From pure FPP to mixed FPP and CAAX competitive inhibitors of farnesyl protein transferase

Article abstract of DOI:10.1016/S0960-894X(03)00171-9

Starting from a FPP analogue with nanomolar inhibitory activity against isolated FPTase, yet lacking activity in cellular assays, structural modifications were performed to enhance cellular activity by removing all acidic functionalities. Overall, these changes resulted in the transformation of a pure FPP to a mixed FPP and CAAX competitive inhibitor with nanomolar activity on isolated FPTase and micromolar inhibitory activity in the farnesylation of H-Ras in cultured DLD-1 cells.

Full text of DOI:10.1016/S0960-894X(03)00171-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1459–1462
From Pure FPP to Mixed FPP and CAAX Competitive Inhibitors
of Farnesyl Protein Transferase
Marc Lannuzel,a Marie Lamothe,a,* Philippe Schambel,b Chantal Etievant,c Bridget Hillc
and Michel Pereza
aDepartment of Medicinal Chemistry 4, Centre de Recherche Pierre Fabre, 17, Avenue Jean Moulin, 81106 Castres Cedex, France
bDepartment of Molecular Modeling, Centre de Recherche Pierre Fabre, 17, Avenue Jean Moulin, 81106 Castres Cedex, France
cDepartment of Cancerology, Centre de Recherche Pierre Fabre, 17, Avenue Jean Moulin, 81106 Castres Cedex, France
Received 15November 2002; revised 20 January 2003; accepted 4 February 2003
Abstract—Starting from a FPP analogue with nanomolar inhibitory activity against isolated FPTase, yet lacking activity in cellular
assays, structural modifications were performed to enhance cellular activity by removing all acidic functionalities. Overall, these
changes resulted in the transformation of a pure FPP to a mixed FPP and CAAX competitive inhibitor with nanomolar activity on
isolated FPTase and micromolar inhibitory activity in the farnesylation of H-Ras in cultured DLD-1 cells.
# 2003 Elsevier Science Ltd. All rights reserved.
Farnesyl protein transferase (FPTase) is a zinc metallo-
enzyme that has emerged as a potential target in cancer
chemotherapy.1 Using farnesylpyrophosphate (FPP),
FPTase catalyzes the farnesylation of several proteins
involved in tumorigenesis, particularly Ras proteins2
whose oncogenic forms can be found in about 30% of
all human cancers, Rho B3 involved in receptor traf-
ficking and centromeric proteins4 implicated in mitosis.
FPTase recognizes the last four C-terminal amino acids
of these proteins that match the CAAX formula, where
A’s are essentially aliphatic amino acids and X is a wild
card. Farnesylation occurs at the cysteine of these
CAAX sequences and is essential for the function of
these proteins.
turned our efforts towards the preparation of some of
the most common prodrugs of phosphonic acids such as
bis POM ester7 1b and bis SATE ester8 1c. However
both compounds failed to provide any trace of cellular
activity9 even at high concentration (10 mM). Herein our
second approach is described, consisting of structural
modifications of 1a.
Rationally-designed FPTase inhibitors5 can be divided
into three categories, namely FPP competitive inhibitors
which occupy the FPP site of FPTase, CAAX competi-
tive inhibitors which can displace the protein substrate
and bisubstrate analogues which are designed to occupy
both sites simultaneously. Following our early work on
FPP analogues,6 which identified farnesylphosphonic
acid derivative 1a (Fig. 1) as a potent inhibitor of
FPTase in vitro (IC50=83 nM), structural modifications
were necessary to provide cellular activity. We first
Figure 1.
The first X-ray analyses of rat FPTase complexed with
FPP10 showed that the head of the farnesyl group is
only a few angstroms away from the zinc ion. We deci-
ded to take advantage of this close proximity and to
replace the phosphonic acid moiety of 1a by a func-
tional group capable of chelating the zinc cation. It was
also postulated that this strategy would probably lead
to a bisubstrate instead of a pure FPP analogue.
The syntheses performed in standard solution chemistry
are described in Scheme 1. Direct farnesylation of bis
protected glycine was successful and gave easy access to
*Corresponding author. Fax: +33-5-63-71-42-96; e-mail: marie.
lamothe@pierre-fabre.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00171-9

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M. Lannuzel et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1459–1462
Table 1. Farnesyltransferase inhibitory activities of compounds 1,
2a–d and 3a–d
Compd
Structure
IC50, mM
In vitroa
Cellb
1a
2a
2b
2c
2d
3a
3b
0.083
0.47
0.7
>10
10
nd
nd
1
Scheme 1. Reagents and conditions: (a) (i) LDA, Farnesyl-Br, THF,
À78 ꢀC to rt; (ii) HCl 2N, 85%. (b) (i) 1-Methyl-3H-imidazole-4-
carbaldehyde, DIEA, 1,2-DCE, rt; (ii) AcOH, NaBH(OAc)3, rt, 57%.
(c) PhSO2Cl, pyridine, rt, 84% from 5, 20% from 3c. (d) LiOH, THF,
H2O, rt, 98% for 6, 38% for 2a, 84% from 4, 48% from 5. (e) H-Phe-
OMe.HCl, EDC, HOOBT, DIEA, DCM, rt, 52%; (f) PhCH2NH2,
EDC, HOOBT, DIEA, DCM, rt, 70% from 6, 82% from 7. (g) (i)
PhCH2NH2, PS-carbodiimide, HOBT, DMF; (ii) MP-carbonate,
65%. (h) FmocOSu, Na2CO3, dioxane, H2O, 0 ꢀC to rt, 94%. (i)
Piperidine, DMF, rt, 93%. (j) 4-(5-Formyl-imidazol-1-ylmethyl)-ben-
zonitrile, AcOH, NaBH(OAc)3, 1,2-DCE, rt, 46%.
>10
0.67
0.091
0.2
5
nd
3c
3d
0.006
0.04
1
>10
Scheme 2. Reagents and conditions: (a) Piperidine, DMF. (b) 7, BOP,
DIEA, NMP, 97%. (c) 4-(5-Formyl-imidazol-1-ylmethyl)-benzonitrile,
AcOH, NaBH(OAc)3, 1,2-DCE, 97%. (d) LiOH, THF, MeOH, H2O,
55 ꢀC, 37% for 2b, 93% for 3a, 65% for 3b. (e) PhCH2NCO, DCM. (f)
1-Methyl-3H-imidazole-4-carbaldehyde, AcOH, NaBH(OAc)3, 1,2-
DCE, 82%.
aFPTase inhibitory assay was performed as described in ref 15, using
purified bovine FPTase, 2 mM dansyl-GCVLS and 2mM FPP.
bInhibition of farnesylation of H-Ras in DLD-1 cells as described in
ref 9.
the central core of all molecules, as a racemic mixture.
Reductive amination with 1-methyl-3H-imidazole-4-
carbaldehyde11 could then be performed in two steps to
give 5, followed by saponification and amidation to
yield the benzylamide derivative 2c. Sulfonylation of 5
was achieved in pyridine using 1.5equiv of phenyl-
sulfonyl chloride. It was followed by sequences of
saponifications and amidations to give compounds 2a
and 2d. The cyanobenzyl analogue 3d was prepared
using the same chemistry as that developed for 2d.
However, saponification of the ethyl ester had to be
carried out before the reductive amination with 4-(5-
formyl-imidazol-1-ylmethyl)-benzonitrile12 so as to
avoid hydrolysis of the nitrile group. Several attempts
were made to keep the diphenylmethylene amino pro-
tecting group of the starting protected glycine, however
it was hydrolyzed under saponification conditions.
Therefore two steps of protection and deprotection had
to be incorporated in order to prepare compound 3d.
Other modifications were performed on solid phase
(Scheme 2), leading to the preparation of analogues 2b,
3a and 3b. Yields of the intermediate reactions were
determined after cleavage of a small fraction of the resin
under LiOH conditions. Loading of 7 on the depro-
tected fmoc-Phe-Wang13 resin was completed in one
cycle using 1.5equivalents of the farnesylated glycine.

M. Lannuzel et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1459–1462
1461
Then, reductive amination could be performed in one
step using 3 equiv of the corresponding aromatic alde-
hyde to give 9 and 10 with no trace of dialkylation pro-
ducts. Both resins were treated with phenyl isocyanate
to form the supported urea intermediates. Unexpect-
edly, under cleavage conditions these intermediates
underwent cyclization into hydantoins 2b and 3b,14 an
event resulting in loss of the phenylalanine residue. Com-
pound 3a was obtained by direct cleavage of resin 10.
inhibitory potencies (Table 2). While FPTase inhibition
by 1a was only affected by FPP concentration, enzyme
inhibition by 3c was markedly affected by the peptide
concentration and only partially by FPP concentration.
From these results, we can conclude that 3c behaves as a
mixed FPP and CAAX competitive inhibitor.
Docking studies using the flexible docking program
FlexX16 suggested two docking hypotheses for 3c that
can explain the pharmacological behavior observed.
Using the coordinates of two published X-ray crystal
structures of human FPTase,17 two models were built to
allow the docking of 3c in the FPP competitive inhibitor
hypothesis (active site empty) and in the CAAX com-
petitive inhibitor hypothesis (active site containing
FPP). In the first model, the imidazole ring was used as
the anchor fragment, then FlexX positioned the rest of
the molecule, fragment by fragment, into the active site
searching for favorable interactions between the ligand
and the amino acid residues. The same protocol was
used to test the second hypothesis using the cyanobenzyl
group as the anchor fragment. 3D conformations gen-
erated from these calculations are represented in Figure
2. In both cases, the docking program calculated an
interaction between the imidazole ring and the zinc
cation. In the FPP competitive inhibitor model, no
solution was found that could allow both imidazole
chelation with the zinc cation and farnesyl full occu-
pancy of the FPP hydrophobic pocket. Indeed, the best
solution shows a partial occupancy of this lipophilic site
where only the central isoprene unit can fit. This model
hypothesis fits well with the partial competitive nature
of 3c with regard to FPP, since 3c cannot interact with
the ionic site of FPP due to the removal of all acidic
residues, and can only partially occupy the hydrophobic
site. In the second model, compound 3c can adopt a
conformation that will allow strong interactions
between the three parts of the molecule and the enzyme.
The cyanobenzyl group can adopt the same position as
the cyanobenzyl in the crystallized reference complex,17
making stacking with FPP isoprenoid and pointing
towards Arg202b and Tyr166a. The flexible farnesyl
unit can fold to find hydrophobic interactions in one of
the CAAX substrate lipophilic sites and the benzyl resi-
due comes across a new hydrophobic pocket (far aryl
binding site18) made of Leu295b, Lys294b and Tyr300b.
In the methyl imidazole series, removal of the phos-
phonic acid was possible, but at the cost of a 5-fold
decrease in activity (Table 1, compare 1a with 2a). None
of the modifications performed thereafter improved
activity (2b, 2c and 2d). However, for the first time in
this farnesyl series, some activity could be detected
using the cellular assay (2d). In the cyanobenzyl imida-
zole series very potent inhibitors of FPTase in vitro were
obtained. Replacement of the phosphonic acid present
in 1a by the cyanobenzyl imidazole group (3a) main-
tained the inhibitory activity, and further removal of the
carboxylic acid (3c) gave a 15-fold decrease in IC50
value. In an attempt to further improve the cellular
activity, substitution of the newly introduced nitrogen
was performed, but this gave a less potent inhibitor (3d).
However, the most striking difference between these two
imidazole series is exemplified by the in vitro results
obtained with compounds 2c and 3c. Hence the mode of
inhibition of the cyanobenzyl imidazole derivatives was
investigated, choosing 3c as a representative example and
comparing it with 1a. The pharmacological behavior of
both compounds was determined qualitatively by obser-
vation of FPP- and CAAX-dependent modulations of
Table 2. Mode of FPTase inhibition by 1 and 3c
FPP (mM)
0.51
2
510
37
20
20
1a (100 nM)a
3c (10 nM)a
80
54
72
55
9
38
9
36
10
32
42
Dansyl-GCVLS (mM)
0.51
2
510
1a (100 nM)b
3c (10 nM)b
43
49
42
41
41
38
34
27
33
14
38
3
aPer cent inhibitions at fixed concentrations of peptide substrate (2 mM).
bPer cent inhibitions at fixed concentrations of FPP (2 mM).
Figure 2. 3D conformations of 3c generated from docking in either empty FPTase (left view) or FPTase-FPP complex (right view). The dark blue
ball represents the zinc ion. The right view (CAAX competitive inhibitor hypothesis) shows 3c interacting with FPP.

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M. Lannuzel et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1459–1462
This FPP analogue approach has allowed the identifi-
cation of very potent inhibitors of FPTase in vitro.
However, results still need to be improved with regard
to their cellular activities. Compound 3c which has
nanomolar inhibitory potency on the enzyme and does
not bear any highly acidic or basic functionalities, still
remains active only in the micromolar range in the cel-
lular assay. It can be questioned whether such behavior
could be attributed to the presence of the very lipophilic
farnesyl moiety. Several groups have been working on
the replacement of this isoprenoid chain with aromatic
residues,19 but it still has to be demonstrated whether
such structural modifications could convey some
improvement in cellular activity. Overall it has been
shown that, removal of all acidic functionalities of 1a
and introduction of 4-cyanobenzylimidazolyl group as a
strong zinc chelator and FPP interacting fragment,
resulted in the transformation of a pure FPP competi-
tive inhibitor into a mixed FPP and CAAX competitive
inhibitor.
Gras, S.; Bonnet, D.; Hill, B. T.; Halazy, S. Bioorg. Med.
Chem. Lett. 1996, 6, 1291.
7. Starrett, E. J.; Tortolani, D. R.; Russell, J.; Hitchcock,
M. J. M.; Whiterock, V.; Martin, J. C.; Mansuri, M. M. J.
Med. Chem. 1994, 37, 1857.
8. Benzaria, S.; Pelicano, H.; Johnson, R.; Maury, G.;
Imbach, J.-L.; Aubertin, A.-M.; Obert, G.; Gosselin, G. J.
Med. Chem. 1996, 39, 4958.
9. Gibbs, J. B.; Pompliano, D. L.; Mosser, S. D.; Rands, E.;
Lingham, R. B.; Singh, S. B.; Scolnick, E. M.; Kohl, N. E.;
Oliff, A. J. Biol. Chem. 1993, 268, 7617.
10. (a) Dunten, P.; Kammlott, U.; Crowther, R.; Weber, D.;
Palermo, R.; Birktoft, J. Biochemistry 1998, 37, 7907. (b) Long,
S. B.; Casey, P. J.; Beese, L. S. Biochemistry 1998, 37, 9612.
11. Daninos-Zeghal, S.; Mourabit, A. A.; Ahond, A.; Poupat,
C.; Potier, P. Tetrahedron 1997, 53, 7605.
12. Dinsmore, C. J.; Williams, T. M.; O’Neill, T. J.; Liu, D.;
Rands, E.; Culberson, J. C.; Lobell, R. B.; Koblan, K. S.; Kohl,
N. E.; Gibbs, J. B. Bioorg. Med. Chem. Lett. 1999, 9, 3301.
13. Fmoc-Phe-Wang was purchased from Senn Chemical
(Dielsdorf Switzerland).
14. Lamothe, M.; Lannuzel, M.; Perez, M. J. Comb. Chem.
2002, 4, 73.
15. Pompliano, D. L.; Gomez, R. P.; Anthony, N. J. J. Am.
Chem. Soc. 1992, 114, 7945.
16. FlexX version 1.11 part of SYBYL molecular modeling
software from Tripos Inc.
Acknowledgements
The authors wish to thank Dr. S. Vispe, Dr. D. Perrin
and S. Gras from the Department of Cancerology for
their technical help and fruitful discussions. Marc Lan-
nuzel wishes to acknowledge Dr. C. Perigaud and Dr.
G. Gosselin for their continuous support on his work.
17. (a) Long, S. B.; Hancock, P. J.; Kral, A. M.; Hellinga,
H. W.; Beese, L. S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
12948. (b) Bell, I. M.; Gallicchio, S. N.; Abrams, M.; Beese,
L. S.; Beshore, D. C.; Bhimnathwala, H.; Bogusky, M. J.;
Carolyn, A.; Culberson, J. C.; Davide, J.; Ellis, H.; Fernandes,
C.; Gibbs, J. B.; Graham, S. L.; Hamilton, K. A.; Hartman,
G. D.; Heimbrook, D. C.; Homnick, C.; Huber, H. E.; Huff,
J. R.; Kassahun, K.; Koblan, K. S.; Nancy, E.; Lobell, R. B.;
Lynch, J. J., Jr.; Robinson, R.; Rodrigues, A. D.; Taylor, J. S.;
Walsh, E. S.; Williams, T. M.; Zartman, C. B. J. Med. Chem.
2002, 45, 2388.
References and Notes
1. Cox, A. D.; Der, C. J. Curr. Opin. Pharmacol. 2002, 2,
388.
2. Barbacid, M. Annu. Rev. Biochem. 1987, 56, 779.
3. Prendergast, G. C.; Rane, N. Expert Opin. Investig. Drugs
2001, 10, 2105.
4. Ashar, H. R.; James, L.; Gray, K.; Carr, D.; Black, S.;
Armstrong, L.; Bishop, W. R.; Kirschmeier, P. J. Biol. Chem.
2000, 275, 30451.
5. Gibbs, R. A.; Zahn, T. J.; Sebolt-Leopold, J. S. Curr. Med.
Chem. 2001, 8, 1437.
`
6. Lamothe, M.; Perrin, D.; Blotieres, D.; Leborgne, M.;
18. Schlitzer, M. Curr. Pharm. Des. 2002, 8, 1713.
19. (a) Moreno Barber, A.; Hardcastle, I. R.; Rowlands,
M. G.; Nutley, B. P.; Marriott, J. H.; Jarman, M. Bioorg.
Med. Chem. Lett. 1999, 9, 623. (b) Schlitzer, M.; Satter, I.
Bioorg. Med. Chem. 1999, 7, 2391. (c) Chehade, K. A. H.;
Andres, D. A.; Morimoto, H.; Spielmann, H. P. J. Org. Chem.
2000, 65, 3027. (d) Turek, T. C.; Gaon, I.; Distefano, M. D. J.
Org. Chem. 2001, 66, 3253. (e) Zhou, C.; Shao, Y.; Gibbs,
R. A. Bioorg. Med. Chem. Lett. 2002, 12, 1417.