Novel conformationally extended naphthalene-based inhibitors of farnesyltransferase [1]

Full text of DOI:10.1021/jm9701177

© Copyright 1997 by the American Chemical Society
Volume 40, Number 12
J une 6, 1997
Communications to the Editor
the two internal amino acids of the CAAX sequence are
Novel Con for m a tion a lly Exten d ed
Na p h th a len e-Ba sed In h ibitor s of
F a r n esyltr a n sfer a se
replaced by surrogates such as benzodiazepines,6b ami-
nobenzoic acids, and aminomethylbenzoic acids.6e,i More
recently, several studies have appeared in which the
entire A1A2X portion of the CA1A2X box has been
replaced by non-peptidic units.8
Christopher J . Burns,*,† J ean-Dominique Guitton,*
Bernard Baudoin, Yves Lelie`vre, Marc Duchesne,
Fabienne Parker, Nadine Fromage, and
Alain Commerc¸on
Largely as a consequence of these efforts, discussion
surrounding the likely active conformation of the CAAX-
based inhibitors has been increasingly fruitful. Early
opinion favored the possibility of a “turn-like” backbone
conformation, based upon analogy to Cys-X-X-Cys tet-
rapeptide motifs found in aspartate transcarbamylase
and “zinc finger” domains,9 as well as on solution NMR
studies using moderately potent CAAX-based deriva-
tives.10 More recent evidence, particularly that from the
aminomethylbenzoic acid based CAAX mimetics, has led
to the suggestion that an “extended” (or nonturned)
conformation is more accurate.4
Our initial molecular modeling simulation studies on
the conformationally constrained Tic-containing pep-
tides and pseudopeptides demonstrated a direct cor-
relation between FTase inhibitory activity and the
proclivity of these inhibitors to adopt an extended
conformation.7 These studies precipitated a search for
hydrophobic scaffolds capable of orienting the cysteine
(or modified cysteine) and methionine units according
to a pharmacophore model derived from the potent
prototype Cys-(N-Me)Val-Tic-Met (Figure 1). We in-
tentionally restricted this search to scaffolds that were
incapable of accessing low-energy turned conformations.
The 1,5-naphthyl scaffold proved to be worthy of
further investigation.11 It satisfies the CO(Met)-NH-
(Cys) distance relationship outlined in Figure 1 and also
provides a rigid hydrophobic template that directs the
Cys and Met amino acids unambiguously into an
extended conformation. Indeed, a modeling simulation
calculates a difference of at least 15-20 kcal between
the low-energy extended conformations and the lowest
energy “pseudocurved” conformation. In no case was a
true turned arrangement manifested.12
Rhoˆne-Poulenc Rorer S. A., Centre de Recherches de
Vitry-Alfortville, 13, Quai J ules Guesde,
B. P. 14, 94403 Vitry-sur-Seine, France
Received February 27, 1997
Oncogenic mutations of the mammalian ras proto-
oncogene have been implicated in 20-30% of all human
cancers including more than 50% of colon and 90% of
pancreatic carcinomas.1,2 A key step in a series of
posttranslational modifications of the oncogene product
Ras is the S-farnesylation of a cysteine residue near the
C-terminus in a reaction catalyzed by the enzyme
farnesyltransferase (FTase). This step is important for
the association of the GTP-binding Ras protein to the
inner surface of the plasma membrane, where it medi-
ates cellular transformation.3 Inhibition of this farnes-
ylation event may lead to novel anti-cancer agents, and
thus has been the subject of much recent research.4
It has been appreciated for several years now that
conformationally flexible tetrapeptides corresponding to
the C-terminal CA1A2X sequence (where A is an ali-
phatic amino acid and X is Met or Ser) of Ras can serve
as effective inhibitors of Ras farnesylation.5 From
efforts to increase not only enzyme-inhibitory activity
but also cellular activity, a number of effective CA1A2X-
based peptidomimetics have emerged.6 While initial
reports focused on flexible pseudopeptidic (ψ[CH2X])
variants, other inhibitors have been designed to incor-
porate conformationally constrained A1A2 replacements,
particularly for CA1A2M peptides and pseudopeptides.
These have ranged from classical amino acid replace-
ments such as CVTicM (where Tic ) L-1,2,3,4-tetrahy-
droisoquinolin-3-ylcarbonyl),6l,7 to inhibitors in which
Synthesis of the dipeptide mimetic scaffold and
several analogues with the cysteine and methionine
residues affixed is described in Scheme 1. Thus 5-nitro-
1-naphthoic acid13 was coupled to L-methionine-OMe
† Author to whom correspondence should be addressed. Present
address: Rhoˆne-Poulenc Rorer, 500 Arcola Rd, SW 8, Collegeville, PA
19426. E-Mail: burnscj@rpr.rpna.com.
S0022-2623(97)00117-9 CCC: $14.00 © 1997 American Chemical Society

1764 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Communications to the Editor
Sch em e 1a
well. We hypothesized therefore that both the naph-
thalene-based inhibitors and the CAAX-based peptides
adopt similar conformations when bound in the active
site of the enzyme. This hypothesis precipitated a
retrospective analysis of both in-house and published
SAR data surrounding the methionine terminus to
ascertain whether the naphthalene-based inhibitors
exhibited the same global binding profile as that re-
ported for the conformationally-flexible peptide-based
inhibitor, CVFM.15 This comparison is described in
Table 1 and displayed graphically in Figure 2.
Using the IC50 values obtained for inhibitors 5 and 8
as a reference, conversion of the Met C(O) to the
thioamide (9 and 10) maintained the same level of
inhibitory activity in both series as the parent com-
pounds 5 and 8. Replacement of Met with the isostere
norleucine (11 and 12) provided a modest decrease in
activity within both series, albeit somewhat greater in
the peptide-based series than in the naphthyl series.
Methylation of the methionine nitrogen (13 and 14) gave
a 40-fold loss of activity in both series, perhaps sug-
gesting the loss of an important hydrogen bond. Modi-
fication of the Met amide to the aminomethyl (ψ[CH2-
NH]Met) analogues 15 and 16 resulted in a significant
loss (>50-fold) of activity in both series as did esterifi-
cation of methionine to the methyl esters (7 and 17;
>50-fold loss for both). While not exhaustive, the data
clearly suggest a direct correlation between the two
series of inhibitors, implying that similar inhibitor-
enzyme interactions are involved for both this new rigid
naphthyl-based scaffold and the flexible peptide-based
scaffolds. Additional comparative analysis will be re-
quired to fully substantiate this postulate.
a
(a) H-Met-OMe, DCC, HOBT, Et3N, CH2Cl2, DMF, 20 h (72%);
(b) SnCl2, EtOH, EtOAc, 0.5 h, 70 °C (90%); (c) Boc-Cys(Trt)-OH,
NMM, i-BuOC(O)Cl, THF, 2 d (71%); (d) L-N-Boc-S-tritylcysteine
aldehyde, NaCNBH3, AcOH, MeOH, 3 Å sieves, 24 h (24%); (e)
LiOH, THF, H2O; (f) TFA, EDT, H2O.
and then reduced with SnCl2 to afford 1. Naphthyl-
amine 1 was then either coupled to Boc-Cys(Trt)-OH to
afford amide 2 or reductively alkylated with L-N-Boc-
S-tritylcysteine aldehyde6f to give the aminomethyl
(Cys-ψ[CH2NH]) derivative 3. Saponification of esters
2 and 3 followed by removal of the Boc and Trt
protective groups with TFA then afforded carboxylic
acids 4 and 5. Alternatively, independent treatment of
2 and 3 directly with TFA provided esters 6 and 7,
respectively.
Further examination of the naphthalene scaffold by
molecular modeling suggested that the isomeric 1,6-
naphthalene-substituted analogue would also be capable
of maintaining the approximate distances and confor-
mational constraints between the Cys and Met termini
projected from the earlier analysis. The parent diamide
18 and the aminomethyl derivatives 19 (RPR 113829)
and 20 (RPR 114334) were prepared according to the
methodology outlined in Scheme 1, starting from 6-ni-
tro-1-naphthoic acid.13 Both carboxylic acids 18 and 19
exhibited potent FTase inhibitory activity (Table 2), the
latter demonstrating significant inhibition of both Ha-
Ras and Ki-Ras in vitro. This result is particularly
pertinent since activated Ki-Ras tumors are much more
prevalent than activated Ha-Ras tumors16 and because
Ki-Ras exhibits a 50-fold higher affinity for FTase than
does Ha-Ras.17
The parent structure 4 demonstrated potent (IC50
)
48 nM) inhibitory activity against FTase. The amino-
methyl variant 5 showed an increase in potency by
almost 10-fold (IC50 ) 5.6 nM, see Tables 1 and 2).
Neither compound served as a substrate for FTase, and
both were competitive with respect to the Ras substrate
used in the assay.7 The phenomenon of augmented
FTase activity upon reduction of the cysteine amide
bond, initially reported by Kohl6a and by Garcia6c for
nonconstrained CAAX peptides, has been observed for
a number of inhibitor series despite considerable diver-
sity in the scaffolding for each class.6,8
It is believed that the cysteinyl sulfur atom of the
CAAX-based inhibitors is involved in an important
binding interaction with a Zn2+ atom of FTase, based
on binding studies of the CAAX-containing proteins.14
Our observation that the 1,5-naphthalene-substituted
CAAX-based inhibitor demonstrated an SAR correlation
with the nonconstrained CAAX peptides implies that
there are similarities in their binding interactions, at
least in the region of the cysteine residue. If, in fact,
these two classes of inhibitors were bound to FTase in
two distinct conformations (extended for one, and turned
for the other), then it would appear unlikely that the
SAR correlation observed at the cysteine terminus
would be maintained at the methionine terminus as
Historically, one of the major challenges in the
research on CAAX peptides and peptidomimetics has
been the translation of potent isolated-enzyme inhibi-
tory activity to activity in cells. Considerable progress
has been made in this area due largely to the structural
backbone modifications alluded to earlier and the
employment of ester “prodrugs” on the methionine
carboxylate.6 Application of the ester prodrug approach
to the 1,5- and 1,6-naphthyl regioisomers (Table 2)
revealed a modest improvement in inhibition of Ha-Ras
processing in THAC cells for the 1,5-substituted ana-
logue 7 compared to the free carboxylic acid 5. A more
dramatic improvement was observed for the 1,6-
substituted ester RPR 114334 (20). The cellular activity
of 20 is many orders of magnitude greater than that

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1765
F igu r e 1. Development of extended pharmacophore model from Cys-(N-Me)Val-Tic-Met.
Ta ble 1. Comparison of 1,5-Naphthalene and CVFM-Based Inhibitors of FTase
FTase
compounda
series Ab
IC50 (nM)
series Bc
IC50 (nM)
series A
series B
X
R1
Z
R2
(no./5)d
(no./8)e
5
9
11
13
15
7
8
10
12
14
16
17
O
S
O
O
H,H
O
H
H
H
Me
H
S
S
CH2
S
S
S
H
H
H
H
H
Me
5.6
4.8
44
210
2570f
325
1.0
0.9
8
37.5
54g
58
37
17
950
1400
2220
3400
1.0
0.5
26
38
60
H
92
a
b
Preparations of 9, 11, 13, and 15 are provided in the Supporting Information. IC50 values for FTase-catalyzed transfer of [3H]FPP
to human Ha-Ras protein; values are means of two or more IC50 determinations. Assay conditions are described in ref 7. c IC50 values are
d
reproduced from ref 15, except for the value for 17 (BMS-187912, Dr. K. Leftheris, Bristol-Myers Squibb, personal communication). Ratio
of FTase IC50 values for series A compounds relative to 5. e Ratio of FTase IC50 values for series B compounds relative to 8. f IC50 value
g
for ψ[CH2NH]Met derivative of 4. Ratio of FTase IC50 values is obtained using value for 15 divided by that for 4 (IC50 48 nM).
exhibited by the peptide derivative Cys-(N-Me)Val-Tic-
Met (21),7 which displayed similar in vitro activity in
the FTase enzyme assay. The improvement in cellular
activity of 20 when compared to that of 21 may be due
to better cell membrane penetration and/or enhanced
resistance to proteolytic degradation due to the replace-
ment of two amino acids and two amide bonds.
RPR 114334 (20) potently inhibits the anchorage-
independent growth of several cell lines (Table 3).
Activated Ha-ras and Ki-ras transformed cell lines were
both inhibited in their ability to form colonies in soft
agar, an important finding in light of the prevalence of
cells harboring mutations in Ki-ras in human carcino-
mas. Several recent studies have pointed out the lack
of a direct correlation between inhibition of activated
Ras processing and inhibition of cell growth,18 a phe-
nomenon which appears to be dependent on cell type
and class of FTase inhibitor. A number of explanations
(parallel processing by other prenyltransferases, mul-
tiple gene mutations, inhibition of non-Ras targets, etc.)
were proposed, some or all of which might be operative
for a given cell line. In addition, it has also been shown
that cells harboring activated upstream tyrosine kinases
and wild-type Ras can also be inhibited by some FTase
inhibitors,18 potentially expanding the therapeutic ap-
plication of these inhibitors. These phenomena are also
observed within this new series of FTase inhibitors
(Table 3). Indeed, the sensitivity toward growth inhibi-
tion in soft agar by RPR 114334 (20) appears to be cell-
dependent (cf., NIH 3T3, H 460, and HCT 116 cells all
bearing mutant forms of Ki-Ras); however, in all of these
cases, the inhibitor is able to significantly block the
anchorage-independent cell growth. Interestingly, we
have found that 19 (the acid form of 20) demonstrates
good inhibitory activity against GGTase in vitro (Ki-
Ras, IC50 73 nM). This additional enzyme activity may
be contributing to the cellular activity of 20, particularly
for Ki-ras transformed cells.19 Finally, RPR 114334 (20)
also potently inhibited the anchorage-independent growth
of c-src-transformed NIH 3T3 cells, which possess the
activated tyrosine kinase Src and wild-type Ras.
In summary, we report here on two new series of
potent, conformationally fixed, naphthalene-based in-
hibitors of FTase. These series were designed based on
the premise that the active conformation of the inhibi-
tory CAAX-based peptides and pseudopeptides is “ex-

1766 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Communications to the Editor
Ta ble 2. In Vitro and Intracellular Activity of Regioisomeric
Naphthalene Inhibitors
Ras processing and in clonogenic assays of tumor cell
growth in soft agar (activated Ha-ras and Ki-ras, as well
as activated tyrosine kinase with wild-type Ras) and has
formed the basis for further research which will be
reported in due course.
Ack n ow led gm en t. The authors thank N. Baudouy,
R. Boulay, F. Clerc, C. Colonna, H. Dubois, T. X. Q. K.
Faitg, M. Leroux, M.-F. Marzin, P. Mouton, and B. Sans
for their expert technical assistance. The financial
support of the Bioavenir program is gratefully acknowl-
edged.
IC50 (nM)
% inhibition
Ha-
Ras
Ki-
of Ras
Ras
processing
compd
position
Y
R
FTasea FTaseb THAC (10 µM)c
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and characterization data for compounds 1-7, 9, 11, 13,
15, and 18-20 (7 pages). Ordering information is given on
any current masthead page.
21 (Cys-(N-Me)Val-Tic-Met)
5
48
5.6
0 at 1 mM
0
0
4
5
7
18
5
5
5
6
6
6
O
H,H
H,H Me 325
O
H,H
H,H Me
H
H
20
0
10
>50
H
H
60
1.8
80
1250
17.5
500
Refer en ces
19 (RPR 113829)
20 (RPR 114334)
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a
b
See Table 1, footnote b. IC50 using Ki-Ras as substrate; value
is the average of two or more IC50 measurements by SPA of FTase
using as first substrate a biotinylated fragment of the C-terminus
of Ki-Ras: [Biotin]-(A)3-SKDG-(K)6-SKTKCVIM (at a concentra-
tion equal to its Km, 55 nM) and as second substrate [3H]FPP (at
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F igu r e 2. Graphical illustration of ratios derived from Table
1.
Ta ble 3. Inhibition of Anchorage-Independent Cell Growth by
RPR 114334 (20)
inhibition of cell growth in soft agar (IC50 µM)a
NIH 3T3 NIH 3T3 NIH 3T3 H 460 HCT 116
compound
Ha-Rasb Ki-Rasc
Srcd
5
Ki-Rase Ki-Rasf
RPR 114334
5
10
5
50
a
b
Assay conditions are described in ref 20. NIH 3T3 cells
transformed by Ha-ras (Val-12). c NIH 3T3 cells transformed by
d
Ki-ras (Val-12). NIH 3T3 cells transformed by c-src (Y527F).
e Human lung carcinoma cells containing a point mutation in Ki-
Ras (Gln61 to His61). f Human colon carcinoma cells containing a
point mutation in Ki-Ras (Gly13 to Asp13).
tended” rather than “turned”. Correlation of the SAR
trends for the 1,5-naphthalene-based series compared
with a representative nonconstrained peptide, CVFM,
provides further evidence that the biologically active
conformations for both the new series and CVFM are
closely related and are therefore extended. The new
prototypes, particularly members of the 1,6-naphthalene-
based series, demonstrate potent cellular activity against

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1767
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