Solid-phase synthesis of prenylcysteine analogs

Article abstract of DOI:10.1021/jo8021692

Prenylcysteine derivatives are of interest for a variety of different biological reasons, including probing the CaaX protein processing pathway. A solid-phase synthesis protocol for the preparation of prenylcysteines using 2-chlorotrityl chloride resin as

Full text of DOI:10.1021/jo8021692

Solid-Phase Synthesis of Prenylcysteine Analogs
James L. Donelson,† Heather B. Hodges-Loaiza,‡ Brian S. Henriksen,†
Christine A. Hrycyna,‡ and Richard A. Gibbs*,†
Department of Medicinal Chemistry and Molecular Pharmacology, Department of Chemistry, and
the Purdue Cancer Center, Purdue UniVersity, West Lafayette, Indiana 47907
rgibbs@purdue.edu
ReceiVed October 14, 2008
Prenylcysteine derivatives are of interest for a variety of different biological reasons, including probing
the CaaX protein processing pathway. A solid-phase synthesis protocol for the preparation of
prenylcysteines using 2-chlorotrityl chloride resin as a solid support has been developed. A series of
novel amide-modified farnesylcysteine analogs were synthesized in both high purity and yield under
mild conditions. The farnesylcysteine analogs were evaluated using human isoprenylcysteine carboxyl
methyltransferase as a biological target, and several new inhibitors, one with significantly enhanced potency,
were identified.
Introduction
Many proteins contain a C-terminal sequence motif called a
CaaX box, where C is cysteine, aa are generally aliphatic
residues, and X can be one of several amino acid residues.1
This motif signals many of these proteins for a series of at least
three post-translational modifications (Figure 1). The first step
is attachment of a 15-carbon farnesyl or 20-carbon geranylgera-
nyl group to the CaaX cysteine by either protein farnesyltrans-
ferase or protein geranylgeranyltransferase I, respectively.
Following prenylation, the last three amino acids are removed
proteolytically by the enzyme Rce1, and last, the newly exposed
R-carboxyl group is methylated by the enzyme isoprenylcysteine
carboxyl methyltransferase (Icmt).2 Recent estimates suggest
that approximately 120 human proteins are farnesylated or
monogeranylgeranylated, including the Ras family of oncopro-
teins.3 Importantly, mutations in the ras oncogene are respon-
sible for approximately 15-20% of all human cancers, leading
to intense interest in inhibitors of the CaaX processing pathway
as potential anticancer agents.4
FIGURE 1. Post-translational processing of CaaX proteins.
Because of the biological importance of isoprenylated pro-
teins,1 there have been numerous efforts to develop chemical
tools to interrogate their function.5 In particular, prenylcysteine
analogs6 and prenylated peptides7 have proven to be valuable
agents for biological studies. We are interested in developing
(4) Basso, A. D.; Kirshmeyer, P. T.; Bishop, W. R. J. Lipid Res. 2006, 46,
15–31.
(5) Krzysiak, A. J.; Rawat, D. S.; Scott, S. A.; Pais, J. E.; Handley, M.;
Harrison, M. L.; Fierke, C. A.; Gibbs, R. A. ACS Chem. Biol. 2007, 2, 385–
389.
(6) Kloog, Y.; Cox, A. D. Semin. Cancer Biol. 2004, 14, 253–261.
(7) Watzke, A.; Brunsveld, L.; Durek, T.; Alexandrov, K.; Rak, A.; Goody,
R. S.; Waldmann, H. Org. Biol. Chem. 2005, 3, 1157–1164.
† Department of Medicinal Chemistry and Molecular Pharmacology and the
Purdue Cancer Center.
‡ Department of Chemistry and the Purdue Cancer Center.
(1) McTaggert, S. J. Cell. Mol. Life Sci. 2006, 63, 255–267.
(2) Winter-Vann, A. M.; Casey, P. J. Nat. ReV. Cancer 2005, 5, 405–411.
(3) Reid, T. S.; Terry, K. L.; Casey, P. J.; Beese, L. S. J. Mol. Biol. 2004,
343, 417–423.
10.1021/jo8021692 CCC: $40.75
Published on Web 03/25/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 2975–2981 2975

Donelson et al.
CHART 1. Amide-Modified Farnesylcysteine Analogs Synthesized on the 2-Chlorotritylchloride Solid Support
prenylcysteine analogs as potential Icmt inhibitors. Icmt is a
novel potential chemotherapeutic target, and inhibitors of Icmt
are thus of particular interest as potential cancer chemothera-
peutic agents.8 Recently, the Casey laboratory has reported that
an Icmt inhibitor, cysmethynil, identified from a compound
library, exhibits activity against cancer cells.9
Early studies on the synthesis of prenylated peptides focused
on the solid-phase synthesis of nonprenylated peptides followed
by the selective prenylation of a free cysteine residue in
solution.10 Fragment condensation routes have also been em-
ployed.11 Other groups have developed methods for the solid-
phase synthesis of prenylated peptides on a variety of linkers
such as oxime,12 PTMSEL,13 and sulfonamide.14 However, we
wished to develop a method that would allow the on-resin
attachment of prenyl groups in order to synthesize diverse
libraries of prenylcysteine derivatives. Here we report both the
development of such a method and its use to prepare a library
of prenylcysteine analogs. One of these analogs exhibits
significantly enhanced inhibitory potency against human Icmt.
AMFCs based on the previous library and the adamantyl lead
(Chart 1) was targeted for synthesis initially.
The solid-phase synthesis of farnesylcysteine derivatives is
complicated by the acid lability of the allylic thioether bond
connecting cysteine to the prenyl group. It has been shown that
the solid-phase synthesis of lipidated peptides requires mild,
nonacidic cleavage conditions. In 2002, the Waldmann group
reported an elegant solid-phase method for the synthesis of
prenylated peptides terminating in a methyl ester on a hydrazine
resin.16 Unfortunately, this method did not work well in our
hands for the generation of peptides that end in a free
carboxylate, a necessary feature for this class of Icmt inhibitors.
Subsequently, Waldmann also reported problems arising from
radical side reactions that occurred during cleavage from the
hydrazine resin.14 Thus, for the purposes of synthesizing the
desired prenylcysteine library, a new approach to the preparation
of prenylcysteine derivatives on solid phase was developed.
The 2-chlorotrityl chloride resin has proven to be a versatile
and useful tool for the synthesis of a variety of peptides,17,18
with certain specific advantages over other solid supports. First,
because of the SN1 mechanism of the loading step, attaching
the cysteine to the chlorotrityl resin does not require activation
of the cysteine carboxyl group, thereby decreasing the likelihood
of racemization. Second, the cleavage conditions are very mild
and result in the rapid, quantitative release of the modified
cysteine from the bead. Third, the resin is commercially
available and relatively inexpensive.
Results and Discussion
We have previously reported the solution-phase synthesis of
a 23-member library of amide-modified farnesylcysteine deriva-
tives (AMFCs).15 This initial library was screened against Icmt,
and six inhibitors possessing bulky aromatic and aliphatic
carboxylic acids attached to the R-nitrogen of the cysteine were
identified. The farnesyl cysteine analog containing an N-1-
adamantylcarbonyl group, 4j, (Chart 1) was identified as the
most potent inhibitor of Icmt with an IC50 of 12 µM.15
Encouraged by these findings, a method was developed for the
solid-phase synthesis of prenylcysteine analogs to facilitate the
synthesis of a more comprehensive library of AMFCs. A set of
Prior to beginning the synthesis of the AMFC set, several
steps of the proposed solid-phase synthetic route to prenylcys-
teine analogs required optimization. These included the loading
of the protected cysteine derivative, the deprotection of the
cysteine sulfur, and in particular the cleavage of the analog from
the resin. We found that using dithiothreitol as a reducing agent,
together with diisopropylamine, for cleavage of the cysteine
protecting group resulted in fewer side products than the
alternative procedure using tributylphosphine and water.19
Interestingly, when collidine was used as a base instead of
diisopropylethylamine, we observed no cleavage of the disulfide
bond.
(8) Bergo, M. O.; Gavino, B. J.; Hong, C.; Beigneux, A. P.; McMahon, M.;
Casey, P. J.; Young, S. G. J. Clin. InVest. 2004, 113, 539–550.
(9) Winter-Vann, A. M.; Baron, R. A.; Wong, W.; de la Cruz, J.; York, J. D.;
Gooden, D. M.; Bergo, M. O.; Young, S. G.; Toone, E. J.; Casey, P. J. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 4336–4341.
(10) Naider, F. R.; Becker, J. M. Biopolymers 1997, 43, 3–14.
(11) Xie, H.; Shao, Y.; Becker, J. M.; Naider, F.; Gibbs, R. A. J. Org. Chem.
2000, 65, 8552–8563.
(12) Dolence, E. K.; Dolence, J. M.; Poulter, C. D. J. Comb. Chem. 2000, 2,
522–536.
(13) Lumbierres, M.; Palomo, J. M.; Kragol, G.; Waldmann, H. Tetrahedron
Lett. 2006, 47, 2671–2674.
(14) Palomo, J. M.; Lumbrierres, M.; Waldmann, H. Angew. Chem., Int. Ed.
2006, 45, 477–481.
(15) Donelson, J. L.; Hodges, H. B.; MacDougall, D. D.; Henriksen, B. S.;
Hrycyna, C. A.; Gibbs, R. A. Bioorg. Med. Chem. Lett. 2006, 16, 4420–4423.
(16) Ludolph, B.; Eisele, F.; Waldmann, H. J. Am. Chem. Soc. 2002, 124,
5954–5955.
(17) Sohma, Y.; Hayashi, Y.; Kimura, M.; Chiyomori, Y.; Taniguchi, A.;
Sasaki, M.; Kimura, T.; Kiso, Y. J. Pept. Sci. 2005, 11, 441–451.
(18) Kitagawa, K.; Aida, C.; Fujiwara, H.; Yagami, T.; Futaki, S. Tetrahedron
Lett. 1997, 38, 599–602.
2976 J. Org. Chem. Vol. 74, No. 8, 2009

Synthesis of Prenylcysteine Analogs
SCHEME 1. Solid-Phase Synthesis of Amide-Modified Farnesylcysteines
TABLE 1. Inhibition Data for Selected AMFC Analogs
A number of methods have been reported for the cleavage
of compounds from 2-chlorotrityl resin.20,21 We compared a
variety of cleavage cocktails including 1:4 hexafluoroiso-
propanol/dichloromethane, 1:2:7 acetic acid/trifluoroethanol/
dichloromethane, 0.5:99.5 trifluoroacetic acid/dichloromethane,
1:99 trifluoroacetic acid/dichloromethane, and 3:97 trifluo-
roacetic acid/dichloromethane. Although none of these condi-
tions resulted in significant loss of the farnesyl group from
the cysteine, the 0.5:99.5 mixture of trifluoroacetic acid/
dichloromethane was chosen for subsequent use because of
its speed and nearly quantitative release of the AMFCs from
the resin. The 1:4 hexafluoroisopropanol/dichloromethane
conditions were attractive because of the essentially neutral
nature of this mixture, but it did not always result in efficient
release of the analog from the bead. Additionally, the higher
cost of hexafluoroisopropanol combined with the requirement
that it be used in higher proportion to TFA made this option
less desirable. While the 1:2:7 acetic acid/trifluoroethanol/
dichloromethane solution worked well, it was much slower
than the TFA solution, which required only 2-3 min for
complete product release.
The solid-phase synthetic route to prenylcysteine analogs is
outlined in Scheme 1. The commercial 2-chlorotritylchloride
resin is coupled to Fmoc-Cys(SStBu)-OH, using 1% collidine
in dichloromethane. Any unreacted trityl moieties were capped
as methoxides by addition of a 1% collidine/MeOH solution to
the reaction mixture. Reductive removal of the dithio-tert-butyl
protecting group from 1 with dithiothreitol was followed by
coupling of the desired farnesyl side chain to the free thiol, using
farnesyl chloride as the electrophile and collidine as the base
to afford intermediate 2. Next, the Fmoc protecting group was
removed with 20% piperidine/DMF. Coupling of the selected
carboxylic acid with the resulting free amine was accomplished
using HBTU/HOBt.22 The polymer-bound prenylcysteine analog
3 was then released from the resin using our optimized cleavage
compound
human Icmt IC50 (µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
18.8 ( 2.7
16.7 ( 2.6
nda
16.7 ( 3.6
15.6 ( 1.3
12.1 ( 2.1
nda
22.9 ( 2.5
nda
4j
12.4 ( 1.0
a nd: IC50 values not determined due to lack of activity in the initial
SPA24 screen.
conditions. Purification by either normal-phase chromatography
or C18 reversed-phase chromatography afforded prenylcysteine
analogs in purity suitable for characterization and biochemical
evaluation, in 45-65% yield based on resin loading.
The 10 prenylcysteine analogs 4a-j (Chart 1) were screened
against recombinant human Icmt23 using our recently developed
Icmt scintillation proximity assay.14,24 The carboxyl substituents
chosen were primarily bulky hydrophobic moieties similar to
the lead adamantyl compound 4j. The octanoyl derivative 4i is
an exception and was prepared as a control for the effect of
hydrophobic moieties on activity. Seven of the analogs, which
included our original lead 4j, resulted in a 50% or greater
decrease in Icmt activity in the SPA at a concentration of 50
µM. Three of the 10 compounds-the norbornyl analog 4c, the
2-napthyl analog 4g, and the octanoyl analog 4i-were inactive
in the SPA. The seven active analogs were then further evaluated
using a vapor diffusion assay (VDA)21 to determine IC50 values
(Table 1). It should be noted that the “hit” rate in this small
collection was significantly higher than in our original study of
AMFCs as Icmt inhibitors.12
None of the compounds were more potent than the lead
adamantyl analog 4j. However, the o-biphenyl analog 4f is
equivalent in potency (IC50 ) 12.1 µM), and the biphenyl
scaffold is significantly more amenable to derivatization than
the adamantyl scaffold. Note that the biological findings here
further confirm that the inhibitory activity of AMFCs does not
(19) (a) Ludolph, B.; Waldmann, H. Chem. Eur. J. 2003, 9, 3683–3691. (b)
Other workers have had difficulties with the tributylphosphine/water protocol
for cysteine disulfide deprotection: Rijkers, D. T. S.; John, A. W.; Kruijtzer,
J. A. W.; Killian, J. A.; Liskamp, R. M. J. Tetrahedron Lett. 2005, 46, 3341–
3345.
(20) Barlos, K.; Chatzi, O.; Gatos, D.; Stavroppoulos, G. Int. J. Pept. Protein
Res 1991, 37, 513–520.
(21) Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. J. Chem. Soc.,
Chem. Commun. 1994, 22, 2559–2560.
(22) Dourtoglou, V.; Bernard, G.; Lambropoulou, V.; Zioudrou, C. Synthesis
1984, 7, 572–574.
(23) Anderson, J. A.; Henriksen, B. S.; Gibbs, R. A.; Hrycyna, C. A. J. Biol.
Chem. 2005, 280, 29454–29461.
(24) Whyte, D., McGuirk, M., Nunez-Oliva, I., Hockenberry, T., Pai, J. Ras
ConVerting Endoprotease (RCE) and Methods, 2001, U.S. Patent 6,261,793.
J. Org. Chem. Vol. 74, No. 8, 2009 2977

Donelson et al.
CHART 2. Subsequent AMFC Library
correlate simply with the hydrophobicity of these compounds25
but is related to steric bulk.
pound containing both the 2-phenoxyphenyl N-substitution
and a farnesyl moiety known to lead to a modest inhibition
of Icmt23 may be a more potent Icmt inhibitor. The synthesis
of this “bidentate” potential inhibitor is shown in Scheme 2.
This compound exhibited no substrate activity and inhibited
Icmt with an IC50 of 2.5 µM, significantly more potent than
the parent o-phenoxyphenyl analog 5e (by ∼ 40%). This
initial finding suggests that disubstituted cysteines can have
increased affinity for Icmt and thus supports the further
investigation of “bidentate” prenylcysteine analog libraries.
An additional 16 analogs depicted in Chart 2, based
primarily on the biphenyl scaffold, were synthesized as shown
in Scheme 1. A preliminary screen indicated that only 5 of
the 16 analogs were Icmt substrates. The 11 non-substrates
were assayed for inhibitory activity using the VDA, and all
11 were found to inhibit Icmt by at least 30% at a
concentration of 10 µM. The four indicated compounds in
Chart 2 were clearly more potent than the o-biphenyl analog
4f. The o-phenoxy derivative (5e) inhibited the ability of Icmt
to methylate AFC by nearly 80% at 10 µM and is nearly
3-fold more potent (IC50 ) 4.3 µM) than the previous
adamantyl- or biphenyl-modified lead Icmt inhibitors. Eleven
of the 16 analogs evaluated in the compound library based
on the 2-biphenyl scaffold were inhibitors. This result
represents a substantial increase in both potency and hit rate
from the previous libraries.
In summary, a solid-phase method for the synthesis of
amide-modified farnesylcysteine analogs was developed using
the 2-chlorotritylchloride resin as a solid support. Using this
method, two libraries of AMFCs were synthesized and
evaluated as inhibitors of Icmt. The products were obtained
in 45-65% yield based on resin loading and in good purity
(>90%). The potential to split the prenylcysteine mixture and
introduce additional diversity in the penultimate step maxi-
mizes the efficiency of the process and greatly accelerates
the synthesis of the library. This procedure may also be more
broadly useful for the synthesis of other prenylcysteine and
prenylpeptide derivatives.
With a suitable lead compound (5e) identified, we next
determined if this protocol could be used to generate
prenylcysteine analogs modified on both the amide and the
prenyl moieties. It was hypothesized that a cysteine com-
Combining the data from the two solid-phase libraries and
the previously described AMFC library,15 trends in activity
begin to emerge. Flexible linkers between the amide group
and the R-group of the AMFCs tend to lead to Icmt
substrates; both compounds 5f and 5l are more efficient Icmt
(25) (a) Using data from our previously published set of AMFCs,15 we have
compared IC50 values for seven analogs with their calculated Log P values and
found no significant correlation (R2 ) 0.18). (b) We have not tested these
compounds versus FTase; however, note that the prenylcysteine peptide product
of this enzyme binds very poorly to it: Cassidy, P. B.; Poulter, C. D. J. Am.
Chem. Soc. 1996, 118, 8761–8762.
2978 J. Org. Chem. Vol. 74, No. 8, 2009

Synthesis of Prenylcysteine Analogs
SCHEME 2. Solid-Phase Synthesis of N,S-Disubstituted AMFC Analog 7
collidine (37 µL, 0.281 mmol). After 5 min the solvent was removed
substrates than the prototypical N-acetylfarnesylcysteine.
Conversely, AMFCs containing sterically bulky substituents
such as quaternary centers adjacent to the amide group tend
to lead to inhibitors of Icmt. The most potent AMFC inhibitor
was the 2-phenoxyphenyl compound 5e. It exhibits a 3-fold
increase in potency over that of the adamantyl and 2-biphenyl
compounds. The 2-anilinophenyl compound 5h was also a
potent analog indicating that a spacer between the two phenyl
rings confers increased potency to the 2-biphenyl scaffold
of Icmt inhibitors. With each of the three libraries synthe-
sized, both the inhibitor hit rate and the potency of the
inhibitors identified increased, indicating the effectiveness
of AMFCs as lead Icmt inhibitors. Note that the preparation
of the third AMFC library led to four compounds with single-
digit micromolar IC50’s, whereas the first two libraries
contained none.
from the resin, and the solution of Fmoc-Cys(StBu)-OH was
transferred to the resin. The mixture was gently agitated for 3 h on
a peptide shaker under argon. Then, 5 mL of a 1% solution of 2,4,6-
collidine in MeOH was then added, and the mixture was agitated
for an additional 10 min. The mixture was drained, and the resin
was washed 3× each with DMF, CH2Cl2, and DMF. The resin was
then dried under high vacuum overnight. By weight, an 80% loading
efficiency was achieved.
General Procedure for Reduction of -StBu Group. Dithio-
threitol (100 mg, 0.65mmol) was dissolved in a 2% diisopropyl-
ethylamine/DMF solution and added to the resin-bound cysteine,
and the reaction vessel was gently agitated overnight. The solvent
was drained, and the resin washed 3× each with DMF, CH2Cl2,
and DMF. A small aliquot of resin was removed, cleaved, and
subjected to reverse-phase HPLC analysis, which determined the
reaction to be complete.
General Procedure for Farnesylation of Resin-Bound Fmoc-
Cysteine. 2,4,6-Collidine (95 µL, 0.72 mmol) was added to a
solution of farnesyl chloride (0.36 mmol) in 4 mL of CH2Cl2. This
solution was added to the resin-bound cysteine under argon, and
the reaction vessel was gently agitated for 4 h at ambient
temperature. The solvent was then drained, and the resin was
washed as before. HPLC analysis following cleavage of a small
portion of resin determined the coupling to be quantitative.
Cleavage of N-Fmoc Group from Cysteine. A 4 mL portion
of a 20% solution of piperidine in DMF was added to the resin-
bound farnesylcysteine, and the vessel was agitated for 10 min.
The solvent was drained and rinsed once with DMF, and the process
was repeated. The solvent was drained, and the resin was rinsed as
before.
General Procedure for Coupling of Carboxylic Acids. 1-Ada-
mantaneacetic acid, hydroxybenzotriazole (HOBt), and HBTU (0.36
mmol of each) were dissolved in 4 mL of DMF, and 2,4,6-collidine
(0.40 mmol) was then added. After 5 min this solution was then
added to the resin and agitated for 3 h. The solvent was drained,
and the resin was rinsed as before.
We have synthesized >50 farnesylcysteines with amide
modifications and identified a lead Icmt inhibitor with an IC50
of 4.3 µM (5e). It was hypothesized that replacing the farnesyl
group with a synthetic farnesyl analog might further enhance
the potency of 5e. An amide-modified and farnesyl-modified
cysteine was synthesized to evaluate the effect of modifica-
tions at two positions on the cysteine. The chimeric AMFC
contained the 2-phenoxyphenylcarbonyl group on the nitrogen
and a previously identified p-biphenyl farnesyl mimic23 on
the sulfur of the cysteine. This compound inhibited Icmt with
an IC50 of 2.5 µM. This represents a nearly 40% increase in
potency over 5e, indicating that disubstituted cysteines
(modified on both the N and S) can be more effective
inhibitors than cysteine derivatives substituted on either the
N or S alone. In summary, we have developed a new solid-
phase route to prenylcysteine analogs and utilized it to
generate an effective Icmt inhibitor.
General Procedure for Cleavage of AMFC from Resin. The
resin-bound 4b was treated twice with a 0.5% solution of
trifluoroacetic acid in CH2Cl2 for 3 min. The solution was drained
into a round-bottom flask, and the resin was washed twice with
anhydrous CH2Cl2. In selected cases, the crude mixture was
analyzed by HPLC (C8 reversed-phase analytical columm; 1.0
mL/min flow rate; 90% A/10% B to 100% B over 20 min (A,
0.025% trifluoroacetic acid/water; B, acetonitrile)); the crude
Experimental Section
Solid-Phase Synthesis of AMFC Analogs. General Procedure
for Loading Cysteine onto Resin. 2-Chlorotrityl chloride resin
(103 mg, 0.114 mmol) was placed in a 25 mL fritted peptide vessel
under argon. To this was added 3 mL of anhydrous CH2Cl2. In a
separate vial, Fmoc-Cys(StBu)-OH (110 mg, 0.255 mmol) was
dissolved in 4 mL of anhydrous CH2Cl2 to which was added 2,4,6-
J. Org. Chem. Vol. 74, No. 8, 2009 2979

Donelson et al.
AMFC was obtained in good purity (70-90%). The solvent was
removed in vacuo, and the crude material was loaded directly
onto a silica column (mobile phase 0.5% MeOH, 0.5% AcOH,
99% CH2Cl2) and purified to give 28 mg of 4b, 65% (based on
starting cysteine derivative loaded onto the resin). Alternatively,
in certain cases, the AMFC was purified by C18 reversed-phase
sep pak syringe cartridges (Fisher Scientific), using a gradient
of 20:80 acetonitrile/water to 100% acetonitrile (with 0.5% (v/
v) trifluoroacetic acid).
N-1-Adamantylacetylcarbonyl-L-cysteine[S-(2E,6E)-3,7,11-tri-
methyldodeca-2,6,10-trienyl]-OH (4a). This compound was syn-
thesized in 65% yield following the general procedure for the solid-
phase synthesis of AMFCs.
N-3-Noradamantanecarbonyl-L-cysteine[S-(2E,6E)-3,7,11-tri-
methyldodeca-2,6,10-trienyl]-OH (4b). This compound was syn-
thesized in 58% yield following the general procedure for the solid-
phase synthesis of AMFCs.
N-2-((2R,4S)-Bicyclo[2.2.1]heptan-2-yl)acetyl-L-cysteine[S-
(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-OH (4c). This
compound was synthesized in 55% yield following the general
procedure for the solid-phase synthesis of AMFCs.
N-4-Pentylbicyclo[2.2.2]octane-1-carbonyl-L-cysteine[S-(2E,6E)-
3,7,11-trimethyldodeca-2,6,10-trienyl]-OH (4d). This compound
was synthesized in 60% yield following the general procedure for
the solid-phase synthesis of AMFCs.
N-(1S)-7,7-Dimethyl-2-oxobicyclo[2.2.1]heptane-1-carbonyl-
L-cysteine[S-(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-
OH (4e). This compound was synthesized in 62% yield following
the general procedure for the solid-phase synthesis of AMFCs.
N-2-Phenylbenzoyl-L-cysteine[S-(2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trienyl]-OH (4f). This compound was synthesized in 48%
yield following the general procedure for the solid-phase synthesis
of AMFCs.
N-1-Napthylacetylcarbonyl-L-cysteine[S-(2E,6E)-3,7,11-tri-
methyldodeca-2,6,10-trienyl]-OH (4g). This compound was syn-
thesized in 52% yield following the general procedure for the solid-
phase synthesis of AMFCs.
N-2-((1R,2S,5R)-2-Isopropyl-5-methylcyclohexyloxy)acetyl-L-
cysteine[S-(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-OH
(4h). This compound was synthesized in 65% yield following the
general procedure for the solid-phase synthesis of AMFCs.
N-Octylcarbonyl-L-cysteine[S-(2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trienyl]-OH (4i). This compound was synthesized in 62%
yield following the general procedure for the solid-phase synthesis
of AMFCs.
N-Adamantylcarbonyl-L-cysteine[S-(2E,6E)-3,7,11-trimethyl-
dodeca-2,6,10-trienyl]-OH (4j). This compound was synthesized
in 60% yield following the general procedure for the solid-phase
synthesis of AMFCs.
N-9-Fluorenylcarbonyl-L-cysteine[S-(2E,6E)-3,7,11-trimeth-
yldodeca-2,6,10-trienyl]-OH (5a). This compound was synthesized
in 47% yield following the general procedure for the solid-phase
synthesis of AMFCs.
N-9,10-Dihydro-9-oxoacridine-4-carbonyl-L-cysteine[S-(2E,6E)-
3,7,11-trimethyldodeca-2,6,10-trienyl]-OH (5b). This compound
was synthesized in 48% yield following the general procedure for
the solid-phase synthesis of AMFCs.
N-2-Phenyl-quinolinoyl-L-cysteine[S-(2E,6E)-3,7,11-trimeth-
yldodeca-2,6,10-trienyl]-OH (5c). This compound was synthesized
in 53% yield following the general procedure for the solid-phase
synthesis of AMFCs.
N-2-(4-Phenoxyphenyl)-benzoyl-L-cysteine[S-(2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trienyl]-OH (5d). This compound was
synthesized in 55% yield following the general procedure for the
solid-phase synthesis of AMFCs.
N-(2-Oxyphenyl)benzoyl-L-cysteine[S-(2E,6E)-3,7,11-trimeth-
yldodeca-2,6,10-trienyl]-OH (5e). This compound was synthesized
in 62% yield following the general procedure for the solid-phase
synthesis of AMFCs.
N-2-((S)-4-Phenyl-2-oxo-oxazolidin-3-yl)acetyl-L-cysteine[S-
(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-OH (5f). This
compound was synthesized in 60% yield following the general
procedure for the solid-phase synthesis of AMFCs.
N-3-Phenylbenzoyl-L-cysteine[S-(2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trienyl]-OH (5g). This compound was synthesized in 58%
yield following the general procedure for the solid-phase synthesis
of AMFCs.
N-2-(Aminophenyl)benzoyl-L-cysteine[S-(2E,6E)-3,7,11-tri-
methyldodeca-2,6,10-trienyl]-OH (5h). This compound was syn-
thesized in 47% yield following the general procedure for the solid-
phase synthesis of AMFCs.
N-3,5-Di-tert-butylbenzoyl-L-cysteine[S-(2E,6E)-3,7,11-tri-
methyldodeca-2,6,10-trienyl]-OH (5i). This compound was syn-
thesized in 55% yield following the general procedure for the solid-
phase synthesis of AMFCs.
N-1-(4-Chlorophenyl)cyclopentanecarbonyl-L-cysteine[S-
(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-OH (5j). This
compound was synthesized in 46% yield following the general
procedure for the solid-phase synthesis of AMFCs.
N-3,3-Diphenylpropanoyl-L-cysteine[S-(2E,6E)-3,7,11-tri-
methyldodeca-2,6,10-trienyl]-OH (5k). This compound was syn-
thesized in 65% yield following the general procedure for the solid-
phase synthesis of AMFCs.
N-3-(Phenylsulfonyl)propanoyl-L-cysteine[S-(2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trienyl]-OH (5l). This compound was
synthesized in 60% yield following the general procedure for the
solid-phase synthesis of AMFCs.
N-(R)-6-Hydroxy-2,5,7,8-tetramethylchroman-2-oyl-L-cys-
teine[S-(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-OH (5m).
This compound was synthesized in 47% yield following the general
procedure for the solid-phase synthesis of AMFCs.
N-4-Phenylbenzoyl-L-cysteine[S-(2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trienyl]-OH (5n). This compound was synthesized in 63%
yield following the general procedure for the solid-phase synthesis
of AMFCs.
N-4-Phenylazobenzoyl-L-cysteine[S-(2E,6E)-3,7,11-trimethyl-
dodeca-2,6,10-trienyl]-OH (5o). This compound was synthesized
in 57% yield following the general procedure for the solid-phase
synthesis of AMFCs.
N-4-Oxo-4H-chromene-2-carbonyl-L-cysteine[S-(2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trienyl]-OH (5p). This compound was
synthesized in 51% yield following the general procedure for the
solid-phase synthesis of AMFCs.
N-2-Phenoxybenzoyl-S-3-(3-methyl-but-2-enyl)-5-(4-biphenyl)pent-
2-enyl-L-cysteine (7). This compound was synthesized in 40% yield
following the general procedure for the solid phase synthesis of
AMFCs, with the exception that farnesyl chloride was replaced as
the prenylating reagent with the previously prepared bromide 8.23
1H NMR (500 MHz, CDCl3) δ 1.64 (s, 3 H), 1.69 (s, 3 H), 2.29
(app t, 2 H), 2.70 (app t, 2 H), 2.75 (d, 1 H, J ≈ 7 Hz), 2.95 (dd,
1H, J ≈ 14 Hz, 6.5 Hz), 3.00 (dd, 2 H, J ≈ 14 Hz, 5.2 Hz), 3.17
(m (complex AB pattern), 2 H), 4.98 (m, 2 H), 5.16 (t, 1 H, J ≈
7.5 Hz), 6.86 (d, 1 H, J ≈ 8.5 Hz), 7.10 (d, 2 H, J ≈ 8.0 Hz), 7.19
(m, 2 H), 7.21 (d, 2 H, J ≈ 8.0 Hz), 7.33 (t, 1 H, J ≈ 7.0 Hz), 7.37
(app t, 3 H), 7.43 (t, 2 H, J ≈ 8.0 Hz), 7.51 (d, 2 H, J ≈ 8.0 Hz),
7.58 (d, 2 H, J ≈ 7.0 Hz), 8.15 (bs, 1 H), 8.27 (d, 1 H, J ≈ 7.0
Hz), 8.75 (d, 1 H, J ≈ 7.0 Hz). 13C NMR (125 MHz, CDCl3) δ
17.9, 25.7, 29.2, 29.7, 32.6, 34.1, 38.6, 52.8, 117.9, 119.8, 120.1,
121.7, 122.4, 123.4, 124.8, 126.9, 127.0, 128.7, 128.7, 130.0, 132.6,
133.3, 138.7, 141.0, 141.1, 142.9, 155.1, 156.0, 165.4, 174.3. HRMS
for C38H39NO4SNa calcd 628.2497, found 628.2503.
Determination of IC50 Values. The compounds were assayed
for Icmt inhibition as previously described.23 Briefly, in a 60
µL reaction, yeast membranes expressing hIcmt (5 µg) were
incubated in 100 mM Tris-HCl in the presence of 25 µM AFC,
20 µM 14C-SAM and various concentrations of AMFC analog
dissolved in DMSO. The assay mixtures were mixed gently and
incubated for 30 min at 30 °C. Reactions were terminated with
2980 J. Org. Chem. Vol. 74, No. 8, 2009

Synthesis of Prenylcysteine Analogs
the addition of 50 µL of 1 M NaOH/1% SDS solution (50 µL).
The reaction mixture (100 µL) was spotted onto a folded piece
of filter paper. The filter paper was lodged in the neck of a
scintillation vial containing 10 mL of scintillation fluid. The vials
were capped and allowed to sit for 2.5 h to allow diffusion of
the [14C]methanol into the scintillation fluid. After 2.5 h, the
filter paper was removed, and the vials were shaken and then
counted in a scintillation counter. Icmt activity (% control) was
fitted versus AMFC concentration to determine the reported IC50
values.
Acknowledgment. This work was supported by the NIH
(RO1CA112483 (R.A.G.) and P30CA21368 (Purdue Cancer
Center Support Grant)).
Supporting Information Available: Synthetic experimental
details and spectroscopic characterization of 4a-j, 5a-p, and
7. This material is available free of charge via the Internet at
http://pubs.acs.org.
JO8021692
J. Org. Chem. Vol. 74, No. 8, 2009 2981