Synthesis, pharmacological evaluation, and molecular modeling studies of novel peptidic CAAX analogues as farnesyl-protein-transferase inhibitors

Article abstract of DOI:10.1021/jm0506165

Fifteen analogues of the C-terminal CA₁A₂X motif were synthesized and evaluated for their inhibition potency against farnesyltransferase (FTase). Replacement of the A₂ residue by phenylalanine or tyrosine-derived analogues, in which a different number of methyl groups were introduced on the aromatic ring, resulted in compounds less active than the reference compound CVFM against FTase except for compounds I and VI (IC₅₀ = 1 μM and 2.5 μM, respectively) that were comparable to CVFM and compound IV (IC₅₀ = 0.1 μM), which was 6-fold more active than the reference compound. Because pseudopeptidic derivatives I-IX were inactive in the cellular assays, the N-formyl- and methyl-ester derivatives (compounds X-XV) were synthesized and tested on different cell lines, showing, in some cases, activity and appreciable selectivity against transformed cells. To rationalize the obtained results, molecular modeling experiments were carried out suggesting the molecular basis of FTase inhibition by these products.

Full text of DOI:10.1021/jm0506165

1882
J. Med. Chem. 2006, 49, 1882-1890
Synthesis, Pharmacological Evaluation, and Molecular Modeling Studies of Novel Peptidic
CAAX Analogues as Farnesyl-Protein-Transferase Inhibitors
Vincenzo Santagada,† Giuseppe Caliendo,† Beatrice Severino,† Antonio Lavecchia,† Elisa Perissutti,† Ferdinando Fiorino,†
Angela Zampella,‡ Valentina Sepe,‡ Daniela Califano,§ Giovanni Santelli,§ and Ettore Novellino*,†
Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli ,Federico II. Via D. Montesano 49, 80131 Napoli, Italy,
Dipartimento di Chimica delle Sostanze Naturali, UniVersita` di Napoli ,Federico II. Via D. Montesano 49, 80131 Napoli, Italy, and
Dipartimento di Oncologia Sperimentale, Istituto dei Tumori ,Fondazione Pascale., Via Semmola 80131, Napoli, Italy
ReceiVed June 29, 2005
Fifteen analogues of the C-terminal CA1A2X motif were synthesized and evaluated for their inhibition potency
against farnesyltransferase (FTase). Replacement of the A2 residue by phenylalanine or tyrosine-derived
analogues, in which a different number of methyl groups were introduced on the aromatic ring, resulted in
compounds less active than the reference compound CVFM against FTase except for compounds I and VI
(IC50 ) 1 µM and 2.5 µM, respectively) that were comparable to CVFM and compound IV (IC50 ) 0.1
µM), which was 6-fold more active than the reference compound. Because pseudopeptidic derivatives I-IX
were inactive in the cellular assays, the N-formyl- and methyl-ester derivatives (compounds X-XV) were
synthesized and tested on different cell lines, showing, in some cases, activity and appreciable selectivity
against transformed cells. To rationalize the obtained results, molecular modeling experiments were carried
out suggesting the molecular basis of FTase inhibition by these products.
Introduction
tion; for this reason, the development of farnesyltransferase
inhibitors, a novel approach to noncytotoxic anticancer therapy,
has been an active area of research over the past decade.
Although advances in medical science have greatly reduced
the toll of many human diseases, cancer still presents challenges
to effective treatment. Many signal transduction proteins,
involved in cell growth and differentiation, have been identified
as possible targets to develop new anticancer treatments.1 One
of the most studied proteins is Ras, a small GTP-binding protein
that is an important switch in signal-transduction pathways that
mediate growth-factor-stimulated cell proliferation.2 Ras proteins
are found mutated in 30-40% of all human cancers, and the
mutation persistently causes a constitutive activation of the
proteins that bind GTP. The three mammalian ras genes yield
four Ras proteins: H-Ras, N-Ras, K-Ras4A, and K-Ras4B.
Several evidences indicate that these different isoforms serve
distinct functions. In this context, a very important aspect is
that cancers arising in different organs are most often associated
exclusively with a mutation in only one ras gene.3 The
attachment of an isoprenoid lipid (prenylation) to the C-terminus
is the first and essential step in a series of posttranslational
modification events that lead to the mature and activated Ras.
Proteins farnesyltransferase (FTase) and geranyl-geranyltrans-
ferase (GGTase), collectively known as CAAX prenyltrans-
ferase, attach their isoprenoid groups, farnesyl and geranylger-
anyl, to the C-terminal cysteine of the Ras and the Rab families
of proteins, respectively.4 Farnesylation is usually followed by
additional modifications of the prenylated protein, such as the
proteolytic removal of the last three residues of the CAAX box
and the carboxymethylation of the new C-terminal-farnesylated
cysteine.5 Farnesylation is required for the subcellular localiza-
tion and tranformation activity of the oncogenic variants of Ras.
Consequently, the inhibition of isoprenylation prevents localiza-
tion at the cell membrane, thereby prohibiting cell transforma-
Enzymatic and crystallographic techniques have explained
the mechanistic details of FTase catalysis, demonstrating that
farnesylation proceeds via an ordered mechanism with farnesyl
pyrophosphate (FPP) binding first, followed by the CA1A2X
substrate (where C is cysteine, A1 and A2 are aliphatic amino
acids, and X is preferably serine or methionine), and then the
cysteine of CA1A2X is farnesylated by the enzyme. During the
search for farnesyltransferase inhibitors some simple tetrapep-
tides, based on CA1A2X, have shown to be able to work as
substrate-analogue inhibitors of FTase, initiating a lot of research
activity. It has been demonstrated that the introduction of an
aromatic residue at the A2 position has a positive effect on
inhibitory potency against the enzyme, as exemplified by
CVFM, CVWM, and CVYM, used as basis for further studies.6
These compounds were found to be very interesting because of
their capability to bind the FTase without being the substrate
for the enzyme. The analysis of their ternary complexes
suggested that the aromatic rings of the peptidic inhibitors did
not allow the access of FPP to the binding site, preventing the
farnesylation of these peptidic inhibitors and that there might
be space for an additional hydrophobic interaction by the side
chain of A2 into the FPP binding site.7 In an effort to further
develop structure-activity relationships, in this article, we
describe the synthesis and the biological activity of a third series8
of nonsubstrate-tetrapeptidic inhibitors (CVFM and CVYM),
where the aromatic residue in the A2 position has been replaced
with uncoded amino acids,9 such as (2S,3aS,7aS)-octahydroin-
dole-2-carboxylic acid (Oic),10 2′,4′-dimethylphenylalanine,
2′,3′-dimethyltyrosine, 2′,5′-dimethyltyrosine, and 2′-methylty-
rosine, to improve the chemical stability of the resulting
compounds and verify the possibility of additional hydrophobic
interactions with the FTase binding site (Chart 1). Moreover, a
molecular modeling study highlighted the site specificity and
the inhibition mechanism for the examined derivatives.
* To whom correspondence should be addressed. Tel: 0039+81678643.
Fax: 0039+81678649. E-mail: novellin@unina.it.
† Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` di
Napoli.
‡ Dipartimento di Chimica delle Sostanze Naturali, Universita` di Napoli.
§ Dipartimento di Oncologia Sperimentale.
10.1021/jm0506165 CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/24/2006

Farnesyl-Protein-Transferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1883
Chart 1. General Structures of the Synthesized Compounds
Scheme 1. Synthesis of the Unnatural Amino Acids (1f-4f)a
a Reagents and conditions: (a) ClCOOEt, pyridine; (b) CH2O in concentrated HCl; (c) CH3CONHCH(COOEt)2 in EtOH/Na; (d) 6N HCl under reflux;
(e) Fmoc-OSu, NaHCO3 in dioxane.
Chemistry
The starting materials (1a-3a) were reacted with ethyl
chlorocarbonate to obtain the corresponding O-carbethoxy
intermediates (1b-3b). The chloromethylation of the O-
carbethoxy derivatives (1b-3b) by CH2O/HCl always gave the
4-chloromethyl derivatives (1c, 2c, and 3c). In addition, when
chloromethylation was applied to m-xylene (4b), we obtained
the derivative 2,4-dimethylbenzyl chloride (4c). Intermediates
Oic was prepared according to the procedure of Vincent
et al.;10 2′,4′-dimethylphenylalanine (Dmp), 2′,3′-dimethylty-
rosine (2,3-Dmt), 2′,5′-dimethyltyrosine (2,5-Dmt), 2′-methyl-
tyrosine, as racemic mixtures, were prepared according to the
method of Abrash et al. ad hoc modified and summarized in
Scheme 1.9

1884 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Scheme 2. Synthesis of Compounds XIV and XV
Santagada et al.
analyses and were characterized by ESI mass spectrometry. The
reference compound, CVFM, was synthesized according to the
literature.8a All of the conventional amino acids and Oic,
employed for the synthesis of compounds I-XV, belonged to
the L series; the uncoded aromatic amino acids, inserted in
position A2, were synthesized as racemic mixtures. The resulting
peptides were obtained as diastereoisomeric mixtures that were
separated using preparative HPLC because they had different
retention times.
The absolute configuration of the unconventional aromatic
amino acid units (2′,4′-dimethylphenylalanine, 2′,3′-dimethyl-
tyrosine, 2′,5′-dimethyltyrosine and 2′-dimethyltyrosine) in the
diastereomeric couples II/III, IV/V, VI/VII, and VIII/IX was
determined by the application of a nonempirical chromato-
graphic method, referred to as advanced Marfey’s method.12
This method, which relies on the elution order of the amino
acids derivatized with (1-fluoro-2,4-dinitrophenyl)-5-L-alanina-
mide (FDAA), was tested on a series of protein and nonprotein
amino acids13 and invariably, the L-amino acid-FDAA derivative
was eluted from a C18 column before its corresponding D-isomer.
It is well established by NMR and UV measurements that the
resolution between L- and D-amino acid derivatives is due to
different hydrophobicities, which are derived from the cis- or
trans-type arrangements of two more hydrophobic substituents
at the R-carbon of the analyzed amino acid and L-Ala-NH2.
Therefore peptides II, V, VI, and VIII were subjected under
vacuum to vapor-phase hydrolysis to obtain a better recovery
of the amino acid derivatives.14 The hydrolizated peptides and
racemic mixtures 1e, 2e, 3e, and 4e were derivatized with
Marfey’s reagent (FDAA) and subjected to HPLC analysis.
Concerning fraction 4e (the racemic mixture of 2′,4′-dimeth-
ylphenylalanine), the HPLC trace of the FDAA derivatives gave
two well-resolved peaks at tR ) 37.1 and tR ) 40.1 min, whose
identity was confirmed by ES/MS (m/z: 468, 2′,4′-dimethylphe-
nylalanine-FDAA + Na+). On the basis of the literature
precedents,13 we assumed that the first eluted peak had the L
configuration. The presence of a peak at tR ) 37.1 min in the
HPLC trace of the FDAA derivatives of peptide II allowed us
to assign the L configuration to the 2′,4′-dimethylphenylalanine
unit in the above peptide. The same procedure was applied to
the FDAA derivatives of fractions 1e, 2e, and 3e (Experimental
Section) that showed, in their HPLC traces, two coupled peaks
at tR ) 57.9 and tR ) 65.3 min (m/z: 484, 2′,3′-dimethyltyrosine-
FDAA + Na+), at tR ) 59.6 and tR ) 65.4 min (m/z: 484,
2′,5′-dimethyltyrosine-FDAA + Na+), and at tR ) 54.2 and tR
) 60.5 min (m/z: 470, 2′-methyltyrosine-FDAA + Na+),
respectively. Comparisons of the HPLC traces of the FDAA
derivatives of peptides V, VI, and VIII allowed us to establish
the configurations of the two residues of dimethyltyrosine as D
and L and the configuration of the methyltyrosine residue as L.
Reagents and conditions: (a) H-Met-OCH3, WSCD/HOBt/TEA; (b) 4N
HCl in dioxane; (c) Boc-D/L-2′,3′-dimethyltyrosine, WSCD/HOBt/TEA; (d)
Boc-Cys(Trt)-OH, WSCD/HOBt/TEA
1c-4c were reacted with diethyl acetamidomalonate in the
presence of EtOH/Na affording the corresponding benzyl-
malonate derivatives (1d-4d), which were successively hydro-
lyzed and decarboxylated by treatment with 6N HCl to give
the desired amino acids (1e-4e). The amino acids (1e-4e) were
treated by Fmoc-OSu in dioxane/water (1/1), in the presence
of NaHCO3, to obtain the corresponding Fmoc derivatives (1f-
4f). N-Formyl-cysteine was obtained by the formylation of
H-Cys(Trt)-OH with formic acid and acetic anydride.11 The
tetrapeptides (I-XIII) were prepared by solid-phase peptide
synthesis on an Fmoc-Met-Wang resin (0.49 mmol/g substitution
grade) using a Milligen 9050 peptide synthesizer. Fluorenylm-
ethyloxy-carbonyl (Fmoc) was employed as the R-amino
protecting group and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetram-
ethyluronium tetrafluoroborate (TBTU)/1-hydroxy-benzotriazole
(HOBt)/N-methylmorpholine (NMM) was applied to the cou-
pling reactions. Removal of the N-Fmoc group was obtained
using 25% piperidine in dimethylformamide (DMF); the cleav-
age of peptides from the resin was effected using a mixture of
trifluoroacetic acid (TFA)/triethylsilane/thioanisole/anisole (90/
5/3/2). Peptides XIV and XV (methyl-ester derivatives) were
synthesized, step by step, by the standard solution-phase
synthesis outlined in Scheme 2.
Results and Discussion
Biological Evaluation. The pseudopeptides prepared in this
study have been tested for their in vitro FTase-inhibitory activity.
Compounds with IC50 values lower than 100 µM have also been
tested for their ability to inhibit cell proliferation. Compounds
I-IX showed IC50 values between 11.4 and 0.1 µM (Table 1).
Compounds I (where Phe was replaced with the sterically
constrained amino acid Oic), II (where Phe was replaced with
2′,4′-L-dimethylphenylalanine), and VI (where Phe was replaced
with 2′,5′-L-dimethyltyrosine) were found to be as active as
CVFM with IC50 values of 1.0, 2.2, and 2.5 µM, respectively;
compound IV (where Phe was replaced with 2′,3′-L-dimethyl-
1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochlo-
ride (WSCD)/HOBt/triethylamine (TEA) was applied to the
coupling reactions; removal of the N-Boc group was obtained
using 4N HCl in dioxane.8a All of the final compounds, purified
by reversed-phase high-performance liquid chromatography (RP-
HPLC) to greater than 98% purity, gave satisfactory elemental

Farnesyl-Protein-Transferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1885
Table 1. Structures and IC50 (µM) ( SD Values of I-XV and Reference Compound CVFM in FTase Bioassay
tyrosine) is the most active of this series with an IC50 value of
0.1 µM (6-fold more active than CVFM). Considering the
diastereoisomeric couples, we have verified that in all of the
cases compounds containing the L aromatic amino acid were
more active than the corresponding diastereisomer incorporating
the D enantiomer: compound II is 2-fold more active than
compound III; compound IV is 40-fold more active than
compound V; compound VI is 4-fold more active than
compound VII; and compound VIII is about 3-fold more active
than compound IX. These results could suggest that there is an
enantioselective interaction with the enzyme where the L-
enantiomer orientation is preferred to the D orientation. Among
compounds embodying the tyrosine analogues, those supporting
two methyl groups and one -OH group were more active than
those supporting just one methyl and one -OH group; peptides
IV and VI and peptides V and VII, in fact, showed IC50 values

1886 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Santagada et al.
Figure 1. Stereoview of the active site showing FTase inhibitor IV (bluishgreen) and the FPP analogue (violet). The protein residues within 4 Å
from the docked ligand are shown and labeled. The three aromatic side chains that define a hydrophobic pocket (Trp102, Trp106, and Tyr361) are
shown in yellow. The zinc ion is shown as a sphere (magenta). The hydrogen bonds are represented with yellow dashed lines. Nonpolar hydrogen
atoms were removed for clarity.
lower than those of VIII and IX, respectively. Replacement of
Phe with 2′,3′-dimethyltyrosine gave the best results (IV and
V); compounds with 2′,5′-dimethyltyrosine (VI and VII) are,
respectively, 25- and 2.5-fold less active than the corresponding
2′,3′-isomer, suggesting the existence of an additional hydro-
phobic interaction with the enzyme that is stronger when the
methyl group is located in the 3′ position. Compounds I-IX,
resulted active from the enzymatic assay, were tested to evaluate
whether they were able to inhibit cell proliferation. The
pharmacological activity of these compounds were determined
in an in vitro assay on a panel of cell lines: FRTL-5, a normal
rat-epithelial-thyroid cell line along with a derived clone
transformed by v-H-Ras, NIH3T3 mouse embryo fibroblasts,
MCF10, a normal human-breast-epithelial cell line, and the
human-tumor cell lines MCF7, MDA MB-231, T47D, and
LoVo. All of the peptides (I-IX) did not exhibit any cell-growth
inhibition; this disappointing result could be due to the fact that
these peptides are not able to penetrate the cell membrane
because of their zwitterionic nature. Among many factors that
affect the activity of compounds in a cell-based assay, a
compound’s ability to penetrate the cell membrane is certainly
important. For this reason, we decided to synthesize the
N-formyl and the methyl-ester derivatives of the most active
compounds from the enzymatic assay, aiming to facilitate
cellular uptake. The newly obtained compounds (X-XV) were
first evaluated in the enzymatic assay against FTase, showing
IC50 values higher than those reported for the corresponding
amino- and carboxy-free parent compounds. As for compounds
I-IX, peptides containing the levo enantiomer of the Tyr-
derived unnatural amino acids were found to be more active
than those containing the dextro enantiomer. Compound X is
10-fold more active than XI (IC50 ) 10.3 and 100 µM,
respectively); compound XII is ≈2-fold more active than XIII
(IC50 ) 5.5 and 10.2 µM, respectively); and compound XIV is
≈3-fold more active than compound XV (IC50 ) 20.4 and 70.5
µM, respectively). It is interesting to note that the difference in
the activity of compounds IV/V and X/XI (IV is 40-fold more
active than V and X is 10-fold more active than XI) is higher
than the other diastereoisomeric couples (II-III, VI-VII,
VIII-IX, and XII-XIII); this could suggest that although the
presence of a methyl group in position 3′ is very important in
the L-enantiomeric orientation, it could be disturbing when the
residue is in the D configuration. N-Formylated derivatives (X-
XIII) showed lower IC50 values compared with those of the
-NH2-free parent compounds (IV-VII). This modification
caused a dramatic loss of affinity when performed on the
peptides embodying 2′,3′-dimethyltyrosine. The methyl-ester
derivatives, XIV and XV, showed IC50 values of 20.4 and 70.5
µM, respectively. This lower enzymatic affinity is consistent
with previous observations that the presence of a free carboxy-
late at the C-terminus is important for a strong inhibition of the
enzyme.7 Nevertheless, compounds X-XV, tested on cell lines,
gave interesting results. Compound X inhibited cell growth in
two human-breast-cancer cell lines (50% inhibition at 100
µM): MDA MB-231 and T47D. Moreover, this product was
not active on MCF10, normal human-breast epithelial cells,
suggesting a selective action on tumor cells. It is difficult to
justify the lack of activity on the breast-cancer cell line MCF7.
Several attempts to correlate FTase activity with cytotoxicity
have been reported, but the results obtained are conflicting.15
Regarding the methyl-ester derivatives, compound XV was
substantially inactive on all of the tested cell lines; however,
compound XIV resulted active on all of the human tumor cell
lines with IC50 values of 65, 83, and 90 µM against MCF7,
MDA MB-231, and T47D respectively, and showed the most
significant effect on the LoVo cells, with an IC50 of 9.1 µM.
Molecular Modeling. To rationalize the above-described
results, molecular modeling experiments were carried out. To
this end, the X-ray structure of rat FTase in complex with
farnesyl pyrophosphate (FPP) and the substrate peptide CVFM
(pdb ref code 1JCR) were employed.4 Compound IV, which
presents the best inhibition potency (IC50 ) 0.1 µM), was
manually docked into the active site by superimposing it onto
the enzyme-bound conformation of the CVFM tetrapeptide.
Figure 1 displays the proposed binding mode into the active
site of FTase for IV, confirming the good fit of the molecule
into the peptide binding site of the enzyme. The peptide binds
in an extended conformation and spans the large active-site
cavity, contacting both the enzyme side chains and the FPP
atom.
The terminal carboxylate of the peptidomimetic participates
in hydrogen bonds with Gln167R (NH-O distance ) 2.0 Å)
similar to those in other peptide substrates.4,16 The C-terminal
Met (X position in CA1A2X sequence) residue of IV binds
isosterically (L-isomer) in a hydrophobic specificity pocket made
up by Ala98, Ala129, Tyr131, Ala151, and Pro152, where it

Farnesyl-Protein-Transferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1887
forms both van der Waals and electrostatic interactions. In
particular, the methionine side chain is oriented in such a way
that the thioether can accept a weak hydrogen bond from the
Ser99â hydroxyl group (OH-S distance ) 2.2 Å).17 The main
chain carbonyl of 2′,3′-dimethyltyrosine (A2 residue of the
CA1A2X motif) interacts directly with FTase through a hydrogen
bond with the guanidinium group of Arg202â (NH-O distance
) 2.0 Å). The 2′,3′-dimethyltyrosine side chain binds isosteri-
cally (L-isomer) in the hydrophobic A2 binding pocket defined
by residues Leu96â, Trp102â, Trp106â, Tyr361â, and the third
FPP isoprene unit, causing hydrophobic interactions. Particu-
larly, residues Trp102â, Trp106â, and Tyr361â form face-on-
face or edge-on-face aromatic-stacking interactions with the
2′,3′-dimethyltyrosine ring. Aromatic-stacking interactions can
contribute significantly to ligand-binding energy, and aromatic
rings that form interactions with the FTase A2 binding site
appear to be an important component of FTase chemistry.1,4
Moreover, the extra methyl group at position 3′ of 2′,3′-
dimethyltyrosine is properly oriented to make hydrophobic
contacts with Leu96â and Trp106â, which easily explains the
enhancement in the FTase-inhibitory activity of IV with respect
to the peptides with the same group in position 2′ (II, VI, and
VIII).
Figure 2. Docking of inhibitor IV displayed into the active site of
FTase. The molecular surface of the active site has been calculated
using the program MOLCAD (implemented in the molecular modeling
software package SYBYL). Hydrophobic (brown), neutral (white), and
hydrophilic (cyan) properties are displayed on the enzyme’s surface.
The FPP analogue is displayed in magenta, whereas enzyme-bound
zinc is shown as a magenta sphere.
Although the A2 binding pocket is mainly dominated by
hydrophobic interactions, a hydrogen bond between the OH
hydrogen of Ser99â and the OH oxygen of the 2′,3′-dimethyl-
tyrosine (OH-O distance ) 1.9 Å) is also observed, which
further contributes to the stabilization of the peptide/FTase
complex.
It is important to note that the lower activity of VIII, which
also has a hydroxyl group at the 4′-position able to form a
hydrogen bond with Ser99â, seems to be due to the lack of the
extra methyl at position 3′, which is involved in hydrophobic
interactions with Leu96â and Trp106â. The Val side chain,
corresponding to the A1 residue of the CA1A2X motif, is oriented
toward the solvent, which is consistent with the wide variety
of residues accommodated at this position in protein and peptide
substrates.18
The Cys residue of IV, which is thought to bind as a thiolate,19
directly coordinates the catalytic zinc ion at a sulfur-zinc
distance of 1.9 Å, whereas its positively charged N-terminus
forms two ion pairs with O and the phosphate oxygen of the
FPP substrate (NH-O distance of 2.5 and 2.2 Å, respectively).
According to this, the low inhibitory potency observed for
N-formyl derivatives X-XIII seems to be due to the inability
of the HCONH-function to form ion pairs with the FPP
substrate.
ability to compete with this natural substrate seems to be
independent of its capacity to achieve a stable interaction with
the Zn2+ ion, but it depends on the possibility of forming an
ion pair between the peptide N-terminus and the R-phosphate
of the FPP substrate as well as the stacking interactions and/or
H bonds between the 2′,3′-dimethyltyrosine side chain and the
FTase A2 binding site (Figure 2).
Conclusions
In conclusion, our study describes the synthesis and the
pharmacological evaluation of a series of tetrapeptidic com-
pounds containing unconventional amino acids derived from
tyrosine and phenylalanine to verify the chance of an additional
hydrophobic interaction with the FTase binding site. Molecular
modeling studies, carried out on the most potent compound of
the series (IV, IC50 ) 0.1 µM), have supported the hypothesis
derived from the biological data, suggesting the molecular basis
of the FTase inhibition by IV. It is clear that the stereochemistry
as well as the presence of a hydrophobic group in position 3′
of the aromatic residue A2 strongly influences the interaction
with the FTase binding site. This product, then, can be
considered to be a peptidomimetic inhibitor that selectively
occupies the binding site of tetrapeptide substrate CVLS, which
corresponds to the C-terminal sequence of p21 Ras protein.
In an effort to elucidate the reasons for the difference in
activity observed when comparing the different diastereoiso-
meric couples, manual superposition of low active compound
V (D-isomer) on the docked structure of IV was carried out.
From a visual inspection of the FTase/V complex, it seems clear
that the extra methyl at the 3′-position of the dimethyltyrosine
moiety causes a steric repulsion with the walls of the binding
cleft and changes the optimal binding mode of the ligand, thus
decreasing the relative stability of the complex. This justifies
the lower enantioselectivity observed in diastereoisomeric
couples II-III, VI-VII, VIII-IX, and XII-XIII, which do
not possess the 3′-methyl group.
These results suggest the molecular basis of FTase inhibition
by IV. In particular, this compound inhibits the FTase selectively
occupying the binding site of tetrapeptide substrate CVLS,
corresponding to the C-terminal sequence of the p21 Ras protein,
and thus it can be defined as a peptidomimetic inhibitor. The
Experimental Section
Materials and Methods. All solvents were purchased from Carlo
Erba (Rodano, Milan, Italy). Extraction solvents were dried over
sodium sulfate. The solvents used for the reactions were dried over
3-Å-molecular sieves. All solvents were filtered and degassed prior
to use. Reagent grade materials were purchased from Bachem
(Bubendorf, Switzerland) and from Aldrich (Milan, Italy) and were
used without further purification. Thin-layer chromatography was
performed on precoated silica gel Kieselgel 60F254 (E.Merck, A.
G.; Darmstadt, Germany) plates. The compounds were detected on
thin-layer chromatography plates by UV light and either chlorination
followed by a solution of 1% starch-15; KI (1:1, v/v) or ninhydrin.
Amino acids analyses were performed in a Carlo Erba 3A-29 amino
acid analyzer, and the Cys values were not detected. Molecular
weights of final peptides were assessed by electrospray-ionization
mass spectrometry (ESI/MS) on a ThermoFinnigan LCQ Ion-Trap.
Where analyses are indicated only by the symbols of the elements,
results obtained are within (0.4% of the theoretical values.

1888 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Santagada et al.
The 1H NMR spectra were recorded on a Bruker AM-500
spectrometer. All NMR spectra were obtained in dilute CDCl3 or
CD3OD solutions. Reversed-phase purification was routinely
performed on a Waters Delta-Prep 4000 system equipped with a
Waters 484 multiwavelength detector on a Vydac C18 silica (15-
20 µm, 50 × 250 mm) high-performance liquid chromatography
(HPLC) column. The operational flow rate was 60 mL/min. The
homogeneity of the products was assessed by analytical reversed-
phase HPLC using both a Vydac C18 column (5 µm, 4.6 × 250
mm) and a Beckman C18 column (5 µm, 4.6 × 250 mm) employing
the following conditions: eluent A, 0.05% TFA (v/v) in water;
eluent B, 0.05% TFA(v/v) in acetonitrile; gradient 0-50% B over
30 min on the Vydac C18 column and 5-35% B over 30 min on
the Beckman C18 column; UV detection at 220 nm, and a flow
rate of 1 mL/min. The column was connected to a Rheodyne model
7725 injector, a Waters 600 HPLC system, a Waters 486 tunable
absorbance detector set to 220 nm, and a Waters 746 chart recorder.
Amino Acid Synthesis. The unconventional amino acids (1e-
4e) were prepared according to the method of Abrash et al. ad hoc
modified and summarized in Scheme 1.9 Here we report the
synthetic procedure and NMR data only for the Fmoc derivatives
(1f-2f) of the unconventional amino acids that were not previously
described.
2(R/S)-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxy-
2,3-dimethyl phenyl)-propionic acid (Fmoc-D,L-2′,3′-dimethyl-
tyrosine-OH) (1f). H-D,L-2′,3′-Dimethyltyrosine-OH‚HCl (1e, 1.6
g) was suspended in 25 mL of 9% Na2CO3 and cooled in ice water.
A solution of 2.4 g of Fmoc-OSu in 25 mL of dioxane was then
added dropwise, and the mixture was stirred at room temperature
for 3 h. The solvent was evaporated, then ethyl acetate was added,
and the water and organic phases were separated. The organic phase
was evaporated, and the residue, loaded onto a silica gel column,
was purified using diethyl ether-hexane (8:2) as eluent. Fractions
containing the product were pooled and concentrated to obtain a
white solid: yield 2.3 g (75%). 1H NMR (MeOD) δ: 1.90 (s, 3H),
2.10 (s, 3H), 2.68-2.78 (m, 1H), 3.10-3.21 (m, 1H), 3.79-3.86
(m, 1H), 4.46-4.70 (m, 3H), 6.37-7.84 (m, 10H, aromatic); MS/
ESI (+), m/z: 432 (M + H+). Anal. (C26H25NO5) C, H, N.
Using the procedure described above for the preparation of 1f,
the following additional intermediates were synthesized using, as
starting material, unconventional amino acids 2e, 3e, and 4e,
respectively:
absorbance of the liberated N-(9-fluorenylmethyl)piperidine. The
complete protected peptide resins were treated with 20 mL of TFA/
triethylsilane/thioanisole/anisole (90/5/3/2) at room temperature for
2 h; the mixture was filtered and the resin washed with TFA/CH2-
Cl2 (1:1). The combined filtrate and washings were evaporated and
the free peptides, I-XIII, precipitated with diethyl ether. The crude
material, containing the diastereoisomeric couples and several minor
impurities as judged by analytical HPLC, were purified by
preparative HPLC. Peptides XIV and XV (methyl-ester derivatives)
were synthesized, step by step, by the standard solution-phase
synthesis outlined in Scheme 2. The intermediates, obtained using
procedures previously reported,8a were characterized by ESI/MS,
furnishing the expected molecular weights.
Compound I and diastereoisomeric couples II-III, IV-V, VI-
VII, VIII-IX, X-XI, XII-XIII, and XIV-XV were purified by
reversed-phase HPLC using a two solvent system: A: 0.1% TFA
(v/v) in water and B: 0.1% TFA (v/v) in acetonitrile (linear gradient
from 0 to 55% B over 60 min, UV detection at 220 nm, and a flow
rate of 60 mL/min). The purified peptides were examined for
homogeneity by analytical HPLC determinations that were carried
out using both a Vydac C18 column (5 µm, 4.6 × 250 mm) and a
Beckman C18 column (5 µm, 4.6 × 250 mm) employing the
following conditions: eluent A, 0.05% TFA (v/v) in water; eluent
B, 0.05% TFA(v/v) in acetonitrile; gradient 0-50% B over 30 min
on the Vydac C18 column and 5-35% B over 30 min on the
Beckman C18 column, UV detection at 220 nm, and a flow rate of
1 mL/min. The final HPLC purity of the peptides was always
>98%. The analytical parameters of the purified peptides are listed
in Table 2 (Supporting Information).
Determination of Absolute Stereochemistry. (a) General
Procedure for Peptide Hydrolysis. Peptide samples (200 µg) were
dissolved in degassed 6 N HCl (0.5 mL) in an evacuated glass tube
and heated at 160 °C for 16 h. The solvent was removed in vacuo,
and the resulting material was subjected to further derivatization.
(b) General Procedure for Obtaining FDAA Derivatives. A
portion of the hydrolyzed mixture (800 µg) or the amino acid
enantiomeric mixture (500 µg) was dissolved in 80 µL of a 2:3
solution of TEA-MeCN and treated with 75 µL of 1% N-(3-fluoro-
4, 6-dinitrophenyl)-L-alaninamide (FDAA) in 1:2 MeCN-acetone.
The vials were heated at 70 °C for 1 h, and the contents were
neutralized with 0.2 N HCl (50 µL) after cooling to room
temperature.
2(R/S)-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxy-
2,5-dimethylphenyl)-propionic acid (Fmoc-D,L-2′,5′-dimethyl-
tyrosine-OH) (2f). Yield 87%. 1H NMR (MeOD) δ: 2.12 (s, 3H),
2.35 (s, 3H), 3.10-3.13 (m, 1H), 3.29-3.35 (m, 1H), 4.19-4.22
(m, 1H), 4.40-4.60 (m, 3H), 6.37-7.84 (m, 10H, aromatic); MS/
ESI (+), m/z: 432 (M + H+). Anal. (C26H25NO5) C, H, N.
2(R/S)-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxy-
2-methylphenyl)-propionic acid (Fmoc -D,L-2′-methyltyrosine-
HPLC and Mass Analysis of Marfey’s (FDAA) Derivatives.
Peptide II and Racemic Amino Acid Mixture 4e. An aliquot of
the L-FDAA derivative was dried under vacuum, diluted with
MeCN-5% HCOOH in H2O (1:1), and separated on a Vydac C18
(25 × 1.8 mm i.d.) column. A linear gradient (H2O (0.2% TFA)/
acetonitrile (0.1% TFA) (90:10 to 50:50)) over 45 min at 2 mL/
min was used, and the FDAA derivatives were detected by UV at
340 nm. Peak identity was confirmed by ESI/MS analysis. The
mass spectra were acquired in positive-ion detection mode (m/z
interval of 320-900), and the data was analyzed using the Xcalibur
(ThermoQuest, San Jose´, California) suite of programs; all masses
were reported as average values. Capillary temperature was set at
280 °C, capillary voltage at 37 V, tube lens offset at 50 V, and ion
spray voltage at 5 V. The retention times of authentic FDAA amino
acids (min): L-2′,4′-dimethylphenylalanine (37.1) and D-2′,4′-
dimethylphenylalanine (40.1). The hydrolyzate of peptide II
contained L-Met (46.8), l-Val (53.8), and L-2′,4′-dimethylpheny-
lalanine (37.1).
1
OH) (3f). Yield 60%. H NMR (MeOD) δ: 2.30 (s, 3H), 3.00-
3.05 (m, 1H), 3.23-3.27 (m, 1H), 4.10-4.13 (m, 1H), 4.46-4.58
(m, 3H), 6.68-7.84 (m, 11H, aromatic); MS/ESI (+), m/z: 418
(M + H+). Anal. (C25H23NO5) C, H, N.
2(R/S)-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2,4-di-
methylphenyl)-propionic acid (Fmoc-D,L-2′,4′-dimethylphenyl-
alanine) (4f). Yield 68%. 1H NMR (MeOD) δ: 2.15 (s, 3H), 2.20
(s, 3H), 3.08-3.10 (m, 1H), 3.25-3.28 (m, 1H), 4.15-4.18 (m,
1H), 4.41-4.73 (m, 3H), 6.81-7.75 (m, 11H, aromatic); MS/ESI
(+), m/z: 416 (M + H+). Anal. (C26H25NO4) C, H, N.
Peptides V, VI, and VIII and Racemic Amino Acid Mixture
1e, 2e, and 3e. To obtain a better resolution in the HPLC traces,
the FDAA derivatives were analyzed on a Vydac C18 (25 × 1.8
mm i.d.) column by means of a linear gradient from 85 to 15%
H2O (0.2% TFA)/4:1 acetonitrile/2-propanol (0.1% TFA) over 115
min at 1 mL/min (UV detection at 340 nm). Peak identity was also
confirmed by ESI/MS analysis.
The retention times of authentic FDAA amino acids (min):
L-2′,3′-dimethyltyrosine (57.9), d-2′,3′-dimethyltyrosine (65.3),
L-2′,5′-dimethyltyrosine (59.6), D-2′,5′-dimethyltyrosine (65.4), l-3′-
methyltyrosine (54.2), and D-2′-methyltyrosine (60.5).
Peptide Synthesis. Compounds I-XIII were prepared by solid-
phase peptide synthesis on a 0.1 mmol scale using an Fmoc-Met-
Wang resin (0.49 mmol/g substitution grade) using a Milligen 9050
peptide synthesizer. Fmoc was employed as the R-amino protecting
group, and Trt was used as the side-chain protecting group for amino
terminal Cys; TBTU/HOBt/NMM was applied to the coupling
reactions. N-Formyl-cysteine, obtained by the formylation of H-Cys-
(Trt)-OH with formic acid and acetic anydride,11 was employed as
the N-terminal residue for the synthesis of compounds X-XIII.
The cleavage of the N-Fmoc protecting group, using 25% piperidine
(v/v) in DMF, was monitored at each stage by measuring the

Farnesyl-Protein-Transferase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1889
The hydrolyzate of peptide V contained: L-Met (46.8), L-Val
(53.8), and D-2′,3′-dimethyltyrosine (65.3).
The hydrolyzate of peptide VI contained: L-Met (46.8), L-Val
(53.8), and L-2′,5′-dimethyltyrosine (59.6).
The hydrolyzate of peptide VIII contained: L-Met (46.8), L-Val
(53.8), and L-2′-methyltyrosine (54.2).
thyroid stimulating hormone (TSH) in the medium. In vitro
experiments on FRTL-5 rat thyroid cells have shown that v-H-Ras
is able, in a single step, to malignantly transform this cell line.24
The FRTL-5 cells were grown in Coon’s modified Ham F12
medium supplemented with 5% calf serum and a mixture of six
hormones: TSH 1 × 10-10 M, insulin 10 mg/mL, somatostatin 10
ng/mL, Glycyl-Histidyl-Lysine acetate 10ng/mL, hydrocortisone 1
× 10-8 M, and transferrin 5 mg/mL. FRTL-5 cells fully transformed
by the v-H-Ras oncogene were grown in Coon’s modified Ham
F12 medium supplemented with 5% calf serum.
NIH 3T3 mouse embryo fibroblasts were grown in DMEM
medium with 10% calf serum. The normal breast-epithelial cells
MCF10 were cultured in 1:1 DMEM/Ham’s-F12 medium (Life
Technologies, Inc.) with 5% horse serum supplemented with 20
ng/mL EGF, 100ng/mL cholera toxin, 0.01 bovine insulin, and 500
ng/mL hydrocortisone. Human tumor cell lines MCF7, T47D, and
MDA MB-231 derived from breast carcinomas and LoVo from a
colon carcinoma were all grown as monolayers in RPMI-1640 (Life
Technologies, Inc., NY) containing 10% fetal bovine serum (Life
Technologies, Inc., NY).
Compound Treatment. Cells were grown in a volume of 100
µL at approximately 10% confluency in 96-well multititer plates
and were allowed to attach and recover for another 24 h. Varying
concentrations of compounds alone were then added to each well,
and the plates were incubated in an atmosphere of 5% CO2 and
95% air at 37 °C for an additional 24 h. Finally, the plates were
washed to remove the drug and incubated for 48 h. The control
cultures included equivalent amounts of the vehicle used to
solubilize each molecule. The experimental agents were solubilized
in DMSO.
Computational Chemistry. All molecular modeling was per-
formed using the software package SYBYL20 running on a Silicon
Graphics Octane 2 R12000 workstation. Molecular models of
compound IV and its derivatives were built according to SYBYL-
standard bond lengths and valence angles. Geometry optimizations
were realized with the SYBYL/MAXIMIN2 minimizer by applying
the BFGS algorithm21 and setting a root-mean-square gradient of
the forces acting on each atom at 0.05 kcal/mol Å as a convergence
criterion. Molecular graphics, root-mean-squares (rms) fit, and
calculations of ring centroids of substructures were also carried out
using SYBYL.
Docking simulations were carried out using the X-ray structure
of rat FTase in complex with farnesyl pyrophosphate (FPP) and
the substrate peptide CVFM(pdb ref code 1JCR).4 Water molecules
were deleted. Hydrogen atoms were added to the unfilled valences
of the amino acids, and the Lys, Asp, and Glu side chains were
modeled in their ionized forms.
To build IV, the experimentally determined FTase-bound
conformation of CVFM was modified by replacing the phenyla-
lanine residue with the 2′,3′-dimethyltyrosine side chain. Hydrogen
atoms were added to the unfilled valences and their positions
optimized while keeping the rest of the molecule fixed.
The position of the docked tetrapeptide CVFM in the enzyme
crystal structure was taken as a starting point. Peptide IV was
docked into the FTase binding site by means of the following
steps: (i) superposition on the enzyme-bound conformation of
CVFM by minimizing the root-mean-square distance between their
common backbone atoms; (ii) removal of CVFM from the receptor;
(iii) manual adjustment of the torsion angles determining the
orientation of the 2′,3′-dimethyltyrosine side chain of IV to avoid
steric clashes with the enzyme; and (iv) energy-minimization of
the resulting IV/FTase complex on the basis of the molecular
mechanics Tripos force field.22 During the minimization step, the
side chains and the ligand itself were allowed to optimize their
position and conformation by keeping the protein backbone atoms
fixed.
Sulforhodamine B Assay. At the end of the treatment, cell
viability was assessed by the sulforhodamine B (SRB) assay.25 Data
were expressed as %T/C ) (OD of treated cells/OD of control cells)
× 100, and the concentration of the test compound causing 50%
inhibition of cell growth (IC50) was calculated from the dose/effect
curve for each tested compound. Every assay was performed in
triplicate, and the drug-IC50 value of each cell line was the average
of at least three independent experiments.
Acknowledgment. This work was supported by the Italian
MIUR fund (PRIN 2005).
Biological Assays. Farnesyltransferase Protein. The enzyme
was a partially purified fraction from a bovine brain homogenate,
prepared essentially as described by Reiss et al.23
Supporting Information Available: Analytical data (Table 2)
and elemental analysis (Table 3) for all compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
Farnesyltransferase Inhibition Assay. To evaluate the farne-
syltransferase inhibition by our compounds, we have used Amer-
sham’s farnesyl transferase kit based on the scintillation proximity
principle. A human lamin-B carboxy-terminus sequence peptide
(biotin-YRASNRSCAIM) is 3H-farnesylated at the cysteine residue
when processed by farnesyl transferase. The resultant complex is
captured by streptavidin-linked SPA beads. In a 100 µL final
reaction volume, the test compound was added in 10 µL of DMSO
to a 70 µL reaction mixture containing 20 µL of 1:20 diluted [3H]-
farnesyl pyrophosphate (0.2 µCi), 46 µL of assay buffer (50 mM
HEPES, 30 mM MgCl2, 20 mM KCl, 5 mM DTT, and 0.01% Triton
X-100), and 20 µL of 0.5 mM biotin-lamin B peptide in a buffer
at pH 7.5 (50 mM HEPES, 25 mM Na2HPO4, 20 mM KCl, 5 mM
DTT, and 0.01% Triton X-100). This mixture was allowed to stand
at 37 °C for 5 min. The reaction was started by adding 4 µL of
diluted enzyme (∼2 µg of protein) to the mixture that was incubated
at 37 °C for 1 h. To stop the reaction, 150 µL of the stop/bead
reagent was added. The samples were counted in a Beckman LS
1801 scintillation counter. Every test compound was assayed at
least twice, and the mean and standard deviation were calculated.
Cell Lines. FRTL-5 is a continuous line of differentiated
epithelial cells derived from normal Fisher rat thyroid that have
retained the typical markers of thyroid differentiation such as
thyroglobulin (TG), thyroperoxidase (TPO), and thyrotropin receptor
(TSH-R) and depend for growth upon the continuous presence of
References
(1) Reid, S. T.; Beese L. S.. Crystal structures of the anticancer clinical
candidates R115777 (Tipifarnib) and BMS-214662 complexed with
protein farnesyltransferase suggest a mechanism of FTI selectivity.
Biochemistry, 2004, 43, 6877-6884.
(2) El Oualid, F.; Burm, B. E. A.; Leroy, I. M.; Cohen, L. H.; van Boom,
J. H.; van den Elst, H.; Overkleeft, H. S.; van der Marel, G. A.;
Overhand, M. Design, synthesis, and evaluation of sugar amino acid
based inhibitors of protein prenyl transferases PFT and PGGT-1. J.
Med. Chem. 2004, 47, 3920-3923.
(3) Brunner, T. B.; Hahn, S. M.; Gupta, A. K.; Muschel, R. J.; McKenna,
W. G.; Bernhard, E. J. Farnesyltransferase inhibitors: an overview
of the results of preclinical and clinical investigations. Cancer Res.
2003, 63, 5656-5668.
(4) Long, S. B.; Hancock, P. J.; Kral, A. M.; Hellinga, H. W.; Beese, L.
S. The crystal structure of human protein farnesyltransferase reveals
the structural basis for inhibition by CaaX tetrapeptides and their
mimetics. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12948-12953.
(5) Bell, I. M. Inhibitors of farnesyltransferase: a rational approach to
cancer chemotherapy? J. Med. Chem. 2004, 47, 1869-1878.
(6) Leftheris, K.; Kline, T.; Natarajan, S.; DeVirgilio, M. K.; Cho, Y.
H.; Pluscec, J.; Ricca, C.; Robinson, S. Peptide based P21RAS farnesyl
transferase inhibitors: systematic modification of the tetrapeptide
CA1A2X motif. Bioorg. Med. Chem. Lett. 1994, 4, 887-892.
(7) Lee, H. Y.; Sohn, J. H.; Kwon, B. M. Development of tripeptidyl
farnesyltransferase inhibitors. Bioorg. Med. Chem. Lett. 2002, 12,
1599-1602.

1890 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Santagada et al.
(8) (a) Caliendo, G.; Fiorino, F.; Grieco, P.; Perissutti, E.; Santagada,
V.; Ramunno, A.; Albrizio, S.; Califano, D.; Giuliano, A.; Santelli,
G. Phenol-derived CVFM analog inhibitors of Ras Farnesyltransferase
possessing cellular in vitro activity Eur. J. Med. Chem. 1998, 33,
725-732. (b) Caliendo, G.; Fiorino, F.; Grieco, P.; Perissutti, E.;
De Luca, S.; Giuliano, A.; Santelli, G.; Califano, D.; Severino, B.;
Santagada, V. Synthesis and biological activity of pseudopeptides
inhibitors of Ras farnesyl transferase containing unconventional amino
acids. Farmaco, 1999, 54, 785-790.
(9) Santagada, V.; Caliendo, G.; Severino, B.; Perissutti, E.; Ceccarelli,
F.; Giusti, L.; Mazzoni, M. R.; Salvadori, S.; Temussi, P. A. Probing
the shape of a hydrophobic pocket in the active site of delta-opioid
antagonists. J. Pept. Sci. 2001, 7, 374-385.
(16) (a) Strickland, C. L.; Windsor, W. T.; Syto, R.; Wang, L.; Bond, R.;
Wu, R.; Schwartz, J.; Le, H. V.; Beese, L. S.; Weber, P. C. Crystal
structure of farnesyl protein transferase complexed with a CaaX
peptide and farnesyl diphosphate analogue. Biochemistry 1998, 37,
16601-16611. (b) Long, S. B.; Casey, P. J.; Beese, L. S. The basis
for K-Ras4B binding specificity to protein farnesyltransferase
revealed by 2 Å resolution ternary complex structures. Structure 2000,
8, 209-222.
(17) Desiraju, G. R.; Steiner, T. Monographs on Crystallography; Oxford
University Press/International Union of Crystallography: Oxford,
1999; Vol. 9.
(18) Reiss, Y.; Stradley, S. J.; Gierasch, L. M.; Brown, M. S.; Goldstein,
J. L. Sequence requirement for peptide recognition by rat brain
p21Ras protein farnesyltransferase. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 732-736.
(19) Hightower, K. E.; Huang, C.-C.; Casey, P. J.; Fierke, C. A. H-Ras
peptide and protein substrates bind protein farnesyltransferase
as an ionized thiolate. Biochemistry, 1999, 37, 15555-
15562.
(20) SYBYL Molecular Modeling System, version 6.9.1; Tripos Inc.: St.
Louis, MO, 2003.
(21) Head, J.; Zerner, M. C. A Broyden-Fletcher-Goldfarb-Shannon
optimization procedure for molecular geometries. Chem. Phys. Lett.
1985, 122, 264-274.
(22) Vinter, J. G.; Davis, A.; Saunders, M. R. Strategic Approaches to
Drug Design. 1. An integrated software framework for molecular
modeling. J. Comput.-Aided Mol. Des. 1987, 1, 31-55.
(23) Reiss, Y.; Goldstein, J. L.; Seabra, M. C.; Casey, P. J.; Brown, M.
S. Inhibition of purified p21Ras farnesyl protein transferase by Cys-
AAX tetrapeptides. Cell 1990, 62, 81-88.
(10) (a) Vincent, M.; Remond, G.; Portevin, B.; Serkiz, B.; Laubie, M.
Stereoselective synthesis of a new perhydroindole derivative of chiral
iminodiacid, a potent inhibitor of angiotensin converting enzyme.
Tetrahedron Lett. 1982, 23, 1677-1680.
(11) Sheehan, J. C.; Yang, D. D. H. The use of N-formylamino acids in
peptide synthesis. J. Am. Chem. Soc. 1958, 80, 1154-1158.
(12) (a) Harada, K.; Fujii, K.; Mayumi, T.; Hibino, Y.; Suzuki, M. A
method using LC/MS for determination of absolute configuration of
constituent amino acids in peptide advanced Marfey’s method.
Tetrahedron Lett. 1995, 36, 1515-1518. (b) Fujii, K.; Ikai, Y.;
Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K. A nonempirical method
using LC/MS for determination of the absolute configuration of
constituent amino acids in a peptide: elucidation of limitations of
Marfey’s method and of its separation mechanism. Anal. Chem. 1997,
69, 3346-3352.
(13) Pe´ter, A.; Olajos, E.; Casimir, R.; Tourwe´, D.; Broxterman, Q. B.;
Kaptein, B.; Armstrong, D. W. High-performance liquid chromato-
graphic separation of the enantiomers of unusual amino acid
analogues. J. Chromatogr., A 2000, 871, 105-113.
(14) Davidson, I. Protein Sequencing Protocol. In Methods in Molecular
Biology, 2nd ed.; Smith, B. J., Ed.; Humana Press: Totowa, NJ, ;
Vol. 211.
(24) Fusco, A.; Berlingieri, M. T.; Di Fiore, P. P.; Portella, G.; Grieco,
M.; Vecchio, G. One- and two-step transformations of rat thyroid
epithelial cells by retroviral oncogenes. Mol. Cell. Biol. 1987, 7,
3365-3370.
(25) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer-drug screening.
J. Natl. Cancer Inst. 1990, 82, 1107-1112.
(15) (a) Smith, V.; Rowlands, M. G.; Barrie, E.; Workman, P.; Kelland,
L. R. Establishment and characterization of acquired resistance to
the farnesyl protein transferase inhibitor R115777 in a human colon
cancer cell line. Clin. Cancer Res. 2002, 8, 2002-2009. (b) Moasser,
M. M.; Rosen, N. The use of molecular markers in farnesyltransferase
inhibitor (FTI) therapy of breast cancer. Breast Cancer Res. Treat.
2002, 73, 135-144.
JM0506165