Hydroxamic acid-based bisubstrate analog inhibitors of Ras farnesyl protein transferase

Article abstract of DOI:10.1021/jm960190h

The rational design, synthesis, and activity of novel, hydroxamic acid- based, collective bisubstrate analog inhibitors of farnesyl protein transferase (FPT) is described. This class of compounds differ structurally from the conventional FPT inhibitors by

Full text of DOI:10.1021/jm960190h

J . Med. Chem. 1996, 39, 4197-4210
4197
Hyd r oxa m ic Acid -Ba sed Bisu bstr a te An a log In h ibitor s of Ra s F a r n esyl P r otein
Tr a n sfer a se
Dinesh V. Patel,*,† Marian G. Young, Simon P. Robinson,‡ Lisa Hunihan, Brenda J . Dean, and Eric M. Gordon†
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New J ersey 08543-4000
Received March 8, 1996X
The rational design, synthesis, and activity of novel, hydroxamic acid-based, collective
bisubstrate analog inhibitors of farnesyl protein transferase (FPT) is described. This class of
compounds differ structurally from the conventional FPT inhibitors by being non-sulfhydryl
and by being bisubstrate based rather than peptide or FPP derived inhibitors. Whereas
replacement of the sulfhydryl group of tetrapeptide CVLS (I50 ) 1 µM) by an N-methylhydrox-
amic acid had a deleterious effect (10, I50 > 360 µM), moderate inhibition was realized with 16
(I50 ) 42.5 µM), a bisubstrate analog involving anchorage of farnesyl and tripeptide groups by
a hydroxamic acid-embedded linker. Starting from 16, a 1 order of magnitude improvement
in in vitro potency was obtained by optimization of the linker (20, I50 ) 4.35 µM). An additional
13-fold enhancement was achieved by substituting the tripeptide moiety VLS in 20 by VVM
(23, I50 ) 0.33 µM). The dependence of these inhibitors on their peptide and farnesyl subunits
is suggestive of their bisubstrate nature. Compound 23 (I50 ) 0.33 µM) is 2 orders of magnitude
better in activity compared to the initial lead 16 (I50 ) 42.5 µM) and is effective in blocking
prenylation of protein in whole cells including p21ras
.
In tr od u ction
proteins, further modification occurs in the form of
palmitoylation of one of the upstream cysteine resi-
dues.11
Uncontrolled cellular proliferation and differentiation
is the primary mechanism for cancer. This event has
now been recognized to be triggered by two main
pathways, namely, activation of cellular protooncogenes
and/or inactivation of tumor-suppressor genes.1 The
most common and potent members in these two catego-
ries are ras oncogenes2 and p53 tumor supressor genes,3
respectively, as evident by their frequent detection in a
wide variety of human tumors. Intervention of the
biological pathway leading to the oncogenic activity of
the corresponding oncoproteins forms the basis for
rational design of novel and specific “anti-cancer” agents.
To date, blockade or attenuation of the ras pathway has
received most attention from the medicinal community
in their search for antitumor drugs.4 A prerequisite to
the transforming activity of the cytosolic ras proteins
is their localization to the plasma membrane, an event
that is carried out by a well-defined sequence of post-
translational modifications.5 The first and obligatory
step in this cascade is farnesylation of a cysteine residue
of the CAAX motif present at the C-terminal of ras.6
This consensus sequence wherein C is cysteine, A is an
aliphatic amino acid, and X is preferably serine or
methionine is present in various proteins subject to post-
translational prenylation.7 Following prenylation, the
protein is acted upon by a protease which cleaves the
carboxyl terminal tripeptide fragment AAX and forms
the protein bearing an S-farnesylated cysteine at the
C-terminus.8 Carboxymethylation of the free carboxyl
group of cysteine then completes the conversion of pro-
One approach for blocking the oncogenic ras activity
would be to use specific inhibitors to interrupt the
activity of any one or more of the enzymes catalyzing
these post-translational modifications.4 However, far-
nesylation is the only true mandatory step in this
cascade, since proteolysis and carboxymethylation have
been shown to be nonobligatory for ras cell transforming
activity.12 Not surprisingly, most attention has been
focused toward the design and synthesis of inhibitors
of farnesyl protein transferase (FPT), the enzyme
catalyzing farnesylation of p21ras 13
Either of the two
.
reacting substrates, namely, the ras protein or the
prenyl group donor farnesyl pyrophosphate (FPP), can
form the basis for design of such inhibitors.14 Thus,
examples of both peptide-15 and FPP-based16 inhibitors
of FPT have been reported. The CAAX tetrapeptide
sequence renders enough specifity for recognition by the
enzyme, and various peptidomimetic variants of this
motif have recently emerged as very novel and potent
inhibitors of FPT.17 With the exception of the recently
reported imidazole zinc chelator-based compounds, a
mandatory feature of almost all peptide-based inhibitors
with good potency has been the presence of a cysteine-
like free mercaptan group.15,17 FPP analogs bear the
advantage of being small sized and non-peptidic, but
face the challenge of reducing the overall charge of
pyrophosphate surrogates in order to confer adequate
cell permeability to such molecules.16 Recently, we
reported on a third class of FPT inhibitors which differ
from the conventional inhibitors in two important
aspects.18 First, these are collective bisubstrate analog
inhibitors, and second, the sulfhydryl group has been
replaced by a phosphinyl or carboxylic acid pharma-
cophore. Besides imparting structural novelty, these
features may display a different degree of selectivity and
specificity profile from the conventional CAAX- or FPP-
based inhibitors in the intervention of these ubiquitous
p21ras to the hydrophobic c-p21ras 9
. The protein can now
become anchored to the membrane and function as a
transforming, mature protein.10 In some but not all ras
† Current address: Versicor Inc., 270 East Grand Ave, South San
Francisco, CA 94080.
‡ Current address: BASF Bioresearch Corp., 100 Research Dr,
Worcester, MA 01605.
X Abstract published in Advance ACS Abstracts, September 15, 1996.
S0022-2623(96)00190-2 CCC: $12.00 © 1996 American Chemical Society

4198 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Patel et al.
F igu r e 1.
post-translational pathways.19 Non-sulfhydryl inhibi-
tors would also bypass toxicological and metabolic issues
that may be specifically associated with a mercaptan
moiety.20 In this study, we discuss our results on a new
class of hydroxamic acid pharmacophore-based, non-
sulfhydryl bisubstrate analog inhibitors of FPT.
tide H-Val-Leu-Ser-OMe 5 yielded tetrapeptide 6 (68%),
which contained the entire framework of the final target
molecules. Base hydrolysis with NaOH gave 72% of the
desired compound 7, but it was also accompanied by
20% of the free aspartic acid derivative resulting from
cleavage of hydroxamic functionality from 7. Addition-
ally, 7 was also accompanied by a substantial amount
of an epimeric contaminant (ca. 20%, most probably at
the serine center) as revealed by HPLC. This prompted
us to try out milder, epimerization-free conditions
recently highlighted in the literature.25 Indeed, when
hydrolysis was performed with Na2CO3, production of
aspartic acid byproduct was not observed, and the
chirality of desired 7 was essentially intact (95%).
Hydrogenolysis of 7 gave 8 (97%), the Boc-protected
tetrapeptide with an N-methyl substituent on the hy-
droxamic acid side chain. Alternatively, the sodium salt
of 7 was converted to the free acid and then treated with
anhydrous HCl to to yield 9 (75%), which was hydro-
genolyzed to the hydroxamic acid 10 (95%).
Preparation of the hydroxamic bisubstrate analogs is
outlined in Scheme 2 and commences with alkylation
of farnesyl bromide with NH2-OTHP 11 to obtain the
THP-protected alkoxyamine intermediate 12 in moder-
ate yields (49%).26 Acylation of 12 with 3-carbomethoxy-
propionyl chloride was uneventful (85%). Hydrolysis of
the methyl ester 13 and coupling of resulting acid 14
with 5 using EDC/HOBt27 gave the fully assembled
bisubstrate intermediate in 58% overall yield for two
steps. Removal of the THP protecting group in the
presence of a farnesyl chain was a questionable step in
this synthetic sequence. Fortunately it was removed
with p-TsOH to give 15 in moderate yields (40%), which
was subjected to basic hydrolysis with 1 N NaOH to
provide 16 in 57% yield. Similarly, acylation of 12 with
ethylmalonyl chloride gave 17 in 82% yield which was
efficiently hydrolyzed to the acid 18 (78%). However,
EDC/HOBt coupling of 18 with tripeptide 5 proceeded
in modest yields (20%) and was accompanied by sub-
stantial amounts of unreacted 18 (30%). The carboxylic
In h ibitor Design . The functional equivalence of
hydroxamate and mercaptan moieties in terms of metal
chelation is well precedented, especially in the area of
metalloproteases.21 Thus, the hydroxamic group is
expected to benefit from ionic and/or metal chelation
interactions prevailing at the active site of FPT during
the farnesylation reaction. A key feature in bisubstrate
design is the assembly and interconnection of critical
binding components of both the reacting partners (FPP
and ras) in a chemically and biologically stable form.
An adequate degree of information about the enzyme
FPT and its catalytic machinery is reported in the
literature,16a,22 and this can serve as a guideline in this
design process. Thus, the farnesyl group of FPP and
the tripeptide groups (AAX) of the C-terminal CAAX
motif are anchored together via a hydroxamic acid-
bearing linker (Figure 1). Since an N-terminal amino
group of simple CAAX type tetrapeptides is not a critical
requirement for activity, it was deleted in the bisustrate
inhibitor design.20 By introducing the hydroxamate
group as a linear component of the linker instead of a
branched functional group, the issue of additional
chirality in that region of the molecule is also avoided.
These features led to substantial simplicity in the
syntheses of these molecules.
Ch em istr y
A successful preparation of the simple N-methyl-
substituted hydroxamic acids 7-10 is outlined in Scheme
1.23
Thus, N-methylbenzyloxyamine 2 was prepared ac-
cording to literature procedure24 and coupled with
aspartic acid derivative 1 to afford intermediate 3 (95%).
Hydrolysis of 3 (90%) followed by coupling with tripep-

Hydroxamic Acid-Based Inhibitors of Ras FPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4199
Sch em e 1a
a
Reagents: (a) CDl, iPr2NEt, 95%; (b) 1 N NaOH, MeOH, 90%; (c) EDC, HOBT, iPr2NEt, H-VLS-OCH3 5, 68%; (d) Na2CO3 (1.0), 2:1
MeOH/H2O, 95%; (e) H2, 10% Pd/C, MeOH, 97%; (f) anhydrous HCl/dioxane, EtOAc, 75%; (g) H2, 10% Pd/C, MeOH, 95%.
Sch em e 2a
a
Reagents: (a) NH2OTHP, THF, 49%; (b) iPr2NEt, THF, ClCO(CH2Me, 85%; (c) 1 N NaOH, MeOH, 100% for 14, 78% for 18; (d) EDC,
HOBT, iPr2NEt, H-VLS-OCH3 5, 58%; (e) pTsOH, MeOH, 40% for 15, 54% for 19, 50% for 22; (f) 1 N NaOH, MeOH, 57% for 16, 66% for
20, 57% for 23, 43% for 26 from 18; (g) iPr2NEt, THF, ClCOCH2CO2Et, 82%; (h) EDC, HOBT, iPr2NEt, H-VLS-OCH3 5, 20%; (i) EDC,
HOBT, iPr2NEt, H-VVM-OCH3 21, 22% for 22, 57% for 25.
acid group of a malonic monester monoacid like 18 is
deactivated because of the electron-withdrawing nature
of the adjacent ester moiety and can be expected to be
less reactive compared to normal acids. Treatment of
the coupled product with p-TsOH gave 19 (54%), which
was hydrolyzed to the hydroxamic bisubstrate 20 (66%).
Having realized inferior coupling yields in the malonyl
series with EDC/HOBT, we utilized the BOP27 reagent
during preparation of the VVM analog 23, but observed
only a marginal improvement upon BOP-mediated
coupling of acid 18 with tripeptide H-Val-Val-Met-OMe
21 (32%). The rest of the sequence leading to prepara-
tion of VVM analog 23 was uneventful. BOP-coupling
did, however, result in substantial improvement during
reaction of 18 with D-methionine bearing tripeptide
H-Val-Val-(D-Met)-OMe 24 (57%). Treatment of the
coupled product with p-TsOH gave 25 which was
isolated but not purified, since in previous instances,

4200 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Patel et al.
Sch em e 3a
a
Reagents: (a) (i) TsCl, pyridine, DMAP, CH2Cl2, 70%; (ii) LiBr, THF, 72%; (b) H2NOTHP, K2CO3, DMF, 31%; (c) KOH, BzOH, 95%;
(d) BOP, iPr2NEt, HCl‚H-VVM-OCH3 21, CH3CN/DMF (1:1), 79%; (e) Pd(OH)2, H2, iPr2NEt, DMF/H2O (4:1), 88%; (f) BOP, iPr2NEt,
CH3CN/DMF (9:4), 64%; (g) p-TsOH, THF, 32%; (h) 1 N NaOH, dioxane/CH3OH/H2O (6:2:1), 79%.
isolation of similar intermediates (e.g. 15, 19, and 22)
had led to moderate overall yields due to low recovery
from silica gel columns. Hydrolysis of the ester 25 with
1 N sodium hydroxide in dioxane and purification of the
final product on CHP-20P gave 26 in 43% yield for two
steps.
very similar to that of an SH group (pKa ) 10). This
led to our attempts at discovering hydroxamic acid-
based bisubstrate analogs specifically designed as in-
hibitors of FPT.
Str u ctu r e-Activity Rela tion sh ip (SAR) of F P T
In h ibitor s. Our efforts in the project commenced with
the preparation of N-methylhydroxamic acid analogs 8
and 10. These baseline compounds were found to be
devoid of any significant activity (I50 >360 µM) (Table
1), suggesting the inability of a simple hydroxamic
moiety in itself to derive appreciable binding and/or the
need for additional recognition sites in these molecules.
A benzyl group may be able to benefit from hydrophobic
interactions in the lipid binding domain of the enzyme
that may normally be occupied by the prenyl moiety of
FPP during the farnesylation event. A similar rational
was recently applied and found to be partly successful
in the area of phenol-based FPT inhibitors.18d This line
of reasoning prompted the testing of penultimate O-
benzylhydroxamic ether precursors 7 and 9 for inhibi-
tory potency, but these were also found to be inactive
(I50 >360 µM).
Synthesis of the homofarnesyl analog of 23 is shown
in Scheme 3. Here, we opted for an alternate route that
is more convergent with respect to farnesyl group
replacements. This also circumvented the problem of
low yields observed previously in the coupling of malonyl
residues to the tripeptides. Thus, homofarnesol 27 was
prepared according to literature procedure28 and con-
verted to its bromide by tosylation of 27 (70%) followed
by treatment with LiBr (72%). Upon reaction of homo-
farnesyl bromide 28 with H2NOTHP, the desired
alkoxyamine 29 (31%) was accompanied by formation
of the N-dialkylated side product (42%). In a separate
step, dibenzyl malonate was selectively saponified to its
monoester (95%)29 and coupled with 21 to provide the
tripeptide 31 (77%). It is noteworthy that hydrogenoly-
sis of the benzyl protecting group in methionine-bearing
intermediate 31 with palladium hydroxide proceeded
smoothly to give the acid 32 (88%). Coupling of inter-
mediates 29 and 32 with BOP (64%), followed by the
usual deprotection with p-TsOH (32%) and hydrolysis
(79%), gave the desired analog 34.
Ta ble 1. Summary of in Vitro Activity of Inhibitors in the Ras
Farnesyl Protein Transferase Assaya
b
b
compd no.
I50 (µM)
compd no.
I50 (µM)
7
8
9
>360
>360
>360
>360
16
20
23
26
34
42.5 ( 2.5
4.3 ( 2.7
0.33 ( 0.17
30.5 ( 10.5
5.6 ( 2.3
Biology a n d Discu ssion
10
It is becoming increasingly clear that intervention of
farnesylation of ras proteins may be an effective way of
attenuating the cell proliferation process and that FPT
inhibitors are likely to emerge as novel, rationally
designed, antitumor agents.7-18 In regular peptide-
based inhibitors, the sulfhydryl group is critically
required for biological activity.15,17 We have initiated
a program based on novel replacements for the thiol
group with functionalities that may benefit from binding
interactions normally enjoyed by a sulfhydryl moiety.18
Ideally, such a group will not act as a substrate but
rather compete with the substrate and function as a
competitive inhibitor for the enzyme. An appropriately
substituted hydroxamic acid group seemed to meet the
above mentioned requirements. It is a good metal ion
chelator, and the acidity of its NH and/or OH proton is
a
See the general Experimental Section for a description of the
methods for determining the IC50 values of these inhibitors.
b
Values are the mean ( SE for two individual estimates of the
.
I50
At this stage, attention was diverted to the prepara-
tion of bisusbtrate analog inhibitors involving the
embodiment of a full farnesyl group on the hydroxamic
ether functionality of these molecules. This reasoning
was based to some extent on our initial success with
carboxylic and phosphonic acid-based bisubstrate in-
hibitors of FPT.18a-c The first bisubstrate analog 16 was
a moderately active FPT inhibitor (I50 ) 42 µM), thereby
validating the hypothesis of collective bisubstrate hy-
droxamic acid based inhibition of FPT. Next, the
possibility of enhancing the in vitro potency of this class

Hydroxamic Acid-Based Inhibitors of Ras FPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4201
P r otein P r en yla tion Stu d ies. The effect of some
of these inhibitors on prenylation of cellular proteins
in general, and specifically on p21ras, was examined in
H-ras-transformed NIH-3T3 cells. Treatment of cells
with [3H]mevalonolactone results in the radiolabeling
of prenylated cellular proteins. Autoradiography of SDS
gels following electrophoresis of proteins from radiola-
beled cells consistantly indicated prenylated protiens of
70, 50, 46, 35, 26-21, and 18 kDa with the majority of
labeling at the 21-26 kDa region. Treatment with the
hydroxamic bisubstate 23 (IC50 ) 0.33 µM on the
isolated enzyme) partly prevented radiolabeling of some
proteins at 10 µg/mL (14 µM) and more completely
inhibited the labeling of the same proteins at 100 µg/
mL (140 µM; see Figure 3A).
In contrast the less active analog 16 (IC50 ) 42 µM
on the isolated enzyme) failed to inhibit whole cell
prenylation even up to 100 µg/mL (see Figure 3B).
Interestingly inhibition of the radiolabeling of cellular
protiens by 23 was not apparent for the proteins in the
21-26 kDa range. These findings are consistent with
the effects recently reported for a series of benzodiaz-
epine peptidomimetic farnesyl transferase inhibitors
that inhibited farnesylation of a number of higher
molecular weight protiens in the whole cell but not the
large number of small G protiens that are geranylger-
ylated.13 To look more precisely at protien farnesylation,
p21 ras was immunoprecipitated from the radiolabeled
cell lysates and the level of prenylation of ras examined
specifically. This indicated a reduced level of p21 ras
prenylation at 100 µM (see Figure 4).
F igu r e 2.
of compounds was investigated. For this purpose,
optimization of three structural componentssthe linker,
the peptide, and the farnesyl groupsof these compounds
was undertaken. To explore the optimal length of the
linker connecting the farnesyl and peptide cosubstrates,
the malonyl analog 20 was prepared. A 10-fold im-
provement in activity over 16 was obtained by shorten-
ing the linker by one methylene unit (20, I50 ) 4.3 µM).
Since 16 and 20 bear the VLS tripeptide sequence, there
was good opportunity for activity enhancement by
replacement with a VVM sequence. Such a substitution
has led to 1 order of magnitude improvement in activity
in the case of simple CAAX-based inhibitors.15,17 It was
gratifying to realize that compound 23, the VVM analog
of 20, did register a 13-fold improvement in activity (I50
) 0.33 µM). The dependency of these inhibitors on the
nature of their peptide moiety was further evidenced
by the 100-fold inferior activity of 26 (I50 ) 30 µM), the
D-methionine analog of 23. Having identified the
adequate linker and tripeptide for these inhibitors as
exemplified by 23, we next worked at altering the
farnesyl group in these molecules. We reasoned that
the transition state of the enzymatic reaction involving
attack of the cysteine thiol group on the allylic, pyro-
phosphate (PP)-bearing carbon atom of FPP may be
mimicked more closely if the hydrophobic side chain of
an inhibitor like 23 had an additional methylene unit
(Figure 2).
Wh ole-Cell Stu d ies. The best hydroxamate bisub-
strate analog 23 was evaluated in our previously
described ras transformation inhibition (RTI) assay.16c
Essentially, NIH-3T3 cells transfected with oncogenic
H-ras DNA were treated with compound 23 24 h after
transfection, and the effect on transformation was
evaluated after 14 days. A 20% inhibition was observed
at 100 µM, and no inhibition could be observed at 10
µM concentration of 23.
This led to the preparation of the homofarnesyl analog
34. Unfortunately, 34 was found to be more than 1
order of magnitude less potent (I50 ) 5.5 µM) than the
parent molecule 23. This suggests that the extra carbon
atom is most likely forcing the farnesyl group of 34 in
an orientation unfavorable for adequate binding at the
active site.
Con clu sion s
The rational design, synthesis, and activity of novel,
hydroxamic acid-based bisubstrate analog inhibitors of
FPT is described. This adds to the list of recently
described non-sulfhydryl inhibitors for the enzyme,
namely, the carboxylic acids,18a phosphinyl acids,18b,c
and phenols.18d Whereas replacement of the sulfhydryl
group of tetrapeptide CVLS (I50 ) 1 µM) by an N-
methylhydroxamic acid was unsuccessful (10, I50 >360
A farnesyl group is a critical component for the
potency of these inhibitors, as evidenced by inactivity
of non-farnesylated analogs 8 and 10 (I50 >360 µM).
Additionally, the SAR with respect to alterations in the
tripeptide portion of these farnesylated compounds
parallels the trend observed for simple CAAX peptide-
based inhibitors. Such dependency and sensitivity on
the farnesyl and peptide moieties of the molecule is an
indirect illustration of the bisubstrate nature of this
class of FPT inhibitors.
µM), moderate inhibition was realized with 16 (I50
)
42.5 µM), a bisubstrate analog involving anchorage of
farnesyl and tripeptide groups by a hydroxamic acid
embedded linker. Starting from 16, a 1 order of
magnitude improvement in in vitro potency was ob-
tained by optimization of the linker (20, I50 ) 4.35 µM).
An additional 13-fold enhancement was realized by
substituting the tripeptide moiety VLS in 20 by VVM
(23, I50 ) 0.33 µM). The dependence of these inhibitors
on their peptide and farnesyl subunits is suggestive of
their bisubstrate nature. Compound 23 is 2 orders of
magnitude better in activity compared to the initial lead
16 and is effective in blocking prenylation of protein in
Bisubstrate analog 23, the best FPT inhibitor for this
series of compounds [I50(FPT) ) 0.33 µM), was evaluated
against the related enzyme geranylgeranyl transferase
and found to be 20-fold less active [I50(GGT-1) ) 6.2 µM).
Additional modifications are probably required to achieve
better levels of selectivity that have been typically
observed with other classes of bisubstrate FPT
inhibitors.18b
whole cells including p21ras
.

4202 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Patel et al.
F igu r e 4. Autoradiograph of a 10/20% SDS-polyacrylamide
gel of immunoprecipites of p21 ras from the lysates of cell
shown in Figure 3A. The bisubstrate 23 has partially inhibited
prenylation of p21 ras at 100 µg/mL. See the general Experi-
mental Section for details of methods.
for flash columns. Reverse phase chromatographic purification
of final compounds was carried out using CHP20P gel, a 75-
150 µm polystyrene-divinyl benzene copolymer purchased
from Mitsubishi Chemical Industries. Analytical HPLC was
performed using two Shimadzu LC-6A pumps with an SCL-
6B system controller and a C-R4AX chromatopac, and an SPD-
6AV UV-vis spectrophotometric detector. HPLC columns
were commercially available from either Whatman or YMC
Corp.
Melting points were determined on an electrothermal
Thomas Hoover capillary melting point apparatus and are
uncorrected. NMR spectra were recorded on one of the
following instruments: J OEL GX-400 operating at 400 (1H)
or 100 MHz (13C), J OEL FX-270 operating at 270 (1H) or 67.8
(13C) MHz, and J OEL FX-60Q operating at 15 MHz (13C).
Chemical shifts are reported in parts per million (ppm)
downfield from tetramethylsilane (TMS), and coupling con-
stants (J ) are in hertz (Hz). IR spectra were recorded on a
Mattson Sirius 100 FT-IR spectrophotometer, and the absorp-
tion maxima are reported in cm-1. Mass spectra were recorded
on a Finnigan MAT TSQ-4600 mass spectrometer (chemical
ionization, CI) or a VG-ZAB-2F mass spectrometer (fast atom
bombardment, FAB). High-resolution mass spectra (HRMS)
were determined using peak-matching techniques versus PEG
standards on a VG-ZAB-2F spectrometer. Optical rotations
were measured using a Perkin-Elmer model 241 polarimeter
and a 10 cm path length optical cell. Microanalysis results
were adjusted to obtain the best fit assuming nonstoichiometric
hydration.
F igu r e 3. Autoradiographs of 10/20% SDS polyacrylamide
gels of H-ras transfected NIH3T3 cell lysates following radio-
labeling of prenylated proteins by treatment with [3H]meva-
lonolactone in the presence of increasing concentrations of the
potent hydroxamic bisubstrate 23, (A, top) or the less active
diastereomer bisubstrate 16 (B, bottom).. See the general
Experimental Section for details of methods.
Exp er im en ta l Section
En zym e In h ibition Stu d ies. Farnesyl protein transferase
was isolated from pig brain as described by Manne et al.13a
and further purified by a 30-55% ammonium sulfate precipi-
tation and subsequent FPLC using a DE52 column with a
linear 0-400 mM NaCl gradient, Hydoxylapatite column with
a 10-110 mM potassium phosphate gradient, and Mono Q
column with a 0-1000 mM NaCl gradient. Fractions contain-
ing farnesyl protein transferase were identified on the basis
of enzyme activity using the assay described below. Active
fractions were combined and dialyzed overnight into 20 mM
Tris-HCl, pH 7.4 (for DE52 and Mono Q), or 10m M potassium
phosphate, pH 7.6 containing 100 mM NaCl (for hydroxlapa-
tite). All dialysis buffers contained 1 mM DTT, 5% glycerol,
0.1-0.25 mM EDTA, 0.1-0.25 mM EGTA, 1 mM benzamidine,
and 10 µg/mL soybean trypsin inhibitor and for the DE52 and
Mono Q dialysis buffers, 1 µg/mL leupeptin and 0.25-0.5 mM
Gen er a l. All reactions were carried out under a positive
pressure of dry argon, unless otherwise specified. Tetrahy-
drofuran (THF) and diethyl ether were distilled from sodium
or potassium benzophenone ketyl prior to use. Acetonitrile,
benzene, dichloromethane, diisopropylamine, hexane, metha-
nol, pyridine, and toluene were distilled from calcium hydride
prior to use.
TLC was performed using EM Science (E. Merck) 5- × 10-
cm plates precoated with silica gel 60 F254 (0.25 mm thickness),
and the spots were visualized by any of the following: UV,
iodine, phosphomolymdic acid (PMA), ceric ammonium sulfate,
anisaldehyde, vanillin, or Rydons stain. EM Science’s silica
gel 60 (230-400 mesh ASTM) was used for flash chromatog-
raphy. A ratio of 25-100:1 silica gel/crude product by weight
and a nitrogen pressure of 5-25 psi was normally employed

Hydroxamic Acid-Based Inhibitors of Ras FPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4203
PMSF. Enzyme activity was purified approximately 2000-fold
relative to the initial crude pig brain cytosol. The PXCR
expression vector containing H-ras and bacterial strain PR13Q
were kindly provided by Dr. Larry Feig (Farnsworth, C. L.;
Marshall, M. S.; Gibbs, J . B.; Stacey, D. W.; Feig, L. A.
Preferential inhibition of the oncogenic form of Ras by muta-
tions in the GAP binding/“effector” domain. Cell 1991, 64, 625-
633). Recombinant p21 H-ras was express in the Escherichia
coli strain PR13Q and processed as described by Farnsworth
et al.28 Following processing and ammonium sulfate precipita-
tion, the pellet was resuspended in 50 mM Tris-HCl, pH 7.5,
20 mM NaCl, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine,
10 µg/mL soybean trypsin inhibitor, 10 µM E64, and 1 µM
pepstatin and dialyzed overnight. The recombinant p21 H-ras
was then partially purified by FPLC using a DE52 column and
was examined by incubating 2 × 106 cells as with [3H]-
mevalonolactone as described above. After overnight incuba-
tion cells were washed with 1 mL of cold PBS and then
incubated for 10 min with 0.7 mL of cold PBSTDS (PBS
containing 1% triton-X-100, 0.5% sodium deoxycholate, 0.1%
SDS, and 0.2% sodium azide). This was transferred to a
microfuge tube together with a 0.8 mL of PBSTDS rinse of
the well. Cells were lysed by sonication and microfuged at
15 000 rpm for 1 h at 4 °C. To 1 mL of cell lysate were added
15 µL of goat anti rat IgG/ protein-A agarose complex (Onco-
gene Sciences) and 10 µL of ras antibody Y13-259 (Oncogene
Sciences), and the mixture was incubated at 4 °C overnight.
The immunoprecipitate was collected by microfuge at 2500
rpm at 4 °C for 15 min. Following removal of the supernatant,
the pellet was washed 4 times with 1 mL of cold PBSTDS.
The pellet was resuspended in 30 µL of loading lysis buffer
and boiled for 10 min. Samples were processed as for whole
cell lysates described above.
a
linear NaCl gradient from 20 to 320 mM. Fractions
containing the p21 H-ras were visualized by coomassie blue-
stained SDS-polyacrylamide gels and assayed for substrate
capacity using the farnesylation assay described below. p21
H-ras with a purity of >60% was obtained with this single-
column purification. Additional processing often led to a more
pure protein with a reduced capacity to be farnesylated
(assumed to be a consequence of the carboxyl terminus
cleavage described in Farnsworth et al (Farnsworth, C. L.;
Marshall, M. S.; Gibbs, J . B.; Stacey, D. W.; Feig, L. A.
Preferential inhibition of the oncogenic form of Ras by muta-
tions in the GAP binding/“effector” domain. Cell 1991, 64, 625-
633)). Farnesyl protein transferase assays were run in 96-
well dishes in a reaction volume of 20 µL. The final reaction
mixture contained 1 µM [3H] FPP (NEN Dupont), 7 µM p21
H-ras, 25 mM MgCl2, 10 mM DTT, 100 mM HEPES, 7.4, and
serial dilutions of inhibitor usually ranging from 360 to 0.02
µM. Reactions were started by adding sufficient enzyme to
produce approximately 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 by adding 90 µL of 4% SDS followed
by 90 µL of 30% TCA. Plates were incubated overnight at 4
°C, and then the precipitates were transferred to Millipore
multiscreen filtration 96-well plates with 0.65 PVDF mem-
branes. Following filtration using the multiscreen vacuum
manifold, the wells were washed once with 200 µL of 4% SDS/
6% TCA and five times with 200 µL of 6% TCA. Following
removal of the bottom seal, excess washing fluid was blotted
and the plates were allowed to dry before the filters were
punched into 4 mL vials using the multiscreen punch. After
incubation at 60-70 °C with 300 µL of Solvable (NEN Dupont),
3 mL of Formula 989 (Dupont) scintillation fluid was added
and radioactivity determined by scintillation counting. Dose-
response curves for inhibitors used triplicate estimates at each
drug concentration and the IC50 estimations were made from
percent control versus log drug concentration plots. Each
compound was tested at least twice.
Wh ole-Cell Assa y. The ras transformation inhibition (RTI)
assay that measures whole-cell activity and cytotoxicity of FPT
inhibitors has been described before in detail by us.16c
P r ep a r a tion of 3. CDI (1.16 g, 7.15 mmol) was added in
one portion to a 0 °C solution of acid 1 (2.1 g, 6.5 mmol) in
THF (20 mL). After 15 min at 0 °C and 30 min at room
temperature, it was recooled to 0 °C, and amine 2 (1.128 g,
6.5 mmol) was added in one portion. iPr2NEt (1.245 mL, 7.15
mmol) was added to this suspension, and the mixture was
stirred with gradual warming to room temperature. After 4h
at room temperature, the reaction mixture was concentrated
by rotary evaporation to remove THF. The residue was taken
up in EtOAc (200 mL) and washed sequentially with 10%
aqueous HCl (2 × 200 mL), saturated aqueous NaHCO3, and
saturated aqueous NaCl (1 × 100 mL), dried, and concentrated
to provide 3 (2.722 g). TLC Rf ) 0.28 (2:1 hexane/EtOAc,
1
visualized by PMA); MS (M + H)+ 443; H NMR (400 MHz,
CDCl3) δ 1.42 (s, 9 H, Boc), 2.88 (d, 1 H, J ) 14), 3.14 (s, 3 H,
NMe), 3.16 (d, 1 H, J ) 14), 4.75 (s, 2 H), 5.16 (app q, 2 H, J
) 12.4), 5.73 (d, 1 H, J ) 8), 7.25-7.37 (m, 10 H): 13C NMR
(100 MHz, CDCl3) δ 28.2 (Me3C), 34.23 (CH2CO), 34.99 (NMe),
49.8 ((CH), 67.0 (PhCH2OC(O)), 76.2 (PhCH2ON(Me)), 79.7
(Me3C), 128.0, 128.1, 128.3, 128.6, 128.8, 129.0, 129.2, 134.0,
135.5, 155.6 (Boc CdO), 171.4, 172.1. Anal. (C24H30N2O6) C,
H, N.
P r ep a r a tion of 4. NaOH (1 N, 10 mL, 10 mmol) was added
in one portion to a 0 °C solution of ester 3 (2.652 g, 6.0 mmol)
in MeOH (24 mL). The reaction mixture was stirred with
gradual warming to room temperature, and TLC revealed total
disappearance of starting material after 2 h. The reaction
mixture was concentrated on the rotary evaporator to remove
MeOH. The residue was suspended in water (25 mL) and
extracted with hexane (50 mL) and ethyl acetate (2 × 50 mL),
and the organic extracts were discarded. EtOAc (50 mL) was
added to the aqueous solution and acidified to pH ) 2.0 with
1 N HCl, and the two layers were separated. The acidic
solution was reextracted with EtOAc (2 × 5 mL). The
combined organic extracts were dried and concentrated in
vacuo to provide 4 as a white solid (1.894 g, 89.6%): mp 135-
137 °C: TLC Rf ) 0.33 (90:20:2.5:1.0 CHCl3/MeOH/H2O/AcO
H); MS (M + H)+ 353; 1H NMR (400 MHz, CDCl3) δ 1.45 (s, 9
H, Boc), 2.75 (dd, 1 H, J ) 7, 17), 3.25 (s, 3 H, NMe), 3.24-
3.33 (m, 1 H), 4.53, (br s, 1 H), 4.89 (s, 2 H), 5.76 (d, 1 H, J )
7), 7.39 (s, 5 H). Anal. (C17H24N2O6) C, H, N.
P r ep a r a tion of 6. iPr2NEt (453 uL, 2.6 mmol) and HOBt
(337 mg, 2.2 mmol) were sequentially added to a 0 °C solution
of acid 4 (704 mg, 2.0 mmol) and HCl‚H-VLS-OMe 5 (735 mg,
2.0 mmol) in THF (6 mL). After 5 min at 0 °C, EDC (403 mg,
2.1 mmol) was added followed by DMF (2 mL). The reaction
mixture was stirred overnight with gradual warming to room
temperature. The next day (total reaction time ) 12 h), the
reaction mixture was concentrated by rotary evaporation to
remove THF, and the residue was taken up in EtOAc (200 mL).
The organic solution was washed sequentially with 10%
aqueous LiCl (2 × 75 mL), phosphate buffer (pH ) 4, 2 × 75
mL), saturated aqueous NaHCO3 (2 × 75 mL), and saturated
aqueous NaCl (1 × 75 mL). Drying and concentration provided
the crude product (1.377 g) which was purified by silica gel
GGT-1 inhibition assays were carried out as described16c
except that recombinant GST-CIIL protein was used as the
geranylgeranyl acceptor substrate.
Cell P r en yla tion Assa ys. NIH-3T3 cells (1.5 × 106)
transformed with H-ras (kindly provided by Dr. R. Weinburg,
Whitehead Ins., Cambridge, MA) were plated in 6-well dishes
in 3 mL of 10% DMEM media and allowed to attach for 3-4
h. The media was removed, and 0.7 mL of DMEM containing
100 µCi/mL of [3H]mevalonolactone (NEN Dupont) and 25 µM
lovastatin was added. Following overnight incubation the
media was removed and discarded and the wells were rinsed
and incubated with trypsin. The cells were harvested by
tapping the plate and rinsing with fresh media. After cen-
trifugation the pellet was washed and transferred to a mi-
crofuge tube and centrifuged again. The pellet was resus-
pended in 35 µL of 1x PBS and 35 µL of loading buffer (10 mL
of Seprasol (Integrated Separation Systems), 100 µL of B-
mercaptoethanol, and 0.2 g of SDS). Samples were boiled for
10 min, and 20 µL samples were loaded on a 10-20% SDS-
PAGE (Integrated Separation Systems). Low- and mid-range
radiolabeled molecular weight markers (Amersham) were also
run with all gels. After electrophoresis the gels were soaked
in Entensify (NEN Dupont), dried under vacuum, and exposed
to film at -76 °C for 5 days. Prenylation of P21 ras in cells

4204 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Patel et al.
chromatography, eluting with 7:3 hexane/acetone to yield pure
6 (896 mg, 67.4%). mp 173-175 °C; TLC Rf ) 0.11 (2:1
hexane/acetone); MS: (M + H)+ 666; 1H NMR (270 MHz,
CDCl3) δ 0.89-1.25 (m, 12 H), 1.44 (s, 9 H), 1.5-1.9 (m, 3 H),
2.5 (m, 1 H), 2.8-3.0 (m, 2 H), 3.17 (s, 3 H), 3.75 (s, 3 H), 3.75-
4.0 (m, 2 H), 4.18-4.70 (m, 4 H), 4.87 (s, 2 H), 5.35 (d, 1 H, J
) 7), 6.8 (m, 1 H), 7.22 (d, 1 H, J ) 7), 7.28 (d, 1 H, J ) 7),
7.39 (s, 5 H); 13C NMR (67.8 MHz, CDCl3) δ 17.4, 19.6, 21.5,
23.7, 25.1, 28.5, 29.2, 33.8, 34.1, 39.3, 51.5, 51.6, 52.7, 55.5,
60.4, 63.2, 76.6, 81.1, 129.1, 129.5, 129.6, 134.3, 156.1, 170.8,
171.3, 172.2, 172.5, 173.6. Anal. (C32H51N5O10) C, H, N.
P r ep a r a tion of 7. Na2CO3 (23 mg, 0.217 mmol) was added
in one portion to a 0 °C solution of ester 6 (134 mg, o.2 mmol)
in MeOH/H2O (2:1, 3 mL). The reaction mixture was stirred
with gradual warming to room temperature, and TLC revealed
total disappearance of starting material after 4 h. The reaction
mixture was concentrated by rotary evaporation to remove
MeOH, and the residue was chromatographed on a CHP20
resin eluting first with H2O and then with 60% aqueous
MeOH. The appropriate fractions were combined and con-
centrated, millipore-filtered, and lyophilized to obtain pure 7
(125 mg, 93%): mp 135-142 °C; TLC Rf ) 0.13 (90:20:2.5:1.0
3 H, J ) 6.0), 1.68-1.85 (m, 2 H), 2.25 (m, 1 H), 3.22 (m, 2 H),
3.30 (s, 3 H), 3.85-4.05 (m, 2 H), 4.25-4.60 (m, 4 H), 8.05 (d,
1 H, J ) 7), 8.37 (d, 1 H, J ) 8). HRMS (M + H)+ calculated
462.2564, found 462.2557. Anal. (C19H35N5O8‚1.2H2O, 1.0
MeOH) C, H, N.
P r ep a r a tion of 11. To a solution of N-hydroxyphthalimide
(20 g, 0.2123 mol, 1 equiv) in THF (350 mL) were added
dihyropyran (17.0 mL, 0.187 mol, 1 equiv) and p-TsOH‚H2O
monohydrate (400 mg) under N2. Additional dihydropyran (5.6
mL, 0.062 mol, 0.3 equiv) was added after 8 and 24 h. The
reaction mixture was filtered and concentrated using a Vigreux
column to 150 mL. Then dihydropyran (11.2 mL, 0.125 mol,
0.6 equiv) and p-toluenesulfonic acid monohydrate (800 mg)
were added. After being stirred for 16 h, the reaction mixture
was concentrated in vacuo. The residue was dissolved in ethyl
acetate (400 mL) and washed with saturated NaHCO3
150 mL), dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was titurated with hexane (3 × 200 mL)
to give O-tetrahydropyranylhydroxyphthalimide (23.9 g,
(3 ×
78%): mp >210 °C; TLC Rf
) 0.81 (3:7 hexane/EtOAc,
visualized by UV, 254 mm); MS (M + H)+ 248; IR (KBr) 1788,
1738 cm-1; 1H NMR (CDCl3) δ 1.70-2.22 (m, 6 H), 3.32 (m, 1
H), 4.52 (m, 1 H), 5.42 (m, 1 H), 7.71-7.84 (m, 4 H). 13C NMR
MHz (CDCl3) δ 18.2, 25.5, 28.3, 62.9, 103.7, 124.0, 129.8, 134.9,
163.0. To a solution of this intermediate (23.64 g, 0.095 mol,
1 equiv) in toluene (60 mL) was added methylhydrazine (5.59
mL, 0.105 mol, 1.1 equiv). After being heated at 80 °C for 1
h, the solution was cooled and the solvent removed in vacuo.
The residue was titurated with ether (20 mL) and filtered. The
solid was dissolved in hexane, treated with charcoal, filtered
through Celite, and concentrated to 50 mL. The crystals were
filtered and dried to give 11 (5.542 g, 50%): mp 33-38 °C;
TLC Rf ) 0.67 (1:4 hexane/EtOAc, visualized by ceric am-
monium sulfate); MS (M + H)+ 118; 1H NMR (400 MHz,
CDCl3) δ 1.53-1.78 (m, 6 H), 3.57 (m, 1 H), 3.91 (m, 1 H),
4.71 (m, 1 H), 5.49 (br s, 2 H); 13C 100 MHz (CDCl3) δ 19.6,
25.3, 28.8, 62.5, 102.5. Anal. (C5H11NO2) C, H, N.
CHCl3/MeOH/H2O/AcOH, visualized by Rydons stain); [R]D
)
-38.3° (c ) 0.58, MeOH); 1H NMR (400 MHz, CD3OD) δ 0.89-
1.01 (m, 12 H), 1.44 (s, 9 H), 1.65-1.73 (m, 3 H), 2.28 (m, 1
H), 2.8-3.1 (m, 2 H), 3.19 (s, 3 H), 3.72-3.85 (m, 2 H), 4.15-
4.55 (m, 4 H), 4.95 (s, 2 H), 7.37-7.45 (m, 5 H). HRMS (M +
H)+ calculated 652.3522, found 652.3518. Anal. (C31H48N5O10
-
Na‚1.8 H2O) C, H, N.
P r ep a r a tion of 8. 10% Pd/C (20 mg) was added in one
portion to a solution of hydroxamic ether 7 (75 mg, 0.111 mmol)
in MeOH (6 mL). The reaction mixture was stirred under an
atmosphere of hydrogen (balloon) for 1 h at room temperature,
at which stage TLC revealed completion of reaction. The
reaction mixture was filtered through Celite, and the catalyst
was washed with MeOH (3 × 10 mL). The combined filtrate
was concentrated by rotary evaporation, and the residue was
dissolved in water (3 mL), millipore-filtered, and lyophilized
to obtain pure 8 (63 mg, 97%): mp 180-185 °C; TLC Rf )
0.63 (4:1:1 BuOH/AcOH/H2O, visualized by Rydons stain); [R]D
) -43.3° (c ) 0.45, MeOH); 1H NMR (400 MHz, CD3OD) δ
0.85-1.05 (m, 12 H), 1.44 (s, 9 H), 1.6-1.8 (m, 2 H), 2.15-
2.30 (m, 1 H), 2.85-3.02 (m, 2 H), 3.17 (s, 3 H), 3.79 (m, 2 H),
4.15-4.30 (m, 2 H), 4.37-4.55 (m, 2 H); HRMS calculated for
(M + Na)+ calculated 584.2907, found 584.2881. Anal.
(C24H43N5O10Na‚1.2H2O, 1.0 MeOH) C, H, N.
P r ep a r a tion of 12. To a solution of NH2OTHP (3.28 g, 28
mmol, 1.5 equiv) in dimethylformamide (50 mL) at 0 °C under
N2 was added farnesyl bromide 11 (5.1 mL, 18.7 mmol, 1.0
equiv) in dimethylformamide (15 mL) and potassium carbonate
(10.3 g, 75 mmol, 4 equiv). After being stirred for 20 h at room
temperature, the reaction mixture was filtered, and the
precipitate was washed with ethyl acetate (30 mL). The
organic layers were concentrated in vacuo to 25 mL and then
diluted with ethyl acetate (70 mL) and washed with 10%
lithium chloride (3 × 30 mL). The organic layer was dried
over sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified on Merck silica gel (400 mL), eluting with
hexane/ethyl acetate (6:1) to give dialkylated material [2.32
P r ep a r a tion of 9. Anhydrous HCl in dioxane (Aldrich, 4
M, 0.25 mL, 1mmol) was added to a 0 °C solution of 7 (116
mg, 0.178 mmol) in EtOAc (1 mL). After 15 min at 0 °C and
3 h at room temperature, the reaction was judged to be
complete by TLC. It was concentrated by rotary evaporation
to remove the solvents, and the residue was triturated with
EtOAc. The precipitated solid was washed with Et2O and
dried in vacuo to give 9 (82 mg, 75.4%): TLC Rf ) 0.39 (4:1:1
BuOH/AcOH/H2O, visualized by Rydons stain); MS (M + H)+
552; [R]D ) -26.3° (c ) 0.65, MeOH); 1H NMR (400 MHz, CD3-
OD) δ 0.88 (d, 3 H, J ) 5.8), 0.93 (d, 3 H, J ) 5.8), 0.98 (d, 3
H, J ) 7.0), 1.02 (d, 3 H, J ) 6.5), 1.5-1.8 (m, 2 H), 2.10-2.28
(m, 1 H), 2.95-3.4 (m, 2 H), 3.25 (s, 3 H), 3.75-4.0 (m, 2 H),
4.15-4.60 (m, 4 H), 4.95 (s, 2 H), 7.41 (m, 5 H). Anal.
(C26H42N5O8Cl‚0.8H2O, 0.1 dioxane) C, H, N.
P r ep a r a tion of 10. 10% Pd/C (25 mg) was added in one
portion to a solution of hydroxamic ether 9 (176 mg, 0.3 mmol)
in MeOH (3 mL). The reaction mixture was stirred under an
atmosphere of hydrogen (balloon) for 3 h at room temperature,
at which stage TLC revealed completion of reaction. It was
filtered through Celite, and the catalyst was washed with
MeOH (3 × 10 mL). The combined filtrate was concentrated
by rotary evaporation to remove MeOH, and the residue was
chromatographed on a CHP20 resin by stepwise gradient
elution with 0%, 25%, and 60% aqueous MeOH. The appropri-
ate fractions were combined and concentrated, millipore-
filtered, and lyophilized to obtain pure 10 (138 mg, 95%); mp
175-190 °C; TLC Rf ) 0.15 (4:1:1 BuOH/AcOH/H2O, visualized
by Rydons stain); 1H NMR (400 MHz, CD3OD) δ 0.98 (d, 3 H,
J ) 6.5), 1.02 (d, 3 H, J ) 6.5), 1.06 (d, 3 H, J ) 6.0), 1.08 (d,
g, 24%, Rf
) 0.55 (1:1 hexane/EtOAc)] and 12 [2.93 g, 47%, Rf
) 0.93 (1:1 hexane/EtOAc]: MS: (M + H)+ 526; 1H NMR (270
MHz, CDCl3) δ 1.59 (s, 6 H), 1.67 (s, 6 H), 1.54-1.74 (m, 6 H),
2.02 (br m, 8 H), 3.60 (m, 3 H), 3.93 (m, 1 H), 4.81-5.30 (m, 4
H);
13C NMR (68 MHz, CDCl3) δ 16.0, 16.4, 17.8, 20.2, 25.4,
25.7, 26.4, 26.7, 29.3, 50.0, 63.1, 101.4, 118.9, 123.7, 123.9,
124.4, 131.2, 135.2, 140.2.
P r ep a r a tion of 13. To a solution of 12 (385 mg, 1.2 mmol,
1 equiv) and DIPEA (730 mL, 4.2 mmol, 3.5 equiv) in THF (2
mL) was added 3-carbomethoxypropionyl chloride (295 mL, 2.4
mmol, 2.0 equiv) at 0 °C. After the reaction mixture was
stirred for 0.5 h at 0 °C and 2 h at room temperature, it was
diluted with ethyl acetate (50 mL) and washed with saturated
sodium bicarbonate. The aqueous layer was reextracted with
ethyl acetate (3 × 50 mL). The organic extracts were com-
bined, dried over sodium sulfate, filtered, and concentrated
in vacuo. The residue was purified on Merck silica gel (100
mL) eluting with hexane/acetone (20:1) to give 13 (435 mg,
85%): TLC Rf ) 0.6 (4:1 hexane/acetone visualized by ceric
ammonium sulfate); IR (CH2Cl2 film) 1742, 1670, 1437 cm-1
;
HRMS (M + H)+ calculated 436.3063, found 436.3072 for
1
C25H42O5N; H NMR (400 MHz, CDCl3) δ 0.59 (s, 6 H), 1.67
(s, 3 H), 1.68 (s, 3 H), 1.60-1.83 (m, 6 H), 1.94-2.17 (m, 8 H),
2.54-2.88 (m, 4 H), 3.60 (m, 1 H), 3.67 (s, 3 H), 3.99 (m, 1 H),
4.22 (dd, 1 H, J ) 6.84, J ) 15.81), 4.45 (dd, 1 H, J ) 6.24,
15.81), 4.95-5.30 (m, 4 H); 13C NMR (100 MHz, CDCl3) δ 16.0,

Hydroxamic Acid-Based Inhibitors of Ras FPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4205
16.4, 17.7, 19.8, 25.0, 25.7, 26.4, 26.5, 26.8, 28.1, 28.6, 28.9,
29.2, 39.6, 39.7, 46.8, 51.6, 63.7, 104.5, 118.9, 123.9, 124.4,
131.1, 135.2, 139.2, 139.4, 173.4, 173.7.
62.7, 62.9, 117.2, 117.8, 123.6, 123.8, 124.3, 131.3, 135.3, 135.5,
140.6, 170.4, 170.6, 170.9, 172.0, 172.2, 172.4, 172.8, 173.7,
174.3.
P r ep a r a tion of 16. To a solution of 15 (134 mg, 0.206
mmol, 1 equiv) in methanol/water (1:1, 3 mL) at 0 °C was
added 1 N sodium hydroxide (289 mL, 0.289 mmol, 1.4 equiv).
After the mixture was stirred at 0 °C for 2 h and room
temperature for 2 h, TLC (hexane:acetone, 1:1) indicated that
some 15 remained. Additional 1 N NaOH (100 mL, 100 mmol,
0.5 equiv) was added and after 1 more h TLC indicated the
reaction was complete. Methanol was removed in vacuo, and
the residue was purified on CHP-20P, eluting with water/
acetonitrile (100:0 to 20:80), to afford 16 (78 mg, 57%): mp
120-121 °C; TLC Rf ) 0.26 (9:1:0.05 CHCl3/CH3OH/HOAc
visualized by ceric ammonium sulfate); MS (M + Na)+ 659;
P r ep a r a tion of 14. To a solution of 13 (435 mg, 1.0 mmol,
1 equiv) in methanol/water (1:1, 10 mL) was added 1 N NaOH
(1.4 mL, 1.4 mmol, 1.4 equiv) at 0 °C. After 2 h at room
temperature, additional 1 N NaOH (0.2 mL, 0.2 mmol, 0.2
equiv) was added since the TLC (hexane/acetone, 4:1) indicated
that some unreacted starting material remained. After an
additional 2 h, the reaction was judged to be complete by TLC.
Methanol was removed in vacuo, and the aqueous layer was
extracted with ethyl acetate (15 mL). The organic layer was
discarded, and the aqueous solution was acidified with 1 N
HCl (pH ) 3) in the presence on ethyl acetate (50 mL). After
the two layers were separated, the aqueous solution was
reextracted with ethyl acetate (2 × 20 mL). The combined
organic layers were dried over sodium sulfate, filtered, and
concentrated in vacuo to give 14 (480 mg, 100%): TLC Rf )
0.0 (4:1 hexane/EtOAc visualized by ceric ammonium sulfate);
MS (M + H)+ 422; HRMS (M + H)+ calculated 422.2906, found
422.2888 for C24H40O5N; 1H NMR (400 MHz, CDCl3) δ 1.60 (s,
6 H), 1.67 (s, 3 H), 1.69 (s, 3 H), 1.60-1.81 (m, 6 H), 1.94-
2.10 (m, 8 H), 2.58-2.89 (m, 4 H), 3.59 (m, 1 H), 4.0 (m, 1 H),
4.24 (dd, 1 H, J ) 6.84, 15.81), 4.45 (dd, 1 H, J ) 6.42, 15.81),
5.00-5.30 (m, 4 H); 13C NMR (100 MHz, CDCl3) δ 16.0, 16.4,
17.7, 19.8, 24.9, 25.7, 26.4, 26.8, 28.0, 28.8, 29.1, 39.6, 39.7,
46.8, 63.7, 104.4, 118.6, 119.4, 123.9, 124.3, 131.2, 135.2, 135.5,
139.5, 139.6, 173.9, 177.9.
1
[R]D ) -21.48° (c ) 0.27, H2O); H NMR (400 MHz (D2O) δ
1.05 (br m, 12 H), 1.60 (s, 3 H), 1.61 (s, 3 H), 1.68 (s, 3 H),
1.73 (s, 3 H), 1.60-1.73 (m, 3 H), 1.99-2.30 (9 H), 2.66-2.92
(m, 4 H), 3.93-4.56 (m, 7 H), 5.13-5.32 (m, 3 H); 13C 100 MHz
(D2O) δ 17.6, 17.9, 19.1, 20.5, 22.5, 24.8, 26.3, 27.2, 28.3, 28.5,
29.9, 31.5, 32.3, 41.3, 41.5, 47.7, 53.9, 58.9, 61.6, 64.2, 119.8,
125.9, 126.3, 132.3, 136.4, 141.9, 174.9, 175.1, 175.2, 177.6,
177.8. Anal. (C33H55N4O8Na‚1.25H2O) C, H, N.
P r ep a r a tion of 17. To a solution of 12 (405 mg, 1.26 mmol,
1 equiv) in tetrahydrofuran (2 mL) were added DIPEA (657
mL, 3.78 mmol, 3 equiv) and ethylmalonyl chloride (322 mL,
2.52 mmol, 2 equiv) at 0 °C under N2. After the mixture was
stirred for 30 min at 0 °C and 2 h at room temperature, the
reaction mixture was diluted with ethyl acetate (50 mL) and
washed with saturated sodium bicarbonate (30 mL). The
aqueous solution was reextracted with ethyl acetate (2 × 30
mL). The organic layers were combined, washed with satu-
rated sodium chloride (10 mL), dried over sodium sulfate,
filtered, and concentrated. The residue was purified on Merck
silica gel (100 mL), eluting with hexane/ethyl acetate (8:1) to
afford 17 (423 mg, 82%): TLC Rf ) 0.4 (4:1hexane/EtOAc
visualization by ceric ammonium sulfate); HRMS (M + H)+
P r ep a r a tion of 15. To a solution of 14 (480 mg, 1.0 mmol,
1 equiv) in THF (50 mL) were added EDC (219 mg, 1.14 mmol,
1.14 equiv) and HOBT (174 mg, 1.14 mmol, 1.14 equiv). After
the mixture was stirred for 30 min, HCl-H-VLS-OCH3 (419
mg, 1.14 mmol, 1.14 equiv) and DIPEA (198 mL, 1.14 mmol,
1.14 equiv) were added. After the mixture was stirred for 24
h at room temperature, the solvents were removed and the
residue was dissolved in ethyl acetate (50 mL). The organic
layer was washed sequentially with saturated sodium bicar-
bonate (50 mL), 10% sodium bisulfate (50 mL) and saturated
sodium chloride (3 × 50 mL). The organic layer was dried
over sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified on Merck silica gel (100 mL), eluting with
hexane/ethyl acetate (1:3 to 0:100) to afford the coupled
intermediate (427 mg, 58%): TLC Rf ) 0.5 (EtOAc, visualized
by ceric ammonium sulfate); MS (M + H)+ 735; 1H NMR (270
MHz (CDCl3) δ 0.89, 0.93, 0.98, 1.04 (d,d,d,d, 12 H, J ) 6.45,
5.86, 7.03, 7.03), 1.60 (s, 6 H), 1.66 (s, 3 H), 1.67 (s, 3 H), 1.60-
1.90 (m, 9 H), 2.04 (m, 9 H), 2.45-2.94 (m, 4 H), 3.57 (m, 1
H), 3.72 (s, 3 H), 3.90-4.88 (m, 9 H), 5.09-5.22 (m, 3 H), 6.81
(m, 1 H), 7.21-7.68 (m, 2 H); 13C 68MHz (CDCl3) δ 16.0, 16.4,
16.5, 17.2, 17.7, 19.5, 19.8, 20.2, 21.0, 23.4, 25.7, 26.4, 26.8,
28.5, 28.8, 28.9, 29.1, 30.7, 31.0, 38.8, 39.0, 39.5, 39.7, 46.8,
51.3, 51.4, 52.3, 55.3, 55.5, 60.4, 63.0, 63.1, 63.9, 64.4, 103.7,
104.7, 118.1, 118.2, 123.7, 124.3, 131.3, 135.3, 139.7, 139.9,
170,4, 170.6, 171.1, 172.3, 172.4, 174.4, 175.6.
1
calculated 436.3063, found 436.3076 for C25H42O5N; H NMR
(400 MHz (CDCl3) δ 1.28 (t, 3 H, J ) 6.84), 1.60 (s, 6 H), 1.68
(s, 3 H), 1.69 (s, 3 H), 1.57-1.76 (m, 6 H), 1.97-2.18 (m, 8 H),
3.50 (dd, 2 H, J ) 5.82), 3.58 (m, 1 H), 4.0 (m, 1 H), 4.19 (q, 2
H, J ) 6.84), 4.20-4.48 (m, 2 H), 4.92-5.32 (m, 4 H); 13C NMR
(100 MHz, CDCl3) δ 14.1, 16.0, 17.7, 19.9, 24.8, 25.7, 26.4, 27.2,
29.1, 39.6, 39.7, 41.4, 46.4, 61.1, 63.9, 104.5, 118.4, 124.0, 124.3,
131.2, 135.3, 139.6, 167.5.
P r ep a r a tion of 18. To a solution of 17 (423 mg, 1.03 mmol,
1 equiv) in ethanol/water (10 mL, 2:1) was added 1 N sodium
hydroxide (1.44 mL, 1.44 mmol, 1.2 equiv). After being stirred
for 2 h, the reaction mixture was concentrated to ca. 4 mL,
diluted with water (20 mL), and extracted with ethyl acetate
(30 mL). The organic layer was discarded, and the aqueous
layer was acidified with 1 N hydrochloric acid (pH ) 2) in the
presence of ethyl acetate (50 mL). The layers were separated,
and the aqueous solution was reextracted with ethyl acetate
(3 × 50 mL). The organic extracts were combined, dried over
sodium sulfate, filtered, and concentrated in vacuo to give 18
(316 mg, 78%): TLC Rf ) 0.41 (9:1:0.05 CHCl3/CH3OH/HOAc
visualized by ceric ammonium sulfate); MS (M + H)+ 408;
HRMS (M + H)+ calculated 408.2750, found 408.2763 for
To a solution of above intermediate (377 mg, 0.514 mmol, 1
equiv) in methanol (4 mL) under N2 was added p-toluene-
sulfonic acid monohydrate (107 mg, 0.565 mmol, 1.1 equiv).
After the mixture was stirred 4 h at room temperature, the
solvents were removed, and the residue was dissolved in ethyl
acetate (40 mL). The organic layer was washed sequentially
with 10% sodium bicarbonate (40 mL) and saturated sodium
chloride (20 mL), dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified on Merck
silica gel (50 mL) eluting with hexane/acetone (4:1 to 1:1) to
afford 15 (121 mg, 40%): TLC Rf ) 0.63 (hexane/acetone, 1:1,
visualized by ceric ammonium sulfate); MS (M + H)+ 651;
HRMS (M + H)+ calculated 651.4333, found 651.4347 for
1
C23H38O5N; H NMR (270 MHz, CDCl3) δ 1.59 (s, 6 H), 1.68
(s, 3 H), 1.70 (s, 3 H), 1.60-2.18 (m, 14 H), 3.42-3.95 (m, 4
H), 4.36 (d, 2 H, J ) 7.01), 4.91-5.29 (m, 4 H); 13C NMR (68
MHz, CDCl3) δ 16.0, 16.4, 17.7, 20.2, 24.7, 25.7, 26.3, 26.7,
29.1, 45.9, 64.7, 104.2, 117.3, 123.6, 124.3, 131.3, 135.4, 140.9,
169.5, 170.7.
P r ep a r a tion of 19. To a solution of 18 (316 mg, 0.78 mmol,
1 equiv) in THF (40 mL) at room temperature under N2 was
added HOBT•H2O (119 mg, 0.78 mmol, 1 equiv) and EDC (149
mg, 0.78 mmol, 1 equiv). The reaction mixture was stirred at
room temperature for 1 h after which HCl‚H-VLS-OCH3 5 (287
mg, 0.78 mmol, 1 equiv) and DIPEA (136 µL, 0.78 mmol, 1
equiv) were added, and the reaction mixture was stirred for
an additional 20 h. The solvents were removed in vacuo, and
the residue was dissolved in ethyl acetate (50 mL) and washed
sequentially with saturated sodium bicarbonate (35 mL), 10%
sodium bisulfate (35 mL), and saturated sodium chloride (35
C
34H59O8N4; 1H NMR (400 MHz, CDCl3) δ 0.95 (m, 12 H), 1.60
(s, 6 H), 1.68 (s, 6 H), 1.60-1.68 (m, 3 H), 2.05 (br m, 9 H),
2.52-2.95 (m, 4 H), 3.75 (s, 3 H), 3.90-4.75 (m, 7 H), 5.02-
5.31 (m, 3 H), 7.32-8.10 (m, 3 H); 13C NMR (100 MHz, CDCl3)
δ 16.0, 16.4, 17.7, 18.2, 19.2, 19.3, 19.4, 21.9, 22.0, 22.7, 22.9,
24.8, 25.7, 26.5, 26.7, 28.1, 29.3, 30.2, 305, 30,7, 30.8, 39.6,
39.7, 40.2, 45.9, 51.6, 51.8, 51.9, 52.6, 55.1, 59.0, 59.5, 62.2,

4206 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Patel et al.
mL). The organic layer was dried over sodium sulfate, filtered,
and concentrated in vacuo. The residue was purified on Merck
silica gel (30 mL) and eluted with ethyl acetate/hexane (3:1 to
100:0) to give the coupled intermediate (113 mg, 20%): TLC
Rf ) 0.45 (EtOAc, visualized by ceric ammonium sulfate); MS
After the mixture was stirred for 3 h, additional p-toluene-
sulfonic acid monohydrate (42 mg, 0.214 mmol, 0.65 equiv)
was added. After the mixture was stirred an additional 4 h,
the solvents were removed in vacuo and the residue was
dissolved in ethyl acetate (40 mL) and washed sequentially
with 10% sodium bicarbonate (10 mL) and saturated sodium
chloride (10 mL). The organic solution was dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was
purified on Merck silica gel (30 mL), eluting with hexane/
acetone (2:1) to give 22 (110 mg, 50%): TLC Rf ) 0.48 (1:1
hexane/acetone visualized by ceric ammonium sulfate); MS (M
+ H)+ 667; HRMS (M + H)+ calculated 667.4104, found
667.4082 for C34H59O7N4S; 1H NMR (270 MHz, CDCl3/CD3OD,
10:1) δ 0.92 (m, 12 H), 1.59 (s, 6 H), 1.67 (s, 3 H), 1.68 (s, 3 H),
2.07 (s, 3 H), 1.90-2.22 (m, 12 H), 2.51 (br m, 2 H), 3.57 (br
m, 2 H), 3.73 (s, 3 H), 4.20-4.65 (m, 5 H), 5.10-5.27 (m, 3 H);
13C 68 MHZ (CDCl3:CD3OD 10:1) δ 15.2, 160, 16.4, 17.7, 18.4,
18.5, 19.1, 19.2, 25.7, 26.5, 26.8, 29.2, 30.0, 30.6, 31.1, 39.7,
45.8, 51.4, 51.5, 52.5, 53.8, 53.9, 58.8, 59.2, 117.4, 123.8, 124.4,
131.4, 135.4, 141.1, 168.7, 171.7, 172.3.
1
(M + H)+ 721; H NMR (400 MHz, CDCl3) δ 0.95 (m, 12 H),
1.60 (s, 6 H), 1.67 (s, 3 H), 1.70 (s, 3 H), 1.60-2.33 (m, 18 H),
3.55 (m, 3 H), 3.73 (s, 3 H), 3.97 (m, 2 H), 4.25-4.75 (m, 6 H),
4.91-5.27 (m, 4 H); 13C NMR (100 MHz, CDCl3) δ 16.0, 16.4,
17.6, 17.7, 19.4, 20.2, 21.3, 23.2, 24.7, 24.8, 25.7, 26.3, 26.4,
26.7, 29.1, 29.4, 29.5, 37.5, 39.5, 39.6, 39.7, 40.4, 46.0, 51.7,
52.4, 55.2, 55.3, 60.3, 60.4, 62.2, 62.6, 64.7, 67.9, 104.2, 117.5,
117.7, 117.8, 123.6, 123.7, 124.3, 131.3, 135.4, 140.5, 140.6,
140.8, 169.0, 160.2, 170.6, 171.3, 172.4, 172.5.
To a solution of of the above intermediate (113 mg, 0.157
mmol, 1 equiv) in THF (1 mL) at room temperature under N2
was added p-toluenesulfonic acid monohydrate (33 mg, 0.173
mmol, 1.1 equiv). After the mixture was stirred at room
temperature for 3 h, more p-toluenesulfonic acid monohydrate
(16 mg, 0.086 mmol, 0.55 equiv) was added. After the mixture
was stirred an additional 2 h, the solvents were removed in
vacuo, and the residue was dissolved in ethyl acetate (20 mL)
and washed sequentially with saturated sodium bicarbonate
(10 mL) and saturated sodium chloride (10 mL). The organic
solution was dried over sodium sulfate, filtered, and concen-
trated in vacuo. The residue was purified on Merck silica gel
(14 mL), eluting with hexane/acetone (4:1 to 1:4) to give 19
(54 mg, 54%): TLC Rf ) 0.40 (EtOAc, visualized by ceric
P r ep a r a tion of 23. To a solution of 22 (110 mg, 0.165
mmol, 1 equiv) in dioxane/methanol/water (3.5 mL, 4:2:1) was
added 1 N NaOH (231 µL, 0.231 mmol, 1.4 equiv). After the
mixture was stirred for 6 h, the solvents were removed in
vacuo. The residue was purified on CHP-20P (15 mL), eluting
with water/acetonitrile (0:100 to 100:0) to give 23 after
lyophilization (63 mg, 57%): mp 135-139 °C; TLC Rf ) 0.67
(CHCl3/CH3OH/HOAc, 5:1:0.05, visualized by ceric ammonium
1
1
ammonium sulfate); H NMR (270 MHz, CDCl3) δ 0.80-1.05
sulfate); MS (M + H)+ 653; [R]D ) -27.8° (c ) 0.78, H2O); H
(m, 12 H), 1.60 (s, 6 H), 1.67 (s, 3 H), 1.69 (s, 3 H), 1.60-2.30
(m, 12 H), 3.72 (s, 3 H), 3.5-4.5 (m, 9 H), 5.02-5.3 (m, 3 H),
7.60-8.00 (m, 3 H); 13C NMR (68 MHz, CDCl3) δ 16.0, 16.4,
17.6, 17.7, 19.3, 21.1, 23.2, 24.8, 25.7, 26.4, 26.8, 29.3, 39.7,
40.1, 41.1, 41.3, 46.0, 52.0, 52.5, 53.9, 55.1, 60.9, 62.4, 69.6,
117.2, 123.7, 124.3, 131.3, 135.4, 141..3, 168.1, 170.5, 170.8,
171.1, 171.9, 173,2
NMR (400 MHz (CD3OD) δ 0.96 (m, 12 H), 1.60 (s, 6 H), 1.66
(s, 3 H), 1.71 (s, 3 H), 2.05 (s, 3 H), 1.88-2.21 (m, 12 H), 2.50
(m, 2 H), 4.18-4.30 (m, 5 H), 5.10-5.32 (m, 3 H); 13C NMR
(68 MHz, CD3OD) δ 15.3, 16.1, 16.5, 17..8, 18.3, 18.9, 19.9,
25.9, 27.4, 27.8, 31.1, 31.6, 24.1, 40.7, 40.8, 47.2, 48.0, 48.4,
48.6, 55.7, 60.3, 60.7, 119.3, 125.1, 125.4, 136.2, 141.4, 172.6,
173.8, 177.7. HRMS (M + Na)+ calculated 675.3767, found
675.3759. Anal. (C33H53N4O7SNa‚2.65H2O) C, H, N.
P r ep a r a tion of HCl‚H-Va l-Va l-(D-Met)-OMe (24): HCl‚
H-(D-Met)-OMe. To a suspension of D-methionine (5g, 0.335
mmol, 1 equiv) in 2,2-dimethoxypropane (330 mL) was added
11.7 M hydrochloric acid (33 mL). After the mixture was
stirred for 16 h, the solvents were removed in vacuo. The
residue was dissolved in methanol (50 mL) and diluted with
ether (400 mL), and the resulting precipitate was filtered and
washed with ether (50 mL). The precipitate was redissolved
in methanol (30 mL) and slowly precipitated with ether (200
mL). The precipitate was filtered, washed with ether (40 mL),
and dried in vacuo to give the HCl salt of H-(D-Met)-OMe (5.44
g, 84%): mp 147-148 °C; TLC Rf ) 0.48 (9:1:0.05 chloroform/
methanol/acetic acid visualized by ceric ammonium sulfate);
IR (KBr) 1748 cm-1; MS (M + H)+ 164; [R]D ) -23.58° (c )
0.85, CH3OH); 1H NMR (270 MHz, CD3OD) δ 2.21 (s, 3 H),
2.31 (m, 2 H), 2.76 (t, 2 H, J ) 7.03), 3.95 (s, 3 H), 4.32 (t, 1 H,
J ) 6.45); 13C NMR (68 MHz, CD3OD) δ 14.9, 30.0, 30.7, 52.7,
53.7, 170.7.
P r ep a r a tion of 20. To a solution of 19 (54 mg, 0.085 mmol,
1 equiv) in 2:1 methanol/water (1 mL) at 0 °C was added 1 N
sodium hydroxide (125 µL, 0.125 mmol, 1.4 equiv). After the
mixture was stirred for 1 h, TLC indicated completion of the
reaction. The solvents were removed, and the residue was
purified on CHP-20P (10 mL) eluting with acetonitrile/water
(1:9 to 8:2). Appropriate fractions were combined and lyo-
philized to give 20 (39 mg, 66%): mp 136-139 °C; TLC Rf )
0.15 (9:1:0.05 CHCl3/CH3OH/HOAc visualized by ceric am-
monium sulfate); MS (M + H)+ 623; [R]D ) -19.6° (c ) 1.0,
1
H2O); H NMR (400 MHz, D2O) δ 0.90-1.11 (m, 12 H), 1.58
(s, 6 H), 1.65 (s, 3 H), 1.71 (s, 3 H), 1.58-1.71 (m, 3 H), 1.96-
2.04 (m, 9 H), 3.87-4.51 (m, 9 H), 5.09-5.32 (m, 3 H); 13C NMR
(68 MHz, D2O) δ 1.6.7, 17.2, 18.2, 18.4, 19.8, 21.7, 23.9, 26.4,
27.5, 27.7, 30.3, 40.7, 47.2, 52.3, 58.2, 60.7, 62.4, 117.2, 125.2,
125.5, 131.3, 135.4, 141.3, 168.1, 170.5, 175.1, 176.2. HRMS
(M + Na)+ calculated 645.3839, found 645.3841. Anal.
(C32H53N4O8Na‚0.75H2O) C, H, N.
P r ep a r a tion of 22. To a solution of 18 (415 mg, 1.02 mmol,
1.0 equiv) in dimethylformamide (12 mL) was added H-VVM-
OCH3 21 (368 mg, 1.02 mmol, 1.0 equiv), BOP (451 mg, 1.02
mmol, 1.0 equiv), and iPr2NEt (522 µL, 3.06 mmol, 3.0 equiv).
After the reaction mixture was stirred for 20 h at room
temperature, it was concentrated in vacuo. The residue was
purified on Merck silica gel (100 mL), eluting with hexane/
ethyl acetate (1:1 to 1:2) to give the coupled product (247 mg,
32%): TLC Rf ) 0.62 (ethyl acetate, visualized by ceric
ammonium sulfate); MS (M + H)+ 751; HRMS (M + H)+
calculated 751.4679, found 751.4614 for C39H67O8N4S; 1H NMR
(400 MHz, CDCl3) δ 0.95 (m, 12 H), 1.59 (s, 6 H), 1.67 (s, 6 H),
2.0 (s, 3 H), 1.60-2.25 (m, 18 H), 2.48 (m, 2 H), 3.72 (s, 3 H),
3.42-4.00 (m, 4 H), 4.22-4.71 (m, 5 H), 4.97-5.29 (m, 4 H),
7.53 (br m, 3 H); 13C NMR (100 MHz, CDCl3) δ 15.3, 16.0, 16.4,
17.7, 18.2, 19.2, 19.4, 199, 24.9, 25.7, 26.4, 26.7, 30.1, 30.4,
30.5, 30,8, 31.2, 39.6, 39.7, 40.2, 46.2, 51.5, 52.3, 58.6, 59.0,
64.1, 104.5, 118.3, 118.4, 123.8, 124.3, 131.2, 135.3, 139.8,
140.0, 167.3, 170.1, 171.4, 172.2.
Boc-Va l-(D-Met)-OMe. To a suspension of D-methionine
methyl ester (5 g, 25 mmol, 1 equiv), Boc-valine (5.51 g, 25
mmol, 1 equiv), and HOBT (3.88 g, 25 mmol, 1 equiv) in THF/
DMF (160 mL, 3:1) was added iPr2NEt (4.85 mL, 28 mmol,
1.1 equiv). The suspension was cooled to 0 °C, and EDC (4.87
g, 25 mmol, 1 equiv) was added. The ice bath was removed,
and after being stirred for 1 h, the reaction mixture was a clear
solution. The solution was stirred for 16 h, and the reaction
mixture was concentrated to ∼40 mL. The concentrate was
diluted with ethyl acetate (200 mL) and washed with saturated
sodium bicarbonate (100 mL), phosphate buffer (pH ) 4, 100
mL), and 10% lithium chloride (3 × 100 mL), dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was
triturated with petroleum ether (50 mL) to give Boc-Val-(D-
Met)-OMe (7.72 g, 84%): mp 94-95 °C; TLC Rf ) 0.91 (4:1:1
butanol/acetic acid/water visualized by ceric ammonium sul-
1
fate); IR (KBr) 1750 cm-1; MS (M + H)+ 363; H NMR (400
MHz (CDCl3) δ 0.91 (d, 3 H, J ) 6.84), 0.98 (d, 3 H, J ) 6.84),
1.45 (s, 9 H), 2.01 (m, 1 H, J ) 7.26), 2.09 (s, 3 H), 2.82 (m, 2
H), 2.49 (t, 2 H, J ) 7.26), 3.75 (s, 3 H), 4.0 (m, 1 H), 4.71 (dd,
1 H, J ) 7.26), 5.0 (m, 1 H), 6.81 (d, 1 H, J ) 7.69); 13C NMR
To a solution of above intermediate (247 mg, 0.329 mmol, 1
equiv) in tetrahydrofuran (2 mL) was added p-toluenesulfonic
acid monohydrate (81 mg, 0.428 mmol 1.3 equiv) under N2.

Hydroxamic Acid-Based Inhibitors of Ras FPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4207
(100 MHz, CDCl3) δ 15.4, 17.5, 19.4, 28.3, 29.9, 30.6, 31.5, 51.4,
52.5, 59.9, 80.0, 155.8, 171.5, 172.2. Anal. (C16H30N2O5S) C,
H, N.
29.1, 29.4, 29.5, 29.6, 29.7, 30.2, 31.4, 39.4, 39.6, 39.7, 46.0,
51.5, 51.6, 52.2, 58.5, 60.0, 60.1, 64.5, 104.3, 104.4, 117.8, 123.7,
124.3, 131.3, 135.5, 140.5, 140.6, 168.1, 168.3, 171.2, 171.3,
172.2, 172.3. Anal. (C39H66N4O8S‚0.17H2O) C, H, N.
Boc-Va l-Va l-(D-Met)-OMe. To a solution of 1.56 M hy-
drochloric acid (60 mL, in acetic acid) and dimethyl sulfide (6
mL) at 0 °C under N2 was added Boc-Val-(D-Met)-OMe (7.72
g, 21 mmol). The ice bath was removed, and after 0.5 h the
reaction mixture was concentrated in vacuo. The residue was
triturated with ether (3 × 100 mL) and petroleum ether (100
mL). The slightly tacky solid was dissolved in acetone (100
mL) and concentrated in vacuo to give the hydrochloride salt
(5.84 g, 92%): TLC Rf ) 0.38 (9:1:0.05 chloroform/methanol/
acetic acid visualized by ceric ammonium sulfate); HRMS
calculated 263.1429, found 263.1433 for C11H23O3N2S.
To a solution of Boc-Val-OH (3.35 g, 15.4 mmol, 1 equiv)
and HOBT (2.35 g, 15.4 mmol 1 equiv) in THF/DMF (190 mL,
5:3) was added EDC (2.96 g, 15.4 mmol, 1 equiv) at 0 °C under
N2. The ice bath was removed, and the reaction mixture was
stirred for 30 min. The reaction mixture was recooled to 0
°C, and HCl‚H-Val-Val-(D-Met)-OMe prepared above (4.59 g,
15.4 mmol, 1 equiv) was added. Then the ice bath was
removed, and iPr2NEt (3.57 mL, 20.0 mmol, 1.3 equiv) was
added in four portions over 4 h. After the reaction mixture
To a solution of the above intermediate (445 mg, 0.593
mmol, 1 equiv) in tetrahydrofuran (6 mL) was added p-
toluenesulfonic acid monohydrate (113 mg, 0.593 mmol, 1
equiv). After being stirred for 6 h, the reaction mixture was
concentrated in vacuo, and the residue was dissolved in ethyl
acetate (300 mL). The organic solution was washed with
saturated sodium bicarbonate (100 mL) and saturated sodium
chloride (100 mL), dried over sodium sulfate, filtered, and
concentrated in vacuo to give 25 (386 mg, 0.593 mmol): TLC
Rf ) 0.47 (9:1 chloroform/methanol visualized by ceric am-
monium sulfate); IR (KBr) 1742 cm-1; MS (M + H)+ 667;
HRMS calculated 667.4104, found 667.4116 for C34H59O7N4S;
1H NMR (400 MHz, CDCl3/CD3OD, 10:1) δ 0.99 (m, 12 H), 1.60
(s, 6 H), 1.67 (s, 3 H), 1.70 (s, 3 H), 2.09 (s, 3 H), 1.95-2.57
(m, 14 H), 3.55 (q, 2 H, J ) 14.1), 3.71 (s, 3 H), 4.23-4.62 (m,
5 H), 5.11-5.30 (3 H); 13H 100 MHz (CDCl3/CD3OD, 10:1) δ
15.23, 16.1, 16.4, 17.8, 18.1, 18.3, 19.4, 19.5, 19.6, 25.8, 26.8,
27.1, 29.5, 30.2, 30.5, 30.8, 31.2, 37.8, 40.1, 40.7, 46.4, 51.9,
52.5, 52.7, 59.3, 59.6, 60.0, 60.2, 117.8, 118.9, 124.2, 124.3,
124.7, 131.6, 135.6, 135.7, 141.2, 141.5, 167.7, 168.8, 169.8,
172.5, 172.7, 172.8.
1
was stirred for 36 h, the solvents were concentrated to ca. /3
volume. The concentrate was diluted with ethyl acetate (250
mL) and washed with saturated sodium bicarbonate (100 mL),
phosphate buffer (100 mL, pH ) 4), and 10% lithium chloride
(3 × 100 mL). The oragnic layer was dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was
triturated with ether (50 mL) and filtered to give Boc-Val-Val-
(D-Met)-OMe (5.73 g, 82%): TLC Rf ) 0.92 (butanol/acetic acid/
water, 4:1:1, visualized by ceric ammonium sulfate); mp 157-
158 °C; IR (KBr) 1740, 1694, 1643, 1522 cm-1; MS (M + H)+
P r ep a r a tion of 26. To a solution of 25 (180 mg, 0.270
mmol, 1 equiv) in dioxane (4 mL) was added 1 N sodium
hydroxide (324 mL, 0.324 mmol, 1.2 equiv). After being stirred
for 6 h, the reaction mixture was cool to -40 °C for 16 h. The
reaction mixture was warmed to room temperature, and water
(5 mL) was added. The solution was concentrated to ∼3 mL
and purified on CHP-20P (40 mL), eluting with water/aceto-
nitrile (100:0 to 0:100) to give 26 (lyophilized, 78 mg, 43%):
mp 122-130 °C; TLC Rf ) 0.50 (chloroform/methanol/acetic
acid, 9:1:0.05, visualized by ceric ammonium sulfate); IR (KBr)
1
462; [R]D ) -16.25° (c ) 0.80, CH3OH); H NMR (400 MHz,
CDCl3) δ 0.97 (m, 12 H), 1.45 (s, 9 H), 2.09 (s, 3 H), 2.01-2.60
(m, 6 H), 3.71 (s, 3 H), 3.91 (dd, 1 H, J ) 6.0, 6.4), 4.35 (m, 1
H), 4.67 (m, 1 H), 5.18 (m, 1 H), 6.66 (m, 1 H), 7.20 (m, 1 H);
13C NMR (100 MHz, CDCl3) δ 15.5, 17.3, 17.8, 19.4, 19.5, 28.3,
30.2, 31.4, 51.6, 52.3, 58.4, 60.9, 156.4, 171.1, 171.8, 172.1.
Anal. (C21H39N3O6S) C, H, N.
1
1634 cm-1; [R]D ) -27.54° (c ) 0.43, CH3OH); H NMR (400
MHz, CD3OD) δ 0.97 (m, 12 H), 1.60 (s, 6 H), 1.66 (s, 3 H),
1.71 (s,3 H), 2.05 (s, 3 H), 1.96-2.49 (m, 14 H), 3.35 (s, 2 H,
partially exchanged with deuterium), 4.26 (m, 5 H), 5.10-5.30
(m, 3 H); 13C NMR (100 MHz, CD3OD) δ 15.5, 16.3, 16.8, 18.0
18.9, 20.1, 20.2, 26.1, 27.6, 28.0, 31.5, 31.7, 34.1, 40.9, 41.0,
47.3, 55.9, 60.8, 61.0, 119.4, 125.3, 125.6, 132.3, 136.4, 141.6,
169.2, 170.7, 172.8, 174.1, 178.2. HRMS (M + Na)+ calculated
675.3767, found 675.3716. Anal. (C33H53N4O7SNa‚1.51H2O) C,
H, N.
HCl‚H-Va l-Va l-(D-Met)-OMe (24). A solution of Boc-Val-
Val-(D-Met)-OMe (2 g, 4.34 mmol) in 1.56 M HCl in acetic acid
(20 mL) and dimethyl sulfide (2 mL) under argon was stirred
for 30 min while being warmed from 0 °C to room temperature.
The solvents were removed in vacuo, and the residue was
triturated with ether (3 × 25 mL). The residue was filtered
and washed with ether (2 × 20 mL) to give 24 (1.65 g, 96%):
mp >210 °C; TLC Rf ) 0.10 (chloroform/methanol/acetic acid,
9:1:0.05, visualized by ceric ammonium sulfate); IR (KBr) 1748,
1649, 1553 cm-1; MS (M + Na)+ 675; [R]D ) -24.39° (c ) 0.52,
P r ep a r a tion of 28. To a solution of homofarnesol 27 (600
mg, 2.54 mmol, 1 equiv) in dichloromethane (30 mL) was added
pyridine (1.02 mL, 12.7 mmol, 5.0 equiv), DMAP (62 mg, 0.508
mmol, 0.2 equiv), and tosyl chloride (1.06 g, 5.59 mmol, 2.2
equiv) under N2. Additional tosyl chloride (200 mg, 0.4 equiv)
was added after 20 and 28 h. After the mixture was stirred a
total of 44 h, the reaction was quenched with saturated
aqueous sodium bicarbonate (15 mL). This was stirred vigor-
ously for 45 min, and the layers were separated. The water
layer was extracted with dichloromethane (2 × 15 mL). The
organic layers were combined and washed with 5% copper
sulfate (15 mL) and water (15 mL), dried over magnesium
sulfate, filtered, and concentrated in vacuo. The residue was
purified on Merck silica (100 mL), eluting with hexane/ethyl
acetate (85:15) to give homofarnesyl tosylate (840 mg, 86%):
TLC Rf ) 0.33 (95:5 hexane/EtOAc, visualized by ceric am-
1
CH3OH); H NMR (400 MHz, CD3OD) δ 1.00 (m, 12 H), 2.07
(s, 3 H), 1.94-2.24 (m, 4 H), 2.42-2.63 (m, 2 H), 3.71 (s, 3 H),
3.80 (d, 1 H, J ) 5.56), 4.26 (d, 1 H, J ) 8.12), 4.54 (m, 1 H);
13C NMR (100 MHz, CD3OD) δ 15.1, 17.9, 18l.9, 19.0, 25.0,
31.2, 31.5, 31.7, 32.0, 52.7, 52.8, 59.5, 60.5, 169.4, 173.4, 173.5.
Anal. (C16H32N3O4SCl‚0.23H2O) C, H, N.
P r ep a r a tion of 25. To a solution of HCl‚H-Val-Val-D-Met-
OMe (24) (562 mg, 1.55 mmol, 1.03 equiv) in DMF (25 mL)
were added sequentially 18 (614 mg, 1.50 mmol, 1 equiv) in
DMF (5 mL), BOP (667 mg, 1.50 mmol, 1 equiv), and iPr2NEt
(262 mL, 1.50 mmol, 1 equiv). After being stirred for 6 h, the
reaction mixture was diluted with ethyl acetate (300 mL) and
washed sequentially with saturated sodium bicarbonate (150
mL), phospate buffer (pH ) 4, 100 mL), and 10% lithium
chloride (3 × 150 mL). The organic solution was dried over
sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified on Merck silica gel (400 mL), eluting with
hexane/acetone (4:1 to 3:1) to give the coupled product (639
mg, 57%): mp 147-150 °C; TLC Rf ) 0.22 (hexane/acetone,
2:1 visualized by ceric ammonium sulfate); IR (KBr) 1738, 1636
cm-1; MS (M + H)+ 751; [R]D ) -8.99° (c ) 1.00, CH3OH); 1H
NMR (400 MHz, CDCl3) δ 0.97 (m, 12 H), 1.60 (s, 6 H), 1.68
(s, 3 H), 1.70 (s, 3 H), 2.12 (s, 3 H), 1.60-2.6 (m, 20 H), 3.55-
3.71 (m, 3 H), 3.70 (s, 3 H), 3.91-4.65 (m, 6 H), 4.90-5.30 (m,
4 H), 7.05 (m, 2 H); 13C NMR (100 MHz, CDCl3) δ 15.5, 16.0,
16.4, 17.3, 17.4, 17.7, 19.5, 19.6, 20.1, 24.8, 25.7, 26.4, 26.7,
1
monium sulfate); MS (M + NH4)+ 408; H NMR (270, CDCl3)
δ 1.56 (s, 3 H), 1.58 (s, 3 H), 1.60 (s, 3 H), 1.68 (s, 3 H), 2.02
(br m, 8 H), 2.34 (dd, 2 H, J ) 7.04), 2.44 (s, 3 H), 3.97 (d of d,
2 H, J ) 7.04), 4.92-5.13 (m, 3 H), 7.33 (d, 2 H, J ) 8.6), 7.79
(d, 2 H, J ) 8.60); 13C NMR (68 MHz (CDCl3) δ 15.9, 16.1,
17.6, 21.5, 26.3, 26.6, 27.7, 39.5, 69.9, 117.3, 123.7, 124.2, 127.8,
129.7, 131.2, 133.2, 135.1, 139.3, 144.5.
To a solution of of the above tosylate intermediate (2.25 g,
57.7 mol, 1 equiv) in tetrahydrofuran (40 mL) was added
lithium bromide (5.6 g, 64.5 mmol, 1.1 equiv) under N2. After
the mixture was refluxed for 4 h, saturated ammonium
chloride (40 mL) was added, and the solution was extracted
with ether (3 × 100 mL). The organic extracts were combined,
washed with saturated sodium bicarbonate (100 mL), dried
over magnesium sulfate, filtered, and concentrated in vacuo.

4208 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Patel et al.
The residue was purified on Merck silica gel (300 mL) eluting
with hexane to give 28 (1.24g, 72%): TLC Rf ) 0.23 (95:5
hexane/EtOAc, visualized by ceric ammonium sulfate); MS (M
chloroform and filtered through Celite and 0.2 mm filter paper.
The material was concentrated in vacuo and triturated with
ether to give 32 (2.4 g, 88% yield).: TLC Rf ) 0.3 (9:1:0.05
CHCl3/MeOH/HOAc visualized by ceric ammonium sulfate);
+ H)+ 299; IR (CHCl3 film) 1446 cm-1 1H NMR (400 MHz,
;
1
CDCl3) δ 1.59(s, 6 H), 1.63 (s, 3 H), 1.67 (s, 3 H), 1.90-2.18
(m, 8 H), 2.55 (dd, 2 H, J ) 7.62), 3.31 (dd, 2 H, J ) 7.63),
5.12 (br m, 3 H); 13C NMR (100 MHz, CDCl3) δ 15.9, 16.1, 17.6,
25.6, 26.3, 26.6, 31.6, 32.5, 39.5, 39.6, 120.8, 123.8, 124.3, 131.0,
135.0, 138.3. Anal. (C16H27Br‚0.40H2O) C, H, N.
MS (M + H)+ 448; IR (KBr) 1746, 1640 cm-1; H NMR (400
MHz, CDCl3/CD3OD, 10:1) δ 0.93-0.97 (m, 12 H), 2.09 (s, 3
H), 1.93-2.17 (m, 4 H), 2.5 (m, 2 H), 3.37 (s, 2 H), 3.74 (s, 3
H), 4.20 (dd, 1 H, J ) 8.55), 4.29 (d, 1 H J ) 6.42), 4.62 (m, 1
H), 7.81 (d, 1 H, J ) 8.55), 7.98 (d, 1 H, J ) 7.69); 13C NMR
(100 MHz, CDCl3/CD3OD, 10:1) δ 15.2, 18.1, 18.3, 19.2, 19.3,
30.2, 30.7, 30.9, 31.2, 41.3, 51.6, 51.7, 52.5, 59.1, 59.3, 167.8,
170.9, 172.1, 172.2, 172.6. Anal. (C19H33N3O7S‚0.14H2O) C,
H, N.
P r ep a r a tion of 29. To a solution of homofarnesyl bromide
28 (900 mg, 3 mmol, 1 equiv) in dimethylformamide (7 mL) at
0 °C under N2 were added NH2OTHP (527 mg, 4.5 mmol, 1.5
equiv) and potassium carbonate (1.65 g, 12 mmol, 4 equiv).
After being stirred for 4 days at room temperature, the reaction
was filtered, diluted with ethyl acetate (75 mL), and washed
with 10% lithium chloride (4 × 70 mL), dried over sodium
sulfate, and concentrated in vacuo. The residue was purified
on Merck silica gel (100 mL), eluting with hexane/ethyl acetate
(9:1) to give unreacted 28 (382 mg, 42%) and the desired 29
(316 mg, 31%): TLC Rf ) 0.36 (hexane/ethyl acetate, 4:1,
visualized by ceric ammonium sulfate); MS (M + H)+ 336; IR
P r ep a r a tion of 33. To a solution of 29 (445 mg, 1.33 mmol,
1 equiv) in acetonitrile/dimethylformamide (9:4, 13 mL) under
N2 were added 32 (594 mg, 1.33 mmol, 1 equiv) and diisopro-
pylethylamine (462 mL, 2.66 mmol, 2 equiv). After the
solution was cooled to 0 °C, BOP (588 mg, 1.33 mmol, 1 equiv)
was added. The reaction mixture was warmed to room
temperature and stirred for 16 h. More acetonitrile (5 mL)
was added, and the reaction mixture was stirred for 6 h. After
the solvents were removed in vacuo, the residue was dissolved
in ethyl acetate (150 mL) and washed sequentially with
saturated sodium bicarbonate (75 mL) and 10% lithium
chloride (4 × 75 mL). The organic layer was dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was
purified on Merck silica gel (106 mL), eluting with hexane/
acetone (4:1 to 3:1) to give the coupled intermediate (655 mg,
64%): mp 151-152 °C; TLC Rf ) 0.23 (2:1 hexane/acetone
visualized by ceric ammonium sulfate); MS (M + H)+ 765; IR
1
(neat) 1441 cm-1; H NMR (400 MHz, CDCl3) δ 1.59 (s, 6 H),
1.63 (s, 3 H), 1.67 (s, 3 H), 1.45-2.33 (m, 16 H), 2.98 (m, 2 H),
3.56 (m, 1 H), 3.91 (m, 1 H), 4.80 (m, 1 H), 5.12 (br m, 3 H),
5.72 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ 16.01, 16.1, 17.7,
20.2, 25.4, 25.7, 26.0, 26.6, 26.8, 29.3, 39.7, 39.8, 52.1, 52.3,
63.0, 101.4, 121.2, 124.1, 124.4, 131.1, 135.0, 137.6. Anal.
(C21H37NO2) C, H, N.
P r ep a r a tion of 31. KOH (6.5 g, 0.1 mol, 1 equiv) in benzyl
alcohol (125 mL) was added dropwise to a solution of dibenzyl
malonate (24.98 mL, 0.1 mmol, 1 equiv) in benzyl alcohol (250
mL). After being stirred for 3 h, the reaction mixture was
diluted with water (500 mL) and extracted with ether (1.5 l
and 2 × 200 mL). The organic extracts were discarded, and
the aqueous layer was acidified with 6 N HCl (pH ) 2.3) in
the presence of ether (200 mL). The layers were separated,
and the aqueous solution was reextracted with ether (2 × 300
mL). The organic extracts were combined, dried over MgSO4,
filtered, and concentrated in vacuo to give the monoester
monoacid (18.57 g, 95%): TLC Rf ) 0.34 (4:1hexane/EtOAc
visualized by ceric ammonium sulfate); MS (M + NH4)+ 212;
1H NMR (270 MHz, CDCl3) δ 3.47 (s, 2 H), 5.19 (s, 3 H), 7.35
(s, 5 H); 13C NMR (68 MHz, CDCl3) δ 40.9, 67.6, 128.4, 128.6,
128.6, 166.4, 171.8.
To a solution of H-Val-Val-Met-OMe (21) (3.26 g, 8.19 mmol,
1 equiv) in acetonitrile/dimethylformamide (1:1, 54 mL) at 0
°C under N2 was added the above monoacid intermediate (1.59
g, 8.19 mmol, 1 equiv), iPr2NEt (2.84 mL, 16.38 mmol, 2 equiv),
and BOP (3.62 g, 8.19 mmol, 1 equiv). The reaction mixture
was stirred for 20 h at room temperature, and the precipitate
(A) was filtered (filtrate B). The precipitate A was dissolved
in chloroform (80 mL) and was washed sequentially with 0.02
N hydrochloric acid (40 mL), saturated sodium bicarbonate (40
mL), and 10% lithium chloride (2 × 40 mL), dried over sodium
sulfate, filtered, and concentrated in vacuo to give 31 (2.40 g).
The filtrate B was concentrated to 9 mL, diluted with ethyl
acetate (150 mL), washed with the same solvents as above,
dried over sodim sulfate, filtered, and concentrated in vacuo.
The residue was triturated with hexane (50 mL) and ether
(50 mL), filtered, and dried in vacuo to give additional 31 (1.1
g) which was combined with the material from above to give
3.5 g (79%) overall yield: TLC Rf ) 0.6 (9:1:0.05 CHCl3/MeOH/
HOAc visualized by ceric ammonium sulfate); MS (M + H)+
538; IR (KBr) 1642 cm-1; 1H NMR (270 MHz, CDCl3) δ 0.85-
0.98 (m, 12 H), 2.01 (s, 3 H), 1.95-2.15 (m, 4 H), 2.45 (m, 2
H), 3.46 (dd, 2 H, J ) 16.42), 3.67 (s, 3 H), 4.42-4.75 (m, 3
H), 5.11 (s, 2 H), 7.3 (m, 5 H); 13C NMR (68 MHz, CDCl3) δ
15.2, 18.7, 18.9, 19.1, 30.1, 30.8, 31.9, 42.3, 51.37, 52.2, 58.4,
58.6, 66.9, 128.2, 128.5, 135.5, 165.7, 168.2, 171.5, 172.0, 172.2.
Anal. (C26H39N3O7S‚0.64H2O) C, H, N.
1
1755, 1636 cm-1; H NMR (400 MHz, CDCl3/CD3OD, 10:1) δ
0.96 (m, 12 H), 1.60 (s, 6 H), 1.64 (s, 3 H), 1.67 (s, 3 H), 2.07and
2.08 (s, 3 H), 1.60-2.53 (m, 22 H), 3.72 and 3.73 (s, 3 H), 3.44-
4.00 (m, 6 H), 4.25-4.61 (m, 3 H), 4.98-5.09 (m, 4 H); 13C NMR
(100 MHz, CDCl3/CD3OD, 10:1) δ 15.2, 16.1, 17.7, 18.0, 18,4,
19.4, 20.3, 25.2, 25.8, 26.0, 26.9, 27.1, 29.5, 30.4, 30.6, 30.8,
30.9, 31.3, 40.1, 48.5, 48.7, 48.9, 49.1, 49.4, 51.8, 52.5, 59.2,
59.6, 64.6, 104.8, 105.0, 120.3, 124.4, 124.7, 131.5, 135.4, 138.5,
168.6, 170.8, 172,4, 172.6. Anal. (C40H68N4O8S‚0.16H2O) C,
H, N.
To a solution of of this intermediate (640 mg, 0.835 mmol,
1 equiv) in tetrahydrofuran (8 mL) was added p-toluenesulfonic
acid monohydrate (317 mg, 1.67 mmol, 2 equiv). After the
mixture was stirred for 6 h, more p-toluenesulfonic acid
monohydrate (158 mg, 0.835 mmol, 1 equiv) was added, and
the reaction mixture was stirred an additional 3 h. The
solvents were removed in vacuo, and the residue was dissolved
in 100 mL of ethyl acetate and washed sequentially with 10%
sodium bicarbonate (50 mL) and saturated sodium chloride
(50 mL). The organic layer was dried over sodium sulfate,
filtered, and concentrated in vacuo. The residue was purified
on Merck silica gel (110 mL), eluting with hexane/acetone (4:1
to 1:2) to give 33 (183 mg, 32%): mp 185-186 °C dec; TLC Rf
) 0.11 (hexane/acetone, 2:1, visualized by ceric ammonium
sulfate); MS (M + H)+ 681; IR (KBr) 1751, 1638 cm-1; [R]D
)
-40.6° (c ) 0.20, CH3OH); 1H NMR (270 MHz, CD3OD) δ 0.98
(m, 12 H), 1.54 (s, 6 H), 1.64 (s, 3 H), 1.66 (s, 3 H), 2.07 (s, 3
H), 1.96-2.54 (m, 16 H), 3.71 (s, 3 H), 3.42-3.71(m, 4 H), 4.23-
4.55 (m, 3 H), 5.10 (br m, 3 H); 13C NMR (100 MHz, CD3OD)
δ 15.3, 16.2, 17.8, 18.3, 18.8, 19.7, 25.9, 26.2, 27.3, 27.6, 30.8,
31.2, 31.3, 31.6, 40.6, 41.2, 48.0, 48.4, 48.7, 49.0, 49.3, 49.6,
49.9, 52.3, 52.4, 52.7, 60.0, 60.3, 120.9, 125.0, 125.2, 131.8,
135.7, 138.7, 169.6, 170.1, 173.2, 173.3, 173.4. Anal. (C35H60
-
N4O7S‚0.42H2O) C, H, N.
P r ep a r a tion of 34. To a solution of 33 (113 mg, 0.166
mmol, 1 equiv) in dioxane/methanol/water (3:1:0.5, 4.5 mL)
was added 1 N sodium hydroxide (0.232 mL, 0.232 mmol, 1.4
equiv). After the mixture was stirred for 16 h, the solvents
were removed in vacuo, and the residue was purified on CHP-
20P (15 mL), eluting with water/acetonitrile (100:0 to 0:100)
to give 34 (90 mg, 79%): mp 169-171 °C; TLC Rf ) 0.27 (9:
1:0.05 CHCl3/MeOH/HOAc, visualized by ceric ammonium
sulfate); IR (KBr) 1572 cm-1; [R]D ) -23.7° (c ) 0.29, CH3-
OH); 1H NMR (270 MHz, CD3OD) δ 0.86-0.90 (m, 12 H), 1.51
(s, 6 H), 1.56 (s, 3 H), 1.57 (s, 3 H), 1.96 (s, 3 H), 1.88-2.41
(m, 16 H), 3.49 (m, 2 H), 4.10 (d, 1 H, J ) 7.63), 4.20 (m, 2 H),
P r ep a r a tion of 32. To a solution of 31 (3.3 g, 6.14 mmol,
1 equiv) in dimethylformamide/water (4:1, 165 mL) were added
iPr2NEt (9.61 mL, 55.3 mmol, 9 equiv) and palladium hydrox-
ide on carbon (350 mg). The reaction mixture was stirred
under an H2 atmosphere for 16 h, filtered through Celite, and
concentrated in vacuo. The residue was dissolved in methanol/

Hydroxamic Acid-Based Inhibitors of Ras FPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4209
4.8 (m, 2 H, D2O also), 5.03 (br m, 3 H); 13C NMR (68 MHz,
CD3OD) δ 15.3, 16.1, 16.2, 17.7, 18.4, 18.9, 19.9, 25.9, 26.4,
27.6, 27.8, 31.1, 34.1, 40.8, 48.1, 48.4, 48.7, 49.0, 49.3, 49.6,
49.9, 50.3, 55.7, 60.4, 60.7, 121.5, 125.4, 125.5, 132.1, 136.0,
138.8, 169.4, 170.1, 172.5, 173.7, 177.7. HRMS (M + H)+
calculated 667.4105, found 667.4099. Anal. (C34H57N4O7-
SNa‚1.5H2O) C, H, N.
sylated and geranylgeranylated substrates. Biochem. J . 1992,
284, 835-840. (f) Shi, Y.-Q.; Rando, R. R. Kinetic mechanism of
isoprenylated protein methyltransferase. J . Biol. Chem. 1992,
267, 9547-9551. (g) Gilbert, B. A.; Tan, E. W.; P-Sala, D.; Rando,
R. R. Structure-activity studies on the retinal rod outer segment
isoprenylated protein methyltransferase. J . Amer. Chem. Soc.
1992, 114, 3966-3973.
(10) Hancock, J . F.; Cadwallader, K.; Marshall, C. J . Methylation
and proteolysis are essential for efficient membrane binding of
prenylated p21K-ras(B). EMBO J . 1991, 10, 641-646.
Ack n ow led gm en t. We are very thankful to Dr.
Veeraswamy Manne for the GGT-1 in vitro and RTI
whole-cell evaluation of inhibitor 23 and for purification
of p21 ras and farnesyl protein transferase.
(11) (a) Hancock, J . F.; Paterson, H.; Marshall, C. J . A Polybasic
domain or Palmitoylation is required in addition to the CAAX
motif to localize p21ras to the plasma membrane. Cell 1990, 63,
133-139. (b) Hancock, J . F.; Cadwallader, K.; Paterson, H.;
Marshall, C. J . A CAAX or a CAAL motif and a second signal
are sufficient for plasma membrane trageting of ras proteins.
EMBO J . 1991, 10, 4033-4039.
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