.5 (S)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-1-(dimethoxyphosphoryl)-4,8-dimethylnona-3,7-dien-1-yl)phosphonate (10)

Freshly dried 2,2,6,6-tetramethylpiperidine (5.35 mL, 31.7 mmol) was combined with n-butyllithium (12.68 mL, 31.7 mmol as a 2.46M solution in hexanes) and THF (50 mL) at dry ice/acetone temperatures. After 15 minutes, compound 9 (5.54 g, 14.4 mmol) was added slowly to the solution and allowed to react for 20 minutes. Then the solution was transferred by cannula into a solution of dimethyl chlorophosphate (3.42 mL, 31.7 mmol) in THF (15 mL) and the resulting solution was allowed to react and reach room temperature overnight (9 hours). The reaction was then diluted with diethyl ether, quenched by addition of deionized water, dried (Na2SO4), and vacuum filtered through celite. The filtrate was concentrated to a yellow oil, which was purified further via flash chromatography (silica gel, 10% EtOH in EtOAc) to give the desired product (2.72 g, 38%, 52% based on recovered 9): 1H NMR (300 MHz, CDCl3) δ 5.28 (t, J = 6.6 Hz, 1H), 5.11–5.06 (m, 2H), 4.20–4.09 (m, 2H), 3.81 (d, JPH = 11.1 Hz, 9H), 2.72–2.56 (m, 2H), 2.36 (tt, JPH = 24.0 Hz, J = 6.2 Hz 1H), 2.08–1.90 (m, 6H), 1.67 (s, 6H), 1.64 (s, 3H), 1.60 (s, 6H), 1.55–1.30 (m, 4H), 1.24–1.12 (m, 1H), 0.91 (d, J = 6.4 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 137.3, 131.5, 131.4, 124.6, 124.2, 121.6 (t, JCP = 7.4 Hz), 65.0, 53.4–53.0 (m, 3 C overlap), 40.1 (t, JCP = 105.8 Hz), 39.8, 38.7, 37.6–37.5 (m), 37.1–36.9 (m), 35.2, 29.1 (d, JCP = 1.4 Hz), 26.7, 25.8, 25.4, 24.0 (m), 19.4, 17.8, 16.3; 31P NMR (121 MHz, CDCl3) δ +26.3–26.2 (m), +24.8–24.6 (m); HRMS (ES+, m/z) calcd for (M+H)+ C24H47O6P2: 493.2848; found: 493.2845.

5.6 ((((E)-1((((S)-3,7-Dimethyloct-6-en-1-yl)oxy)((pialoxy)methoxy)phosphoryl)-4,8-dimethylnona-3,7-dien-1-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate) (11)

Compound 10 (340 mg, 0.7 mmol) was dissolved in acetonitrile (3 × 3 mL) and then concentrated under reduced pressure to remove any residual water. Compound 10 was then added to a solution of chloromethyl pivalate (0.31 mL, 2.16 mmol) and sodium iodide (330 mg, 2.2 mmol) in acetonitrile (5 mL). This reaction mixture was then heated at reflux for approximately 5 hours. The acetonitrile was removed under reduced pressure and the resulting material was dissolved in diethyl ether. The solution was washed with brine (4 × 10 mL) and the organic layer was dried (Na2SO4), vacuum filtered through celite, and concentrated to a yellow oil. Final purification by flash chromatography (silica gel, 25% EtOAc in hexanes + 1% TEA) afforded the desired product 11 as a faint yellow oil (60 mg, 11%, 20% based on recovered 10): 1H NMR (300 MHz, CDCl3) δ 5.71–5.7 (m, 6H), 5.28–5.23 (m, 1H), 5.10–5.06 (m, 2H), 4.20–4.12 (m, 2H), 2.70–2.60 (m, 2H), 2.50 (tt, JPH = 24.0 Hz, J = 6.3 Hz, 1H), 2.07–1.95 (m, 6H), 1.68 (s, 6H), 1.62 (s, 3H), 1.60 (s, 6H), 1.54-1.29 (m, 5H), 1.24 (s, 27H), 0.91 (d, J = 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 176.9, 176.8, 176.6, 137.9, 131.5, 131.4, 124.5, 124.0, 120.7, 82.8–81.8 (m, 3 C overlap), 65.2, 39.7, 38.8, 38.7, 37.3–37.2 (m), 37.00, 36.98 29.1, 26.93, 26.91, 26.88, 26.85, 26.81, 26.59, 26.58, 25.70, 25.68, 25.4, 23.71, 23.66, 19.19, 19.14, 17.68, 17.65 16.13, 16.11, 15.3; 31P NMR (121 MHz, CDCl3) δ +23.1 (d, JPP = 2.9 Hz), +23.0 (d, JPP = 2.8 Hz), +22.4–22.3 (m), +22.2–22.1 (m); HRMS (ES+, m/z) calcd for (M+H)+ C39H71O12P2: 793.4421; found: 793.4411.

5.7 (S)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-2-(dimethoxyphosphoryl)-5,9-dimethyldeca-4,8-dien-2-yl)phosphonate (12)

Compound 10 (302 mg, 0.6 mmol) was dissolved in THF (5 mL) and the solution was cooled in an ice bath for 15 minutes. After NaH (37 mg, 0.9 mmol, 60% dispersion in mineral oil) was added to the solution, it was allowed to react for 30 minutes. Iodomethane (0.06 mL, 0.9 mmol) was then added and the reaction was allowed to proceed for 1.5 hours. The solution was then diluted with diethyl ether, quenched by addition of deionized water, and washed with water (3 × 5 mL). The organic layer was dried (Na2SO4) and vacuum filtered through celite. The filtrate was concentrated to a yellow oil which was purified by flash chromatography (silica gel, 5% EtOH in EtOAc) yielding the desired compound 12 as a yellow oil (0.30 g, 97%): 1H NMR (300 MHz, CDCl3) δ 5.34 (t, J = 7.2 Hz, 1H), 5.12–5.05 (m, 2H), 4.20–4.12 (m, 2H), 3.75 (d, JPH = 10.8 Hz, 3H), 3.74 (d, JPH = 10.5 Hz, 3H), 3.73 (d, JPH = 10.2 Hz, 3H), 2.60 (td, JPH = 15.3 Hz, J = 7.4 Hz, 2H), 2.10–1.93 (m, 6H), 1.73–1.70 (m, 2H), 1.67 (s, 6H), 1.61 (s, 3H), 1.60 (s, 6H), 1.39 (t, JPH = 15.6 Hz, 3H), 1.30–1.12 (m, 3H), 0.92 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 138.2, 131.4, 131.3, 124.6, 124.3, 118.8 (t, JCP = 6.9 Hz), 65.0, 53.6–53.2 (m, 3C overlap), 41.5 (t, JCP = 133.1 Hz), 40.1, 39.7, 37.6 (t, JCP = 3.6 Hz), 37.1–37.0 (m), 31.1 (t, JCP = 6.2 Hz), 29.0, 26.7, 25.7, 25.4, 19.3, 19.3, 17.7, 17.7, 16.2; 31P NMR (121 MHz, CDCl3) δ +29.8 (d, JPP = 7.6 Hz), +28.3– 28.1 (m); HRMS (ES+, m/z) calcd for (M+H)+ C25H49O6P2: 507.3004; found: 507.3000.

5.8 (S)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-4-(dimethoxyphosphoryl)-7,11-dimethyldeca-1,6,10-trien-4-yl)phosphonate (13)

According to the procedure described for preparation of compound 12, compound 10 (300 mg, 0.6 mmol) was dried, dissolved in THF (8 mL), and treated with NaH (40 mg, 0.9 mmol, 60% dispersion in mineral oil) and allyl bromide (0.08 mL, 0.9 mmol). Standard work-up and concentration in vacuo gave a light yellow oil. Final purification by flash chromatography (silica gel, 5% EtOH in EtOAc) afforded the desired compound 13 (97 mg, 30% yield, 74% based on recovered 10) 1H NMR (300 MHz, CDCl3) δ 6.03–5.92 (m, 1H), 5.40–5.36 (m, 1H), 5.11–5.07 (m, 4H), 4.17–4.12 (m, 2H), 3.775 (d, JPH = 10.5 Hz, 3H), 3.767 (d, JPH = 10.8 Hz, 3H), 3.760 (d, JPH = 10.8 Hz, 3H), 2.69–2.58 (m, 4H), 2.10–1.96 (m, 6H), 1.74–1.71 (m, 2H), 1.67 (s, 6H), 1.61 (s, 3H), 1.60 (s, 6H), 1.55–1.30 (m, 3H), 0.91 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 137.9, 133.7 (t, JCP = 7.3 Hz) 131.5, 131.5, 124.7, 124.4, 118.8 (t, JCP = 7.7 Hz), 118.1, 65.1–64.9 (m), 53.6–53.2 (m, 3C overlap), 46.3 (t, JCP = 130.7 Hz), 40.2, 37.7–37.6 (m), 37.2–37.1 (m), 35.3–35.2 (m), 29.2, 27.4, 26.7, 25.8, 25.5, 25.1, 19.4–19.3 (m), 17.8, 17.8, 16.5; 31P NMR (121 MHz, CDCl3) δ +28.8–28.7 (m), 27.4–27.1 (m); HRMS (ES+, m/z) calcd for (M+H)+ C27H51O6P2: 533.3161; found: 533.3164.

5.9 (S)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-5-(dimethoxyphosphoryl)-2,8,12-trimethyltrideca-2,7,11-trien-5-yl)phosphonate (14)

According to the procedure described for preparation of compound 12, compound 10 (360 mg, 0.7 mmol) was dried, dissolved in THF (5 mL), and treated with NaH (40 mg, 1.1 mmol, 60% dispersion in mineral oil) and freshly prepared prenyl bromide (0.13 mL, 1.1 mmol). After stirring overnight, standard work-up gave a faint yellow oil purified further by flash chromatography (silica gel, 5% EtOH in EtOAc) to afford the product 14 (270 mg, 65% yield). 1H NMR (300 MHz, CDCl3) δ 5.39–5.37 (m, 2H), 5.12–5.10 (m, 2H), 4.14–4.13 (m, 2H), 3.80 (d, JPH = 11.1 Hz, 9H), 2.67–2.57 (m, 4H), 2.08–1.97 (m, 6H), 1.73 (s, 3H), 1.69 (s, 6H), 1.63 (s, 6H), 1.61 (s, 6H), 1.58–1.34 (m, 5H), 0.93 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 137.7, 133.9, 131.5, 131.4, 124.7, 124.4, 119.3–119.0 (m, 2C overlap), 65.0–64.8 (m), 53.5–53.1 (m, 3C overlap), 46.5 (t, JPH = 130.8 Hz), 40.2, 37.7, 37.7, 37.2, 37.1, 29.3–29.2 (m), 26.8, 26.3, 25.8, 25.5, 19.4, 19.3, 18.0, 18.0, 17.8, 16.4. 31P NMR (121 MHz, CDCl3) δ +29.3–29.3 (m), 27.8–27.6 (m); HRMS (ES+, m/z) calcd for (M+H)+ C29H55O6P2: 561.3474; found: 561.3479

5.10(S)-3,7-Dimethyloct-6-en-1-yl methyl ((6E,11E)-9-(dimethoxyphosphoryl)-2,6,12,16-tetramethylheptadeca-2,6,11,15-tetraen-9-yl)phosphonate (15)

According to the procedure described for preparation of compound 12, compound 10 (290 mg, 0.6 mmol) was dissolved in THF (5 mL), and treated with NaH (40 mg, 0.9 mmol, 60% dispersion in mineral oil), and freshly prepared geranyl bromide (0.18 mL, 0.9 mmol). After the reaction was allowed to stir overnight, standard work-up gave a light yellow oil (0.265 mg, 71%): 1H NMR (300 MHz, CDCl3) δ 5.38 (m, 2H), 5.10–5.08 (m, 3H), 4.15–4.13 (m, 2H), 3.78 (d, JPH = 10.8 Hz, 9H), 2.66–2.56 (dt, JPH = 16.2 Hz, J = 8.7 Hz 4H), 2.07–2.04 (m, 10H), 1.67 (s, 9H), 1.62 (s, 6H), 1.60 (s, 9H), 1.48–1.43 (m, 5H), 0.92 (d, J = 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 137.6, 131.43, 131.38, 124.7, 124.4, 119.16 (t, JPC = 7.6 Hz), 65.0–64.8 (m), 53.4–53.1 (m, 3 C overlap), 46.5 (t, JPC = 130.7 Hz), 40.2, 37.8–37.7 (m), 37.2–37.1 (m), 34.4, 29.3, 26.7, 25.8, 25.5, 19.4, 19.3, 17.8, 17.7, 16.4; 31P NMR (121 MHz, CDCl3) δ 29.4–29.3 (m), +27.8–27.7 (m); HRMS (ES+, m/z) calcd for (M+Na)+ C34H62O6P2Na: 651.3919; found: 651.3922.

5.11 ((((E)-2-((((S)-3,7-dimethyloct-6-en-1-yl)oxy)((pivaloyloxy)methoxy)phosphoryl)-5,9-dimethyldeca-4,8-dien-2-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate) (16)

Compound 12 (0.12 g, 0.2 mmol) was dissolved in acetonitrile (3 × 3mL) and then concentrated under reduced pressure to remove any residual water. Compound 12 was then added to a solution of chloromethyl pivalate (0.11 mL, 0.76 mmol) and sodium iodide (0.11 g, 0.6 mmol) in acetonitrile (5 mL), and this reaction mixture was heated at reflux for approximately 24 hours. The acetonitrile was removed under reduced pressure and the resulting material was dissolved in diethyl ether. The solution was washed with brine (4 × 10 mL) and the organic layer was dried (Na2SO4), vacuum filtered through celite, and concentrated to a yellow oil. Final purification by flash chromatography (silica gel, 20% EtOAc in hexanes + 1% TEA) afforded the final product as a faint yellow oil (0.07 g, 34%): 1H NMR (300 MHz, CDCl3) δ 5.77–5.66 (m, 6H), 5.29 (t, JPH = 7.5 Hz, 1H), 5.08–5.07 (m, 2H), 4.16–4.11 (m, 2H), 2.60 (dt, JPH = 15.7, J = 6.9 Hz, 2H), 2.06–1.95 (m, 6H), 1.80–1.71 (m, 2H), 1.70 (s, 3H), 1.67 (s, 6H), 1.59 (s, 6H), 1.54–1.48 (m, 3H), 1.39 (t, JPH = 16.5 Hz, 3H), 1.23 (s, 27H), 0.90 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 176.9–176.8 (m, 3C overlap), 139.2, 131.5, 131.5, 124.6, 124.3, 124.3, 83.0–82.1 (m, 3C overlap), 65.1–65.0 (m), 41.5 (t, JPC = 133.3 Hz), 40.2, 38.8, 38.8, 37.4–37.3 (m), 37.11–37.06 (m), 30.6, 30.5, 29.22, 29.18, 27.04, 27.00, 26.99, 26.97, 26.7, 25.8, 25.5, 19.35, 19.31, 19.29, 17.80, 17.76, 16.30, 16.27, 15.7; 31P NMR (121 MHz, CDCl3) δ +26.8–26.6 (m), +25.9–25.8 (m); HRMS (ES+, m/z) calcd for (M+Na)+ C40H72O12P2Na: 829.4397; found: 829.4404.

5.12 ((((E)-4-((((S)-3,7-Dimethyloct-6-en-1-yl)oxy)((pivaloyloxy)methoxy)phosphoryl)-7,11-dimethyldodeca-1,6,10-trien-4-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate) (17)

According to the procedure described for preparation of compound 16, compound 17 (97 mg, 0.2 mmol) was added to a solution of chloromethyl pivalate (0.30 mL, 1.99 mmol), sodium iodide (300 mg, 1.99 mmol), and acetonitrile (5 mL) and the solution was heated at reflux for 8 hours. Standard workup and purification by flash chromatography (silica gel, 15% EtOAc in hexanes + 1% TEA) gave a clear oil (34 mg, 23%): 1H NMR (300 MHz, CDCl3) δ 6.01–5.85 (m, 1H), 5.79–5.66 (m, 6H), 5.35–5.31 (m, 1H), 5.10–5.07 (m, 4H), 4.18–4.08 (m, 2H), 2.69–2.57 (m, 4H), 2.08–1.95 (m, 6H), 1.80–1.70 (m, 2H), 1.67 (s, 6H), 1.59 (s, 9H), 1.53–1.45 (m, 3H), 1.23 (s, 27H), 0.92 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 176.8–176.7 (m, 3C overlap), 138.5, 138.5, 134.7, 131.4, 131.3, 124.5 124.2, 124.2, 82.9, 82.5, 81.8, 64.8, 47.6, 40.1, 40.0, 38.7, 38.4, 37.3, 37.3, 37.0, 36.3, 29.2, 29.2, 27.1, 27.0, 26.9, 26.9, 26.8, 26.7, 25.7, 25.7, 25.4, 19.2, 19.1, 17.9, 17.7, 17.6, 16.2, 16.2; 31P NMR (121 MHz, CDCl3) δ +25.5–25.4 (m), 24.8–24.7 (m); HRMS (ES+, m/z) calcd for (M+Na)+ C42H74O12P2Na: 855.4553; found: 855.4559.

5.13 ((((E)-5-((((S)-3,7-Dimethyloct-6-en-1-yl)oxy)((pivaloyloxy)methoxy)phosphoryl)-2,8,12-trimethyltrideca-2,7,11-trien-5-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate) (18)

According to the procedure described for preparation of compound 16, compound 18 (267 mg, 0.41 mmol) was added to a solution of chloromethyl pivalate (0.37 mL, 2.55 mmol), sodium iodide (380 mg, 2.55 mmol), and acetonitrile (7 mL) and the solution was heated at reflux for 8 hours. Standard workup and purification by flash chromatography (silica gel, 15% EtOAc in hexanes + 1% TEA) gave a clear oil (133 mg, 32%): 1H NMR (300 MHz, CDCl3) δ 5.78–5.62 (m, 6H), 5.35–5.27 (m, 2H), 5.10–5.02 (m, 2H), 4.20–4.03 (m, 2H), 2.66–2.56 (m, 4H), 2.06–1.94 (m, 6H), 1.81–1.76 (m, 2H), 1.71 (s, 3H), 1.66 (s, 6H), 1.59 (s, 12H), 1.52–1.40 (m, 3H), 1.23 (s, 27H), 0.90 (d, 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 176.8–176.7 (m, 3C overlap), 138.5, 138.5, 134.7, 131.4, 131.4, 124.5, 124.2, 124.2, 82.9, 82.6, 81.8, 64.7, 47.6, 40.1, 40.1, 38.7, 38.4, 37.3, 37.2, 37.0, 36.3, 29.2, 29.2, 27.1, 26.9, 26.9, 26.9, 26.9, 26.7, 26.1, 25.7, 25.7, 25.4, 19.2, 19.1, 17.9, 17.7, 17.6, 16.2, 16.2; 31P NMR (121 MHz, CDCl3) δ +26.0–25.9 (m), 25.3–25.1 (m); HRMS (ES+, m/z) calcd for (M+Na)+ C44H78O12P2Na: 883.4866; found: 883.4872.

5.14 ((((6E,11E)-9-((((S)-3,7-Dimethyloct-6-en-1-yl)oxy)((pivaloyloxy)methoxy)phosphoryl)-2,6,12,16-tetramethylheptadeca-2,6,11,15-tetraen-9-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate) (19)

According to the procedure described for preparation of compound 16, compound 19 (265 mg, 0.42 mmol) was added to a solution of chloromethyl pivalate (0.30 mL, 2.08 mmol), sodium iodide (312 mg, 2.08 mmol), and acetonitrile (5 mL) and the solution was heated at reflux for 10 hours. Standard workup and flash chromatography (silica gel, 10% EtOAc in hexanes + 1% TEA) gave a yellow oil (36 mg, 9%): 1H NMR (500 MHz, CDCl3) δ 5.78–5.65 (m, 6H), 5.36–5.31 (m, 2H), 5.10–5.08 (m, 3H), 4.22–4.05 (m, 2H), 2.68–2.57 (m, 4H), 2.09–1.95 (m, 10H), 1.80–1.70 (m, 2H), 1.68 (s, 9H), 1.62 (s, 6H), 1.60 (s, 9H), 1.52–1.40 (m, 3H), 1.23 (s, 27H), 0.91 (d, J = 5.7 Hz, 3H) 13C NMR (125 MHz, CDCl3) δ 176.8–176.7 (m, 3C overlap), 138.4, 138.4, 131.4, 131.3, 124.5, 124.2, 82.9, 82.6, 81.8, 64.8, 46.3 (t, JPC = 130.6 Hz), 40.2, 40.1, 38.7, 37.5, 37.4, 37.3, 37.3, 37.0, 29.7, 29.2, 29.2, 28.8, 26.9, 26.9, 26.9, 26.7, 25.7, 25.6, 25.4, 19.3, 19.2, 17.7, 17.6, 16.3, 16.2; 31P NMR (202 MHz, CDCl3) δ +26.0 (d, JPP = 9.9 Hz), +25.3 (d, JPP = 9.9 Hz); HRMS (ES+, m/z) calcd for (M+Na)+ C49H86O12P2Na: 951.5492; found: 951.5507.

5.15 (R)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-4,8-dimethylnona-3,7-dien-1-yl)phosphonate (20)

According to the procedure described for preparation of compound 9, the acid chloride 8 (2.63 g, 9.9 mmol) was allowed to react with (R)-(+)-citronellol (1.55 g, 9.9 mmol). Parallel workup and purification provided the mixed ester 20 (2.21 g, 58%): 1H NMR (400 MHz, CDCl3) δ 5.12–5.08 (m, 3H), 4.10–4.05 (m, 2H), 3.74 (d, JPH = 11.2 Hz, 3H), 2.30–2.26 (m, 2H), 2.07–1.96 (m, 6H), 1.80–1.70 (m, 2H), 1.67 (s, 6H), 1.61 (s, 3H), 1.60 (s, 6H), 1.59–1.44 (m, 2H), 1.37–1.21 (m, 2H), 1.20–1.17 (m, 1H), 0.92 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 136.4, 131.4, 131.6, 124.5, 124.1, 122.9 (d, JCP = 17.0 Hz), 64.0 (d, JCP = 6.6 Hz), 52.0 (d, JCP = 6.5 Hz), 39.5, 37.5 (d, JCP = 6.7 Hz), 36.9, 29.0 (d, JCP = 2.5 Hz), 26.5, 25.3 (d, JCP = 137.2 Hz), 25.6, 25.3, 20.9, 20.9, 19.2, (d, JCP = 1.7 Hz), 19.2, 17.6, 15.9; 31P NMR (161 MHz, CDCl3) δ +33.25, +33.20; HRMS (ES+, m/z) calcd. for (M + H)+ C22H42O3P: 385.2872; found: 385.2864.

5.16 (R)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-1-(dimethoxyphosphoryl)-4,8-dimethylnona-3,7-dien-1-yl)phosphonate (21)

According to the procedure described for preparation of compound 10, the mixed ester 9 (1.02 g, 2.6 mmol) was treated with a solution of LTMP, formed from 2,2,6,6-tetramethylpiperidine (0.99 mL, 5.8 mmol) and n-butyl lithium (2.32 mL, 5.8 mmol), followed by reaction with dimethyl chlorophosphate (0.65 mL, 5.8 mmol). Standard work-up and purification gave the desired bisphosphonate 21 (0.46 g, 36%): 1H NMR (400 MHz, CDCl3) δ 5.20 (t, J = 6.0 Hz, 1H), 5.03–5.00 (m, 2H), 4.10–4.05 (m, 2H), 3.73 (d, JPH = 14.8 Hz, 9H), 2.62–2.50 (m, 2H), 2.29 (tt, JPH = 23.6 Hz, J = 6.0 Hz 1H), 2.01–1.85 (m, 6H), 1.60 (s, 6H), 1.57 (s, 3H), 1.53 (s, 6H), 1.50–1.35 (m, 2H), 1.32–1.23 (m, 2H), 1.18–1.06 (m, 1H), 0.85 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 137.1, 131.4, 131.2, 124.5, 124.1, 121.5 (t, JCP = 7.0 Hz), 65.0–64.9 (m), 53.2–52.9 (m, 3 C overlap), 39.6, 36.8 (t, JCP = 132.2 Hz), 37.5–37.4 (m), 37.0, 36.9, 28.9 (d, JCP = 2.0 Hz), 26.5, 25.7, 25.6, 25.3, 23.9 (t, JCP = 5.1 Hz), 19.2, 17.6, 16.0; 31P NMR (121 MHz, CDCl3) δ +26.3, +24.7–24.6 (m); HRMS (ES+, m/z) calcd for (M + H)+ C24H47O6P2: 493.2848; found: 493.2850.

5.17 (R)-3,7-Dimethyloct-6-en-1-yl methyl ((E)-2-(dimethoxyphosphoryl)-5,9-dimethyldeca-4,8-dien-2-yl)phosphonate (22)

According to the procedure described for methylation of compound 12, the bisphosphonate 21 (0.160 g, 0.3 mmol) was treated with NaH (20 mg, 0.5 mmol) and methyl iodide (0.05 mL, 0.8 mmol) to give the expected product 22 (152 mg, 92%): 1H NMR (300 MHz, CDCl3) δ 5.26 (t, J = 6.8 Hz, 1H), 5.03–4.99 (m, 2H), 4.12–4.03 (m, 2H), 3.75 (d, JPH = 10.8 Hz, 3H), 3.74 (d, JPH = 10.8 Hz, 3H), 3.73 (d, JPH = 10.8 Hz, 3H), 2.53 (td, JPH = 15.5 Hz, J = 7.5 Hz, 2H), 2.03–1.86 (m, 6H), 1.69–1.63 (m, 2H), 1.60 (s, 6H), 1.54 (s, 3H), 1.52 (s, 6H), 1.32 (t, JPH = 16.7 Hz, 3H), 1.26–1.07 (m, 3H), 0.85 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 138.3, 131.5, 131.4, 124.7, 124.3, 118.8 (t, JCP = 7.3 Hz), 65.1 (m), 53.7–53.3 (m, 3C overlap), 41.6 (t, JCP = 133.3 Hz), 40.2, 38.9, 37.7–37.6 (m), 37.2–37.1 (m), 31.1 (t, JCP = 5.2 Hz), 29.1, 26.7, 25.8, 25.5, 19.4, 19.3, 17.8, 17.7, 16.3; 31P NMR (121 MHz, CDCl3) δ +29.8 (d, JPP = 7.9 Hz), +28.3– 28.2 (m); HRMS (ES+, m/z) calcd for (M + H)+ C25H49O6P2: 507.3004; found: 507.3006.

5.18 ((((E)-2-((((R)-3,7-dimethyloct-6-en-1-yl)oxy)((pivaloyloxy)methoxy)phosphoryl)-5,9-dimethyldeca-4,8-dien-2-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate) (23)

According to the procedure described for preparation of compound 16, compound 22 (165 mg, 0.3 mmol) was treated with POMCl (0.15 mL, 1.0 mmol) and NaI (151 mg, 1.0 mmol) in acetonitrile at reflux. Standard work-up and purification provided the desired POM ester 23 (108 mg, 41%): 1H NMR (300 MHz, CDCl3) δ 5.79–5.66 (m, 6H), 5.29 (t, JPH = 7.5 Hz, 1H), 5.09–5.06 (m, 2H), 4.19–4.08 (m, 2H), 2.61 (dt, JPH = 15.8, J = 7.4 Hz, 2H), 2.08–1.91 (m, 6H), 1.77–1.70 (m, 2H), 1.67 (s, 6H), 1.59 (s, 9H), 1.56–1.47 (m, 3H), 1.39 (t, JPH = 17.3 Hz, 3H), 1.24 (s, 9H), 1.23 (s, 18H), 0.90 (d, J = 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 176.8–176.7 (m, 3C overlap), 139.1, 131.5, 131.5, 124.5, 124.3, 118.0, 83.1–81.8 (m, 3C overlap), 65.2–64.9 (m), 41.6 (t, JPC = 133.4 Hz), 40.1, 38.8, 38.8, 37.4–37.3 (m), 37.1–37.0 (m), 30.6, 30.5, 29.21, 29.18, 27.1, 27.00, 26.98, 26.97, 26.6, 25.7, 25.5, 19.4, 19.32, 19.28, 17.79, 17.77, 16.30, 16.27, 15.7; 31P NMR (121 MHz, CDCl3) δ +26.8–26.6 (m), +25.9–25.8 (m); HRMS (ES+, m/z) calcd for (M + Na)+ C40H72O12P2Na: 829.4397; found: 829.4395.

5.19 Immunoblot analysis

RPMI-8226 (ATCC, Manassas, VA) cells were incubated (37 °C and 5% CO2) with test compounds for 48 hrs in RPMI-1640 media containing 10% fetal bovine serum and penicillin-streptomycin. Whole cell lysate was obtained using RIPA buffer (0.15 M NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton (v/v) X-100, 0.05 M Tris HCl) containing protease and phosphatase inhibitors. Protein content was determined using the bicinchoninic acid (BCA) method (Pierce Chemical, Rockford, IL). Equivalent amounts of cell lysate were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, probed with the appropriate primary antibodies, and detected using HRP-linked secondary antibodies and Amersham Pharmacia Biotech ECL Western blotting reagents per manufacturer's protocols.

5.20 Lambda light chain ELISA

Human lambda light chain kit (Bethyl Laboratories, Montgomery, TX) was used to quantify intracellular monoclonal protein levels of whole cell lysate. Lambda light chain levels were normalized to total protein levels (as determined by BCA).

5.21 MTT cytotoxicity assay

RPMI-8226, MM.1S (ATCC, Manassas, VA), or ALMC-2 (obtained from Dr. Diane Jelinek, Mayo Clinic, Rochester, MN) cells were plated (2.5 ×104 cells/100 μL/well) in 96-well plates in the presence or absence of the novel compounds and incubated for 48 hrs. MTT solution (35 μL/well; 5 mg/mL PBS) was subsequently added and cells were incubated for an additional 4 hrs. MTT solubilizing solution (0.01 M HCl/10% SDS) was then added (100 μL/well). After overnight incubation at 37 °C, plates were analyzed on a microplate spectrophotometer at 540 nm. Compounds were tested in quadruplicate and three independent experiments were performed. The absorbance for control cells was defined as an MTT activity of 100%.

5.22 Statistics

Two-tailed t-testing was used to calculate statistical significance. An α of 0.05 was set as the level of significance. CalcuSyn software (Biosoft, Cambridge, UK) was used to analyze the concentration response curves and determine the EC50 for the MTT cytotoxicity assays.

Scheme 1
Synthesis of bisphosphonate mixed esters.

Supplementary Material

Acknowledgements

Financial support from the Roy J. Carver Charitable Trust (01-224) and the National Institutes of Health (R01CA-172070) is gratefully acknowledged.

Footnotes

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Supplementary data

Supplementary data associated with this article, including NMR spectra, can be found in the online version, at

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