Peptidomimetic inhibitors of farnesyltransferase with high in vitro activity and significant cellular potency

Article abstract of DOI:10.1016/j.bmcl.2007.09.015

2-o-Tolyl or 2-o-anisyl substituted 4-hydroxy- and 4-carboxybenzamides of methionine, etherified and amidified with 2-hydroxymethyl- and 2-aminomethylpyridodioxane, respectively, are described as inhibitors of Ras protein farnesyltransferase (FTase). Of t

Full text of DOI:10.1016/j.bmcl.2007.09.015

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6192–6196
Peptidomimetic inhibitors of farnesyltransferase with high
in vitro activity and significant cellular potency
Cristiano Bolchi,a Marco Pallavicini,a Chiara Rusconi,a Luisa Diomede,b
Nicola Ferri,c Alberto Corsini,c Laura Fumagalli,a Alessandro Pedretti,a
Giulio Vistolia and Ermanno Valotia,*
a
`
Istituto di Chimica Farmaceutica e Tossicologica ‘‘Pietro Pratesi’’, Universita di Milano, via Mangiagalli 25, I-20133 Milano, Italy
bDipartimento di Biochimica e Farmacologia Molecolare, Istituto di Ricerche Farmacologiche ‘‘Mario Negri’’,
via La Masa 19, I-20156 Milano, Italy
c
`
Dipartimento di Scienze Farmacologiche, Universita di Milano, via Balzaretti 9, I-20133 Milano, Italy
Received 30 July 2007; revised 5 September 2007; accepted 5 September 2007
Available online 8 September 2007
Dedicated to Professor Luigi Villa in memory of his outstanding teaching and research contribution.
Abstract—2-o-Tolyl or 2-o-anisyl substituted 4-hydroxy- and 4-carboxybenzamides of methionine, etherified and amidified with 2-
hydroxymethyl- and 2-aminomethylpyridodioxane, respectively, are described as inhibitors of Ras protein farnesyltransferase
(FTase). Of the sixteen compounds, resulting from the substitution pattern of benzamide and the configuration of the two stereo-
centers, seven inhibited FTase activity with potencies in the nanomolar range. They were all 2-oxymethylpyridodioxane ethers and,
among them, the four o-tolyl substituted stereoisomers also showed micromolar antiproliferative effect on human aortic smooth
muscle cells interfering with Ras farnesylation. The docking analysis enlightened significant differences in enzyme interaction
between oxymethylpyridodioxane and aminomethylpyridodioxane derivatives.
Ó 2007 Elsevier Ltd. All rights reserved.
Farnesyltransferase enzyme (FTase) catalyzes the cova-
lent transfer of the isoprenoid farnesyl from prenyl
donor farnesyldiphosphate (FPP) to a cysteine residue
of protein substrates, such as Ras proteins, containing
a C-terminal CAAX motif, in which C is the cysteine that
is farnesylated, AA are usually aliphatic aminoacids, and
X is the terminal aminoacid, normally methionine, ser-
ine, alanine, or glutamine. Prenylation of Ras proteins
by FTase is essential for these proteins to take part in
the transduction of extracellular mitogenic signals to
the nucleus. Because mutant Ras proteins are responsible
for about 30% of all human cancers and their malignant
activity is also dependent on FTase catalyzed prenyla-
tion, farnesyltransferase inhibitors (FTIs) have emerged
as a new class of extremely promising anticancer drugs.1
More recently, FTIs have been thought to be effective in
preventing proliferation of smooth muscle cells (SMCs)
in the arterial wall,2,3 a prominent feature of atheroscle-
rosis and restenosis after angioplasty and coronary stent
implantation.4 Finally, FTIs that specifically target FTa-
ses of protozoan parasites, such as Plasmodium falcipa-
rum or Trypanosome brucei, have been proposed for
the treatment of parasitic infections.5–10
The discovery that CAAX tetrapeptides can potently inhi-
bit FTase in cell-free assays,11,12 though totally inactive in
whole cells, has prompted the development of CAAX
mimetics with progressively minimized peptidic features
in order to obtain FTIs with higher metabolic stability
and improved cell permeability. These objectives were
achieved through modifications such as (a) the isosteric
replacement of the internal dipeptide (AA) with 4-amino
or 4-hydroxy substituted 2-biphenylcarboxylic acid and
the ortho methylation or methoxylation of the flanking
phenyl ring of the biphenyl spacer and (b) the replacement
of the cysteine residue (C), bearing the problematic thiol
group, by 3-pyridyl, optimally linked to the biphenyl core
by an oxymethylene bridge.13–15
Based on such observations, we designed the structures
1–16, whose salient feature is the presence of pyridodi-
oxane in lieu of 3-pyridyl ether to mimic the cysteine
Keywords: Farnesyltransferase; Peptidomimetic inhibitors; Antiproli-
ferative agents; Antitumors.
*
Corresponding author. E-mail: ermanno.valoti@unimi.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.015

C. Bolchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6192–6196
6193
residue of CAAX tetrapeptides. The replacement was
Br
Br
undertaken in order to restrict the conformational free-
dom of this portion of the molecules, while improving its
hydrogen bond acceptor capability, thanks to the addi-
tional oxygen, and making it chiral by the presence of
the novel dioxane stereocenter.
COOH
COOEt
a
O2N
O2N
b or e
21
Y
R
X
O
COOH
Y
Y
CCOEt
CCOEt
CCOEt
*
N
H
O
O
S
O2N
H2N
HO
*
N
d
d
c
c
23 Y = Me
24 Y = OMe
25 Y = Me
26 Y = OMe
27 Y = Me
28 Y = OMe
1-4
5-8
R = Me X = O
= OMe
= Me
= OMe
= O
= NHCO
= NHCO
9-12
13-16
Scheme 2. Synthesis of the ethyl esters of 2-o-tolyl- and 2-o-anisyl-4-
hydroxybenzoic acid. Reagents and conditions: (a) ethanol, ethyl
orthoformate, H2SO4 concd, reflux, 12 h, 79%; (b) tolyl boronic acid,
Pd(PPh3)4, 2 M Na2CO3, ethanol, toluene, reflux, 12 h, 90%; (c) H2-
Pd/C, methanol, 3 h, 73% (25) and 86% (26); (d) NaNO2, 2 N H2SO4,
acetone, À10 °C and then rt, 12 h, 91% (27) and 89% (28); (e) o-
methoxyphenyl boronic acid, Pd(PPh3)4, 2 M Na2CO3, ethanol,
toluene, reflux, 12 h, 81%.
The synthetic accessibility to the pyridodioxane synthon
as unichiral 2-hydroxymethyl and 2-aminomethyl deriv-
atives led us to conceive its connection to the biphenyl
core through a short methyleneoxy and methyleneami-
nocarbonyl chain, respectively.
sequence led to ethyl 2-(2-methoxyphenyl)-4-hydroxy-
benzoate 28 starting from 21, but using o-methoxyphe-
nyl boronic acid in place of tolyl boronic acid
(Scheme 2).
To synthesize compounds 1–8, we firstly prepared the
two non-aminoacidic building blocks, namely the mesyl-
oxymethylpyridodioxane 18, in both the enantiomeric
forms, and ethyl 4-hydroxybenzoate, o-tolyl or o-anisyl
substituted at position 2 (27 and 28, respectively).
Nucleophilic displacement of mesylate group of (R)-18
by the sodium salts of 27 and 28 yielded (S)-32 and
(S)-33, respectively. Both these ethyl esters were hydro-
lyzed and the resultant carboxylic acids (S)-34 and (S)-
35 condensed with L-methionine methyl ester to give
the corresponding amides 36 and 37, whose ester func-
tion was hydrolyzed yielding the target compounds 1
and 5 with S configuration at both the stereocenters
(Scheme 3). The corresponding RR enantiomers 2 and
6 were synthesized from (S)-18 and D-methionine methyl
ester by the same sequence of reactions illustrated in
Scheme 3. Identically, the stereoisomers 3 and 7, having
the benzodioxane stereocenter in S configuration and
the aminoacid residue in R configuration, and the
respective enantiomers 4 and 8 were prepared using
(R)-18 and D-methionine methyl ester in the former case
and (S)-18 and L-methionine methyl ester in the latter.
As outlined in Scheme 1, the R enantiomer of intermedi-
ate 18 was obtained by mesylation of (S)-2-hydroxy-
methyl-2,3-dihydro[1,4]dioxino-[2,3-b]pyridine (S)-17,
whose univocal synthesis from (S)-benzylglycerol we
have recently reported together with that of its antipode
from (R)-benzylglycerol.16 Analogously, (R)-17 was
transformed into mesylate (S)-18.
As shown in Scheme 2, compound 27 was prepared from
2-bromo-4-nitrobenzoic acid 21,17 which was converted
into ethyl ester 22 and then coupled with tolyl boronic
acid using Suzuki conditions to give biphenyl derivative
23. Successive reduction of nitro to amino group
afforded 25, which was submitted to diazotation and
diazonium salt decomposition yielding ethyl 2-(2-meth-
ylphenyl)-4-hydroxybenzoate 27. The same synthetic
The synthesis of compounds 9–16 required unichiral
aminomethylpyridodioxane 20, readily accessible in
both the enantiomeric forms from (R)-18 and (S)-18
(see Scheme 1 for (S)-20) and, as a biphenyl derivative,
methyl 2-(2-methylphenyl)-4-carboxybenzoate18 or its
methoxy analogue 31, which was prepared from com-
mercially available dimethyl 2-iodoterephthalate 29 as
shown in Scheme 4. To prepare 9 and 13, these two
methyl carboxybenzoates were reacted with (S)-20
obtaining (S)-44 and (S)-45, respectively (Scheme 5).
Successive hydrolysis of methyl ester afforded the car-
boxylic acids (S)-46 and (S)-47, which were condensed
with L-methionine methyl ester to give 48 and 49, respec-
N
O
N
O
N
O
N
O
a
b
c
O
O
O
O
OH
N3
OMs
NH2
(R)-18
(S)-19
(S)-20
(S)-17
Scheme 1. Synthesis of the S enantiomer of aminomethylpyridodiox-
ane. Reagents and conditions: (a) MsCl, TEA, DCM, rt, 1 h, 98%; (b)
NaN3, DMF, H2O, reflux, 6 h, 91%; (c) N2H4, PdO, MeOH, reflux,
2 h, 92%.

6194
C. Bolchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6192–6196
tively. Finally, the methionine ester function was hydro-
(R)-18
lyzed yielding the target compounds 9 and 13 with S
configuration at both the stereocenters. The same syn-
thetic route shown in Scheme 5 was followed to prepare
the corresponding RR enantiomers 10 and 14, the ster-
eoisomers 11 and 15, with S and R configuration at pyr-
idodioxane and methionine stereocenter, respectively,
and the enantiomers of these latter 12 and 16 using ami-
nomethylpyridodioxane 20 and methionine methyl ester
in the required configuration.
a or e
XOOC
O
NH
S
COOX
R
R
O
O
O
O
O
O
Initially, compounds 1–16 were tested for their ability to
inhibit FTase activity and the respective IC50 values are
listed in Table 1. Successively, the same compounds
were examined in a cellular assay that measured inhibi-
tion of human aortic SMC proliferation. The most ac-
tive FTase inhibitors, namely the o-methylated
biphenyl ethers 1–4, efficiently inhibited SMC prolifera-
tion in a concentration dependent manner with an IC50
value in the micromolar range (Table 2), whereas the
other compounds did not show any significant effect at
concentrations ranging from 50 to 250 lM.
N
d
N
b
c
(S)-32 R=Me
X=Et
1 R=Me
X=H
5 R=OMe
X=H
(S)-34 R=Me
X=H
(S)-35 R=OMe
X=H
36 R=Me
X=Me
b
c
d
(S)-33 R=OMe
X=Et
37 R=OMe
X=Me
Scheme 3. Synthesis of compounds 1 and 5. Reagents and conditions:
(a) 27, NaH, DME, À15 °C and then reflux, 48 h, 56%; (b) 3 N KOH,
methanol, 50 °C, 12 h, 86% ((S)-34) and 70% ((S)-35); (c) L-methionine
methyl ester hydrochloride, HOBt, EDAC, DMF, 15 min, rt and then
TEA, 24 h, rt, 69% (36) and 72% (37); (d) 3 N KOH, methanol, 12 h,
rt, 92% (1) and 91% (5); (e) 28, NaH, DME, À15 °C and then reflux,
48 h, 86%.
To directly evaluate the effect of the compounds with
antiproliferative activity, in particular of 2 and 4, on
protein prenylation, Ras farnesylation process was
COOMe
COOMe
COOMe
COOH
I
Table 1. Inhibition of FTase activity and interaction energies with
FTase of compounds 1–16 (in brackets the absolute configuration of
methionine residue and then that of the pyridodioxanemethyl
substituent)
b
a
OMe
OMe
COOMe
COOMe
29
30
31
Compound
IC50 (lM)
Energy (kcal/mol)
1 (SS)
2 (RR)
3 (RS)
4 (SR)
5 (SS)
0.050
0.053
0.075
0.045
0.053
0.112
>1000
0.055
12
À55.15
À55.04
À57.62
À58.02
À53.38
À53.56
À54.60
À58.48
À37.61
À49.24
À43.83
À38.69
À52.81
À52.24
À44.52
À52.83
Scheme 4. Synthesis of compound 31. Reagents and conditions: (a) o-
methoxyphenyl boronic acid, Pd(PPh3)4, 2 M Na2CO3, ethanol,
toluene, reflux, 3 h, 75%; (b) 1.7 N KOH, 1/1 THF/methanol, rt,
12 h, 72%.
6 (RR)
7 (RS)
8 (SR)
9 (SS)
(S)-20
a or e
10 (RR)
11 (RS)
12 (SR)
13 (SS)
14 (RR)
15 (RS)
16 (SR)
>1000
>1000
16
XOOC
O
NH
NH
S
COOX
NH
302
>1000
843
151
R
N
R
N
O
O
O
O
The inhibition curves were fitted using ‘one-site competition’ equation
built into Prism version 4.0 for Windows (GraphPad Software, San
Diego, CA). This analysis gives the IC50 (the drug concentration
inhibiting the enzyme activity by 50%) with a 95% of confidence
interval. The energy values were obtained by molecular docking
calculation.
O
O
c
b
d
(S)-44 R=Me
(S-)46 R = Me
X = H
(S)-47 R = OMe
X = H
48 R=Me
9 R=Me
X=Me
(S)-45 R=OMe
X=Me
X=Me
X=H
49 R=OMe d 13 R=OMe
b
c
X=Me
X=H
Table 2. Antiproliferative effect of compounds 1–4 on human aortic
SMCs
Scheme 5. Synthesis of compounds 9 and 13. Reagents and conditions:
(a) methyl 2-o-tolyl-4-carboxybenzoate, HOBt, EDAC, DMF, 15 min,
rt and then TEA, 12 h, rt, 63%; (b) 3 N KOH, methanol, 4 h, rt, 95%
((S)-46) and 96% ((S)-47); (c) L-methionine methyl ester hydrochloride,
HOBt, EDAC, DMF, 15 min, rt and then TEA, 24 h, rt, 64%; (d) 3 N
KOH, methanol, 12 h, rt, 96% (9) and 91% (13); (e) 31, HOBt, EDAC,
DMF, 15 min, rt and then TEA, 12 h, rt, 65%.
Compound
1
2
3
4
IC50 (lM)
157.9
16.8
237.8
58.8
The IC50 values were determined by linear regression analysis of the
logarithm of the concentration versus the percentage of the inhibitory
effect.

C. Bolchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6192–6196
6195
estimated by Western blot analysis from a total protein
extract of cells incubated under the same experimental
conditions utilized for evaluating cell proliferation.
Compounds 2 and 4, at 250 lM, proved to directly
interfere with Ras farnesylation as indicated by the
appearance of a slower migrating band also after incu-
bation with HMG-CoA reductase inhibitor simvastatin
(Fig. 1).
Compounds 1–16 were docked in the crystal structure of
human FTase. The resultant inhibitor–enzyme interac-
tion energies are reported in Table 1. It is significant that
the phenyl ethers 1–8, all much more potent FTIs, with
the exception of 7, than the benzamides 9–16, show low-
er interaction energies than these latter occupying the
first eight positions in the energy rank. Furthermore,
the o-methyl substituted compounds 1–4, the most po-
tent inhibitors of FTase and of cell proliferation in the
series, are also the top interacting ones in terms of
energy.
The configuration of the two stereocenters does not ex-
ert significant influence on the activity of these com-
pounds. Excepting 7, all the enantiomers and the
diastereomers are virtually equiactive in the series of
the most active phenyl ethers 1–8 and this is consistent
with the modest energy differences resulting from
molecular docking. On the contrary, molecular docking
enlightens the importance of the linker between biphe-
nyl core and pyridodioxane for the role played by the
biheterocycle in the interaction with the enzyme. In
the FTase complexes with methyleneoxy compounds
1–8, the pyridodioxane system shields the enzyme zinc
ion, which is conversely engaged in an ionic interaction
with the methionine carboxyl of the inhibitor in the
FTase complexes with methyleneaminocarbonyl com-
pounds 9–16 (Fig. 2). Such a different binding mode
could explain the great difference in FTase inhibiting
activity between the phenylethers 1–8 and the benza-
mides 9–16.
Figure 2. Complexes of the human FTase with 4 (A) and 9 (B) showing
the two different binding modes typical of oxymethylpyridodioxane
ethers 1–8 and aminomethylpyridodioxane amides 9–16. In the former
(A), the ligand is placed in a hydrophobic pocket lined by several
aromatic residues of both a and b FTase subunits and the carboxyl
group interacts with the Arg-202b and the Gln-167a residues. In the
latter (B), the main interactions involve the residues of the b subunit
and the ligand carboxyl group interacts with the zinc ion.
In conclusion, we have demonstrated that CAAX
mimetics, in which cysteine is replaced by a pyridodiox-
anemethyl residue etherifying the known AA bioisostere
2-o-tolyl-4-hydroxybenzoic acid, inhibit FTase activity
both in cell-free and in whole cell tests at nanomolar
and micromolar level, respectively. Consistently with
docking results, critical features of such new FTIs are
the nature of the short linker between pyridodioxane
and biphenyl core and the o-substitution on this latter,
and not the configuration of the two stereocenters.
Acknowledgment
KDa
Unprenylated Ras
Financial support provided by the Italian Ministero
`
dell’Istruzione, dell’Universita e della Ricerca Scientifica
is gratefully acknowledged.
Prenylated Ras
-21
Cnt. Simva. 2
4
Figure 1. Experimental conditions are as in Table 2. After 72 h
incubation of the cells with 250 lM concentration of compounds 2 and
4 and 2 lM simvastatin (Simva), total cell lysates were prepared and
Ras prenylation evaluated by Western blotting analysis with a specific
antibody (anti Ras clone, Upstate). The slower migrating band
represents the unprenylated form of Ras (Unpre. Ras), while the
faster migrating band is prenylated Ras (Pre. Ras).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.09.015.

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C. Bolchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6192–6196
Lombardo, L. J.; Williams, D. K.; Hamilton, A.; Sebti, S.;
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