α-Methylation enhances the potency of isoprenoid triazole bisphosphonates as geranylgeranyl diphosphate synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.10.023

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.

Full text of DOI:10.1016/j.bmc.2017.10.023

Accepted Manuscript
α-Methylation Enhances the Potency of Isoprenoid Triazole Bisphosphonates
as Geranylgeranyl Diphosphate Synthase Inhibitors
Robert A. Matthiesen, Michelle L. Varney, Pauline C. Xu, Alex S. Rier, David
F. Wiemer, Sarah A. Holstein
PII:
S0968-0896(17)31775-3
https://doi.org/10.1016/j.bmc.2017.10.023
BMC 14028
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 September 2017
12 October 2017
18 October 2017
Please cite this article as: Matthiesen, R.A., Varney, M.L., Xu, P.C., Rier, A.S., Wiemer, D.F., Holstein, S.A., α-
Methylation Enhances the Potency of Isoprenoid Triazole Bisphosphonates as Geranylgeranyl Diphosphate
Synthase Inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.10.023
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α-Methylation Enhances the Potency of Isoprenoid
Triazole Bisphosphonates as Geranylgeranyl
Diphosphate Synthase Inhibitors
Robert A. Matthiesen,a Michelle L. Varney,b Pauline C. Xu,c Alex S. Riera
David F. Wiemera,d and Sarah A. Holsteinb*
aDepartment of Chemistry, University of Iowa, Iowa City, IA 52242-1294, US
bDepartment of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, US
cCollege of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, US
cDepartment of Pharmacology, University of Iowa, Iowa City, IA 52242-1109, US
sarah.holstein@unmc.edu
KEYWORDS. GGDP synthase, inhibition, isoprenoid biosynthesis, triazole, bisphosphonate

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.
2

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
3

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.
O
O
O
(NaO)2P
(NaO)2P
(NaO)2P
N
N
2
N
3
(NaO)2P
O
(NaO)2P
O
(NaO)2P
O
C-12 substituents
N
N
N
N
N
N
C-10 substituents
C-11 substituents
1 E isomer
GGDPS IC50 = 17 µM
4 E isomer
GGDPS IC50 = 0.17 µM
7 n = 2 (E)
GGDPS IC50 = 2.56 µM
2 ~2:1 E:Z
GGDPS IC50 = 2.2 µM
5 ~3:1 E:Z
GGDPS IC50 = 0.045 µM
8 n = 2 (Z)
GGDPS IC50 = 1.29 µM
3 Z isomer
GGDPS IC50 = 0.38 µM
6 Z isomer
GGDPS IC50 = 0.075 µM
Figure 1. Reported triazole bisphosphonates and their activity in GGDPS enzyme assays.
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
4

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.
(MeO)2(O)P
P(O)(OMe)2
9
1) paraformaldehyde
Et2NH, MeOH
(MeO)2(O)P
P(O)(OMe)2
71%
13
NaH
CH3I
reflux, 2 h
2) p-TsOH, toluene
reflux, 16 h
NaCCH, THF
-15 °C to rt
16 h
THF
0 °C to rt
5 h
(MeO)2(O)P
P(O)(OMe)2
60%
54% (4:1, 13:14)
10
(MeO)2(O)P
P(O)(OMe)2
N3
14
NaI, POMCl
ACN, 80 °C 16 h
sodium ascorbate
CuSO4 H2O
28%
tBuOH/H2O (4:1)
(POMO)2(O)P
P(O)(OPOM)2
11
(MeO)2(O)P
NaCCH, THF
-15 °C to rt 16 h
X
N
(MeO)2(O)P
POMCl, NaI
ACN, reflux
N N
15
steps
X
(POMO)2(O)P
(POMO)2(O)P
POM = (CH3)3CCO2CH2-
N
12
N N
Scheme 1. Attempted preparation of a triazole bisphosphonate prodrug.
5

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.
1) TMSBr, Collidine
DCM, 0 °C to rt
16 h
1) NaH, 15-C-5
THF, 0 °C
30 min
(EtO)2(O)P
(EtO)2(O)P
(EtO)2(O)P
(EtO)2(O)P
(NaO)2(O)P
(NaO)2(O)P
N
N
N
N
N
N
N
N N
2) 2M NaOH, rt 16 h
2) MeI, 0 °C 5 h
16 3:1 E/Z
17 Z
19 3:1 E/Z (88%)
20 Z (66%)
21 E (41%)
22 3:1 E/Z (67%)
23 Z (29%)
24 E (43%)
18 E
1) NaH, 15-C-5
THF, 0 °C
30 min
1) TMSBr, Collidine
DCM, 0 °C to rt
16 h
(EtO)2(O)P
(EtO)2(O)P
(EtO)2(O)P
(EtO)2(O)P
(NaO)2(O)P
(NaO)2(O)P
N
N
N
N
N
N
2) MeI, 0 °C 5 h
N
N N
2) 2M NaOH, rt 16 h
25 2.6:1 E/Z
26 Z
28 2.6:1 E/Z (81%)
29 Z (96%)
30 E (96%)
31 2.6:1 E/Z (35%)
32 Z (56%)
33 E (78%)
27 E
Scheme 2. Preparation of a methylated triazole bisphosphonates.
6

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.
1) NaH, THF, -78 °C
2) propargyl bromide
-78 °C to rt
H2, 10% Pd/C
EtOAc, 3 atm
14
10
(MeO)2(O)P
P(O)(OMe)2
41%
95%
34
POMCl, NaI
ACN, reflux
94%
sodium ascorbate
CuSO4 H2O
tBuOH/H2O (4:1)
(POMO)2(O)P
(POMO)2(O)P
72%
N
(POMO)2(O)P
P(O)(OPOM)2
2
N
N
H
35
37
36
N3
Scheme 3. Preparation of the triazole bisphosphonate prodrug 37.
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.
7

1) NaH, 15-C-5
THF, 0 °C
2) ethyl iodide
0 °C to rt
(EtO)2(O)P
(EtO)2(O)P
N
2
N
N
36%
38
1) TMSBr, collidine
DCM, 0 °C to rt
2) 2N NaOH,
29%
(NaO)2(O)P
(NaO)2(O)P
N
N
N
39
Scheme 4. Synthesis of the ethyl-substituted bisphosphonate 39.
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.
8

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).
9

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).
10

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).
11

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
Fold-selectivity
for GGDPS
compared to
FDPS
Cellular LEC1
GGDPS IC50 (µM) FDPS IC50 (µM)
(µM)
1.27 + 0.303
0.920 + 0.089
20.5 + 2.30
62.2 + 7.22
>100
49
0.1
0.1
5.0
22
23
24
>108
3.5
71.4 + 24.2
0.100 + 0.019
0.086 + 0.022
0.125 + 0.027
5.85 + 1.30
24.1 + 3.15
59.6 + 15.0
43.1 + 2.98
94.3 + 20.6
241
693
345
16
0.03
0.025
0.02
2.5
31
32
33
39
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.
12

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).
13

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.
Acknowledgements
Financial support from the Roy J. Carver Charitable Trust (01-224 to D. F. W.) and the
National Institutes of Health (R01CA-172070 to S. A. H.) is gratefully acknowledged.
14

Supplementary data
Supplementary data associated with this article, including NMR spectra can be found in the online
version, at
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
1
13
magnetic stirring. All NMR spectra were obtained at 300 MHz for H, and 75 MHz for C, 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 x 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.
15

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 x 20 mL). The combined organic layers were dried (MgSO4)
1
and filtered, and the filtrate was concentrated in vacuo to afford compound 14 as a yellow oil: H
NMR (300 MHz, CDCl3) δ 3.76 (dd, JHP = 10.8 Hz, JHP = 1.5 Hz, 12H), 2.79–2.68 (td, JHP = 14.7
31
Hz, J = 2.8 Hz, 2H), 2.05 (t, J = 2.8 Hz, 1H), 1.47 (t, JHP = 16.4 Hz, 3H); P 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 x 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
1
1
isomers for the H NMR spectrum and the major isomer from the 13C NMR spectrum: H 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, JHP = 9.4 Hz, JHP = 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, JHP = 16.8 Hz, J
13
= 1.9 Hz, 3H); C NMR (75 MHz, CDCl3) 142.9, 142.4 (t, JCP = 9.0 Hz), 132.3, 123.5, 123.3,
117.4, 53.9 (t, JCP = 3.3 Hz, 2C), 53.5 (t, JCP = 3.3 Hz, 2C), 47.9, 42.2 (t, JCP = 134.5 Hz), 39.6,
16

28.8 (t, JCP = 4.2 Hz), 26.3, 25.8, 17.8, 16.6, 16.4 (t, JCP = 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 x 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
1
mixture of E and Z isomers for the H NMR data and the major isomer from the 13C NMR
1
spectrum: H 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, JHP = 15.3 Hz, JHP = 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, JCP =9.7 Hz), 142.3, 131.7, 123.3, 123.2, 117.3, 62.6 (t, JCP
= 3.2 Hz, 2C), 62.3 (t, JCP = 3.2 Hz, 2C), 47.5, 41.6 (t, JCP = 132.7 Hz), 39.3, 29.4, 28.7 (t, JCP = 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
17

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, JHP = 15.5 Hz, JHP = 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, JHP = 16.7
Hz, 3H), 1.27 (td, J = 7.1 Hz, JHP = 3.0 Hz, 12H); 13C NMR (75 MHz, CDCl3) δ 142.5 (t, JCP = 9.9
Hz), 142.4, 132.4, 123.4, 123.1, 118.2, 62.8 (t, JCP = 3.3 Hz, 2C), 62.5 (t, JCP = 3.3 Hz, 2C), 47.5,
41.7 (t, JCP = 133.9 Hz), 32.0, 28.7 (t, JCP = 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, JHP = 15.5 Hz, JHP = 15.5 Hz, 2H),
2.12–2.02 (m, 4H), 1.77 (d, JHP = 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, JHP = 3.1 Hz, 12H); 13C NMR (75 MHz, CDCl3) δ 142.7 (t, JCP
=
10.0 Hz), 142.7, 132.2, 123.5, 123.5, 117.4, 63.0 (t, JCP = 3.3 Hz, 2C), 62.6 (t, JCP = 3.3 Hz, 2C),
47.8, 41.9 (t, JCP = 133.9 Hz), 39.5, 28.7 (t, JCP = 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
18

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
(3x). 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
1
mixture of E and Z isomers for the H 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, JHP = 13.1 Hz, 2H), 2.26–2.05 (m, 4H), 1.76 (s, 3H), 1.62 (s, 3H), 1.57–
13
1.55 (m, 3H), 1.06 (t, JHP = 14.5 Hz, 3H); C NMR (75 MHz, D2O) δ 146.8 (t, JCP = 8.9 Hz),
143.2, 133.8, 125.8, 124.0, 117.5, 47.9, 41.0 (t, JCP = 120.1 Hz), 38.7, 30.1, 25.6, 25.0, 18.8 (t, JCP
= 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, JHP = 15.0 Hz, 3H); 13C NMR (75 MHz, D2O) δ 146.7 (t, JCP = 9.0 Hz), 143.6, 134.0,
125.6, 123.7, 117.9, 47.7, 41.0 (t, JCP = 119.5 Hz), 31.4, 30.0, 25.8, 25.0, 22.6 18.7 (t, JCP = 4.0
19

31
Hz), 17.1; P 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
13
(t, JHP = 15.4 Hz, 3H); C NMR (75 MHz, D2O) δ 144.1 (m), 143.7, 133.8, 125.3, 124.0, 117.2,
31
47.9, 40.3 (t, JCP = 116.3 Hz), 38.6, 28.0, 25.5, 24.9, 17.0, 16.8 (t, JCP = 4.1 Hz), 15.7; P 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
1
1
the H NMR spectrum and the major isomer in the 13C NMR spectrum: H 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, JHP = 16.7
13
Hz, 3H), 1.33–1.26 (m, 12H); C NMR (75 MHz, CDCl3) δ 145.2 (t, JCP = 9.6 Hz), 139.2, 131.5,
124.0, 123.8, 118.6, 62.8 (t, JCP = 3.4 Hz, 2C), 62.5 (t, JCP = 3.4 Hz, 2C), 49.9, 41.5 (t, JCP = 134.7
31
Hz), 39.5, 29.6, 29.1, 28.7 (t, JCP = 4.3 Hz), 26.4, 25.6, 17.6, 16.3 (m, 4H), 16.0; P (121 MHz,
CDCl3) 26.0 ppm; HRMS (ES+) m/z calcd for C24H46N3O6P2 (M + H)+ 534.2862, found 534.2870.
20

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
1
bisphosphonate 29 (685 mg, 96%) as a yellow oil: H 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,
13
J =0.7 Hz, 3H), 1.48 (t, JHP = 16.7 Hz, 3H), 1.32–1.26 (m, 12H); C NMR (75 MHz, CDCl3) δ
142.1 (t, JCP = 9.6 Hz), 138.8, 131.5, 123.8, 123.6, 119.4, 62.6 (t, JCP = 3.4 Hz, 2C), 62.3 (t, JCP
=
3.3 Hz, 2C), 49.9, 41.4 (t, JCP = 132.8 Hz), 31.6, 28.8, 28.6 (t, JCP = 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
1
afforded bisphosphonate 30 (394 mg, 96%) as a yellow oil: H 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, JHP = 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, JHP = 16.6 Hz, 3H),
13
1.12–1.03 (m, 12H); C 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, JCP = 132.2 Hz), 39.5, 29.5, 29.0, 28.6, 26.4, 25.5,
31
17.5, 16.2 (4C). 15.8; P (121 MHz, CDCl3) 25.8 ppm; HRMS (ES+) m/z calcd for C24H46N3O6P2
(M + H)+ 534.2862, found 534.2864.
21

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
1
in the 13C NMR spectrum: H NMR (300 MHz, D2O) δ 7.91 (s, 1H), 5.19–5.07 (m, 2H), 4.40–4.33
(m, 2H), 3.18 (t, JHP = 13.2 Hz, 2H), 2.59 (dt, J = 7.0 Hz, J = 7.0 Hz, 2H), 2.09–1.90 (m, 4H), 1.67
13
(s, 3H), 1.59 (s, 3H), 1.44 (3H), 1.15 (t, JHP = 14.9 Hz, 3H); C NMR (75 MHz, D2O) δ 145.6 (t,
JCP = 9.3 Hz), 140.1, 133.7, 125.8, 124.4, 119.3, 50.1, 40.4 (t, JCP = 115.0 Hz), 38.9, 28.7, 28.3,
25.8, 25.0, 18.6 (t, JCP = 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: 1H 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, JHP = 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, JHP = 15.0 Hz, 3H); 13C NMR (75 MHz, D2O) δ 146.8 (t, JCP = 9.1 Hz),
140.6, 134.3, 126.7, 124.7, 120.6, 50.7, 41.3 (t, JCP = 118.2), 31.5, 30.1, 28.9, 26.3, 25.5, 23.1, 19.2
31
(t, JCP = 3.8 Hz), 17.6; P (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: 1H NMR (300 MHz, D2O) δ 7.78 (s, 1H), 5.09–4.94 (m, 2H), 4.24 (t, J
22

= 6.9 Hz, 2H), 3.16 (t, JHP = 13.3 Hz, 2H), 2.57 (dt, J = 6.8 Hz, J = 6.8 Hz, 2H), 1.98–1.88 (m, 4H),
13
1.65 (s, 6H), 1.58 (s, 3H), 1.08 (t, JHP = 15.0 Hz, 3H); C NMR (75 MHz, D2O) δ 146.3 (t, JCP
=
9.0 Hz), 140.0, 133.6, 126.0, 124.3, 119.3, 50.0, 40.7 (t, JCP = 117.3 Hz), 38.9, 29.6 (m), 28.4, 25.8,
25.0, 18.7 (t, JCP = 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
1
concentrated in vacuo to afford compound 34 (379 mg, 95%) as a yellow oil: H 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, JCP = 6.9 Hz, 4C), 30.2 (t, JCP = 136.8 Hz), 9.96 (t, JCP = 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 x 30 mL) and the
combined organic phases were dried (MgSO4) and filtered, and the filtrate was concentrated in
23

vacuo. Final purification by column chromatography (10/90 ethanol/hexanes) afforded compound
1
14 (830 mg, 41%) as a yellow oil: H NMR (300 MHz, CDCl3) δ 3.87–3.82 (m, 12H) 2.82 (dt, J =
13
2.8 Hz, JHP = 14.8 Hz, 2H), 2.10 (t, J = 2.8 Hz, 1H), 1.56 (t, JCP = 16.4 Hz, 3H); C NMR (75
MHz, CDCl3) δ 79.0 (t, JCP = 9.7 Hz), 71.6, 53.8 (d, JCP = 6.8 Hz, 2C), 53.8 (d, JCP = 6.8 Hz, 2C),
31
40.5 (t, JCP = 136.1 Hz), 23.2 (m), 16.8 (t, JCP = 6.0 Hz); P 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 x 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
1
the following reaction without further purification: H NMR (300 MHz, CDCl3) δ 5.72–5.63 (m,
8H) 2.82 (dt, J = 2.8 Hz, JHP = 15.5 Hz, 2H), 2.05 (t, J = 2.8 Hz, 1H), 1.50 (t, JCP = 16.1 Hz, 3H),
1.18 (s, 36H); 13C NMR (75 MHz, CDCl3) δ 176.8 (d, JCP = 0.6 Hz, 4C), 82.5–82.3 (m, 4C), 77.7 (t,
JCP = 11.0 Hz), 72.5, 40.5 (t, JCP = 136.6 Hz), 38.8, 26.9 (s, 12C), 22.7 (t, JCP = 4.2 Hz), 16.1 (t, JCP
31
= 5.8 Hz); P 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
24

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 x 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
1
bioassays: H 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, JHP = 16.0 Hz, JHP =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, JCP = 2.8 Hz, 2C), 82.2 (t, JCP = 2.8 Hz, 2C), 50.4, 42.4 (t, JCP
=
133.7 Hz), 39.9 (2C), 38.9 (2C), 32.0, 29.0, 28.2 (t, JCP = 4.1 Hz), 27.0 (12C), 26.5, 25.9, 23.5, 17.8,
15.7 (t, JCP = 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 x
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
1
mixture of E and Z isomers for the H NMR spectrum and the major isomer in the 13C NMR
1
spectrum: H 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, JHP = 16.1 Hz, J = 12.4 Hz, 2H), 2.16–1.81 (m,
25

6H), 1.71 (s, 3H), 1.61 (s, 3H), 1.51 (s, 3H), 1.20 (dt, JHP = 10.4 Hz, J = 3.4 Hz, 12H), 1.14 (dt, JHP
13
= 3.0 Hz, J = 7.7 Hz, 3H); C NMR (75 MHz, CDCl3) δ 142.8 (t, JCP = 10.7 Hz), 142.7, 132.2,
123.5, 123.3, 117.4, 62.7 (t, JCP = 3.4 Hz, 2C), 62.4 (t, JCP = 3.4 Hz, 2C), 47.8, 47.2 (t, JCP = 131.2
Hz), 39.5, 26.3, 26.5 (t, JCP = 4.3 Hz), 25.7, 23.8 (t, JCP = 4.8 Hz), 23.5, 17.7, 16.6–16.4 (m, 4C),
9.9 (t, JCP = 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, D2O) δ 7.81 (s, 1H), 5.42–5.28 (m, 1H), 5.03–4.95 (m,
1H), 4.81–4.76 (m, 2H), 3.06 (t, JHP = 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, JHP = 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, JCP = 120.2 Hz), 39.2, 29.0 (m), 26.1, 25.6 (m), 25.5, 23.1, 17.6, 11.4
31
(m); P 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,
26

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 significance. An α of 0.05 was set as
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
27

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