Potent, cell active, non-thiol tetrapeptide inhibitors of farnesyltransferase.

Article abstract of DOI:10.1021/jm9507284

All previously reported CAAX-based farnesyltransferase inhibitors contain a thiol functionality. We report that attachment of the 4-imidazolyl group, via 1-, 2-, or 3-carbon alkyl or alkanoyl spacers, to Val-Tic-Met or tLeu-Tic-Gln provides potent FT inhibitors. (R*)-N-[[1,2,3,4-Tetrahydro-2-[N-[2-(1H-imidazol-4-yl)ethyl] -L-valyl]-3-isoquinolinyl]carbonyl]-L-methionine ([imidazol- 4-yl-ethyl]-Val-Tic-Met), with FT IC50 = 0.79 nM, displayed potent cell activity in the absence of prodrug formation (SAG EC50 = 3.8 muM).

Full text of DOI:10.1021/jm9507284

J . Med. Chem. 1996, 39, 353-358
353
Expedited Articles
P oten t, Cell Active, Non -Th iol Tetr a p ep tid e In h ibitor s of F a r n esyltr a n sfer a se
J ohn T. Hunt,*,† Ving G. Lee,† Katerina Leftheris,† Bernd Seizinger,‡ J oan Carboni,‡ J ohn Mabus,‡ Carol Ricca,‡
Ning Yan,‡ and Veeraswamy Manne‡
Departments of Chemistry and BioMolecular Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New J ersey 08543-4000
Received October 3, 1995X
All previously reported CAAX-based farnesyltransferase inhibitors contain a thiol functionality.
We report that attachment of the 4-imidazolyl group, via 1-, 2-, or 3-carbon alkyl or alkanoyl
spacers, to Val-Tic-Met or tLeu-Tic-Gln provides potent FT inhibitors. (R*)-N-[[1,2,3,4-
Tetrahydro-2-[N-[2-(1H-imidazol-4-yl)ethyl]-L-valyl]-3-isoquinolinyl]carbonyl]-L-methionine ([im-
idazol-4-yl-ethyl]-Val-Tic-Met), with FT IC50 ) 0.79 nM, displayed potent cell activity in the
absence of prodrug formation (SAG EC50 ) 3.8 µM).
Mutated ras genes are found in a wide variety of
human tumors, with the highest rates observed in
cancers of the pancreas (90%), colon (50%), and lung
(30%).1,2 This incidence has prompted considerable
efforts at elucidating the pathways of Ras transforma-
tion and developing therapeutic agents which might
interfere with this pathway. These latter efforts have
focused primarily on the development of inhibitors of
the enzyme farnesyltransferase (FT).
of a non-thiol CAAX-based inhibitor, and these analogs
were weak inhibitors.18
Farnesyltransferase requires for its function the
presence of two metal ions, zinc and magnesium.19 In
one plausible catalytic mechanism, the magnesium
coordinates to the oxygens of FPP, and the CAAX thiol
of the Ras protein coordinates to the zinc, perhaps with
the formation of a zinc thiolate assisted by a nearby
general base. In considering alternate zinc ligands, we
were drawn to consider imidazole because of the preva-
lence of histidine as a key ligand in metalloenzymes.20
Despite its ubiquitous use by enzymes as a metal ligand,
the use of the imidazole ring as a metal ligand in
metalloenzyme inhibitors has received scant attention.21
In this report, we describe the discovery of extremely
potent imidazole-based tetrapeptide inhibitors of FT.
These compounds display cellular effects in the absence
of prodrug formation.
In order to perform both its normal and oncogenic
functions, the Ras protein must be membrane associ-
ated. The carboxy terminus of proteins of the Ras
family consists of a tetrad referred to as a CAAX box,
where C is cysteine, A is an aliphatic amino acid, and
X is a member of a limited set of amino acids. Mem-
brane localization of Ras is the result of a three-step
posttranslational modification of its CAAX box, involv-
ing farnesylation of the cysteine by farnesyl pyrophos-
phate (FPP), hydrolysis of the three C-terminal amino
acid residues, and methyl esterification of the resulting
C-terminal farnesylcysteine. Of these steps, farnesy-
lation appears to be a necessary and sufficient step for
membrane localization, implicating FT as a preferred
target for interrupting oncogenic Ras signaling.3
Ch em istr y, Biologica l Resu lts, a n d Discu ssion
We have discovered that cysteine-based tetrapeptides
which contain (S)-1,2,3,4-tetrahydro-3-isoquinolinecar-
boxylic acid (Tic) in place of the A2 residue of the CAAX
box provide potent FT inhibitors.22 We have also
previously demonstrated that the imidazole-containing
tetrapeptide His-Val-Phe-Met was a poor inhibitor (FT
IC50 ) 6.8 µM).8 Nevertheless, we were interested in
determining whether this poor activity was due to
inappropriate spacing of the imidazole ring with respect
to the remainder of the tripeptide. Direct coupling of
commercially available 4-imidazoleacetic acid to the HCl
salt of Val-Tic-Met-OMe using 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride, HOBt, and
N-methylmorpholine in DMF, followed by ester hydroly-
sis, afforded 2, which inhibited FT with an IC50 value
of 6 nM (Table 1). Compound 2 displayed 300-fold
selectivity for inhibition of FT versus GGTI; in fact, all
of the imidazole tetrapeptides were more potent inhibi-
tors of FT versus GGTI, with selectivities ranging from
300- to 40 000-fold. Compound 2 was also active in
cell-based assays, as demonstrated by its ability to
inhibit the anchorage independent growth of H-Ras
A number of groups have reported thiol FT inhibitors
based on the CAAX sequence. These include tetrapep-
tides,4 reduced tetrapeptides,5-8 constrained tetrapep-
tides,9,10 and tetrapeptide-like molecules in which spac-
ers have been used to replace one or more of the central
amides or amino acid residues.11-15 While many of
these thiol-containing compounds are effective inhibitors
of both the isolated enzyme and cellular Ras processing,
the presence of the oxidizable thiol functionality confers
disadvantages on the development of such compounds
as therapeutic agents. Although non-thiol inhibitors
based upon FPP analogs (see ref 16) or bisubstrate
analogs17 have been reported, there is only one report
* To whom correspondence should be addressed at Bristol-Myers
Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000.
Tel: (609) 252-4989. Fax: (609) 252-6804. E-mail: hunt@bms.com.
† Department of Chemistry.
‡ Department of Biomolecular Drug Discovery.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0353$12.00/0 © 1996 American Chemical Society

354 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Ta ble 1. Imidazole-Containing Inhibitors of Farnesyltransferase
Hunt et al.
compd
n
R1
R2
X
FT IC50 (nM)
GGTI IC50 (nM)
SAG IC50 (µM)
MTD (µM)
1a
2a
3a
4a
5a
6a
7b
8b
0
1
0
1
1
2
1
2
H
H
H
H
Me
Me
Me
Me
SMe
SMe
SMe
SMe
CONH2
CONH2
CONH2
CONH2
O
O
H,H
H,H
O
O
H,H
H,H
84 ( 10
6.6 ( 1
114 ( 25
0.79 ( 0.07
28 ( 0.5
4.4 ( 0.25
3.4 ( 0.05
20 ( 3.5
25 800 ( 5600
2020 ( 700
100
50
100
3.8
3.9
27
>150
>150
NT
>150
>150
>150
NT
17 400 ( 330
234 ( 41
28% (360 µM)
168 000 ( 54 000
30 500 ( 5300
106 000 ( 42 000
3.2
32
>150
a
b
Elemental analysis for C, H, N as hydrated TFA salts. Elemental analysis for C, H, N as hydrated Li salts. NT ) not tested.
transformed rat-1 cells in soft agar (SAG EC50 ) 50 µM).
Compound 2 and the other imidazole tetrapeptide
inhibitors did not display cytotoxicity to untransformed
NIH 3T3 cells. The high MTD values (no toxicity
observed at 150 µM, the highest concentration tested)
indicate that SAG activity was not due to general
cytotoxicity. Encouraged by these findings, we synthe-
sized other imidazole-containing tetrapeptides. Com-
pound 1 was prepared by coupling the tripeptide with
1-(triphenylmethyl)imidazole-4-carboxylic acid and sub-
sequent deprotection. Compound 3 was prepared by
reductive amination (NaCNBH3) of the tripeptide with
[1-(triphenylmethyl)imidazole-4-carboxaldehyde and sub-
sequent deprotection. Compound 4 was prepared by
reductive amination of the tripeptide with [1-(ethoxy-
carbonyl)imidazol-4-yl]acetaldehyde and subsequent
deprotection. Both the amide and methyleneamine
1-carbon linkers afforded moderate, equipotent inhibi-
tors. However, the 2 carbon methyleneamine analog 4
afforded a subnanomolar inhibitor of FT. More impor-
tantly, 4 was a potent inhibitor in the SAG assay.
Almost complete (95%) inhibition of the growth of
H-Ras-transformed cells occurred with 25 µM 4 (Figure
1B), and in three independent assays, the EC50 value
for 4 was 3.8 ( 0.1 µM. This potent cell activity was
observed despite the fact that 4 contains an unesterified
and therefore charged carboxylic acid group.
We have previously demonstrated that Tic-containing
tetrapeptides in which t-Leu and Gln occupied the A1
and X sites, respectively, show increased cell activity
relative to their FT inhibitory potency (decrease in the
SAG EC50/FT IC50 ratio).22 These changes were incor-
porated into imidazole tetrapeptides with the more
potent 2-carbon linker. In addition, the effect of length-
ening the linker to three atoms was also investigated
in this series. These compounds were prepared by
procedures analogous to those described above. As
shown in Table 1, the acetamide analog 5 was a
moderately potent FT inhibitor. The 4-imidazolylethyl
analog 7, while 4-fold less potent than 4 as an FT
inhibitor, was equipotent in the SAG assay. The
3-carbon linkers also afforded good FT inhibitors,
although in this case the 4-imidazolylpropionyl analog
6 was 5-fold more potent than the 4-imidazolylpropyl
analog 8.
F igu r e 1. Whole cell activity of 4. A and B: Effect on growth
of H-Ras-transformed NIH 3T3 cells (44-911 cells) in soft
agar. Experimental conditions used to determine anchorage
independent growth in soft agar were as described.26 After 14
days of growth at 37 °C, colonies were photographed at 40×
magnification using phase contrast ring 2. Colonies larger than
0.1 mm were counted in the untreated (A) and treated (B)
samples to determine the percent inhibition of colony growth.
Colony growth was inhibited by 95% with 4 at 25 µM (B). The
total number of colonies per 35 mm well in untreated samples
was 1856. C and D: Effect on morphology of H-Ras-trans-
formed rat-1 cells. The initial plating conditions and the
treatment protocol with inhibitor were essentially as de-
scribed.26 Representative areas of the wells were photographed
on day 4 at 40× magnification: C, no treatment; D, 25 µM 4.
tripeptide spacing as histidine, suggested that optimal
spacing was not the reason for the effectiveness of these
compounds as FT inhibitors compared to His-Val-Phe-
Met. To further confirm this, the L-histidine analog 9
(Figure 2) was prepared and found to be a relatively
poor inhibitor (FT IC50 ) 520 nM; SAG EC50 ) 100 µM;
30% inhibition of GGTI at 360 µM; MTD >150 µM).
Similarly, the D,L-imidazol-4-ylglycine analog 10 (FT
IC50 ) 180 nM; SAG EC50 ) 100 µM; 6% inhibition of
GGTI at 180 µM) was also a moderate inhibitor com-
pared to its desamino analog 4. These results indicate
that the presence of the R-amine is detrimental to the
inhibitory potency of imidazole-based tetrapeptide FT
inhibitors. One possible explanation for this effect is
The high inhibitory potency of the 3-carbon amide
analog 6, which maintains the same imidazole to

Non-Thiol Tetrapeptide Inhibitors of FT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 355
mL, 7.21 mmol) was added followed by N-hydroxybenzotriazole
(HOBt; 974 mg, 7.21 mmol). The mixture was stirred for 5
min, and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hy-
drochloride (EDC; 1.38 g, 7.21 mmol) was added. The mixture
was allowed to come to room temperature, stirred overnight,
and partitioned between methylene chloride and brine. The
organic phase was washed successively with citric acid,
NaHCO3, and brine, dried, and concentrated under vacuum
to give 2.64 g (86%) of 11: MS (M + H)+ 423; 1H-NMR (CDCl3,
270 MHz) δ 7.25-7.13 (4H, m), 4.86-4.40 (4H, m), 3.69 (3H,
s), 3.32 (2H, m), 3.05 (2H, m), 1.93 (3H, m), 1.90-1.78 (2H,
m), 1.50 (9H, 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 (12). A solution of 11 (2 g, 6.22
mmol) in methylene chloride (10 mL) was treated at room
temperature with trifluoroacetic acid (TFA; 10 mL) and
dimethyl sulfide (0.5 mL) and stirred for 0.5 h. The mixture
was concentrated under vacuum, dissolved in methylene
chloride, and concentrated. This procedure was repeated five
times to yield 12 (99%) as a clear glass: 1H-NMR (CDCl3, 270
MHz) δ 7.24-7.10 (4H, m), 4.53-4.39 (4H, m), 3.64 (3H, s),
3.26 (2H, m), 2.52 (1H, m), 2.44 (1H, m), 1.98 (5H, m).
(R*)-N-[[2-[N-[(1,1-Dim eth yleth oxy)ca r bon 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 (13). To a solution of 12 (4.73 mmol) in
methylene chloride (20 mL) at 0 °C was added N-(tert-
butyloxycarbonyl)-L-valine (2.06 g, 9.47 mmol), bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (BOP-Cl, 1.2 g, 4.7 mmol) and
DIEA (1.2 g, 1.6 mL, 9.5 mmol). The mixture was stirred for
24 h at 0 °C. Additional BOP-Cl; (1.2 g, 4.7 mmol), and DIEA
(590 mg, 0.8 mL, 4.73 mmol) were added, and the mixture was
stirred for an additional 12 h at 0 °C. The mixture was
concentrated, and the residue was chromatographed (silica gel,
50% ethyl acetate/hexane). Appropriate fractions were col-
lected and concentrated to yield 13 as a clear oil (2.4 g, 97%):
F igu r e 2. Structures of R-amine-containing imidazole tet-
rapeptides.
that the R-ammonium ion may provide an internal
hydrogen bond to the imidazole, tying up the lone pair
of electrons which would otherwise be available for the
putative zinc coordination.
The most potent imidazole tetrapeptide inhibitor, 4
(BMS-193269), was further characterized to ensure that
its effects in the SAG assay were due to mechanism-
based inhibition of FT. The posttranslational processing
status of the Ras protein in cells can be monitored by
SDS-PAGE of lysed cell extracts. In the presence of an
FT inhibitor, Ras protein farnesylation is blocked, and
the unprocessed protein migrates as a slower band.
Treatment of H-Ras-transformed rat-1 cells with 100
µM 4 led to complete inhibition of Ras processing, and
the IC50 for this inhibition was found to be about 5 µM.
Compared to their untransformed counterparts, H-Ras-
transformed rat-1 cells round up, appear refractile, and
pile up due to a loss of contact inhibition (Figure 1C).
Treatment of these cells with 25 µM 4 for 4 days led to
a complete restoration of the untransformed phenotype,
with the cells flattening and growing in a monolayer
(Figure 1D). The IC50 for reversion of the transformed
phenotype by 4 was about 5 µM.
In conclusion, we have discovered potent tetrapeptide
inhibitors of FT in which the key thiol functionality is
replaced by the chemically stable imidazole group. In
the absence of prodrug formation, these compounds are
potent inhibitors in cell-based assays, leading to inhibi-
tion of Ras processing, restoration of the untransformed
phenotype, and inhibition of anchorage independent
growth in soft agar.
1
MS (M + H)+ 522; H-NMR (CD3OD, 400 MHz) δ 7.25-7.18
(4H, m), 5.29 (1H, m), 5.01 (1H, m), 4.91 (1H, m), 4.70-4.54
(2H, m), 3.71 (3H, s), 3.32 (1H, m), 3.09 (1H, m), 2.25-2.02
(4H, m), 2.00 (3H, s), 1.82 (1H, m), 1.42 (9H, s), 1.14-0.90 (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 , Hyd r och lor id e
(14). A solution of 13 (100 mg, 0.192 mmole) in 4 N HCl in
dioxane was stirred at 0 °C for 1 h. The solution was
concentrated under vacuum to afford a pink oil which was
chased with diethyl ether (3 × 20 mL) and triturated with
diethyl ether to afford 81 mg (91%) of 14 as a pink solid: MS
1
(M + H)+ 422; H-NMR (CDCl3, 270 MHz) δ 7.32-7.23 (4H,
m), 4.83 (1H, d, J ) 15.0 Hz), 4.72 (1H, m), 4.61 (1H, d, J )
15.0 Hz), 4.58 (1H, m), 4.52 (1H, d, J ) 4.7 Hz), 3.70 (3H, s),
3.25 (1H, m), 3.11 (1H, m), 2.58-2.46 (2H, m), 2.37 (1H, m),
2.06 (3H, s), 1.94 (1H, m), 1.20 (3H, m), 1.08 (3H, m); 13C-
NMR (CD3OD, 100 MHz) 173.64, 173.44, 169.60, 135.11,
129.10, 128.47, 128.16, 126.88, 56.94, 52.97, 52.59, 47.54,
32.61, 32.24, 31.19, 30.96, 19.07, 16.20, 15.23 ppm.
Exp er im en ta l Section
4-(Hyd r oxym eth yl)-1-(tr ip h en ylm eth yl)im id a zole (15).
A solution of triphenylmethyl chloride (2.2 g, 7.9 mmol) in
DMF (5 mL) was added to a mixture of 4-(hydroxymethyl)-
imidazole (1 g, 7.4 mmol) and triethylamine (2.5 mL, 18 mmol)
in DMF (7.5 mL). The mixture was stirred for 18 h and
filtered, and the solid was washed with water and dried under
vacuum to afford 2.33 g (93%) of 15 as a white solid: MS (M
+ H)+ 341.
1-(Tr ip h en ylm eth yl)im id a zole-4-ca r boxa ld eh yd e (16).
A mixture of 15 (2.33 g, 6.85 mmol) in dioxane (100 mL) was
heated to 85 °C to obtain a homogenous solution. The heat
source was removed, solid MnO2 (5 g, 57.5 mg) was added all
at once, and the mixture was stirred until the temperature
began to drop. The mixture was heated for 6 h at 85 °C and
filtered through a pad of Celite, and the filtrate was concen-
trated. The resulting white solid was dissolved in methylene
chloride (20 mL), hexane was added, and the mixture was
allowed to stand overnight. The solid was filtered and dried
to afford 1.86 g (80%) of 16 as a white solid. 1H-NMR (CDCl3)
δ 9.9 (s, 1H), 7.63 (s, 1H), 7.55 (s, 1H), 7.38 (m, 9H), 7.13 (m,
6H).
Gen er a l Ch em ica l P r oced u r es. Melting points were
recorded on a Thomas-Hoover capillary apparatus and are
reported uncorrected. IR spectra were recorded on a Mattson
Sirius 100 spectrometer. Proton NMR (1H-NMR) and carbon
NMR (13C-NMR) spectra were obtained on J OEL FX-270 and
GX-400 spectrometers and are reported relative to tetrameth-
ylsilane (TMS) reference. Analytical and preparative HPLC
were performed on YMC columns (A-302, S-5, 120A ODS, 4.6
× 150 mm; SH-345-15, S-15, 120A ODS, 20 × 500 mm) with
acetonitrile:water gradients containing 0.1% trifluoroacetic
acid. Chromatography was performed under flash conditions
using EM Science silica gel, 0.040-0.063 mm particle size.
THF was distilled from Na/benzophenone. Solutions were
dried with magnesium sulfate unless otherwise noted.
(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 (11). A solution of (S)-3,4-dihydro-2,3(1H)-isoquino-
linedicarboxylic acid, 2-(1,1-dimethylethyl) ester (2.0 g, 7.21
mmol) and L-methionine, methyl ester, hydrochloride (1.44 g,
7.21 mmol) in 5:15 N-methylpyrrolidinone-methylene chloride
was stirred at 4 °C. N,N-Diisopropylethylamine (DIEA; 1.23

356 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Hunt et al.
1-(Tr ip h en ylm eth yl)im id a zole-4-ca r boxylic Acid (17).
To a suspension of Ag2O (0.68 g, 3.0 mmol) in water (4 mL) at
55 °C was added NaOH (570 mg, 14.4 mmol) followed by 16
(1 g, 3.0 mmol). The mixture was stirred at 55 °C for 15 min,
filtered hot through a pad of Celite, and the filtrate was
acidified to pH 3.5 with 6 N HCl. The resulting tan solid was
collected, rinsed with water, dried under vacuum to afford 110
mg (10%) of 17: MS (M + H)+ 355.
(S)-N-[[1,2,3,4-Tetr a h yd r o-2-[N-[[1-(tr ip h en ylm eth yl)-
im idazol-4-yl]car bon yl]-L-valyl]-3-isoqu in olin yl]car bon yl]-
L-m eth ion in e, Meth yl Ester (18). To a solution of 17 (89
mg, 0.192 mmol), HOBt (25.9 mg, 0.192 mmol), and 14 (74.6
mg, 0.21 mmol), in 5 mL of dichloromethane at 0 °C was added
N-methylmorpholine (NMM; 21 mL, 0.192 mmol) followed by
EDC (40 mg, 0.21 mmol). The mixture was stirred at room
temperature for 18 h and partitioned between water and
dichloromethane. The organic phase was extracted with
saturated sodium bicarbonate (2×), 3 N aqueous HCl, water,
and brine and dried. The solvent was removed under vacuum
to afford 110 mg (76%) of 18: MS (M + H)+ 780.
(S)-N-[[1,2,3,4-Tetr a h yd r o-2-[N-[[1-(tr ip h en ylm eth yl)-
im idazol-4-yl]car bon yl]L-valyl]-3-isoqu in olin yl]car bon yl]-
L-m eth ion in e (19). To a cooled solution of 18 (110 mg, 0.145
mmol) in methanol (5 mL) was added a solution of 1 N aqueous
NaOH (5 mL). The mixture was stirred for 2 h and concen-
trated to remove methanol, and the residue was partitioned
between water and dichloromethane. The aqueous layer was
acidified to pH 4 with 6 N HCl and extracted with methylene
chloride (4×). The combined organic phases were washed with
water and brine and dried. The solvent was removed under
vacuum to afford 106 mg (98%) of crude 19.
palladium on carbon (100 mg). The mixture was stirred for 1
h
and filtered through a pad of Celite and the filtrate
concentrated to afford 0.37 g (98%) of 21 as an oil: MS (M +
H)+ 183.
(R*)-N-[[1,2,3,4-Tetr a h yd r o-2-[N-[2-(1H-im id a zol-4-yl)-
et h yl]-L-va lyl]-3-isoq u in olin yl]ca r b on yl]-L-m et h ion in e,
Tr iflu or oa ceta te (2:5) Sa lt (4). To a solution of 14 (83 mg,
0.18 mmol) and 21 (100 mg) in methanol (5 mL) was added
solid NaBH3CN (23 mg, 0.36 mmol). After 10 min, additional
amounts of NaBH3CN (23 mg, 0.36 mmol) and acetic acid (21
µL, 0.36 mmol) were added, and the mixture was stirred for
30 min. Two additional portions of 21 (150 mg), NaBH3CN
(46 mg, 0.72 mmol), and acetic acid (41 µL, 0.72 mmol) were
added over 1 h. The mixture was stirred for 18 h and
evaporated, and the residue was dissolved in methanol (5 mL).
Sodium hydroxide (1 N, 4 mL) was added, the solution was
stirred for 1 h, and the methanol was evaporated. The aqueous
phase was diluted to 10 mL with water, and 1 mL of TFA was
added. The mixture was stirred for 10 min and concentrated,
and the residue was dissolved in 25 mL of (80:20) water/
acetonitrile containing 0.1% TFA. The mixture was centri-
fuged and decanted to remove insoluble materials. The
solution was subjected to preparative HPLC on an octadecyl-
silane column (S-10, 30 × 500) using a gradient system from
80% water:20% acetonitrile:0.1% TFA to 55% water:45%
acetonitrile:0.1% TFA over 50 min at 40 mL/min. Fractions
containing product with a minimum purity of 99% were pooled
and lyophilized to afford 22 mg of 4 (24%) as a white solid:
MS (M + H)+ 502. 1H-NMR (CD3OD) δ 8.79 (d, 1H, J ) 9.3
Hz), 7.26 (m, 5H), 4.85 (m, 2H), 4.65 (m, 1H), 4.28 (m, 1H),
3.4 (m, 1H), 3.2 (m, 4H), 2.55 (m, 2H), 2.2 (m, 2H), 2.06-1.94
(d, 3H), 1.9 (m, 1H), 1.2 (m, 6H); 13C-NMR (CD3OD) 173.25,
171.74, 170.57, 167.65, 133.96, 133.89, 133.68, 132.05, 131.33,
128.74, 127.78, 127.68, 126.97, 126.71, 125.91, 125.26, 116.71,
62.92, 56.02, 55.81, 50.89, 45.25, 31.08, 30.99, 30.17, 29.53,
20.86, 17.80, 16.52, 16.51, 13.64 ppm; IR (KBr) 2976, 1674,
(S)-N-[[1,2,3,4-Tet r a h yd r o-2-[N-(1H -im id a zol-4-ylca r -
b on yl)-L-va lyl]-3-isoq u in olin yl]ca r b on yl]-L-m e t h ion -
in e, Tr iflu or oa ceta te (1:1) Sa lt (1). To a degassed solution
of triethylsilane (0.23 mL, 1.4 mmol) and TFA (5 mL) in
methylene chloride (5 mL) was added 19. The mixture was
stirred for 4 h and concentrated, and the residue was dissolved
in 25 mL of (80:20) water/acetonitrile/0.1% TFA. The mixture
was centrifuged and decanted to remove insoluble materials.
The solution was subjected to preparative HPLC on an
octadecylsilane column (S-10, 30 × 500) using a gradient
system from 80% water:20% acetonitrile:0.1% TFA to 55%
water:45% acetonitrile:0.1% TFA over 50 min at 40 mL/min.
Fractions were analyzed by analytical reversed phase HPLC,
and those containing product with a minimum purity of 99%
were pooled and lyophilized to afford 14 mg (19.6%) of 1 as a
1433, 1204, 1136, 835, 721 cm-1
.
N -M e t h y l-N -m e t h o x y [2-a m i n o -N -[(1,1-d i m e t h y l-
et h oxy)ca r b on yl]-2-N-[(1,1-d im et h ylet h oxy)ca r b on yl]-
im id a zol-4-yl]a ceta m id e (22). To a solution of 2-amino-N-
[(1,1-dimethylethoxy)carbonyl]-2-N-[(1,1-dimethylethoxy)-
carbonyl]imidazole-4-acetic acid23,24 (330 mg, 0.97 mmol), N,O-
dimethyl hydroxylamine hydrochloride (97 mg, 1 mmol), and
bromotris(pyrrolidinophosphonium) hexafluorophosphate (466
mg, 1 mmol) in methylene chloride (10 mL) at 0 °C were added
DIEA (0.523 mL, 3 mmol) and DMAP (12.2 mg, 0.1 mmol).
The solution was stirred at room temperature for 2 h and
concentrated, the residue was dissolved in ethyl acetate (40
mL), and the solution was washed with 1 N KHSO4 (3 × 30
mL) and water (1 × 30 mL). The organic layer was dried,
filtered, and concentrated. The residue was chromatographed
(silica gel, 1:1 ethyl acetate:hexanes). Fractions containing the
product were pooled and concentrated to afford 260 mg (70%)
of 22 as a white solid: MS (M + H)+ 385+; 1H-NMR (CDCl3) δ
8.08 (1H, s), 7.41 (1H, s), 5.81 (1H, s), 3.72 (1H, s), 3.21 (1H,
s), 1.61 (9H, s), 1.42 (9H, s).
1
white solid: MS (M + H)+ 502; H-NMR (CD3OD) δ 8.74 (d,
1H, J ) 9.3 Hz), 8.05 (d, 1H, J ) 9.3 Hz), 7.21 (m, 5H), 4.88
(m, 2H), 4.69 (m, 1H), 3.3 (m, 1H), 3.2 (m, 4H), 2.55 (m, 2H),
2.2 (m, 2H), 2.06-1.94 (d, 3H), 1.9 (m, 1H), 1.1-1.04 (m, 6H);
13C-NMR (CD3OD) 173.27, 172.22, 171.58, 172.0, 170.3, 135.52,
134.02, 133.80, 132.12, 127.78, 127.38, 126.90, 126.78, 126.49,
125.78, 125.42, 120.27, 120.00, 55.71, 55.44, 55.00, 50.99,
46.14, 31.24, 31.02, 30.91, 30.59, 29.44, 18.39, 17.25, 13.70
ppm; IR (KBr) 3435, 3032, 2971, 2926, 1645, 1549, 1415, 1206,
1140, 801, 750, 723 cm-1
.
S-E t h yl[1-(et h oxyca r b on yl)im id a zol-4-yl)t h ioa cet ic
Acid (20). To a mixture of 4-imidazoleacetic acid (2 g, 12
mmol) and triethylamine (5.1 mL, 37 mmol) in methylene
chloride (40 mL) at 0 °C was added a solution of ethyl
chloroformate (2.5 mL, 26 mmol) in methylene chloride (10
mL) over 20 min to maintain the reaction temperature between
0 and 5 °C. The mixture was stirred for 15 min, and
ethanethiol (2.0 mL, 28 mmol) and (N,N-dimethylamino)-
pyridine (DMAP; 100 mg) were added consecutively. The
mixture was stirred for 30 min at 0 °C, warmed to room
temperature, washed with water and brine, and dried. The
solvent was evaporated, the residue was adsorbed on a plug
of silica gel, and the plug was eluted with dichloromethane to
remove the unreacted ethanethiol followed by 10% methanol:
dichloromethane. Fractions containing product were combined
and evaporated to yield 0.55 g of 20 as an oil: MS (M + H)+
243.
(S)-1,2,3,4-Tetr ah ydr o-2-[3-m eth yl-N-[(ph en ylm eth oxy)-
ca r bon yl]-L-va lyl]-3-isoqu in olin eca r boxylic Acid , Meth yl
E st er (23). To a solution of 3-methyl-N-[(phenylmethoxy)-
carbonyl]-L-valine (5.31 g, 20 mmol) in methylene chloride (80
mL) at 0 °C under argon were sequentially added DIEA (10.6
mL, 60 mmol), BOP (5.08 g, 20 mmol), and (S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid, methyl ester, hydro-
chloride (5.68 g, 25 mmol). The mixture was allowed to warm
to 5 °C over 2 h, stirred for 16 h, and washed with 1 N
hydrochloric acid, saturated sodium bicarbonate and brine. The
organic layer was dried, filtered, and concentrated to afford
an oil. Chromatography (silica gel, 20% ethyl acetate/hexanes)
afforded 23 (4.0 g, 45%): MS (M + H)+ 439.
(S)-1,2,3,4-Tetr ah ydr o-2-[3-m eth yl-N-[(ph en ylm eth oxy)-
ca r bon yl]-L-va lyl]-3-isoqu in olin eca r boxylic Acid (24). To
a solution of 23 (830 mg, 1.9 mmol) in THF/methanol (4 mL/5
mL) was added lithium hydroxide (1 N, 1.9 mL). After 1 h,
additional lithium hydroxide (1 N, 1.5 mL) was added. After
2 h, sodium hydroxide (1 N, 1.9 mL) was added. After another
1 h, solvent was removed and the residue was partitioned
[1-(E t h oxyca r b on yl)im id a zol-4-yl]a cet a ld eh yd e (21).
To a degassed solution of 20 (0.5 g, 2.1 mmol) and triethylsi-
lane (1.64 mL, 10.3 mmol) in acetone (15 mL) was added 10%

Non-Thiol Tetrapeptide Inhibitors of FT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 357
between 1 N hydrochloric acid and ethyl acetate. The organic
layer was dried and concentrated to afford 24 (820 mg).
(R*)-N2-[[1,2,3,4-Tet r a h yd r o-2-[3-m et h yl-N-[(p h en yl-
m eth oxy)ca r bon yl]-L-va lyl]-3-isoqu in olin yl]ca r bon yl]-L-
glu ta m in e, 1,1-Dim eth yleth yl Ester (25). To a solution of
24 (0.9 g, 2.1 mmol), L-glutamine, tert-butyl ester, hydrochlo-
ride (0.50 g, 2.1 mmol), and BOP (0.93 g, 2.1 mmol) in 3:1 CH3-
CN:DMF (26 mL) was added DIEA (1.1 mL, 6.3 mmol). The
solution was stirred for 16 h, the reaction quenched with 1 N
HCl (100 mL), and the mixture extracted four times with ethyl
acetate, and the combined organic extracts were washed three
times with 10% LiCl, dried, filtered, and concentrated. The
residue was chromatographed (silica gel, 1:1 hexanes:acetone)
to afford 25 (1.1 g, 87%) as a white solid: mp 60-68 °C; MS
(M + H)+ 609.
was determined spectrophotometrically at 450 nm. The
results are expressed as MTD (maximum tolerated dose), the
highest tested nontoxic concentration of the inhibitor.
Ack n ow led gm en t. We thank Dr. Gregory Vite for
a critical reading of the manuscript. Microanalyses, IR
spectra, and mass spectra were kindly provided by the
Bristol-Myers Squibb Department of Analytical Re-
search and Development.
Refer en ces
(1) Barbacid, M. ras Genes. In Annual Review of Biochemistry;
Richardson, C., Ed.; Annual Reviews Inc.: Palo Alto, CA, 1987;
Vol. 56, pp 779-827.
(2) Bos, J . L. ras Oncogenes in Human Cancer: A Review. Cancer
Res. 1989, 49, 4682-4689.
(R*)-N2-[[1,2,3,4-Tetr a h yd r o-2-(3-m eth yl-L-va lyl)-3-iso-
qu in olin yl]ca r bon yl]-L-glu ta m in e, 1,1-Dim eth yleth yl Es-
ter , Hyd r och lor id e (26). Palladium hydroxide on carbon
(10%, 91 mg) was added to a solution of 25 (0.91 g, 1.5 mmol)
in THF (9.1 mL) with 1 N HCl (1.5 mL). A balloon containing
hydrogen was attached to the flask, and the mixture was
stirred for 3 h. The mixture was filtered through Celite and
the filtrate concentrated under vacuum to afford 26 (0.77 g,
100%) which was used without further purification: MS (M
+ H)+ 475 (free base).
(3) Cox, A. D.; Der, C. J . Protein Prenylation: More Than J ust Glue?
Curr. Opin. in Cell Biol. 1992, 4, 1008-1016.
(4) 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.
(5) 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.
(6) 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.
(7) 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.
(8) 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.
(9) J ames, G. L.; Goldstein, J . L.; Brown, M. S.; Rawson, T. E.;
Somers, T. C.; McDowell, R. S.; Crowley, C. W.; Lucas, B. K.;
Levinson, A. D.; Marsters, J . Benzodiazepine Peptidomimetics:
Potent Inhibitors of Ras Farnesylation in Animal Cells. Science
1993, 260, 1937-1942.
(10) Marsters, J .; 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) Nigam, M.; Seong, C. M.; Qian, Y.; Hamilton, A. D.; Sebti, S. M.
Potent Inhibition of Human Tumor p21ras Farnesyltransferase
by A1A2-lacking p21ras CA1A2X Peptidomimetics. J . Biol. Chem.
1993, 268, 20695-20698.
(12) 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.
(13) Wai, J . S.; Bamberger, D. L.; Fisher, T. E.; Graham, S. L.; Smith,
R. L.; Gibbs, J . B.; Mosser, S. D.; Oliff, A. I.; Pompliano, D. L.;
Rands, E.; Kohl, N. E. Synthesis and Biological Activity of Ras
Farnesyl Protein Transferase Inhibitors. Tetrapeptide Analogs
with Amino Methyl and Carbon Linkages. Bioorg. Med. Chem.
1994, 2, 939-947.
(14) 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.
(15) 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.
(16) Gibbs, J . B.; Oliff, A.; Kohl, N. E. Farnesyltransferase Inhibi-
tors: Ras Research Yields a Potential Cancer Therapeutic. Cell
1994, 77, 175-178.
(17) Bhide, R. S.; Patel, D. V.; Patel, M. M.; Robinson, S. P.; Hunihan,
L. W.; Gordon, E. M. Rational Design of Carboxylic Acid Based
Bisubstrate Inhibitors of Ras Farnesyl Protein Transferase.
Bioorg. Med. Chem. Lett. 1994, 4, 2107-2112.
(18) Patel, D. V.; Patel, M. M.; Robinson, S. S.; Gordon, E. M. Phenol
based Tripeptide Inhibitors of Ras Farnesyl Protein Transferase.
Bioorg. Med. Chem. Lett. 1994, 4, 1883-1888.
(19) Reiss, Y.; Brown, M. S.; Goldstein, J . L. Divalent Cation and
Prenyl Pyrophosphate Specificities of the Protein Farnesyltrans-
ferase from Rat Brain, A Zinc Metalloenzyme. J . Biol. Chem.
1992, 267, 6403-6408.
N2-[[(S)-2-[N-[2-Am in o-N-[(1,1-d im eth yleth oxy)ca r bo-
n yl]-2-[N-[(1,1-d im eth yleth oxy)ca r bon yl]im id a zol-4-yl]-
eth yl]-3-m eth yl-L-valyl]-1,2,3,4-tetr ah ydr o-3-isoqu in olin yl]-
ca r bon yl]-L-glu ta m in e, 1,1-Dim eth yleth yl Ester (27). To
a solution of 22 (0.18 g, 0.47 mmol) in THF (20 mL) at 0 °C
under argon was added 1 M lithium aluminum hydride in THF
(0.47 mL, 0.47 mmol) dropwise. The solution was stirred for
30 min followed by dropwise addition of 1 M KHSO4 to pH 4.
The mixture was stirred for 1 h at 0 °C, ether (40 mL) and
water (40 mL) were added, and the layers were separated. The
aqueous layer was washed with ether, and the organic layers
were pooled, dried, and concentrated. The highly unstable
residue was immediately dissolved in dry methanol (20 mL),
along with 3 Å molecular sieves (0.5 g), acetic acid (0.2 mL),
and 26 (300 mg, 0.58 mmol). The solution was stirred for 10
min followed by the portionwise addition of NaBH3CN (29 mg,
0.47 mmol) over 1 h. The solution was stirred for 4 h and
concentrated, and the residue was chromatographed (silica gel,
19:1 chloroform:methanol). Fractions containing the product
were pooled and concentrated to afford 90 mg (25%) of 27 as
a slightly yellow oil. Due to instability of the Boc groups, this
compound was minimally characterized and carried on to the
next step: MS (M + H)+ 784+.
N 2-[[(S)-2-[N -[2-Am in o-2-(1H -im id a zol-4-yl)e t h 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-glu ta m in e, Tr iflu or oa cta te (1:3) Sa lt (10). A solu-
tion of 27 (40 mg, 0.05 mmol) in TFA (5 mL) and methylene
chloride (5 mL) was stirred for 2.5 h and concentrated. The
residue was dissolved in 3 mL of a 50/50 mixture of 0.1% TFA
in methanol and 0.1% TFA in water and subjected to HPLC
purification (YMC C18 column (S-10, ODS 30 × 500 mm);
solvent A, 0.1% TFA in 90% water, 10% methanol; solvent B,
0.1%TFA in 90% methanol, 10% water; 10-35% B in A over
60 min). Fractions containing the major peak were pooled and
lyophilized to yield 20 mg (45%) of 10 as a fluffy white solid:
MS (M + H)+ 528+; HRMS (M + H)+ 528.2939; MS/MS 110+,
178+, 195+, 256+, 273+, 306+, 365+, 511+, 528+; 1H-NMR
(CDCl3) δ 8.40 (1H, m), 7.46 (1H, m), 7.22 (4H, m), 5.10 (1H,
m), 4.8-4.3 (5H, m), 4.19 (1H, m), 3.3-3.0 (2H, m), 2.28 (1H,
m), 1.92 (2H, m), 1.80 (1H, m), 1.10 (9H, 2s).
Biologica l Testin g. Assays for FT and GGTI inhibition,25
Ras processing inhibition,26 inhibition of soft agar growth,26
and phenotypic reversion26 were performed as previously
described. The gross in vitro cytotoxicity of FT inhibitors was
assessed using untransformed NIH 3T3 (“normal”) cells. NIH
3T3 cells were plated at 2500 cells/well in a 96-well microtiter
plate, and 24 h later serial dilutions of drugs were added. After
incubation at 37 °C for 48 h, the tetrazolium dye XTT (2,3-
bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-
2H-tetrazolium hydroxide) containing phenazine methosulfate
was added. The reduction of XTT as a measure of live cells

358 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Hunt et al.
(20) Christianson, D. W. Structural Biology of Zinc. In Metallopro-
teins: Structural Aspects; Edsall, J . T., Anfinsen, C. B., Richards,
F. M., Eisenberg, D. S., Eds.; Academic Press: San Diego, CA,
1991; Vol. 42, pp 281-355.
(21) Cecchi, R.; Ciabatti, R.; Favara, D.; Barone, D.; Baldoli, E.
Imidazolyl Derivatives of Enalapril as Potential Angiotensin
Converting Enzyme Inhibitors. Farmaco, Ed. Sci. 1985, 40, 541-
554.
(22) Leftheris, K.; Kline, T.; Vite, G.; Cho, Y. H.; Bhide, R. S.; Patel,
M. M.; Schmidt, R.; Carboni, J . M.; Yan, N.; Ricca, C.; Gullo-
Brown, J . L.; Andahazy, M. L.; Barbacid, M.; Seizinger, B. R.;
Meyers, C. A.; Hunt, J . T.; Manne, V. Highly Potent Nonprodrug
Inhibitors of Ras Farnesyl Transferase Possessing Whole Cell
Activity. J . Med. Chem., in press.
(24) J anusz, J . M.; Young, P. A.; Blum, R. B.; Riley, C. M. High-
Potency Dipeptide Sweeteners. 2. L-Aspartylfuryl-, Thienyl-, and
Imidazolylglycine Esters. J . Med. Chem. 1990, 33, 1676-1682.
(25) 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.
(26) 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.
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.
(23) Schneider, F. Synthesis of Alpha-(4-Imidazolyl)glycine and its
Derivatives. Z. Physiol. Chem. 1961, 324, 206-210.
J M9507284