Development of highly potent inhibitors of Ras farnesyltransferase possessing cellular and in vivo activity

Article abstract of DOI:10.1021/jm950642a

Analogs of CVFM (a known nonsubstrate farnesyltransferase (FT) inhibitor derived from a CA₂A₂X sequence where C is cysteine, A is an aliphatic residue, and X is any residue) were prepared where phenylalanine was replaced by (Z)-dehyd

Full text of DOI:10.1021/jm950642a

224
J . Med. Chem. 1996, 39, 224-236
Develop m en t of High ly P oten t In h ibitor s of Ra s F a r n esyltr a n sfer a se P ossessin g
Cellu la r a n d in Vivo Activity
Katerina Leftheris,*,§ Toni Kline,‡ Gregory D. Vite,§ Young H. Cho,§ Rajeev S. Bhide,§ Dinesh V. Patel,§
Manorama M. Patel,§ Robert J . Schmidt,§ Harold N. Weller,§ Mary L. Andahazy,† J oan M. Carboni,†
J ohnni L. Gullo-Brown,† Francis Y. F. Lee,† Carol Ricca,† William C. Rose,† Ning Yan,† Mariano Barbacid,†
J ohn T. Hunt,§ Chester A. Meyers,‡ Bernd R. Seizinger,† Robert Zahler,§ and Veeraswamy Manne†
Departments of Oncology Chemistry, Peptide and Protein Research, and Oncology Drug Discovery, The Bristol-Myers Squibb
Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New J ersey 08543-4000
Received August 30, 1995X
Analogs of CVFM (a known nonsubstrate farnesyltransferase (FT) inhibitor derived from a
CA1A2X sequence where C is cysteine, A is an aliphatic residue, and X is any residue) were
prepared where phenylalanine was replaced by (Z)-dehydrophenylalanine, 2-aminoindan-2-
carboxylate, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Tic), and indoline-2-carboxylate. The
greatest improvement in FT inhibitory potency was observed for the Tic derivative (IC50 ) 1
nM); however, this compound was ineffective in blocking oncogenic Ras-induced transformation
of NIH-3T3 fibroblast cells. A compound was prepared in which both the Cys-Val methylene-
amine isostere and the Tic replacement were incorporated. This derivative inhibited FT with
an IC50 of 0.6 nM and inhibited anchorage-independent growth of stably transformed NIH-
3T3 fibroblast cells by 50% at 5 µM. Replacing the A1 side chain of this derivative with a
tert-butyl group and replacing the X position with glutamine led to a derivative with an IC50
of 2.8 nM and an EC50 of 0.19 µM, a 26-fold improvement over (S*,R*)-N-[[2-[N-(2-amino-3-
mercaptopropyl)-L-valyl]-1,2,3,4-tetrahydro-3-isoquinolinyl]carbonyl]-L-methionine. This deriva-
tive, (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-tert-leucyl]-1,2,3,4-tetrahydro-3-isoquinolinyl]-
carbonyl]-L-glutamine, was evaluated in vivo along with (S*,R*)-N-[[2-[N-(2-amino-3-
mercaptopropyl)-L-tert-leucyl]-1,2,3,4-tetrahydro-3-isoquinolinyl]carbonyl]-L-methionine methyl
ester for antitumor activity in an athymic mouse model implanted ip with H-ras-transformed
rat-1 tumor cells. When administered by injection twice a day at 45 mg/kg for 11 consecutive
days, both compounds showed prolonged survival time (T/C ) 142-145%), thus demonstrating
efficacy against ras oncogene-containing tumors in vivo.
In tr od u ction
(where C is cysteine, A is an aliphatic residue, and X is
any residue) at the C-terminus of p21ras triggers a series
of post-translational processing events.4 The initial
step, when X ) Met or Ser, involves farnesylation of
Cys186 by the enzyme farnesyltransferase (FT). Subse-
quently, the three amino acids distal to the prenyl
cysteine are removed by proteolysis.5 Finally, the
C-terminal carboxyl is capped by methylation. Other
modifications such as palmitoylation and phosphoryla-
tion occur in certain p21ras proteins.6,7 The initial
prenylation appears to be a prerequisite for all subse-
quent modifications which enable p21ras to localize to
the plasma membrane.8
Inhibiting the S-farnesylation of p21ras may block the
growth of Ras-mediated tumors, and much effort has
been directed toward the design of inhibitors of FT.9-20
Our efforts have focused on the design of inhibitors
based on the CA1A2X tetrapeptide sequence.12 As shown
by others, the presence of an aromatic amino acid in
the A2 position, as in CVFM (compound 1), is a major
determinant for nonsubstrate tetrapeptide inhibitor
activity.19 Nonsubstrate inhibitors are not subject to
inactivation through prenylation and are therefore well
suited as FT inhibitor leads. The distance between the
aromatic ring and the amide backbone is critical for
preventing farnesylation of these inhibitors,12 suggest-
ing a defined binding site may exist for the aromatic
ring. We and others have observed that replacing
selected amide bonds with a reduced amide isostere in
ras genes encode a family of GTP-binding proteins
(p21ras) known to play a major role in controlling cell
growth and differentiation.1 Cycling between the active
GTP-bound form and inactive GDP-bound form, p21ras
acts as a regulatory switch in cell signaling. Upstream
elements associated with transmembrane receptors
facilitate GTP binding by p21ras thus propagating an
extracellular stimulus downstream to intracellular and
ultimately intranuclear targets. In normal cell signal-
ing, inactivation of p21ras occurs through hydrolysis of
bound GTP to GDP by the protein’s intrinsic GTPase
activity coupled with strong stimulation by GTPase-
activating protein (GAP). Specific mutations in the ras
gene impair GTPase activity and response to GAP of
p21ras, rendering it constituitively active and resulting
in uncontrolled cell growth and morphologic transfor-
mation of normal cells. Mutated ras genes have been
found in 15% of all human carcinomas, with much
higher occurrences in pancreatic adenocarcinomas (90%)
and human colon tumors (50%).1
Ras proteins must be membrane associated2,3 to
function, and post-translational modification is critical
to this localization. A conserved CA1A2X sequence
* To whom correspondence should be addressed.
† Department of Oncology Drug Discovery.
‡ Department of Peptide and Protein Research.
§ Department of Oncology Chemistry.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0022-2623/96/1839-0224$12.00/0 © 1996 American Chemical Society

Potent Inhibitors of Ras Farnesyltransferase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 225
Sch em e 1a
amide bond isostere are described in the Experimental
Section. Compounds 34 and 35 were prepared as a
mixture of diastereomers starting from racemic 1,2,3,6-
tetrahydropyridine-2-carboxylic acid24 utilizing the ap-
proach outlined for compound 21. The isomers were
separated by HPLC in the final purification step.
Compound 36 was prepared starting from 3-amino-2-
naphthoic acid utilizing the approach outlined for
compound 25. Modifications of the methionine (X)
position were accomplished by utilizing a convergent
approach (outlined in Scheme 6) in which 22 was
converted to the partially protected tripeptide interme-
diate 30 followed by coupling to the desired amino acid
ester and protecting group removal. Derivatives modi-
fied at the A1 position were prepared as described for
25.
a
(a) Fmoc-OSu, 10% Na2CO3, 1,4-dioxane, °C; (b) Bop/HOBt,
Met-Wang resin followed by piperidine; (c) Fmoc-Val-OH, HOBt,
Bop followed by piperidine; (d) Boc-Cys(S-Tr)-OH, Bop, HOBt,
DIEA; (e) TFA, Et3SiH.
a CAAX-based FT inhibitor leads to significant improve-
ment in inhibitory potency and whole cell activity.9,11,12,20
This isosteric replacement prevents proteolytic degrada-
tion, thereby improving the stability of peptide-based
inhibitors. Herein, we report the development of
CA1A2X-based inhibitors of FT where the aromatic ring
at the designated A2 position is conformationally re-
stricted. These inhibitors are cell-permeable in non-
prodrug form and are efficacious in vivo.
Resu lts a n d Discu ssion
Starting with 1 as the lead, we initially determined
if conformationally constraining the aromatic ring at the
A2 position could lead to improved FT inhibition. Thus,
the compounds prepared in this study were initially
screened as inhibitors of FT in an in vitro assay using
recombinant p21H-ras protein and FT isolated from
porcine brain.25
Ch em istr y
The unnatural amino acid 2 was prepared via a
Strecker synthesis starting from indanone as described
previously.21 Following Fmoc protection of 2, 3 was
coupled to Met-Wang resin using standard coupling
techniques. Fmoc deprotection of the resin-bound dipep-
tide using piperidine was followed by assembly of the
remaining residues using solid phase peptide synthesis
techniques. The cysteine residue was added as N-[(tert-
butyloxy)carbonyl]-S-(triphenylmethyl)-L-cysteine (Boc-
Cys(S-Tr)-OH) which allowed both resin cleavage and
protecting group removal on treatment with trifluoro-
acetic acid (TFA). This approach afforded 4 in five steps
(Scheme 1).
To prepare a tetrapeptide containing dehydrophenyl-
alanine at the A2 position, ring opening of oxazolone 522
with methionine methyl ester (Met-OMe) afforded 6. Boc
deprotection of 6 using TFA followed by coupling of Boc-
Cys(S-Tr)-OH and protecting group removal gave 7
(Scheme 2). Compound 14 was prepared by amide bond
coupling of commercially available (S)-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acid to Met-OMe using
standard coupling techniques followed by acidic cleavage
of the Boc group. Of the amide bond-coupling methods
attempted, bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(Bop-Cl) was superior in providing 10 from N-[(tert-
butyloxy)carbonyl]-L-valine (Boc-Val) and 9 (>98% yield).
Following Boc group removal of 10, 11 was coupled with
Boc-Cys(S-Tr)-OH and protecting groups were removed
to afford 14 (Scheme 3). To provide the indoline
derivative 21, 15 was coupled to Boc-Val using Bop-Cl.
After saponification and coupling to L-methionine tert-
butyl ester (Met-OtBu) to give 18, selective removal of
the Boc group using formic acid followed by coupling of
19 with Boc-Cys(S-Tr)-OH and subsequent protecting
group removal afforded 21 (Scheme 4). Scheme 5 shows
a representative synthesis of compounds containing the
reduced amide bond isostere. Aldehyde 22 was pre-
pared via formation and reduction of the N,O-dimeth-
ylhydroxamate.23 Reductive coupling of 22 with 11
followed by protecting group removal afforded 25.
Other derivatives in this series containing the reduced
As shown in Table 1, orienting the aromatic ring
perpendicular to the backbone (compound 4) is strongly
disfavored, leading to a 100-fold reduction in enzyme
inhibitory potency compared to 1 (IC50 ) 37 nM).
Structures containing the A2 aromatic ring oriented
parallel to the amide backbone (7 and 21) are slightly
less potent than 1. By far, the greatest improvement
in inhibitory activity was obtained by replacing Phe with
(S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Tic) (14),
leading to a 37-fold enhancement in potency. The
aromatic ring of Tic and the appended side chains are
clearly adapting a highly favorable orientation for
interaction with the enzyme. Others have found that
Tic is a suitable replacement for Phe in CA1A2X-based
FT inhibitors.10,18 This observation demonstrates that
alkylation of the amine at the A2 position is not
detrimental to inhibitory potency provided the proper
orientation of the aromatic ring is maintained. Prior
studies demonstrated that N-methylation at the A2
amine of 1 leads to a 10-fold reduction in inhibitory
potency,12 conceivably because the aromatic ring is less
able to orient in a favorable position.
Having identified 14 as an important lead, compounds
25 and 31-33 were prepared to examine the effects of
amide bond modifications on FT inhibition. If tolerated,
such modifications were expected to lead to improved
whole cell and in vivo activity by stabilizing the peptide
bonds to enzyme-mediated proteolysis. As found for
other CAAX-based inhibitors, incorporating the meth-
yleneamine amide bond isostere either maintains or
slightly improves FT inhibitory potency (31 and 33)
except when the methyleneamine is situated between
Tic and Met (32).11,12 These results and the improve-
ment in whole cell activity that was realized (vide infra)
led us to substitute a methyleneamine in place of the
C-A1 amide bond (i.e., 25) in the remainder of our
structure-activity development.
In an effort to understand the role of the Tic hetero-
cyclic ring, 1,2,3,6-tetrahydropyridine-2-carboxylic acid
stereoisomers 34 and 35 were prepared and determined

226 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Leftheris et al.
Sch em e 2a
a
(a) Met-OMe/THF; (b) TFA/CH2Cl2; (c) Boc-Cys(S-Tr)-OH, Bop, HOBt, DIEA, THF; (d) LiOH, MeOH, THF; (e) TFA, Et3SiH.
Sch em e 3a
a
(a) Met-OMe, EDC, HOBt, DIEA, NMP, CH2Cl2; (b) 4 N HCl/dioxane; (c) Boc-Val-OH, Bop-Cl, DIEA, CH2Cl2; (d) Boc-Cys(S-Tr)-OH,
EDC, HOBt, DMF; (e) LiOH, MeOH, THF; (d) TFA, Et3SiH.
Sch em e 4a
a
(a) Bop-Cl, NMM, Boc-Val-OH, CH2Cl2; (b) LiOH, MeOH, THF; (c) Bop, NMM, Met-OtBu, CH2Cl2; (d) Et3SiH, HCO2H; (e) Boc-Cys(S-
Tr)-OH, Bop, NMM, CH2Cl2; (f) Et3SiH, TFA.
Sch em e 5a
highly selective for FT over GGT1 with the greatest
selectivity found for 33 (25 times better than 1).
The whole cell activity profile of FT inhibitors was
evaluated utilizing two assays. The ras transformation
inhibition assay (RTI) measures the ability of FT
inhibitors to inhibit the transformation of normal NIH-
3T3 cells transfected with oncogenic H-ras DNA.14 The
cells were visually evaluated for percent inhibition of
transformation and gross toxicity at 10 and 100 µM
concentrations of inhibitors (Table 3). In a second assay,
compounds were evaluated for their ability to inhibit
the anchorage-independent growth in soft agar (SAG
assay) of NIH-3T3 cells stably transformed with onco-
genic H-ras.15 The inhibitor was added to the growth
medium every 2 days for an 8 day duration. The
number of colonies >0.1 mm in size was measured at
different concentrations of inhibitor and an EC50 value
determined (Table 3).
a
(a) 11, NaBH3CN, AcOH, MeOH; (b) NaOH, MeOH; (c) TFA,
CH2Cl2, Et3SiH.
to be significantly less active than 25 against FT.
Hence, the main function of the heterocyclic ring of Tic
appears to be in anchoring the aromatic ring in an
optimal binding orientation rather than orienting the
rest of the molecule in a favorable bioactive conforma-
tion. Replacing Tic with 3-amino-2-naphthoic acid (36)
leads to a significant reduction in inhibitory activity,
suggesting the existence of a well-defined inhibitor-
binding pocket.
To determine if aromatic ring alterations affect non-
substrate character, several inhibitors were tested using
a known substrate farnesylation assay (data not shown).19
None of these compounds are substrates, suggesting
that they probably bind in a manner similar to that of
1. In addition, the selectivity of inhibitors for FT over
geranylgeranyltransferase 1 (GGT1) is an important
issue, since these enzymes share a common subunit and
catalyze similar reactions. The three compounds in
Table 1 (1, 25, and 33) tested against both enzymes are
Compound 14 is not active at 100 µM in the RTI assay
probably because of proteolytic cleavage, as has been
shown by others for a similar compound.11 Of the
compounds tested in the Tic series, 25 and 33 display
the best whole cell activity. Both are capable of inhibit-
ing ras transformation in whole cells by 80% at 10 µM,
and both inhibit the soft agar growth of H-ras-trans-
formed cells with an EC50 of 5 µM. Compound 25 was
evaluated for inhibition of Ras processing in H-Ras-
transformed cells. Following treatment for 24 h, un-
processed precursor Ras proteins accumulated and
localized to the cytosolic fraction. Inhibition of Ras
processing was dose dependent with an IC50 value of 5

Potent Inhibitors of Ras Farnesyltransferase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 227
Sch em e 6a
a
(a) HCl‚Val-OMe, NaBH3CN, MeOH; (b) Cbz-Cl, DIEA, CH2Cl2; (c) 2 N LiOH; (d) HCl‚Tic-OMe, Bop-Cl, DIEA, CH2Cl2; (e) 1 N LiOH,
THF, MeOH; (f) H2NCHRCO2Me, Bop, DIEA, DMF; (g) 1 N LiOH, THF; (h) TFA, Et3SiH; (i) TFA, thioanisole, 1,2-ethanedithiol, TMSBr.
Ta ble 1. FT in Vitro Activity of Derivatives Constrained at the
Ta ble 2. In Vitro Inhibition of FT Inhibitors Modified at the
A2 Position
A1 and X Positions
IC50 (nM)a
no. R1
R2
R3
FT
GGT1
25 iPr -CH2CH2SCH3
37 Me -CH2CH2SCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
0.60 ( 0.040 110 ( 17
45 ( 3.0
38 Et
-CH2CH2SCH3
14 ( 2.3
41 iPr n-butyl
42 iPr -CH2CONH2
770 ( 190
1100 ( 540
90 ( 11
1.3 ( 0.5
43 iPr -CH2CH2SO2NH2
44 iPr -CH2CH2SO2CH3
45 iPr -CH2CH2CONH2
46 tBu -CH2CH2CONH2
47 tBu -CH2CH2CON(CH3)2
48 tBu -CH2CH2OCH3
49 tBu -CH2CH2SCH3
50 tBu -CH2CH2SO2CH3
51 tBu -CH2CH2SCH3
0.92 ( 0.020 400 ( 12
4.4 ( 0.55 1000 ( 240
2.8 ( 0.070 1400 ( 370
1.8 ( 0.060
1.4 ( 0.50
0.57 ( 0.11
0.46 ( 0.080
85 ( 35
22 ( 1.5
4.8 ( 1.6
14 ( 1.1
200 ( 23
a
n ) 3 unless otherwise indicated.
Ta ble 3. Whole Cell Profile of FT Inhibitors
RTI inhibition (%)
no.
100 µM
10 µM
SAG EC50 (µM)
25
31
33
37
38
41
42
43
44
45
46
47
48
49
50
51
100
70
100
NT
NT
80
>80
0.0
80
NT
NT
20
5.0
5.0
15
15
26
NT
75
NT
15
8.3
1.0
2.0
0.19
1.8
2.6
0.5
0.57
0.41
100
100
90
100
a
n ) 3 unless otherwise indicated.
100
100
90
µM. This inhibition of ras processing correlates well
with the EC50 for SAG inhibition. Although both
compounds show activity in whole cells, the EC50/IC50
ratios are high (>6000) suggesting that 25 and 33 are
poorly permeable. In an effort to address these issues,
we chose to optimize both the X and A1 positions.
Modifying the methionine side chain to norleucine
(Nle) (41) leads to a large drop in FT inhibitory potency
and an increase in GGT1 inhibitory potency, resulting
in a highly selective GGT1 inhibitor. This is an intrigu-
ing result considering that Nle is often used as a
chemically stable isosteric replacement for Met.26 Al-
though both are unbranched hydrophobic residues, they
are clearly not interchangeable at the active site of
either FT or GGT1.27 Perhaps differences in flexibility
100
or polarizability account for these differences.26 The
preference for Nle by GGT1 is also surprising, consider-
ing that GGT1 primarily recognizes CAAX sequences
containing branched aliphatic side chains at the X
position.28 Another interesting finding is that replace-
ment of the hydrophobic Met residue with a variety of
polar side chains leads to analogs which retain sub-
stantial FT inhibitory potency. Methyl ether (48)-,
carboxamide (45)-, and sulfone (44)-containing side
chains provide potent inhibitors, while a sulfonamide
(43) leads to a moderately potent inhibitor. The differ-
ence in FT inhibitory potency between 42 and 45

228 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Leftheris et al.
Ta ble 4. Effect of FT Inhibitors on Mice Bearing ip Rat-1
model against rat-1 tumor cells indicates that these
derivatives are efficacious against ras oncogene-contain-
ing tumors in vivo.
Tumorsa
dose
compd
(mg/kg/injection)
schedule
MST
%T/C
Exp er im en ta l Section
46
51
control
45
45
2qd×11
2qd×11
18.5
17.0
12.0
16.0
154
142
100
133
Ra s Biologica l Assa ys. En zym es. FT was isolated from
pig brain as described by Manne et al.25 and further purified
at least 2000-fold relative to the initial crude pig brain cytosol.
GGT1 was purified 400-fold from porcine brain tissue. Each
of the final enzyme preparations is free of the other enzyme.
P r otein P r ep a r a tion s. p21ras proteins, from bacteria
carrying expression vectors coding for v-H-ras gene sequences,
were purified as described28 to g80% purity. p21ras CVLL
proteins were purified as described29 to g80% purity from an
expression vector30 kindly provided by Dr. Channing Der,
University of North Carolina at Chapel Hill.
F a r n esyltr a n sfer a se Assa y. FT assays were run in 96-
well dishes in a reaction volume of 20 µL as described in detail
earlier.14 Briefly, the reaction mixtures contained 1 µM [3H]-
FPP (NEN DuPont), 7 µM p21 H-Ras, 25 mM MgCl2, 10 mM
DTT, 100 mM Hepes (pH 7.4), and serial dilutions of inhibitors
usually ranging from 360 µM to 0.02 nM. Reactions were
started by adding sufficient enzyme to produce 1-2 pmol of
[3H]FPP incorporation in 1 h in the control wells. Following
incubation at 37 °C for 1 h, the reactions were stopped and
the samples processed as described.14 Dose-response curves
for inhibitors used triplicate estimates at each drug concentra-
tion, and the IC50 estimations were made from percent control
versus log drug concentration plots. Unless otherwise indi-
cated, each compound was tested at least twice.
Ger a n ylger a n yltr a n sfer a se (GGT1) In h ibition Assa y.
To determine the IC50 values for inhibition, the GGT1 assay
was also adapted to 96-well microtiter plate format as de-
scribed.14 Briefly, 12 µM p21ras CVLL, 0.5 µM [3H]GGPP (19.3
Ci/mmol; NEN), 10 mM dithiothreitol (DTT), 5 mM MgCl2, 5
µM ZnCl2, 100 mM Hepes, pH 7.4, and partially purified GGT1
were incubated in a total volume of 20 µL at 37 °C for 1 h,
and samples were processed as described for the FT assay.
All assays were carried out in triplicate. Unless otherwise
indicated, each compound was tested at least twice.
r a s Tr a n sfor m a tion In h ibition (RTI) Assa y. The whole
cell activity of FT inhibitors was evaluated by a ras transfor-
mation inhibiton (RTI) assay. The RTI assay is based on
transformation of mouse NIH-3T3 cells by oncogenic ras DNA
transfection. NIH-3T3 cells (2.5 × 104/35 mm well) were
seeded and allowed to attach overnight. The cells were
transfected with linearized plasmid carrying oncogenic H-ras31
using the calcium phosphate precipitation technique. One day
following the transfection, cells were washed and inhibitors
added at the indicated concentrations into the medium.
Inhibitors were replenished every 48 h for 8 days along with
the change of medium, for a total of four treatments. The
degree of transformation was scored after 14 days. In the
absence of an inhibitor, transfected cells grow aggressively and
initially give the appearance of abnormal “foci” surrounded
by normal cells. In the presence of a cell-permeable inhibitor,
either the number of foci is reduced or transformation is
completely inhibited. The percent inhibition and the gross
cytotoxicity of inhibitors were evaluated visually by light
microscopy.
1/10 control
a
106 cells implanted on day 0 (except 105 cells in 1/10 control);
treatments began on day 1.
indicates that the polar carboxamide is an effective
replacement for the methionine sulfide. The Gln analog
45 is slightly more potent than 25 in whole cell assays,
despite its lower potency versus FT. The sulfone analog
44, while equipotent to 25 versus FT, also shows
improved SAG activity over 25.
At the A1 position, the Ala analog 37 is a modest
inhibitor. Increasing the number of methyl groups leads
to increased potency (i.e., 37 < 38 < 25 ) 49). Although
the isopropyl (25) and tert-butyl (49) side chains were
equipotent as FT inhibitors, the tert-butyl side chain led
to substantial improvement in cell activity. For ex-
ample, 49 is 10-fold more potent than 25 in the SAG
assay, even though it is equipotent as an FT inhibitor.
The reason for the increased cell potency of these
analogs is uncertain, although conformational effects of
the tert-butyl side chain may play some undefined role.
Increased hydrophobicity may also be a contributing
factor.
Combining the two effects which independently lead
to increased cell activity, namely, a tert-butyl side chain
at A1 and a polar X side chain, provides analog 46, which
displays an EC50 in soft agar of 0.19 µM (25-fold more
potent than 25). The very low EC50/IC50 ratio for 46 (70-
fold) suggests that this tetrapeptide is reasonably
membrane permeable, despite the presence of an unes-
terified carboxyl group. These compounds showed no
cytotoxicity up to 100 µM, indicating that the SAG
activity observed is from cellular penetration of FT
inhibitors and not due to general cytotoxicity (data not
shown).
Two FT inhibitors were evaluated in vivo for their
antitumor activity in athymic mice implanted ip with
H-ras-transformed rat-1 tumor cells (Table 4). When
administered ip twice a day at 45 mg/kg/injection for
11 consecutive days, both 46 and 51 (the methyl ester
of 49) were active, as demonstrated by prolonged
survival time (T/C ) 142-154%, T/C g 125% considered
an active result; see experimental for detailed descrip-
tion). Lower doses of either compound (15 mg/kg/
injection) did not produce an active result. The efficacy
of 51 at 45 mg/kg/injection using this animal model was
confirmed by subsequent investigations with 2qd×14
(T/C ) 180) and 2qd×7 (T/C ) 140) treatment schedules.
In summary, we have designed potent FT inhibitors
capable of penetrating cells and reversing morphological
transformation at submicromolar concentrations. Op-
timizing the orientation of the side chain aromatic ring
at the A2 position leads to a significant improvement
in inhibitory potency. Whole cell activity is improved
by replacing amide bonds with the methyleneamine
isostere and modifying the A1 and X positions. As
shown in one example, the observed effects in whole
cells can be correlated with inhibition of ras processing.
Evaluation of two compounds in an athymic mouse
An ch or a ge-In d ep en d en t Gr ow th in Soft Aga r Su sp en -
sion . Anchorage-independent growth was measured in soft
agar suspension as described earlier.15 Briefly, single cell
suspensions of H-ras-transformed NIH-3T3 cells (44-911 cells)
were obtained, mixed into a top soft agar mixture (0.3%), and
laid on a 0.6% bottom agar layer. Initially, inhibitors at the
indicated concentrations were incorporated into the soft agar
mixture. FT inhibitors were replenished every 2 days for 8
days by overlaying 100 µL of 20-fold concentrated stock in PBS
on the surface of the top agar layer. All the assays were
carried out in parallel duplicate wells. After 14 day growth,
colonies larger than 0.1 mm size in diameter were counted.
In Vivo Stu d ies. Athymic Balb/c-background female mice,
18-22 g, were purchased from Harlan Sprague-Dawley
(Indianapolis, IN). Rat-1 (H-ras-transformed rat fibroblast

Potent Inhibitors of Ras Farnesyltransferase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 229
line) tumors were sourced from in vitro propagated cells. A
detailed description of the general assay and evaluation
methods has been reported.32 Briefly, experiments were
initiated by the implantation, ip, of 1 × 106 cells, except in
control groups given titrated cell inocula. Group sizes con-
sisted of six mice in treatment groups and eight mice in
untreated control groups. All experiments began on day 1
post-tumor implant. Compounds 46 and 51 were dissolved in
sterile water and injected ip within 1 h of dissolution. Both
compounds were administered ip, usually twice a day (see
below) for 11 consecutive days (2qd×11) at varying dose levels.
Additionally, 51 was evaluated at 45 mg/kg/injection, ip, twice
daily for 7 and 14 days (2qd×7 and 2qd×14, respectively).
Twice a day injections were given 6-8 h apart on weekdays;
on weekends, a single injection was given at twice the
indicated dose (i.e., the total intended daily amount of com-
pound was given all at once, not as a split dosage as was done
on weekdays). Therapeutic results are presented in terms of
increases in life span reflected by the relative median survival
time (MST) of treated (T) versus control (C) groups (%T/C
values) and any long-term survivors. The activity criterion
for increased life span was a T/C of g125%. At the highest
twice daily dose level tested, 45 mg/kg/injection, both com-
pounds produced an active result. Control mice had a MST
of 12 days, and the mice treated with compound 46 or 51 had
MST’s of 18.5 and 17.0 days, respectively. Thus, these
therapies resulted in %T/C values of 154% and 142%, respec-
tively.
Ch em istr y. Gen er a l. Partially protected amino acids
were obtained from Bachem California, Novabiochem, Sigma,
and Schweizerhall Inc. Other reagents were obtained from
Aldrich. THF was distilled from Na/benzophenone, and meth-
ylene chloride was distilled from CaH2 prior to use. IR spectra
were recorded on a Mattson Sirius 100 spectrometer. Proton
NMR (1H-NMR) and carbon NMR (13C-NMR) spectra were
obtained on J eol GSX 400 or 270 MHz spectrometers and are
reported in parts per million (ppm) relative to tetramethylsi-
lane as the internal standard. Fast atom bombardment mass
spectrometry (FAB-MS) was conducted on a J EOL lSX 102
instrument; chemical ionization mass spectra (CI-MS) were
obtained using NH3 desorption chemical ionization (scan rate
50-900 µm/1.6 s; dynode voltage ) 1000 (V) on a Finnigan
TSQ 4600 mass spectrometer. High-resolution mass measure-
ments were made using a J EOL HX-110 double-focusing mass
spectrometer at a resolution of M/∆M ) 10 000 equipped with
a fast atom bombardment (FAB) ionization source. The FAB
matrix used was 50/50 thioglycerol/glycerol. Poly(ethylene
glycol)s were used as the internal calibrant. Analytical and
preparative HPLC were performed on YMC columns (YMC S-3
ODS 4.6 × 150 mm, S-5 ODS 4.6 × 250 mm, and YMC S-10
30 × 500 mm) with methanol/water and acetonitrile/water
gradients containing 0.1% TFA or 0.2% H3PO4. Thin-layer
chromatography (TLC) analyses of reactions were run on silica
gel 50 F254 plates (Merck). Flash chromatography was run on
230-400 mesh silica gel 60 (EM Science). Unless otherwise
noted, solutions were dried using MgSO4.
method on a Milligen Biosearch Model 9600 peptide synthe-
sizer using the Fmoc chemistry program supplied with the
instrument. Starting with Fmoc-methionine-substituted p-
alkoxybenzyl alcohol resin (Wang, polystyrene, 1% divinyl-
benzene copolymer, 1.0 g, 0.31 mmol; Bachem), amino acids
(including 3) were coupled as their NR-Fmoc derivatives with
the exception of the final residue, cysteine, which was blocked
as the N-Boc(S-Trt) derivative. Fmoc groups were removed
at each cycle by a 10 min treatment with 30% piperidine/35%
DMF/35% toluene. Each amino acid derivative was coupled
(1 h, room temperature), in 6-7-fold molar excess, using 1
equiv of (benzotriazol-1-yloxy)tris(dimethylamino)phospho-
nium hexafluorophosphate (Bop) and 1 equiv of hydroxyben-
zotriazole (HOBt) in DMF/CH2Cl2 mixtures. The dried, pro-
tected peptidyl-resin weighed 1.13 g (weight gain, 90% of
theory). Simultaneous deprotection and cleavage of the pep-
tide from the resin (0.4 g) was performed by stirring TFA (9
mL, 120 mmol), 1,2-ethanedithiol (1.0 mL), dimethyl sulfide
(0.5 mL), phenol (0.7 g), and thioanisole (0.25 mL) for 1.5 h.
The resin was filtered and washed with TFA (2 mL). The
combined filtrate was concentrated, and cold ether was added
to solidify the oily residue. The solution was filtered and the
solid was washed with ether, dissolved in a small amount of
methanol and 0.1% TFA/water, and purified by preparative
HPLC (YMC S-5 ODS 22 × 250 mm column eluting over 40
min with 25-35% B in A (A, 90% aqueous acetonitile contain-
ing 0.1% TFA; B, 10% aqueous acetonitrile containing 0.1%
TFA), flow rate 9 mL/min, UV monitored at 220 nm). The
appropriate fractions were pooled, concentrated and lyophi-
lized to yield 4 as a white fluffy powder (16 mg, 20%): MS (M
+ H)+ 511; mp 129-130 °C; [R]25 ) -18.3° (c 0.5, MeOH);
D
1H-NMR (DMSO J , 270 MHz) δ 7.63 (1H, m), 7.12 (4H, m),
4.52 (1H, m), 3.98 (2H, m), 3.42-3.13 (2H, m), 2.90 (2H, m),
2.44 (2H, m), 2.08 (1H, m), 2.00 (3H, s), 1.92 (1H, m), 1.80
(1H, m), 0.70-0.80 (6H, m). Anal. (C23H34N4O5S2‚2C2-
HF3O2‚H2O) C, H, N.
N-[2-[[(1,1-Dim eth yleth oxy)ca r bon yl]-L-va lyl]-1-oxo-3-
p h en yl-2-p r op en yl]-L-m eth ion in e Meth yl Ester (6).
A
solution of L-methionine methyl ester hydrochloride (950 mg,
5.8 mmol) in THF (20 mL) was added dropwise to an ice-cooled
solution of oxazolinone 522(2.1 g, 5.8 mmol) in THF (20 mL).
The mixture was allowed to equilibrate to room temperature,
and stirring was continued for 72 h. The solution was
concentrated in vacuo and the residue dissolved in ethyl
acetate. The organic phase was washed successively with 5%
aqueous citric acid, half-saturated aqueous NaHCO3, and brine
followed by drying and concentration to give 2.25 g (77%) of 6
as an oil: accurate mass measurement (M + H)+ calcd for
C25H38N3O6S 508.2481, found 508.2470 (∆ppm ) 2.3); 1H-NMR
(CHCl3, 270 MHz) δ 7.31-7.4 (5H, m), 7.26 (1H, s), 7.00-7.10
(1H, br s), 4.82 (1H, m), 4.0 (1H, m), 3.78 (3H, s), 2.57-2.63
(2H, m), 2.20-2.40 (2H, m), 2.05-2.16 (5H, s over m), 1.50-
1.76 (1H, br s), 1.45 (9H, s), 0.95-1.03 (6H, m).
N-[2-(N-L-Cystein yl-L-valyl)-1-oxo-3-ph en yl-2-pr open yl]-
L-m eth ion in e Tr iflu or oa ceta te (7). Intermediate 6 (1.0 g,
2.0 mmol) was treated with TFA:CH2Cl2 (1:1, 15 mL) at 4 °C
for 1 h followed by concentration under vacuum. Ethyl ether
was added, and the resulting precipitate was filtered, washed
with ethyl ether, and dried. The free base was generated by
dissolving the residue in a solution of half-saturated aqueous
NaHCO3 and extracting the free amine using ethyl acetate.
The organic layer was dried and concentrated, and the residue
was dissolved in THF (5 mL) and added to an ice-cooled
solution of Boc-Cys(S-Tr)-OH (2.8 g, 6.0 mmol), Bop (2.7 g, 6.0
mmol), HOBt (810 mg, 6.0 mmol), and diisopropylethylamine
(1.0 mL, 6.0 mmol) in THF (60 mL). The reaction mixture
was stirred under nitrogen for 16 h followed by concentration
in vacuo. The residue was dissolved in ethyl acetate and
washed with 5% citric acid, half-saturated NaHCO3, and brine,
dried, and concentrated in vacuo. Purification on flash silica
gel (4 × 18 cm column) using 25-40% ethyl acetate in hexane
provided 1.1 g (64%) of ester. A solution of LiOH (130 mg, 3.2
mmol) in 2 mL of H2O and 10 mL of methanol was added to
the above ester (1.0 g, 1.2 mmol) in 10 mL of THF, and the
mixture was stirred for 16 h. The base was neutralized with
3.5 mmol of citric acid (dissolved in H2O), the solution was
[N-(F lu or en ylm et h oxyca r b on yl)a m in o]-2,3-d ih yd r o-
1H-in d en e-2-ca r boxylic Acid (3). A solution of N-[(9-
fluorenylmethoxycarbonyl)oxy]succinimide (4.5 g, 14 mmol) in
1,4-dioxane (50 mL) at 0 °C was added all at once to a solution
of 221 (2.0 g, 12 mmol) in 10% sodium carbonate solution (70
mL). The mixture was stirred at 0 °C for 2 h and for an
additional 20 h at room temperature. The mixture was
concentrated to remove 1,4-dioxane, and the remaining aque-
ous solution was washed with ether (3 × 100 mL), acidified to
pH 2, and extracted with ethyl acetate (3 × 100 mL). The
combined ethyl acetate extract was washed with brine (100
mL), dried, and concentrated to yield 3 as a white solid (3.8 g,
81%): Rf 0.5 (CH2Cl2:CH3OH:CH3CO2H, 85:10:5); MS (M +
H)+ 400; 1H-NMR (DMSO-d6, 270 MHz) δ 12.58 (1H, br s), 7.92
(1H, s), 7.86 (2H, d, J ) 7.6 Hz), 7.68 (2H, d, J ) 7.0 Hz), 7.40
(2H, t), 7.30 (2H, t), 7.16 (4H, m), 4.27 (2H, d, J ) 6.4 Hz),
3.45 (2H, d, J ) 16.4 Hz), 3.22 (2H, d, J ) 17.1 Hz).
N-[[2-[(N-L-Cyst ein yl-L-va lyl)a m in o]-2,3-d ih yd r o-1H -
in d en -2-yl]ca r bon yl]-L-m eth ion in e Tr iflu or oa ceta te (4).
Compound 4 was assembled by the stepwise solid phase

230 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Leftheris et al.
concentrated under vacuum, and the residue was dissolved in
ethyl acetate. The organic solution was washed with saturated
aqueous NaCl, dried, and concentrated in vacuo to give 920
mg of the acid (95%). To a solution of the acid in TFA:
methylene chloride (1:1, 15 mL) was added triethylsilane (0.2
mL), and the solution was stirred for 2.6 h. The solvents were
removed in vacuo, and the oily residue was washed with
hexanes, dissolved in 1:1 methanol:water containing 0.1% TFA,
and purified by preparative HPLC (YMC ODS column (22 ×
250 mm, 5 µm, 120 Å); solvent A, 0.1% TFA in water; solvent
B, 0.1% TFA in acetonitrile; 5-50% B over 40 min; flow rate
9 mL/min; UV monitored at 220 nm). The appropriate
fractions were collected, concentrated, and lyophilized to yield
m eth ion in e Meth yl Ester (12). To a cooled solution of 11
(1.1 g, 2.3 mmol) in DMF (30 mL) was added Boc-Cys(S-Tr)-
OH (3.2 g, 6.9 mmol), Bop (3.1 g, 6.9 mmol), HOBt (940 mg,
6.9 mmol), and diisopropylethylamine (1.2 mL, 6.9 mmol). The
mixture was stirred for 16 h under nitrogen. The solvent was
removed in vacuo and the residue dissolved in ethyl acetate.
The organic solution was washed successively with 5% aqueous
citric acid, half-saturated aqueous NaHCO3, and brine. The
extract was dried and concentrated. The residue was chro-
matographed (flash silica gel, 5 × 16 cm, 25-40% ethyl acetate
in hexane) to give 1.34 g (67%) of 12 as a yellow viscous oil:
accurate mass measurement (M + H)+ calcd for C48H59N4O7S2
1
867.3825, found 867.3843 (∆ppm ) 2.1); H-NMR (CDCl3, 270
24 mg of 7 as a white fluffy powder: MS (M + H)+ 497; [R]20
MHz) δ 8.10-7.90 (1H, d, J ) 5 Hz), 7.45-7.17 (19H, m), 6.80-
6.70 (1H, br d, J ) 5 Hz), 6.55-6.45 (1H, d, J ) 5 Hz), 5.20-
4.10 (6H, m), 3.70, 3.57 (3H, 2s, rotamers), 3.25-2.85 (2H, m),
2.80-2.40 (2H, m), 2.13-1.94 (5H, m), 1.98-1.95 (3H, 2s,
rotamers), 1.43, 1.40 (9H, 2s, rotamers), 1.06-0.92 (6H, ddd,
J ) 5, 5, 20 Hz).
D
1
) +90° (c 0.5, methanol); H-NMR (D2O, 270 MHz) δ 7.34-
7.47 (5H, m), 4.55-4.58 (1H, dd, J ) 5 Hz), 4.29-4.30 (1H, t,
J ) 6 Hz), 4.21-4.23 (1H, t, J ) 6 Hz), 2.93-3.02 (2H, m),
2.49-2.59 (2H, m), 2.00-2.02 (5H, s over m), 0.85-0.93 (6H,
dd, J ) 7, 26 Hz). Anal. (C22H33N4O5S2‚C2HF3O2) C, H, N, F,
S.
(R*,S*)-N-[[2-[N-[[2-[[(1,1-Dim et h ylet h oxy)ca r b on yl]-
a m in o]-3-[(tr ip h en ylm eth yl)th io]p r op yl]ca r bon yl]-L-va -
ly l]-1,2,3,4-t e t r a h y d r o -3-is o q u in o lin y l]c a r b o n y l]-L -
m eth ion in e (13). A solution of 12 (1.3 g, 1.6 mmol) and 1 M
LiOH (3.1 mL, 3.1 mmol) in methanol (20 mL) and THF (10
mL) was stirred for 3 h. Citric acid (3.1 mmol) was added,
and the solution was concentrated, diluted with water (25 mL),
and washed with ethyl acetate. The ethyl acetate layer was
washed with saturated aqueous NaCl solution and dried, and
the solvent was removed in vacuo to give 650 mg (0.76 mmol,
50%) of 13 as a glassy solid: accurate mass measurement (M
+ Na)+ calculated for C47H56N4O7S2Na 875.3489, found 875.3513
(R*)-N-[[2-[(1,1-Dim eth yleth oxy)ca r bon yl]-1,2,3,4-tet-
r a h yd r o-3-isoqu in olin yl]ca r bon yl]-L-m eth ion in e Meth yl
Ester (8). A solution of NR-Boc-L-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid (Boc-L-Tic) (2.0 g, 7.2 mmol) and meth-
ionine methyl ester hydrochloride (1.4 g, 7.2 mmol) was stirred
in 5:15 NMP:CH2Cl2 at 4 °C. Diisopropylethylamine (1.2 mL,
7.2 mmol) was added followed by HOBt (970 mg, 7.2 mmol).
The reaction mixture was stirred for 5 min; then 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDCI; 1.4 g, 7.2 mmol) was added. The reaction mixture
stirred at room temperature, under nitrogen for 16 h, followed
by partitioning between CH2Cl2 and saturated aqueous NaCl.
The organic phase was washed successively with 5% citric acid,
half-saturated NaHCO3, and brine, dried, filtered, and con-
centrated in vacuo to give 2.6 g (86%) of 8 as a white solid:
accurate mass measurement (M + H)+ calcd for C21H31N2O5S
423.1954, found 423.1968 (∆ ppm ) 3.1); 1H-NMR (CDCl3, 270
MHz) δ 7.22-7.26 (4H, br m), 4.52-4.57 (4H, br m), 3.69 (3H,
s), 3.36-3.41 (2H, t, J ) 7Hz), 2.38 (2H, t, J ) 7 Hz), 1.93-
2.02 (5H, s over m), 1.51 (9H, br s).
(R*)-N-[(1,2,3,4-Tetr a h yd r o-3-isoqu in olin yl)ca r bon yl]-
L-m eth ion in e Meth yl Ester (9). A solution of 8 (2.0 g, 4.7
mmol) in CH2Cl2 (2 mL), dimethyl sulfide (0.6 mL), and 4 N
HCl in dioxane (10 mL) was stirred for 1 h under nitrogen.
The mixture was concentrated, and diethyl ether was added.
The product precipitated and was filtered, washed with diethyl
ether, and dried under vacuum to give 1.6 g (99%) of 9 as a
glassy white solid which was used directly in the next step.
(R*)-N-[[2-[N-[(1,1-Dim eth yleth oxy)ca r bon yl]-L-va lyl]-
1,2,3,4-t et r a h yd r o-3-isoq u in olin yl]ca r b on yl]-L-m et h io-
n in e Meth yl Ester (10). To a solution of 9 in CH2Cl2 at 0 °C
(20 mL) was added Boc-L-valine (2.1 g, 9.5 mmol), Bop-Cl (1.2
g, 4.7 mmol), and N,N-diisopropylethylamine (1.6 mL, 9.5
mmol). The mixture was stirred for 16 h at 0 °C. Additional
Bop-Cl (1.2 g, 4.7 mmol) and N,N-diisopropylethylamine (0.80
mL, 4.7 mmol) were added, and the mixture was stirred for
an additional 8 h at 0 °C. The mixture was concentrated and
chromatographed (flash silica gel, 4.1 × 20 cm, 1:1 ethyl
acetate:hexane) to give 10 as a clear oil (2.4 g, 97%): accurate
mass measurement (M + H)+ calcd for C26H40N3O6S 522.2638,
found 522.2623 (∆ppm ) 2.6); 1H-NMR (CDCl3) δ 7.20-7.13 (4H,
m), 5.3-4.7 (3H, m), 4.7-4.5 (2H, m), 4.4-4.2 (1H, m), 3.64-
3.59 (3H, 2s), 3.35-3.15 (2H, d, J ) 7 Hz), 3.05-2.90 (2H, d,
J ) 7 Hz), 2.22-1.89 (7H, m), 1.34 (9H, s), 1.02-0.9 (6H, m).
(R*)-N-[(2-L-Va lyl-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl)-
ca r bon yl]-L-m eth ion in e Meth yl Ester (11). A solution of
10 (1.2 g, 2.3 mmol) in CH2Cl2 (3 mL), 4 N HCl/dioxane (10
mL), and 0.5 mL of dimethyl sulfide was stirred for 1 h
followed by concentration under vacuum. Diethyl ether was
added, and the precipitated product was filtered, washed with
diethyl ether and dried under vacuum to give 1.1 g (99%) of
11 as a white solid: MS (M + H)+ 422; this was used without
purification.
1
(∆ppm ) 2.8); H-NMR (CO(CD3)2, 270 MHz) δ 7.5-6.9 (19 H,
m), 6.2-5.9 (1H, m), 4.9-4.7 (2H, m), 4.3-4.7 (1H, m), 3.9-
3.7 (2H, m), 3.0-2.8 (2H, m), 2.7-2.3 (2H, m), 2.2-2.1 (2H,
m), 2.0-1.7 (2H, m), 1.3 (9H, s), 0.9-0.6 (6H, m).
(R*,S*)-N-[[2-[N-[(2-Am in o-3-t h iop r op yl)ca r b on yl]-L-
va lyl]-1,2,3,4-t e t r a h yd r o-3-isoq u in olin yl]ca r b on yl]-L-
m eth ion in e Tr iflu or oa ceta te (14). A solution of 13 (330
mg, 0.38 mmol) in 15 mL of 1:1 TFA:CH2Cl2 (15 mL) was
stirred at room temperature for 2.6 h. The solvents were
removed in vacuo, and the oily residue was washed with
hexanes, dissolved in 1:1 methanol:water containing 0.1% TFA,
and purified by preparative HPLC (YMC C-18 column (22 ×
250 mm, 5 µm, 120 Å) using a gradient of 25-60% B in A over
60 min (A ) 0.05% TFA in H2O; B ) 0.05% TFA in CH3CN))
to give, after lyophilization, 120 mg (49%) of 14 as a white
1
fluffy powder: MS (M + H)+ 511; H-NMR (D2O, 270 MHz) δ
7.21-7.30 (4H, m), 4.8-5.1 (2H, d, J ) 42 Hz), 4.80-4.84 (1H,
m), 4.64-4.65 (1H, m), 4.32-4.34 (1H, t, J ) 4 Hz), 4.19-
4.23 (1H, t, J ) 5 Hz), 2.91-3.22 (4H, m), 2.30-2.32 (2H, m),
2.20-2.29 (2H, m), 1.98 (3H, d, J ) 1 Hz), 1.81-1.85 (2H, m),
0.91-1.00 (6H, dd, J ) 6, 28 Hz). Anal. (C23H34N4O5S2‚
C2HF3O2‚H2O) C, H, N, F, S.
(S)-1-[N-[(1,1-Dim et h ylet h oxy)ca r b on yl]-L-va lyl]-2,3-
d ih yd r o-1H-in d ole-2-ca r boxylic Acid Eth yl Ester (16). To
a solution of Bop-Cl (2.5 g, 10 mmol), indoline-2-carboxylic acid
ethyl ester (15) (1.2 g, 5 mmol), and NMM (2.2 mL, 20 mmol)
in CH2Cl2 (20 mL) was added a solution of Boc-Val-OH (2.2 g,
10 mmol) in CH2Cl2 (30 mL) containing DMF (1 mL) at 0 °C
under argon. The reaction was allowed to warm to 5 °C over
2 h, and stirring was continued for 20 h at 5 °C. The mixture
was washed with 1 N HCl (30 mL) and 10% LiCl (30 mL),
dried, filtered, and concentrated. The oil obtained was purified
by flash chromatography eluting with 10% acetone in hexane
to afford 16 contaminated with 15. The oil was dissolved in
diethyl ether (30 mL), and the solution was washed with 1 N
HCl (3 × 20 mL), dried, and concentrated to afford pure ester
16 (880 mg, 47%) as an oil. An additional 200 mg (17%) of 16
was recovered from the acidic fraction: TLC Rf 0.33 (20%
1
acetone in hexane); MS (M + H)+ 391; H-NMR (CDCl3, 270
MHz) δ 8.3 (1H, d, J ) 6.0 Hz), 7.2-7.0 (3H, m), 5.5-5.4 (1H,
m), 5.1-4.95 (1H, m), 4.3-4.1 (2H, m), 3.65-3.25 (2H, m),
2.25-2.2 (1H, m), 1.4 (9H, s), 1.25 (3H, t, J ) 7.0 Hz), 1.21
(1.5H, d, J ) 7.0 Hz, rotamers), 1.17 (1.5H, d, J ) 7.0 Hz),
1.01 (1.5H, d, J ) 7.0 Hz), 0.94 (1.5H, d, J ) 7.0 Hz); 13C-
NMR (CDCl3, 67.8 MHz) δ 171.2, 171.0, 170.7, 155.9, 155.3,
(R*,S*)-N-[[2-[N-[[2-[[(1,1-Dim et h ylet h oxy)ca r b on yl]-
a m in o]-3-[(tr ip h en ylm eth yl)th io]p r op yl]ca r bon yl]-L-va -
ly l]-1,2,3,4-t e t r a h y d r o -3-is o q u in o lin y l]c a r b o n y l]-L -

Potent Inhibitors of Ras Farnesyltransferase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 231
142.2, 130.9, 128.72, 128.1, 127.8, 125.7, 125.0, 124.3, 123.8,
122.3, 120.5, 117.3. 113.7, 111.9, 108.3, 79.5, 79.1, 77.2, 62.3,
61.3, 60.5, 60.3, 57.5, 56.4, 33.3, 32.6, 31.0, 30.2, 28.2, 19.6,
19.4, 17.2, 15.6, 14.3, 13.9, 13.8.
removed and the residue was purified by preparative HPLC
(YMC C-18 column (30 × 500 mm, 10 µm, 120 Å) using a
gradient of 10-90% B in A over 60 min (A ) 0.1% TFA in
H2O; B ) 0.1% TFA in CH3CN)) to give, after lyophilization,
21 (56 mg, 52%): mp 72-74 °C; MS (M + H)+ 497; [R]D
)
(S)-1-[N-[(1,1-Dim et h ylet h oxy)ca r b on yl]-L-va lyl]-2,3-
d ih yd r o-1H-in d ole-2-ca r boxylic Acid (17). To a solution
of 16 (840 mg, 2.2 mmol) in THF (9 mL) was added 1 N LiOH
(3 mL, 3 mmol) and sufficient methanol (0.3 mL) for a
homogeneous solution. After 3 h, 1 N HCl (5 mL) was added
and the mixture was extracted with ethyl acetate (2 × 5 mL).
The organic extracts were combined, dried, filtered, and
concentrated to afford acid 17 (650 mg, 83%): MS (M + H)+
-49.8° (c 0.49, methanol); 1H-NMR (CD3OD, 400 MHz) δ 8.6,
8.1 (1H, 2d, 1:3 ratio, J ) 9.0 Hz), 7.3-6.9 (3H, m), 5.0 (1H,
m), 4.7-4.5 (2H, m), 4.0 (1H, m), 3.6-3.5 (1H, m), 3.3-3.2 (1H,
m), 2.95-2.8 (2H, m), 2.8-2.6 (2H, m), 2.2-1.9 (3H), 2.0 (3H,
s), 0.95 (3H, d, J ) 7.0 Hz), 0.92 (3H, d, J ) 7.0 Hz); IR (KBr)
1674, 2969 cm-1. Anal. (C22H32N4O5S2‚1.3CHF3O2‚1.8‚H2O) C,
H, N, F, S.
1
363; H-NMR (CDCl3, 270 MHz) δ 8.27 (1H, d, J ) 8.0 Hz),
(R*,S*)-N-[[2-[N-[2-[[(1,1-Dim eth yleth oxy)car bon yl]am i-
n o]-3-[(tr ip h en ylm eth yl)th io]p r op yl]-L-va lyl]-1,2,3,4-tet-
r a h yd r o-3-isoqu in olin yl]ca r bon yl]-L-m eth ion in e Meth yl
Ester (23). A solution of 11 (460 mg, 1.0 mmol) and 22 (0.55
g, 1.2 mmol)12 in dry methanol (15 mL) containing 0.5 g of 3 Å
molecular sieves was stirred at room temperature for 0.5 h.
Glacial acetic acid (0.4 mL) was added followed by portionwise
addition of NaBH3CN (126 mg, 2.0 mmol) over 30 min under
nitrogen. Additional compound 22 (0.28 g, 0.6 mmol) was
added, and the mixture was stirred for 16 h. On cooling the
mixture to 0 °C, saturated NaHCO3 (100 mL) was slowly
added. The product was then extracted into ethyl acetate (60
mL). The ethyl acetate layer was washed with water (100 mL)
and brine (100 mL), dried, concentrated, and chromatographed
(silica gel, 4.1 × 15 cm, 1:1 ethyl acetate:hexane) to yield 514
mg of 23 as a clear oil (67%): MS (M + H)+ 853; 1H-NMR (400
MHz, CDCl3) δ 8.10-7.90 (1H, m), 7.45-7.17 (19 H, m), 6.80-
6.70 (1H, m), 6.55-6.45 (1H, m), 5.20-4.10 (6H, m), 3.70, 3.57
(3H, 2s, rotamers), 3.25-2.85 (2H, m), 2.80-2.40 (2H, m),
2.13-1.94 (5H, m), 1.98-1.95 (3H, 2s, rotamers), 1.43, 1.40
(9H, 2s, rotamers), 1.06-0.92 (6H, m).
(R*,S*)-N-[[2-[N-[2-[[(1,1-Dim eth yleth oxy)car bon yl]am i-
n o]-3-[(tr ip h en ylm eth yl)th io]p r op yl]-L-va lyl]-1,2,3,4-tet-
r a h yd r o-3-isoqu in olin yl]ca r bon yl]-L-m eth ion in e (24). A
solution of 23 (300 mg, 0.35 mmol) in methanol (10 mL), THF
(5 mL), and 1 M LiOH (0.4 mmol, 0.4 mL) was stirred for 24
h. The mixture was neutralized by addition of 1 N HCl (0.4
mL, 0.4 mmol) and concentrated. The residue was redissolved
in water (30 mL), and the solution was washed with ethyl
acetate. The organic layer was washed with brine (50 mL),
dried, filtered, and concentrated to yield 290 mg (99%) of 24
as a white solid: MS (M + H)+ 840.
(R*,S*)-N-[[2-[N-(2-Am in o-3-th iop r opyl)-L-va lyl]-1,2,3,4-
tetr a h yd r o-3-isoqu in olin yl]ca r bon yl]-L-m eth ion in e Tr i-
flu or oa ceta te (25). A solution of 24 (120 mg, 0.14 mmol) in
CH2Cl2 (5 mL), trifluoroacetic acid (5 mL), and triethylsilane
(0.2 mL) was stirred for 1 h at room temperature. The mixture
was concentrated, redissolved in methylene chloride (50 mL),
and concentrated. The crude solid was washed with hexanes,
dissolved in 2 mL of 0.1% aqueous TFA containing 20%
methanol, and purified by preparative HPLC (YMC C-18
column, 2.2 × 25 cm, 5 µm, 120 Å; solvent A, 0.1% TFA in
water; solvent B, 0.1% TFA in acetonitrile; 25-35% B in 40
min, flow rate 9 mL/min; UV monitored at 220 nm) to yield,
after lyophilization, 35 mg of 25 (33%) as a white fluffy solid:
mp 103-104 °C; [R]25D ) -44.2° (c 0.5, CH3OH); MS (M + H)+
497; 1H-NMR (CD3OD) δ 7.17 (4H, m), 4.72 (4H, m), 4.48 (1H,
m), 4.29 (1H, m), 3.92 (1H, m), 3.25 (2H, m), 2.69 (2H, m),
2.43 (1H, m), 2.12 (3H, m), 1.95 (3H, 2s), 1.83 (2H, m), 0.98
(6H, m). Anal. (C23H36N4O4S2‚0.65‚CHF3O2‚0.93H2O) C, H,
N, S, F.
7.3-7.0 (3H, m), 5.72 (1H, d, J ) 9.4 Hz), 5.15-5.05 (1H, m),
4.7-4.60 (1H, m), 3.6-3.4 (2H, m), 2.1-2.0 (1H, m), 1.4, 1.3
(9H, 2s, 1:3 ratio), 1.05 (3H, d, J ) 7.0 Hz), 0.92 (3H, d, J )
7.0 Hz). Anal. (C28H43N3O6S) C, H, N.
(S)-N-[[1-[N-[(1,1-Dim et h ylet h oxy)ca r b on yl]-L-va lyl]-
2,3-d ih yd r o-1H-in d ol-2-yl]ca r bon yl]-L-m eth ion in e Meth -
yl Ester (18). To a solution of 17 (300 mg, 0.83 mmol) in
CH2Cl2 (4 mL) at 0 °C under argon were added sequentially)
HCl‚Met-O-tBu (242 mg, 1.0 mmol), NMM (0.24 mL, 2.2
mmol), and Bop (442 mg, 1.0 mmol). The mixture was allowed
to warm to room temperature over 2 h, stirred overnight, and
then washed with 1 N HCl (5 mL) and saturated NaHCO3 (5
mL). The two aqueous washes were extracted separately with
CHCl3 (5 mL each). The organic extracts were combined,
dried, and concentrated. Purification by silica gel column
chromatography eluting with 30% ethyl acetate in hexane
afforded 18 (340 mg, 74%): TLC Rf 0.19 (30% ethyl acetate in
1
hexane); mp 79-81 °C; MS (M + H)+ 550; H-NMR (CDCl3,
270 MHz) δ 8.1 (0.5 H, d, J ) 2.0 Hz, rotamers), 7.9 (0.5H, d,
J ) 2.0 Hz), 7.25 (1H, d, J ) 9.0 Hz), 7.15 (2H, d, J ) 15 Hz),
5.45 (1H, d, J ) 2 Hz), 5.3-5.15 (1H, m), 5.1-4.9 (1H, m),
4.6-4.4 (1H, m), 4.3-4.2 (1H, m), 3.6-3.3.3 (2H, m), 2.4-1.8
(5H, m), 1.98 and 1.87 (3H, 2s, rotamers), 1.43 (9H, s), 1.2-
0.95 (6H, m).
(S)-N-[(1-L-Va lyl-2,3-d ih yd r o-1H-in d ol-2-yl)ca r bon yl]-
L-m eth ion in e Meth yl Ester (19). To a solution of 18 (130
mg, 0.24 mmol) in formic acid (98%, 0.5 mL) at room temper-
ature under argon was added triethylsilane (0.08 mL), and the
progress of the reaction was monitored by TLC analysis. After
9 h, saturated aqueous NaHCO3 (5 mL) was added and the
mixture was extracted with CHCl3 (2 × 5 mL). The organic
extracts were combined, dried, filtered, and concentrated to
1
afford free amine 19 (100 mg, 92%): MS (M + H)+ 450; H-
NMR (CDCl3, 270 MHz) δ 8.3 (1H, d, J ) 2.0 Hz, rotamers),
7.25 (2H, m), 7.15 (1H, m), 5.0 (1H, d, J ) 2 Hz), 5.45 (1H, m),
3.7-3.1 (4H, m), 2.4-2.2 (2H, m), 2.1-1.8 (7H, m), 1.45 (9H,
s), 1.01 (3H, d, J ) 7.0 Hz), 0.93 (3H, dd, J ) 8.0 Hz); 13C-
NMR (CDCl3, 67.8 MHz) δ 175.3, 171.2, 170.4, 141.9, 129.4,
127.8, 124.6, 117.8, 82.7, 62.0, 59.2, 52.2, 34.2, 32.2, 30.5, 29.8,
29.6, 27.9, 19.9, 17.4, 15.2, 6.7, 6.3.
(R*,S*)-N-[[2-[N-[[2-[[(1,1-Dim et h ylet h oxy)ca r b on yl]-
a m in o]-3-[(tr ip h en ylm eth yl)th io]p r op yl]ca r bon yl]-L-va -
lyl]-2,3-dih ydr o-1H-in dol-2-yl]car bon yl]-L-m eth ion in e (20).
To a solution of 19 (100 mg, 0.22 mmol) in CH2Cl2 (1 mL) at
0 °C under argon were added sequentially Boc-Cys(S-Tr)-OH
(120 mg, 0.26 mmol), NMM (0.033 mL, 0.3 mmol), and Bop
(120 mg, 0.26 mmol). The reaction mixture was allowed to
warm to room temperature over 2 h and then stirred for 16 h.
The reaction mixture was diluted with CH2Cl2 (5 mL) and
washed sequentially with 1 N HCl, saturated aqueous NaH-
CO3, and brine (5 mL each). Purification by flash silica gel
column chromatography eluting with 30% ethyl acetate in
Compounds 31-33, 36-38, and 49 were prepared using
amide bond-coupling and reductive amination procedures
described for the preparation of 25 starting with the appropri-
ate amino acid or aldehyde. Final products were purified by
reverse phase HPLC as described for 25.
(R*,R*)-N-[[2-[2-(L-Cyst ein yla m in o)-3-m et h ylb u t yl]-
1,2,3,4-t et r a h yd r o-3-isoq u in olin yl]ca r b on yl]-L-m et h io-
n in e Tr iflu or oa ceta te (31): mp 112-113 °C; [R]25D ) -55.7°
(c 0.5, CH3OH); MS (M + H)+ 497; 1H-NMR (CD3OD, 270 MHz)
δ 7.28 (4H, m), 4.57 (2H, m), 4.39 (1H, m), 4.22 (2H, m), 4.11
(1H, m), 3.14 (4H, br m), 2.55 (2H, m), 2.21 (2H, m), 2.10 (3H,
s), 1.97 (3H, m), 0.98 (6H, m). Anal. (C23H36N4O4S2‚2.20‚
CHF3O2‚0.80H2O) C, H, N.
1
hexanes afforded 20 (160 mg, 81%): MS (M + H)+ 895; H-
NMR (CDCl3, 270 MHz) δ 8.1-7.9 (1H, br m), 7.5-7.0 (18H,
m), 5.0 (1H, d, J ) 2 Hz), 5.3-5.1 (1H, m), 5.0-4.6 (1H, m),
4.5-4.2 (1H, m), 4.1-3.8 (1H, m), 3.6-3.2 (2H, m), 2.8-2.7
(1H, m), 2.6-2.2 (2H, m), 2.1-1.9 (7H), 1.43 (18H, s), 1.2-0.9
(6H, m).
(S)-N-[[1-(N-L-Cystein yl-L-va lyl)-2,3-d ih yd r o-1H-in d ol-
2-yl]ca r bon yl]-L-m eth ion in e Tr iflu or oa ceta te (21). To
solution of 20 (140 mg, 0.16 mmol) in CH2Cl2 (1.5 mL) at room
temperature under argon were added triethylsilane (0.2 mL,
1.25 mmol) and TFA (0.5 mL). After 2 h, the volatiles were

232 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Leftheris et al.
(S)-N-[(N-L-Cystein yl-L-va lyl)-1,2,3,4-tetr a h yd r o-3-iso-
qu in olin yl]-L-m eth ion in e Tr iflu or oa ceta te (32): mp 129-
(m, 15H); 13C-NMR (CD3OD) δ 19.1, 19.6, 28.8, 30.7, 32.6, 36.0,
51.6, 51.9, 52.3, 53.0, 67.6, 68.5, 80.1, 127.8, 128.9, 130.8, 146.2,
130 °C; [R]25 ) +110 ° (c 0.1, CH3OH); MS (M + H)+ 495;
157.7, 169.0, 169.9, 171.2, 176.7, 202.9. Anal. (C33H42-
N2O4S‚0.17H2O) C, H, N.
D
1H-NMR (CD3OD, 400 MHz) δ 7.74 (1H, m), 7.66 (1H, m), 7.44
(1H, m), 7.33 (1H, m), 5.01 (1H, m), 4.68 (1H, m), 4.28 (2H,
m), 4.14 (1H, m), 3.99 (1H, m), 3.73 (1H, m), 3.45 (1H, m),
2.94 (1H, m), 2.81 (1H, m), 2.54 (2H, m), 2.00 (3H, s), 2.02
(1H, m), 1.91 (2H, m), 0.94 (6H, m); 13C-NMR (CD3OD, 100
MHz) δ 174.1, 172.7, 160.84, 160.43, 160.03, 159.6, 116.50,
113.6, 61.03, 53.50, 47.4, 44.4, 41.5, 39.7, 33.4, 29.2, 26.1, 24.3,
23.0, 21.2; IR 3434, 2928, 2556, 1680, 1576, 1429, 1204, 1138,
837, 801, 723 cm-1. Anal. (C22H32N4O5S2‚2.03CHF3O2‚1.8H2O)
C, H, N.
(R*)-N-[[2-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-3-
[(tr ip h en ylm eth yl)th io]p r op yl]ca r bon yl]-N-(ben zyloxy-
ca r bon yl)-L-va lin e Meth yl Ester (27). Benzyl chlorofor-
mate (90%, 10% BzCl, 3.2 mL, 20 mmol) was added to a
solution of 26 (3.8 g, 6.8 mmol) and DIEA (3.8 mL, 22 mmol)
in CH2Cl2 (20 mL) at 0 °C. The mixture was warmed to room
temperature and stirred for 16 h. The reaction mixture was
diluted with CH2Cl2 (100 mL) and washed sequentially with
1 N HCl (2 × 100 mL) and 10% NaHCO3 (2 × 100 mL). The
organic layer was dried, filtered, and concentrated under
vacuum. The residue was purified by flash chromatography
(eluting with 8:1 hexane:acetone) to afford 27 (4.3 g, 92%) as
a white solid: mp 48-52 °C; MS (M - H)- 695; 1H-NMR (CD3-
OD, 270 MHz) δ 0.73 (d, 3H, J ) 6.45 Hz), 0.93 (d, 3H, J )
6.45 Hz), 1.42 (s, 9H), 2.27 (m, 3H), 3.14 (m, 6H), 4.97-5.10
(m, 3H), 7.14-7.36 (m, 20H); 13C-NMR (CD3OD) δ 19.6, 20.8,
28.8, 29.5, 29.9, 36.2, 50.6, 51.0, 51.8, 52.7, 65.2, 67.5, 68.8,
80.3, 127.8, 128.0, 128.2, 128.9, 129.2, 129.5, 130.7, 137.5,
146.1, 157.4, 158.1, 172.9; IR (KBr) 1709 cm-1; [R]D ) -9.7° (c
1.5, CH3OH). Anal. (C40H46N2O6S) C, H, N, S.
(R*)-N-[[2-[[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-3-
[(tr ip h en ylm eth yl)th io]p r op yl]ca r bon yl]-N-(ben zyloxy-
ca r bon yl)-L-va lin e (28). Lithium hydroxide (2 N, 1.5 mL,
3.0 mmol) was added dropwise to a solution of 27 (0.70 g, 1.0
mmol) in THF (5 mL) and methanol (3.5 mL). The homoge-
neous reaction mixture was warmed to room temperature and
stirred for 16 h. The mixture was concentrated under vacuum,
diluted with 1 N HCl (30 mL), and extracted with CH2Cl2 (3
× 50 mL). The combined organic extracts were dried, filtered,
and concentrated under vacuum to afford 28 (0.68 g, 100%):
mp 66-72 °C; TLC Rf 0.61 (9:1:0.05 chloroform:methanol:acetic
acid, visualization by UV); MS (M - H)- 681; [R]D ) -0.2° (c
0.83, CH3OH); 1H-NMR (CD3OD) δ 0.83 (m, 3H), 0.93 (m, 3H),
1.50 (m, 9H), 1.90-1.92 (m, 1H), 2.35-2.60 (m, 2H), 3.30-
3.55 (m, 2H), 3.77-4.20 (m, 2H), 4.95-5.22 (m, 2H), 7.24-
7.45 (m, 20H); 13C-NMR (CD3OD) δ 19.9, 21.1, 21.4, 28.8, 29.2,
29.5, 29.9, 36.2, 51.1, 51.6, 51.7, 52.0, 54.8, 61.8, 65.2, 67.7,
67.8, 68.7, 80.3, 127.7, 127.9, 128.2, 128.9, 129.1, 129.3, 129.5,
130.7, 137.4, 146.0, 157.4, 157.9, 158.4, 173.9, 178.2; IR (KBr)
1653, 1707 cm-1. Anal. (C40H46N2O6S) C, H, N, S.
(R*,R*)-N-[[2-[2-[(2-Am in o-3-th iop r op yl)a m in o]-3-m e-
th ylbu tyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bon yl]-
L-m eth ion in e Tr iflu or oa ceta te (33): mp 72-73 °C; [R]25
D
) -30.0° (c 0.18, CH3OH); MS (M + H)+ 483; H-NMR (CD3-
1
OD, 270 MHz) δ 7.28 (4H, m), 4.68 (1H, m), 4.19 (1H, m), 3.66
(1H, m), 3.42 (2H, m), 3.24 (2H, m), 3.03 (2H, m), 2.94 (2H, d,
J ) 4.28 Hz), 2.60 (2H, m), 2.20 (2H, m), 2.10 (3H, s), 2.07
(3H, m), 1.01 (3H, d, J ) 7.03 Hz), 0.93 (3H, d, J ) 7.04 Hz).
Anal. (C22H32N4O5S2‚1.9CHF3O2‚1.5H2O) C, H, N, F.
(R)-N-[[3-[[N-(2-Am in o-3-m er ca p top r op yl)-L-va lyl]a m i-
n o]-2-n a p h th a len yl]ca r bon yl]-L-m eth ion in e Tr iflor oa ce-
ta te (36): mp 101-102 °C; [R]25 ) -28.7° (c 0.15, CH3OH);
D
MS (M + H)+ 507; 1H-NMR (CD3OD, 270 MHz) δ 8.80 (1H, s),
8.24 (1H, s), 7.80 (2H, m), 7.42 (2H, m), 4.74 (2H, m), 3.41
(1H, m), 3.05 (1H, m), 2.91 (1H, m), 2.81 (2H, m), 2.62 (2H,
m), 2.17 (3H, m), 2.05 (3H, s), 0.97 (6H, m). Anal. (C24H34
-
N4O5S2‚2.5‚CHF3O2‚1.9H2O) C, H, N, F.
(S*,R*)-N-[[2-[N-(2-Am in o-3-m er captopr opyl)-L-alan yl]-
1,2,3,4-t et r a h yd r o-3-isoq u in olin yl]ca r b on yl]-L-m et h io-
n in e Tr iflu or oa ceta te (37): mp 89-90 °C; [R]25 ) -32.1°
D
(c 1.0, CH3OH); MS (M + H)+ 469; 1H-NMR (CD3OD, 270 MHz)
δ 7.16 (4H, m), 4.71 (2H, m), 4.57 (2H, m), 4.43 (1H, m), 4.32
(1H, m), 3.98 (1H, m), 3.56 (1H, m), 3.33-3.02 (2H, m), 2.84
(2H, m), 2.37 (1H, m), 1.98 (2H, m), 1.89 (1H, m), 2.10-1.76
(3H, m), 1.57 (2H, m), 1.43 (1H, m). Anal. (C21H32N4O4S2‚
1.9CHF3O2‚3.17H2O) C, H, N, F.
[R*-[R*-(S*)]]-N-[[2-[2-[(2-Am in o-3-m er ca p t op r op yl)-
a m in o]-1-oxob u t yl]-1,2,3,4-t et r a h yd r o-3-isoq u in olin yl]-
ca r bon yl]-L-m eth ion in e Tr iflu or oa ceta te (38): mp 85-
95 °C; [R]D ) -31° (c 0.1, CH3CH2OH); IR (KBr) 3435, 2924,
1674, 1431 cm-1; MS (M + H)+ 483; 1H-NMR (CD3OD, 270
MHz) δ 7.25 (4H, m), 1.8-4.9 (20H, m), 1.97, 2.07 (3H, 2s,
rotamers), 1.1 (6H, m). Anal. (C22H34N4O4S2‚1.9CHF3O2‚
1.5H2O) C, H, N, S, F.
[R-(R*,S*)]-N-[[2-[N-(2-Am in o-3-m e r ca p t op r op yl)-3-
m eth yl-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bo-
n yl]-L-m eth ion in e Tr iflu or oa ceta te (49): mp 87-90 °C;
MS (M + H)+ 511; IR (KBr) 1676, 1204 cm-1; [R]D ) -27° (c
0.46, CH3OH); 1H-NMR (CD3OD, 400 MHz) δ 7.35-7.20 (4H,
m), 5.0-4.55 (5H, m), 3.45-3.1 (2H, m), 2.90-2.45 (5H, m),
2.07, 2.03 (3H, 2 s, rotameric), 2.2-1.95 (2H, m), 1.12, 1.06
(9H, 2s). Anal. (C24H38N4O4S2‚1.9CHF3O2‚1.5H2O) C, H, N,
S, F.
(R*)-N-[N-[2-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-
3-[(tr iph en ylm eth yl)th io]pr opyl]-N-(ben zyloxycar bon yl)-
L-va lyl]-1,2,3,4-tetr a h yd r oisoqu in olin e-2-ca r boxylic Acid
Meth yl Ester (29). N,N-Diisopropylethylamine (20 mL, 110
mmol) was added to a solution of 28 (24 g, 35 mmol), Tic-OMe‚
HCl (9.7 g, 43 mmol), and Bop-Cl (11 g, 43 mmol) in CH2Cl2
(200 mL). The mixture was stirred at 0 °C for 16 h. The
reaction was quenched with 1 N HCl (300 mL) and the mixture
extracted with CH2Cl2 (3 × 100 mL). The combined organic
extracts were washed with 10% NaHCO3 (100 mL), dried,
filtered, and concentrated under vacuum. The residue was
purified by flash chromatography (eluting with 4:1 hexane:
acetone) to afford 29 (22 g, 72%) as a white solid: mp 66-70
°C; TLC Rf 0.28 (4:1 hexane:acetone, visualization by UV); MS
N-[[2-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-3-[(t r i-
p h en ylm eth yl)th io]p r op yl]ca r bon yl]-L-va lin e Meth yl Es-
ter (26). Acetic acid (2.6 mL, 45 mmol) was added to a solution
of 22 (20 g, 45 mmol) and L-valine methyl ester hydrochloride
(9.0 g, 54 mmol) in methanol (50 mL). The mixture was stirred
at room temperature for 30 min. NaBH3CN (2.8 g, 45 mmol)
in THF (50 mL) was added dropwise, and the mixture was
stirred at room temperature for 2 h. The reaction was
quenched with NaHCO3 (3.8 g, 45 mmol) in water (20 mL) and
the mixture concentrated under vacuum. The residue was
dissolved in 10% NaHCO3 (10 mL) and extracted with CH2Cl2
(3 × 100 mL). The combined organic extracts were dried,
filtered, and concentrated under vacuum. The residue was
purified by flash chromatography (eluting with 9:1 hexane:
acetone) to afford 26 (18 g, 70%) as a viscous oil: TLC Rf 0.25
(8:1 hexane:acetone, visualization by UV); MS (M + H)+ 563;
1
(M + H)+ 856; [R]D ) +21.0° (c 1.0, CH3OH); H-NMR (CD3-
OD, 400 MHz) δ 0.76-0.98 (m, 6H), 1.14-1.31 (m, 9H), 2.16-
2.40 (m, 3H), 2.96-3.59 (m, 8H), 3.93 (m, 1H), 4.50-5.30 (m,
5H), 7.12-7.42 (m, 24H); 13C-NMR (CD3OD) δ 14.4, 18.7, 18.8,
19.6, 23.7, 28.4, 28.7, 29.5, 31.7, 32.2, 32.7, 36.4, 36.7, 36.9,
41.4, 45.0, 46.2, 46.5, 47.0, 47.4, 47.6, 50.2, 50.8, 52.7, 55.0,
62.2, 62.4, 65.2, 67.7, 69.2, 72.9, 80.4, 93.1, 127.3, 127.5, 127.8,
128.0, 128.1, 128.3, 128.9, 129.1, 129.2, 129.3, 129.6, 129.9,
130.3, 130.7, 133.9, 134.4, 137.4, 146.0, 157.3, 158.6, 172.8,
173.2, 219.3; IR (KBr) 1638, 1711 cm-1. Anal. (C51H75N3O7S)
C, H, N.
(R*)-N-[N-[2-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-
3-[(tr iph en ylm eth yl)th io]pr opyl]-N-(ben zyloxycar bon yl)-
L-va lyl]-1,2,3,4-tetr a h yd r oisoqu in olin e-2-ca r boxylic Acid
(30). Lithium hydroxide (1 N, 54 mL, 54 mmol) was added
dropwise to a solution of 29 (13 g, 15 mmol) in THF (100 mL)
and MeOH (50 mL). The homogeneous reaction mixture was
warmed to room temperature and stirred for 16 h. The
IR (CH2Cl2 film) 1491, 1715 cm-1; [R]D ) -3.8° (c 1.3, CH -
3
OH); 1H-NMR (CD3OD) δ 0.86, 0.87 (d, 6H, J ) 7.04, 6.45Hz),
1.43 (s, 9H), 1.80-1.82 (m, 1H), 2.29-2.31 (m, 2H), 2.53-2.70
(m, 2H), 2.89 (m, 1H), 3.60 (m, 1H), 3.66 (s, 3H), 7.16-7.40

Potent Inhibitors of Ras Farnesyltransferase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 233
mixture was concentrated under vacuum, diluted with 1 N HCl
(100 mL), and extracted with CH2Cl2 (3 × 150 mL). The
combined organic extracts were dried, filtered, and concen-
trated under vacuum to afford 30 (12 g, 100%) as a white
solid: mp 88-94 °C; TLC Rf 0.52 (9:1:0.05 chloroform:metha-
nol:acetic acid, visualization by UV); MS (M - H)- 840; [R]D
) +8.0° (c 1.68, CH3OH); 1H-NMR (CD3OD, 270 MHz) δ 0.71-
0.92 (m, 6H), 1.26-1.35 (m, 9H), 1.86 (m, 1H), 2.19-2.40 (m,
2H), 2.80-3.51 (m, 5H), 3.65-3.98 (m, 1H), 4.51-5.40 (m, 5H),
7.12-7.41 (m, 24H); 13C-NMR (CD3OD, 67.5 MHz) δ 18.7, 18.9,
19.6, 26.5, 28.7, 29.5, 31.8, 32.3, 36.6, 46.5, 47.0, 47.4, 50.8,
53.8, 54.4, 62.2, 62.3, 65.2, 67.6, 68.8, 69.2, 80.4, 127.2, 127.7,
127.9, 128.2, 128.5, 128.9, 129.1, 129.6, 129.8, 130.7, 133.4,
133.9, 134.2, 137.4, 146.0, 157.2, 158.6, 172.6, 173.1, 173.9;
IR (KBr) 1638, 1711 cm-1. Anal. (C50H55N3O7S‚0.37H2O) C,
H, N, S.
The reaction mixture was diluted with diethyl ether (50 mL)
and centrifuged and the solid triturated, discarding the liquid
portion. Trituration was repeated with diethyl ether (2 × 50
mL), ethyl acetate (2 × 50 mL), and diethyl ether (1 × 50 mL).
The solid was dried under vacuum and then purified by
preparative HPLC (YMC S-10 ODS 30 × 500 mm, 220 nm, 28
mL/min, 10-90% aqueous methanol with 0.1% TFA). The
appropriate fractions were concentrated under vacuum (at 35
°C), dissolved in water (20 mL), and lyophilized to afford 41
(0.25 g, 33%) as a white lyophilate: mp 80-90 °C; TLC Rf 0.69
(6:3:1 n-propanol:ammonium hydroxide:water, visualization by
ceric ammonium sulfate); MS (M + H)+ 479; [R]D ) -27.7° (c
0.43, CH3OH); 1H-NMR (CD3OD, 400 MHz) δ 7.29-7.22 (4H,
m), 4.89-4.51 (3H, m), 4.34-4.06 (2H, m), 3.77-2.75 (5H, m),
2.21-2.10 (1H, br m), 1.82-1.49 (4H, m), 1.31-0.83 (13H, m);
IR (KBr) 1674, 3435 cm-1
1.29H2O) C, H, N, S, F.
.
Anal. (C24H38N4O4S.CHF3O2‚
(S*,R*)-N-[[2-[N-[2-[[(1,1-Dim eth yleth oxy)car bon yl]am i-
n o]-3-[(t r ip h en ylm et h yl)t h io]p r op yl]-N-(b en zyloxyca r -
bon yl)-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bo-
n yl]-L-n or leu cin e Met h yl E st er (39). N,N-Diisopropyl-
ethylamine (0.86 mL, 5.0 mmol) was added to a solution of 30
(1.4 g, 1.7 mmol), norleucine methyl ester hydrochloride (0.3
g, 1.7 mmol), and Bop (0.73 g, 1.7 mmol) in CH3CN (9 mL)
and DMF (3 mL). The mixture was stirred for 16 h, the
reaction quenched with 1 N HCl (50 mL), and the mixture
extracted with ethyl acetate (3 × 60 mL). The combined
organic extracts were washed with 10% NaHCO3 (1 × 50 mL)
and 10% LiCl (2 × 50 mL), dried, filtered, and concentrated
under vacuum. The residue was purified by flash chromatog-
raphy (eluting with 4:1 hexane:acetone) to afford 39 (1.3 g,
79%) as a white solid: mp 58-68 °C; TLC Rf 0.37 (4:1 hexane:
acetone, visualization by UV); MS (M + H)+ 969; [R]D ) -21.8°
(c 1.35, CH3OH); 1H-NMR (CDCl3, 400 MHz) δ 7.41-7.07 (m,
24H), 5.27-4.51 (m, 6H), 4.40-3.80 (m, 1H), 3.67, 3.65 (3H,
2s), 3.70-2.98 (m, 5H), 2.34-2.16 (m, 3H), 1.65-1.61 (m, 1H),
1.48-1.38 (m, 9H), 1.50-0.7 (m, 14H); 13C-NMR (CDCl3, 100
MHz) δ 172.5, 170.0, 157.2, 155.1, 144.6, 135.9, 133.8, 132.7,
129.5, 128.7, 128.4, 128.0, 127.9, 126.9, 125.8, 79.2, 68.0, 66.4,
61.0, 54.5, 52.1, 50.0, 46.3, 45.0, 35.6, 35.2, 32.0, 31.2, 28.3,
Compounds 42-45 were prepared and purified in an
analogous manner to 41 starting from 30 and the appropriate
amino acid methyl ester.
[R-(R*,S*)]-N2[[2-[N-(2-Am in o-3-m er ca p top r op yl)-L-va -
lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bon yl]-L-a sp a r -
a gin e Tr iflu or oa ceta te (42): mp 95-110 °C; TLC Rf 0.50
(6:3:1 n-propanol:ammonium hydroxide:water, visualization by
ceric ammonium sulfate); MS (M + H)+ 480; [R]D ) -16.1° (c
0.54, CH3OH); 1H-NMR (CD3OD) δ 7.24-7.22 (4H, m), 4.89-
4.65 (4H, m), 3.51-3.08 (5H, m), 2.89-2.51 (5H, m), 2.20-1.9
(1H, br m), 1.02, 1.04, 1.15 (6H, dd, J ) 6.84, 7.27 Hz); IR
(KBr) 1204, 1674, 3424 cm-1. Anal. (C22H33N5O5S‚1.8CHF3O2‚
1.8H2O) C, H, N, S, F.
(S*,R*)-N-[[2-[N-(2-Am in o-3-m er ca p top r op yl)-L-va lyl]-
1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bon yl ]-γ-(a m in o-
su lfon yl)-L-r-a m in obu tyr ic Acid Tr iflu or oa ceta te (43):
Rf 0.6 (6:3:1 nPrOH:NH4OH:H2O, visualized by UV, PMA); MS
(M + H)+ 530; [R]D ) -16.3° (c 0.19, CH3OD); 1H-NMR (CDCl3,
400 MHz) δ 7.4-7.2 (4H, m), 4.9-4.4 (5H, m), 3.8-3.2 (5H,
m), 2.9-2.2 (7H, m), 1.1 (6H, d, J ) 8 Hz); IR (KBr) 3439,
1678, 1653, 1437, 1206, 1142, 723 cm-1. Anal. (C22H35N5O6S2‚
.
2.5CHF3O2 0.75H2O) C, H, N.
26.6, 22.1, 19.5, 19.2, 18.5; IR (KBr) 1696, 1653, 1499 cm-1
.
(S*,R*)-N-[[2-[N-(2-Am in o-3-m er ca p top r op yl)-L-va lyl]-
1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bon yl]-3-[(m eth yl-
su lfon yl)m eth yl]-L-a la n in e Tr iflu or oa ceta te (44): mp
105-115 °C; TLC Rf ) 0.52 (6:3:1 n-propanol:ammonium
hydroxide:water, visualization by ceric ammonium sulfate); MS
(M + H)+ 529; [R]D ) -10.0° (c 0.12, CH3OH); 1H-NMR (CD3-
OD, 400 MHz) δ 1.07-1.19 (m, 6H), 2.15 (br m, 2H), 2.25 (br
m, 1H), 2.42 (br m, 2H), 2.80-3.02 (m, 5H), 2.92, 2.97 (s, 3H),
3.09-3.48 (m, 3H), 4.25 (m, 1H), 4.45-4.80 (m, 3H), 7.22-
Anal. (C57H68N4O8S‚0.27H2O) C, H, N, S.
(S*,R*)-N-[[2-[N-[2-[[(1,1-Dim eth yleth oxy)car bon yl]am i-
n o]-3-[(t r ip h en ylm et h yl)t h io]p r op yl]-N-(b en zyloxyca r -
bon yl)-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bo-
n yl]-L-n or leu cin e (40). Lithium hydroxide (2 N, 2.3 mL, 4.7
mmol) was added to a solution of 39 (1.1 g, 1.2 mmol) in THF
(10 mL) at 0 °C, warmed to room temperature, and stirred for
4 h. The reaction mixture was concentrated under vacuum
and dissolved in water (50 mL), the reaction quenched with 1
N HCl to pH of 4.0, and the mixture extracted with dichlo-
romethane (3 × 50 mL). The combined organic extracts were
dried, filtered, and concentrated under vacuum to afford 40
(1.1 g, 98%) as a hygroscopic solid: mp 86-94 °C; MS (M +
H)+ 955; [R]D ) -17° (c 1.15, CH3OH); 1H-NMR (CD3OD, 400
MHz) δ 7.12-7.42 (24H, m), 4.59-5.31 (5H, m), 4.23-4.31 (1H,
m), 3.70-3.85 (1H, m), 2.99-3.31 (5H, m), 2.02-2.23 (3H, m),
0.66-0.91, 1.04-1.71 (24H, m); 13C-NMR (CD3OD, 100 MHz)
175.2, 173.3, 172.6, 158.5, 158.0, 157.4, 146.0, 137.3, 135.2,
135.0, 130.7, 130.6, 129.9, 129.6, 129.1, 128.9, 128.2, 128.0,
127.7, 127.1, 80.4, 69.2, 68.8, 67.5, 62.6, 56.6, 56.4, 53.3, 50.5,
47.4, 46.7, 37.1, 36.8, 33.0, 32.6, 29.3, 29.1, 28.7, 28.3, 26.5,
23.3, 20.1,19.9, 18.6; IR (KBr) 1653, 1696, 2932 cm-1. Anal.
(C56H66N4O8S‚0.43H2O) C, H, N, S.
(S*,R*)-N-[[2-[(2-Am in o-3-m er ca p t op r op yl)-L-va lyl]-
1,2,3,4-t et r a h yd r o-3-isoq u in olin yl]ca r b on yl]-L-n or leu -
cin e Tr iflu or oa ceta te (41). Trifluoroacetic acid (5.3 mL, 68
mmol) was added dropwise to a solution of 40 (1.0 g, 1.05
mmol) and triethylsilane (1.7 mL, 11 mmol) in CH2Cl2 (10 mL)
at 0 °C. The reaction mixture was warmed to room temper-
ature and stirred for 0.75 h. The reaction mixture was
concentrated under vacuum and the residue triturated with
hexane (3 × 20 mL) and dried under vacuum. A solution at 0
°C of trifluoroacetic acid (18 mL, 220 mmol), thioanisole (1.2
mL, 11 mmol), and ethanedithiol (0.88 mL, 11 mmol) was
added to the residue. Bromotrimethylsilane (0.96 mL, 7.3
mmol) was added at 0 °C, and the mixture was stirred for 1 h.
7.31 (m, 4H); IR (KBr) 2567, 1676, 1530, 1294 cm-1
. Anal.
(C23H36N4O6S2‚2.21CHF3O2‚1.16H2O) C, H, N, S, F.
[R-(R*,S*)]-N2[[2-[N-(2-Am in o-3-m er ca p top r op yl)-L-va -
ly l]-1,2,3,4-t e t r a h y d r o -3-is o q u in o lin y l]c a r b o n y l]-L -
glu ta m in e Tr iflu or a ceta te (45): mp 65-75 °C; TLC Rf 0.69
(6:3:1 n-propanol:ammonium hydroxide:water, visualization by
ceric ammonium sulfate); MS (M + H)+ 494; [R]D ) -21.4° (c
1
0.69, CH3OH); H-NMR (400 MHz, CD3OD) δ 7.33-7.22 (m,
4H), 4.64-4.91 (m, 3H), 4.38-4.23 (br m, 2H), 3.49-2.78 (m,
5H), 2.12-2.31 (m, 6H), 1.93 (br m, 1H), 1.07, 1.08, 1.09 (d,
6H, J ) 7.6 Hz); IR (KBr) 1202, 1670, 2567 cm-1
(C23H35N5O5S‚2.25CHF3O2‚1.63H2O) C, H, N, S, F.
. Anal.
N-[N-[(1,1-Dim et h ylet h oxy)ca r b on yl]-L-va lyl]-1,2,3,6-
tetr a h yd r op yr id in e-2-ca r boxylic Acid Meth yl Ester (52).
Bop-Cl (2.2 g, 8.5 mmol) was added to a stirred solution of
tetrahydropyridinecarboxylic acid methyl ester (1.0 g, 7.1
mmol), Boc-Val-OH (1.7 g, 7.8 mmol), and DIEA (3.0 g, 4.1
mL, 23 mmol) in DMF (25 mL) at 5 °C under argon. After
stirring for 15 h at 5 °C, the mixture was partitioned between
ethyl acetate (30 mL) and 10% HCl (10 mL). The aqueous
layer was re-extracted (2 × 20 mL) with ethyl acetate. The
combined organic extracts were washed sequentially with 10%
HCl (2 × 10 mL), water (2 × 30 mL), saturated aqueous
NaHCO3 (3 × 10 mL), and brine (2 × 20 mL). The organic
layer was dried, filtered, and concentrated to afford 52 (0.86
g, 36%) as a clear viscous oil: MS (M + H)+ 341; 1H-NMR (CD3-
OD, 400 MHz) δ 5.9-5.1 (6H, m), 4.8-4.1 (3H, m), 2.9-2.5

234 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Leftheris et al.
(2H, m), 2.5-1.7 (8H, m), 1.5-1.4 (18H, m), 1.04-0.91 (6H,
m). Anal. (C17H28N2O5‚0.75H2O) C, H, N.
lyophilate; TLC Rf 0.16 (9:1:0.1 CHCl3:CH3OH:CH3COOH,
visualized by UV, PMA); IR (KBr) 2924, 1672, 1427, 1202, 1134
cm-1; MS (M + H)+ 447; 1H-NMR (CDCl3, 400 MHz) δ 8.09
(1H, m), 5.8-5.7 (2H, m), 5.46-5.45 (1H, m), 4.55 (1H, m),
4.18 (2H, m), 3.6 (1H, m), 3.03-2.4 (12H, m), 2.1-2.0 (6H, m),
1.02-1.00 (6H, m). Anal. (C19H34N4O4S2‚1.5CHF3O2‚1.24
H2O) C, H, N.
(R*)-N-[[1-[N-[(1,1-Dim eth yleth oxy)ca r bon yl]-L-va lyl]-
1,2,3,6-t et r a h yd r o-2-p yr id in yl]ca r b on yl]-L-m et h ion in e
ter t-Bu tyl Ester (53). To a solution of 52 (0.86 g, 2.5 mmol)
in methanol (10 mL) at room temperature was added 1 N
NaOH (2.8 mL, 2.8 mmol). The reaction mixture was stirred
for 2 h at room temperature, concentrated, and diluted with 1
N HCl (4 mL)/ethyl acetate (10 mL). The aqueous layer was
re-extracted with ethyl acetate (2 × 20 mL). The combined
organic layers were washed with brine, dried, filtered, and
concentrated to afford the free acid (0.80 g, 97%). To this acid
(0.8 g, 2.7 mmol) was added Bop (1.3 g, 2.9 mmol), H-Met-O-
tBu (0.65 g, 2.7 mmol), and N,N-diisopropylethylamine (0.66
mL, 3.7 mmol) in DMF (8 mL) at room temperature under
argon. After stirring for 15 h, the reaction mixture was
partitioned between ethyl acetate (20 mL) and 10% HCl (10
mL). The aqueous layer was extracted (2 × 20 mL) with ethyl
acetate. The combined organic extracts were washed sequen-
tially with 10% HCl (2 × 10 mL), water (2 × 20 mL), saturated
aqueous NaHCO3 (2 × 10 mL), and brine (2 × 20 mL). The
organic layer was dried, filtered, and concentrated to afford
53 (0.87 g, 78%) as a yellow oil: accurate mass measurement
(M + H)+ calcd for C25H44O6N3S 514.2927, found 514.2939
(∆ppm ) 2.1); TLC Rf 0.57 (ethyl acetate, visualized by UV,
PMA); 1H-NMR (400 MHz) δ 5.9-5.5 (2H, m), 4.8-2.7 (4H,
m), 2.5-2.3 (5H, m), 2.0-1.9 (6H, m), 1.5-1.4 (9H, m), 1.3-
0.9 (6H, m).
(R*)-2-[N-(Ben zyloxycar bon yl)-3-m eth yl-L-valyl]-1,2,3,4-
tetr a h yd r oisoqu in olin e-3-ca r boxylic Acid Meth yl Ester
(55). To a solution of N-(benzyloxycarbonyl)-L-tert-leucine (5.3
g, 20 mmol) in CH2Cl2 (80 mL) at 0 °C under argon were
sequentially added N,N-diisopropylethylamine (11 mL, 60
mmol), Bop-Cl (5.1 g, 20 mmol), and Tic-OMe‚HCl (5.7 g, 25
mmol). The mixture was allowed to warm to 5 °C over 2 h
and then stirred overnight (16 h). The mixture was washed
with 1 N HCl, saturated aqueous NaHCO3, and brine (50 mL
each). The organic layer was dried, filtered, and concentrated
to afford an oil. Purification by flash silica gel column
chromatography eluting with 20% ethyl acetate in hexanes
afforded 55 (4.0 g, 45%): TLC Rf 0.28 (30% ethyl acetate in
hexanes, visualized by UV and ceric ammonium molybdate);
MS (M + H)+ 439; 1H-NMR (CDCl3, 400 MHz) δ 7.4-7.1 (9H,
m), 5.6 (1H, d, J ) 10 Hz), 5.3 (1H, t, J ) 5 Hz), 5.06 (2H, dd,
J ) 12, 16 Hz), 5.1-5.0 (1H, m), 4.75 (1H, d, J ) 10 Hz), 3.6,
3.5 (3H, 2s, rotamers), 3.2 (1H, dq, J ) 5, 21 Hz), 1.1, 1.0 (9H,
2s); 13C-NMR (CDCl3, 100.4 MHz) δ 171.6, 171.3, 156.2, 136.2,
132.4, 132.2, 128.4, 128.1, 128.0, 127.4, 127.1, 126.2, 66.9, 57.2,
52.3, 52.2, 46.6, 36.3, 30.8, 26.3.
N-[[1-[N-(R)-[2-[[(1,1-Dim eth yleth oxy)car bon yl]am in o]-
3-[(tr ip h en ylm eth yl)th io]p r op yl]-L-va lyl]-1,2,3,6-tetr a h y-
d r o-2-p yr id in yl]ca r bon yl]-L-m eth ion in e ter t-Bu tyl Ester
(54). Triethylsilane (0.18 mL, 1.0 mmol) was added to a
solution of 53 (0.26 g, 0.50 mmol) in formic acid (3 mL) at room
temperature under argon. After stirring for 2 h, the reaction
mixture was allowed to stand for 2 days at 4 °C. The reaction
mixture was diluted with NaHCO3 (saturated, to pH 8-9) and
ethyl acetate (40 mL). The aqueous layer was extracted with
ethyl acetate (2 × 30 mL). The combined organic extracts were
washed with brine, dried, filtered, and concentrated to afford
the amine (0.076 g, 36%). The product was used in the next
step without purification. A solution of sodium cyanoborohy-
dride (0.012 g, 0.20 mmol) in methanol (0.5 mL) was added
dropwise to a stirred solution of the above amine (0.075 g, 0.16
mmol) and aldehyde 22 (0.11 g, 0.24 mmol) in acetic acid (0.76
mL) and methanol (0.5 mL) at 0 °C. The mixture was slowly
warmed to room temperature and stirred for 15 h. The
reaction mixture was diluted with NaHCO3 at 0 °C and
extracted with ethyl acetate (3 × 20 mL). The combined
extracts were washed with brine, dried, filtered, and concen-
trated. The product was purified by flash chromatography
eluting with 30-50% ethyl acetate/hexane to afford 54 (0.14
g, 97%) as a highly viscous oil: TLC Rf 0.27 (1:1 hexane:ethyl
acetate, visualized by PMA); MS (M + H)+ 845; 1H-NMR (270
MHz) δ 7.5-7.2 (16H, m), 6.9 (1H, m), 6.8 (H, m), 5.9-5.6 (2H,
m), 4.9-3.3 (8H, m), 2.8-2.1 (6H, m), 2.0-1.9 (3H, s), 1.5-
1.4 (18H, m), 1.0-0.8 (6H, m).
(R*)-2-[N-(Ben zyloxycar bon yl)-3-m eth yl-L-valyl]-1,2,3,4-
tetr a h yd r oisoqu in olin e-3-ca r boxylic Acid Meth yl Ester
(56). Sodium hydroxide (1 N, 13 mL, 13 mmol) was added in
three portions to a solution of 55 (1.9 g, 4.3 mmol) in methanol
(30 mL), and the resulting solution was stirred at room
temperature for 2 h. The reaction mixture was concentrated
under vacuum, dissolved in HCl (1 N, 70 mL), and extracted
with CH2Cl2 (3 × 150 mL). The combined organic extracts
were dried, filtered, and concentrated under vacuum to afford
56 (1.8 g, 100%) which was used without further purification:
mp 78-88 °C; TLC Rf 0.32 (9:1 CHCl3:methanol, visualization
1
by UV); MS (M + H)+ 425; [R]D ) +15.6° (c 0.9, CH3OH); H-
NMR (CD3OD, 400 MHz) δ 1.09 (s, 9H), 3.07-3.22 (m, 2H),
4.67-5.20 (m, 6H), 7.16-7.30 (m, 9H); 13C-NMR (CD3OD,
100.4 MHz) δ 26.8, 26.9, 31.8, 32.4, 36.8, 45.0, 47.7, 50.0, 54.0,
54.8, 56.7, 58.6, 58.8, 67.8, 127.1, 127.5, 127.8, 128.0, 128.3,
129.0, 129.4, 132.7, 133.2, 134.2, 138.0, 158.5, 173.3, 173.8,
173.9; IR (KBr) 3430, 1719, 1518 cm-1. Anal. (C24H28N2O5)
C, H, N.
(R*)-N-[[2-[N-(Ben zyloxyca r b on y)-3-m et h yl-L-va lyl]-
1 ,2 ,3 ,4 -t e t r a h y d r o -3 -i s o q u i n o l i n y l ] c a r b o n y l ] -L -
glu t a m in e ter t-Bu t yl E st er (57). N,N-Diisopropylethyl-
amine (1.1 mL, 6.3 mmol) was added to a solution of 56 (0.9 g,
2.1 mmol), L-glutamine tert-butyl ester hydrochloride (0.50 g,
2.1 mmol), and Bop (0.93 g, 2.1 mmol) in 3:1 CH3CN:DMF (26
mL). The mixture was stirred at room temperature for 16 h.
The reaction was quenched with HCl (1 N, 100 mL), the
mixture was extracted with ethyl acetate (4 × 75 mL), and
the combined organic extracts were washed with 10% LiCl (3
× 150 mL), dried, filtered, and concentrated under vacuum.
The residue was purified by flash chromatography (eluting
with 1:1 hexane:acetone) to afford 57 (1.1 g, 87%) as a white
solid: mp 60-68 °C; TLC Rf 0.62 (2:1 acetone:hexane, visu-
alization by UV); [R]D ) -37.2° (c 0.88, CH3OH); MS (M +
H)+ 609; 1H-NMR (CD3OD, 400 MHz) δ 7.32-7.17 (m, 9H),
5.09-4.52 (m, 7H), 3.71, 3.58 (s, 3H), 2.95-2.30 (m, 4H), 2.93,
2.87 (s, 3H), 2.45-2.20 (m, 1H), 2.07-1.98 (m, 1H), 1.06, 1.02
(s, 9H); 13C-NMR (CD3OD, 100.4 MHz) δ 174.0, 173.1, 172.6,
138.0, 135.8, 135.5, 129.4, 129.2, 128.9, 128.6, 128.1, 128.0,
127.3, 126.9, 67.8, 61.5, 59.2, 58.9, 57.7, 57.3, 53.1, 52.5, 52.1,
51.8, 45.7, 40.7, 36.8, 36.3, 32.8, 27.1, 26.9, 26.1, 20.9, 14.4;
N-[[1-[N-(R)-(2-Am in o-3-m er captopr opyl)-L-valyl]-1,2,3,6-
tetr a h yd r o-2-p yr id in yl]ca r bon yl]-L-m eth ion in e Tr iflu o-
r oa ceta te (34). Triethylsilane (0.05 mL, 0.037 g) was added
to a solution of 54 (0.14 g, 0.16 mmol) and TFA (1.0 mL) in
CH2Cl2 (1 mL). After stirring for 13 h, the reaction mixture
was concentrated to afford a solid (0.6 g). A portion of this
residue (0.2 g) was purified by prep HPLC (YMC S-10 ODS
30 × 500 mm column, eluting over 30 min with 0-90%
aqueous methanol containing 0.1% TFA) to afford, after
lyophilization, 34 (23 mg, 32%) as a white solid: accurate mass
measurement (M + H)+ calcd for C19H35N4O4S2 447.1200, found
447.2112 (∆ppm ) 2.3); 1H-NMR (400 MHz CD3OD) δ 8.04-
8.02 (H, d, J ) 8 Hz), 5.8-5.7 (2H, m), 5.45-5.43 (H, d, J ) 7
Hz), 4.5-3.6 (6H, m), 3.2-2.4 (12H, m), 2.09-2.05 (6H, m),
1.58-0.9 (6H, m, rotamers); IR (KBr) 2972, 1676, 1204, 1138
IR (KBr) 1669, 1701, 1717, 3428 cm-1
1.95H2O) C, H, N.
. Anal. (C33H44N4O7‚
cm-1
.
(R*)-N-[[2-[3-Met h yl-L-va lyl]-1,2,3,4-t et r a h yd r o-3-iso-
qu in olin yl]ca r bon yl]-L-glu ta m in e ter t-Bu tyl Ester (58).
Palladium hydroxide on carbon (10%, 0.091 g) was added to a
solution of 57 (0.91 g, 1.5 mmol) in THF (9.1 mL) and 1 N
HCl (1.5 mL). A hydrogen balloon was attached to the
N-[[1-[N-(S)-(2-Am in o-3-m er captopr opyl]-L-valyl]-1,2,3,6-
tetr a h yd r o-2-p yr id in yl]ca r bon yl]-L-m eth ion in e tr iflu o-
r oa ceta te (35). Compound 35 was obtained as a second
component in the purification of 34: yield 17 mg of white

Potent Inhibitors of Ras Farnesyltransferase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 235
reaction, and the mixture was stirred for 3 h. The reaction
mixture was filtered through Celite and concentrated under
vacuum to afford 58 (0.77 g, 100%) which was used without
further purification: mp foams and liberates gas above 128
°C; TLC Rf 0.77 (6:3:1 n-propanol:ammonium hydroxide:water,
visualization by ninhydrin); MS (M + H)+ 475; [R]D ) -35.9°
(c 0.49, CH3OH); 1H-NMR (CD3OD, 400 MHz) δ 7.45-7.27 (m,
4H), 5.00-4.60 (m, 3H), 4.35 (br m, 1H), 3.35-3.25 (m, 1H),
3.20-3.05 (m, 2 H), 2.35 (t, 2H, J ) 7.04 Hz), 2.30-1.85 (m,
2H), 1.47, 1.46 (s, 9H), 1.19 (s, 9H); 13C-NMR (CD3OD, 100.4
MHz) δ 177.9, 172.1, 169.2, 135.7, 135.6, 129.2, 128.3, 128.2,
127, 83.1, 58.8, 58.0, 53.7, 36.0, 32.8, 32.6, 29.6, 28.9, 28.2,
4.25 (br m, 1H), 3.18 (s, 3H), 3.45-3.10 (m, 6H), 3.05-2.96
(m, 1H), 2.80-2.70 (m, 1H), 2.46-2.42 (m, 2H), 1.93-1.90 (m,
1H), 1.88-1.77 (m, 1H), 1.01 (s, 9H); IR (KBr) 1204, 1431,
1676, 2967 cm-1
. Anal. (C28H40N4O9SF6‚0.61H2O) C, H, N.
[3S-[2(S*),3R*(R*)]]-2-[[[2-[N-(2-Am in o-3-m er ca p top r o-
pyl)-3-m eth yl-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]-
ca r bon yl]a m in o]-4-(m et h ylsu lfon yl)b u t a n oic Acid Tr i-
flu or oa ceta te (50): mp 120-130 °C; TLC Rf 0.64 (6:3:1
n-propanol:ammonium hydroxide:water, visualization by ceric
ammonium sulfate or UV); MS (M + H)+ 543; [R]D ) -18.9°
(c 0.62, CH3OH); 1H-NMR (DMSO-d6, 125 °C, 400 MHz) δ 7.18
(s, 4H), 4.96-4.85 (m, 1H), 4.59-4.56 (m, 2H), 4.35-4.25 (m,
1H), 3.32 (br m, 1H), 3.20-3.12 (m, 3 H), 3.05-2.98 (m, 4H),
2.87 (s, 3H), 2.70 (br m, 1H), 2.47-2.43 (m, 1H), 2.18 (br m,
1H), 2.01 (br m, 1H), 1.01 (s, 9H); IR (KBr) 2971, 1678, 1433,
1204 cm-1. Anal. (C28H40N4O10S2‚1.88H2O) C, H, N.
26.8, 26.7; IR (KBr) 1370, 1663, 1728, 3416 cm-1
. Anal.
(C25H39N4O5Cl‚0.2CH3OH‚0.2C2H4O) C, H, N.
(S*,R*)-N-[[2-[N-[2-[[(1,1-Dim eth yleth oxy)car bon yl]am i-
n o]-3-[(tr ip h en ylm eth yl)th io]p r op yl]-3-m eth yl-L-va lyl]-
1 ,2 ,3 ,4 -t e t r a h y d r o -3 -i s o q u i n o l i n y l ] c a r b o n y l ] -L -
glu ta m in e ter t-Bu tyl Ester (59). Sodium cyanoborohydride
(0.077 g, 1.2 mmol) was added portionwise to a solution of 22
(0.46 g, 0.91 mmol), 58 (0.61 g, 1.4 mmol), and acetic acid
(0.135 mL) in methanol (4.5 mL), and the resulting solution
was stirred at room temperature for 2.25 h. The reaction was
quenched with water (100 mL) and the mixture extracted with
CH2Cl2 (3 × 100 mL). The combined organic extracts were
dried, filtered, and concentrated under vacuum. The residue
was purified by flash chromatography (eluting with ethyl
acetate) to afford 59 (0.55 g, 67%) as a gum: TLC Rf 0.41 (1:1
(R*)-N-[[2-[N-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-
3-m eth yl-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r -
bon yl]-L-m eth ion in e Meth yl Ester (60). To a solution of
N-(tert-butyloxycarbonyl)-L-tert-leucine (0.4 g, 1.7 mmol) in
CH2Cl2 (5 mL) at 0 °C under argon was added sequentially
PyBroP (0.79 g, 1.7 mmol), N,N-diisopropylethylamine (0.72
mL, 5.1 mmol), and (dimethylamino)pyridine (22 mg, 0.2
mmol). After 5 min, 9 (0.6 g, 1.7 mmol) was added. The
reaction mixture was allowed to warm to room temperature.
After 3 h, the reaction mixture was washed successively with
1 N HCl, saturated aqueous NaHCO3, and brine (5 mL each).
Each aqueous layer was extracted with chloroform (5 mL). The
organic layers were combined, dried, filtered, and concentrated.
The residue was purified by flash silica gel column chroma-
tography eluting with 30% ethyl acetate in hexanes to afford
60 (430 mg, 48%) as a viscous oil: MS (M + H)+ 536; TLC Rf
0.41 (50% ethyl acetate in hexane, visualized by UV and ceric
1
hexane:acetone, visualization by UV); MS (M + H)+ 906; H-
NMR (DMSO-d6, 400 MHz) δ 7.28-7.12 (m, 19H), 5.00 (m,
1H), 4.89, 4.84 (s, 2H), 4.56 (br m, 1H), 4.06 (m, 1H), 3.35 (br
m, 1H), 3.09 (br m, 2H), 2.81 (m, 2 H), 2.40-2.10 (m, 2 H),
2.04 (t, 2 H, J ) 7.62 Hz), 1.95-1.74 (m, 2 H), 1.35 (s, 9H),
1.33 (s, 9H), 0.92 (s, 9H); 13C-NMR (CDCl3, 100.4 MHz) δ 175.4,
171.6, 170.8, 144.5, 143.1, 133.9, 129.4, 127.9, 127.0, 126.6,
125.7, 82.6, 66.4, 60.4, 56.0, 51.9, 50.0, 49.7, 49.3, 49.0, 48.7,
47.2, 35.6, 34.2, 31.3, 29.0, 28.2, 27.8, 26.7, 20.9, 14.0; IR (KBr)
1491, 1628, 1676, 2976 cm-1. Anal. (C52H67N5O7S‚1.33H2O)
C, H, N.
1
ammonium molybdate); H-NMR (CDCl3, 270 MHz) δ 7.25-
7.1 (4H, m), 6.8 (1H, m), 5.44-5.4 (1H, m), 5.3-4.8 (2H, m),
4.7-4.6 (2H, m), 4.5-4.3 (1H, m), 3.7, 3.66 (3H, 2s, rotamers),
3.8-3.6 (1H, m), 3.3-3.2 (0.5H, m), 3.05-2.95 (0.5H, m), 2.3-
2.2 (2H, m), 2.1-1.9 (2H, m), 2.0, 1.97 (3H, 2s, rotamers), 1.41
(9H, s), 1.07 (9H, s).
(S*,R*)-N2-[[2-[N-(2-Am in o-3-m er ca p top r op yl)-3-m eth -
yl-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bon yl]-
L-glu ta m in e Tr iflu or oa ceta te (46). Trifluoroacetic acid (5
mL, 65 mmol) was added to a solution of 59 (0.49 g, 0.54 mmol)
and triethylsilane (0.84 mL, 5.4 mmol) in CH2Cl2 (5 mL) at 0
°C, and the resulting solution was stirred at room temperature
for 3 h. Chloroform (10 mL) was added, and the mixture was
concentrated under vacuum. The residue was triturated with
hexane and dried under vacuum. The residue was purified
by preparative HPLC (EM S-12 Lichrosphere Select B, 50 ×
250 mm, 220 nm, 40 mL/min, gradient 10-90% aqueous
methanol with 0.1% TFA) to afford, after lyophilizing, 46 (0.28
g, 71%) as a white solid: mp decomposition above 97 °C; TLC
Rf 0.55 (6:3:1 n-propanol:ammonium hydroxide:water, visual-
[R-(R*,S*)]-N-[[2-[N-[2-[[(1,1-Dim eth yleth oxy)ca r bon y-
l]a m in o]-3-[(t r ip h en ylm et h yl)t h io]p r op yl]-3-m et h yl-L-
va lyl]-1,2,3,4-t e t r a h yd r o-3-isoq u in olin yl]ca r b on yl]-L-
m eth ion in e Meth yl Ester (61). Anhydrous HCl (4 N in
dioxane, 1 mL, 4 mmol) and triethylsilane (0.24 mL, 1.5 mmol)
were added to solid 60 (400 mg, 0.75 mmol) at room temper-
ature under argon. After stirring for 2 h, the mixture was
concentrated and the residue was triturated with diethyl ether
and dried in vacuo to afford tLeu-Tic-Met-OMe (325 mg). To
a solution of the tripeptide (310 mg, 0.66 mmol) in methanol
(2 mL) was added a solution of 22 (0.3 g, 0.6 mmol) in methanol
(1 mL) and acetic acid (0.1 mL). After 5 min, solid NaCNBH3
(33 mg, 0.6 mmol) was added. After 1 h, 22 (0.3 g, 0.6 mmol)
in methanol (1 mL) and NaCNBH3 (67 mg, 1.2 mmol) were
added. After 30 min, 22 (0.3 g, 0.6 mmol) in methanol (1 mL)
was added. After another 1 h, the mixture was diluted with
ethyl acetate (5 mL) and diethyl ether (5 mL) and washed with
saturated aqueous NaHCO3 (10 mL). The organic layer was
washed with brine (10 mL), dried, filtered, and concentrated
in vacuo. The residue was purified by flash column chroma-
tography eluting with a step gradient of 30-50% ethyl acetate
in hexane to afford the white foam 56 (540 mg, 95%): TLC Rf
0.27 (1:2, ethyl acetate in hexanes, visualized by UV and ceric
ization by ceric ammonium sulfate); MS (M + H)+ 508; [R]D
)
-17.6° (c 0.33, CH3OH); 1H-NMR (DMSO-d6, 400 MHz) δ 7.17
(s, 4H), 5.02-4.85 (m, 3H), 4.59-4.52 (m, 1H), 4.20-4.18 (m,
1H), 3.34 (br m, 2H), 2.96 (br m, 2H), 2.72 (br m, 1H), 2.47-
2.35 (m, 2H), 2.13-2.08 (m, 2H), 1.96-1.81 (m, 2H), 1.00 (s,
9H); IR (KBr) 1431, 1674, 2972, 3437 cm-1. Anal. (C28H39N5O9-
SF6‚1.07H2O) C, H, N.
Compounds 47, 48, and 50 were prepared and purified in a
similar manner to 46 starting from 58 and the appropriate
amino acid tert-butyl ester.
1
[R-(R*,S*)]-N2-[[2-[N-(2-Am in o-3-m er ca p t op r op yl)-3-
m eth yl-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bo-
n yl]-N5,N5-d im et h yl-L-glu t a m in e Tr iflu or oa cet a t e (47):
MS (M + H)+ 536; [R]D ) -15° (c 0.22, CH3OH); IR (KBr) 2969,
1680, 1638, 1427, 1206, 1138, 723 cm-1. Anal. (C26H41N5O5S‚
2.83CF3COOH) C, H, N.
ammonium molybdate); MS (M + H)+ 867; H-NMR (CDCl3,
400 MHz) δ 7.4-7.1 (19H, m), 6.7 (1H, d, J ) 7.0 Hz), 5.0-4.4
(6H, m), 3.7, 3.6 (3H, 2s), 3.7-3.5 (2H, m), 3.32 (1H, dd, J )
7, 14 Hz), 3.0 (1H, dd, J ) 7, 14 Hz), 2.5-2.1 (2H, m), 2.0-1.7
(3H, m), 1.98, 1.85 (3H, 2s), 1.44, 1.35 (9H, 2s, 1:4 ratio,
rotamers), 1.0, 0.93 (9H, 2s, 4:1 ratio, rotamers).
[R-(R*,S*)]-N-[[2-[N-(2-Am in o-3-m e r ca p t op r op yl)-3-
m eth yl-L-va lyl]-1,2,3,4-tetr a h yd r o-3-isoqu in olin yl]ca r bo-
n yl]-O-m eth yl-L-h om oser in e Tr iflu or oa ceta te (48): mp
melts with decomposition at 85-95 °C; TLC Rf 0.41 (6:3:1
n-propanol:ammonium hydroxide:water, visualization by ceric
ammonium sulfate); MS (M + H)+ 495; [R]D ) -23.7° (c 0.27,
CH3OH); 1H-NMR (DMSO-d6, 125 °C, 400 MHz) δ 7.17 (s, 4H),
5.03 (t, 1H, J ) 5.98 Hz), 4.90 (br m, 1H), 4.59 (br m, 1H),
[R-(R*,S*)]-N-[[2-[N-(2-Am in o-3-m e r ca p t op r op yl)-3-
m et h yl-L-va lyl]-1,2,3,4-t et r a h yd r o-3-isoq u in olin yl]ca r -
bon yl]-L-m eth ion in e Meth yl Ester Tr iflu or oa ceta te (51).
Triethylsilane (0.066 mL, 0.42 mmol) was added to a solution
of 56 (0.18 g, 0.21 mmol) and TFA (0.4 mL) in dichloromethane
(0.8 mL) at room temperature. After stirring for 3 h, the
reaction mixture was concentrated to afford a solid which was
purified by preparative HPLC (YMC S-10 ODS 30 × 500 mm

236 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Leftheris et al.
M.; Barbacid, M.; Seizinger, B. R. Bisubstrate Inhibitors of
Farnesyltransferase: A Novel Class of Specific Inhibitors of Ras
Transformed Cells. Oncogene 1995, 10, 1763-1779. Patel, D. V.;
Gordon, E. M.; Schmidt, R. J .; Weller, H. N.; Young, M. G.;
Zahler, R.; Barbacid, M.; Carboni, J . M.; Gullo-Brown, J . L.;
Hunihan, L.; Ricca, C.; Robinson, S.; Seizinger, B. R.; Toumari,
A. V.; Manne, V. Phosphinyl Acid- Based Bisubstrate Analog
Inhibitors of Ras Farnesyl Protein Transferase. J . Med. Chem.
1995, 38, 435-442.
column, eluting with 30-90% aqueous methanol containing
0.1% TFA, gradient over 30 min) to afford, after lyophilization,
51 (0.036 g, 33%): [R]D ) -32° (c 0.25, MeOH): MS (M + H)+
525; 1H-NMR (400 MHz, CDCl3) δ 7.19-7.13 (4H, m), 6.9 (1H,
s), 4.8-4.4 (5H, m), 3.9-3.4 (7H, m), 3.1-2.3 (7H, m), 1.97-
1.66 (7H, m), 1.07-1.06 (9H, s, rotamers); IR (KBr) 3435, 2924,
1437, 1682, 1636, 1208, 1134 cm-1
2.0CHF3O20.73H2O) C, H, N.
. Anal. (C25H40N4O4S2‚
(16) Vogt, A.; Qian, Y.; Blaskovich, M. A.; Fossum, R. D.; Hamilton,
A. D.; Sebti, S. M. A Non-peptide Mimetic of Ras-CAAX:
Selective Inhibition of Farnesyltransferase and Ras Processing.
J . Biol. Chem. 1995, 270, 660-664.
(17) Qian, Y.; Blaskovich, M. A.; Saleem, M.; Seong, C. M.; Wathen,
S. P.; Hamilton, A. D.; Sebti, S. M. Design and Structural
Requirements of Potent Peptidomimetic Inhibitors of p21ras
Farnesyltransferase. J . Biol. Chem. 1994, 269, 12410-12413.
(18) Clerc, F. F.; Guitton, J . L.; Fromage, M.; Lelievre, Y.; Duchesne,
M.; Tocque, B.; J ames-Surcouf, E.; Commercon, A.; Becauart,
J . Constrained Analogs of KCVFM With Improved Inhibitory
Properties Against Farnesyl Transferase. Bioorg. Med. Chem.
Lett. 1995, 1779-1784.
(19) Goldstein, J . L.; Brown, M. S.; Stradley, S. J .; Reiss, Y.; Gierasch,
L. M. Nonfarnesylated Tetrapeptide Inhibitors of Protein Far-
nesyltransferase. J . Biol. Chem. 1991, 266, 15575-15578.
(20) Kohl, N. E.; Mosser, S. D.; deSolms, S. J .; Giulani, E. A.;
Pompliano, D. L.; Graham, S. L.; Smith, R. L.; Scolnick, E. M.;
Oliff, A.; Gibbs, J . B. Selective Inhibition of ras-Dependent
Transformation by a Farnesyltransferase Inhibitor. Science
1993, 260, 1934-1937.
(21) Pinder, R. M.; Butcher, B. H.; Buxton, D. A.; Howells, D. J .
2-Aminoindan-2-carboxylic acids. Potential Tyrosine Hydroxy-
lase Inhibitors. J . Med. Chem. 1971, 14, 892-893.
(22) Kaur, P.; Uma, K.; Balaram, P.; Chauhan, V. S. Synthetic and
conformational studies on dehydrophenylalanine containing
model peptides. Int. J . Pep. Prot. Res. 1989, 33, 103-109.
(23) Fehrentz, J . A.; Heitz, A.; Castro, B. Synthesis of aldehydic
peptides inhibiting renin Int. J . Pept. Protein Res. 1985, 26, 236-
241.
(24) Leete, E.; Mueller, M. E. Biomimetic Synthesis of Anatabine
from 2,5-Dihydropyridine Produced by the Oxidative Decarboxy-
lation of Baikiain. J . Am. Chem. Soc. 1982, 104, 6440-6444.
(25) Manne, V.; Roberts, D.; Tobin, A.; O’Rourke, E.; DeVirgilio, M.;
Meyers, C. A.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, H. F.;
Barbacid, M. Identification and Preliminary Characterization
of protein-cysteine farnesyltransferase. Proc. Natl. Acad. Sci.
U.S.A. 1990, 87, 7541-7545.
(26) Gelman, S. H. On the Role of Methionine Residues in the
Sequence- Independent Recognition of Nonpolar Protein Sur-
faces. Biochemistry 1991, 30, 6633-6636.
(27) Tucker, J .; Sczakiel, G.; Feuerstein, J .; J ohn, J .; Goody, R. S.;
Wittinghofer, A. Expression of p21 proteins in Escherichia coli
and stereochemistry of the nucleotide-binding site. EMBO J .
1986, 5, 1351-1358.
(28) Cox, A. D.; Der, C. The ras/cholesterol connection: Implications
for ras oncogenicity. Crit. Rev. Oncogenesis 1992, 3, 365-400.
(29) Hattori, S.; Ulsh, L. S.; Halliday, K.; Shih, T. Y. Biochemical
properties of a highly purified v-rasH p21 protein overproduced
in Escherichia coli and inhibition of its activities by a monoclonal
antibody. Mol. Cell. Biol. 1985, 5, 1449-1455.
(30) Cox, A. D.; Hisaka, M. M.; Buss, J . E. Specific Isoproenoid
Modification is required for function of normal, but not oncogenic
ras function. Mol. Cell. Biol. 1992, 12, 2606-2615.
(31) Sukumar, S.; Carney, W. P.; Barbacid, M. Independent molecular
pathways in initiation and loss of hormone responsiveness of
breast carcinomas. Science 1988, 240, 524-526.
(32) Geran, R. I.; Greenberg, N. H.; MacDonald, M. M.; Schumacher,
A. M.; Abbot, B. J . Protocols for screening chemical agents and
natural products against animal tumor and other biological
systems. Cancer Chemother. Rep. 1972, 3, 1-103.
Ack n ow led gm en t. We would like to thank the
Analytical R&D Department at Bristol-Myers Squibb
for obtaining mass spectra and elemental analyses.
Refer en ces
(1) Barbacid, M. In Annual Review of Biochemistry; Richardson, C.,
Ed.; Annual Reviews Inc.: Palo Alto, CA, 1987; Vol. 56, pp 779-
827.
(2) Kato, K.; Cox, A. D.; Hisaka, M. M.; Graham, S. M.; Buss, J . E.;
Der, C. J . Isoprenoid addition to Ras protein is the critical
modification for its membrane association and transforming
activity. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6403-6407.
(3) J ackson, J . H.; Cochrane, C. G.; Bourne, J . R.; Solski, P. A.; Buss,
J . E.; Der, C. J . Farnesol modification of Kirsten-ras exon 4B
protein is essential for transformation. Proc. Natl. Acad. Sci.
U.S.A. 1990, 87, 3042-3046.
(4) Gibbs, J . B. Ras C-Terminal Processing Enzymes-New Drug
Targets? Cell 1991, 65, 1-4.
(5) Gutierrez, L.; Magee, A. I.; Marshall, C. J .; Hancock, J . F. Post-
translational processing of p21ras is two-step and involves
carboxyl-methylation and carboxy-terminal proteolysis. EMBO
J . 1989, 8, 1093-1098.
(6) Newman, C. M.; Magee, A. I. Posttranslational processing of the
ras superfamily of small GTP-binding proteins. Biochim. Bio-
phys. Acta 1993, 1155, 79-96.
(7) Schafer, W. R.; Rine, J . Protein prenylation: Genes, Enzymes,
Targets and Functions. Annu. Rev. Genet. 1992, 26, 209-237.
(8) Hancock, J . F.; Magee, A. I.; Childs, J . E.; Marshall, C. J . All
Ras Proteins Are Polyisoprenylated but Only Some are Palmi-
toylated. Cell 1989, 57, 1167-1177.
(9) Garcia, A. M.; Rowell, C.; Ackermann, K.; Kowalczyk, J . J .;
Lewis, M. D. Peptidomimetic Inhibitors of Ras Farnesylation and
Function in Whole Cells. J . Biol. Chem. 1993, 268, 18415-18418.
(10) Marsters, J . C.; McDowell, R. S.; Reynolds, M. E.; Oare, D. A.;
Somers, T. C.; Stanley, M. S.; Rawson, T. E.; Struble, M. E.;
Burdick, D. J .; Chan, K. S.; Duarte, C. M.; Paris, K. J .; Tom, J .
Y. K.; Wan, D. T.; Xue, Y.; Burnier, J . P. Benzodiazepine
Peptidomimetic Inhibitors of Farnesyltransferase. Bioorg. Med.
Chem. 1994, 2, 949-957.
(11) Graham, S. L.; deSolms, S. J .; Giuliani, E. A.; Kohl, N. E.; J osser,
S. D.; Oliff, A. I.; Pompliano, D. L.; Rands, E.; Breslin, M. J .;
Deana, A. A.; Garsky, V. M.; Scholz, T. H.; Gibbs, J . B.; Smith,
R. L. Pseudopeptide Inhibitors of Ras Farnesyl-Protein Trans-
ferase. J . Med. Chem. 1994, 37, 725-732.
(12) Leftheris, K.; Kline, T.; Natarajan, S.; DeVirgilio, M. K.; Cho,
Y. H.; Pluscec, J .; Ricca, C.; Robinson, S.; Seizinger, B. R.;
Manne, V.; Meyers, C. A. Peptide Based P21RAS Farnesyl
Transferase Inhibitors: Systematic Modification of the Tet-
rapeptide CA1A2X Motif. Bioorg. Med. Chem. Lett. 1994, 4, 887-
892.
(13) Harrington, E. M.; Kowalczyk, J . J .; Pinnow, S. L.; Ackermann,
K.; Garcia, A. M.; Lewis, M. D. Cysteine and Methionine Linked
by Carbon Pseudopeptides Inhibit Farnesyl Transferase. Bioorg.
Med. Chem. Lett. 1994, 4, 2775-2780.
(14) Manne, V.; Ricca, C. S.; Brown, J . G.; Tuomari, A. V.; Yan, N.;
Patel, D. V.; Schmidt, R.; Lynch, M. J .; Ciosek, J .; Carboni, J .
M.; Robinson, S.; Gordon, E. M.; Barbacid, M.; Seizinger, B. R.;
Biller, S. A. Ras Farnesylation as a Target for Novel Antitumor
Agents: Potent and Selective Farnesyl Diphosphate Analog
Inhibitors of Farnesyltransferase. Drug Dev. Res. 1995, 34, 121-
137.
(15) Manne, V.; Yan, N.; Carboni, J . M.; Tuomari, A. V.; Ricca, C. S.;
Brown, J . G.; Andahazy, M. L.; Schmidt, R. J .; Patel, D.; Zahler,
R.; Weinmann, R.; Der, C. J .; Cox, A. D.; Hunt, J . T.; Gordon, E.
J M950642A