Solid-phase synthesis and pharmacological evaluation of a library of peptidomimetics as potential farnesyltransferase inhibitors: an approach to new lead compounds

Article abstract of DOI:10.1016/j.ejmech.2006.03.017

Oncogenic Ras proteins whose activation is farnesylation by farnesyltransferase have been seen as important targets for novel anticancer drugs. Inhibitors of this enzyme have already been developed as potential anti-cancer drugs, particularly by rational

Full text of DOI:10.1016/j.ejmech.2006.03.017

European Journal of Medicinal Chemistry 41 (2006) 745–755
http://france.elsevier.com/direct/ejmech
Original article
Solid-phase synthesis and pharmacological evaluation of a library
of peptidomimetics as potential farnesyltransferase inhibitors:
an approach to new lead compounds
P. Gilleron a, R. Millet a, R. Houssin a, N. Wlodarczyk a, A. Farce b, A. Lemoine a
J.-F. Goossens a, P. Chavatte b, N. Pommery a, J.-P. Hénichart a,*
a Institut de chimie pharmaceutique Albert-Lespagnol, EA 2692, université de Lille-II, rue du professeur-Laguesse, BP 83, 59006 Lille, France
b Laboratoire de chimie thérapeutique, faculté des sciences pharmaceutiques et biologiques,
université de Lille-II, rue du professeur-Laguesse, BP 83, 59006 Lille, France
Received in revised form 16 March 2006; accepted 20 March 2006
Available online 02 May 2006
Abstract
Oncogenic Ras proteins whose activation is farnesylation by farnesyltransferase have been seen as important targets for novel anticancer
drugs. Inhibitors of this enzyme have already been developed as potential anti-cancer drugs, particularly by rational design based on the structure
of the CA1A2X carboxyl terminus of Ras. Synthesis of a peptidomimetics library via solid-phase synthesis using the MultipinTM method is
described here. The most active hits on cellular assays were resynthesized and enzymatic activity was measured. Compounds A1, A5 and A7
present significant activity on the isolated enzyme (IC50 = 117, 57.3 and 28.5 nM) and their molecular docking in the active site of the enzyme
provides details on key interactions with the protein.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Farnesyltransferase; Inhibitors; Peptidomimetics; Solid phase synthesis
1. Introduction
metalloenzyme farnesyltransferase (FTase) prevents membrane
localization of Ras and so constitutes a valid target for the con-
ception of new cytostatic anticancer drugs [5]. Farnesyltransfer-
ase inhibitors (FTIs) have thus been developed as a new class of
extremely promising drugs for cancer treatment, either by ra-
tional design based on the structure of the CA1A2X carboxyl
terminus of Ras or by random screening of chemical libraries
or natural products [6,7]. Three of these inhibitors (Fig. 1) are
being assessed in phase II/III of clinical trials [8,9]: R-115777
(for colorectal, non-small cell lung and breast cancers [10–12]),
SCH-66336 and BMS-214662 (for leukemia [8,13]).
Cancer is an increasingly common disease for which treat-
ments are not always selective of tumor cells. One of the pos-
sible targets would be to inhibit the function of the oncogene
ras which is among the most frequently activating mutated
genes in human tumors (30% on average, reaching up to 50%
in colon cancer and up to 90% in pancreas cancer) [1,2]. Ras
proteins play a key role in signal transduction pathway, cell
growth and cell proliferation [3]. Activation of Ras proteins re-
quires association to the plasma membrane which is mainly de-
pendent on the attachment of a farnesyl (15-carbon) or of a
geranylgeranyl (20-carbon) group to the cysteine residue lo-
cated in a tetrapeptidic CA1A2X sequence at the carboxyl ter-
minus of Ras proteins (A1 and A2: aliphatic amino acids; X:
methionine or serine for farnesyltransferase, leucine or isoleu-
cine for type I geranylgeranyltransferase) [4]. Inhibition of Zn-
We report here the design and synthesis of a library of 64
peptidomimetics, potent specific inhibitors based on the struc-
ture of the CA1A2X sequence. The aim of this work was to
rapidly discover new lead compounds. Compounds were pre-
pared using solid-phase parallel synthesis by the MultipinTM
method. These inhibitors were built on the basis of a zinc che-
lator linked to a selectivity factor (FTase vs. GGTase-I) by a
spacer which correctly positioned these elements in the enzyme
active site.
* Corresponding author. Tel.: +33 3 20 96 43 74; fax: +33 3 20 96 49 06.
E-mail address: jean-pierre.henichart@univ-lille2.fr (J.-P. Hénichart).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.017

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P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
Fig. 2. Structure of aldehydes A–H.
the farnesylated molecule after moving the farnesyl moiety into
the exit groove [16].
The terminal residue X was Phe-NH2 which was more
stable than Met-NH2, prevented any risk of oxidation and al-
lowed recognition by FTase or GGTase-I. In addition, amide
group was found more effective than a C-terminal carboxylic
function for cellular penetration of inhibitors, as found in one
of our previous work.
Fig. 1. Inhibitors in clinical trials.
Eight heterocycles A–H (Fig. 2) were chosen to vary the
zinc chelator C of the CA1A2X box according to three criteria:
● Ability of nitrogen heterocycles, such as imidazole or pyr-
idine, to coordinate the enzyme-bound zinc ion. Furan and
thiophene were selected because of their property to main-
tain the enzymatic activity despite their lacking of metal co-
ordination [14].
2. Chemistry
Solid-phase peptide synthesis methodology is the quickest
and most efficient way to prepare a library of peptidomimetics
through parallel multi-step synthesis [17]. This method allows
for limited purification difficulties.
● Chemical opportunity: each compound possessed an alde-
hyde function in order to incorporate the spacer.
● Stability of compounds: our interest was focused on aro-
matic aldehydes generally more stable than hydroxamates
or aliphatic aldehydes.
The library of 64 compounds was built on a Geysen’s pins
apparatus (Chiron MultipinTM method) [18,19]. The com-
pounds were prepared as C-terminal amides using a Rink
amide resin. Couplings on the pin apparatus were from the C-
to the N-terminus (as is standard for solid phase peptide synth-
esis). The synthesis was performed according to the general
reaction pathway outlined in Scheme 1.
So as to control the coupling of the two amino acids, the
backbone amine (N-terminal position) was protected by an
Fmoc residue which is stable with regard to acidic reagents
and tertiary amines but can be readily cleaved under basic con-
ditions. Fmoc deprotection and washing cycles were performed
between each step. Fmoc deprotection of the pins using piper-
idine (compound 9) was followed by coupling Fmoc-(S)-Phe-
Seven of them (B–H) were commercially available whereas
aldehyde A was synthesized according to a previously de-
scribed procedure [15]. Besides these aldehydes, eight Fmoc-
protected amino acids 1–8 (Fig. 3) were selected according to
their diversity in varying the spacer A1A2 and their aliphatic,
alicyclic or aromatic structure associated with their hydropho-
bic character required for enzyme recognition. These could be
linear or branched so as to give either an extended conforma-
tion mimicking the position of the CA1A2X box in the sub-
strate site or a bent conformation mimicking the location of
Fig. 3. Structure of protected amino acids 1–8.

P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
747
Scheme 1. Reagents and conditions: (a) Piperidine/DMF (1:4), 20 min; (b) Fmoc-(S)-Phe-OH, HBTU, DIEA, DMF, 6 h; (c) Fmoc-R1-OH (1–8, see Fig. 3), HBTU,
DIEA, DMF, 6 h; (d) i. R2-CHO (A–H, see Fig. 2), MeOH, 2 h; ii. NaBH3CN, 16 h; (e) TFA/H2O/CH2Cl2/thioanisole/anisole/EDT (45:1:1:1:1:1), 3 h.
OH with the amino resin residue using standard HBTU cou-
pling methodology. Removal of the Fmoc protecting group
gave intermediate 10. The second amino acid Fmoc-R1-OH
(1–8) was introduced using the same coupling method. After
Fmoc deprotection giving 11, the final step consisted in the
introduction of the heterocycle R2 via reductive amination of
its appended aldehyde function (R2-CHO, compounds A–H)
and gave the solid-phase compound 12. This reaction was car-
ried out using a two-step procedure to limit the formation of
the disubstituted amine: i) reaction between amine and alde-
hyde to give imine ii) reduction by NaBH3CN. The reaction
was optimized by varying several parameters: i) aldehyde con-
centration (10–500 mmol l–1) ii) reaction time (30–180 min)
iii) solvent polarity (MeOH or CH2Cl2/DMF) and iv) pH (by
adding CH3COOH or diisopropylethylamine). The best con-
version rates were observed when aldehyde concentration was
200 mmol l–1, reaction time was 2 h, solvent was MeOH and
pH was that of the methanolic solution. Apart from A1, forma-
tion of the by-product resulting from double reductive amina-
tion could be explained by adsorption of the aldehyde on the
resin.
TFA (in the presence of scavengers) mediated cleavage of
peptidomimetic 12 from the resin and gave dipeptide 13 as C-
terminal amide. Purity of each dipeptide was checked by LC-
MS analysis (> 80% purity). Peptidomimetics synthesized with
this method proved to be suitable for biological studies.
The whole library was tested for antiproliferative activity on
L1210 (murine leukemia) and DU145 (prostate) cell lines.
Only eight molecules showed inhibition > 80% at 100 μM on
L1210 or DU145: compounds A1, A5–A7, D7, E7, G7, H7
were selected for solution-phase resynthesis on a larger scale
and for additional biological assessment (Fig. 4).
Fig. 4. Structure of compounds A1, A5–A7, D7, E7, F7, G7.

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P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
Scheme 2. Reagents and conditions: (a) Boc-(S)-β-homoPhe-OH, PyBOP, DIEA, CH2Cl2, rt, 16 h, 65%; (b) i. HCl/Et2O, MeOH, rt, 4 h; ii. 10% NaHCO3, 90%; (c)
R2-CHO (A, D, E, G or H, see Fig. 2), NaBH3CN, MeOH, 3 Å molecular sieves, rt, 16 h, 20–50%; (d) Isonipecotic acid, HBTU, HOBt, DIEA, CH2Cl2, rt, 16 h,
80%; (e) TFA/CH2Cl2 (1:1), rt, 4 h, 95%; (f) i. 1-(4-Cyanobenzyl)-5-formylimidazole, TEA, NaBH3CN, MeOH, 3 Å molecular sieves, 50 °C, 16 h; ii. HCl/
isopropanol, 48–60%; (g) Boc-(S)-β-homoproline, HBTU, HOBt, DIEA, CH2Cl2, rt, 16 h, 86%.
The solution-phase synthesis of the selected compounds is
illustrated on Scheme 2.
3. Results and discussion
H-(S)-Phe-NH2·(HCl) was coupled with Boc-(S)-β-
homoPhe-OH, isonipecotic acid or Boc-(S)-β-homoPro to yield
dipeptides 14, 16 or 18. The Boc protecting group was re-
moved under standard conditions (HCl or TFA) and the result-
ing amines were subjected to reductive amination with alde-
hydes A, D, E, G or H to give final compounds A5–A7, D7,
E7, G7 or H7.
Another synthetic pathway was considered for the synthesis
of A1 (Scheme 3) since its isolation and purification failed
using the previous synthesis strategy.
H-(S)-β-Ala-OMe·HCl was then subjected to reductive ami-
nation with 1-cyanobenzyl-1H-5-formylimidazole A to give
secondary amine 20. After protection of amine as carbamate
(Boc) and saponification of the ester function of 21 with 2 N
aqueous NaOH, carboxylic acid 22 was coupled with H-(S)-
Phe-NH2 to produce amide 23; the final deprotection of amine
(TFA) provided compound A1.
The eight resynthesized compounds were tested for their
ability to inhibit FTase catalyzed transfer of the FPP moiety
to dansyl-CVIM and GGTase-I (which catalyses the transfer
of the GGPP moiety to dansyl-GCVLL) [20,21]. This assay
was performed by fluorescence spectrometry using rat brain
cytosol fractions as an enzyme source [22]. Cellular prolifera-
tion was investigated on L1210, DU145, PC3 and LNCaP
cells. Their biological activities are summarized in Tables 1
and 2.
Compounds A1, A5 and A7 inhibit FTase at submicromolar
concentrations and exhibit good selectivity against GGTase-I.
A1 blocks growth of L1210, DU145 and PC3 cells at micro-
molar concentrations, A7 blocks growth of L1210 and LNCaP
in the same concentration range and A5 is the most potent
compound on L1210 cellular assay with an IC50 of 42.25 μM.
The best enzymatic activities were observed with 1-(4-cya-
nobenzyl)-5-methylimidazole containing compounds. This

P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
749
Scheme 3. Reagents and conditions: (a) 1-(4-Cyanobenzyl)-5-formylimidazole, TEA, NaBH3CN, MeOH, 3 Å molecular sieves, rt, 16 h, 65%; (b) (Boc)2O, 2 N
NaOH, dioxane/H2O 4:1, rt, 16 h, 30%; (c) i. 2 N NaOH, MeOH, rt, 3 h; ii. 1 N HCl, 60%; (d) H-(S)-Phe-NH2•HCl, HBTU, HOBt, DIEA, CH2Cl2, rt, 18 h, 62%; (e)
TFA/CH2Cl2 (1:1), rt, 4 h, 98%.
confirmed previous results [15] where this heterocyclic group
constituted an effective cysteine substitute for FTIs. No other
heterocycle was found to have better enzymatic activity in
binding Zn2+.
The inhibition of compound A7 with a β-homoPhe spacer
was four times more potent (IC50 = 28.5 nM) than that of A1
with a β-Ala spacer (IC50 = 117 nM). Thus, a supplementary
benzyl group led to an increase in FTase inhibition which can
be explained by its satisfactory position and favorable interac-
tions on the FTase active site. Moreover, inhibitors A1 and A7
exhibited their competitive nature (Table 1).
Table 1
a
FTase and GGTase I activities of compounds A1, A5–A7, D7, E7, F7, G7
Finally, molecular docking, using the modeling program
GOLD, was performed to study the interaction of the most po-
tent compounds A1, A5 and A7 (Figs. 5–7) with the enzyme
and to provide a reasonable explanation for the inhibitory ac-
tivity of these structures.
Compound
IC50 (nM)
GGTase-I
Kapp (nM)
FTase
A1
A5
A6
A7
D7
E7
G7
H7
a
117 10
57.3 9.0
> 40 000
123 11
b
b
nd
nd
nd
nd
b
b
b
nd
28.5 4.2
1200 112
> 40 000
328 21
254 18
> 40 000
54.5 9.2
In addition to coordination observed between the distal ni-
trogen of imidazole and Zn2+ used as starting fragments for this
b
b
nd
nd
nd
nd
nd
nd
b
b
b
> 40 000
> 40 000
b
Data are given as mean S.E.M. of three independent experiments.
Not determined.
b
Table 2
a
Biological activities of compounds A1, A5–A7, D7, E7, F7, G7 on cell
growth
b
Compound
IC50 (μM) or % inhibition
L1210
11%
42.25
3%
41%
14%
10%
17%
31%
DU145
5%
PC3
4%
LNCaP
c
A1
A5
A6
A7
D7
E7
G7
H7
a
ns
nd
nd
c
c
d
ns
nd
ns
ns
ns
nd
ns
d
d
d
c
c
27%
38%
15%
17%
11%
c
5%
c
39%
33%
11%
ns
ns
c
14%
Fig. 5. Docking of inhibitor A1 (in black) in the FTase binding site. The FPP,
Arg202β, Tyr166α and the hydrophobic pocket defined by the aromatic side
chains of Trp102β, Trp106β, Tyr361β are shown in grey. The hydrogen bond
between N–H and C=O is highlighted by a dotted line.
Data are given as mean S.E.M. of three independent experiments.
Compounds tested at the concentration of 10 μM.
Not significant.
b
c
d
Not determined.

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P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
docyclic conformation, as does the piperidinic residue in A5,
and then satisfactorily positioning the aryl residue of Phe-NH2
in the A2 binding site and favoring the fit of the supplementary
benzyl of A7 in the exit groove. No interaction of the C-term-
inal amide residue with the active site was observed.
A1, A5 and A7 contained respectively a β-Ala, a piperidinyl
or an (S)-β-homoPhe spacer. Thus, these scaffolds constitute
interesting patterns to position the FTase recognition elements
in the FTase binding site. These results could be used to design
new FTIs to improve enzymatic and cellular activities.
4. Conclusion
We have reported herein the preparation of a library of po-
tent CA1A2X-based FTIs using parallel solid-phase synthesis.
MultipinTM methodology proved to be a very convenient meth-
od for parallel multi-step synthesis. Although these peptidomi-
metics are unlikely to be drug candidates themselves, they
could be valuable leads for developing new FTIs. This
CA1A2X-based library provides an interesting new tool to im-
prove structure-activity relationships and thus to propose new
efficient structures. We identified three molecules, A1, A5 and
A7, containing respectively a β-Ala, a piperidinyl or an (S)-β-
homoPhe spacer. Optimization of these hits through non-pepti-
dic inhibitors will be described in a future paper.
Fig. 6. Docking of inhibitor A5 (in black) in the FTase binding site. The FPP,
Arg202β, Tyr166α and the hydrophobic pocket defined by the aromatic side
chains of Trp102β, Trp106β, Tyr361β are shown in grey.
Recently, protein farnesylation has been identified in trypa-
nosomatids and in the malaria parasite; FTIs have been shown
to be toxic towards these parasites [24,25]. Results of antipar-
asitic activity on the chloroquine-resistant FcB1R strain of
Plasmodium falciparum revealed no significant activity for
the components of this library. In vitro antiplasmodial activity
was determined using a previously described assay [26].
Recent studies have, however, suggested that the cytotoxic
actions of FTIs are not exclusively due to the inhibition of Ras
proteins and have indicated that other farnesylated protein tar-
gets, other than Ras, have to be considered [27,28]. These can-
didate targets include other Ras-family GTPases such as RhoB
[29] and Rheb [30], the centromere-binding proteins CENP-E
and CENP-F [31,32], the phosphatases PRL-1, -2 and -3, [33]
or an unidentified protein that functions as activator of the PI3-
K/Akt pathway [34]. These arguments may explain the ob-
served discordance between the enzymatic and cellular activ-
ities of the inhibitors described here.
Fig. 7. Docking of inhibitor A7 (in black) in the FTase binding site. The FPP,
Arg202β, Tyr166α, Cys95β, Leu96β and the hydrophobic pocket defined by
the aromatic side chains of Trp102β, Trp106β, Tyr361β are shown in grey. The
hydrogen bond between N–H and C=O is highlighted by a dotted line.
5. Experimental protocols
study, docking revealed an interaction between Phe-NH2 and a
hydrophobic pocket, called the A2 binding site, delineated by
Trp102β, Trp106β and Tyr361β in the β-subunit [23]. The X
residue was positioned in the hydrophobic pocket where the A2
residue fits: A1, A5 and A7 constituted three mimetic inhibi-
tors of the CA1A2 sequence. This study also revealed an addi-
tional interaction between the A7 homoPhe spacer and the exit
groove (partly defined by Cys95β and Leu96β) that could en-
hance inhibitory activity. Docking also revealed a hydrogen
bonding between the secondary amine and the amide oxygen
of these compounds A1 and A7, giving the molecules a pseu-
5.1. Chemistry
SynPhaseTM crowns, deepwell microtiter plates (8 × 12),
and MultipinTM stems were purchased from Chiron Technolo-
gies (San Diego, CA). Analytical thin-layer chromatography
was performed on precoated Kieselgel 60F254 plates from
Merck; the spots were located by UV (254 and 366 nm). Silica
gel 60 (230–400 mesh) purchased from Merck was used for
column chromatography. The structures of resynthesized com-
pounds were supported by IR (FT-Bruker Vector 22 instru-
1
ment, neat) and by H NMR at 300 MHz (Bruker DRX-300

P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
751
spectrometer). Chemical shifts were reported in ppm using tet-
ramethylsilane as a standard, J values are in hertz, and the
splitting patterns were designated as follows: s, singlet; d,
doublet; t, triplet; q, quadruplet; m, multiplet; bs, broad singlet;
dd, doublet of doublet. Melting points were determined with a
Büchi 530 capillary melting point apparatus and remain uncor-
rected. Mass spectra were recorded on a quadripolar Finnigan
Mat SSQ 710 instrument. Elemental analyses of resynthesized
compounds were performed by the “Service Central d’Ana-
lyses” at the CNRS, Vernaison (France).
were decanted. LC-MS were recorded for all the products. The
corresponding molecular ion was observed for each peptidomi-
metic, and the HPLC indicated one major product in each case.
5.1.2. Compounds resynthesized in solution-phase
for biological evaluation
5.1.2.1. N-tert-butoxycarbonyl-(S)-β-homoPhe-(S)-Phe-NH2
(14). Diisopropylethylamine (1.90 ml, 10.7 mmol) and PyBOP
(2.79 g, 5.37 mmol) were added to a solution of Boc-(S)-β-
homoPhe-OH (1 g, 3.58 mmol) in 50 ml of dry CH2Cl2 at
room temperature. After 2 h, H-(S)-Phe-NH2·HCl (719 mg,
3.58 mmol) was added and the mixture was stirred for 16 h
at room temperature. The solvent was then removed under re-
duced pressure. The residue was dissolved in AcOEt and
washed with 5% aqueous NaHCO3, 0.25 N HCl and H2O.
The organic layer was dried over MgSO4 and the solvent was
evaporated. The residue was triturated with CH2Cl2/Et2O (1:1)
and the white solid was filtered and dried to give 14 (65%). m.
p. 180–181 °C; IR (cm–1): 1687, 1677, 1647; 1H NMR
(DMSO-d6): 1.39 (s, 9H), 1.72 (m, 2H), 2.48–2.52 (m, 1H),
2.80–2.87 (m, 1H), 2.99–3.03 (m, 1H), 3.32–3.34 (m, 1H),
3.80–3.83 (m, 1H), 4.43–4.49 (m, 1H), 7.12–7.29 (m, 12H),
7.41 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H); MS (CI): 426 (MH+).
Anal. (C24H31N3O4) C, H, N.
5.1.1. Library solid-phase synthesis
Solid-phase synthesis was performed using SynPhase™-
MD (methacrylic acid/dimethylacrylamide) I-series crowns
(pins) derivatized with Fmoc-protected rink amide linkers.
The 64 products were synthesized in deepwell microtiter plates
(8 × 12) using Multipin™ stems to support the pins. Peptido-
mimetic synthesis was carried out at room temperature without
agitation. The following standard purification and deprotection
procedures were used: the stems were suspended in a plastic
wash bath, the attached pins were washed successively with
DMF (3 × 5 min), MeOH (1 × 5 min) and CH2Cl2
(3 × 5 min) and the pins were then air-dried to give the solid-
phase intermediates. Deprotection of the Fmoc protected
amines was obtained using 25% piperidine in DMF for
20 min. After deprotection and purification of 64 pins by the
standard procedure, the first coupling of Fmoc-(S)-Phe-OH was
achieved by immersing each pin in the well of a microtiter
plate. Each well contained 400 μl of a DMF solution of
Fmoc-(S)-Phe-OH (120 mmol l–1), HBTU (120 mmol l–1),
and DIEA (144 mmol l–1). After 6 h, the pins were removed
from the microtiter wells, washed by the standard procedure
and allowed to air-dry. The pins were deprotected following
the above conditions, washed and allowed to air-dry. The sec-
ond coupling reaction and deprotection were performed using
the same methodology. Each pin was then immersed in a mi-
crotiter plate wherein each well contained 400 μl of a solution
of the appropriate Fmoc-(S)-amino acid 1–8 (120 mmol l–1),
HBTU (120 mmol l–1) and DIEA (144 mmol l–1) in DMF.
After 6 h, the pins were removed from the microtiter wells
and deprotected by washing and air-drying. The pins were de-
protected using the above conditions, washed and allowed to
air-dry. Reductive amination was then obtained by immersing
each pin in a microtiter plate wherein each well contained 400
μl of an MeOH solution of the appropriate aldehyde A–H
(200 mmol l–1). After 2 h, the pins were removed from the
microtiter wells, washed with MeOH (3 × 5 min), CH2Cl2
(2 × 5 min) and allowed to air-dry. The pins were then im-
mersed in 400 μl of an MeOH solution of NaBH3CN
(240 mg in 30 ml MeOH). After 16 h, the pins were removed
from the microtiter plate, washed with MeOH (3 × 5 min),
CH2Cl2 (2 × 5 min), and allowed to air-dry. The peptidomi-
metics were cleaved from the pins using TFA/H2O/CH2Cl2/
thioanisole/anisole/1,2-ethanedithiol (90:2:2:2:2:2) for 3 h,
and the solvents were removed under reduced pressure. A sol-
vent mixture containing Et2O/petroleum ether (1:2) was added
to each well, the solutions were centrifuged, and the solvents
5.1.2.2. H-(S)-β-homoPhe-(S)-Phe-NH2 (15). An HCl-saturated
Et2O solution was added to a solution of carbamate 14 (1 g,
2.35 mmol) in 20 ml of MeOH. The mixture was stirred for 4 h
at room temperature, and the solvent evaporated. The residue
was dissolved in 10% aqueous NaHCO3 and extracted with
AcOEt. The organic layer was dried over MgSO4 and evapo-
rated to give 15 as a colorless oil (90%). IR (cm–1): 3393,
1
1632; H NMR (CDCl3): 1.69–1.71 (m, 2H), 1.96–2.08 (m,
2H), 2.52–2.57 (m, 2H), 2.81–3.08 (m, 1H), 3.14–3.16 (m,
1H), 3.19–3.38 (m, 1H), 4.63–4.70 (m, 1H), 5.48 (s, 1H),
6.20 (s, 1H), 7.12–7.31 (m, 10H), 7.88 (d, J = 7.8 Hz, 1H),
MS (CI): 326 (MH+). Anal. (C19H23N3O2) C, H, N.
5.1.2.3. General procedure for the synthesis of A7, D7, E7,
G7 and H7. Molecular sieves (3 Å) and NaBH3CN (0.55
mmol) were added to a stirred solution of amine 15 (0.2 g,
0.55 mmol) and appropriate aldehyde A, D, E, G or H (0.44
mmol) in 30 ml of dry MeOH. The reaction mixture was stirred
at room temperature for 16 h in a dry nitrogen atmosphere. The
molecular sieves were filtered off and the solvent was removed
under reduced pressure. The residue was dissolved in AcOEt
and washed successively with 5% aqueous NaHCO3, H2O and
brine. The organic layer was dried over MgSO4 and evapo-
rated. The residue was purified by column chromatography
(CH2Cl2/MeOH 9:1).
5.1.2.3.1. [N-(4-cyanobenzyl-5-imidazolylmethyl)]-(S)-β-
homoPhe-(S)-Phe-NH2 (A7). White solid; yield 50%; m.p.
61–62 °C; IR (cm−1): 2229, 1654; 1H NMR (DMSO-d6):
2.32–2.40 (m, 2H), 2.72–2.91 (m, 2H), 3.01–3.28 (m, 2H),
3.54–3.59 (m, 2H), 4.28–4.33 (m, 1H), 4.53–4.65 (m, 1H),

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P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
5.35 (s, 2H), 6.68 (s, 1H), 6.81–7.85 (m, 18H), 8.02 (d,
J = 9.1 Hz, 1H); MS (CI): 521 (MH+). Anal. (C31H32N6O2)
C, H, N.
5.1.2.4.2. 1-tert-Butoxycarbonyl-(S)-β-homoPro-(S)-Phe-NH2
(18). White solid; yield 86%; m.p. 96–97 °C; IR (cm–1): 1682,
1680, 1633; H NMR (CDCl3): 1.46 (s, 9H), 1.50–2.25 (m,
1
4H), 2.55–2.65 (m, 2H), 2.90–3.12 (m, 2H), 3.18–3.35 (m,
2H), 3.98–4.15 (m, 1H), 4.70–4.78 (m, 1H), 5.90 (bs, 1H),
6.78 (bs, 2H), 7.19–7.35 (m, 5H); MS (CI): 376 (MH+). Anal.
(C20H29N3O4) C, H, N.
5.1.2.3.2. [N-(3-quinolylmethyl)]-(S)-β-homoPhe-(S)-Phe-NH2
(D7). White solid; yield 20%; m.p. 273–274 °C; IR (cm–1):
3312, 1644; H NMR (DMSO-d6): 2.77–2.83 (m, 2H), 3.18–
1
3.22 (m, 2H), 3.30–3.37 (m, 2H), 3.60–3.69 (m, 2H), 4.20–
4.26 (m, 1H), 4.65–4.75 (m, 1H), 7.40–8.10 (m, 18H), 8.20
(d, J = 8.2 Hz, 1H), 8.81 (s, 1H); MS (CI): 466 (MH+). Anal.
(C29H30N4O2) C, H, N.
5.1.2.5. General procedure for the preparation of amines 17
and 19. Carbamate 16 or 18 was dissolved in 20 ml of a solu-
tion of TFA/CH2Cl2 (1:1). The reaction mixture was stirred at
room temperature for 4 h. The solvent was then evaporated.
The residue was triturated in Et2O and the solid was filtered
and dried.
5.1.2.3.3. [N-(3-thienylmethyl)]-(S)-β-homoPhe-(S)-Phe-NH2
(E7). White solid; yield 20%; m.p. 75–76 °C; IR (cm–1): 3312,
1649; 1H NMR (DMSO-d6): 2.35–2.60 (m, 2H), 2.72–2.88 (m,
2H), 2.89–3.07 (m, 2H), 3.35–3.42 (m, 3H), 4.45–4.72 (m,
1H), 7.10–7.25 (m, 14H), 7.44–7.64 (m, 2H), 8.10 (d,
J = 8.0 Hz, 1H); MS (EI): 421 (M+). Anal. (C24H27N3O2S) C,
H, N.
5.1.2.5.1. Piperidine-4-carbonyl-(S)-Phe-NH2 trifluoroacetate
(17). White solid; yield 95%; m.p. 206–207 °C; IR (cm–1):
1
1672, 1640; H NMR (DMSO-d6): 1.38–1.50 (m, 1H), 1.61–
1.79 (m, 3H), 2.39–2.47 (m, 1H), 2.69–2.90 (m, 3H), 3.00–
3.07 (m, 1H), 3.13–3.26 (m, 2H), 4.41–4.49 (m, 1H), 7.09 (s,
1H), 7.17–7.28 (m, 5H), 7.52 (s, 1H), 8.11 (d, J = 8.8 Hz, 1H),
8.34 (bs, 1H), 8.69 (bs, 1H); MS (CI): 276 (MH+). Anal.
(C15H21N3O2·CF3COOH) C, H, N.
5.1.2.3.4. {N-[4-(1H-imidazol-1-yl)benzyl]}-(S)-β-homoPhe-
(S)-Phe-NH2 (G7). Yellow solid; yield 40%; m.p. 166–167 °C;
IR (cm–1): 3301, 1666, 1635; 1H NMR (DMSO-d6): 2.30–2.51
(m, 2H), 2.75–2.86 (m, 2H), 2.89–3.07 (m, 2H), 3.21–3.35 (m,
1H), 3.45–3.58 (m, 2H), 4.57–4.73 (m, 1H), 6.96–7.82 (m,
20H), 8.10 (d, J = 8.2 Hz, 1H); MS (CI): 482 (MH+). Anal.
(C29H31N5O2) C, H, N.
5.1.2.5.2. (S)-H-β-homoPro-(S)-Phe-NH2 trifluoroacetate (19).
White solid; yield 95%; m.p. 198–200 °C; IR (cm–1): 1680,
1643; 1H NMR (DMSO-d6): 1.60–1.90 (m, 4H), 2.45–2.70
(m, 4H), 3.18–3.30 (m, 2H), 3.45–3.62 (m, 1H), 4.45–4.55
(m, 1H), 7.15 (s, 1H), 7.18–7.30 (m, 5H), 7.56 (s, 1H), 8.42
(d, J = 8.4 Hz, 1H), 8.70 (bs, 1H), 9.02 (bs, 1H); MS (CI): 276
(MH+). Anal. (C15H21N3O2·CF3COOH) C, H, N.
5.1.2.3.5. {N-[(1H-indol-3-ylmethyl)]}-(S)-β-homoPhe-(S)-
Phe-NH2 (H7). Brown solid; yield 35%; m.p. 187–189 °C; IR
1
(cm–1): 1664, 1633; H NMR (DMSO-d6): 2.28–2.49 (m, 2H),
2.68–2.87 (m, 2H), 2.91–3.07 (m, 2H), 3.27–3.38 (m, 1H),
3.83–3.98 (m, 2H), 4.49–4.68 (m, 1H), 7.11–7.92 (m, 19H),
8.07 (d, J = 8.2 Hz, 1H); MS (CI): 455 (MH+). Anal.
(C28H30N4O2) C, H, N.
5.1.2.6. General procedure for the preparation of amines A5
and A6. 1-Cyanobenzyl-5-formylimidazole (361 mg, 1.71
mmol), 3 Å molecular sieves were added to a stirred solution
of amines 17 or 19 (2.57 mmol) and triethylamine (0.36 ml,
2.57 mmol) in 30 ml of dry MeOH before NaBH3CN was
added (118 mg, 1.88 mmol) after 2 h at 50 °C in a dry nitrogen
atmosphere. The reaction mixture was stirred at 50 °C for 16 h.
The molecular sieves were filtered off and the solvents were
removed under reduced pressure. The residue was dissolved
in AcOEt and washed successively with 5% aqueous
NaHCO3, H2O and brine. The organic layer was dried over
MgSO4 and evaporated. The residue was purified by column
chromatography (CH2Cl2/MeOH 95:5) and hydrochloride was
obtained by adding a 5–6 N isopropanolic HCl solution. The
salt was then washed with Et2O and AcOEt and dried.
5.1.2.4. General procedure for the synthesis of carbamates 16
and 18. Diisopropylethylamine (4.5 ml, 26.2 mmol), HBTU
(4.97 g, 13.1 mmol) and HOBt (590 mg, 4.36 mmol) were
added to a solution of isonipecotic acid or Boc-(S)-β-homoPro
(8.72 mmol) in 60 ml of dry CH2Cl2. After 2 h, H-(S)-Phe-
NH2·HCl (1.75 g, 8.72 mmol) was added and the mixture
was stirred for 16 h at room temperature. The salts were fil-
tered off and the filtrate was washed with 0.1 N HCl, H2O
and brine. The organic layer was dried over MgSO4 and the
solvent was then removed under reduced pressure. The residue
was purified by column chromatography (CH2Cl2/MeOH
49:1).
5.1.2.4.1. 1-tert-Butoxycarbonylpiperidine-4-carbonyl-(S)-Phe-
5.1.2.6.1. 1-{[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl}pi-
peridine-4-carbonyl-(S)-Phe-NH2 hydrochloride (A5). White
solid; yield 60%; m.p. 235–237 °C; IR (cm−1): 2237, 1673,
1628; 1H NMR (DMSO-d6): 1.69–1.78 (m, 2H), 2.05–2.12
(m, 2H), 2.59–2.77 (m, 4H), 3.00 (dd, J = 6.5 Hz, 1H), 3.16–
3.20 (m, 2H), 3.36 (s, 2H), 4.39–4.49 (m, 1H), 5.34 (s, 2H),
7.05 (s, 1H), 7.19–7.28 (m, 5H), 7.43 (s, 1H), 7.53 (d,
J = 8.4 Hz, 2H), 7.91 (d, J = 8.5 Hz, 2H), 8.15 (s, 1H), 8.64
NH2 (16). White solid; yield 80%; m.p. 165–166 °C; IR
1
(cm–1): 1685, 1670, 1636; H NMR (CDCl3): 1.46 (s, 9H),
1.51–1.53 (m, 1H), 1.56–1.62 (m, 1H), 2.17–2.30 (m, 1H),
2.66–2.74 (m, 2H), 2.99–3.22 (m, 3H), 3.66–3.76 (m, 1H),
4.02–4.11 (m, 2H), 4.71 (q, J = 7.3 Hz, 1H), 5.79 (bs, 1H),
6.33 (bs, 1H), 6.52 (d, J = 8.5 Hz, 1H), 7.19–7.33 (m, 5H);
MS (CI): 376 (MH+). Anal. (C20H29N3O4) C, H, N.

P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
753
(d, J = 8.5 Hz, 1H), 9.23 (s, 1H), 11.56 (bs, 1H); MS (CI): 471
1 N aqueous HCl until pH 3–4 and the organic layer was ex-
tracted, dried over MgSO4 and evaporated. The residue was
purified by column chromatography (CH2Cl2/MeOH 9:1) to
give 22 as a white solid (60%); m.p. 123–124 °C; IR (cm–1):
3396, 2231, 1695, 1686; 1H NMR (CDCl3): 1.37 (s, 9H),
2.44–2.47 (m, 2H), 3.35–3.37 (m, 2H), 4.45 (s, 2H), 5.31–
5.33 (m, 2H), 7.13–7.19 (m, 3H), 7.62–7.65 (m, 3H); MS
(CI): 385 (MH+). Anal. (C20H24N4O4) C, H, N.
(MH+). Anal. (C27H30N6O2·2.5 HCl·5 H2O) C, H, N.
5.1.2.6.2. 1-{[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl}-
(S)-β-homoPro-(S)-Phe-NH2 hydrochloride (A6). White solid;
yield 48%; m.p. 224–226 °C; IR (cm–1): 2231, 1665, 1636;
1H NMR (DMSO-d6): 1.70–2.00 (m, 4H), 2.45–2.75 (m, 2H),
2.90–3.20 (m, 2H), 3.30–3.50 (m, 2H), 3.72–3.84 (m, 1H),
4.29–4.49 (m, 2H), 4.53–4.69 (m, 1H), 5.77 (s, 2H), 7.15–
7.32 (m, 7H), 7.52 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.5 Hz,
2H), 8.12 (s, 1H), 8.64 (d, J = 8.5 Hz, 1H), 9.22 (s, 1H),
11.73 (bs, 1H); MS (CI): 471 (MH+). Anal. (C27H30N6O2·2
HCl·3 H2O) C, H, N.
5.1.2.10. 3-[(tert-Butoxycarbonyl)-3-{[1-(4-cyanobenzyl)-1H-
imidazol-5-yl]methyl}-β-Ala-(S)-Phe-NH2 (23). Diisopropy-
lethylamine (0.27 ml, 1.56 mmol), HBTU (296 g, 0.78 mmol)
and HOBt (35.1 mg, 0.26 mmol) were added to a solution of
acid 22 (200 mg, 0.52 mmol) in 20 ml of dry CH2Cl2. After
2 h, H-(S)-Phe-NH2·HCl (104 mg, 0.52 mmol) was added and
the mixture was stirred for 18 h at room temperature. The salts
were filtered off and the filtrate was washed with 0.1 N HCl,
H2O and brine. The organic layer was dried over MgSO4 and
the solvent was then removed under reduced pressure. The re-
sidue was purified by column chromatography (CH2Cl2/MeOH
91:9) to give 23 as a white solid (62%); m.p. 130–131 °C; IR
5.1.2.7. Methyl 3-({[1-(4-cyanobenzyl)-1H-imidazol-5-yl]
methyl}-β-Ala-OMe (20). 1-(4-Cyanobenzyl)-5-formylimida-
zole (3 g, 14.2 mmol) and 3 Å molecular sieves were added
to a stirred solution of H-β-Ala-OMe·HCl (4.96 g, 35.5 mmol)
and triethylamine (5 ml, 35.5 mmol) in 80 ml of dry MeOH,
before NaBH3CN was added (983 mg, 15.6 mmol) after 2 h at
room temperature in a dry nitrogen atmosphere. The reaction
mixture was stirred at room temperature for 16 h. The molecu-
lar sieves were filtered off and the solvent was removed under
reduced pressure. The residue was dissolved in AcOEt and
washed successively with 5% aqueous NaHCO3, H2O and
brine. The organic layer was dried over MgSO4 and evapo-
rated. The residue was purified by column chromatography
(CH2Cl2/MeOH 95:5) to give 20 as a colorless oil (65%); IR
1
(cm–1): 2230, 1695, 1678, 1672; H NMR (CDCl3): 1.32 (s,
9H), 2.28–2.33 (m, 2H), 3.03–3.11 (m, 2H), 3.26–3.30 (m,
2H), 4.21–4.32 (m, 2H), 4.59–4.66 (m, 1H), 5.29–5.31 (m,
2H), 5.53 (bs, 1H), 6.39 (bs, 1H), 7.07–7.11 (m, 3H), 7.21–
7.33 (m, 6H), 7.55 (s, 1H), 7.61 (d, J = 8.2 Hz, 2H); MS
(CI): 531 (MH+). Anal. (C29H34N6O4) C, H, N.
1
(cm–1): 2230, 1732; H NMR (CDCl3): 2.42 (t, J = 6.0 Hz,
5.1.2.11. 3-{[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl}-β-
Ala-(S)-Phe-NH2 trifluoroacetate (A1). Carbamate 23 (50 mg,
0.12 mmol) was dissolved in 10 ml of a solution of
TFA/CH2Cl2 (1:1). The reaction mixture was stirred at room
temperature for 4 h. The solvent was then evaporated. The re-
sidue was triturated in Et2O and the white solid was filtered
and dried to give A1 (98%); m.p. 86–89 °C; IR (cm–1): 2230,
2H), 2.81 (t, J = 6.0 Hz, 2H), 3.59 (s, 2H), 3.67 (s, 3H), 5.35
(s, 2H), 7.00 (s, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.52 (s, 1H),
7.63 (d, J = 8.2 Hz, 2H); MS (CI): 299 (MH+). Anal.
(C16H18N4O2) C, H, N.
5.1.2.8. Methyl 3-[(tert-butoxycarbonyl)-3-{[1-(4-cyano-
1
benzyl)-1H-imidazol-5-yl]methyl}-β-Ala-OMe (21). Di-tert-bu-
tyl dicarbonate (1.65 g, 7.55 mmol) and 2 N aqueous NaOH
(3.77 ml, 7.55 mmol) were added to a solution of amine 20
(1.5 g, 5.03 mmol) in 50 ml of a mixture dioxane/H2O (4:1).
The reaction mixture was stirred at room temperature for 16 h.
The solvents were then evaporated, the residue was dissolved
in AcOEt and washed with H2O and brine. The organic layer
was dried over MgSO4 and concentrated under reduced pres-
sure. The residue was purified by column chromatography
(CH2Cl2/MeOH 96:4) to give 21 as a yellow oil (30%); IR
1695, 1680; H NMR (DMSO-d6): 2.69–2.82 (m, 2H), 2.98–
3.15 (m, 4H), 4.15–4.35 (m, 2H), 4.38–4.50 (m, 1H), 5.62 (bs,
2H), 7.14 (s, 1H), 7.20–7.25 (m, 7H), 7.40 (d, J = 8.2 Hz, 2H),
7.58 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 8.43 (d, J = 8.1 Hz, 1H),
9.05 (bs, 2H); MS (CI): 545 (MH+). Anal.
(C24H26N6O2·CF3COOH) C, H, N.
5.2. Biology
1
(cm–1): 2230, 1735, 1686; H NMR (CDCl3): 1.34 (s, 9H),
5.2.1. Preparation of FTase from the cytosolic fraction of rat
brain [22]
2.38–2.46 (m, 2H), 3.27–3.31 (m, 2H), 3.70 (s, 3H), 4.44 (s,
2H), 5.26–5.30 (m, 2H), 7.07–7.10 (m, 3H), 7.53–7.63 (m,
3H); MS (CI): 399 (MH+). Anal. (C21H26N4O4) C, H, N.
Sprague-Dawley male rats (9–10 weeks old) were sacri-
ficed; brains were removed and immediately placed in an ice-
cold 0.1 M HEPES buffer, pH 7.4, 25 mM MgCl2 and 10 mM
DTT (dithiothreitol). The brains were cleaned and homoge-
nized in a 0.1 M HEPES buffer, pH 7.4, 1 mM MgCl2 and
1 mM DTT. The homogenates were centrifuged at 10 000 g
for 20 min at 4 °C. The supernatants were centrifuged at 100
000 g for 1 h at 4 °C. The cytosolic fraction (the 100 000 g
supernatants) was stored at –80 °C. Protein concentration was
determined by the bicinchoninic acid (BCA) protein assay
5.1.2.9. 3-[(tert-Butoxycarbonyl)-3-{[1-(4-cyanobenzyl)-1H-
imidazol-5-yl]methyl}amino]-β-Ala-OH (22). 2 N Aqueous
NaOH (0.80 ml, 1.60 mmol) was added to a solution of ester
21 (320 mg, 0.80 mmol) in 5 ml of MeOH. The reaction mix-
ture was stirred at room temperature for 3 h and then neutra-
lized with 1 N aqueous HCl until pH 5–6. MeOH was evapo-
rated and replaced by AcOEt. The mixture was acidified with

754
P. Gilleron et al. / European Journal of Medicinal Chemistry 41 (2006) 745–755
method (Pierce) using bovine serum albumin as protein stan-
dard.
tor 66 described by Bell [37] was obtained from its X-ray crys-
tal structure in the RCSB Protein Data Bank (1LD7). Flexible
docking of A1, A5 and A7 into the enzyme active site was
performed using GOLD software [38]. The distance observed
in the crystal structure between distal nitrogen imidazole and
the zinc cation, was applied as a constraint. For each com-
pound, the most stable docking model was selected from the
best conformation predicted by the GoldScore [38] and X-
Score [39] scoring functions. The complexes were energy-
minimized using the Powell method available in the Maximin2
procedure with the Tripos force field and a dielectric constant
of 4.0 until the gradient value reached 0.01 kcal mol–1 Å–1.
5.2.2. Protein prenyl transferase assay
FTase activity was determined by a continuous fluorescence
assay (modified by us [20]), as previously described [21]. The
data was collected on a Spex Fluoro Max spectrofluorimeter.
Optimal parameters are an excitation wavelength at 340 nm
and an emission at 505 nm (slit 10,10) for dansyl-CVIM and
dansyl-GCVLL. The standard reaction mixture containing
50 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 10 μM ZnCl2,
5 mM DTT, 0.01% n-dodecyl-β-D-maltoside, 5.4 mg ml–1 of
cytosolic protein, 1.3 μM dansyl-CVIM and 25 μM FPP for the
FTase assay, and 4 μM dansyl-GCVLL and 10 μM GGPP for
the GGT-I assay, was incubated at 25 °C, and fluorescence
intensity was recorded for 10 min. A study of cross prenylation
of dansyl-CVIM by GGTase with FPP or GGPP was per-
formed. No variation in fluorescence was observed in any case.
Graphical representations of fluorescence variation ΔF/Δt = f
[ligand] gave sigmoid plots. Analysis of cell curves made it
possible to calculate the IC50 values of each compound by
using a theoretical equation (Graphpad Prism 3.03, Graphpad
software, San Diego, CA).
Acknowledgements
The authors are grateful to the “Association pour la Re-
cherche contre le Cancer” for their financial support.
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Molecular modeling studies were performed using SYBYL
software version 6.92 [35] running on a Silicon Graphics work-
station. Three-dimensional models of compounds A1, A5 and
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