Synthesis and activity of fluorescent isoprenoid pyrophosphate analogues

Article abstract of DOI:10.1021/jo049101w

New fluorescent analogues of farnesol and geranylgeraniol have been prepared and then converted to the corresponding pyrophosphates. These analogues incorporate anthranylate or dansyl-like groups anchored to the terpenoid skeleton through amine bonds that

Full text of DOI:10.1021/jo049101w

Synthesis and Activity of Fluorescent Isoprenoid Pyrophosphate
Analogues
MeeKyoung Kim,† Troy S. Kleckley,† Andrew J. Wiemer,‡ Sarah A. Holstein,‡
Raymond J. Hohl,‡ and David F. Wiemer*,†
Departments of Chemistry, Pharmacology, and Internal Medicine, University of Iowa,
Iowa City, Iowa 52242-1294
david-wiemer@uiowa.edu
Received May 28, 2004
New fluorescent analogues of farnesol and geranylgeraniol have been prepared and then converted
to the corresponding pyrophosphates. These analogues incorporate anthranylate or dansyl-like
groups anchored to the terpenoid skeleton through amine bonds that would be expected to be
relatively stable to metabolism. After addition of the alcohols or the pyrophosphates to the culture
medium, their fluorescence is readily observed inside a human-derived leukemia cell line. Enzyme
assays have revealed that the farnesyl pyrophosphate analogue is an inhibitor of FTase, while the
corresponding alcohol is not. These results, together with Western blot analyses of cell lysates,
indicate that the farnesyl pyrophosphate analogue penetrates the cells as an intact pyrophosphate
and that it does so at a biologically relevant concentration.
Introduction
that the majority of studies on masking phosphate and
phosphonate anions have focused on nucleoside ana-
logues and derivatives, and the extent to which these
studies apply to more lipophilic organophosphorus com-
pounds is not completely clear.
For some time, we have been interested in isoprenoid
metabolism, including both synthesis of modified farne-
sols8 and preparation of phosphonic acid analogues of
farnesyl pyrophosphate (FPP, 1) (Figure 1).9-13 Several
other research groups have reported preparation of
pyrophosphate derivatives of farnesol analogues, some
of which serve as serve either as alternate substrates or
as inhibitors for the enzyme farnesyl:protein transferase
(FTase, EC 2.5.1.58).14-25 One of the simplest FTase
inhibitors, R-hydroxy farnesylphosphonate (2), has been
Phosphate and pyrophosphate esters are ubiquitous
intermediates in metabolism, and numerous efforts have
been directed at preparation of analogues that may
display useful biological activity.1 In many cases, phos-
phonic acid analogues of the phosphate monoester have
been prepared to provide increased metabolic stability2
because phosphonates would not be expected to undergo
hydrolysis catalyzed by common phosphatases. However,
phosphonic acids, phosphate esters, and pyrophosphate
esters all bear substantial negative charge at physiologi-
cal pH, and it has long been recognized that this may
limit the compounds’ ability to penetrate the cell
membrane.2-4 In an effort to circumvent this perceived
limitation, various programs have developed biodegrad-
able protecting groups that will mask a negative charge
until after penetration of the cell membrane.5,6 For
example, the pivaloyloxymethyl (POM) group has been
used to generate neutral derivatives of acyclic nucleoside
phosphonates that have anti-viral activity. The resulting
derivatives have an improved profile of biological activity
at least in part because of an improved ability for these
nucleotide analogues to enter the body through the
intestinal mucosa.7 However, it would be easy to conclude
(7) Farquhar, D.; Chen, R.; Khan, S. J. Med. Chem. 1995, 38, 488-
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* Corresponding author. Tel: 319-335-1365. Fax: 319-335-1270.
† Department of Chemistry.
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114.
‡ Departments of Internal Medicine and Pharmacology.
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10.1021/jo049101w CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/05/2004
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J. Org. Chem. 2004, 69, 8186-8193

Fluorescent Isoprenoid Pyrophosphate Analogues
of compound 3, while the Waldmann group has reported
preparation of a fluorescent anthranilate ester of geranyl
pyrophosphate (i.e., compound 4) that can be viewed as
an FPP analogue. Out of concern that the ester linkage
of compound 4 might be cleaved by nonspecific esterases,
which would release the fluorescent label from the
isoprenoid chain, the presumably more stable amine 5
became our initial target.
To begin the synthesis of the anthranylate derivative
5, geranyl acetate (6) was oxidized with selenium dioxide
and tert-butyl hydroperoxide as described by Sharp-
less.31,34 Both the alcohol 7 and the aldehyde 8 were
isolated after column chromatography, and the alcohol
7 was oxidized with PDC to afford additional aldehyde.
Compound 8 could be used in a reductive amination with
methyl anthranylate (10), but the yield was moderate and
the resulting product proved difficult to isolate. After
removal of the acetate group and treatment of the
resulting alcohol with TBDMSCl gave the TBDMS-
protected aldehyde 9, reductive amination proceeded
smoothly to afford the new amine 11. This product was
much less polar than the starting material 10, so it was
more readily purified by column chromatography and the
yield was improved. Final reaction with TBAF gave the
farnesol analogue 12 in 90% yield.35
The farnesol analogue 12 was converted to its corre-
sponding chloride 13 through reaction with N-chlorosuc-
cinimide and dimethyl sulfide in CH2Cl2.36,37 The result-
ing allylic chloride (13) was not isolated because it may
be unstable, so it was used directly for the next step.
Reaction with tris(tetra-n-butylammonium) hydrogen
pyrophosphate38,39 produced compound 14. Purification
by sequential ion exchange chromatography and C18
reversed-phase column chromatography afforded the
farnesyl pyrophosphate analogue 5 in 30% yield from
alcohol 12 (Scheme 1). Because compound 5 decomposes
upon prolonged storage at rt, as monitored by 31P NMR
spectroscopy, it has been stored at 0 °C in a buffer
solution (MeOH/10mM aq. NH4OH, 7:3). As expected,
compound 5 is highly fluorescent with an emission
maximum of 422 nm.
FIGURE 1. Structures of FPP and analogues.
shown to be very effective in enzyme assays,26 but our
studies have suggested that it is less effective in cell-
based assays.9 Whether this apparent decrease in activity
is due to a limited ability of the phosphonic acid to
penetrate the cell membrane, a heightened instability in
the culture medium, or some other reason is not clear.
However, it is known that under conditions of mevalonate
depletion induced by an HMG-CoA reductase inhibitor,
addition of FPP or GGPP can restore protein farnesyla-
tion or geranylgeranylation, respectively.27 In addition,
radiolabeled FPP or GGPP can become incorporated into
isoprenylated proteins.28 Such experiments can only be
successful if the isoprenoid pyrophosphates penetrate the
cell membrane at a level sufficient to restore a significant
internal concentration. To provide more information on
the ability of charged isoprenoids to enter cells at
biologically relevant concentrations, we have prepared
fluorescent analogues of FPP and GGPP and have
studied their impact on isoprenoid metabolism in a
human-derived leukemia cell line (RPMI-8402 cells).
Both the synthesis of these compounds and select studies
on their activity in cells are presented here.29
A geranylgeranyl pyrophosphate analogue 27 related
to the dansyl group also has been prepared as a prototype
for a second family of fluorescent isoprenoids.40,41 For this
synthesis, commercially available 5-aminonaphthalene
(29) Presented in part at the 221st National Meeting of the
American Chemical Society, April 4, 2001, San Diego (302-MEDI). See
also: US Patent No. 6,727,234 B2.
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Spielmann, H. P. Biochemistry 2001, 40, 12254-12265.
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Results and Discussion
Design of the initial target compound was guided by
recent studies of Spielmann30,31 and Waldmann.32,33 The
Spielmann group has shown that an aniline ring can
serve as a surrogate for a prenyl unit through preparation
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J. Org. Chem, Vol. 69, No. 24, 2004 8187

Kim et al.
SCHEME 1. Synthesis of FPP Analogue 5
SCHEME 3. Byproduct Formation upon BOC
Hydrolysis of Compound 18
SCHEME 4. Synthesis of Geranylgeranyl
Pyrophosphate Analogue 27
SCHEME 2. Synthesis of N,N-Dimethyl-5-
aminonaphthalenesulfonamide (19)
BOC cleavage and subsequent reaction of the amine 19
with trace amounts of Br2.43 When compound 18 was
treated with HBr at 0 °C, the deprotection was greatly
improved and compound 19 was obtained as a single
product (Scheme 4).
Reductive amination of compound 19 with aldehyde 8
gave the desired product 22, but the parallel reaction of
compound 19 with the TBDMS-protected aldehyde 9 gave
a better yield and allowed more facile separation of the
product 23 (Scheme 4). Hydrolysis of the TBDMS group
by treatment of compound 23 with TBAF in THF gave
the geranylgeraniol analogue 24 in 98% yield. Alcohol
24 was a highly fluorescent green-yellow oil with an
excitation maximum at 394 nm and an emission at 537
nm in MeOH.
sulfonic acid (15) was first protected as its BOC deriva-
tive 16 through reaction with di-tert-butyl carbonate
(Scheme 2). The sulfonic acid 16 then was converted to
the sulfonyl chloride 17 by treatment with triphenylphos-
phine and thionyl chloride, and the acid chloride 17 was
treated with dimethylamine to generate compound 18.42
Cleavage of the BOC protecting group of compound 18
initially was problematic. Treatment with HCl/dioxane
generated the desired compound as well as the unex-
pected byproduct 20 (Scheme 3). A similar byproduct (21)
also was obtained by treatment of compound 18 with
HBr/HOAc at room temperature and may result from
Conversion of the alcohol 24 to the corresponding
allylic chloride 25 was achieved upon treatment with
N-chlorosuccinimide and DMS in CH2Cl2,36,37 and the
pyrophosphate 26 was obtained upon subsequent reaction
with (NBu4)3HP2O7 in CH3CN. The product pyrophos-
phate 26 was converted to the NH4+ salt by loading on a
(40) Hartwig, J. F.; Pil, P. M.; Lippard, S. J. J. Am. Chem. Soc. 1992,
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+
(41) Yamamoto, Y.; Katow, H.; Sofuku, S. Chem. Lett. 1994, 8, 1379-
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8188 J. Org. Chem., Vol. 69, No. 24, 2004

Fluorescent Isoprenoid Pyrophosphate Analogues
SCHEME 5. Synthesis of Geranylgeranyl
Pyrophosphate Analogue 36
buffer solution (iPrOH:25 mM aq NH4HCO3, 1:49) to
yield the desired geranylgeranyl pyrophosphate analogue
27 in 39% overall yield from the allylic alcohol 24
(Scheme 4).
The terminal methyl groups of FPP fit the FTase
pocket near the bottom of the active site.44-46 If the larger
steric demand of a terminal anthranylate or dansyl-like
group limits the ability of the pyrophosphate analogues
to fit into the prenyl transferases, either fluorescent
group could be moved in to the middle of the prenyl
chains where it might be better accommodated. This
strategy is supported by a very recent study that con-
trasts the active site of GGTase with the FTase active
site.47
Preparation of a GGPP analogue with a fluorescent
reporter group in the interior of the chain began with
conversion of compound 16 to the sulfonyl chloride,
followed by condensation with methylamine to afford the
sulfonamide 28 (Scheme 5). The sulfonamide 28 then was
treated with NaH in DMF followed by reaction with
5-bromo-2-methyl-3-pentene to yield the dialkyl sulfona-
mide 29 in 55% yield. The BOC group was cleaved by
treatment with 30% HBr/HOAc to afford the amine 30
in quantitative yield.
After removal of the BOC protecting group, reductive
amination of compound 30 with the TBDMS-protected
aldehyde 3148 generated compound 32 in good yield.
Removal of the TBDMS group through reaction with
TBAF afforded the geranylgeraniol analogue 33 as a
green-yellow fluorescent oil in 41% yield after final puri-
fication by column chromatography. The geranylgeranyl
pyrophosphate analogue 36 was prepared from alcohol
33 via allylic chloride 34 through standard reactions as
described for compound 27.36,37
The fluorescent farnesol/farnesyl pyrophosphate ana-
logues and geranylgeraniol/geranylgeranyl pyrophos-
phate analogues have been the subject of a variety of bio-
assays. Using an in vitro enzyme assay, the ammonium
salt of compound 5 was shown to inhibit FTase with an
IC50 value of ∼50 nM. This is comparable to the effects
of compound 2 (IC50 ) 39 nM).26 As a control, anthranilic
acid alone was shown not to have an inhibitory effect on
FTase (data not shown) and the farnesol analogue 12 also
did not inhibit FTase (IC50 >50 µM). Parallel studies
assessing the ability of the geranylgeranyl analogues 24,
27, 33, and 36 to inhibit GGTase are planned.
Consistent with the known cytotoxicity of farnesol,49
the farnesol analogue 12 proved to be more toxic to cells
than did the pyrophosphate 5. DNA synthesis assays
demonstrated that while the pyrophosphate 5 did not
reach an IC50 at 100 µM, compound 12 inhibited
[3H]-thymidine incorporation with an IC50 of ∼10 µM in
RPMI-8402 human leukemia cells.
in Figure 2, intracellular accumulation of compounds 5
and 12 occurred in a time-dependent manner, with
greater uptake of the alcohol 12. Similarly, the fluores-
cent geranylgeranyl analogues (compounds 24 and 27)
also could be visualized within cells (Figure 3). Uptake
of the GGOH analogue 24 is more facile, presumably
because it is neutral. However, microscopy clearly shows
that the GGPP analogue 27 also penetrates cells. The
mechanism by which the charged pyrophosphates enter
the cell is not well-understood. Previous studies have
shown that the uptake of radiolabeled FPP or GGPP is
not significantly affected by the temperature of the
incubation medium or by metabolic poisons,28 suggesting
that the process is energy-independent. It should be noted
Fluorescent microscopy was performed to assess the
cellular uptake of the fluorescent compounds. As shown
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J. Org. Chem, Vol. 69, No. 24, 2004 8189

Kim et al.
FIGURE 2. Uptake of farnesyl analogues 5 and 12 by RPMI-8402 cells. Cells were incubated with compound 12 (10 µM) or 5
(100 µM) for 3 or 18 h and were subsequently examined via fluorescent microscopy. Control cells under phase-contrast microscopy
and under fluorescent microscopy are also shown.
that in plants, there appears to be a unidirectional
transport system in the chloroplast envelope membrane
which mediates the export of isoprenoid pyrophosphates
(IPP, GPP > FPP, DMAPP) from the chloroplast to the
cytosol.50
phosphorylate farnesol and geranylgeraniol to yield FPP
and GGPP.51-53 However, there are several findings in
our studies which support the hypothesis that the activity
of the pyrophosphate 5 is not a consequence of hydrolysis
followed by phosphorylation once inside the cell. First,
compounds 5 and 12 displayed differential toxicities in
the DNA synthesis assays; the alcohol 12 is greater than
10 times more cytotoxic than the pyrophosphate 5.
Second, treatment of intact cells with the pyrophosphate
5 results in inhibition of Ras farnesylation but treat-
ment with the alcohol 12 does not. It is interesting to
note that while levels of FPP in human plasma have been
estimated to be in the range of 5-7 ng/mL (∼15 nM),
levels of farnesol were below the detection limit of 0.5
ng/mL (∼2 nM)54 indicating stability of FPP under
physiologic conditions. While these results do not bear
on the potential clinical utility of isoprenoid pyrophos-
phate analogues, they do suggest a valuable role for such
compounds as metabolic probes in cell cultures and
encourage further efforts to design pyrophosphate-based
inhibitors and alternate substrates of the prenyltrans-
ferase enzymes.
Further evidence of cellular uptake and biological
activity of compound 5 was obtained through examina-
tion of Ras protein processing via western blot analysis
(Figure 4). As a control, cells were treated with lovastatin,
which resulted in the accumulation of unmodified Ras
(the more slowly migrating band). Incubation of cells with
the pyrophosphate 5 led to the appearance of unmodified
Ras, consistent with the enzyme studies which demon-
strated that compound 5 is an FTase inhibitor. In con-
trast, treatment of cells with the alcohol 12 alone did not
increase unmodified Ras. The increase in unmodified Ras
induced by compound 5 could be prevented by coincuba-
tion with FPP (1), indicating that compound 5 is likely a
competitive inhibitor of FTase with respect to FPP (data
not shown).
In summary, we have prepared fluorescent analogues
of both FPP and GGPP. The FPP analogue 5 is a potent
inhibitor of FTase in both enzymatic assays and cul-
tured cells. These experiments provide additional evi-
dence that isoprenoid pyrophosphates can cross cell
membranes intact. It has been shown that cells can
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8190 J. Org. Chem., Vol. 69, No. 24, 2004

Fluorescent Isoprenoid Pyrophosphate Analogues
FIGURE 3. Uptake of geranylgeranyl analogues 24 and 27 by RPMI-8402 cells. Cells were incubated with compound 24 (10 µM)
or 27 (100 µM) for 3 or 18 h and were subsequently examined via fluorescent microscopy. Control cells under phase-contrast
microscopy and under fluorescent microscopy are also shown.
additional 2 h. The mixture then was diluted with ether, and
a white precipitant was removed by filtration. The filtrate
was concentrated under reduced pressure, and the residue
was washed with water and brine and dried (MgSO4). Final
purification by column chromatography (hexane/EtOAc,
95:5) gave compound 9 (3.89 g, 69%) as a colorless oil.56
Amine 11. Methyl anthranilate hydrochloride (1.00 g, 5.33
mmol) and aldehyde 9 (4.39 g, 15.5 mmol) were dissolved in
ClCH2CH2Cl (21 mL). Acetic acid (2.5 mL, 43.2 mmol) and 4
Å molecular sieves were added, and the reaction solution was
stirred for 5 min at rt. After NaBH(OAc)3 (6.89 g, 32.5 mmol)
was added, the solution was stirred for 2.7 h at rt and then
quenched by addition of 5% aq NaHCO3 dropwise at 0 °C. The
product was extracted with ether, and the combined extracts
were washed with brine, dried (MgSO4), and concentrated to
afford a colorless oil. Final purification by flash column
chromatography (hexane/EtOAc, 95:5) gave compound 11 (2.51
g, 89%) as a colorless oil: 1H NMR δ 7.89 (dd, J ) 7.5, 1.6 Hz,
1H), 7.85 (br s, 1H), 7.31 (dq, J ) 7.7, 1.4 Hz, 1H), 6.63 (d, J
) 7.7 Hz, 1H), 6.58 (dq, J ) 7.5, 1.4 Hz, 1H), 5.40 (dt, J ) 6.8,
1.3 Hz, 1H), 5.29 (dt, J ) 6.4, 1.4 Hz, 1H), 4.18 (d, J ) 6.4 Hz,
2H), 3.85 (s, 3H), 3.73 (d, J ) 5.3 Hz, 2H), 2.20-2.12 (m, 2H),
2.06-2.00 (m, 2H), 1.67 (s, 3H), 1.61 (s, 3H), 0.90 (s, 9H), 0.07
(s, 6H); 13C NMR (100 MHz) δ 169.1, 151.4, 136.6, 134.4, 131.5,
131.5, 125.8, 124.6, 114.4, 111.7, 109.8, 60.3, 51.4, 50.6, 39.2,
26.1, 26.0 (3C), 18.4, 16.3, 14.5, -5.0 (2C); HRMS (FAB) calcd
for C24H40NO3Si (M + H)+ 418.2777, found 418.2780.
FIGURE 4. Effect of farnesyl analogues on Ras processing.
Cells were treated for 24 h with (A) compound 5 (100 µM) or
(B) compound 12 (10 µM) in the presence or absence of 10 µM
lovastatin.
Experimental Section
8-(tert-Butyldimethylsilyloxy)-2,6-dimethylocta-2,6-di-
enal (9). Solid K2CO3 (7.13 g, 51.6 mmol) was added to a
solution of compound 834,55 (4.20 g, 20.0 mmol) in MeOH (40
mL) at rt, and the reaction mixture was stirred for 2.5 h and
then was diluted with EtOAc. The aqueous layer was ex-
tracted with EtOAc, and the combined organic layers were
dried (MgSO4). After concentration in vacuo, the residue was
dissolved in THF (10 mL), the solution was cooled to 0 °C,
i-Pr2NEt (13.9 mL, 79.9 mmol) was added, and the resulting
mixture was stirred for 10 min. TBDMSCl (8.37 g, 0.12 mol)
was added, and the reaction mixture was stirred for an
Farnesol Analogue 12. Compound 11 (1.55 g, 3.70 mmol)
was treated with TBAF (26.6 mL, 26.7 mmol) in THF (31 mL),
and the reaction solution was stirred for 2 h at 0 °C. The
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J. Org. Chem, Vol. 69, No. 24, 2004 8191

Kim et al.
reaction solution was quenched by addition of saturated
NH4Cl (aq) and extracted with EtOAc. The combined organic
layers were washed with brine and dried (MgSO4). Final
purification by column chromatography afforded the farnesol
analogue 12 (1.01 g, 90%) as a colorless oil: 1H NMR δ 7.89
(d, J ) 8.0 Hz, 1H), 7.82 (br s, 1H), 7.31 (dd, J ) 8.6, 7.1 Hz,
1H), 6.63 (d, J ) 8.6 Hz, 1H), 6.58 (dd, J ) 8.0, 7.1 Hz, 1H),
5.42-5.36 (m, 2H), 4.12 (d, J ) 6.8 Hz, 2H), 3.85 (s, 3H), 3.72
(d, J ) 5.1 Hz, 2H), 2.22-2.15 (m, 2H), 2.08-2.03 (m, 2H),
1.68 (s, 3H), 1.66 (s, 3H); 13C NMR δ 169.2, 151.3, 138.7, 134.5,
131.6, 131.5, 125.3, 124.0, 114.4, 111.6, 109.5, 59.3, 51.5, 50.3,
39.0, 25.7, 16.1, 14.7; HRMS (FAB) calcd for C18H26NO3 (M +
H)+ 304.1913, found 304.1907.
Chloride 13. N-Chlorosuccinimide (399 mg, 2.99 mmol) was
dissolved in CH2Cl2 (11 mL), the solution was allowed to cool
to -30 °C in an acetonitrile/dry ice bath and dimethyl sulfide
(0.24 mL, 3.26 mmol) was added dropwise by syringe. The
reaction mixture was allowed to warm to 0 °C in an ice bath
and then stirred for 5 min. After the reaction mixture was
cooled to -40 °C, a solution of the farnesol analogue 12 (824
mg, 2.72 mmol) in CH2Cl2 (2.5 mL) was added dropwise by
syringe at -40 °C. The suspension was allowed to warm to 0
°C in an ice bath and then stirred for 3 h. The reaction mixture
was allowed to warm to rt, stirred an additional 15 min, then
poured into a separatory funnel containing cold brine and
extracted with CH2Cl2. The organic layers were combined,
dried (MgSO4), and filtered. After concentration on a rotary
evaporator, the residue was dried in vacuo for 1 h to afford
compound 13 as a colorless oil. This material was carried to
the next step without further purification: 1H NMR δ 7.9 (dd,
J ) 8.0, 1.6 Hz, 1H), 7.32 (ddd, J ) 8.6, 7.1, 1.6 Hz, 1H), 6.63
(dd, J ) 8.6, 0.8 Hz, 1H), 6.59 (ddd, J ) 8.0, 7.1, 0.8 Hz, 1H),
5.40 (m, 2H), 4.07 (d, J ) 7.8 Hz, 2H), 3.83 (s, 3H), 3.74 (d, J
) 4.4 Hz, 2H), 2.17 (m, 2H), 2.08 (m, 2H), 1.72 (s, 3H), 1.68 (s,
3H); 13C NMR δ 169.0, 151.3, 142.2, 134.3, 131.9, 131.4, 125.0,
120.6, 114.4, 111.6, 109.7, 51.4, 50.3, 41.0, 39.0, 25.8, 16.0, 14.6;
HRMS (FAB) calcd for C18H25NO2Cl (M + H)+ 322.1574, found
322.1558.
solid: 1H NMR (400 MHz, D2O) δ 8.56 (d, J ) 8.4 Hz, 1H),
8.13 (dd, J ) 7.2, 1.0 Hz, 1H), 8.08 (d, J ) 8.6 Hz, 1H), 7.65
(dd, J ) 8.4, 8.1 Hz, 1H), 7.54 (dd, J ) 8.6, 7.2 Hz, 1H), 7.51
(dd, J ) 8.1, 1.1 Hz, 1H), 3.04 (q, J ) 7.3 Hz, 6H), 1.50 (s,
9H), 1.16 (t, J ) 7.4 Hz, 9H); 13C NMR (100 MHz, D2O) δ 159.4,
141.2, 135.8, 132.3, 131.5, 129.9, 129.1, 129.0, 127.6, 126.4,
126.3, 84.7, 49.3 (3C), 30.3 (3C), 10.9 (3C). Anal. Calcd for
C21H32N2O5S: C, 59.41; H 7.60; N, 6.60. Found: C, 59.31; H
7.51; N, 6.60.
Sulfonamide 18. Triphenylphosphine (2.60 g, 9.92 mmol)
in CH2Cl2 (104 mL) was cooled in an ice bath to 0 °C, thionyl
chloride (0.79 mL, 10.9 mmol) was added, and the reac-
tion mixture was stirred for 20 min. The ice bath was re-
moved, and compound 16 (2.03 g, 4.77 mmol) was added. The
reaction mixture was stirred for 1 h at rt and cooled to 0 °C,
and then dimethylamine hydrochloride (1.17 g, 14.30 mmol)
and triethylamine (4.2 mL, 30.5 mmol) were added drop-
wise over 5 min. After the solution was stirred for 2 h at rt, it
was diluted with water and extracted with CH2Cl2. The
combined organic layers were washed with 0.5 N HCl and
brine and dried (MgSO4). After concentration in vacuo, final
purification by column chromatography (hexane/EtOAc, 7:3)
gave compound 18 (1.50 g, 4.29 mmol, 90%) as a white
solid: 1H NMR δ 8.52 (d, J ) 8.6 Hz, 1H), 8.15 (d, J )
9.2 Hz, 1H), 8.13 (dd, J ) 7.5, 1.1 Hz, 1H), 7.79 (d, J ) 7.6 Hz,
1H), 7.52 (dd, J ) 9.2, 7.5 Hz, 1H), 7.49 (dd, J ) 8.6, 7.5 Hz,
1H), 6.94 (br s, 1H), 2.76 (s, 6H), 1.51 (s, 9H); 13C NMR δ
153.6, 133.6, 133.2, 130.3, 129.6, 128.6, 127.7, 127.3, 124.0,
122.2, 121.3, 80.9, 37.3 (2C), 28.3 (3C). Anal. Calcd for
C17H22N2O4S: C, 58.27; H 6.33; N, 7.99. Found: C, 57.93;
H 6.24; N, 7.88.
5-Amino-N,N-dimethyl-1-naphthalenesulfonamide (19).
To a solution of compound 18 (2.87 g, 8.91 mmol) in HOAc (11
mL) was added 30% HBr/HOAc (22 mL) at 0 °C. The reaction
mixture was stirred for 20 min and then allowed to warm to
rt and stand for 40 min. The reaction mixture was diluted with
ether to obtain a white precipitate, which was collected by
filtration and crystallized from ethanol/ether to generate com-
pound 19 (2.92 g, 99%) as a white solid: 1H NMR (CD3OD) δ
8.34 (d, J ) 8.5 Hz, 1H), 8.12 (dd, J ) 7.5, 1.0 Hz, 1H), 8.05
(d, J ) 8.7 Hz, 1H), 7.50 (dd, J ) 8.5, 7.5 Hz, 1H), 7.39 (dd,
J ) 8.7, 7.5 Hz, 1H), 6.91 (dd, J ) 7.5, 1.0 Hz, 1H), 2.78 (s,
6H); 13C NMR (CD3OD) δ 145.3, 132.9, 130.5, 130.4, 128.9,
128.5, 125.1, 122.4, 114.7, 110.4, 36.9 (2C). Anal. Calcd for
C12H14N2O2S: C, 57.58; H 5.64; N, 11.19. Found: C, 57.58; H
5.64; N, 11.03.
5-Amino-4-chloro-N,N-dimethyl-1-naphthalenesulfona-
mide (20). Compound 18 (153 mg, 0.44 mmol) was treated
with 6 N HCl in dioxane (1.3 mL) for 1 h at rt. The reaction
was quenched by addition of 1 N NaOH and extracted with
CH2Cl2. The combined organic layers were washed with water
and brine and dried (MgSO4). After removal of solvent under
reduced pressure, the product was purified by column chro-
matography (CHCl3/EtOAC, 9:1) to afford compound 19 (74
mg, 68%) and the byproduct 20 (6 mg, 5%) as yellow oils. For
compound 20: 1H NMR δ 8.16 (d, J ) 7.3 Hz, 1H), 8.13 (d, J
) 9.3 Hz, 1H), 8.05 (d, J ) 8.6 Hz, 1H), 7.51 (dd, J ) 8.6, 7.3
Hz, 1H), 7.49 (d, J ) 9.3 Hz, 1H), 4.69 (br s, 2H, exchanges
with D2O), 2.80 (s, 6H); 13C NMR δ 138.9, 133.2, 130.2, 129.0,
128.5, 126.8, 124.8, 123.7, 116.0, 115.2, 37.4 (2C). Anal. Calcd
for C12H13N2O2SCl: C, 50.62; H 4.60; N, 9.84; Cl, 12.45.
Found: C, 50.56; H 4.55; N, 9.76; Cl, 12.69.
Pyrophosphate 5. Tris(tetra-n-butylammonium) hydrogen
pyrophosphate (3.32 g, 3.68 mmol) was dissolved in CH3CN
(11 mL), compound 13 in CH3CN (2 mL) was added, and the
resulting mixture was stirred for 3 h at rt. After concentration
in vacuo, the resulting residue was dissolved in ion exchange
buffer (1/49 v/v, isopropyl alcohol and 25 mM NH4HCO3) and
loaded onto a column (2.5 × 8.6 cm) containing 140 mequiv of
cation-exchange resin (100-200 mesh 1.7 mequiv/mL). The
eluant was collected, frozen, and lyophilized to give compound
5 as a white solid. The initial product was purified by C18
reverse phase column chromatography (25 mM NH4HCO3/
CH3CN, 10:0 to 4:6) to afford pyrophosphate 5 (422 mg, 30%
from compound 12) as a white solid: 1H NMR (400 MHz, D2O)
δ 7.81 (d, J ) 7.6 Hz, 1H), 7.29 (dd, J ) 8.3, 7.4 Hz, 1H), 6.63
(d, J ) 8.3 Hz, 1H), 6.58 (dd, J ) 7.6, 7.4 Hz, 1H), 5.40 (br s,
1H), 5.29 (br s, 1H), 4.44 (br s, 2H), 3.80 (s, 3H), 3.63 (s, 2H),
2.14-2.10 (m, 2H), 2.01-1.99 (m, 2H), 1.66 (s, 3H), 1.57 (s,
3H); 13C NMR (100 MHz, D2O) δ 170.3, 151.1, 142.7, 135.2,
132.3, 131.9, 125.8, 120.2 (d, JCP ) 8.7 Hz), 115.7, 113, 110.4,
62.8 (d, JCP ) 3.9 Hz), 52.2, 50.0, 39.0, 25.9, 16.0, 14.0; 31P
NMR (120 MHz, D2O) δ -8.6 (d, JPP ) 20.0 Hz, 1P), -10.8 (d,
JPP ) 20.0 Hz, 1P); HRMS (FAB) calcd for C18H28NO9P2 (M -
3NH4 + H)2- 464.1239, found 464.1247.
+
5-[[(1,1-Dimethylethoxy)carbonyl]amino]-1-naphtha-
lenesulfonic Acid, Triethylamine Salt (16). To a solution
of 5-aminonaphthalenesulfonic acid (15, 2.43 g, 10.9 mmol) and
triethylamine (1.74 mL, 12.5 mmol) in anhydrous methanol
(21 mL) was added di-tert-butyl dicarbonate (5.51 mL, 24.0
mmol). The reaction mixture was stirred for 36 h at rt and
then concentrated by rotary evaporation. The residue was
dissolved in water (100 mL) and washed with EtOAc. After
the remaining organic solvent was removed in vacuo, the
aqueous solution was frozen in a dry ice/acetone bath and
lyophilized to afford compound 16 (4.48 g, 97%) as a light pink
Pyrophosphate 27. To a solution of (NBu4)3HP2O7 (6.98
g, 7.74 mmol) in CH3CN (16 mL) was added by syringe a
solution of allylic chloride 25 in CH3CN (3.4 mL), and the
reaction mixture was stirred at rt for 3 h. The solvent was
removed in vacuo, and the resulting residue was dissolved in
an ion- exchange buffer (1/49 v/v, isopropyl alcohol and 25 mM
NH4HCO3) and then loaded onto a column (2.5 × 16 cm)
containing 262 mequiv of a cation-exchange resin (100-200
mesh). The eluant was frozen and lyophilized to give compound
27 as a yellow solid. Final purification by column chromatog-
8192 J. Org. Chem., Vol. 69, No. 24, 2004

Fluorescent Isoprenoid Pyrophosphate Analogues
raphy (cellulose powder) and C18 reversed-phase column
chromatography (25 mM NH4HCO3/CH3CN, 6:4) gave the
pyrophosphate 27 (926 mg, 39% from compound 24) as a yellow
solid: 1H NMR (ND4OD/D2O) δ 8.56 (d, J ) 8.1 Hz, 1H), 8.33
(d, J ) 7.6 Hz, 1H), 8.22 (d, J ) 8.1 Hz, 1H), 7.70 (dd, J ) 8.1,
7.8 Hz, 1H), 7.62 (dd, J ) 8.1, 7.6 Hz, 1H), 6.77 (d, J ) 7.8 Hz,
1H), 5.72 (t, J ) 6.6 Hz, 1H), 5.34 (br s, 1H), 4.83 (m, 2H),
3.92 (s, 2H), 2.93 (s, 6H), 2.31-2.25 (m, 2H), 2.19-2.16 (m,
2H), 1.94 (s, 3H), 1.83 (s, 3H); 13C NMR (ND4OD/D2O) δ 147.8,
144.5, 135.0, 134.7, 133.1, 132.8, 132.3, 130.5, 128.4, 127.2,
125.6, 123.5 (d, JCP ) 9.7 Hz), 115.6, 108.7, 65.3 (d, JCP ) 4.5
Hz), 53.8, 41.9, 40.0 (2C), 29.0, 18.9, 17.0; 31P NMR (D2O) δ
-6.0 (1P, d, JPP) 22.0 Hz), -9.8 (1P, d, JPP ) 22.0 Hz); HRMS
(FAB) calcd for C22H31N2O9P2S (M - 3NH4+ + 2H)- 561.1226,
found 561.1206.
Sulfonamide 28. Dichlorotriphenylphosphorane (3.10 g,
9.30 mmol) and compound 17 (2.01 g, 4.73) were dissolved in
CH2Cl2 (100 mL), and the solution was stirred for 1.5 h at r.
The solution then was cooled to 0 °C in an ice bath, and NEt3
was added dropwise. After the reaction mixture was stirred
for 10 min, it was allowed to warm to rt and then allowed to
stir for 2 h. The reaction mixture was diluted with water and
extracted with CH2Cl2. The combined organic layers were
washed with 0.5 N HCl and brine and diluted with hexane to
precipitate triphenylphosphine oxide as a white solid. The
precipitate was removed by filtration, and the filtrate was
dried (MgSO4). After concentration in vacuo, final purification
by column chromatography (hexane/EtOAc, 8:2) gave the
desired product 28 (1.45 g, 91%) as a white solid: 1H NMR
(CD3OD) δ 8.56 (dt, J ) 8.3, 1.0 Hz, 1H), 8.35 (dt, J ) 8.6, 1.0
Hz, 1H), 8.24 (dd, J ) 7.5, 1.1 Hz, 1H), 7.74-7.62 (m, 3H),
2.49 (s, 3H), 1.57 (s, 9H); 13C NMR (CD3OD) δ 156.8, 136.2,
136.1, 131.2, 130.9, 130.5, 129.7, 128.9, 125.3, 124.1, 123.4,
81.6, 29.1, 28.8 (3C); HRMS (FAB) calcd for C16H20N2O4NaS
(M + Na)+ 359.1041, found 359.1055.
the solvent was removed under reduced pressure, the resulting
residue was dissolved in an ion-exchange buffer (1/49 v/v,
isopropyl alcohol and 25 mM NH4HCO3) and loaded onto a
column (2 × 5 cm) containing 26 mequiv of a cation-exchange
resin (100-200 mesh 1.7 mequiv/mL). The eluant was col-
lected, frozen, and lyophilized to give compound 36 as a yellow
solid. The initial product was purified by C18 reversed-phase
column chromatography (25 mM NH4HCO3/CH3CN, 7:3) to
afford the pyrophosphate 36 (20 mg, 18% from compound 33)
as a yellow solid: 1H NMR (ND4OD/D2O) δ 8.89 (d, J ) 8.7
Hz, 1H), 8.58 (d, J ) 7.3 Hz, 1H), 8.21 (d, J ) 8.8 Hz, 1H),
8.07-7.96 (m, 2H), 7.25 (d, J ) 7.9 Hz, 1H), 6.17 (t, J ) 6.6
Hz, 1H), 5.25 (t, J ) 6.5 Hz, 2H), 4.97 (t, J ) 6.5 Hz, 1H), 4.37
(s, 2H), 3.65-3.58 (m, 2H), 3.32 (s, 3H), 2.60-2.2.53 (m, 2H),
2.23 (s, 3H), 1.88 (s, 3H), 1.87 (s, 3H); 13C NMR (ND4OD/D2O)
δ 147.9, 140.7, 138.5, 135.7, 133.2, 132.7, 132.4, 131.0, 127.6,
126.1, 124.8 (d, JCP ) 9.2 Hz), 123.2, 115.9, 109.6, 65.2 (d, JCP
) 5.0 Hz), 53.5, 52.4, 36.5, 28.8, 28.0, 20.0, 17.3; 31P NMR (D2O)
δ -5.3 (1P, d, JPP) 22.5 Hz), -9.4 (1P, d, JPP ) 22.5 Hz);
HRMS (FAB) calcd for C22H31N2O9P2S (M - 3NH4 + 2H)-
+
561.1226, found 561.1211.
Cell Culture. RPMI-8402 cells57 were cultured at 37 °C in
the presence of 5% CO2 in RPMI-1640 media containing 10%
heat-inactivated fetal bovine serum.
Enzyme Assays. FTase assays were performed using a
method modified from Harwood.58 The reaction mixture con-
tained Tris (50 mM, pH7.5), DTT (5 mM), ZnCl2 (20 µM), MgCl2
(5 mM), H-Ras (4 µM), FPP (500 nM), and bovine brain
homogenate (30 µg) containing partially purified FTase.59,60
[3H]-Thymidine Assay. RPMI cells (2 × 105 cells/200 µL)
were incubated in a 96-well plate with varying concentrations
of test compounds for a total of 24 h. During the last four hrs,
cells were labeled with [3H]-thymidine (0.04 µCi/well). Cells
were harvested, and the amount of [3H]-thymidine incorpo-
rated into cellular DNA was determined by scintillation
spectrophotometry as previously described.61
Sulfonamide 29. A mixture of compound 28 (405 mg,
1.20 mmol) and NaH (60% dispersion in mineral oil; 86.4 mg,
2.16 mmol) was suspended in DMF (3.3 mL) and stirred for
40 min at 60 °C. 5-Bromo-2-methyl-2-pentene (0.4 mL, 2.99
mmol) was added slowly, and the reaction mixture was stirred
for 40 min at 60 °C. After the reaction mixture was allowed to
cool to rt, it was diluted with water and extracted with
chloroform, and the combined organic layers were dried
(MgSO4). After concentration in vacuo, purification of the
residue by column chromatography (hexane/EtOAc, 8:2) af-
forded compound 29 as a colorless oil (274 mg, 55%): 1H NMR
δ 8.51 (d, J ) 8.7 Hz, 1H), 8.21-8.16 (m, 2H), 7.86 (d, J )
7.4 Hz, 1H), 7.63-7.52 (m, 2H), 6.85 (s, 1H, exchanges with
D2O), 4.98-4.93 (m, 1H), 3.17 (t, J ) 7.4 Hz, 2H), 2.84 (s, 3H),
2.24-2.17 (m, 2H), 1.61 (s, 3H), 1.55 (s, 12H); 13C NMR δ 153.8,
135.1, 134.6, 133.8, 130.1, 129.7, 128.7, 128.0, 127.2, 124.3,
122.4, 121.3, 120.0, 81.2, 49.6, 34.3, 28.5 (3C), 26.9, 25.8, 17.9;
HRMS (FAB) calcd for C22H31N2O4S (M + H)+ 419.2005, found
419.1986.
Sulfonamide 30. A solution of compound 29 in HOAc (0.8
mL) was treated with 30% HBr/HOAc (1.6 mL) at 0 °C. The
reaction mixture was stirred for 5 min at 0 °C and then allowed
to warm to rt and stand for 20 min. The reaction mixture was
diluted with ether to precipitate the product. The white
precipitate was collected by filtration, and the crude compound
was crystallized from ethanol/ether to afford compound 30 (251
mg, 100%) as a white solid: 1H NMR δ 8.20 (m, 3H), 7.53 (m,
2H), 7.03 (d, J ) 6.9 Hz, 1H), 4.96 (t, J ) 6.3 Hz, 1H), 3.22-
3.17 (m, 2H), 2.83 (s, 3H), 2.24-2.19 (m, 2H), 1.65 (s, 3H), 1.60
(s, 3H); 13C NMR δ 134.3, 134.7, 134.5, 130.2, 130.1, 129.1,
128.6, 127.1, 125.2, 124.1, 123.4, 120.1, 50.2, 36.6, 26.9, 25.8,
17.9; HRMS (FAB) calcd for C17H23N2O2S (M + H)+ 319.1480,
found 319.1484.
Fluorescence Microscopy. Following incubation of cells
with the test compounds for 3 or 18 h, cells were examined
and photographed using a fluorescence microscope with a CCD
camera.
Western Blotting. Cells (5 × 106 cells/5 mL) were incu-
bated for 24 h in the presence or absence of test compounds
and lovastatin. Western blotting was performed as previously
described.57
Acknowledgment. We thank Prof. Lei Geng and
Gufang Wang for their assistance in obtaining the
fluorescence spectra, Chris Wohlford-Lenane and the
University of Iowa Central Microscopy Research Facility
for their assistance with the fluorescence microscopy,
and the University of Iowa Mass Spectrometry Facility.
This project was supported by the Roy J. Carver
Charitable Trust as a Research Program of Excellence
and the Roland W. Holden Family Program for Experi-
mental Cancer Therapeutics.
Supporting Information Available: General experimen-
tal protocols; the 1H, 13C, and (where relevant) 31P NMR
spectra for compounds 5, 11, 12, 19, 23, 24, 27, 29, 33, and
36; and the experimental procedures for preparation of com-
pounds 22-25 and 32-34. This material is available free of
charge via the Internet at http://pubs.acs.org.
JO049101W
(57) Hohl, R. J.; Lewis, K. J. Biol. Chem. 1995, 270, 17508-17512.
(58) Harwood, H. J., Jr. Anal. Biochem. 1995, 226, 268-278.
(59) Moores, S. L.; Schaber, M. D.; Mosser, S. D.; Rands, E.; O’Hara,
M. B.; Garsky, V. M.; Marshall, M. S.; Pompliano, D. L.; Gibbs, J. B.
J. Biol. Chem. 1991, 266, 14603-14610.
(60) Khan, S. G.; Mukhtar, H.; Agarwal, R. J. Biochem. Biophys.
Methods 1995, 30, 133-144.
(61) Larson, R. A.; Yachnin, S. Blood 1984, 64, 257-262.
Pyrophosphate 36. (NBu4)3HPO7 (264 mg, 0.29 mmol) was
dissolved in CH3CN (0.5 mL) at rt. Compound 34 in CH3CN
(0.5 mL) was added, and the resulting mixture was stirred
for 3 h at rt, when the milky suspension became clear. After
J. Org. Chem, Vol. 69, No. 24, 2004 8193