α-Methylation Enhances the Potency of Isoprenoid Triazole Bisphosphonates as Geranylgeranyl Diphosphate Synthase Inhibitors

Abstract

Disruption of protein geranylgeranylation via inhibition of geranylgeranyl diphosphate synthase (GGDPS) represents a novel therapeutic strategy for a variety of malignancies, especially those characterized by excessive protein secretion such as multiple myeloma. Our work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. Here we present the synthesis and biological evaluation of a new series of isoprenoid triazoles modified by incorporation of a methyl group at the α-carbon. These studies reveal that incorporation of an α-methyl substituent enhances the potency of these compounds as GGDPS inhibitors, and, in the case of the homogeranyl/homoneryl series, abrogates the effects of olefin stereochemistry on inhibitory activity. The incorporation of the methyl group allowed preparation of a POM-prodrug, which displayed a 10-fold increase in cellular activity compared to the corresponding salt. These studies form the basis for future preclinical studies investigating the anti-myeloma activity of these novel α-methyl triazole bisphosphonates.

Graphical abstract

1. Introduction

The mammalian isoprenoid biosynthetic pathway (IBP) is responsible for the synthesis of both steroidal and non-steroidal isoprenoids. The synthesis of the linear intermediates farnesyl diphosphate (FDP) and geranylgeranyl diphosphate (GGDP) is catalyzed by the enzymes FDP synthase (FDPS) and GGDP synthase (GGDPS), respectively. These two enzymes have been of interest from a therapeutic perspective, because both FDP and GGDP serve as isoprenoid donors for protein prenylation reactions. Several FDPS inhibitors have found clinical use in the treatment of bone disorders, including osteoporosis, myeloma bone disease, and metastatic bone disease.¹ Due to their in vitro anti-cancer activity as well as antiparasitic activity, there is interest in the further development of FDPS inhibitors.^2,3 While GGDPS inhibitors have not yet been examined in clinical trials, these agents also have potential as anti-cancer therapies.^2,4,5 We have focused on the utility of GGDPS inhibitors as anti-myeloma agents. Inhibitors of GGDPS, by virtue of their ability to disrupt Rab GTPase geranylgeranylation, impair protein trafficking processes. This in turn can result in induction of the unfolded protein response pathway and ultimately apoptosis.^6,7

Recently, our efforts to develop potent and selective GGDPS inhibitors have focused on triazole bisphosphonates that carry isoprenoid chains.⁵ Structure-function analysis has revealed that the chain length of the alkyl substituent as well as the olefin stereochemistry affects inhibitory activity.^8–11 When the activity of triazoles derived from 10-carbon (i.e. 1 – 3) vs 11-carbon (i.e. 4 – 6) vs 12-carbon azides (i.e. 7 and 8) has been compared, the 11-carbon derivatives (i.e., homogeranyl and homoneryl) have shown the most potent GGDPS inhibitory activity. In addition, for any given chain length in this set of compounds, the Z-isomer has proven to be more potent than the corresponding E-isomer.^8,10,11 Interestingly, however, a 3:1 mixture of the E:Z isomers for the homogeranyl length (5) is more potent than either isomer alone, and further studies demonstrated that the two isomers interact in a synergistic manner to inhibit the target enzyme.¹⁰ To enhance cell uptake preparation of a prodrug form of the bisphosphonate might be advantageous,¹² but efforts to secure a prodrug form of compound 5 have been frustrated, at least in part, because of the acidity of the α-position. To circumvent this issue, it became important to determine whether incorporation of an alkyl substituent at the α-carbon position could preserve the activity of these agents as GGDPS inhibitors. Here we report the synthesis and biological activity of a novel group of α-methylated isoprenoid triazole bisphosphonates.

2. Synthesis

Our initial goal was to prepare a tetra pivaloyloxymethyl (POM)¹³ derivative of the active agent 5, because this mixture showed attractive potency as the sodium salt⁹ and a prodrug can show enhanced potency in cellular bioassays.^14,15 Such acyloxy derivatives of phosphonic acids are best prepared by treatment of the corresponding methyl ester with a reactive alkylating agent like POMCl,¹² and it is conceivable that the POM groups could be introduced at several different stages of the synthesis. As shown in Scheme 1, commercial tetramethyl methylenebisphosphonate (9) could be readily converted to the olefin 10 under standard conditions,¹⁶ and the subsequent reaction with POMCl gave the tetra POM compound 11 in a reasonable yield.¹² However, efforts to convert this olefin to a terminal acetylene appropriate for a click reaction,¹⁷ which ultimately would lead to the triazole 12, went unrewarded presumably because of competing reaction with the POM groups. Introduction of the alkyne could be accomplished by conjugate addition of sodium acetylide to the tetramethyl ester 10. In this process the desired adduct 13 always was accompanied by a significant amount of the methylated product 14, and separation of these two compounds was not readily accomplished. Treatment of the mixture with base and methyl iodide did result in clean conversion to the methylated compound 14, and this alkyne does undergo click reactions under standard conditions to give the expected product 15. Unfortunately, efforts to convert the tetramethyl ester 15 to the POM compound were not successful, perhaps because the triazole system reacts with POMCl.

Scheme 1.

Attempted preparation of a triazole bisphosphonate prodrug.

Preparation of the target compound 12 proved challenging through both of the reaction sequences described above, and so it was decided to determine whether an α-methyl group would have a significant impact on the biological activity of a triazole bisphosphonate before pursuing preparation of a POM prodrug. If α-methylation could improve, or even simply maintain, the activity of the corresponding bisphosphonates, it would eliminate concerns about the acidic α-hydrogen and allow more options for synthetic sequences to prodrug forms. Therefore, we turned to our small library of triazole bisphosphonates and prepared a set of methylated analogues.

Methylation of the C₁₀ compounds proved to be straightforward as shown in Scheme 2. Upon treatment with sodium hydride followed by methyl iodide, the E/Z-mixture (16) as well as the individual Z- (17) and E-isomers (18) all undergo methylation smoothly, and the methylated products 19, 20, and 21, were obtained in modest to good yields. Hydrolysis under the standard McKenna conditions provided the desired salts 22, 23, and 24. In a parallel fashion, the homologated compounds 25 (an E/Z mixture), 26 (the Z isomer), and 27 (the E isomer) were first methylated to give bisphosphonates 28–30, and then hydrolyzed to give the desired salts 31–33. All six of these new compounds were tested for biological activity, and the results (as explained in detail below) were sufficiently encouraging that we proceeded to prepare a POM prodrug of the Z-isomer 32.

Scheme 2.

Preparation of a methylated triazole bisphosphonates.

Based on feedback from the bioassays (vide infra), the POM derivative of bisphosphonate 32 became the next target, and a revised strategy was employed to prepare the acetylene 14. For this sequence, the olefin 10 first was reduced via catalytic hydrogenation to give the bisphosphonate 34 and circumvent any risk of dialkylation at the α-carbon of compound 9. Subsequent treatment of compound 34 with sodium hydride, followed by reaction with propargyl bromide, gave the desired acetylene 14. Treatment of this tetramethyl ester with excess POMCl in the presence of sodium iodide gave the tetraPOM compound 35 in good yield. However, out of concern for the stability of this material, it was treated with the azide 36 after minimal purification to obtain the tetraPOM triazole 37. This material then was employed in the cell-based bioassays described below.

Finally, to investigate the effect of a larger substituent at the α-position compound 2 was converted to its ethyl derivative. This synthesis was straightforward given preparation of the α-methyl compounds above. Treatment of bisphosphonate 2 with sodium hydride and ethyl iodide gave the alkylated derivative 38 in modest yield, and hydrolysis of the ethyl esters proceeded under standard conditions to afford compound 39. While bioassays with this compound were disappointing, they did support the conclusion that the methyl group was the more interesting substituent.

3. Biological results and discussion

The impact of substituents at the α-carbon of the isoprenoid triazole bisphosphonates on cellular activity was assessed in myeloma cells. Impairment of cellular protein geranylgeranylation was determined via two methods: 1) ELISA for intracellular lambda light chain which is a marker for disruption of Rab GTPase geranylgeranylation⁶ (Figure 2A) and 2) immunoblot analysis for unmodified Rap1a (a substrate of GGTase I) (Figure 2B). Lovastatin, an HMG-CoA reductase inhibitor, was included as a positive control.⁶ In both the geranyl/neryl/mixture series and the homogeranyl/homoneryl/mixture series, the addition of a methyl group at the α-carbon resulted in enhanced cellular activity, with a larger magnitude of change observed in the geranyl/neryl length compounds. The higher homologue which bears an ethyl group (compound 39) at the α-carbon was found to be less potent than either the parent compound 2 or the methyl analogue 22 (Figure 3), and so higher homologues were not pursued further.

Figure 2. Comparison of the effects of the novel α-methylated triazole bisphosphonates to the non-methylated analogues on protein geranylgeranylation.

RPMI-8226 cells were incubated for 48 hours in the presence or absence of lovastatin (Lov, 10 μM) or varying concentrations of the test compounds. A) Intracellular lambda light chain concentrations were determined via ELISA. Data are expressed as a percentage of control (mean + SD, n=3). The * denotes p < 0.05 per unpaired two-tailed t-test. B) Immunoblot analysis of Rap1a (antibody detects only unmodified protein) and β-tubulin (as a loading control).

Figure 3. Cellular activity of an α-ethylated triazole bisphosphonate.

RPMI-8226 cells were incubated for 48 hours in the presence or absence of lovastatin (Lov, 10 μM) or varying concentrations of the test compound. A) Intracellular lambda light chain concentrations were determined via ELISA. Data are expressed as a percentage of control (mean + SD, n=3). The * denotes p < 0.05 per unpaired two-tailed t-test. B) Immunoblot analysis of Rap1a (antibody detects only unmodified protein) and β-tubulin (as a loading control).

Next, the impact of the olefin stereochemistry on biological activity was determined. As shown in Figure 4, the Z-configuration of the geranyl-length 23 was approximately 50-fold more potent than the E-isomer 24. Interestingly, however, the activity of the two C₁₁ compounds (33 and 32) were very similar, with the E-isomer (33) slightly more potent than the Z-isomer (32).

Figure 4. Effects of olefin stereochemistry on activity of the α-methylated triazole bisphosphonates.

RPMI-8226 cells were incubated for 48 hours in the presence or absence of lovastatin (Lov, 10 μM) or varying concentrations of the test compounds. A) Intracellular lambda light chain concentrations were determined via ELISA. Data are expressed as a percentage of control (mean + SD, n=3). The * denotes p < 0.05 per unpaired two-tailed t-test. B) Immunoblot analysis of Rap1a (antibody detects only unmodified protein) and β-tubulin (as a loading control).

Consistent with the cellular assays, the enzymatic assays revealed that the methylated mixture 22 is more potent than its corresponding non-methylated analogue 2,⁸ and that the neryl isomer 23 is more potent than the geranyl isomer 24 (Table 1). In contrast, the GGDPS inhibitory activity of the α-methylated homogeranyl/homoneryl mixture (31) was very similar to the individual isomers (32 and 33). There was specificity for GGDPS over FDPS, with at least 3.5-fold selectivity in the case of the least potent GGDPS inhibitor (24) and 240- to 690-fold selectivity for the methylated homogeranyl/homoneryl series.

Table 1.

Summary of the bioassay results of the novel triazole bisphosphonates.

Compound	GGDPS IC50 (μM)	FDPS IC50 (μM)	Fold-selectivity for GGDPS compared to FDPS	Cellular LEC1 (μM)
22	1.27 ± 0.303	62.2 ± 7.22	49	0.1
23	0.920 ± 0.089	>100	>108	0.1
24	20.5 ± 2.30	71.4 ± 24.2	3.5	5.0
31	0.100 ± 0.019	24.1 ± 3.15	241	0.03
32	0.086 ± 0.022	59.6 ± 15.0	693	0.025
33	0.125 ± 0.027	43.1 ± 2.98	345	0.02
39	5.85 ± 1.30	94.3 ± 20.6	16	2.5

¹Cellular LEC (lowest effective concentration) is defined as the lowest concentration for which an unmodified Rap1a band is visible in the immunoblot and a statistically significant increase in intracellular lambda light chain is observed in the ELISA.

Finally, the activity of the POM-prodrug 37 was assessed in comparison to the corresponding salt. As shown in Figure 5, the POM-version of 32 (37) displayed enhanced cellular potency compared to the corresponding salt with approximately 10-fold improvement in activity.

Figure 5. A POM-prodrug enhances cellular activity compared to the corresponding phosphonate salt.

RPMI-8226 cells were incubated for 48 hours in the presence or absence of lovastatin (Lov, 10 μM) or varying concentrations of the test compounds. A) Intracellular lambda light chain concentrations were determined via ELISA. Data are expressed as a percentage of control (mean + SD, n=3). The * denotes p < 0.05 per unpaired two-tailed t-test. B) Immunoblot analysis of Rap1a (antibody detects only unmodified protein) and β-tubulin (as a loading control).

4. Conclusions

In conclusion, we have prepared and assayed a new series of α-methylated isoprenoid triazole bisphosphonates. These studies demonstrate that incorporation of a methyl group at the α-carbon enhances the activity of these compounds as GGDPS inhibitors. Intriguingly, in the case of the α-methyl C₁₁ homogeranyl/homoneryl series, the incorporation of the α-substituent abrogates the impact of olefin stereochemistry that we had previously observed,^8,10,11 such that the two isomers (32 and 33) are nearly identical in activity. From a drug development perspective, this finding is significant as it allows for a single isomer to be carried forward as a lead compound. Our prior studies with the parent compounds 4 and 6 suggested that the two isomers synergistically inhibit GGDPS by interacting at two sites within the enzyme.¹⁰ Further studies are required to determine the mechanism by which the additional methyl group alters these interactions and it will be important to perform crystallography studies with the enzyme in the presence of 32 or 33. In addition to having multiple potential binding sites within the subunit, there is an additional layer of complexity as the enzyme monomer has been reported to associate into hexamers or octomers in solution.^18–20 Finally, the strategy of masking the negatively charged bisphosphonate groups with POM groups resulted in at least a 10-fold improvement in cellular activity, and future studies will explore additional pro-drug strategies as the preclinical development of these agents proceeds.

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. 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 MHz for ¹H, and 75 MHz for ¹³C, with internal standards of (CH₃)₄Si (1H, 0.00) or CHCl₃ (¹H, 7.27; ¹³C, 77.2 ppm) for non-aqueous samples or H₂O (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 Preparation of 4,4-bis(dimethoxyphosphoryl)pent-1-yne (14)

To a stirred solution of the vinyl bisphosphonate 10 (1.10 g, 4.51 mmol) in THF (13 mL) chilled to −10 °C was added NaCCH (18% w/w) (1.35 mL, 4.51 mmol) dropwise over 60 min. The reaction was allowed to warm to room temperature over 16 h. The reaction then was quenched by addition of 1N HCl (15 mL), and the solution was extracted with EtOAc (3 × 20 mL). The combined organic layers were dried (MgSO₄) and filtered, and the filtrate was concentrated in vacuo. The conjugate addition product was obtained as a 4:1 ratio of compounds 13 and 14 by ³¹P NMR (121 MHz, CDCl₃, 24.0 and 23.8 ppm),¹⁷ and converted to compound 14 without further purification.

To a solution of compounds 13 and 14 as a 4:1 mixture (268 mg) in THF (5 mL) cooled to 0 °C was added NaH (60% in oil, 44 mg, 1.09 mmol), and the mixture was allowed to stir for 20 min. To the resulting solution was added iodomethane (0.08 mL, 1.24 mmol) and the reaction was allowed to stir for 16 h while it warmed to room temp. The reaction was quenched by addition of water and extracted with EtOAc (3 × 20 mL). The combined organic layers were dried (MgSO₄) and filtered, and the filtrate was concentrated in vacuo to afford compound 14 as a yellow oil: ¹H NMR (300 MHz, CDCl₃) δ 3.76 (dd, J_HP = 10.8 Hz, J_HP = 1.5 Hz, 12H), 2.79–2.68 (td, J_HP = 14.7 Hz, J = 2.8 Hz, 2H), 2.05 (t, J = 2.8 Hz, 1H), 1.47 (t, J_HP = 16.4 Hz, 3H); ³¹P NMR (121 MHz, CDCl3) δ 27.5. This material was carried to the next step without further purification.

5.3 4-[2,2-bis(dimethoxyphosphoryl)propyl]-1-(3,7-dimethylocta-2,6-dienyl)triazole (15)

To a stirred solution of compound 14 (85 mg, 0.30 mmol) in tBuOH/H₂O (4:1) (3 mL) was added geranyl azide (83 mg, 0.46 mmol), sodium ascorbate (17 mg, 0.08 mmol) and CuSO₄ (sat, ~0.01 mL), and the reaction was allowed to stir for 16 hr. The solvent was removed in vacuo, and then the residue was diluted with brine (10 mL) and EtOAc (10 mL) and extracted with EtOAc (3 × 10 mL). After the combined organic layers were washed with NH₄OH (5 mL), dried (Na₂SO₄), and filtered, the filtrate was concentrated in vacuo. Final purification by column chromatography (20% EtOH in hexanes) afforded compound 15 (39 mg, 28%) as a cloudy oil. Reported is the mixture of E and Z isomers for the ¹H NMR spectrum and the major isomer from the ¹³C NMR spectrum: ¹H NMR (300 MHz, CDCl₃) δ 7.46 (s, 1H), 5.40–5.32 (m, 1H), 5.10–5.98 (m, 1H), 4.92–4.85 (m, 2H), 3.78–3.71 (dd, J_HP = 9.4 Hz, J_HP = 10.9 Hz, 12H), 3.32–3.22 (dd, J =15.4 Hz, J = 13.6 Hz, 2H), 2.20–2.02 (m, 4H), 1.75 (s, 3H), 1.66–1.63 (m, 3H), 1.58–1.54 (m, 3H), 1.51 – 1.38 (td, J_HP = 16.8 Hz, J = 1.9 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) 142.9, 142.4 (t, J_CP = 9.0 Hz), 132.3, 123.5, 123.3, 117.4, 53.9 (t, J_CP = 3.3 Hz, 2C), 53.5 (t, J_CP = 3.3 Hz, 2C), 47.9, 42.2 (t, J_CP = 134.5 Hz), 39.6, 28.8 (t, J_CP = 4.2 Hz), 26.3, 25.8, 17.8, 16.6, 16.4 (t, J_CP = 5.7 Hz); ³¹P NMR δ 28.2; HRMS (ES⁺) m/z calcd for C₁₉H₃₅N₃O₆P₂ (M + H)⁺ 463.2079, found 464.2080.

5.4 4-[2,2-bis(diethoxyphosphoryl)propyl]-1-(3,7-dimethylocta-2,6-dienyl)triazole (19)

To a stirred solution of bisphosphonate 16 (577 mg, 1.14 mmol) in THF (10 mL) at 0 °C was added NaH (60% in oil, 82 mg, 2.05 mmol) followed by 15-crown-5 (0.04 mL, 0.22 mmol), and the reaction mixture was allowed to stir at 0° C for 30 minutes. To the reaction mixture was added MeI (0.12 mL, 1.94 mmol) and the reaction mixture was allowed to stir for 5 hours while it warmed to room temperature. The reaction was quenched by addition of NH₄Cl and extracted with EtOAc (3 × 15 mL). After the combined organic layers were dried (Na₂SO₄) and filtered, the filtrate was concentrated in vacuo to afford compound 19 (523 mg, 88%) as a yellow oil. Reported is the mixture of E and Z isomers for the ¹H NMR data and the major isomer from the ¹³C NMR spectrum: 1H NMR (300 MHz, CDCl₃) δ 7.20 (s, 1H), 5.12–5.04 (m, 1H), 4.80–4.67 (m, 1H), 4.58 (t, J = 7.1 Hz, 2H), 3.86–3.77 (m, 8H), 2.97 (dd, J_HP = 15.3 Hz, J_HP = 15.3 Hz, 2H), 1.87–1.72 (m, 4H), 1.45 (s, 3H), 1.34–1.32 (m, 3H), 1.27–1.24 (m, 3H), 1.22–1.10 (m, 3H), 0.99–0.91 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 142.4 (t, J_CP =9.7 Hz), 142.3, 131.7, 123.3, 123.2, 117.3, 62.6 (t, J_CP = 3.2 Hz, 2C), 62.3 (t, J_CP = 3.2 Hz, 2C), 47.5, 41.6 (t, J_CP = 132.7 Hz), 39.3, 29.4, 28.7 (t, J_CP = 3.9 Hz), 26.0, 25.5, 17.5, 16.3–16.1 (m, 5C), 16.0; ³¹P NMR (121 MHz, CDCl₃) 25.6 ppm; HRMS (ES⁺) m/z calcd for C₂₃H₄₄N₃O₆P₂ (M + H)⁺ 520.2705, found 520.2695.

5.5 4-[2,2-bis(diethoxyphosphoryl)propyl]-1-[(2Z)-3,7-dimethylocta-2,6-dienyl]triazole (20)

According to the procedure described above for compound 19, a solution of compound 17 (590 mg, 1.17 mmol) in THF (12 mL) at 0 °C was treated with NaH (60% in oil, 84 mg, 2.11 mmol), 15-crown-5 (0.04 mL, 0.22 mmol), and MeI (0.12 mL, 1.99 mmol). A parallel workup and final purification by column chromatography (10/90 ethanol/hexanes) afforded bisphosphonate 20 (402 mg, 66%) as a yellow oil: ¹H NMR (300 MHz, CDCl₃) δ 7.50 (s, 1H), 5.44–5.36 (m, 1H), 5.13–5.05 (m, 1H), 4.90 (d, J = 7.9 Hz, 2H), 4.20–4.08 (t, J = 7.1 Hz, 8H), 3.31 (dd, J_HP = 15.5 Hz, J_HP = 15.5 Hz, 2H), 2.22–2.08 (m, 4H), 1.77 (d, J = 1.1 Hz, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.48 (t, J_HP = 16.7 Hz, 3H), 1.27 (td, J = 7.1 Hz, J_HP = 3.0 Hz, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 142.5 (t, J_CP = 9.9 Hz), 142.4, 132.4, 123.4, 123.1, 118.2, 62.8 (t, J_CP = 3.3 Hz, 2C), 62.5 (t, J_CP = 3.3 Hz, 2C), 47.5, 41.7 (t, J_CP = 133.9 Hz), 32.0, 28.7 (t, J_CP = 4.4 Hz), 26.3, 25.6, 23.3, 17.6, 16.3–16.1 (m, 5C); ³¹P NMR (121 MHz, CDCl₃) 26.0 ppm; HRMS (ES+) m/z calcd for C₂₃H₄₄N₃O₆P₂ (M + H)⁺ 520.2705, found 520.2710.

5.6 4-[2,2-bis(diethoxyphosphoryl)propyl]-1-[(2E)-3,7-dimethylocta-2,6-dienyl]triazole (21)

According to the procedure described above for compound 19, a solution of bisphosphonate 18 (300 mg, 0.59 mmol) in THF (6 mL) at 0 °C was treated with NaH (60% in oil, 42 mg, 1.06 mmol), 15-crown-5 (0.02 mL, 0.11 mmol), and MeI (0.06 mL, 1.00 mmol). A parallel workup and final purification by column chromatography (10/90 ethanol/hexanes) provided bisphosphonate 21 (125 mg, 41%) as a yellow oil: ¹H NMR (300 MHz, CDCl₃) δ 7.51 (s, 1H), 5.44–5.36 (m, 1H), 5.08–5.01 (m, 1H), 4.92 (d, J = 7.3 Hz, 2H), 4.19–4.09 (m, 8H), 3.31 (dd, J_HP = 15.5 Hz, J_HP = 15.5 Hz, 2H), 2.12–2.02 (m, 4H), 1.77 (d, J_HP = 1.1 Hz, 3H), 1.67 (d, J = 1.1 Hz, 3H), 1.58 (s, 3H), 1.49 (t, J = 16.7 Hz, 3H), 1.28 (td, J = 7.1 Hz, J_HP = 3.1 Hz, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 142.7 (t, J_CP = 10.0 Hz), 142.7, 132.2, 123.5, 123.5, 117.4, 63.0 (t, J_CP = 3.3 Hz, 2C), 62.6 (t, J_CP = 3.3 Hz, 2C), 47.8, 41.9 (t, J_CP = 133.9 Hz), 39.5, 28.7 (t, J_CP = 4.3 Hz), 26.3, 25.7, 17.7, 16.6–16.3 (m, 6C); ³¹P NMR (121 MHz, CDCl₃) 26.0 ppm; HRMS (ES+) m/z calcd for C₂₃H₄₄N₃O₆P₂ (M + H)⁺ 520.2705, found 520.2706.

5.7 Sodium salt 22

Under standard conditions,^8,21 to a stirred solution of the ethyl ester 19 (523 mg, 1.01 mmol) in CH₂Cl₂ (17 mL) at 0 °C, collidine (9.4 mL, 7.07 mmol) and TMSBr (97%, 1.1 mL, 8.45 mmol) were added dropwise in succession. The reaction was allowed to stir overnight while it warmed to room temperature, and the solvent then was removed in vacuo. The resulting residue was diluted with toluene (30 mL) and concentrated in vacuo to remove any excess TMSBr (3×). The residue was treated with 2N NaOH (3.4 mL, 6.8 mmol) and the solution was allowed to stir overnight at room temperature. Anhydrous acetone was added and the mixture was placed in the freezer for 20 minutes. The resulting solid was collected by filtration and dissolved in water, and the solution was diluted with anhydrous acetone. This mixture was placed in the freezer for 20 minutes. The resulting solid was collected by filtration, dissolved in water, and lyophilized to provide the desired salt 22 (333 mg, 67%) as a white powder. Reported below is the data for the mixture of E and Z isomers for the ¹H NMR spectrum and the major isomer in the ¹³C NMR spectrum: ¹H NMR (300 MHz, D₂O) δ 7.90 (s, 1H), 5.54–5.40 (m, 1H), 5.17–5.09 (m, 1H), 4.92 (t, J = 6.8 Hz, 2H), 3.16 (t, J_HP = 13.1 Hz, 2H), 2.26–2.05 (m, 4H), 1.76 (s, 3H), 1.62 (s, 3H), 1.57–1.55 (m, 3H), 1.06 (t, J_HP = 14.5 Hz, 3H); ¹³C NMR (75 MHz, D₂O) δ 146.8 (t, J_CP = 8.9 Hz), 143.2, 133.8, 125.8, 124.0, 117.5, 47.9, 41.0 (t, J_CP = 120.1 Hz), 38.7, 30.1, 25.6, 25.0, 18.8 (t, J_CP = 4.0 Hz), 17.1, 15.8; ³¹P NMR (121 MHz, D₂O) δ 23.9; HRMS (ES⁻) m/z calcd for C₁₅H₂₂N₃O₆P₂ (M – H)⁻ 406.1297, found 406.1302.

5.8 Sodium salt 23

As described above for compound 22, the ethyl ester 20 (200 mg, 0.38 mmol) in CH₂Cl₂ (6.4 mL) at 0 °C was treated with, collidine (0.35 mL, 2.66 mmol) and TMSBr (97%, 0.42 mL, 1.62 mmol). A parallel workup provided the desired salt 23 (55 mg, 29%) as a white powder: ¹H NMR (300 MHz, D₂O) δ 7.78 (s, 1H), 5.44–5.37 (m, 1H), 5.05–4.97 (m, 1H), 4.81 (t, J = 7.3 Hz, 2H), 3.09–3.00 (m, 2H), 2.15–1.98 (m, 4H), 1.66 (d, J = 0.8 Hz, 3H), 1.52 (s, 3H), 1.47 (s, 3H), 0.96 (t, J_HP = 15.0 Hz, 3H); ¹³C NMR (75 MHz, D₂O) δ 146.7 (t, J_CP = 9.0 Hz), 143.6, 134.0, 125.6, 123.7, 117.9, 47.7, 41.0 (t, J_CP = 119.5 Hz), 31.4, 30.0, 25.8, 25.0, 22.6 18.7 (t, J_CP = 4.0 Hz), 17.1; ³¹P NMR (121 MHz, D₂O) δ 23.9; HRMS (ES⁻) m/z calcd for C₁₅H₂₃N₃O₆P₂ (M – H)⁻ 406.1297, found 406.1304.

5.9 Sodium salt 24

As described above for compound 22, the ethyl ester 21 (100 mg, 0.19 mmol) in CH₂Cl₂ (3.2 mL) at 0 °C was treated with collidine (0.18 mL, 1.33 mmol) and TMSBr (97%, 0.21 mL, 1.62 mmol). A parallel workup provided the desired salt 24 (41 mg, 43%) as a white powder: ¹H NMR (300 MHz, D₂O) δ 7.72 (s, 1H), 5.37–5.31 (m, 1H), 5.04–4.95 (m, 1H), 4.85 (d, J = 7.3 Hz, 2H), 3.13–3.04 (m, 2H), 2.05–1.96 (m, 4H), 1.66 (s, 3H), 1.52 (s, 3H), 1.44 (m, 3H), 1.16 (t, J_HP = 15.4 Hz, 3H); ¹³C NMR (75 MHz, D₂O) δ 144.1 (m), 143.7, 133.8, 125.3, 124.0, 117.2, 47.9, 40.3 (t, J_CP = 116.3 Hz), 38.6, 28.0, 25.5, 24.9, 17.0, 16.8 (t, J_CP = 4.1 Hz), 15.7; ³¹P NMR (121 MHz, D₂O) δ 23.5; HRMS (ES⁻) m/z calcd for C₁₅H₂₃N₃O₆P₂ (M – H)⁻ 406.1297, found 406.1295.

5.10 4-[2,2-bis(diethoxyphosphoryl)propyl]-1-(4,8-dimethylnona-3,7-dienyl)triazole (28)

According to the procedure described above for compound 19, a solution of compound 25 (522 mg, 1.01 mmol) in THF (8.5 mL) at 0 °C was treated with NaH (60% in oil, 72 mg, 1.81 mmol), 15-crown-5 (0.04 mL, 0.19 mmol), and MeI (0.11 mL, 2.24 mmol). A parallel workup afforded the bisphosphonate 28 (435 mg, 81%) as a yellow oil. Reported is the mixture of E and Z isomers for the ¹H NMR spectrum and the major isomer in the ¹³C NMR spectrum: ¹H NMR (300 MHz, CDCl₃) δ 7.56 (s, 1H), 5.13–5.02 (m, 2H), 4.31–4.25 (m, 2H), 4.20–4.11 (m, 8H), 3.38–3.27 (m, 2H), 2.57 (dt, J = 7.1 Hz, J = 7.4 Hz, 2H), 2.07–1.97 (m, 4H), 1.68–1.55 (m, 9H), 1.48 (t, J_HP = 16.7 Hz, 3H), 1.33–1.26 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 145.2 (t, J_CP = 9.6 Hz), 139.2, 131.5, 124.0, 123.8, 118.6, 62.8 (t, J_CP = 3.4 Hz, 2C), 62.5 (t, J_CP = 3.4 Hz, 2C), 49.9, 41.5 (t, J_CP = 134.7 Hz), 39.5, 29.6, 29.1, 28.7 (t, J_CP = 4.3 Hz), 26.4, 25.6, 17.6, 16.3 (m, 4H), 16.0; ³¹P (121 MHz, CDCl₃) 26.0 ppm; HRMS (ES+) m/z calcd for C₂₄H₄₆N₃O₆P₂ (M + H)⁺ 534.2862, found 534.2870.

5.11 4-[2,2-bis(diethoxyphosphoryl)propyl]-1-[(3Z)-4,8-dimethylnona-3,7-dienyl]triazole (29)

According to the procedure described above for compound 19, a stirred solution of bisphosphonate 26 (691 mg, 1.32 mmol) in THF (11 mL) at 0 °C was treated with NaH (60% in oil, 95 mg, 2.37 mmol), 15-crown-5 (0.05 mL, 0.25 mmol), and MeI (0.14 mL, 2.24 mmol). A parallel workup gave bisphosphonate 29 (685 mg, 96%) as a yellow oil: ¹H NMR (300 MHz, CDCl₃) δ 7.55 (s, 1H), 5.12–5.03 (m, 2H), 4.26 (t, J = 7.4 Hz, 2H), 4.19–4.11 (m, 8H), 3.38–3.26 (m, 2H), 2.56 (dt, J = 7.3 Hz, J = 7.3 Hz, 2H), 2.04–1.92 (m, 4H), 1.69 (d, J = 1.1 Hz, 3H), 1.67 (d, J = 0.7 Hz, 3H), 1.59 (d, J =0.7 Hz, 3H), 1.48 (t, J_HP = 16.7 Hz, 3H), 1.32–1.26 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 142.1 (t, J_CP = 9.6 Hz), 138.8, 131.5, 123.8, 123.6, 119.4, 62.6 (t, J_CP = 3.4 Hz, 2C), 62.3 (t, J_CP = 3.3 Hz, 2C), 49.9, 41.4 (t, J_CP = 132.8 Hz), 31.6, 28.8, 28.6 (t, J_CP = 4.3 Hz), 26.1, 25.4, 23.1, 17.4, 16.2 (m, 5C); ³¹P (121 MHz, CDCl₃) 26.0 ppm; HRMS (ES+) m/z calcd for C₂₄H₄₆N₃O₆P₂ (M + H)⁺ 534.2862, found 534.2859.

5.12 4-[2,2-bis(diethoxyphosphoryl)propyl]-1-[(3E)-4,8-dimethylnona-3,7-dienyl]triazole (30)

According to the procedure described above for compound 19, a stirred solution of compound 27 (400 mg, 0.77 mmol) in THF (6.5 mL) at 0 °C was treated with NaH (60% in oil, 55 mg, 1.39 mmol), 15-crown-5 (0.03 mL, 0.15 mmol), and MeI (0.08 mL, 1.31 mmol). A parallel work-up afforded bisphosphonate 30 (394 mg, 96%) as a yellow oil: ¹H NMR (300 MHz, CDCl3) δ 7.37 (s, 1H), 4.95–4.79 (m, 2H), 4.26 (m, 2H), 4.03–3.87 (m, 8H), 3.12 (t, J_HP = 13.9 Hz, 2H), 2.45–2.28 (m, 2H), 1.89–1.67 (m, 4H), 1.46 (s, 3H), 1.38 (s, 3H), 1.34 (m, 3H), 1.27 (t, J_HP = 16.6 Hz, 3H), 1.12–1.03 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 142.4–141.9 (m), 139.0, 131.2, 123.9, 123.7, 118.6, 62.6 (m, 2C), 62.3 (m, 2C), 49.8, 41.5 (t, J_CP = 132.2 Hz), 39.5, 29.5, 29.0, 28.6, 26.4, 25.5, 17.5, 16.2 (4C). 15.8; ³¹P (121 MHz, CDCl₃) 25.8 ppm; HRMS (ES+) m/z calcd for C₂₄H₄₆N₃O₆P₂ (M + H)⁺ 534.2862, found 534.2864.

5.13 Sodium salt 31

As described above for compound 22, the ethyl ester 28 (144 mg, 0.27 mmol) in CH₂Cl₂ (4.5 mL) at 0 °C was treated with collidine (0.25 mL, 1.89 mmol) and TMSBr (97%, 0.30 mL, 2.27 mmol). Standard workup provided the desired salt 31 (48 mg, 35%) as a white powder. Reported is the mixture of E and Z isomers for the ¹H NMR spectrum and the major isomer in the ¹³C NMR spectrum: ¹H NMR (300 MHz, D₂O) δ 7.91 (s, 1H), 5.19–5.07 (m, 2H), 4.40–4.33 (m, 2H), 3.18 (t, J_HP = 13.2 Hz, 2H), 2.59 (dt, J = 7.0 Hz, J = 7.0 Hz, 2H), 2.09–1.90 (m, 4H), 1.67 (s, 3H), 1.59 (s, 3H), 1.44 (3H), 1.15 (t, J_HP = 14.9 Hz, 3H); ¹³C NMR (75 MHz, D₂O) δ 145.6 (t, J_CP = 9.3 Hz), 140.1, 133.7, 125.8, 124.4, 119.3, 50.1, 40.4 (t, J_CP = 115.0 Hz), 38.9, 28.7, 28.3, 25.8, 25.0, 18.6 (t, J_CP = 3.9 Hz), 17.1, 15.1; ³¹P (121 MHz, D₂O) 23.3 ppm; HRMS (ES−) m/z calcd for C₁₆H₂₈N₃O₆P₂ (M – H)⁻ 420.1453, found 420.1451.

5.14 Sodium salt 32

As described above for compound 22, the bisphosphonate ester 29 (551 mg, 1.03 mmol) in CH₂Cl₂ (17 mL) at 0 °C was treated with collidine (0.95 mL, 7.22 mmol) and TMSBr (97%, 1.15 mL, 8.67 mmol). Standard workup provided the desired salt 32 (293 mg, 56%) as a white powder: ¹ H NMR (300 MHz, D₂O) δ 7.89 (s, 1H), 5.18–5.08 (m, 2H), 4.33 (t, J = 6.7 Hz, 2H), 3.16 (t, J_HP = 13.3 Hz, 2H), 2.57 (dt, J = 6.8 Hz, J = 6.8 Hz, 2H), 1.98–1.88 (m, 4H), 1.65 (s, 6H), 1.58 (s, 3H), 1.08 (t, J_HP = 15.0 Hz, 3H); ¹³C NMR (75 MHz, D₂O) δ 146.8 (t, J_CP = 9.1 Hz), 140.6, 134.3, 126.7, 124.7, 120.6, 50.7, 41.3 (t, J_CP = 118.2), 31.5, 30.1, 28.9, 26.3, 25.5, 23.1, 19.2 (t, J_CP = 3.8 Hz), 17.6; ³¹P (121 MHz, D₂O) 23.8; HRMS (ES−) m/z calcd for C₁₆H₂₈N₃O₆P₂ (M – H)⁻ 420.1453, found 420.1454.

5.15 Sodium salt 33

As described above for compound 22, the bisphosphonate ester 30 (390 mg, 0.73 mmol) in CH₂Cl₂ (12 mL) at 0 °C was treated with collidine (0.68 mL, 5.12 mmol) and TMSBr (97%, 0.79 mL, 6.14 mmol). Standard workup provided the desired salt compound 33 (290 mg, 78%) as a white powder: ¹ H NMR (300 MHz, D₂O) δ 7.78 (s, 1H), 5.09–4.94 (m, 2H), 4.24 (t, J = 6.9 Hz, 2H), 3.16 (t, J_HP = 13.3 Hz, 2H), 2.57 (dt, J = 6.8 Hz, J = 6.8 Hz, 2H), 1.98–1.88 (m, 4H), 1.65 (s, 6H), 1.58 (s, 3H), 1.08 (t, J_HP = 15.0 Hz, 3H); ¹³C NMR (75 MHz, D₂O) δ 146.3 (t, J_CP = 9.0 Hz), 140.0, 133.6, 126.0, 124.3, 119.3, 50.0, 40.7 (t, J_CP = 117.3 Hz), 38.9, 29.6 (m), 28.4, 25.8, 25.0, 18.7 (t, J_CP = 3.8 Hz), 17.1, 15.2 ; ³¹P (121 MHz, D₂O) 23.7 ppm; HRMS (ES−) m/z calcd for C₁₆H₂₈N₃O₆P₂ (M – H)⁻ 420.1453, found 420.1454.

5.16 1,1-bis(dimethoxyphosphoryl)ethane 34

Under conditions parallel to those used for preparation of the corresponding tetraethyl ester,²² a solution of compound 10 (397 mg, 1.63 mmol) in EtOAc (11 mL) in the presence of 10% palladium on carbon (74 mg) was treated with hydrogen under 3 atm at room temperature for 4 h. Then the reaction mixture was filtered, and the filtrate was concentrated in vacuo to afford compound 34 (379 mg, 95%) as a yellow oil: ¹H NMR (300 MHz, CDCl₃) δ 3.70–3.60 (m, 12H), 2.42–2.17 (1H), 1.38–1.20 (m, 3H); ¹³C NMR (75 MHz, CDCl₃) 53.2 (d, J_CP = 6.9 Hz, 4C), 30.2 (t, J_CP = 136.8 Hz), 9.96 (t, J_CP = 6.4 Hz); ³¹P NMR δ 26.1; HRMS (ES⁺) m/z calcd for C₆H₁₇O₆P₂ (M + H)+ 247.0500, found 247.0490.

5.17 Preparation of compound 14 by alkylation of compound 34

Under conditions parallel to those used for preparation of the corresponding tetraethyl ester,²³ a stirred mixture of NaH (261 mg, 60% in oil, 10.9 mmol, previously washed with pentane) in THF (22 mL) at −78 °C was treated with a chilled solution of compound 34 (1.79 g, 7.26 mmol) in THF (10 mL) at −78 °C, and the reaction mixture was allowed to stir for 1 hour. A solution of propargyl bromide (2.33 mL, 80% in toluene, 21.9 mmol) in THF (10 mL) at −78 °C was added dropwise and the reaction mixture was allowed to stir for 16 hours at −78 °C. The reaction then was allowed to warm to room temperature over 8 hours. The mixture was evaporated to dryness in vacuo, and saturated aqueous ammonium chloride was added (15 mL). The solution was extracted with CH₂Cl₂ (3 × 30 mL) and the combined organic phases were dried (MgSO₄) and filtered, and the filtrate was concentrated in vacuo. Final purification by column chromatography (10/90 ethanol/hexanes) afforded compound 14 (830 mg, 41%) as a yellow oil: ¹H NMR (300 MHz, CDCl₃) δ 3.87–3.82 (m, 12H) 2.82 (dt, J = 2.8 Hz, J_HP = 14.8 Hz, 2H), 2.10 (t, J = 2.8 Hz, 1H), 1.56 (t, J_CP = 16.4 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 79.0 (t, J_CP = 9.7 Hz), 71.6, 53.8 (d, J_CP = 6.8 Hz, 2C), 53.8 (d, J_CP = 6.8 Hz, 2C), 40.5 (t, J_CP = 136.1 Hz), 23.2 (m), 16.8 (t, J_CP = 6.0 Hz); ³¹P NMR (121 MHz, CDCl₃) δ 27.6; HRMS (ES⁺) m/z calcd for C₉H₁₉O₆P₂ (M + H)+ 285.0657, found 247.0658.

5.18 Compound 35

To a stirred solution of compound 14 (1.09 g, 3.85 mmol) in acetonitrile (8 mL) was added flame-dried NaI (2.43 g, 16.2 mmol) followed by POMCl (2.38 mL, 16.54 mmol) and the reaction was allowed to stir at reflux for 21 hours. The resulting mixture was concentrated in vacuo and then dissolved in CH₂Cl₂ and water. The aqueous solution was extracted with CH₂Cl₂ (2 × 30 mL) and the combined organic phases were dried (Na₂SO₄) and filtered. The filtrate was concentrated in vacuo to afford compound 35 (2.46 g, 94%) as a dark yellow oil which was used in the following reaction without further purification: ¹H NMR (300 MHz, CDCl₃) δ 5.72–5.63 (m, 8H) 2.82 (dt, J = 2.8 Hz, J_HP = 15.5 Hz, 2H), 2.05 (t, J = 2.8 Hz, 1H), 1.50 (t, J_CP = 16.1 Hz, 3H), 1.18 (s, 36H); ¹³C NMR (75 MHz, CDCl₃) δ 176.8 (d, J_CP = 0.6 Hz, 4C), 82.5–82.3 (m, 4C), 77.7 (t, J_CP = 11.0 Hz), 72.5, 40.5 (t, J_CP = 136.6 Hz), 38.8, 26.9 (s, 12C), 22.7 (t, J_CP = 4.2 Hz), 16.1 (t, J_CP = 5.8 Hz); ³¹P NMR (121 MHz, CDCl₃) δ 23.5; HRMS (ES⁺) m/z calcd for C₂₉H₅₀O₁₄NaP₂ (M + Na)⁺ 707.2574, found 707.2584.

5.19 Compound 37

To a stirred solution of compound 35 (764 mg, 1.12 mmol) and azide 36²⁴ (324 mg, 1.67 mmol) in t-BuOH/H₂O (4:1, 11 mL total), saturated CuSO₄ (0.01 mL) and sodium ascorbate (67 mg, 0.34 mmol) were added in sequence. The resulting reaction mixture was allowed to stir for five days at room temperature, and the solvent then was removed in vacuo. The resulting residue was dissolved in brine and extracted with EtOAc (5x). The combined organic extracts were washed with 5% NH₄OH, dried (Na₂SO₄) and filtered, and the filtrate was concentrated in vacuo to afford the crude triazole 37 (707 mg, 72%) as a yellow oil. Further purification of a portion (55 mg) via HPLC on a C-8 5 μM 250 × 10 mm Restek column scanning at a wave length of 210 nm afforded pure compound 37 (24 mg, 44%) as a light yellow oil material that was used in the bioassays: ¹H NMR (300 MHz, CDCl₃) δ 7.51 (s, 1H), 5.74–5.64 (m, 8H), 5.14 –5.04 (m, 2H), 4.32–4.24 (m, 2H), 3.30 (dd, J_HP = 16.0 Hz, J_HP =16.0 Hz, 2H), 2.58 (dt, J = 7.4 Hz, J = 7.3 Hz, 2H), 2.04–1.97 (m, 4H), 1.69 (d, J = 1.1 Hz, 3H), 1.67 (s, 3H), 1.59 (s, 3H), 1.48 (t, J = 17.7 Hz, 3H), 1.23–1.22 (m, 36H); ¹³C NMR (75 MHz, CDCl₃) δ 177.0 (2C), 176.9 (2C), 141.0, 139.4, 132.0, 124.4, 124.0, 119.7, 82.4 (t, J_CP = 2.8 Hz, 2C), 82.2 (t, J_CP = 2.8 Hz, 2C), 50.4, 42.4 (t, J_CP = 133.7 Hz), 39.9 (2C), 38.9 (2C), 32.0, 29.0, 28.2 (t, J_CP = 4.1 Hz), 27.0 (12C), 26.5, 25.9, 23.5, 17.8, 15.7 (t, J_CP = 5.8 Hz); ³¹P NMR (121 MHz, CDCl₃) δ 24.7; HRMS (ES⁺) m/z calcd for C₄₀H₆₉N₃O1₄P₂ (M + H)⁺ 878.4333, found 878.4346.

5.20 4-[2,2-bis(diethoxyphosphoryl)butyl]-1-(3,7-dimethylocta-2,6-dienyl)triazole 38

To a stirred solution of compound 2 (378 mg, 0.75 mmol) in THF (8 mL) at 0 °C was added NaH (60% in oil, 57 mg, 1.42 mmol) followed by 15-crown-5 (0.03 mL, 0.15 mmol) and the reaction mixture was allowed to stir at 0 °C for 30 minutes. To the reaction mixture was added EtI (0.11 mL, 1.34 mmol) and the reaction mixture was allowed to stir for 5 hours while it warmed to room temperature. The reaction then was quenched by addition of NH₄Cl and extracted with EtOAc (3 × 15 mL) and the combined organic layers were dried (Na₂SO₄) and filtered. The filtrate was concentrated in vacuo to afford bisphosphonate 38 (144 mg, 36%) as a yellow oil. Reported is the mixture of E and Z isomers for the ¹H NMR spectrum and the major isomer in the ¹³C NMR spectrum: ¹H NMR (300 MHz, CDCl₃) δ 7.43 (s, 1H), 5.37–5.29 (m, 1H), 5.05–4.95 (m, 1H), 4.84 (t, J = 6.8 Hz, 2H), 4.12–4.00 (m, 8H), 3.28 (dd, J_HP = 16.1 Hz, J = 12.4 Hz, 2H), 2.16–1.81 (m, 6H), 1.71 (s, 3H), 1.61 (s, 3H), 1.51 (s, 3H), 1.20 (dt, J_HP = 10.4 Hz, J = 3.4 Hz, 12H), 1.14 (dt, J_HP = 3.0 Hz, J = 7.7 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 142.8 (t, J_CP = 10.7 Hz), 142.7, 132.2, 123.5, 123.3, 117.4, 62.7 (t, J_CP = 3.4 Hz, 2C), 62.4 (t, J_CP = 3.4 Hz, 2C), 47.8, 47.2 (t, J_CP = 131.2 Hz), 39.5, 26.3, 26.5 (t, J_CP = 4.3 Hz), 25.7, 23.8 (t, J_CP = 4.8 Hz), 23.5, 17.7, 16.6–16.4 (m, 4C), 9.9 (t, J_CP = 4.8 Hz); ³¹P NMR (121 MHz, CDCl₃) 25.6 ppm. HRMS (ES+) m/z calcd for C₂₄H₄₆N₃O₆P₂ (M + H)+ 534.2862, found 534.2867.

5.21 Sodium salt 39

As described above for compound 22, the ester 38 (144 mg, 0.27 mmol) in CH₂Cl₂ (5 mL) at 0 °C was treated with collidine (0.25 mL, 1.89 mmol) and TMSBr (97%, 0.29 mL, 2.27 mmol). A parallel workup provided the desired salt 39 (40 mg, 29%) as a white powder. Reported is the mixture of E and Z isomers for the ¹H NMR spectrum and the major isomer in the ¹³C NMR spectrum: ¹H NMR (300 MHz, 7.81 (s, 1H), 5.42–5.28 (m, 1H), 5.03–4.95 (m, D₂O) δ 1H), 4.81–4.76 (m, 2H), 3.06 (t, J_HP = 12.8 Hz, 2H), 2.12–1.88 (m, 6H), 1.62 (s, 3H), 1.49 (s, 3H), 1.41 (s, 3H), 0.86 (t, J_HP = 6.6 Hz, 3H); ¹³C NMR (75 MHz, D₂O) δ 147.4 (m), 143.7, 134.3, 126.3, 124.5, 118.1, 48.4, 46.5 (t, J_CP = 120.2 Hz), 39.2, 29.0 (m), 26.1, 25.6 (m), 25.5, 23.1, 17.6, 11.4 (m); ³¹P NMR (121 MHz, D₂O) 22.7 ppm; HRMS (ES⁻) m/z calcd for C₁₆H₂₈N₃O₆P₂ (M – H)⁻ 420.1453, found 420.1447.

5.22 Immunoblot analysis

RPMI-8226 (ATCC, Manassas, VA) cells were incubated (37 ºC and 5% CO₂) with test compounds for 48 hours 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 Bio-Rad Clarity ECL Substrate Western blotting reagents per manufacturer’s protocols.

5.23 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).

5.24 FDPS and GGDPS enzyme assays

Recombinant FDPS was kindly provided by Dr. Raymond Hohl (Penn State Cancer Institute). Recombinant GGDPS was obtained from MyBioSource (San Diego, CA). Enzyme assays were performed as previously described.⁹ Compounds were tested in duplicate at multiple concentrations and three independent experiments were performed. Digeranyl bisphosphate²⁵ and zoledronic acid²⁶ were always included as positive controls for the GGDPS and FDPS assays, respectively.

5.25 Statistics

Two-tailed t-testing was used to calculate statistical of 0.05 was set as significance. An α the level of significance. CompuSyn software (ComboSyn, Inc.,) was used to analyze the concentration response curves and determine the IC₅₀ values. This software is based on the work of Chou and Talalay.^27,28

Figure 1.

Reported triazole bisphosphonates and their activity in GGDPS enzyme assays.

Scheme 3.

Preparation of the triazole bisphosphonate prodrug 37.

Scheme 4.

Synthesis of the ethyl-substituted bisphosphonate 39.