Aromatic farnesyl diphosphate analogues: Vinyl triflate-mediated synthesis and preliminary enzymatic evaluation

Article abstract of DOI:10.1016/S0960-894X(02)00187-7

A stereocontrolled vinyl triflate-based synthetic route has been used to prepare four analogues of farnesyl diphosphate (FPP) where the terminal isoprene units have been replaced with aromatic moieties. Two of these analogues exhibit no productive interac

Full text of DOI:10.1016/S0960-894X(02)00187-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1417–1420
Aromatic Farnesyl Diphosphate Analogues:
Vinyl Triflate-Mediated Synthesis and
Preliminary Enzymatic Evaluation
Chunmei Zhou, Ying Shao and Richard A. Gibbs*
Department of Pharmaceutical Sciences, College of Pharmacy and AHP, Wayne State University,
Detroit, MI 48202, USA
Received 11 January 2002; accepted 12 February 2002
Abstract—A stereocontrolled vinyl triflate-based synthetic route has been used to prepare four analogues of farnesyl diphosphate
(FPP) where the terminal isoprene units have been replaced with aromatic moieties. Two of these analogues exhibit no productive
interaction with protein farnesyltransferase, but the 2-naphthyl derivative 2 is a modest inhibitor of the enzyme, and the para-
biphenyl derivative 4 is a surprisingly effective alternative substrate. # 2002 Elsevier Science Ltd. All rights reserved.
Protein farnesylation is a common post-translational
modification, which plays a key role in a variety of sig-
nal transduction pathways. It is carried out by an
enzyme, protein-farnesyl transferase (FTase), which
recognizes the CAAX box (where X=Ser, Met, Thr, or
Gln) at the carboxyl terminus of a protein substrate and
then attaches the farnesyl groupfrom farnesyl diphos-
phate (FPP, Fig. 1) to the free sulfhydryl of the cysteine
residue.1 Initial studies on this process demonstrated
that the key signal transduction protein and oncogene
product Ras is farnesylated. The potential of farnesyla-
FTase inhibitors, and effective alternative substrates for
this enzyme. Recent structural studies on mammalian
FTase have suggested an alternative route to the
development of FPP-based inhibitors. These structural
studies, by the Duke group,5 the Hoffman-LaRoche
group,6 and the Schering group,7 have demonstrated
that FPP binds to a deep hydrophobic pocket in FTase.
This pocket is primarily lined with the electron-rich
aromatic side chains of Tyr and Trpresidues. This
finding immediately suggested that aromatic analogues
of FPP, particularly where the b- and o-isoprene units
were replaced with aromatic moieties, would be potent
FTase inhibitors. Such compounds could take advan-
tage of favorable aromatic-aromatic interactions that
would promote tight binding to the FTase active site.8
tion inhibitors as anticancer agents led to extensive and
1
successful efforts to developsuch comopunds.
This
intense effort has been rewarded recently with the
introduction of FTase inhibitors into human clinical
trials for the treatment of a variety of neoplasias.1,2
Several other groups in the FTase arena have made the
same observation, and have reported their initial results
directed toward the development of aromatic-substituted
FPP-based FTase inhibitors (vide infra).9 We anticipated
Efforts in the area of FTase inhibitors have con-
centrated on substrate-based and non-substrate-based
compounds that bind to the CAAX peptide site on
FTase. In contrast, previous work in this laboratory has
been directed toward the synthesis and evaluation of
FPP analogues modified in the a-isoprene unit (Fig. 1),3
and isomeric FPP and GGPP analogues.4 These studies
have resulted in the discovery of potent, low nanomolar
*Corresponding author at current address: Department of Medicinal
Chemistry and Molecular Pharmacology, School of Pharmacy and
Pharmacal Sciences, Purdue University, West Lafayette, IN 47907,
USA. Fax: +1-765-494-1414; e-mail: rag@pharmacy.purdue.edu
Figure 1. Structure of FPP, with the a, b, and o isoprene units
indicated.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00187-7

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C. Zhou et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1417–1420
Figure 2. Structures of the four aromatic FPP analogues synthesized
and evaluated versus FTase in this study.
that our stereoselective vinyl triflate-mediated route to
isoprenoid analogues1,3,4 could be readily adapted to the
synthesis of FPP analogues where both the b- and
o-isoprene units were replaced with aromatic moieties.
The four analogues shown in Figure 2 were chosen as
the initial targets for synthesis and evaluation. These
compounds were chosen in part due to the ready avail-
ability of the necessary benzylic starting materials.
However, in addition to addressing the general question
regarding the suitability of aromatic moieties as iso-
prenoid mimics, analogues 1–4 also provide insight into
the hydrophobic bulk and overall geometry necessary
for an FPP analogue to bind to the FTase active site.
The syntheses of 1–4 began with the commercially
available benzyl bromides 5a–c (X=Br) and the benzyl
chloride 5d (X=Cl) (Fig. 3). The selective mono-alkyl-
ation of the ethyl acetoacetate dianion 6 was attempted
following the procedure previously developed by Wei-
ler10 and extensively utilized in our laboratory.3,4 Sur-
prisingly, very poor yields of the desired b-ketoesters
7a–d were obtained. In order to obtain satisfactory
yields of 7a–d, it was necessary to conduct the alkyl-
ation of 6 at room temperature, rather than 0 ꢀC.11 The
kinetic triflation of 7a–d also proved to be more pro-
blematic than in the parent isoprenoid case, and it was
necessary to conduct the enolate formation and trifla-
tion at room temperature to obtain sufficient amounts
of 8a–d. In contrast to the first two steps, we found that
the Pd/CuI-catalyzed methylation of the triflates to
afford 9a–d, and their subsequent reduction to 10a–d,
proceeded smoothly in the same manner as observed in
the parent system. The farnesol analogues 10a–d were
then converted to the desired aromatic FPP analogues
1–4 following the two-step phosphorylation procedure
of Davisson et al.12
Figure 3. Syntheses of FPP analogues 1–4.
(mFTase). A modified version of the continuous spectro-
fluorimetric assay for FTase was employed to test the
ability of 1, 2, 3, and 4 to act as mFTase substrates with
the peptide cosubstrate dansyl-GlyCysValLeuSer-OH.13,14
Initial studies indicated that 1, 2, and 3 were not sub-
strates, while 4 was a substrate for mFTase. More
detailed evaluation demonstrated that the para-biphenyl
analogue 4 is a surprisingly effective mFTase substrate.
Analogue 4 exhibited a submicromolar (800Æ28 nM)
Km value for the enzyme, less than 3-fold higher than
that observed with the natural substrate E,E-FPP under
the same assay conditions (300 nM).4 Moreover, the
approximate Vmax value observed with 4 was only
ꢁthree fold less than that seen with E,E-FPP. Note that
the Km value does not provide a true indication of affi-
nity for the enzyme due to the complexity of the
mFTase kinetic mechanism.15 In contrast to the results
obtained with 4, analogues 1–3 were exceptionally poor
mFTase substrates. Therefore, these compounds were
The four aromatic analogues of FPP, having been pre-
pared, were then evaluated as potential substrates for or
inhibitors of mammalian protein-farnesyl transferase

C. Zhou et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1417–1420
1419
not surprising that 3, which mimics a folded confor-
mation of FPP, does not bind to mFTase. The active
site of mFTase is quite large, with the peptide and FPP
binding sites adjacent to each other,7 and thus one
might anticipate that 3 might utilize a portion of the
peptide binding site and still bind to mFTase, but this
was not the case. The most significant biological result
presented here is the ability of 4 to act as an excellent
substrate for mFTase. The recently reported work of
Spielmann (A and B, Fig. 4) has established that an
aromatic moiety can act as an effective mimic for the
o-isoprene unit, but it is striking that an appropriate
aromatic moiety can effectively replace both the b- and
o-isoprene units of FPP. Prenylation of CAAX box
proteins with 4 and possibly certain related analogues
could lead to the modification of Ras and other key signal
transduction proteins with a prenyl group distinct in
structure from the naturally occuring farnesyl moiety. Such
unnatural prenyl groups may be valuable tools to inter-
rogate the biological functions of protein prenylation.17
Figure 4. Structures of aromatic FPP analogues synthesized and eval-
uated versus FTase in other laboratories.
evaluated as inhibitors of mFTase. The phenyl analogue
1 and the ortho-biphenyl analogue 3 exhibited no inhi-
bitory activity, and thus no apparent interaction with
mFTase, (IC50 ꢂ10 mM; essentially no inhibition of
mFTase was observed at 10 mM with either compound).
However, the naphthyl analogue 2 proved to be a mod-
est inhibitor of mFTase, with an IC50 value of 5.4 mM.
Acknowledgements
Several other groups in the FTase arena have reported
their initial results directed toward the development of
aromatic-substituted FPP-based FTase inhibitors (Fig.
4). In particular, the Spielmann laboratory has descri-
bed several o-substituted FPP analogues and their
interaction with FTase. The aniline-substituted ana-
logue A is not an FTase inhibitor, but instead is a sur-
prisingly effective alternative substrate that is
transferred to Ras with the same efficiency as the nat-
ural substrate FPP.9 The set of analogues represented
by structure B (n=1–5) were found by Spielmann et al.
to be modestly effective alternative substrates
(Vrel=0.022–0.22; Km=56–890 nM).9 Distefano and
co-workers have recently prepared the benzophenone-
derived photoaffinity label C, and found that it is a poor
substrate for mammalian and yeast FTase, but an
effective inhibitor of these enzymes [IC50(mFTa-
se)=2.3 mM].9 They have also determined the structure
of the mFTase–C complex, which confirms that the
aromatic moiety in this analogue serves as a good mimic
of the b- and o-isoprene units of FPP. Note that a novel
synthetic route to other aromatic substituted farnesoids
has been developed by Wiemer and co-workers,9
although no biological data on these analogues has yet
been reported.
This work was supported by NIH grant CA 78819 to
R.A.G.
References and Notes
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