Design and in vivo analysis of potent non-thiol inhibitors of farnesyl protein transferase

Article abstract of DOI:10.1021/jm990080l

Inhibitors of farnesyl protein transferase (FPTase) based upon a pseudotripeptide template are described that comprise an imidazole group substituted with a hydrophobic substituent. (1,5)-Disubstitution of the imidazole group is shown to be the optimal ar

Full text of DOI:10.1021/jm990080l

3356
J . Med. Chem. 1999, 42, 3356-3368
Design a n d in Vivo An a lysis of P oten t Non -Th iol In h ibitor s of F a r n esyl P r otein
Tr a n sfer a se
Neville J . Anthony,*,† Robert P. Gomez,† Micheal D. Schaber, Scott D. Mosser, Kelly A. Hamilton,
Timothy J . O’Neil, Kenneth S. Koblan, Samuel L. Graham,† George D. Hartman,† Daksha Shah, Elaine Rands,
Nancy E. Kohl, J ackson B. Gibbs, and Allen I. Oliff‡
Departments of Medicinal Chemistry and Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486
Received February 18, 1999
Inhibitors of farnesyl protein transferase (FPTase) based upon a pseudotripeptide template
are described that comprise an imidazole group substituted with a hydrophobic substituent.
(1,5)-Disubstitution of the imidazole group is shown to be the optimal array that leads to potent
and selective inhibitors of FPTase. A variety of aryl and isoprenyl substituents are shown to
afford effective inhibitors, and the mechanism by which these compounds inhibit FPTase has
been investigated. The biochemical behavior of these compounds suggests that they bind to
FPTase at the site usually occupied by the protein substrate. In experiments in cell culture,
the methyl ester prodrugs of these inhibitors are cell permeant and potently inhibit the
posttranslational modification of H-Ras protein. Additionally, these molecules revert the
phenotype of ras transformed cells as evidenced by their ability to slow the growth of ras
transformed cell lines in soft agar. One of the inhibitors, as its methyl prodrug, was evaluated
in two in vivo models of tumor growth. The compound selectively inhibited the growth of tumors
derived from H-ras transformed cells, in nude mice, and caused the regression of preexisting
tumors in an H-ras transgenic animal model.
In tr od u ction
steady-state7 and pre-steady-state kinetic analysis.8,9
The reaction occurs via a functionally ordered mecha-
nism in which FPTase initially binds FPP to form a
binary complex. The enzyme subsequently recognizes
and binds its substrate protein through coordination of
the cysteinyl thiol group to an active site zinc atom.10
This interaction in conjunction with an attractive Cou-
lombic interaction between the substrates C-terminal
carboxylate and the guanidinium group from arginine
202 in the proteins â subunit11 are key determinants of
substrate recognition. Nucleophilic activation of the
thiol group occurs upon its coordination to the zinc atom,
and the transfer of the farnesyl residue proceeds from
the ternary complex. The reaction occurs with inversion
of configuration at the allylic carbon through a transi-
tion state that is thought to involve partial ionization
prior to carbon-sulfur bond formation.12,13 The rate-
limiting step in the overall reaction is the slow release
of the product from the enzyme, a process that is
catalyzed by an additional molecule of the FPP cosub-
strate.14 Recently the three-dimensional structure of
the FPTase‚FPP binary complex has been reported.15,16
This information may lead to a more complete under-
standing of the details of substrate recognition and
catalysis.
The current interest17-19 in obtaining inhibitors of
FPTase (FTIs) stems from the discovery that the protein
products of the ras oncogenes are substrates for FPTase.
Importantly, attachment of the farnesyl group to onco-
genic variants of Ras is essential for their ability to
transform cells. Treatment of ras transformed cells with
specific inhibitors of FPTase has been shown to inhibit
the farnesylation of Ras protein and revert the pheno-
type and morphology of ras transformed cell lines in
tissue culture.20,21 Additionally, FTIs have been shown
Approximately 20-30% of a wide variety of human
cancers including 50% of colon and over 90% of pancre-
atic cancers have been found to contain a mutated or
activated ras gene.1,2 This suggests that therapeutic
agents directed against the proteins encoded by these
genes might be effective anticancer agents.3,4 Ras
proteins are synthesized in the cell as cytosolic precursor
molecules that require a series of posttranslational
modifications to acquire full biological activity.5 The first
and obligatory of these posttranslational modifications
is isoprenylation by the enzyme farnesyl protein trans-
ferase (FPTase).
FPTase is a heterodimeric protein that is composed
of 48-kDa (R) and 46-kDa (â) subunits. The enzyme
catalyzes the transfer of the 15-carbon atom isoprenoid
chain, farnesyl, from the prenyl donor farnesyl diphos-
phate (FPP) to the thiol of a cysteine residue in a
substrate protein. The protein substrates for FPTase
contain the consensus sequence motif CAAX at their
carboxy terminus, where C is cysteine, A is generally
an aliphatic amino acid, and X is commonly methionine
or serine. The related enzyme geranylgeranyl protein
transferase type 1 (GGPTase 1) is comprised of the same
48-kDa R subunit and a unique â subunit. This enzyme
catalyzes the transfer of the 20-carbon isoprenoid gera-
nylgeranyl from geranylgeranyl diphosphate (GGPP) to
proteins that contain a CAAX motif in which the carboxy
terminal X residue is most commonly the branched
amino acid leucine.6
The mechanism of the FPTase enzyme-catalyzed
farnesylation reaction has been studied in detail by
† Department of Medicinal Chemistry.
‡ Current address: DuPont Pharmaceuticals Co., Rte 141 and Henry
Clay Rd, Wilmington, DE 19880.
10.1021/jm990080l CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/27/1999

Non-Thiol Inhibitors of FPTase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3357
Ch a r t 1. Pseudotetrapeptide Inhibitors of Farnesyl
Protein Transferase
Sch em e 1. Synthesis of Regioisomeric Benzylimidazole
Derivativesa
to slow the growth of tumors in xenograft experiments
in nude mice.22-24 Similarly, in studies involving trans-
genic animal models, FTIs have been demonstrated to
shrink preexisting tumors.25 While these studies have
suggested the potential utility of FTIs as anticancer
agents, evidence is accumulating that the mechanism
of FTI action is complex and may involve the combined
effect of inhibiting the farnesylation of a variety of
cellular proteins.26-30
The N-(naphthylmethyl)glycine pseudotripeptide tem-
plate 4 has been shown to function as an effective
platform for the discovery of potent cysteinyl,31 1 (Chart
1), and non-cysteinyl,32,33 2 and 3, inhibitors of FPTase.
Comparison of the structures of the cysteinyl inhibitor
1 with those of the imidazole and thioether derivatives
2 and 3 suggested that the thiol and imidazole19,34-39
functionalities share the common property of being
capable of acting as effective ligands to zinc. In contrast,
the thioether 3 lacks a putative zinc ligand and presum-
ably derives its affinity for FPTase from productive
hydrophobic interactions that the benzyl substituent
makes with suitable active site residues. This analysis
suggested that zinc ligation and occupancy of a nearby
hydrophobic binding site might be accomplished simul-
taneously by a single molecule bearing suitable append-
ages. In this paper we describe the synthesis and
biological properties of potent inhibitors of FPTase that
feature a substituted imidazole moiety that appears to
satisfy these requirements.
Ch em istr y
To elucidate which regioisomeric substitution of the
imidazole was most consistent with FPTase inhibitory
potency, substituted imidazoleacetic acid derivatives
were prepared, coupled to the previously described
amine 4,31 and screened for FPTase inhibitory activity.
Thus, imidazoleacetic acid methyl ester was treated
with sodium hydride and alkylated with benzyl bromide
to afford an inseparable (3:1) mixture of the regioiso-
meric benzyl derivatives 6 and 7 (Scheme 1). The major
isomer, the 1,4-substituted imidazole 6, resulted from
preferential alkylation of the least sterically hindered
nitrogen atom.
a
Reagents: (a) HCl, methanol; (b) NaH, PhCH2Br, DMF; (c)
HCl, H2O; (d) EDC, NEt3, HOOBT, 4; (e) LiOH, methanol, H2O.
amine 4 to afford the corresponding amides 8 and 9
which were separated by HPLC. The major isomer was
assigned structure 8 based upon appropriate nuclear
Overhauser effect experiments. Hydrolysis of the car-
boxy terminal methyl esters 8 and 9 afforded the
corresponding acids 10 and 11 in good yield. The ability
of the regioisomeric compounds 10 and 11 to inhibit
FPTase was assessed in an in vitro assay, and the
results are presented in Table 1.
Acidic hydrolysis of the methyl esters 6 and 7 occurred
cleanly to afford the corresponding acids. These acids
underwent efficient EDC coupling reactions with the

3358 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Anthony et al.
Ta ble 1. Dependence of FPTase Inhibitory Potency upon the
Imidazole Substitution Pattern
Sch em e 2. Synthesis of (1,5)-Disubstituted Imidazole
Derivativesa
FPTase IC50
(nM)a
FPTase IC50
(nM)b
R
compd
compd
C6H5CH2
4-C5H4NCH2
4-NO2C5H4CH2
10
12
14
82 ( 2 (2)
74 ( 26 (3)
10 ( 5 (3)
11
13
15
0.15 (1)
2.8 ( 0.8 (3)
0.16 ( 0.05 (3)
a
The concentration of compound required to inhibit 50% of
FPTase-catalyzed incorporation of [3H]FPP into recombinant
[Leu68]RAS1(term.)CVIM protein5 by 50%. Determined using
b
FPTase at a concentration of approximately 1 nM. Determined
using FPTase at a concentration of 10 pM. The assay results are
reported as a concentration ( SEM for the number of determina-
tions shown in parentheses. With one determination, the values
are estimated to be reliable to within 2-fold.
The 1,5-disubstituted imidazole isomer 11 was sig-
nificantly more potent than its regioisomer 10. Impor-
tantly, the benzylimidazole 11 was also more potent
than both the unsubstituted imidazole derivative 2
which has an IC50 of 6.2 nM32 and the benzyl thioether
3 which has an IC50 of 36 nM.33 To assess the generality
of the regiochemical dependence of FPTase inhibitory
potency, the corresponding 4-pyridylmethyl and 4-ni-
trobenzyl derivatives were subsequently prepared. Again
the 1,5-disubstituted imidazole derivatives 13 and 15
were found to have superior FPTase inhibitory activity.
These results are presented in Table 1.
a
Reagents: (a) TrBr, NEt3, DMF; (b) 4-CNC6H4CH2Br, aceto-
nitrile, 55 °C; (c) methanol, reflux; (d) LiOH, THF, H2O; (e) EDC,
NEt3, HOOBT, 4; (f) NaOH, methanol, H2O.
methanolysis. The desired imidazole derivatives could
then be isolated by column chromatography.42 We have
found this synthetic methodology best suited to the use
of activated halides. The chief limitation of the method
is that the intermediate imidazolium salts can undergo
premature loss of the trityl group during the time course
of the alkylation reaction. This can lead to the formation
of the corresponding bis-alkylated imidazolium salts.
The methyl ester 19 was saponified to the corresponding
imidazoleacetic acid derivative, and subsequent EDC
coupling with the amine 4 provided the prodrug methyl
ester 20 in good yield. Base-catalyzed hydrolysis of the
ester 20 to the corresponding carboxylic acid occurred
straightforwardly to afford the FTI 21.
Since the most potent isomer in each of these ex-
amples was derived from the minor product of the
imidazole alkylation reaction, our attention turned
toward securing a regioselective synthesis of the re-
quired 1,5-disubstituted imidazoles. This particular
array formally requires the alkylation of the more
sterically encumbered imidazole nitrogen atom. We,
therefore, adopted a blocking/alkylation strategy similar
to that used previously to prepare regioselectively
alkylated histidine derivatives.40,41 The synthetic route
is outlined in Scheme 2. This methodology has proven
suitable for the preparation of the benzylic and allylic
imidazole derivatives illustrated in Table 2. For ex-
ample, 4-imidazoleacetic acid methyl ester 16 was
protected in a highly regioselective manner with the
sterically demanding triphenylmethyl (trityl) group.
Subsequent alkylation of the imidazole, by heating the
tritylimidazole derivative 17 with 4-cyanobenzyl bro-
mide in acetonitrile, resulted in the formation of the
imidazolium salt 18. In this case, the intermediate
imidazolium salt 18 precipitated from the reaction
mixture and could be isolated in high purity by filtra-
tion. We have found that tritylimidazolium salts of this
general type are stable but chemically reactive species.
Upon warming the imidazolium salt 18 in methanol, the
molecule undergoes regiospecific solvolysis to liberate
the desired 1,5-disubstituted imidazole derivative 19,
as its hydrobromide salt. In cases where the requisite
imidazolium salt did not precipitate from the reaction
mixture, the crude imidazolium salt was subjected to
Str u ctu r e-Activity Rela tion sh ip s
The compounds prepared in this manner were char-
acterized as in vitro inhibitors of FPTase using purified
recombinant human FPTase at a concentration of 1 nM.
To more accurately determine the potency of the more
active compounds, the inhibitors were further tested in
a similar protocol that utilized the enzyme at a concen-
tration of 10 pM.31 The in vitro substrates used in the
enzyme inhibition assay were [1-3H]FPP and [Leu68]-
RAS1(term.)CVIM protein5. The inhibitory activities of
the compounds are presented in Table 2, as an IC50
value. This value corresponds to the concentration of
inhibitor required to reduce the incorporation of radio-
labeled isoprenoid into the Ras protein to one-half of
the level observed in the absence of inhibitor. The
compounds were similarly assayed against the closely
related enzyme GGPTase 1 using either bovine or, in
the case of more potent inhibitors, the recombinant
human enzyme.

Non-Thiol Inhibitors of FPTase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3359
Ta ble 2. In Vitro Activity of FPTase Inhibitors
soft agar MICh
FPTase IC50
GGPTase IC50
(nM)b
CTE
Raspro IC50
H-Ras
(µM)
Raf
(µM)
R
compd
(nM)a
compd
(µM)f
(µM)g
C6H5CH2
11
13
15
21
24
0.15 (1)
8200 (1)
ndc
9
22
23
10-25 (2)
50 (1)
10 (1)
2.5-10 (1)
1-10 (2)
0.1-1 (4)
1 (2)
10 (1)
1 (1)
1 (3)
1 (1)
1 (1)
1 (2)
1 (2)
2.5 (1) >10 (1)
1 (2)
2.5 (2)
1 (1)
10 (2)
4-C5H4NCH2
4-NO2C6H4CH2
4-CNC6H4CH2
4-FC6H4CH2
2.8 ( 0.8 (3)
0.16 ( 0.05 (3)
0.15 (1)
0.45 ( 0.02 (2)
0.55 ( 0.05 (2)
0.12 (1)
0.12 (1)
4.0 ( 0.5 (2)
0.41 (1)
>25 (1)
>10 (1)
>2.5 (3)
10 (1)
>10 (1)
10 (2)
92d (1)
67 ( 39d,e (4) 20
5000 (1)
2200 ( 1000 (2) 32
10000 (1)
10000 (1)
10-25 (2) e1 (9)
31
10 (1)
10 (1)
5-10 (2)
10 (1)
25 (1)
10 (1)
25 (1)
1-10 (1)
1 (1)
4-CH3OC6H4CH2 25
1-C10H7CH2
2-C10H7CH2
2-C9H6NCH2
geranyl
26
27
28
29
30
33
34
1-10 (2)
1 (3)
1-10 (1)
1-10 (1)
1-10 (1)
0.1-1 (1)
10 (2)
17500 ( 2000 (2) 35
10000 (1)
10000 (1)
3000 (1)
36
37
10 (2)
2.5-10 (2)
>10 (2)
farnesyl
2.8 ( 0.8 (3)
L-749,75022 1.8 ( 0.2 (7)
L-744,83225 >50 (2)
a
The concentration of compound required to inhibit 50% of FPTase-catalyzed incorporation of [3H]FPP into recombinant
[Leu68]RAS1(term.)CVIM protein5 by 50%. Determined using FPTase at a concentration of 10 pM. The assay results are reported as a
b
concentration ( SEM for the number of determinations shown in parentheses. The concentration of compound required to inhibit 50%
of GGPTase-catalyzed incorporation of [3H]GGPP into the recombinant human Ha-RasCVLL protein by 50%. Determined using GGPTase
from bovine brain. c nd, not determined. Determined using human recombinant GGPTase. e This compound did not inhibit the
d
posttranslational modification of the GGPTase substrate H-RasCVLL at a concentration of 10 µM in NIH 3T3 cells. f Highest nontoxic
g
concentration for cultured NIH 3T3 cells as assessed by MTT staining. Inhibition of posttranslational processing of v-Ras protein in
h
cultured NIH 3T3 cells. Minimum inhibitory concentration (MIC) required to achieve a reduction in size and number of colonies of
Rat-1 v-ras or Rat-1 v-raf transformed cells in soft agar relative to vehicle-treated control.
As previously stated, the 1,5-disubstituted imidazole
derivatives bearing the benzyl (11), 4-nitrobenzyl (15),
and 4-pyridylmethyl (13) imidazole substituents are
significantly more potent than their corresponding 1,4-
disubstituted isomers (compounds 10, 14, and 12; Table
1). It is interesting that the pyridylmethyl derivative
13 is significantly less active than the benzyl compound
11. This may indicate that these residues occupy a
hydrophobic region within the FPTase active site.
quinoline groups occupy a common site within the
FPTase enzyme.
To address the possibility that the benzylic imidazole
substituent may bind to FPTase by occupying the part
of the enzyme active site that binds FPP, the corre-
sponding farnesyl (30) and geranyl (29) derivatives were
prepared. These compounds were also found to be
selective inhibitors of FPTase. The geranyl derivative
was particularly potent.
To evaluate if the electronic nature of the benzylic
substituent was an important determinant of the po-
tency of this class of inhibitors, a series of benzylimi-
dazole derivatives was prepared in which the electronic
character of the para substituent was varied. Inspection
of Table 2 shows that FPTase inhibitory potency is
relatively insensitive to the electronic influence of this
substituent. In contrast, inhibition of GGPTase 1 was
most effectively achieved by compounds bearing the
strongly electron-deficient 4-nitrobenzyl (10) and 4-cy-
anobenzyl (2l) substituents. Despite this GGPTase
inhibitory activity, these compounds are still selective
inhibitors of FPTase (400-fold). Enlargement of the
imidazole substituent from benzyl to 1- or 2-naphthyl-
methyl was also tolerated by FPTase. The 1- and
2-naphthyl derivatives 27 and 28 are potent and selec-
tive inhibitors of the enzyme. In contrast, the 2-quino-
line derivative 28, which may be regarded as a hybrid
of the 2-naphthylmethyl 27 and the 4-pyridylmethyl 13,
was significantly less potent than the 2-naphthyl de-
rivative 27. This behavior parallels that seen in the
previous comparison of the benzyl and pyridylmethyl
derivatives 11 and 13 and suggests that the pyridyl and
To gain more detailed information about the mecha-
nism by which these compounds inhibit FPTase, the
4-cyanobenzyl derivative 21 was characterized by steady-
state enzyme kinetic analysis. A Lineweaver-Burk
analysis for compound 21 against the protein substrate
K-Ras is shown in Figure 1. The 4-cyanobenzylimidazole
derivative was found to be a competitive inhibitor
with respect to the protein substrate. The Ki for the
inhibitor 21 was 1.8 ( 0.1 nM in this assay. Additional
experiments suggest that the compound is not com-
petitive with respect to the isoprenoid substrate FPP.
These results indicate that the cyanobenzyl group
takes advantage of a binding site that is distinct from
that usually occupied by the isoprene donor FPP. A
similar kinetic analysis of the geranyl derivative 29
showed that this compound was similarly competitive
with protein substrate, was not competitive with FPP,
and therefore does not function as a product-like inhibi-
tor.43,44
Since the identity of the C-terminal residue is an
important determinant for the faithful discrimina-
tion of substrate proteins by FPTase and GGPTase
1, we evaluated the effects of replacing the C-terminal

3360 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Anthony et al.
The cytotoxic endpoint (CTE) defines the highest con-
centration of compound that is tolerated by NIH 3T3
cells in a 48-h assay. The practical significance of the
CTE value is that it defines a concentration of inhibitor
above which the effect the compound has on cells may
be ascribed to nonspecifc toxicity. For the compounds
presented in Table 2 the CTE value ranged from 5 to
50 µM.
To assess the ability of these compounds to inhibit
the posttranslational processing of Ha-Ras in NIH 3T3
cells, an assay based upon the difference in electro-
phoretic mobility of posttranslationally modified and
unmodified Ha-Ras protein on an SDS page gel was
utilized.21 Briefly, cells were incubated in the presense
of test compound or solvent control for 24 h, and the
newly synthesized proteins were labeled using [35S]-
methionine during the following 20 h. The cells were
lysed, and the total Ha-Ras protein was immunopre-
cipitated and subjected to SDS gel electrophoretic
separation. The intensity of the unprocessed and pro-
cessed Ha-Ras bands were then quantitated. An IC50
value in this assay is the concentration of inhibitor that
results in equal amounts of unfarnesylated and post-
translationally modified Ha-Ras. The results from this
assay are shown in Table 2. The HMG CoA reductase
inhibitor lovastatin was used as a positive control in this
assay, and at a concentration of 15 µM, lovastatin causes
>90% inhibition of Ha-Ras processing.
F igu r e 1. FPTase inhibitor 21 is competitive with K-Ras.
methionine residue of the 4-cyanobenzylimidazole
inhibitor 21 with leucine (Table 3). As anticipated,
this substitution causes a turnover in enzyme selectiv-
ity, with the leucinyl derivative 38 now being 4-5-fold
more potent against GGPTase 1 than FPTase. This
behavior is also consistent with the mechanistic clas-
sification of these inhibitors as being CAAX competi-
tive.
Inspection of the data obtained from this study shows
that the 4-nitrobenzyl (23), 4-cyanobenzyl (20), 4-meth-
oxybenzyl (32), and the 2-naphthyl (34) derivatives were
the most efficient inhibitors of Ha-Ras farnesylation in
whole cells. The compounds 23 and 20, bearing the
strongly electron-withdrawing nitro and cyano substit-
uents, were particularly effective. The remaining com-
pounds, while possessing significant activity in this
assay, were less potent with IC50 values in the 1-10
µM range. The efficacy in inhibiting Ha-Ras farnesyla-
tion in whole cells does not straightforwardly correspond
to the potency of the compounds in the in vitro enzyme
inhibition assay. Whether these differences are due to
the differential cell permeability of the compounds or
reflect differences in the cleavage efficacy of the prodrug
esters has not been determined.
The presence of a free carboxylate at the C-terminus
of substrate proteins is also an important determinant
in the recognition of substrate peptides by FPTase.11
This structural feature in a substrate protein appears
to be identified by FPTase through attractive Coulombic
interactions between the C-terminal carboxylate of the
substrate and the guanidine moiety of arginine 202 â
from the enzyme. To test the sensitivity of these
inhibitors to the absense of a free terminal carboxylate,
the carboxylate in 21 was replaced with an isosteric
C-terminal amide 40. In accord with expectation, this
substitution results in a 100-fold decrease in FPTase
inhibitory potency. However, the potency of this amide
derivative (IC50 15 nM) is quite comparable to that of
the carboxylate-containing inhibitors 2 and 3. This
finding is important as it indicates that the 4-cyanoben-
zylimidazole moiety may be incorporated into FPTase
inhibitors that may not require a prodrug strategy to
gain efficient entry into cells.45
Since the 4-cyanobenzyl derivative 21 has significant
GGPTase 1 activity in vitro (IC50 67 nM), the corre-
sponding methyl ester prodrug 20 of this compound was
further investigated to examine its ability to inhibit
GGPTase 1 in whole cells. For this assay, NIH 3T3 cells
expressing a chimeric H-Ras protein that has a CVLL
motif at its carboxy terminus was used. However
compound 20 at a concentration of 10 µM was unable
to significantly inhibit the geranylgeranylation of this
substrate. This result suggests that the effect prodrug
20 has on whole cells is due to its FPTase inhibitory
activity.
To test the facility with which these imidazole deriva-
tives inhibit FPTase in whole cells, the compounds were
examined in a series of cell-based assays. The methyl
ester prodrugs of the FTIs were used in these experi-
ments. While the ester prodrugs are intrinsically weak
inhibitors of FPTase, they appear to be significantly
more cell penetrant than their acid counterparts (see
Table 3, entries 20 and 21). Upon entry into the cell,
the prodrug esters can be hydrolyzed to their corre-
sponding active acid by the action of intracellular
esterases.
One of the phenotypical properties that cells acquire
upon transformation with an oncogenic ras gene is an
ability to grow in an anchorage-independent manner in
soft agar. The ester prodrugs were therefore evaluated
for their ability to inhibit the growth of Rat-1 cells
transformed by H-ras in this medium.21 A minimum
inhibitory concentration (MIC) in this assay is the
lowest concentration of test compound that causes a
The cytotoxicity of the prodrug methyl esters of the
FPTase inhibitors was assessed using a viable staining
assay with MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyl-2H-tetrazolium bromide.21 Cells were incubated
with the test compound over a range of concentrations.

Non-Thiol Inhibitors of FPTase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3361
Ta ble 3. In Vitro Effects of Carboxy Terminal Modifications
soft agar MICg
R
compd
FPTase IC50 (nM)a
0.15 (1)
GGPTase IC50 (nM)b
CTE (µM)e
g50 (1)
Raspro IC50 (µM)f
H-Ras (µM)
2.5-10
Raf (µM)
21
67 ( 39d (4)
10
nd
20
38
ndc
430
nd
10-25 (2)
e1 (9)
1 (3)
nd
>2.5 (3)
90d (1)
nd
nd
nd
39
40
nd
nd
nd
10 (1)
25 (1)
nd
10 (1)
10 (1)
>10 (1)
13 (1)
10 (1)
25 (1)
a
The concentration of compound required to inhibit 50% of FPTase-catalyzed incorporation of [3H]FPP into recombinant[Leu68]-
RAS1(term.)CVIM protein by 50%. Determined using FPTase at a concentration of 10 pM. The assay results are reported as a concentration
b
( SEM for the number of determinations shown in parentheses. The concentration of compound required to inhibit 50% of GGPTase-
catalyzed incorporation of [3H]GGPP into the recombinant [Leu68]RAS1(term.)CVLL protein by 50%. Determined using GGPTase from
d
bovine brain. c nd, not determined. Determined using human recombinant GGPTase. e Highest nontoxic concentration for cultured NIH
g
3T3 cells as assessed by MTT staining. f Inhibition of posttranslational processing of v-Ras protein in cultured NIH 3T3 cells. Minimum
inhibitory concentration (MIC) required to achieve a reduction in size and number of colonies of Rat-1 v-ras or Rat-1 v-raf transformed
cells in soft agar relative to vehicle-treated control.
reduction in the number and size of colonies of trans-
formed cells in soft agar, relative to control cells that
were treated with vehicle. To evaluate the compound’s
ability to specifically inhibit H-Ras-induced cell growth,
the ester prodrugs were also tested for their affect on
the growth of v-raf transformed Rat-1 cells. The under-
lying mechanistic rationale for this counterscreen stems
from the fact that Raf is a downstream effector of Ras
in the signal transduction pathway and can transform
cells independently of Ras.46 The results of this study
are shown in Table 2.
All of the compounds tested in these assays were able
to selectively inhibit the growth of H-Ras transformed
Rat-1 cells. Somewhat surprisingly, there is not a strong
correspondence between the ability of the compounds
to inhibit H-Ras processing in NIH 3T3 cells and their
growth inhibitory effect on H-Ras transformed Rat-1
cells in soft agar. For example, the 4-nitrobenzyl (23)
and 4-cyanobenzyl (20) derivatives inhibit H-Ras far-
nesylation at 0.1-1 µM, whereas the unsubstituted
benzyl (11) derivative requires concentrations of 2.5-
10 µM to be as effective; yet these compounds have
similar potency in the soft agar assay (MIC 1 µM). This
discrepancy may be due to differences between the
cell lines used in the two experiments, or it may indi-
cate that other farnesylated proteins play a significant
role in determining the phenotype of transformed
cells.47
It should be noted that these compounds are some-
what more potent than the prototypical thiol inhibitor
1 which has an MIC in the soft agar assay of 2.5-5
µM.31 Thus, it appears that FPTase inhibitors that
contain a suitably substituted imidazole moiety can
have improved cell-based potency over their thiol coun-
terparts.
Previous reports from these laboratories have dem-
onstrated that the thiol FTI (Table 2) L-744,832 is
efficacious in slowing the growth of tumors derived from
H-ras transformed cells in nude mice.22 Due to its in
vitro potency, favorable tissue distribution,48 and suit-
able physical properties, the non-thiol prodrug 20 was
selected for examination in a similar nude mouse
xenograft study. Briefly, 36 athymic mice were divided
into three treatment groups and innoculated subcutane-
ously with 106 Rat-1 cells transformed with either Zip
H1a-ras or mos 3a2 oncogenes. The following day one
group of 12 mice received 40 mpk subcutaneously49 b.i.d.
of the imidazole prodrug 20, and one group received
vehicle alone (phosphate-buffered saline) once daily. As
a positive control in this assay, the thiol prodrug L-744,-
832 was administered 40 mpk subcutaneously once
daily to the remaining 12 animals. After 12 and 14 days
the animals that received the mos and ras transformed
cells, respectively, were sacrificed and the tumors were
excised and weighed. The efficacy of the two compounds
was measured by comparing the average tumor weight

3362 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Anthony et al.
Ta ble 5. Efficacy of the FPTase Inhibitor 20 in MMTV
Ta ble 4. Effect of the FPTase Inhibitor 20 on the Growth of
Tumors in Nude Micea
v-Ha-ras Transgenic Micec
% difference
(relative to vehicle)
14-day MGR
(mm3/day)
average 14-day MGR
(mm3/day)
average tumor weight (g)
animal no.
Rat-1
Rat-1
Rat-1
Vehicle Groupa
17.1
26.0
16.2
compdb,c,d
ZipH1ae
mos3a2f
ZipH1ag mos3a2
1
2
3
19.8 ( 3.1
vehicle
20
0.302 ( 0.032 0.563 ( 0.050
0.171 ( 0.032 0.534 ( 0.069
L-744,832 0.020 ( 0.007 0.475 ( 0.058
-43
-93
-5
-16
Compound 20b Group
4
5
6
7
-10.2
-5.5
-4.7
1.3
a
12 mice were randomly assigned to each vehicle or treatment
-4.8 ( 2.4
b
group. The vehicle phosphate-buffered saline was administered
by sc injection of a 0.1-mL solution once daily. c Mice received 40
d
mkd of compound 20 by sc injection twice daily. Mice received
40 mkd of L-744,832 by sc injection once daily. e Rat-1 ZipH1a
tumors were harvested after 14 days of dosing. f Rat-1 mos3a2
a
b
10% Ethanol-30% polypropylene glycol. Mice received 120
mkd of compound 20 by sc injection once daily. c Tumor volume
was calculated twice weekly from caliper measurements according
to the approximate formula for a prolate ellipsoid: (W2 × L)/2,
where W and L are in millimeters and L g W. The area under the
curve (AUC) for a particular tumor was calculated according to
the formula: (vol1 - vol2)/2 × (day2 - day1). The mean growth
rate was calculated according to the formula: [(sumAUC) - (vol1
× (dayn - day1))]/(dayn - day1)2.
g
tumors were harvested after 12 days of dosing. Statistical
significance of the difference between the average tumor weight
in the vehicle- and drug-treated groups was evaluated using
Students one-sided t-test (P < 0.01).
from the drug-treated groups of animals with that of
the average tumor weight from the animals that re-
ceived the vehicle. The results from this experiment are
tabulated in Table 4.
MGR of -5.4 ( 1.9 mm3/day following its daily admin-
istration at a dose of 40 mpk.25
As reported previously22 the thiol prodrug L-744,832
was highly effective in inhibiting the growth of H-ras
tumors, showing a 93% reduction in tumor mass com-
pared to vehicle and having little effect (-16%) on the
control tumors that derived from implantation of mos
transformed cells. The non-thiol prodrug 20 was also
effective at slowing the growth of the H-ras tumors,
causing a 43% reduction in the average tumor size.
Again, growth of the mos tumors was not significantly
affected (-5%). No overt toxic effects due the compounds
were noted except for some local scarring at the site of
drug administration.
The tumors that develop in a nude mouse xenograft
experiment are localized and well-encapsulated and are
not wholly reminiscent of those found in naturally
arising cancers. In contrast tumors that arise spontane-
ously in transgenic animals exhibit a pathology that
is more similar to that encountered clinically. Pre-
vious reports from these laboratories with the thiol
inhibitor L-744,832 have demonstrated that this com-
pound caused regression of spontaneously arising tu-
mors in an Ha-ras transgenic mouse model. It was
therefore of interest to evaluate the non-thiol agent 20
in this system.25
Mice harboring the viral Ha-ras oncogene under the
control of the mouse mammary tumor virus long ter-
minal repeat (MMTV) were calipered periodically and
entered into study when a primary tumor of ap-
proximately 100 mm3 was detected. The animals were
divided into two groups: one received daily subcutane-
ous injections of the vehicle (10% EtOH, 30% propylene
glycol), and the other received 120 mpk of compound
20 in the same vehicle, subcutaneously, once a day. The
tumors were measured and a mean daily growth rate
(MGR) calculated. The results of this study are pre-
sented in Table 5. After 14 days of treatment the growth
of the primary tumors in the vehicle group had an
average MGR of +19.8 mm3/day. In contrast, the tumors
in the animals that were receiving the imidzole deriva-
tive 20 shrank in size with an average MGR of -4.1
mm3/day. This result is similar to that seen with the
prototypical thiol inhibitor L-744,832, which had an
Con clu sion
It has been demonstrated that an imidazole, in
combination with a suitable hydrophobic substituent,
may be successfully combined to obtain effective re-
placements for the cysteinyl moiety in the FTI 1. This
finding may be of value since there is some concern that
non-mechanism-based side effects may occur upon
chronic administration of a FPTase inhibitor that
contains a thiol moiety. The successful union of the
imidazole and its hydrophobic substituent is highly
dependent upon the substitution pattern of the imida-
zole moiety. The 1,5-disubstituted imidazole has been
shown to be the optimal array that leads to potent and
selective inhibitors of FPTase. A variety of aryl and
isoprenyl substituents have been shown to afford effec-
tive inhibitors that bind to FPTase at the site usually
occupied by the protein substrate. In experiments in cell
culture, the methyl ester prodrugs of these inhibitors
have been shown to be cell permeant and to potently
inhibit the posttranslational modification of H-Ras
protein. Additionally, these molecules are able to revert
the phenotype of ras transformed cells as evidenced by
their ability to slow the growth of ras transformed cell
lines in soft agar. On the basis of these findings, the
methyl prodrug 20 was evaluated in two in vivo models
of tumor growth. This compound is able to selectively
inhibit the growth of tumors derived from H-ras trans-
formed cells in nude mice at doses that did not affect
the growth of mos transformed xenografts. Similarly,
the prodrug 20 was shown to cause the regression of
preexisting tumors in an H-ras transgenic animal
model. The efficacy of this compound in these models
approaches that of the prototypical thiol inhibitor
L-744,832.
These results demonstrate that an imidazole moiety
in conjunction with a suitably positioned 4-cyanobenzyl
group can function as a highly effective cysteine re-
placement within the N-arylalkyl pseudopeptide class
of FTIs. This new pharmacophore may be of value in
the development of farnesyltransferase inhibitors as
chemotherapeutic agents.

Non-Thiol Inhibitors of FPTase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3363
methyl)-1H-imidazol-4-ylacetic acid hydrochloride and 1-(phe-
nylmethyl)-1H-imidazol-5-ylacetic acid hydrochloride (3:1
mixture, 115 mg, 0.455 mmol), 2(S)-[2-({2(S)-amino-3(S)-
methylpentyl}naphthalen-1-ylmethylamino)acetylamino]-4-
(methylsulfanyl)butyric acid methyl ester bis-hydrochloride (4)
(244 mg, 0.455 mmol), 3-hydroxy-1,2,3-benzotriazin-4(3H)-one
(HOOBT) (74 mg, 0.46 mmol), and triethylamine (0.19 mL,
1.36 mmol) in DMF (5 mL) was added 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) (87 mg, 0.455
mmol), and the solution stirred at room temperature overnight.
The reaction was quenched with saturated aq sodium bicar-
bonate (20 mL) and water (25 mL) and extracted into EtOAc
(2 × 50 mL). The combined organic extracts were washed with
brine (5 mL), and the solvent was evaporated in vacuo. The
residue was purified by flash chromatography (2-5% MeOH/
CH2Cl2 gradient elution) to provide the amides 8 and 9 as a
glass (3:1 mixture (237 mg, 79%)). For use in biological assays
these isomers were separated by preparative HPLC (Nova
Prep 5000 Semipreparative HPLC system and a Waters
PrepPak cartridge, 47 × 300 mm, C18, 15 µm, 100A) eluting
with 5-95% acetonitrile/water (0.1% TFA) at 100 mL/min) to
give after lyophilization pure 8 (121 mg) and 9 (21 mg) as the
bis-trifluoroacetate salts. 8: 1H NMR (CD3OD, 400 MHz) δ
8.95 (1H, s), 8.27 (1H, m), 7.96 (2H, m), 7.68 (1H, d), 7.60-
7.37 (9H, m), 5.38 (2H, s), 5.0-4.8 (1H, m), 4.52 (1H, t, J )
10.6 Hz), 4.42 (1H, dd, J ) 4.0 and 6.6 Hz), 4.14 (1H, m), 3.92
(1H, d, J ) 13.3 Hz), 3.83 (1H, d, J ) 13.3 Hz), 3.70 (1H, s),
3.64 (1H, m), 3.54 (2H, m), 3.22 (1H, dd, J ) 7.0 and 8.0 Hz),
2.37 (1H, m), 2.10 (1H, m), 2.00 (3H, s), 1.98 (1H, m), 1.79
(1H, m), 1.58 (1H, m), 1.42 (1H, m), 1.17 (1H, m) and 0.90
(6H, m) ppm; FAB HRMS exact mass calcd for C37H48N5O4S
658.342702 (MH+), found 658.341278. Anal. (C37H47N5O4S‚
3.0TFA‚0.15H2O) C,H,N. 9: 1H NMR (CD3OD, 400 MHz) δ
8.80 (1H, s), 8.26 (1H, m), 7.89 (2H, m), 7.66-7.24 (8H, m),
7.21 (2H, s), 5.36 (2H, m), 4.37 (3H, m), 4.09 (1H,br s), 3.66
(3H, s), 3.56 (3H, m), 3.50-2.90 (3H, m), 2.27 (1H, br s), 2.20
(1H, br s), 1.96 (3H, s), 1.90 (1H, br s), 1.68 (1H, br s), 1.58
(1H, br s), 1.40 (1H, m), 1.18 (1H, m) and 0.89 (6H, m) ppm;
FAB HRMS exact mass calcd for C37H48N5O4S 658.342702
(MH+), found 658.343754. Anal. (C37H47N5O4S‚1.85TFA‚
0.10H2O) C,H,N.
Exp er im en ta l Section
Gen er a l Meth od s. Proton NMR spectra were run at 400
MHz on a varian Unity 400 or VRX-400 spectrometer, and
chemical shifts are reported in parts per million (δ) downfield
from tetramethylsilane as internal standard. Fast atom bom-
bardment mass spectra were recorded on a VG-ZAB-HF
spectrometer using glycerol as matrix. Elemental analyses
were performed using a Perkin-Elmer 2400 model II elemental
analyzer. Silica gel 60 (230-400) mesh from EM Science was
used for column chromatography, and analytical thin-layer
chromatography was conducted using EM Science Kieselgel
60 F254 plates. Preparative HPLC was performed on a Septech
Novaprep 5000 with either a C-18Vydac or PrepPak reverse-
phase column. For reactions performed under anhydrous
conditions, glassware was either oven- or flame-dried and the
reaction was run under a positive pressure of argon. Solvents
and reagents were obtained from commercial sources and used
without furthur purification. The reported yields are the actual
isolated yields of purified material and are not optimized. The
following examples are illustrative of the procedures employed.
P r ep a r a tion of N-[2(S)-(1-(P h en ylm eth yl)-1H-im id a -
zol-4-yla cetyl)a m in o-3(S)-m eth ylp en tyl]-N-(1-n a p h th yl-
m eth yl)glycylm eth ion in e Bis-tr iflu or oa ceta te Sa lt (10)
a n d N-[2(S)-(1-(P h en ylm eth yl)-1H-im id a zol-5-yla cetyl)-
a m in o-3(S)-m eth ylp en tyl]-N-(1-n a p h th ylm eth yl)glycyl-
m eth ion in e Bis-tr iflu or oa ceta te Sa lt (11). P r ep a r a tion
of 1H-Im id a zole-4-a cetic Acid Meth yl Ester Hyd r och lo-
r id e. A solution of 1H-imidazole-4-acetic acid hydrochloride
(3.89 g, 23.9 mmol) in methanol (100 mL) was saturated with
hydrogen chloride gas. The solution was allowed to stand for
18 h at room temperature, and then the solvent evaporated
in vacuo to give the title compound (4.20 g,100%) as a white
solid: 1H NMR (CDCl3, 400 MHz) δ 8.85 (1H, s), 7.45 (1H, s),
3.89 (2H, s) and 3.75 (3H, s) ppm.
P r ep a r a tion of 1-(P h en ylm eth yl)-1H-im id a zol-4-yla ce-
tic Acid Meth yl Ester (6) a n d 1-(P h en ylm eth yl)-1H-
im id a zol-5-yla cetic Acid Meth yl Ester (7) (3:1 m ixtu r e).
To a solution of sodium hydride (37.3 mg, 1.56 mmol) in
dimethylformamide (DMF) (2 mL) at 0 °C was added, via
cannula, a solution of 1H-imidazole-4-acetic acid methyl ester
hydrochloride (115 mg, 0.71 mmol) in DMF (3 mL). The
reaction was stirred at 0 °C for 15 min. To the resulting
suspension was added benzyl bromide (0.084 mL, 0.71 mmol),
and the mixture was stirred at room temperature for 2 h. The
reaction was quenched with saturated aqueous sodium bicar-
bonate (15 mL) and water (20 mL) and extracted into CH2Cl2
(2 × 50 mL). The combined organic extracts were washed with
brine (20 mL) and dried (MgSO4), and the solvent was
evaporated in vacuo. The residue was purified by flash
chromatography (eluting with acetonitrile) to afford an in-
separable 3:1 mixture of 6 and 7 (106 mg, 65%) as an oil: 1H
NMR (CDCl3, 400 MHz) δ 7.53 (0.25H, s), 7.48 (0.75H, s), 7.35
(3H,m), 7.18 (1.5H, d, J ) 7.4 Hz), 7.06 (0.5H, d, J ) 7.2 Hz),
7.00 (0.25H, s), 6.87 (0.75H, s), 5.16 (0.5H, s), 5.08 (1.5H, s),
3.72 (1.5H, s), 3.65 (2.25H, s), 3.63 (0.75H, s) and 3.48 (0.5H,
s) ppm.
P r ep a r a tion of 2(S)-[2-({2(S)-[2-(1-Ben zyl-1H-im id a zol-
4-yl)a cet yla m in o]-3(S)-m et h ylp en t yl}n a p h t h a len -1-yl-
m et h yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic
Acid Bis-tr iflu or oa ceta te (10) a n d 2(S)-[2-({2(S)-[2-(3-
Ben zyl-3H-im idazol-4-yl)acetylam in o]-3(S)-m eth ylpen tyl}-
n a p h t h a len -1-ylm et h yla m in o)a cet yla m in o]-4-(m et h yl-
su lfa n yl)bu tyr ic Acid Bis-tr iflu or oa ceta te (11). To a
solution of 8 and 9 (2:1 mixture, 50 mg, 0.057 mmol) in
methanol (5 mL) at room temperature was added lithium
hydroxide (0.57 mL of a 1 M aq solution, 0.547 mmol), and
the reaction was stirred for 4 h. After this time, the reaction
was quenched by the addition of trifluoroacetic acid (to pH <3),
and the solvent was evaporated in vacuo. The residue was
purified by preparative HPLC (Nova Prep 5000 Semiprepara-
tive HPLC system and a Waters PrepPak cartridge, 47 × 300
mm, C18, 15 µm, 100A) eluting with 5-95% acetonitrile/water
(0.1% TFA) at 100 mL/min) to give after lyophilization pure
10 (29 mg, 87%) and 11 (12 mg, 72%) as the bis-trifluoroacetate
salts. 10: 1H NMR (CD3OD, 400 MHz) δ 8.83 (1H, s), 8.21 (1H,
d, J ) 9.5 Hz), 7.88 (2H, m), 7.54 (1H, d, J ) 6.9 Hz), 7.50-
7.30 (9H, m), 5.32 (2H, s), 4.56 (1H, br d, J ) 10.0 Hz), 4.36
(2H, m), 4.09 (1H, m), 3.55 (4H, m), 3.17 (1H, br d, J ) 10
Hz), 2.98 (1H, t, J ) 10.0 Hz), 2.29 (1H, m), 2.18 (1H, m), 1.96
(1H, m), 1.95 (3H, s), 1.67 (1H, m), 1.56 (1H, m), 1.37 (1H, m),
1.11 (1H, m) and 0.88 (6H, m) ppm; FAB HRMS exact mass
calcd for C36H46N5O4S 644.327052 (MH+), found 644.326691.
Anal. (C36H45N5O4S‚2.15TFA) C,H,N. 11: 1H NMR (CD3OD,
400 MHz) δ 8.80 (1H, s), 8.29 (1H, m), 7.92 (2H, m), 7.61 (1H,
br), 7.53-7.32 (7H, m), 7.21 (2H, br s), 5.37 (2H, s), 4.37 (2H,
m), 4.08 (1H, m), 3.57 (4H, br m), 3.05 (2H, m), 2.29 (2H, m),
2.20 (1H, m), 1.96 (3H, s), 1.70 (1H, m), 1.62 (1H, m), 1.57
(1H, m), 1.39 (1H, m), 1.13 (1H, m) and 0.88 (6H, m) ppm;
P r ep a r a tion of 1-(P h en ylm eth yl)-1H-im id a zol-4-yla ce-
tic Acid Hyd r och lor id e a n d 1-(P h en ylm eth yl)-1H-im i-
d a zol-5-yla cetic Acid Hyd r och lor id e (3:1 m ixtu r e). A
solution of 6 and 7 (3:1 mixture, 106 mg, 0.046 mmol) in HCl
(3 mL of a 1 M aq solution) was heated at 45 °C for 4 h. The
solution was evaporated in vacuo to afford an inseparable 3:1
mixture of the title compounds (120 mg, 100%) as a glass: 1H
NMR (DMSO-d6, 400 MHz) δ 9.26 (0.75H, s), 9.23 (0.25H, s),
7.60 (0.25H, m), 7.58 (0.75H, s), 7.45-7.26 (5H, m), 5.43 (0.5H,
s), 5.41 (0.5H, s), 3.77 (1.5H, s), 3.75 (0.5H, s) ppm.
P r ep a r a tion of 2(S)-[2-({2(S)-[2-(1-Ben zyl-1H-im id a zol-
4-yl)a c e t yla m in o ]-3(S )-m e t h y lp e n t yl}n a p h t h a le n -1-
y lm e t h y la m i n o )a c e t y la m i n o ]-4-(m e t h y ls u lfa n y l)-
bu tyr ic Acid Meth yl Ester Bis-tr iflu or oa ceta te (8) a n d
2(S )-[2(S )-({2-[2-(3-B e n zy l-3H -im id a zo l-4-y l)a c e t y l-
a m in o]-3-m et h ylp en t yl}n a p h t h a len -1-ylm et h yla m in o)-
a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic Acid Met h yl
E st er Bis-t r iflu or oa cet a t e 9. To a solution of 1-(phenyl-

3364 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Anthony et al.
FAB HRMS exact mass calcd for C36H46N5O4S 644.327052
(MH+), found 644.327917.
1.96 (3H, s), 1.9 (1H, m), 1.67 (1H, m), 1.57 (1H, m), 1.42 (1H,
m), 1.15 (1H, m), 0.89 (6H, m) ppm; FAB HRMS exact mass
calcd for C36H45N6O6S 689.31213 (MH+), found 689.31135.
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(1-p yr id in -4-ylm eth yl-1H-
im id a zol-4-yl)a cetyla m in o]p en tyl}n a p h th a len -1-ylm eth -
yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic a cid
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(3-p yr id in -4-ylm eth yl-3H-
im id a zol-4-yl)a cetyla m in o]p en tyl}n a p h th a len -1-ylm eth -
yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic a cid
1
m eth yl ester tr is-tr iflu or oa ceta te: H NMR (CD3OD, 400
1
MHz) δ 8.99 (1H, s), 8.65 (2H, d, J ) 4.9 Hz), 8.28 (1H, d, J )
9.4 Hz), 7.91 (2H, m), 7.69 (1H, d, J ) 6.5 Hz), 7.61-7.44 (6H,
m), 5.59 (2H, s), 4.90 (1H, m), 4.68 (1H, d, J ) 13.4 Hz), 4.42
(1H, m), 4.16 (1H, m), 3.90 (1H, d, J ) 15.6 Hz), 3.82 (1H, d,
J ) 15.6 Hz), 3.75-3.55 (2H, m), 3.69 (3H, s), 3.50 (1H, d, J )
13.1 Hz), 3.20 (1H, m), 2.37 (1H, m), 2.29 (1H, m), 1.99 (3H,
s), 1.96 (1H, m), 1.77 (1H, m), 1.58 (1H, m), 1.23 (1H, m), 1.19
(1H, m), and 0.91 (6H, m) ppm; FAB HRMS exact mass calcd
for C36H47N6O4S 659.337951 (MH+), found 659.336943. Anal.
(C36H46N6O4S‚4.95TFA‚2.2H2O) C,H,N.
m eth yl ester (22): H NMR (CD3OD, 400 MHz) δ 9.01 (1H,
s), 8.63 (2H, m), 8.28 (1H, m), 7.98 (2H, m), 7.70 (1H, d, J )
6.0 Hz), 7.52 (4H, m), 7.41 (2H, d, J ) 6.2 Hz), 5.62 (2H, s),
4.94 (1H, m), 4.72 (1H, m), 4.42 (1H, m), 4.07 (1H, m), 3.89
(2H, m), 3.68 (1H,m), 3.69 (3H, s), 3.55 (2H, m), 3.24 (1H, m),
2.39 (1H, m), 2.31 (1H, m), 2.00 (3H, s), 1.98 (1H, m), 1.79
(1H, m), 1.58 (1H, m), 1.42 (1H, m), 1.18 (1H, m), and 0.91
(6H, m) ppm; FAB HRMS exact mass calcd for C36H47N6O4S
659.337951 (MH+), found 659.336826.
2(S )-{2-[(3(S )-Me t h yl-2(S )-{2-[3-(4-n it r ob e n zyl)-3H -
im id a zol-4-yl]a cetyla m in o}p en tyl)n a p h th a len -1-ylm eth -
yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic a cid
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(1-p yr id in -4-ylm eth yl-1H-
im id a zol-4-yl)a cetyla m in o]p en tyl}n a p h th a len -1-ylm eth -
ylam in o)acetylam in o]-4-(m eth ylsu lfan yl)bu tyr ic acid tr is-
tr iflu or oa ceta te (12): 1H NMR (CD3OD, 400 MHz) δ 8.96
(1H, s), 8.55 (2H, d, J ) 5.2 Hz), 8.21 (1H, d, J ) 7.2 Hz), 7.97
(2H, m), 7.69 (1H, d, J ) 7.2 Hz), 7.60-7.40 (6H, m), 5.58
(2H, s), 4.91 (1H, d, J ) 13.2 Hz), 4.69 (1H, d, J ) 13.2 Hz),
4.38 (1H, dd, J ) 4.6 and 8.8 Hz), 4.15 (1H, m), 3.89 (1H, d,
J ) 16.1 Hz), 3.81 (1H, d, J ) 16.1 Hz), 3.71 (1H, d, J ) 17
Hz), 3.62 (1H, d, J ) 17 Hz), 3.50 (1H, dd, J ) 3.4 and 12 Hz),
3.21 (1H, m), 2.38 (1H, m), 2.27 (1H, m), 1.99 (1H, m), 1.99
(3H, s), 1.77 (1H, m), 1.58 (1H, m), 1.43 (1H, m), 1.16 (1H, m),
and 0.88 (6H, m) ppm; FAB HRMS exact mass calcd for
1
m eth yl ester bis-tr iflu or oa ceta te (23): H NMR (CD3OD,
400 MHz) δ 8.91 (1H, s), 8.26 (1H, d, J ) 12.8 Hz), 8.21 (2H,
d, J ) 10.7 Hz), 7.91 (2H, m), 7.65-7.36 (7H, m), 5.51 (2H, s),
4.72-3.99 (4H, m), 3.66 (3H, s), 3.66-3.24 (4H, m), 3.20-2.85
(2H, m), 2.29 (1H, m), 2.20 (1H, m), 1.96 (3H, s), 1.91 (1H, br
s), 1.70 (1H, d, J ) 16 Hz), 1.56 (1H, m), 1.38 (1H, m), 1.13
(1H, m) and 0.88 (6H, m) ppm; FAB HRMS exact mass calcd
for C37H47N6O6S 703.32778 (MH+), found 703.32852.
R egioselect ive P r ep a r a t ion of 2(S)-{2-[(2(S)-{2-[3-(4-
Cya n oben zyl)-3H-im id a zol-4-yl]a cetyla m in o}-3(S)-m eth -
ylp en tyl)n a p h th a len -1-ylm eth yla m in o]a cetyla m in o}-4-
(m eth ylsu lfa n yl)bu tyr ic Acid Meth yl Ester Bis-tr iflu or o-
a ceta te Sa lt (20). P r ep a r a tion of 1-(Tr ip h en ylm eth yl)-
1H-im id a zol-4-yla cetic Acid Meth yl Ester (17). To a
suspension of 1H-imidazole-4-acetic acid methyl ester hydro-
chloride (16) (5.11 g, 28.9 mmol) in DMF (60 mL) were added
triethylamine (10.1 mL, 72.3 mmol) and triphenylmethyl
bromide (10.3 g, 31.8 mmol), and the reaction was stirred for
72 h. After this time, the reaction mixture was diluted with
EtOAc (600 mL) and washed with saturated aqueous sodium
bicarbonate (200 mL), water (100 mL), and brine (150 mL).
The organic extract was dried (MgSO4) and evaporated in
vacuo. The residue was purified by flash chromatography (25-
100% ethyl acetate/hexanes gradient elution) to provide 17
(8.30 g, 75%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ
7.35 (1H, s), 7.31 (9H, m), 7.22 (6H, m), 6.76 (1H, s), 3.68 (3H,
s) and 3.60 (2H, s) ppm.
C
35H45N6O4S 645.322301 (MH+), found 645.323649.
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(3-p yr id in -4-ylm eth yl-3H-
im id a zol-4-yl)a cetyla m in o]p en tyl}n a p h th a len -1-ylm eth -
ylam in o)acetylam in o]-4-(m eth ylsu lfan yl)bu tyr ic acid tr is-
tr iflu or oa ceta te (13): 1H NMR (CD3OD, 400 MHz) δ 8.97
(1H, s), 8.58 (2H, s), 8.27 (1H, m), 7.95 (2H, m), 7.64 (1H, m),
7.50 (4H, m), 7.31 (2H, d, J ) 4.4 Hz), 5.57 (2H, s), 4.63 (2H,
m), 4.38 (1H, m), 4.09 (1H, m), 3.78 (2H, m), 3.60 (2H, m),
3.42 (1H, m), 3.15 (1H, m), 2.36 (1H, m), 2.15 (1H, m), 2.01
(1H, m), 1.98 (3H, s), 1.76 (1H, m), 1.55 (1H, m), 1.41 (1H, m),
1.15 (1H, m), and 0.88 (6H, m) ppm; FAB HRMS exact mass
calcd for C35H45N6O4 645.322301 (MH+), found 645.321321.
2(S )-{2-[(3(S )-Me t h yl-2(S )-{2-[1-(4-n it r ob e n zyl)-1H -
im id a zol-4-yl]a cetyla m in o}p en tyl)n a p h th a len -1-ylm eth -
yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic a cid
1
m eth yl ester bis-tr iflu or oa ceta te: H NMR (CD3OD, 400
MHz) δ 8.96 (1H, s), 8.17 (1H, m), 8.23 (2H, d, J ) 8.7 Hz),
7.92 (2H, d, J ) 8.9 Hz), 7.61 (1H, d, J ) 6.9 Hz), 7.56 (2H, d,
J ) 8.9 Hz), 7.50 (2H, m), 7.44 (2H, m), 5.52 (2H, s), 4.70 (1H,
d, J ) 9.4 Hz), 4.49 (1H, d, J ) 11.9 Hz), 4.38 (1H, dd, J ) 4.7
and 8.9 Hz), 4.13 (1H, m), 3.67 (3H, s), 3.65 (4H, m), 3.30 (1H,
m), 3.06 (1H, m), 2.31 (1H, m), 2.23 (1H, m), 1.97 (3H, s), 1.94
(1H, m), 1.71 (1H, m), 1.57 (1H, m), 1.42 (1H, m), 1.17 (1H,
m), 0.90 (3H, d, J ) 6.9 Hz) and 0.87 (3H, t, J ) 7.4 Hz) ppm;
FAB MS calcd for C37H47N6O6S 703 (MH+), found 703. Anal.
(C37H46N6O6S‚2.40TFA‚0.25H2O) C,H,N.
2(S )-{2-[(3(S )-Me t h yl-2(S )-{2-[1-(4-n it r ob e n zyl)-1H -
im id a zo l-4-y l]a c e t y la m in o }p e n t y l)n a p h t h a le n -1-y l-
m et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic
a cid bis-tr iflu or oa ceta te (14): 1H NMR (CD3OD, 400 MHz)
δ 8.86 (1H, s), 8.23 (2H, d, J ) 8.8 Hz), 8.22 (1H, m), 7.90 (2H,
d, J ) 7.3 Hz), 7.55 (2H, d, J ) 8.4 Hz), 7.44-7.28 (5H, m),
5.50 (2H, s), 4.53 (1H, m), 4.35 (2H, m), 4.12 (1H, m), 3.79-
3.25 (4H, m), 3.26-2.86 (2H, m), 2.27 (1H, m), 2.18 (1H, m),
1.96 (3H, s), 1.9 (1H, m), 1.67 (1H, m), 1.57 (1H, m), 1.42 (1H,
m), 1.15 (1H, m), 0.90 (3H, d, J ) 6.9 Hz) and 0.86 (3H, t, J )
7.3 Hz) ppm; FAB HRMS exact mass calcd for C36H45N6O6S
689.31213 (MH+), found 689.31262.
P r ep a r a tion of 1-(4-Cya n op h en ylm eth yl)-1H-im id a zol-
5-yla cetic Acid Meth yl Ester (19). To a solution of 1-(triph-
enylmethyl)-1H-imidazol-4-ylacetic acid methyl ester (17) (8.0
g, 20.9 mmol) in acetonitrile (10 mL) was added 4-cyanobenzyl
bromide (4.10 g, 20.9 mmol) and heated at 55 °C for 3 h. After
this time, the reaction was cooled to room temperature, and
the resulting precipitate 18 was collected by filtration. The
filtrate was collected, and this solution was heated at 55 °C
for 15 h. After this time, the reaction mixture was evaporated
to dryness and treated with EtOAc (70 mL), and the solids
were collected by filtration. The two solids were combined,
suspended in methanol (140 mL), and heated at reflux for 30
min. The resulting solution was evaporated in vacuo. The solid
residue was triturated with EtOAc (75 mL) and then parti-
tioned between CH2Cl2 (100 mL) and saturated aq sodium
bicarbonate (100 mL). The organic extract was washed with
brine and dried (MgSO4), and the solvent evaporated in vacuo
to provide 19 (3.88 g, 70%) as a white solid: 1H NMR (CDCl3,
400 MHz) δ 7.65 (2H, d, J ) 8.1 Hz), 7.53 (1H, s), 7.15 (2H, d,
J ) 8.1 Hz), 7.04 (1H, s), 5.24 (2H, s), 3.63 (3H, s) and 3.46
(2H, s) ppm.
P r ep a r a tion of 1-(4-Cya n op h en ylm eth yl)-1H-im id a zol-
5-yla cet ic Acid . 1-(4-Cyanophenylmethyl)-1H-imidazol-5-
ylacetic acid methyl ester (19) (4.44 g, 17.4 mmol) was
dissolved in THF (100 mL), lithium hydroxide (18.8 mL of a 1
M aq solution, 18.8 mmol) was added, and the resulting
mixture stirred at room temperature for 16 h. The reaction
was neutralized with hydrochloric acid (18.8 mL of a 1 M aq
solution, 18.8 mmol) and the solvent removed by lyophilization.
2(S )-{2-[(3(S )-Me t h yl-2(S )-{2-[3-(4-n it r ob e n zyl)-3H -
im id a zo l-4-y l]a c e t y la m in o }p e n t y l)n a p h t h a le n -1-y l-
m et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic
a cid bis-tr iflu or oa ceta te (15): 1H NMR (CD3OD, 400 MHz)
δ 8.89 (1H, s), 8.25 (1H, m), 8.21 (2H, d, J ) 9.0 Hz), 7.89 (2H,
m), 7.64-7.34 (7H, m), 5.52 (2H, s), 4.59-3.88 (4H, m), 3.77-
3.38 (4H, m), 3.18-2.75 (2H, m), 2.27 (1H, m), 2.18 (1H, m),

Non-Thiol Inhibitors of FPTase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3365
The acid obtained by this protocol was of sufficient quality to
be used in the following reaction without further purification:
1H NMR (CD3OD, 400 MHz) δ 8.22 (1H, s), 7.74 (2H, d, J )
8.3 Hz), 7.36 (2H, d, J ) 8.3 Hz), 7.15 (1H, s), 5.43 (2H, s) and
3.49 (2H, s) ppm.
δ 8.70 (1H, s), 8.27 (1H, m), 7.92 (2H, m), 7.63 (1H, s), 7.56-
7.35 (4H, m), 7.18 (2H, d, J ) 8.6 Hz), 6.93 (2H, d, J ) 8.6
Hz), 5.27 (2H, s), 4.93-4.29 (2H, m), 4.36 (1H, m), 4.12 (1H,
m), 3.79 (3H, s), 3.63 (4H, m), 3.07 (2H, m), 2.28 (1H, m), 2.19
(1H, m), 2.02-1.88 (1H, m), 1.95 (3H, s), 1.70 (1H, m), 1.60
(1H, m), 1.43 (1H, m), 1.18 (1H, m), and 0.91 (6H, m) ppm;
FAB HRMS exact mass calcd for C37H48N5O5S 674.337617
(MH+), found 674.338053.
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(3-n a p h th a len -1-ylm eth yl-
3H -im id a zol-4-yl)a cet yla m in o]p en t yl}n a p h t h a len -1-yl-
m et h yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic
a cid bis-tr iflu or oa ceta te (26): 1H NMR (CD3OD, 400 MHz)
δ 8.41 (1H, s), 8.19 (1H, d, J ) 7.7 Hz), 7.99 (2H, m), 7.87 (3H,
m), 7.64 (1H, m), 7.56 (1H, t, J ) 7 Hz), 7.46 (6H, m), 7.16
(1H, d, J ) 8 Hz), 5.79 (2H, s), 5.04-4.71 (1H, m), 4.61-4.38
(1H, m), 4.38-4.21 (1H, m), 4.14 (1H, m), 3.97-3.51 (4H, m),
3.51-3.21 (1H, m), 3.21-2.85 (1H, m), 2.21 (1H, m), 2.13 (1H,
m), 1.98 (1H, m), 1.91 (3H, s), 1.66 (1H, m), 1.56 (1H, m), 1.40
(1H, m), 1.15 (1H, m), and 0.87 (6H, m) ppm; FAB HRMS
exact mass calcd for C40H48N5O4S 694.342702 (MH+), found
694.342837. Anal. (C40H47N5O4S‚2.70TFA‚0.5H2O) C,H,N.
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(3-n a p h th a len -2-ylm eth yl-
3H -im id a zol-4-yl)a cet yla m in o]p en t yl}n a p h t h a len -1-yl-
m et h yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic
a cid bis-tr iflu or oa ceta te (27): 1H NMR (CD3OD, 400 MHz)
δ 8.88 (1H, s), 8.28 (1H, d, J ) 9 Hz), 7.96-7.78 (5H, m), 7.67
(1H, s), 7.57-7.41 (7H, m), 7.32 (1H, d, J ) 9 Hz), 5.55 (2H,
s), 4.81(1H, m), 4.56 (1H, m), 4.37 (1H, m), 4.06 (1H, m), 3.89-
3.50 (4H, m), 3.42 (1H, m), 3.10 (1H, m), 2.28 (1H, m), 2.19
(1H, m), 2.03-1.86 (1H, m), 1.93 (3H, s), 1.90 (1H, m), 1.71
(1H, m), 1.52 (1H, m), 1.37 (1H, m) and 0.87 (6H, m) ppm;
FAB HRMS exact mass calcd for C40H48N5O4S 694.342702
(MH+), found 694.342837. Anal. (C40H47N5O4S‚2.95TFA‚
0.5H2O) C,H,N.
2(S)-[2-({3(S)-Met h yl-2(S)-[2-(3-q u in olin -4-ylm et h yl-
3H -im id a zol-4-yl)a cet yla m in o]p en t yl}n a p h t h a len -1-yl-
m et h yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic
a cid bis-tr iflu or oa ceta te (28): 1H NMR (CD3OD, 400 MHz)
δ 8.87 (1H, s), 8.82 (1H, d, J ) 5 Hz), 8.28 (1H, m), 8.15 (1H,
d, J ) 8.6 Hz),8.06-7.82 (4H, m), 7.67 (2H, m), 7.58 (1H, s),
7.48 (3H, s), 6.96 (1H, m), 6.03 (2H, s), 4.93-4.57 (2H, m), 4.22
(1H, m), 4.08 (1H, m), 3.72 (4H, m), 3.47 (1H, m), 3.13 (1H,
m), 2.28 (1H, m), 2.21 (1H, m), 1,95 (3H, s), 1.87 (1H, m), 1.70
(1H, m), 1.48 (1H, m), 1.35 (1H, m), 1.09 (1H, m), and 0.84
(6H, m) ppm; FAB HRMS exact mass calcd for C39H47N6O4S
695.33795 (MH+), found 695.33893.
2(S)-{2-[(2(S)-{2-[3-(3,7-Dim et h yloct a -2,6-d ien yl)-3H -
im id a zol-4-yl]a cetyla m in o}-3(S)-m eth ylp en tyl)n a p h th a -
len -1-ylm et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)-
bu tyr ic a cid bis-tr iflor oa ceta te (29): 1H NMR (CD3OD, 400
MHz) δ 8.67 (1H, s), 8.27 (1H, m), 7.92 (2H, m), 7.59 (1H, m),
7.52 (2H, m), 7.46 (1H, t, J ) 7.8 Hz), 7.38 (1H, s), 5.28 (1H,
t, J ) 11.2 Hz), 5.04 (1H, m), 4.96-4.54 (1H, m), 4.72 (2H, s),
4.54-4.31 (1H, m), 4.39 (1H, m), 4.13 (1H, m), 3.82-3.31 (4H,
m), 3.68 (2H, m), 3.31-2.79 (2H, m), 2.30 (1H, m), 2.12 (5H,
m), 1.97 (3H, s), 1.97 (1H, m), 1.73 (1H, m), 1.71 (3H, s), 1.70
(3H, s), 1.60 (3H, s), 1.44 (1H, m), 1.18 (1H, m) and 0.92 (3H,
d, J ) 6.8 Hz), and 0.90 (3H, t, J ) 7.5 Hz) ppm; FAB HRMS
exact mass calcd for C39H56N5O4S 690.405303 (MH+), found
690.405157.
4-(Meth ylsu lfan yl)-2(S)-{2-[(3(S)-m eth yl-2(S)-{2-[3-(3,7,-
11-t r im e t h yld od e ca -2,6,10-t r ie n yl)-3H -im id a zol-4-yl]-
a c e t y la m in o }p e n t y l)n a p h t h a le n -1-y lm e t h y la m in o ]-
a cetyla m in o}bu tyr ic a cid bis-tr iflu or oa ceta te (30): 1H
NMR (CD3OD, 400 MHz) δ 8.68 (1H, s), 8.18 (1H, m), 7.90
(2H, m), 7.52 (3H, m), 7.44 (1H, t, J ) 7.5 Hz), 7.37 (1H, s),
5.29 (1H, br t, J ) 7.0 Hz), 5.08 (2H, m), 4.95-4.64 (1H, m),
4.73 (2H, m), 4.37 (2H, m), 4.12 (1H, m), 3.71 (2H, m), 3.47
(2H, m), 3.11 (1H, m), 2.95 (1H, m), 2.27 (1H, m), 2.23-2.01
(9H, m), 2.01-1.89 (1H, m), 1.97 (3H, s), 1.77-1.54 (2H, m),
1.71 (3H, s), 1.65 (3H, s), 1.60 (3H, s), 1.58 (3H, s), 1.42 (1H,
m), 1.16 (1H, m), 0.91 (3H, t, J ) 7.0 Hz) and 0.87 (3H, d, J )
7.5 Hz) ppm; FAB HRMS exact mass calcd for C44H64N5O4S
758.467903 (MH+), found 758.467591.
P r ep a r a tion of 2(S)-{2-[(2(S)-{2-[3-(4-Cya n oben zyl)-3H-
im id a zol-4-yl]a cetyla m in o}-3(S)-m eth ylp en tyl)n a p h th a -
len -1-ylm et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)-
bu tyr ic Acid Meth yl Ester Bis-tr iflu or oa ceta te Sa lt (20).
To a solution of 1-(4-cyanophenylmethyl)-1H-imidazol-5-ylace-
tic acid (1.30 g, 3.91 mmol), amine hydrochloride 4 (2.08 g,
3.91 mmol), triethylamine (1.71 mL, 12.3 mmol), and HOOBT
(0.67 g, 4.10 mmol) in DMF (30 mL) was added EDC (0.79 g,
4.10 mmol), and the reaction was stirred at room temperature
16 h. The reaction was quenched with saturated aq sodium
bicarbonate (100 mL) and water (100 mL) and extracted into
EtOAc (150 mL × 3). The combined organic extracts were
washed with brine (100 mL), and the solvent was evaporated
in vacuo. The residue was purified by flash chromatography
(4-7% MeOH/CH2Cl2 gradient elution) to provide the amide
20 (1.98 g, 74%) as a white solid. For use in biological assays
this material was furthur purified by preparative HPLC (Nova
Prep 5000 Semipreparative HPLC system and a Waters
PrepPak cartridge, 47 × 300 mm, C18, 15 µm, 100A) eluting
with 5-95% acetonitrile/water (0.1% TFA) at 100 mL/min to
give 20 as its bis-trifluoroacetate salt after lyophilization): 1H
NMR (CD3OD, 400 MHz) δ 8.92 (1H, s), 8.31 (1H, m), 8.01
(1H, d, J ) 8.3 Hz), 7.96 (1H, m), 7.75 (2H, d, J ) 8.2 Hz),
7.72 (1H, m), 7.58-7.48 (3H, m), 7.45 (1H, m), 7.41 (2H, d, J
) 8.3 Hz), 5.51 (2H, s), 4.97 (1H, m), 4.76 (1H, m), 4.41 (1H,
m), 4.10 (1H, m), 3.92 (2H, m), 3.75-3.47 (3H, m), 3.69 (3H,
s), 3.25 (1H, m), 2.37 (1H, m), 2.30 (1H, m), 2.00 (3H, s), 1.97
(1H,m), 1.79 (1H, m), 1.58 (1H, m), 1.43 (1H, m), 1.19 (1H, m)
and 0.93-0.88 (6H, m) ppm; FAB HRMS exact mass calcd for
C
38H47N6O4S 683.337951 (MH+), found 683.338437. Anal.
(C38H46N6O4S‚2.40TFA‚1.90H2O) C,H,N.
P r ep a r a tion of 2(S)-{2-[(2(S)-{2-[3-(4-Cya n oben zyl)-3H-
im id a zol-4-yl]a cetyla m in o}-3(S)-m eth ylp en tyl)n a p h th a -
len -1-ylm et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)-
bu tyr ic Acid Bis-tr iflu or oa ceta te (21). To a solution of the
methyl ester 20 (25.6 mg, 0.028 mmol) in methanol (1 mL)
was added sodium hydroxide (0.28 mL of a 1.0 M solution, 0.28
mmol), and the solution was stirred at room temperature for
2 h. After this time, the reaction was acidified to pH 3 by the
addition of trifluoroacetic acid, and the solvent was evaporated
in vacuo. The residue was purified by preparative HPLC (Nova
Prep 5000 Semipreparative HPLC system and a Waters
PrepPak cartridge, 47 × 300 mm, C18, 15 µm, 100A) eluting
with 5-95% acetonitrile/water (0.1% TFA) at 100 mL/min to
give 21 (21 mg, 83%) as its bis-trifluoroacetate salt after
lyophilization: 1H NMR (CD3OD, 400 MHz) δ 8.87 (1H, s),
8.27 (1H, d, J ) 9.2 Hz), 7.90 (2H, m), 7.73 (2H, d, J ) 8.2
Hz), 7.60 (1H, s), 7.46 (4H, m), 7.36 (2H, d, J ) 8.2 Hz), 5.48
(2H, s), 4.95-4.28 (2H, m), 4.36 (1H, m), 4.09 (1H, m), 3.59
(4H, m), 3.51-2.73 (2H, m), 2.29 (1H, m), 2.19 (1H, m), 2.03-
1.85 (1H, m), 1.97 (3H, s), 1.70 (1H, m), 1.56 (1H, m), 1.39
(1H, m), 1.14 (1H, m) and 0.90-0.79 (6H, m) ppm; FAB HRMS
exact mass calcd for C37H44N6O4S 669.322301 (MH+), found
669.323148. Anal. (C37H44N6O4S‚2.45TFA‚1.3H2O) C,H,N.
2(S)-{2-[(2(S)-{2-[3-(4-F lu or oben zyl)-3H-im id a zol-4-yl]-
a c e t y la m i n o }-3(S )-m e t h y lp e n t y l)n a p h t h a le n -1-y l-
m et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic
a cid bis-tr iflu or oa ceta te (24): 1H NMR (CD3OD, 400 MHz)
δ 8.79 (1H, s), 8.30 (1H, m), 8.00-7.80 (2H, m), 7.65-7.40 (5H,
m), 7.30-7.20 (2H, m), 7.13 (2H, t, J ) 8.7 Hz), 5.35 (2H, m),
4.38 (2H, m), 4.13 (1H, m), 3.80-3.40 (4H, m), 3.10 (1H, m),
2.40-2.15 (2H, m), 1.97 (3H, s), 1.95 (1H, m), 1.70 (1H, m),
1.60 (1H, m), 1.43 (1H, m), 1.07 (1H, m), and 1.00-0.80 (6H,
m) ppm; FAB MS m/z ) 662 (M + 1). Anal. (C36H44N5O4S‚
0.60H2O‚2.30TFA) C,H,N.
2(S)-{2-[(2(S)-{2-[3-(4-Met h oxyben zyl)-3H-im id a zol-4-
yl]a ce t yla m in o}-3(S )-m e t h ylp e n t yl)n a p h t h a le n -1-yl-
m et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic
a cid bis-tr iflu or oa ceta te (25): 1H NMR (CD3OD, 400 MHz)

3366 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Anthony et al.
2(S)-{2-[(2(S)-{2-[3-(4-F lu or oben zyl)-3H-im id a zol-4-yl]-
a c e t y la m i n o }-3(S )-m e t h y lp e n t y l)n a p h t h a le n -1-y l-
m et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic
a cid m eth yl ester bis-tr iflu or oa ceta te (31): 1H NMR (CD3-
OD, 400 MHz) δ 8.77 (1H, s), 8.28 (1H, m), 8.00-7.80 (2H,
m), 7.65-7.40 (5H, m), 7.30-7.20 (2H, m), 7.14 (2H, t, J ) 8.6
Hz), 5.34 (2H, m), 4.39 (2H, m), 4.13 (1H, m), 3.68 (3H, s),
3.65-3.40 (4H, m), 2.95 (1H, m), 2.40-2.15 (2H, m), 1.97 (3H,
s), 1.95 (1H, m), 1.70 (1H, m), 1.60 (1H, m), 1.43 (1H, m), 1.07
(1H, m), and 1.00-0.80 (6H, m) ppm; FAB MS m/z ) 676 (M
+ 1). Anal. (C37H46N5O4S‚0.45H2O‚1.65TFA) C,H,N.
2(S)-{2-[(2(S)-{2-[3-(4-Met h oxyb en zyl)-3H-im id a zol-4-
yl]a ce t yla m in o}-3(S )-m e t h ylp e n t yl)n a p h t h a le n -1-yl-
m et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr ic
a cid m eth yl ester bis-tr iflu or oa ceta te (32): 1H NMR (CD3-
OD, 400 MHz) δ 8.70 (1H, s), 8.27 (1H, m), 7.92 (2H, m), 7.70-
7.35 (5H, m), 7.18 (2H, d, J ) 8.5 Hz), 6.92 (2H, d, J ) 8.5
Hz), 5.27 (2H, s), 4.60-4.00 (4H, m), 3.79 (3H, s), 3.67 (3H, s),
3.61 (4H, m), 3.40-2.75 (2H, m), 2.28 (1H, m), 2.19 (1H, m),
1.96 (3H, s), 1.91 (1H, m), 1.70 (1H, m), 1.60 (1H, m), 1.43
(1H, m), 1.18 (1H, m), and 0.91 (6H, m) ppm. FAB HRMS
exact mass calcd for C38H50N5O5S 688.353267 (MH+), found
688.352186. Anal. Calcd for C38H49N5O5S‚1.75TFA‚1.75H2O:
C, 54.45; H, 5.98; N, 7.67. Found: C, 54.44; H, 5.95; N, 7.85.
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(3-n a p h th a len -1-ylm eth yl-
3H -im id a zol-4-yl)a cet yla m in o]p en t yl}n a p h t h a len -1-yl-
m et h yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic
a cid m eth yl ester bis-tr iflu or oa ceta te (33): 1H NMR (CD3-
OD, 400 MHz) δ 8.42 (1H, s), 8.31 (1H, d, J ) 8.9 Hz), 8.04-
7.80 (5H, m), 7.69 (1H, m), 7.59-7.39 (7H, m), 7.20 (1H, d, J
) 8.2 Hz), 5.80 (2H, s), 5.0-4.5 (2H, m), 4.26 (1H, m), 4.13
(1H, m), 4.0-3.6 (4H, m), 3.64 (3H, s), 3.49 (1H, m), 3.18 (1H,
m), 2.17 (2H, m), 1.91 (3H, s), 1.86 (1H, m), 1.67 (1H, m), 1.55
(1H, m), 1.41 (1H, m), 1.16 (1H, brs), and 0.88 (6H, m) ppm;
FAB HRMS exact mass calcd for C41H50N5O4S 708.358352
(MH+), found 708.357618. Anal. (C41H49N5O4S‚3.10TFA‚
0.55H2O) C,H,N.
2(S)-[2-({3(S)-Meth yl-2(S)-[2-(3-n a p h th a len -2-ylm eth yl-
3H -im id a zol-4-yl)a cet yla m in o]p en t yl}n a p h t h a len -1-yl-
m et h yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic
a cid m eth yl ester bis-tr iflu or oa ceta te (34): 1H NMR (CD3-
OD, 400 MHz) δ 8.89 (1H, s), 8.29 (1H, d, J ) 9 Hz), 7.92 (4H,
m), 7.83 (1H, d, J ) 9 Hz), 7.68 (1H, s), 7.58-7.42 (7H, m),
7.33 (1H, d, J ) 9 Hz), 5.54 (2H, s), 4.90-4.50 (2H, m), 4.38
(1H, m), 4.05 (1H, m), 3.93-3.32 (5H, m), 3.65 (3H, s), 3.12
(1H, m), 2.24 (2H, m), 1.93 (3H, s), 1.87 (1H, brs), 1.72 (1H, br
s), 1.52 (1H, brs), 1.38 (1H, brs), 1.13 (1H, brs) and 0.87 (6H,
1.60 (3H, s), 1.41 (1H, m), 1.15 (1H, m), 0.91 (3H, d, J ) 7.0
Hz) and 0.88 (3H, t, J ) 7.5 Hz) ppm; FAB HRMS exact mass
calcd for C40H58N5O4S 704.420953 (MH+), found 704.420223.
Anal. (C40H57N5O4S‚1.80TFA‚0.25H2O) C,H,N.
4-(Meth ylsu lfan yl)-2(S)-{2-[(3(S)-m eth yl-2(S)-{2-[3-(3,7,-
11-t r im e t h yld od e ca -2,6,10-t r ie n yl)-3H -im id a zol-4-yl]-
a c e t y la m in o }p e n t y l)n a p h t h a le n -1-y lm e t h y la m in o ]-
a cet yla m in o}b u t yr ic a cid m et h yl est er b is-t r iflu or o-
a ceta te (37): 1H NMR (CD3OD, 400 MHz) δ 8.70 (1H, s), 8.26
(1H, m), 7.91 (2H, m), 7.52 (3H, m), 7.48 (1H, m), 7.37 (1H, s),
5.40 (1H, m), 5.08 (2H, m), 4.94-4.72 (3H, m), 4.71 (1H, m),
4.40 (1H, m), 4.13 (1H, m), 3.95-2.80 (6H, m), 3.68 (3H, s),
2.27 (1H, m), 2.21 (1H, m), 2.09 (8H, m), 1.97 (3H, s), 1.92
(2H, m), 1.72 (3H, s), 1.65 (1H, m), 1.65 (3H, s), 1.60 (3H, s),
1.58 (3H, s), 1.42 (1H, m), 1.18 (1H, m) and 0.90 (6H, m) ppm;
FAB HRMS exact mass Calcd for C45H66N5O4S 772.483553
(MH+), found 772.481709.
2(S)-{2-[(2(S)-{2-[3-(4-Cya n oben zyl)-3H-im id a zol-4-yl]-
a c e t y la m i n o }-3(S )-m e t h y lp e n t y l)n a p h t h a le n -1-y l-
m eth yla m in o]a cetyla m in o}-4-m eth ylp en ta n oic a cid bis-
tr iflu or oa ceta te sa lt (38): 1H NMR (CD3OD, 400 MHz) δ
8.89 (1H, s), 8.28 (1H, d, J ) 8.9 Hz), 8.00-7.90 (2H, m), 7.73
(2H, d, J ) 8.1 Hz), 7.70-7.60 (1H, m), 7.58-7.42 (4H, m),
7.37 (2H, d, J ) 8.1 Hz), 5.49 (2H, m), 4.28-4.04 (2H, m),
3.90-3.00 (9H, m), 1.70-1.30 (5H, m), 1.16 (1H, m), and 1.00-
0.70 (12H, m) ppm; FAB HRMS exact mass calcd for C38H47N6O4
651.365879 (MH+), found 651.365115. Anal. (C38H46N6O4‚
2.65TFA‚0.25H2O) C,H,N.
2(S)-{2-[(2(S)-{2-[3-(4-Cya n oben zyl)-3H-im id a zol-4-yl]-
a c e t y la m i n o }-3(S )-m e t h y lp e n t y l)n a p h t h a le n -1-y l-
m eth ylam in o]acetylam in o}-4-m eth ylpen tan oic acid m eth -
yl ester (39): 1H NMR (CD3OD, 400 MHz) δ 8.25 (1H, d, J )
8.8 Hz), 7.87 (1H, d, J ) 7.3 Hz), 7.80 (1H, d, J ) 7.9 Hz),
7.70-7.64 (3H, m), 7.50-7.35 (4H, m), 7.18 (2H, d, J ) 8.2
Hz), 6.90 (1H, s), 5.36 (1H, d, J ) 16.8 Hz), 5.30 (1H, d, J )
16.8 Hz), 4.22 (1H, dd, J ) 9.2 and 5.5 Hz), 4.15 (1H, d, J )
13.2 Hz), 4.10 (1H, d, J ) 13.2 Hz), 4.02 (1H, m), 3.65 (3H, s),
3.30-3.10 (4H, m), 2.83 (1H, dd, J ) 13.3 and 3.5 Hz), 2.69
(1H, dd, J ) 13.3 and 9.6 Hz), 1.55 (1H, m), 1.41-1.11 (4H,
m), 1.10-0.97 (1H, m), and 0.90-0.70 (12H, m) ppm; FAB
HRMS exact mass calcd for C39H49N6O4 665.381530 (MH+),
found 665.380331. Anal. (C39H48N6O4‚0.80H2O) C,H,N.
2(S)-{2-[(2(S)-{2-[3-(4-Cya n oben zyl)-3H-im id a zol-4-yl]-
a c e t y la m i n o }-3(S )-m e t h y lp e n t y l)n a p h t h a le n -1-y l-
m et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)b u t yr a -
1
m id e bis-tr iflu or oa ceta te sa lt (40): H NMR (CD3OD, 400
MHz) δ 8.82 (1H, s), 8.19 (1H, m), 7.83 (2H, m), 7.66 (2H, d, J
) 8.2 Hz), 7.54 (1H, m), 7.50-7.35 (4H, m), 7.30 (2H, d, J )
8.2 Hz), 5.41 (2H, s), 4.13 (1H, m), 3.95 (1H, m), 3.75-3.25
(4H, m), 3.20-2.90 (2H, m), 2.30-2.00 (3H, m), 1.89 (3H, s),
1.90 (1H, m), 1.70-1.40 (3H, m), 1.31 (1H, m), 1.07 (1H, m),
and 0.95-0.75 (6H, m) ppm; FAB HRMS exact mass calcd for
m) ppm; FAB HRMS exact mass calcd for
C41H50N5O4S
708.358352 (MH+), found 708.357618. Anal. (C41H49N5O4S‚
3.20TFA‚0.75H2O) C,H,N.
2(S)-[2-({3(S)-Met h yl-2(S)-[2-(3-q u in olin -4-ylm et h yl-
3H -im id a zol-4-yl)a cet yla m in o]p en t yl}n a p h t h a len -1-yl-
m et h yla m in o)a cet yla m in o]-4-(m et h ylsu lfa n yl)b u t yr ic
a cid m eth yl ester bis-tr iflu or oa ceta te (35): 1H NMR (CD3-
OD, 400 MHz) δ 8.88 (1H, s), 8.83 (1H, d, J ) 4.8 Hz), 8.28
(1H, m), 8.15 (1H, d, J ) 8.6 Hz), 7.99-7.85 (4H, m), 7.67 (2H,
m), 7.57 (1H, s), 7.48 (3H, m), 6.96 (1H, m), 6.02 (2H, s), 4.90
(1H, m), 4.62 (1H, m), 4.18 (1H, m), 4.07 (1H, m), 3.94-3.50
(4H, m), 3.64 (3H, s), 3.45 (1H, m), 3.13 (1H, m), 2.28 (1H, m),
2.21 (1H, m), 1.95 (3H, s), 1.87 (1H, m), 1.69 (1H, m), 1.48
(1H, m), 1.35 (1H, m), 1.11 (1H, m), and 0.84 (6H, m) ppm;
FAB HRMS exact mass calcd for C40H49N6O4S 709.353601
(MH+), found 709.353711.
2(S)-{2-[(2(S)-{2-[3-(3,7-Dim et h yloct a -2,6-d ien yl)-3H -
im id a zol-4-yl]a cetyla m in o}-3(S)-m eth ylp en tyl)n a p h th a -
len -1-ylm et h yla m in o]a cet yla m in o}-4-(m et h ylsu lfa n yl)-
bu tyr ic a cid m eth yl ester bis-tr iflu or oa ceta te (36): 1H
NMR (CD3OD, 400 MHz) δ 8.67 (1H, s), 8.27 (1H, m), 7.92
(2H, m), 7.57 (1H, m), 7.53 (2H, m), 7.46 (1H, dd, J ) 9.0 Hz),
7.36 (1H, s), 5.29 (1H, t, J ) 6.0 Hz), 5.08 (1H, t, J ) 6.0 Hz),
4.71 (1H, m), 4.71-4.12 (1H, m), 4.38 (1H, m), 4.12 (1H, m),
3.80-3.33 (4H, m), 3.68 (3H, s), 3.14 (1H, m), 2.96 (1H, m),
2.29 (1H, m), 2.21 (1H, m), 2.12 (4H, m), 2.11 (1H, m), 1.97
(3H, s), 1.97 (1H, m), 1.70 (3H, s), 1.68 (3H, s), 1.65 (1H, m),
C
37H46N7O3S 683.338286 (MH+), found 683.339559. Anal.
(C37H45N7O3S‚2.60TFA‚0.20H2O) C,H,N.
Ack n ow led gm en t. The authors would like to thank
Kenneth D. Anderson, Patrice A. Ciecko, and Matthew
M. Zrada for analytical chemistry support; Arthur B.
Coddington, Harri Ramjit, and Charles W. Ross III for
mass spectral analyses; Michael J . Bogusky, J oan S.
Murphy, Steven M. Pitzenbuerger, and Sandor L. Varga
for NMR spectroscopy expertise; Donald Burns, Wai-Si
Eng, I-Wu Chen, Yousif Sahly, and Kari Vastag for
providing tissue distribution data; J ohn D. Gilbert and
J oan D. Ellis for LCMS analysis; and J oy M. Hartzell
for her help in preparing this manuscript.
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Anthony et al.
(48) Experiments conducted in rodents have revealed that the methyl
ester prodrug 20 is very rapidly hydrolyzed (<1 min) in nude
mice or rat following iv administration and that approximately
90% of the iv dose of 20 is excreted in the bile as the corre-
sponding acid 21. Despite this rapid hydrolysis, iv administra-
tion of the prodrug 20 to rats (4 mpk) resulted in 2-6-fold
greater concentrations of the active acid 21 in heart, lung, and
spleen tissues (5 min postdosing) than dosing the acid 21. In
these experiments very little parent ester 20 was observed in
the tissues. These results indicate that the prodrug ester 20 is
more tissue penetrant than the acid 21 and that the acid is
additionally subject to efficient biliary excretion.
(49) This dose was selected based upon pharmacodynamic data: sc
dosing of the ester 20 (40 mpk) to nude mice resulted in
relatively constant drug levels in both the plasma (1-2 µM) and
tissues (spleen 0.4-1 µM) for
a period of 6 h. These drug
concentrations were sufficient to significantly (>90%) inhibit the
farnesyltransferase activity present in cytosolic fractions of
homogenized spleen tissue derived from drug-treated animals
verses non-drug-treated control animals.
J M990080L