Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase

Article abstract of DOI:10.1016/j.bmc.2014.03.014

When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase.

Full text of DOI:10.1016/j.bmc.2014.03.014

Bioorganic & Medicinal Chemistry 22 (2014) 2791–2798
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Geranyl and neryl triazole bisphosphonates as inhibitors
of geranylgeranyl diphosphate synthase
a
b
a
b
b
a,c,
⇑
,
Xiang Zhou , Sarah D. Ferree , Veronica S. Wills , Ella J. Born , Huaxiang Tong , David F. Wiemer
b,c,
Sarah A. Holstein
a
Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA
Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
Department of Pharmacology, University of Iowa, Iowa City, IA 52242, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a
significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the
design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a ser-
ies of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds
were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis
of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration
of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers
through parallel reaction sequences. The products were assayed against multiple enzymes as well as
in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor
of geranylgeranyl diphosphate synthase.
Received 3 January 2014
Revised 26 February 2014
Accepted 8 March 2014
Available online 24 March 2014
Keywords:
Isoprenoid biosynthesis
Inhibition
GGDP synthase
Olefin stereochemistry
Ó 2014 Elsevier Ltd. All rights reserved.
1
. Introduction
Biosynthesis of isoprenoids in mammals and higher plants pro-
by administration of statins that inhibit HMG-CoA reductase, and
osteoporosis commonly is treated with bisphosphonates that inhi-
bit farnesyl diphosphate synthase (FDPS). There has been increas-
ing interest in geranylgeranyl diphosphate synthase (GGDPS)
inhibitors because of their anti-proliferative effects and as poten-
ceeds through a series of enzymes that converts 3-hydroxy-3-
methylglutaryl-coenzyme A (HMG-CoA) to products with larger
hydrocarbon chains including farnesyl pyrophosphate (FPP) and
geranylgeranyl pyrophosphate (GGPP). These key compounds are
elaborated to a constellation of more complex derivatives, includ-
ing sesquiterpenes, diterpenes, steroids, and dolichols among
2
–4
tial anti-malarial agents.
Inhibitors of FTase have been investi-
gated extensively as potential chemotherapeutic agents by virtue
5
of their anti-Ras activity.
While there have been a select number of studies of FTase
1
6–8
many others. Furthermore, both FPP and GGPP also are employed
inhibitors that are modeled after the isoprenoid substrates,
the
in post-translational prenylation reactions mediated by farnesyl
transferase (FTase) and geranylgeranyl transferase (GGTase) I and
II. This processing is particularly important to proteins of the Ras
superfamily, because their function requires membrane localiza-
tion that is brought about by incorporation of one or more hydro-
phobic isoprenoid chains.
Given the importance of isoprenoid biosynthesis to metabolism,
it is not surprising that these enzymes are the proximate targets of
therapeutic agents employed for treatment of a number of impor-
tant diseases. For example, hypercholesterolemia often is treated
FTase inhibitors which have been clinically investigated have been
derived through strategies such as peptide mimics or bisubstrate
analogues. The latter approaches, while feasible for FTase and
GGTase I, have not been possible for GGTase II, as this enzyme does
not directly recognize its protein substrate. Instead GGTase II ‘out-
sources’ its substrate specificity to the Rab escort protein (REP)
which delivers Rabs to the active site of the transferase.10
9
We have been interested in the strategy of inhibiting GGTase II
in multiple myeloma cells to disrupt monoclonal protein secretion
and induce apoptosis via the unfolded protein response pathway.11
There has also been increasing interest in Rabs in other malignan-
cies, particularly with respect to the role that Rabs play in mediat-
⇑
Corresponding author. Tel.: +1 319 335 1365.
E-mail address: david-wiemer@uiowa.edu (D.F. Wiemer).
Current address: Departments of Medicine and Immunology, Roswell Park Cancer
1
2
ing tumor cell migration and invasion. We recently reported the
preparation of a small set of triazole bisphosphonate salts repre-
sented by compound 1 (Fig. 1), and found that incorporation of a
Institute, Buffalo, NY 14263, USA.
http://dx.doi.org/10.1016/j.bmc.2014.03.014
0
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2
792
X. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 2791–2798
synthetic route that would avoid the issue of olefin isomerization.
(
NaO)
2
(O)P
(O)P
In this manuscript we report development of a successful strategy
through studies of geranyl and neryl epoxides, as well as the
surprising biological results discovered with one of the new
compounds.
N
N
N
(NaO)
2
1
(
EtO)
2
(O)P
(O)P
2. Synthesis
N
3
and
3
or
(EtO)
2
Given that the equilibration of geranyl (3) and neryl azides (4) is
a sigmatropic process, an apparent way to circumvent the rear-
rangement is to protect the C-2 olefin in a way that allows stereo-
controlled regeneration after formation of the azide and the
cycloaddition. While there may be a number of viable strategies,
we were intrigued by the possibility of epoxidation. Regiospecific
introduction of the epoxide is well established. The only concern
might be regeneration of the olefin with stereocontrol, especially in
the presence of the triazole ring and the bisphosphonate moiety.
2
4
N
3
Figure 1. Synthesis of isoprenoid triazoles via click chemistry.
1
5
longer isoprenoid chain on this heterocyclic template resulted in
13
modest inhibitors of GGTase II. The synthesis was based on ‘click’
chemistry of acetylene 214 with an isoprenoid derived azide. That
approach allowed rapid assembly of the target compounds but it
was limited in that a [3,3]-sigmatropic rearrangement of the allylic
azides allows them to equilibrate and the cycloaddition reaction
then afforded products that were mixtures of olefin isomers. For
example, whether geranyl azide (3) or neryl azide (4) was
employed, a 2:1 mixture of E- and Z-olefin isomers was obtained
and these isomers were not readily separable.13 To support more
detailed biological studies of isoprenoid triazoles we sought a
To begin exploration of this strategy, geraniol (5) was converted
15,16
regioselectively to the corresponding epoxide 6 (Fig. 2).
While
material prepared in this way is racemic, because this intermediate
was viewed primarily as a protected olefin control of the absolute
stereochemistry was of no lasting concern. More importantly, only
one regioisomer of this epoxide was detectable. After formation of
1
6,17
bromide 7
via the mesylate of alcohol 6, reaction with NaN
3
gave the expected product 8. Out of concern for the stability of this
azide, after minimal purification it was used immediately in a
cycloaddition with acetylene 2. This click reaction proceeded
VO(C
5 7 2 2
H O )
O
O
TBHP
NaN3
N
3
X
HO
71%
MsCl
LiBr
52%
8
74%
6 X = OH
5
7
X = Br
5
8%
2
(EtO)
2
2
(O)P
(O)P
O
NaI (EtO)
TFAA
55% (EtO)
(O)P
(O)P
2
N
N
N
(EtO)
N
2
N
N
9
10
75%
TMSBr
TMSBr
collidine
68%
collidine
1
M NaOH
1M NaOH
(
NaO)
2
2
(O)P
(O)P
O
(NaO)
2
(O)P
(O)P
N
N
N
(NaO)
N
(NaO)
2
N
N
12
11
19
(
NaO)
2
(O)P
(O)P
O
(NaO)
2
(O)P
(O)P
N
N
N
N
N
(NaO)
2
N
(NaO)
2
2
0
TMSBr
collidine
TMSBr
collidine
1M NaOH
76%
O
64%
1
M NaOH
NaI
(
EtO)
2
(O)P
(O)P
(EtO)
2
(O)P
(O)P
TFAA
N
N
70%
(EtO)
2
N
(EtO)
2
N
N
N
17
18
2
67%
O
O
VO(C
5 7 2 2
H O )
NaN3
TBHP
60%
85%
MsCl
LiBr
N
3
16
X
14 X = OH
5 X = Br
HO
13
1
6
8%
Figure 2. Synthesis of geranyl and neryl triazole bisphosphonates.

X. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 2791–2798
2793
smoothly to afford the expected triazole 9. Attempted reaction of
this triazole epoxide with TMSCl and NaI under the literature con-
1
8
ditions gave no sign of an olefin product and instead returned
only unreacted starting material. However when treated with NaI
and trifluoroacetic anhydride (TFAA) under the conditions of
1
9
Sonnet, which is believed to result in formation of trifluoroacetyl
iodide in situ, epoxide 9 was converted to a single olefin. This prod-
uct was assigned as the E-isomer 10 on the basis of its NMR data,
and those spectral data matched that of the major isomer formed
through reaction of geranyl (3) or neryl (4) azide with acetylene
1
3
2
3
.
Perhaps the most diagnostic signal was that observed at
1
3
9.6 ppm in the C NMR spectrum, corresponding to the methy-
lene group adjacent to the more substituted carbon of newly
formed olefin. This is very close to the value reported for geraniol
2
0
(
39.7), and quite different from that reported for nerol (32.2).
Once bisphosphonate ester 10 was in hand, hydrolysis under
standard conditions afforded the salt 11. While this salt was the
primary objective of these efforts, intermediate 9 also was viewed
as a potential source of biological activity if the epoxide could be
preserved through the course of phosphonate ester hydrolysis.
Standard conditions for ester hydrolysis appeared to result in diol
formation, but brief treatment with NaOH after reaction with
TMSBr and collidine gave the new epoxide triazole salt 12. Assay
of this second bisphosphonate allows a direct comparison with
the activity of the corresponding olefin 11.
After preparation of the geranyl derivatives 11 and 12, it was of
interest to attempt a parallel series of reactions starting with nerol,
to allow determination of activity as a function of the olefin or
epoxide stereochemistry. Therefore nerol (13) was converted to
1
6
the azido expoxide 16 through the intermediate epoxides 14
1
7
and 15. The click reaction of azide 16 and acetylene 2 proceeded
smoothly to give triazole 17. Treatment of this epoxide isomer with
NaI and TFAA also gave a single olefin product, identified as the
Z-isomer 18 by comparison with the pure E-isomer 10 or the minor
component of the isomer mixture prepared earlier,13 which con-
firms that the reduction itself is a stereospecific process with these
isoprenoid olefins. Again, a diagnostic signal was observed in the
Figure 3. Neryl triazole 19 potently disrupts protein geranylgeranylation in human
myeloma cells. RPMI-8226 cells were incubated for 48 h in the presence or absence
of test compounds. Cells were lysed using RIPA buffer to generate whole cell lysate
or with Triton X-114 to generate aqueous and detergent fractions. Immunoblot
analysis was performed. The Rap1a antibody detects only unmodified protein.
b-Tubulin was used as a loading control for whole cell lysate and aqueous fractions
while calnexin was used the loading control for the detergent fraction. The gels are
representative of two independent experiments. (A) Cells were incubated in the
1
3
C NMR spectrum at 32.2 ppm representing the methylene group
adjacent to the newly formed olefin. This is equal to the value re-
ported for that carbon in nerol (32.2), and quite different from that
reported for geraniol (39.7).20 Finally both esters 18 and 17 were
converted to the corresponding bisphosphonate salts (19 and 20,
respectively).
presence or absence of 10
were incubated in the presence or absence of increasing concentration (0.1, 1,
M) of either DGBP, 11, or 19. (C) Addition of GGPP, but not mevalonate (Mev) or
FPP, prevents the effects of 19 on Rap1a geranylgeranylation. Cells were incubated
in the presence or absence of inhibitor (10 M lovastatin (Lov) or 10 M 19) and/or
isoprenoid (1 mM Mev, 10 M FPP, or 10 M GGPP).
lM DGBP (positive control) or test compound. (B) Cells
10 l
3
. Biological results and discussion
l
l
Our preliminary studies with compound 1 revealed that it dis-
l
l
rupts protein geranylgeranylation in intact cells (unpublished
data). To determine whether this activity is a consequence of the
geranyl and/or neryl stereochemistry, and to begin to determine
the underlying mechanism of action, a series of western blot exper-
iments was performed. Rap1a is a substrate of GGTase I while Rab6
is a substrate of GGTase II. For detection of Rap1a, whole cell lysate
was prepared and an antibody that detects only unmodified Rap1a
was utilized. For Rab6, a Triton X-114 lysis was employed to gen-
erate aqueous and detergent fractions. Under control conditions,
the majority of Rab6 is found in the detergent (membrane) frac-
tion, while in the setting of an agent which disrupts Rab geranyl-
geranylation, Rab6 becomes localized to the aqueous (cytosolic)
isomer 19 effectively disrupted both Rap1a and Rab6 geranylger-
anylation. Interestingly, neither of the epoxide derivatives (12
and 20) displayed activity in this assay, and none of the tested
compounds disrupted Ras farnesylation (data not shown).
To evaluate further the differences in potency between the two
isomers, the geranyl and neryl triazoles were tested at varying
concentrations and compared to the known GGDPS inhibitor DGBP.
As shown in Figure 3B, accumulation of unmodified Rap1a was
observed with 10
geranyl isomer 11 only weakly disrupted Rap1a geranylgeranyla-
tion at 10 M while unmodified Rap1a is detected following
treatment with 1 M of compound 19, indicating that the neryl
lM DGBP but not at lower concentrations. The
l
21
fraction. The known GGDPS inhibitor digeranyl bisphosphonate
l
2
2
(
DGBP) was used as a positive control. As shown in Figure 3A,
stereochemistry confers a potency which is at least an order of
treatment with DGBP results in the accumulation of unmodified
Rap1a and Rab6. A similar pattern was displayed upon treatment
with compound 1 which is a mixture of olefin isomers. Evaluation
of the individual isomers revealed that the geranyl isomer 11 only
weakly disrupted Rap1a geranylgeranylation while the neryl
magnitude greater than the geranyl isomer.
Add-back experiments in which cells were incubated in the
presence of both compound 19 and either mevalonate, FPP, or
GGPP, demonstrated that only GGPP prevents the accumulation
of unmodified Rap1a that is induced by compound 19 (Fig. 3C).

2
794
X. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 2791–2798
Figure 4. Neryl triazole 19 depletes cells of GGPP and increases intracellular FPP levels. RPMI-8226 cells were incubated for 24 h in the presence or absence of 1
compound. DGBP was included as a positive control. Cells were counted and intracellular FPP/GGPP levels were measured. Data are expressed as percentage of control
mean ± SD, n = 2). The ⁄ denotes p <0.05 per unpaired two-tailed t-test and compares treated cells to untreated control cells.
lM test
(
Table 1
IC50 of 375 nM while the geranyl triazole 11 is approximately
0-fold less potent. To determine whether there was any cross-
Evaluation of triazole phosphonates against GGDPS and related enzymes
4
Compound
GGDPS
FDPS
FTase
GGTase I
GGTase II
IC50 M)
reactivity against FDPS or the prenyl transferase enzymes, the
triazole phosphonates were tested against FDPS, FTase, GGTase I,
and GGTase II. The FDPS studies revealed that compound 19 is over
IC50
(
lM)
IC50
(
lM)
IC50
(
l
M)
IC50
(
lM)
(l
1
2.2
17
0.38
23
84
57
79
33
47
500
200
340
500
500
300
>1000
>1000
>1000
>1000
>1000
1
1
1
2
1
9
2
0
>500
400
400
340
2
00-fold less potent as an inhibitor against FDPS than it is against
GGDPS. Consistent with our prior studies demonstrating that com-
pound 1 does not inhibit GGTase II, neither of the individual olefin
isomers (11 or 19) nor the related epoxides (12 or 20) displayed
inhibitory activity against this enzyme at concentrations up to
1 mM. The triazole phosphonates displayed weak activity against
both FTase and GGTase I, with IC50 values in the 300–500 lM
range, which is unlikely to be relevant to the observed activity of
these agents in cell culture.
Finally, the ability of these agents to disrupt a key cellular pro-
cess in myeloma cells was examined. RPMI-8226 cells were incu-
bated with the test compounds and the effects on monoclonal
protein trafficking were determined via lambda light chain ELISA
performed on whole cell lysate. We already have demonstrated
that agents which impair Rab geranylgeranylation disrupt mono-
clonal protein trafficking in myeloma cells, resulting in decreased
secretion and increased intracellular levels of light chain.11 As
shown in Figure 5, the olefin mixture 1 induces an accumulation
of intracellular lambda light chain equivalent to that induced by
the positive control DGBP. This activity is a consequence of the ner-
yl triazole 19 as the geranyl triazole 11 has no effect on intracellu-
lar light chain levels.
17
This is in contrast to the results observed when cells were treated
with the HMG-CoA reductase inhibitor lovastatin, where either
mevalonate or GGPP could prevent lovastatin’s effects. In aggre-
gate, these western blot studies are consistent with the hypothesis
that the neryl isomer 19 disrupts protein geranylgeranylation via
inhibition of GGDPS.
To address this hypothesis further, the effects of the geranyl and
neryl triazoles on intracellular FPP and GGPP levels were exam-
ined. As shown in Figure 4, the isomer mixture represented by
structure 1 (ꢀ2:1 E to Z) decreased GGPP levels and increased
FPP levels, consistent with inhibition of GGDPS. This activity was
a consequence of the neryl component (19) which even more po-
tently decreased GGPP levels and increased FPP levels while the
geranyl isomer did not significantly alter GGPP levels.
The group of triazole compounds was tested for the ability to
directly inhibit GGDPS in an in vitro enzyme assay. As shown in
Table 1, the neryl triazole 19 is the most potent inhibitor with an
Figure 5. Neryl triazole 19 disrupts monoclonal protein trafficking in human myeloma cells. RPMI-8226 cells were incubated for 48 h in the presence or absence of 10 lM
test compound or DGBP. Intracellular lambda light chain concentrations were determined via ELISA. Data are expressed as percentage of control (mean ± SD, n = 3).
The ⁄ denotes p <0.05 per unpaired two-tailed t-test and compares treated cells to untreated control cells.

X. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 2791–2798
2795
We have previously prepared a series of mono- and dialkyl bis-
phosphonates with isoprenoid chains that differed in both chain
length and olefin stereochemistry.23 The most potent GGDPS inhib-
itors were digeranyl bisphosphonate (DGBP) and 2E,6E-farnesyl
bisphosphonate (21). The geranyl/neryl and dineryl bisphospho-
nate versions were approximately 14-fold less potent than DGBP
while the 2E,6Z-(22), 2Z,6E- and 2Z,6Z-isomers were approxi-
mately 6-, 400-, and 700-fold less potent than the 2E,6E-isomer.
effectiveness might be further enhanced through prodrug strate-
2
4
gies, although this has yet to be examined with these triazoles.
DGBP has been demonstrated to be competitive with respect to
2
5
FPP as opposed to IPP. Whether compound 19 inhibits GGDPS
via a similar mechanism also remains to be determined.
4
. Conclusions
6
In addition, in the monoalkyl series the geranyl bisphosphonate
In conclusion, we have developed a new synthetic route which
was at least one order of magnitude more potent than the corre-
sponding neryl bisphosphonate.24 In our present study, we discov-
ered that the neryl triazole bisphosphonate 19 is a significantly
more potent inhibitor of GGDPS than the corresponding geranyl
triazole bisphosphonate 11.
The incorporation of a triazole with a 10-carbon substituent re-
sults in a total chain length which is equivalent to the previously
described farnesyl bisphosphonates. As shown in Figure 6, from
one perspective the triazole ring system can be viewed as a nearly
isosteric replacement for an isoprenoid unit in an E-configuration,
although the C-3 to C-5 bond lengths and angles in compounds 21
and 22 differ slightly from the C-3 to N-5 array in compounds 11
and 19. Even so, the geranyl isomer 11 clearly would be viewed
as most closely parallel to 2E,6E-farnesylbisphosphonate (21)
while the neryl isomer 19 would more closely approximate
affords the preparation of individual olefin isomers of isoprenoid
triazole bisphosphonates. This synthetic approach should be appli-
cable to the preparation of the isomers of the longer chained tria-
zole bisphosphonates which are of interest because of their
potential activity as GGTase II inhibitors. We have demonstrated
that the neryl triazole 19 is a potent and specific inhibitor of
GGDPS in both enzymatic and cellular assays. This potent inhibitor
has important therapeutic implications because of the integral
roles that geranylgeranylated proteins play in diverse cellular
activities.
5. Experimental procedures and methods
5.1. General experimental conditions
2E,6Z-farnesylbisphosphonate (22). Because earlier studies have
shown that the 2E,6E-isomer 21 is about 6-fold more potent than
the 2E,6Z-isomer 22, it would be reasonable to predict that the ger-
anyl derivative 11 would be more active than the neryl isomer 19.
Nevertheless, in the actual experiments the potency of the neryl
compound 19 is actually more than 40-fold greater than the gera-
nyl isomer 11. This experimental data suggests that the triazole
moiety itself may aid in binding to GGDPS. If so, this affords a new
design element that might be exploited in further development
of GGDPS inhibitors. Finally, the importance of the first isoprenoid
olefin in this compound series also is clear as the incorporation of
an epoxide in the cis-configuration (20) reduces the activity against
GGDPS to a level comparable to the geranyl triazole.
Tetrahydrofuran was freshly distilled from sodium/benzophe-
none, 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 reac-
tions in nonaqueous solvents were conducted in flame-dried glass-
ware under a positive pressure of argon and with magnetic stirring.
1
All NMR spectra were obtained at 300, 400, or 500 MHz for H, and
1
3
1
75, 100, or 125 MHz for C, with internal standards of (CH
3
)
4
Si ( H,
1
13
0.00) or CDCl
3
( H, 7.27; C, 77.2 ppm) for non-aqueous samples
1
13
or D
ples. The P chemical shifts were reported in ppm relative to 85%
PO (external standard). High resolution mass spectra were ob-
2
O ( H, 4.80) and 1,4-dioxane ( C, 66.7 ppm) for aqueous sam-
3
1
H
3
4
Of note, the neryl triazole 19 is approximately an order of mag-
nitude more potent in disrupting protein geranylgeranylation in
cell culture studies than DGBP, but is slightly less potent in the
in vitro enzyme assay where DGBP has a reported IC50 of
0.2 lM. This may be a consequence of enhanced cellular uptake
of compound 19 as compared to DGBP by virtue of the presence
tained at the University of Iowa Mass Spectrometry Facility. Silica
gel (60 Å, 0.040–0.063 mm) was used for flash chromatography.
5.2. Tetraethyl (E)-(2-(1-((3-methyl-3-(4-methylpent-3-en-1-
yl)oxiran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)ethane-1,1-
diyl)bis(phosphonate) (9)
2
1
of one isoprenoid chain as opposed to two. In addition, its
Solid NaN
mide 7
3
(171 mg, 2.63 mmol) was added to a solution of bro-
(403 mg, 1.73 mmol) in DMF (7 mL) and the reaction
was allowed to stir overnight at rt while protected from light in
1
6,17
2
6
(
NaO)
2
(O)P
(O)P
a flask wrapped with aluminum foil. Once the reaction was com-
plete, it was diluted with ether. After the filtrate was washed with
water (five times) and then with brine to remove any remaining
3
5
(NaO)
2
2
1
DMF, the organic layer was dried (Na
cuo to obtain the azide as a yellow-orange oil (250 mg, 74%). ( H
NMR (300 MHz, CDCl ) d 5.08 (t, J = 7.1 Hz, 1H), 3.47–3.41 (m,
H), 2.98 (t, J = 6.0 Hz, 1H), 2.15–2.05 (m, 2H), 1.76–1.42 (m, 2H),
.70 (s, 3H), 1.62 (s, 3H), 1.32 (s, 3H)). Without further purification,
2 4
SO ) and concentrated in va-
1
(
NaO)
2
(O)P
(O)P
3
5
3
N
2
1
(NaO)
2
N
N
11
a portion of the azide 8 (140 mg, 0.72 mmol) and tetraethyl-(3-bu-
1
4
tyn-1-ylidene)-1,1-bisphosphonate (2, 180 mg, 0.55 mmol) were
dissolved in a solution of water in t-BuOH (1:4, 3 mL), followed by
addition of CuSO
(
NaO)
2
2
(O)P
(O)P
N
N
(NaO)
N
4
(5 M, 0.01 mL) and sodium ascorbate (33 mg,
.17 mmol) in sequence. After the reaction was allowed to stir
overnight at rt, the solvent was removed under vacuum. The
resulting residue was dissolved in brine and extracted with EtOAc.
The combined organic layer was washed with 5% NH
Na SO ), and concentrated in vacuo. The resulting oil was purified
via flash chromatography (10% EtOH in hexanes) to provide the de-
sired triazole 9 as a colorless oil (170 mg, 58%): H NMR (400 MHz,
0
1
9
(NaO)
2
(O)P
(O)P
4
OH, dried
(NaO)
2
(
2
4
22
1
Figure 6. Comparison of farnesyl and triazole bisphosphonates.

2
796
X. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 2791–2798
CDCl
3
) d 7.63 (s, 1H), 5.04 (t, J = 7.1 Hz, 1H), 4.69 (dd, J = 14.4 Hz,
5.5. Sodium (E)-(2-(1-((3-methyl-3-(4-methylpent-3-en-1-
yl)oxiran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)ethane-1,1-
diyl)bis(phosphonate) (12)
4.1 Hz, 1H), 4.26 (dd, J = 14.8 Hz, 7.2 Hz, 1H), 4.22–4.09 (m, 8H),
3.35 (td, JHP = 16.0 Hz, J = 6.2 Hz, 2H), 3.10 (dd, J = 7.6 Hz, 4.4 Hz,
1H), 2.98 (tt, JHP = 23.5 Hz, J = 6.4 Hz, 1H), 2.07 (td, J = 8.2 Hz,
7.6 Hz, 2H), 1.73–1.63 (m, 1H), 1.67 (s, 3H), 1.60 (s, 3H), 1.54–
1
.45 (m, 1H), 1.42 (s, 3H), 1.33–1.27 (m, 12H); 13C NMR (75 MHz,
An ice cold solution of epoxide 9 (76 mg, 0.15 mmol) in dichlo-
romethane (4 mL) was treated with collidine (0.20 mL, 1.5 mmol)
and TMSBr (0.23 mL, 1.73 mmol) in sequence. After it was allowed
to react overnight at rt, and the reaction was complete based on
CDCl
3
) d 145.5 (t, JCP = 7.8 Hz), 132.5, 122.9, 122.7, 62.8 (d,
JCP = 6.5 Hz, 2C), 62.6 (d, JCP = 6.6 Hz, 2C), 61.4, 60.4, 49.9, 38.1,
3
1
3
6.6 (t, JCP = 132.5 Hz), 25.7, 23.5, 22.1 (t, JCP = 4.0 Hz), 17.7, 17.0,
analysis of the P NMR spectrum, the reaction solvent was re-
moved in vacuo. The resulting residue was then dissolved in tolu-
ene and dried under vacuo, and this process was repeated three
times. The white solid that formed was allowed to stir with 1 N
NaOH (0.75 mL, 0.75 mmol) for 2 min at rt. This material was pre-
cipitated by addition of acetone followed by removal of water on a
lyophilizer to obtain the initial salt. The salt was dissolved in water
and allowed to stir in 1 N NaOH (0.12 mL, 0.12 mmol) for 2 min at
3
1
1
6.4 (d, JCP = 3.1 Hz, 2C), 16.3 (d, JCP = 2.3 Hz, 2C);
P NMR
+
+
(
121 MHz, CDCl
3
) d 22.3; HRMS (ES , m/z) calcd for (M+H)
22 42 3 7 2
C H N O P : 522.2498, found: 522.2488.
5
1
.3. Tetraethyl (E)-(2-(1-(3,7-dimethylocta-2,6-dien-1-yl)-1H-
,2,3-triazol-4-yl)ethane-1,1-diyl)bis(phosphonate) (10)
1
Sodium iodide (60 mg, 0.4 mmol) was weighed in a round bot-
rt to remove residual collidine observed in H NMR spectrum. The
tom flask and dried in an oven overnight. After the salt was dis-
solved in acetonitrile/THF (1:1, 1 mL), trifluoroacetic anhydride
desired product then was precipitated by addition of acetone fol-
lowed by removal of water on a lyophilizer to produce the final
product 12 as a white solid (49 mg, 68%): H NMR (500 MHz,
1
(
0.014 mL, 0.1 mmol) was added. After 5 min, when the solution
had turned to a deep yellow color, it was cooled in an ice bath.
The starting material 9 (50 mg, 0.1 mmol) was then added to the
reaction vessel as a neat oil. After an additional 5 min, the ice bath
was removed and the reaction mixture was allowed to stir over-
night at rt. Once the reaction was complete based on TLC analysis
D
2
O) d 7.93 (s, 1H), 5.15–5.09 (m, 1H), 4.76 (dd, J = 14.9 Hz,
4.3 Hz, 1H), 4.51 (dd, J = 14.7 Hz, 7.4 Hz, 1H), 3.43 (dd, J = 7.4 Hz,
4.6 Hz, 1H), 3.21 (td, HP = 14.9 Hz, J = 7.1 Hz, 2H), 2.21 (tt,
HP = 21.1 Hz, J = 6.7 Hz, 1H), 2.18–2.11 (m, 2H), 1.84–1.74 (m,
1H), 1.74–1.70 (m, 1H), 1.66 (s, 3H), 1.62 (s, 3H), 1.57–1.50 (m,
J
J
1
3
(
5% MeOH/EtO
ous layer was extracted with Et
bined, dried (Na SO ), and concentrated in vacuo. Final purification
by column chromatography (10% MeOH/EtO ) afforded the desired
product 10 as a colorless oil (28 mg, 55%): H NMR (500 MHz,
CDCl ) d 7.44 (s, 1H), 5.40 (tq, J = 7.1 Hz, 1.0 Hz, 1H), 5.06 (t,
J = 5.7 Hz, 1H), 4.92 (d, J = 6.9 Hz, 2H), 4.22–4.07 (m, 8H), 3.32
td, JHP = 15.6 Hz, J = 6.3 Hz, 2H), 3.03 (tt, JHP = 23.6 Hz, J = 6.3 Hz,
2
), it was diluted with saturated NaHSO
3
. The aque-
1H), 1.49 (s, 3H); C NMR (125 MHz, D
123.1, 64.2, 62.0, 49.4, 40.0 (t, JCP = 112.5 Hz), 37.3, 24.9, 23.1,
22.1, 16.9, 15.6; P NMR (202 MHz, D
m/z) calcd for (MÀH)
2
O) d 148.5, 134.0, 124.3,
2
O, the organic extracts were com-
3
1
À
2
4
2
O) d 18.7; HRMS (ES ,
À
2
14 24 3 7 2
C H N O P : 408.1090; found: 408.1101.
1
3
5.6. Tetraethyl (Z)-(2-(1-((3-methyl-3-(4-methylpent-3-en-1-
yl)oxiran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)ethane-1,1-
diyl)bis(phosphonate) (17)
(
1
1
CDCl
H), 2.14–2.04 (m, 4H), 1.78 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H),
.28 (t, J = 7.0 Hz, 6H), 1.28 (t, J = 7.1 Hz, 6H); 13C NMR (125 MHz,
According to the procedure described for preparation of com-
1
7
3
) d 145.2 (t, JCP = 7.8 Hz), 143.1, 132.3, 123.6, 121.7, 117.3,
pound 9, bromide 15 (400 mg, 1.7 mmol) was treated with
NaN (170 mg, 2.6 mmol). The resulting intermediate organic azide
6
2
3.1, 63.0, 62.7, 62.7, 48.0, 39.6, 36.7 (t, JCP = 132.6 Hz), 30.5,
6.3, 25.9, 22.3 (t, JCP = 4.4 Hz), 17.9, 16.5, 16.4, 16.4, 16.4;
3
3
1
P
(16, 260 mg, 1.33 mmol) was then isolated and treated with bis-
phosphonate 2 (330 mg, 1.02 mmol) to afford the desired triazole
+
+
NMR (202 MHz, CDCl
3
) d 22.5; HRMS (ES , m/z) calcd for (M+H)
C
22
H
42
N
3
6
O P
2
: 506.2549; found: 506.2547.
17 (354 mg, 67%) as a colorless oil after purification by flash chro-
1
matography (10% EtOH in hexanes): H NMR (300 MHz, CDCl
3
) d
5
1
.4. Sodium (E)-(2-(1-(3,7-dimethylocta-2,6-dien-1-yl)-1H-
,2,3-triazol-4-yl)ethane-1,1-diyl)bis(phosphonate) (11)
7.68 (s, 1H), 5.14 (t, J = 7.6 Hz, 1H), 4.76 (dd, J = 14.1 Hz, 3.3 Hz,
1H), 4.28–4.05 (m, 9H), 3.34 (td, JHP = 16.5 Hz, J = 6.3 Hz, 2H),
3
.10 (dd, J = 7.4 Hz, 3.7 Hz, 1H), 3.00 (tt, JHP = 23.4 Hz, J = 6.3 Hz,
Collidine (0.21 mL, 1.57 mmol) was added into an ice cold
1H), 2.25–2.13 (m, 2H), 1.82–1.56 (m, 2H), 1.71 (s, 3H), 1.65 (s,
3H), 1.36 (s, 3H), 1.30 (t, J = 7.1 Hz, 12H); C NMR (75 MHz, CDCl )
3
1
3
solution of compound 10 (79 mg, 0.16 mmol) in CH
2
Cl
2
(3.5 mL)
followed by addition of TMSBr (0.25 mL, 1.88 mmol) and the
reaction was allowed to stir overnight at rt. Once the reaction
was complete, based on analysis of the 31P NMR spectrum of the
reaction mixture, it was diluted with toluene. After the solvent
was removed in vacuo, the residue was washed with toluene five
more times and dried to remove any remaining TMSBr. It was then
treated with 1 N NaOH (1.0 mL, 1 mmol) overnight. The reaction
mixture was then dried on a lyophilizer to obtain the salt, which
was then dissolved in a small amount of water, precipitated by
addition of acetone, isolated by filtration, and dried. This material
d 145.0 (t, JCP = 7.6 Hz), 132.2, 122.6, 122.3, 62.4, 62.3, 62.1, 62.0,
61.3, 61.2, 49.4, 36.2 (t, JCP = 132.6 Hz), 32.7, 25.3, 23.6, 21.7 (t,
31
J
CP = 3.8 Hz), 21.5, 17.3, 16.0, 15.9, 15.9, 15.8; P NMR (121 MHz,
+
+
3 22 42 3 7 2
CDCl ) d 22.2; HRMS (ES , m/z) calcd for (M+H) C H N O P :
522.2498; found: 522.2507.
5.7. Tetraethyl (Z)-(2-(1-(3,7-dimethylocta-2,6-dien-1-yl)-1H-
1,2,3-triazol-4-yl)ethane-1,1-diyl)bis(phosphonate) (18)
Bisphosphonate ester 18 was synthesized according to the pro-
cedure employed for the preparation of compound 10 with some
modifications. Sodium iodide (80 mg, 0.53 mmol) was weighed in
a round bottom flask and dried in an oven overnight. After the salt
was dissolved in acetonitrile/THF (1:1, 1.14 mL), trifluoroacetic
anhydride (0.019 mL, 0.14 mmol) was added. After 5 min, the
solution was cooled to 0 °C, and epoxide 17 (69 mg, 0.13 mmol)
in acetonitrile/THF (1:1, 0.5 mL) was added. After an additional
5 min, the ice bath was removed and the reaction was allowed to
stir overnight at rt. Once the reaction was complete based on TLC
was further dissolved in water and lyophilized to produce the pure
1
white salt 11 (59 mg, 75%): H NMR (500 MHz, D
2
O) d 7.81 (s, 1H),
5
.45 (t, J = 6.6 Hz, 1H), 5.12 (s, 1H), 4.97 (d, J = 7.4 Hz, 2H), 3.22 (td,
J
2
HP = 14.7 Hz, J = 6.2 Hz, 2H), 2.42 (tt, JHP = 21.2 Hz, J = 6.4 Hz, 1H),
1
3
.17–2.05 (m, 4H), 1.78 (s, 3H), 1.64 (s, 3H), 1.56 (s, 3H);
O) d 147.0 (t, JCP = 3.7 Hz), 143.7, 133.5, 123.9,
23.6, 117.1, 47.9, 39.6 (t, JCP = 118.6 Hz), 38.7, 25.6, 25.0, 21.8 (t,
C
NMR (125 MHz, D
1
2
31
JCP = 3.4 Hz), 17.1, 15.7; P NMR (202 MHz, D
2
O) d 18.8; HRMS
C H N O P Na : 482.0575; found:
+
+
(
4
ES , m/z) calcd for (M+H)
82.0570.
14 22 3 6 2 4
3
analysis (25% EtOH/hexane), it was diluted with saturated NaHSO .

X. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 2791–2798
2797
The aqueous layer was extracted with Et
were combined, dried (Na SO ), and concentrated in vacuo to afford
the desired product 18 (47 mg, 70%) as a yellow oil which was used
without further purification: 1H NMR (400 MHz, CDCl
) d 7.45 (s,
H), 5.40 (t, J = 7.5 Hz, 1H), 5.13–5.06 (m, 1H), 4.90 (d, J = 7.5 Hz,
H), 4.21–4.06 (m, 8H), 3.34 (td, JHP = 16.2 Hz, J = 6.5 Hz, 2H), 3.04
2
O, the organic extracts
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.
2
4
3
1
2
(
1
tt, JHP = 23.6 Hz, J = 6.2 Hz, 1H), 2.22–2.09 (m, 4H), 1.79 (s, 3H),
1
3
.69 (s, 3H), 1.62 (s, 3H), 1.28 (t, J = 6.9 Hz, 12H); C NMR
) d 145.0 (t, JCP = 11.5 Hz), 142.8, 132.7, 123.3,
5.11. Measurement of intracellular FPP and GGPP levels
(
100 MHz, CDCl
3
1
3
1
21.7, 118.1, 63.0, 62.9, 62.7, 62.6, 47.7, 36.6 (t, JCP = 132.6 Hz),
2.2, 26.4, 25.8, 23.5, 22.2 (t, JCP = 4.0 Hz), 17.8, 16.4, 16.4, 16.3,
Intracellular FPP and GGPP levels were measured using the pre-
2
9
viously reported reversed phase HPLC methodology. Briefly, fol-
lowing incubation with drugs, cells were collected and counted
using Trypan blue staining and a Bio-Rad TC10 automated cell
counter. Isoprenoid pyrophosphates were extracted from cell
pellets with extraction solvent (butanol/75 mM ammonium
hydroxide/ethanol 1:1.25:2.75). After drying down by nitrogen
gas, the FPP and GGPP in the residue were incorporated into
fluorescent GCVLS or GCVLL peptides by FTase or GGTase I. The
prenylated fluorescent peptides were separated and quantified by
reversed phase HPLC with fluorescence detection.
3
1
+
6.3; P NMR (121 MHz, CDCl
3
) d 22.5; HRMS (ES , m/z) calcd for
+
(
M+H) C22
H
42
N
3
O
6 2
P : 506.2549; found: 506.2553.
5
1
.8. Sodium (Z)-(2-(1-(3,7-dimethylocta-2,6-dien-1-yl)-1H-
,2,3-triazol-4-yl)ethane-1,1-diyl)bis(phosphonate) (19)
According to the procedure employed for preparation of com-
pound 11, bisphosphonate 18 (66 mg, 0.13 mmol) was treated with
collidine (0.17 mL, 1.31 mmol), TMSBr (0.21 mL, 1.57 mmol) and
then 1 N NaOH (0.75 mL, 0.75 mmol). A parallel work-up and final
purification by precipitation gave the desired product 19 (40 mg,
5.12. FDPS and GGDPS enzyme assay
6
5
3
4%) as a white solid: 1H NMR (500 MHz, D
.52 (t, J = 7.0 Hz, 1H), 5.19 (t, J = 7.1, 1H), 4.95 (d, J = 7.4 Hz, 2H),
.14 (td, JHP = 14.9 Hz, J = 5.5 Hz, 2H), 2.29–2.13 (m, 4H), 2.05 (tt,
2
O) d 7.81 (s, 1H),
Recombinant FDPS and GGDPS were kindly provided by Dr.
Raymond Hohl, University of Iowa. Recombinant enzyme (10 nM
FDPS, 20 nM GGDPS) was incubated with assay buffer (50 mM
J
HP = 21.7 Hz, J = 5.6 Hz, 1H), 1.80 (s, 3H), 1.67 (s, 3H), 1.62 (s,
H); 1 C NMR (125 MHz, D
3
Tris–HCl, pH 7.7, 20 mM MgCl
compounds for 10 min at room temperature. The reaction was ini-
tiated by the addition of 10 M GPP (for FDPS) or 10 M FPP (for
M [ C]-IPP and was carried out at 37 °C for
0 min. The reaction was stopped by the addition of saturated
3
1
1
2
O) d 150.7, 143.7, 134.2, 123.7, 123.5,
2
, 5 mM TCEP, 5 lg/mL BSA) and test
17.8, 47.8, 41.5 (t, JCP = 118.0 Hz), 31.4, 25.8, 25.0, 24.4, 22.6,
3
1
À
7.0; P NMR (202 MHz, D
2
O) d 19.8; HRMS (ES , m/z) calcd for
l
l
À
14
(
MÀH)
C
14
24
H N
3
O
P
6 2
: 392.1140; found: 392.1135.
GGDPS) and 10 l
3
5
.9. Sodium (Z)-(2-(1-((3-methyl-3-(4-methylpent-3-en-1-
NaCl. Radiolabeled GGPP was extracted with n-butanol and
yl)oxiran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)ethane-1,1-
counted via liquid scintillation counting.
diyl)bis(phosphonate) (20)
5
.13. FTase and GGTase I enzyme assay
To a stirred solution of bisphosphonate 17 (168 mg, 0.32 mmol)
in CH
2
Cl
2
(8.7 mL) at 0 °C, collidine (0.43 mL, 3.2 mmol) and TMSBr
FTase and GGTase I activity was determined using the methods
3
0
31
(
97%, 0.52 mL, 3.9 mmol) were each added dropwise in succession.
of Cassidy et al. and Pickett et al. with some modifications. For
FTase, reaction mixtures were initiated by the addition of 3 nM re-
combinant FTase (Jena Biosciences) to reaction buffer (50 mM
The reaction was allowed to stir overnight while it warmed to
room temperature. Once the reaction was complete, which was
determined by analysis of the 31P NMR spectrum of the reaction
mixture, the solvent was removed in vacuo. The residue then
was diluted with toluene (10 mL) and concentrated in vacuo to re-
move excess TMSBr (3Â). The resulting residue was dried on a vac-
uum line overnight. It then was treated with 1 N NaOH (1.61 mL,
Tris–HCl, pH 7.5, 5 mM DTT, 10 mM MgCl
2
, 10
2
lM ZnCl , 0.04%
n-dodecyl b-d-maltoside (DDM)) containing 10
lM FPP and 5 lM
dansyl-GCVLS (Biosynthesis, Inc.). For GGTase I, 12 nM of recombi-
nant GGTase I (Jena Biosciences) was added to reaction buffer
(50 mM Tris–HCl, pH 7.5, 5 mM DTT, 5 mM MgCl M ZnCl
2
, 50
l
2
,
1
.61 mmol), allowed to stir at room temperature for 2 min, and
10 mM KCl, 0.04% DDM) containing 10 M GGPP and 7 lM dan-
l
immediately precipitated from anhydrous acetone. The resulting
solid was removed by filtration, dissolved in water, and freeze
syl-GCVLS (Biosynthesis, Inc.). Reactions were carried out at 30 °C
in the presence or absence of test compounds. After one hour, reac-
tion mixtures were placed on ice, and the reaction was stopped by
the addition of acetonitrile containing 5% HCl. The mixture was
dried to provide compound 20 (122 mg, 76%) as a white powder:
1
2
H NMR (500 MHz, D O) d 7.94 (s, 1H), 5.26–5.23 (m, 1H), 4.87–
4
1
1
1
1
2
C
.84 (m, 1H), 4.43–4.38 (dd, J = 15.0, 8.5 Hz, 1H), 3.45–3.44 (m,
H), 3.22 (td, JHP = 15.0 Hz, J = 7.0 Hz, 2H), 2.31–2.18 (m, 3H),
.86–1.80 (m, 1H) 1.74 (s, 3H), 1.71–1.70 (m, 1H) 1.68 (s, 3H),
briefly vortexed and then spun at 14,000 rpm for 5 min. 100 lL
of the supernatant was injected into an HPLC with fluorescence
detection and the prenylated fluorescent peptides were quantified.
1
3
2
.42 (s, 3H); C NMR (125 MHz, D O) d 148.3, 133.8, 124.1,
23.1, 64.0, 62.9, 49.6, 39.9 (t, JCP = 111.0 Hz), 32.2, 25.1, 23.5,
5.14. GGTase II enzyme assay
3
1
À
À
2.0, 21.0, 17.1; P NMR d 19.0; HRMS (ES , m/z) calcd for (MÀH)
: 408.1090; found: 408.1114.
14
H
24
N
3
O
7
P
2
GGTase II activity was determined using the method of Seabra
1
3,32
and James with some modifications as previously described.
3
5
.10. Immunoblot analysis
Briefly, reactions were initiated with the addition of 5
GGPP to reaction buffer (50 mM sodium HEPES, 5 mM MgCl
1 mM dithiothreitol, 1 mM NP-40) containing 30 nM recombinant
GGTase II (Jena Biosciences), 2 M recombinant Rab1A, and 1
lM [ H]-
2
,
RPMI-8226 cells were incubated with drugs for 48 hrs. 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. Aqueous and
detergent fractionations were obtained using Triton X-114 as pre-
l
lM
recombinant REP-2 (Jena Biosciences) with or without the test
compounds. After a 20 min incubation at 37 °C, reactions were ter-
minated by the addition of 200
lL 10% HCl/EtOH. Samples were fil-
2
7,28
viously described.
Protein content was determined using the
tered through P-81 filters (Whatman) by a Brandel harvester.

2
798
X. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 2791–2798
Filters were then washed in EtOH, dried, and counted using liquid
scintillation counting. Recombinant Rab1A was prepared by trans-
forming BL21 Star DE3 competent cells with a plasmid containing
N-His-tagged Rab1A (GeneCopoeia). A Qiagen Ni-NTA Fast Spin kit
was used to purify the recombinant protein. Protein content was
determined using the BCA method.
References and notes
1
2
3
.
.
.
Holstein, S. A.; Hohl, R. J. Lipids 2004, 39, 293.
Wiemer, A. J.; Wiemer, D. F.; Hohl, R. J. Clin. Pharmacol. Ther. 2011, 90, 804.
Dudakovic, A.; Wiemer, A. J.; Lamb, K. M.; Vonnahme, L. A.; Dietz, S. E.; Hohl, R.
J. J. Pharmacol. Exp. Ther. 2008, 324, 1028.
4.
Zhang, Y. H.; Zhu, W.; Liu, Y. L.; Wang, H.; Wang, K.; Li, K.; No, J. H.; Ayong, L.;
Gulati, A.; Pang, R.; Freitas-Junior, L.; Morita, C. T.; Oldfield, E. ACS Med. Chem.
Lett. 2013, 4, 423.
5
.15. Monoclonal protein quantitation
5. Holstein, S. A.; Hohl, R. J. Curr. Opin. Pharmacol. 2012, 12, 704.
6.
Holstein, S. A.; Cermak, D. M.; Wiemer, D. F.; Lewis, K.; Hohl, R. J. Bioorg. Med.
Chem. 1998, 6, 687.
Cells were incubated in the presence or absence of test com-
7
8
.
.
Hohl, R. J.; Lewis, K. A.; Cermak, D. M.; Wiemer, D. F. Lipids 1998, 33, 39.
Gibbs, B. S.; Zahn, T. J.; Mu, Y. Q.; Sebolt-Leopold, J. S.; Gibbs, R. A. J. Med. Chem.
1999, 42, 3800.
pounds for specified periods of time. The cells were lysed in RIPA
buffer containing protease and phosphatase inhibitors. Protein
content was determined using the BCA method. Human lambda
light chain kit (Bethyl Laboratories, Montgomery, TX) was used
to quantify intracellular monoclonal protein levels.
9
.
Sousa, S. F.; Fernandes, P. A.; Ramos, M. J. Curr. Med. Chem. 2008, 15, 1478.
1
0. Guo, Z.; Wu, Y. W.; Das, D.; Delon, C.; Cramer, J.; Yu, S.; Thuns, S.; Lupilova, N.;
Waldmann, H.; Brunsveld, L.; Goody, R. S.; Alexandrov, K.; Blankenfeldt, W.
EMBO J. 2008, 27, 2444.
1
1
1
1. Holstein, S. A.; Hohl, R. J. Leuk. Res. 2011, 35, 551.
2. Recchi, C.; Seabra, M. C. Biochem. Soc. Trans. 2012, 40, 1398.
3. Zhou, X.; Hartman, S. V.; Born, E. J.; Smits, J. P.; Holstein, S. A.; Wiemer, D. F.
Bioorg. Med. Chem. Lett. 2013, 23, 764.
5
.16. Statistics
1
1
4. Skarpos, H.; Osipov, S. N.; Vorob’eva, D. V.; Odinets, I. L.; Lork, E.;
Roschenthaler, G. V. Org. Biomol. Chem. 2007, 5, 2361.
5. Sharpless, K. B.; Michaels, R. C. J. Am. Chem. Soc. 1973, 95, 6136.
Two-tailed t-testing was used to calculate statistical signifi-
cance. An
a of 0.05 was set as the level of significance. Concentra-
tion response curves for the enzyme assays were analyzed via
CalcuSyn software (Biosoft, Cambridge, UK) to determine IC50
values.
16. Lempers, H. E. B.; Garcia, A. R. I.; Sheldon, R. A. J. Org. Chem. 1998, 63, 1408.
1
1
1
2
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Financial support from the University of Iowa Graduate College
in the form of an Iowa AGEP/Dean’s Graduate Fellowship (to
V.S.W.), the Center for Biocatalysis and Bioprocessing in the form
of an NIH Predoctoral Fellowship in Biotechnology (to V.S.W.),
the Roy J. Carver Charitable Trust, and the NIH (R01CA-172070)
is gratefully acknowledged.
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Supplementary data (NMR spectra) associated with this article
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j.bmc.2014.03.014.
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