Efficient syntheses, human and yeast farnesyl-protein transferase inhibitory activities of chaetomellic acids and analogues

Article abstract of DOI:10.1016/S0968-0896(99)00312-0

Chaetomellic acids are a class of alkyl dicarboxylic acids that were isolated from Chaetomella acutiseta. They are potent and highly specific farnesyl-pyrophosphate (FPP) mimic inhibitors of Ras farnesyl-protein transferase. We have previously described t

Full text of DOI:10.1016/S0968-0896(99)00312-0

Bioorganic & Medicinal Chemistry 8 (2000) 571±580
Ecient Syntheses, Human and Yeast Farnesyl-Protein
Transferase Inhibitory Activities of Chaetomellic Acids
and Analogues
Sheo B. Singh,* Hiranthi Jayasuriya, Keith C. Silverman, Cynthia A. Bon®glio,
Joanne M. Williamson and Russell B. Lingham
Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 15 August 1999; accepted 28 October 1999
AbstractÐChaetomellic acids are a class of alkyl dicarboxylic acids that were isolated from Chaetomella acutiseta. They are potent
and highly speci®c farnesyl-pyrophosphate (FPP) mimic inhibitors of Ras farnesyl-protein transferase. We have previously described
the ®rst biogenetic type aldol condensation-based total synthesis of chaetomellic acid A. Modi®cation of the later steps of that synthesis
resulted in the ecient syntheses of chaetomellic acids A and B in three steps with 75±80% overall yield. In this report, details of the
original total syntheses of chaetomellic acids A, B and C, the new syntheses of acids A and B and structure±activity relationship of these
compounds against various prenyl transferases including human and yeast FPTase and bovine and yeast GGPTase I are described.
Chaetomellic acids are di€erentially active against human and yeast FPTase. Chaetomellic acid A inhibited human and yeast FPTase
activity with IC50 values of 55 nM and 225 mM, respectively. In contrast, chaetomellic acid C showed only a 10-fold di€erential in inhi-
bitory activities against human versus yeast enzymes. In keeping with molecular modeling-based predictions, the compounds with
shorter alkyl side chains (C-8) were completely inactive against FPTase. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
In 1993 we reported the isolation of chaetomellic acids
A (1a) and B (2a; Fig. 1), as potent inhibitors of
FPTase.7,8 We also subsequently reported the ®rst
synthesis of chaetomellic acid A based on an aldol
condensation approach.9 These acids were character-
ized as alkyl cis-dicarboxylates and appear to mimic
FPP (3; Fig. 1) at the active site of the enzyme.7 Struc-
turally, these acids resemble FPP as demonstrated by
molecular modelling.7 Among the chaetomellic acids, 1a
(IC50=55 nM) is 3 times more active than 2a (IC50=
185 nM) against recombinant human FPTase.7 These
classes of natural products have a propensity to cyclize
and most members were isolated in the anhydride
form.7
The ras oncogenes are mutated in about 25% of human
tumors.1 These genes encode 21kDa proteins called p21
or Ras. The normal and oncogenic activity of this pro-
tein is dependent on its ability to associate with the
inner lea¯et of the plasma membrane that occurs as a
result of post-translational modi®cation. Out of several
post-translational steps, farnesylation of the cysteine
residue of the C-terminal CaaX motif of Ras is essential
and obligatory2 and is catalyzed by the heterodimeric
enzyme
farnesyl±protein
transferase
(FPTase).
Recently, numerous potent inhibitors of this enzyme
have been reported from various laboratories.3,4 These
inhibitors were derived from various sources including
rationally designed peptidomimetics and screening of
natural products and has led to the con®rmation, at
least in animal models, that inhibition of FPTase is a
viable target for the development of drugs for the treat-
ment of cancer.5,6 In fact, a number of FPTase inhibi-
tors are in human clinical trials by several companies
including Merck.
There have been six new reports of syntheses of chaeto-
mellic acid A from a number of groups. These syntheses
include a cobaloxime-mediated synthesis reported10,11
by Branchaud and Slate from the University of Oregon;
a succinate to maleate oxidation method reported by
Kates and Schauble12 from Villanova University; an
elegant two-step synthesis by the groups of Vederas and
Poulter13 from the University of Alberta and the Uni-
versity of Utah involving alkyl organocuprates; three
syntheses by the Argade's group14±16 from the National
Chemical Laboratory of India; and a Barton radical
*Corresponding author. Tel.: +1-732-594-3222; fax: +1-732-594-6880;
e-mail: sheo_singh@merck.com
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00312-0

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S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
Figure 1.
decarboxylation-based synthesis reported by the Samadi
group17 from CNRS, France.
reaction of lithium diisopropylamide with methyl pal-
mitate gave an enolate that was slowly added to the
cooled ( 78 ꢀC) solution of methylpyruvate. This gave a
1:1 diastereomeric mixture of aldol products 5a and 6a in
greater than 80% yield (Scheme 1). These products were
readily separated by silica gel chromatography and their
stereochemistry was determined based on the products
of b-elimination reactions (vide infra).
Our original synthesis was designed to generate ole®nic
isomers to examine the structure activity relationships.
A simple modi®cation of our initial synthesis resulted in
>80% overall yield of the cis-dicarboxylates in three
steps. In the present paper we describe the details of our
original aldol condensation based total syntheses of
chaetomellic acids A and B, the modi®ed synthesis of
both acids, synthesis of chaetomellic acid C, the isola-
tion of new chaetomellic acid anhydrides D±E, and the
biological evaluation of these compounds and isomers
in human and yeast FPTase and GGPTase I assays.
The aldol reaction of methyl oleate with methyl pyr-
uvate was performed in a similar manner to give a 1:1
diastereomeric mixture of 5b and 6b with a greater than
80% yield. Operationally this reaction was easier due to
the increased solubility of the unsaturated enolate at lower
temperature. A similar reaction of methyl myristate with
methyl pyruvate gave 5c and 6c.
ꢀ-Elimination reaction. The direct acid catalyzed elim-
ination of water molecules from the aldol products to
generate the tetra-substituted ole®n failed due to the
diculty of formation of a carbonium ion a- to the
carboxymethyl group. However, it was possible to apply
a base catalyzed b-elimination approach to generate the
necessary ole®n. Thus, separate reactions of 5a and 6a
with four molar equivalents of p-toluenesulfonic anhy-
dride, two molar equivalents of 2,6-di-tert-butyl-4-
methylpyridine in a mixture of CH2Cl2 and pyridine
(1.5:1) gave tosylates 7a and 8a, respectively, which were
used in the next reaction without puri®cation. Methyl-
ene chloride was removed from the reaction mixture
and the tosylate 7a was heated at 130 ꢀC after addition
of DBU to give the citraconate diester 9a (Z-isomer),
the itaconate diester 10a and the mesaconate diester 11a
(E-isomer) in 4:3:0.5 ratio, respectively. The combined
two step yield starting from 5a to 9a±11a was greater
than 85%. Similar reactions with tosylate 8a a€orded
predominantly the E-isomer 11a with minor amounts of
Synthesis of chaetomellic acids
The synthetic strategy of chaetomellic acids involved a
biomimetic-type aldol condensation of the appropriate
fatty acid ester with pyruvate followed by elimination of
an equivalent of water and hydrolysis of the ester
groups. Details of synthesis follows.
Aldol condensation. Aldol condensations of the appro-
priate fatty acid esters with methylpyruvate would give
an aldol product that would have the carbon skeleton of
chaetomellic acids. The aldol reactions in the standard
conditions Ð addition of methylpyruvate to enolate Ð
resulted in very poor yields due to the insolubility of the
enolate at 78 ꢀC. This problem could not be cir-
cumvented by raising the temperature due to the base
catalyzed polymerization of methyl pyruvate at room
temperature. However, performing the aldol reaction in
the reverse order easily overcame this problem. The
Scheme 1. Aldol condensation of fatty acid esters with methyl pyruvate.

S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
573
9a and 10a. The elimination reaction of isomer 7a was
relatively slower than that of isomer 8a. The stereo-
chemistry of aldol products 5a and 6a was assigned as
SÃ,RÃ and SÃ,SÃ, respectively, based on the product dis-
tribution of the elimination reactions just described. The
assumption was made that the anti-periplanar elimina-
tion would be the predominant pathway whenever pos-
sible and would result in a faster rate of reaction. This
would be consistent with the formation of the E-ole®n
11a from 8a (SÃ,SÃ) via anti-periplanar elimination and
formation of 9a via syn or periplanar elimination from
7a (SÃ,RÃ). Formation of the terminal ole®n 10a was
suppressed to less than 10% when the elimination reac-
tion was performed in re¯uxing toluene with 2 equivalent
of DBU using puri®ed tosylate (Scheme 2).
Similar separate hydrolyses of diesters 10b, 10c and 11b,
11c gave the corresponding terminal ole®nic derivatives
of chaetomellic acids B and C, 13b and 13c and E-diacids
14b, 14c, respectively.
Modi®ed synthesis of chaetomellic acids A and B
The mixtures of aldol products 5a/6a and 5b/6b were
hydrolyzed by re¯uxing in either 4 N NaOH (5a/6a) or
LiOH (5b/6b) to give 92 and 99% yields of diacids 15a
and 15b, respectively (Scheme 4). These acids were not
puri®ed and re¯uxed in acetic anhydride for about 2 h
to give anhydrides of chaetomellic acids A (12a) and B
(12b) in almost quantitative yields. This modi®cation
produced chaetomellic acids A and B in >75 and >80%
overall yields, respectively, and is one of the most
ecient synthesis of these compounds reported to date.
Aside from stereochemical assignment, the puri®cation
of the aldol product was not necessary for the prepara-
tion of ole®ns. In fact, the mixture of aldol products 5b/
6b and 5c/6c reacted readily with p-toluenesulfonic
anhydride to give a mixture of tosylate 7b/8b and 7c/8c
which were heated in toluene in the presence of DBU to
give the corresponding mixtures of ole®ns 9b, 10b, 11b
and 9c, 10c, 11c, respectively. These ole®ns were readily
separated by silica gel chromatography.
Hydrolysis of esters
Methyl esters were hydrolyzed by re¯uxing with 1 N
sodium hydroxide in a mixture of methanol±THF±H2O
to give the corresponding acids or anhydrides in 90-
95% yield. Hydrolysis of Z-diesters 9a, 9b and 9c was
rapid and gave the anhydrides of chaetomellic acid A
12a, chaetomellic acid B 12b, and chaetomellic acid C
12c, respectively. We have demonstrated earlier that the
anhydrides can be converted to chaetomellic acid A (1a)
at pH 7.5 in solution. Re¯uxing of 11a with 1 N NaOH
overnight gave the trans-diacid 14a. Hydrolysis of 10a
accordingly produced predominantly 13a with minor
amounts of 12a and 14a (Scheme 3).
Scheme 3. Hydrolysis of esters. Reagents: (i) a. 1 N NaOH, CH3OH±
THF±H2O. 80 ꢀC; b. 4 N HCl.
Scheme 2. b-Elimination reactions of tosylated aldol products. Reagents: (i) p-toluenesulfonic anhydride, CH2Cl2, C5H5N, 2,6-di-tert-butyl-4-
methylpyridine, 40 ꢀC; (ii) toluene, DBU, re¯ux.

574
S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
Scheme 4. Modi®ed synthesis of chaetomellic acids A and B. Reagents: (i) LiOH, THF±H2O; (ii) Ac2O, re¯ux.
(Table 1). We have demonstrated earlier that these
Isolation and structure of chaetomellic acids D (1b) and E
(2b)
reversible inhibitors are competitive with respect to FPP
for their inhibition and are selective for FPTase since
they do not inhibit other prenyl transferases such as
GGPTase-I or other FPP-utilizing enzymes such as
squalene synthase. Chaetomellic acid A is the most
potent (IC50=55 nM) compound of the series while the
longer chain acid B was less potent (IC50=155 nM). The
molecular modelling experiments with chaetomellic acid
A and FPP indicated that a cis-diacid and a C-12 chain
would be optimal for binding assuming an extended
conformation of the side chain. To evaluate this predic-
tion the isomeric diacids (13a, 13b, 14a, 14b) of chaeto-
mellic acids A and B and C-12 chain compound
chaetomellic acid C (4) and its isomers (13c, 14c) were
synthesized and results are summarized below.
Gel permeation chromatography of a methyl ethyl
ketone extract of an unidenti®ed sterile mycelial broth
followed by reverse-phase HPLC yielded minor
amounts chaetomellic acids D (1b) and E (2b) in addi-
tion to signi®cant amounts of chaetomellic acids A and
B. High resolution EI mass spectral analysis of 1b and
2b showed molecular formulae of C19H34O5 and
C21H36O5, respectively. The comparison of these for-
mulae with that of chaetomellic acids A and B indicated
that these compounds contained an additional oxygen
1
atom. H NMR spectrum of both compounds showed
the absence of the ole®nic methyl group in each of the
two compounds and the presence of a hydroxymethyl
1
signal at ꢁd 4.62 ppm. The remainder of the H NMR
spectrum of 1b and 2b was identical to that of 1a and
2a. Based on these observations structures 1b and 2b
were assigned to chaetomellic acids D and E, respec-
tively (see Fig. 1). In contrast to chaetomellic acids A
and B, these compounds tended to exist in the diacid
form under non acidic conditions as indicated by mass
spectral analysis that could be due to the presence of the
polar hydroxymethyl group.
The vinyl diacid analogue of chaetomellic acid A (13a)
is 36 times less active when compared to chaetomellic
acid A and its trans isomer (14a) is completely inactive
up to 100 mM. Both vinyl (13b) and trans (14b) isomers
of chaetomellic acid B were completely inactive up to
100 mM. Surprisingly, the prediction of chain length by
molecular modelling did not hold up and chaetomellic
acid C (4), the acid that was predicted to be the most
active, exhibited an IC50 value of 500 nM and was
approximately 10-fold less active than chaetomellic acid
A. However, the activity pattern of the vinyl (13c) and
trans isomers (14c) of chaetomellic acid C (4) was sig-
ni®cantly di€erent from the corresponding isomers of
Structure±activity relationship of chaetomellic acids
Chaetomellic acids A and B are potent, selective and
speci®c inhibitors of recombinant human FPTase
Table 1. Prenyl transferase activities of chaetomellic acids and analogues
Compounds
rHFPTasea
55 nM
GGPTase Ib
ScFPTasec
ScGGPTased
Chaetomellic acid A (1a)
92 mM
17 mMe
225 mMf
300 mM*
4 mMe
3 mMf
Ð
>300 mMe
Chaetomellic acid B (2a)
Chaetomellic acid C (4)
185 nM
500 nM
54 mM
Ð
Ð
112 mMe
Chaetomellic acid D (1b)
Chaetomellic acid E (2b)
Vinyl acid A (13a)
Vinyl acid B (13b)
Vinyl acid C (13c)
Trans acid A (14a)
Trans acid B (14b)
Trans acid C (14c)
Aldol diacid (15a)
C-8 acid (16)
250 nM
270 nM
2 mM
>100 mM
4 mM
>100 mM
>100 mM
5 mM
>100 mM
>100 mM
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
2.4 mMe
96 mMe
Farnesyl diacid (17)
Geranylgeranyl diacid (18)
277 mMe
11.5 mMe
Ð
aRecombinant human FPTase.
bBovine brain GGPTase I.
cSaccharomyces cerevisiae FPTase.
dSaccharomyces cerevisiae GGPTase.
eData from University of Utah, continuous ¯uorescence assay results.
fData from Merck Research Laboratories, SPA assay results.

S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
575
acids A and B. Compounds 13c and 14c showed IC50
values of 4 and 5 mM, respectively. They showed only 8-
and 10-fold lower activity when compared to the corre-
sponding cis-isomer chaetomellic acid C.
and Poulter originally reported that chaetomellic acid A
was signi®cantly less active against ScFPTase (IC50=17
mM) when compared to mammalian FPTase (IC50=55
nM). The activity of chaetomellic acid A against ScFPTase
was even weaker (IC50=225 mM) when tested in our
laboratories. The reasons for this di€erence in potency
are not readily apparent but could be due to di€erences
in the method of measurement of yeast FPTase activity
in the two laboratories. The University of Utah group
used a continuous ¯uorescence assay21 while the Merck
group made use of Scintillation Proximity Assay (SPA).
Furthermore, the apparent lack of activity of acid A
against yeast FPTase is surprising given that the yeast
and mammalian enzymes share a common a-subunit.
The weaker potency of acid C, compared to acids A and
B, contradicts the prediction made by the modeling
experiments. This prediction was obviously based on the
assumption that the respective alkyl side chains exists in
an extended conformation at the active site of the
enzyme. It is obvious now that this assumption may not
be correct and it is possible that the alkyl side chain
could assume alternative conformations within the
active sites, one of which could be coiled conformation.
This observation is further supported by the complete
lack of inhibitory activity of C-8 acid (16).18
The C-12 chain acid (3)10 is 10 times less active against
human FPTase when compared to the C-14 chain acid
(1a) vide supra. However, it is more active (IC50=3 to 4
mM) than chaetomellic acid A (IC50=17 or 225 mM)
when tested against yeast FPTase. Aside from the
synthesis and activity of chaetomellic acid C, the Poul-
ter and Vederas groups also reported13 the preparation
and the activities of chaetomellic acid analogues con-
sisting of farnesyl (17) and geranylgeranyl (18) chains
(Fig. 2). As expected, the farnesyl acid was active
against yeast FPTase (IC50=2.4 mM) and was 100-fold
selective over yeast GGPTase. In contrast, the ger-
anylgeranyl acid (18) was less active against yeast
GGPTase activity (IC50=11.5 mM) and was also less
selective (10-fold over yeast FPTase).
X-ray co-crystal structure of rat-FPTase with FPP indi-
cates a bend in extended conformation of FPP at the
active site.19 It is possible that a similar bend may occur
for chaetomellic acids thereby facilitating and maximiz-
ing the hydrophobic interactions in the binding pocket.
For chaetomellic acids to exhibit potent inhibitory
activities against FPTase the dicarboxylate groups must
be in register with the divalent zinc atom present at the
active site. It appears that binding of the dicarboxylate
group of chaetomellic acid A coordinate best with the
zinc atom. Chaetomellic acids, with longer or shorter
chains, forces the dicarboxylate away from the zinc
register and leads to poor inhibitor binding and, hence,
inhibitory activity. These results are consistent with the
molecular ruler hypothesis of substrate binding and
speci®city.19
The observation of di€erences in the potency of chae-
tomellic acids between mammalian and fungal (or yeast)
FPTase is noteworthy. Chaetomellic acid A is more
active against both recombinant human and bovine
brain FPTase (IC50=55 nM) and is less active against
yeast FPTase (IC50=17±225 mM), depending on the
assay used. These di€erences in the IC50 values are
similar to the di€erences in the anity of FPP for the
two enzymes. FPP has a higher anity for mammalian
FPTase (KD=12 nM,22,23 than yeast FPTase (KD=75
nM).24,25 Inhibitors that compete with FPP for binding
for their inhibitory activity may be expected to re¯ect
this di€erence of the KD values in their IC50 values.
Substitutions of the ole®nic methyl group with smaller
(for example, H) or larger group (for example, Ph) in the
head unit of chaetomellic acid A resulted in a signi®cant
reduction in inhibitory activity (Singh and Graham,
unpublished results). However, the hydroxymethyl
group substitution did not have any signi®cant e€ect on
the potency of these compounds. For example, chaeto-
mellic acid D (1b) and chaetomellic acid E (2b) (Fig. 1)
displayed IC50 values of 250 and 270 nM, respectively.
There was no signi®cant di€erence in the inhibitory
activities of chaetomellic acids when tested against other
mammalian prenyl transferases such as bovine brain
FPTase.8,20 The esteri®cation of the carboxyl groups of
chaetomellic acids caused the complete loss of inhibi-
tory activities.
In conclusion, we have described the ecient synthesis
of chaetomellic acids A, B, C and several isomers. These
compounds were evaluated in various prenyl transferase
assays including human and yeast FPTase, and bovine
and yeast GGPTase. The activity pro®les of these com-
pounds in these assays were surprisingly di€erent but
not beyond reasonable explanation. The cisoid orienta-
tion of the dicarboxylic acids along with an alkyl side
chain of proper length are critical for binding and hence
inhibitory activities. The trans-, and vinyl-diacids are
These compounds were evaluated against yeast (Sacchar-
omyces cerevisiae) FPTase and GGPTase I enzymes and
the comparative activity pro®les are presented in Table
1. The data is compared to that reported by Poulter's
group of the University of Utah. The groups of Vederas
Figure 2. Structures of synthetic and natural chaetomellic acid analogues (diacid form).

576
S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
signi®cantly less active in human FPTase. cis-Diacids
consisting of shorter alkyl chain were inactive against
human FPTase as predicted by molecular modeling
experiments. All of the chaetomellic acids are signi®cantly
less active against yeast FPTase as would be expected due
to the di€erences in the KD values of the two enzymes.
methyl pyruvate (1.2 mL, 12 mmol) in THF (10 mL).
The progress of the reaction was monitored on TLC
(hexane:EtOAc, 4:1). The solution was stirred for 2 h
and then quenched with 10% aqueous citric acid at
78 ꢀC and poured on to 300 mL EtOAc. The layers
were separated and organic phase was washed with 100
mL water, dried over sodium sulfate, concentrated
under reduced pressure and ®ltered through a bed of
silica gel. Elution with 5% EtOAc in hexane gave 3.1 g
(81.8%) of a 1:1 diastereomeric mixture of aldol pro-
ducts as a gum. A small portion of the mixture was
chromatographed on a ¯ash silica gel (200 cc) column
packed in hexane. The column was eluted with 5%
EtOAc in hexane to a€ord the ®rst diastereomer (5a,
HPLC tR=7.0 min, Whatman ODS-3, C-18 (4.6Â250
mm), CH3OH:H2O, 92:8, at a ¯ow rate of 1.5 mL/min),
followed by the mixture and then the second diastereo-
mer (6a, tR=6.4 min). Both compounds were obtained
Experimental
General procedure
All commercial reagents were obtained from Sigma±
Aldrich and were used without any further puri®cation.
Biotinylated peptides were synthesized by Synpep and
the molecular weights veri®ed by MS by the supplier.
[3H]-Farnesylpyrophosphate was purchased from either
Amersham or NEN; the speci®c radioactivity varied
(15±22 Ci/mM) depending on the lot and supplier. Non-
radioactive farnesylpyrophosphate was purchased from
ARC, Inc. Strepavidin±SPA beads were supplied by
Amersham. Hepes bu€er, dithiothreitol, skim milk,
bovine serum albumin (BSA), and dodecylmaltoside
were from Sigma.
1
as solids. 5a: H NMR (CDCl3, d): 3.75 (3H, s), 3.67
(3H, s), 2.76 (1H, dd, J=10.5, 3.9 Hz), 1.68 (2H, m),
1.41 (3H, s), 1.25 (24H, brm), 0.87 (3H, t, J=6.3 Hz);
FABMS (m/z): 373 (M+H), 340, 322, 313 (100%). 6a:
1H NMR (CDCl3, d): 3.79 (3H, s), 3.71 (3H,s), 2.75
(1H, dd, J=11.7, 3.3 Hz), 1.83 (2H, m), 1.42 (3H, s),
1.23 (24H, m), 0.87 (3H, t, J=6.3 Hz); FABMS (m/z):
373 (M+H), 340, 322, 313 (100%).
Mass spectra were recorded on Finnigan±MAT model
MAT212 (electron impact, EI, 90 eV), MAT 90 (Fast
Atom Bombardment, FAB), TSQ70B (FAB, EI), and
Finnigan FTMS mass spectrometers. Exact mass mea-
surements were performed by either high resolution
(HR-EI) using per¯uorokerosene (PFK) or per¯uoro-
polypropylene oxide (Ultramark U1600F) or PEG600
(FTMSESI) as an internal standard. Trimethyl silyl
derivatives were prepared with a 1:1 mixture of
BSTFA:pyridine at room temperature. 13C NMR spec-
tra were recorded in CD2Cl2 or CD3OD or CDCl3 at 75
MHz on a Varian XL-300 spectrometer or at 100 MHz
on a Varian Unity-400 spectrometer. Chemical shifts
are given in ppm relative to TMS at zero using the sol-
vent peak at 53.8 ppm (CD2Cl2), 49.00 ppm (CD3OD)
and 77.00 ppm (CDCl3), respectively, as an internal
standard. 1H NMR spectra were recorded in CD2Cl2 or
CD3OD or CDCl3 at 300 MHz on a Varian XL-300
spectrometer or at 400 MHz on a Varian Unity-400
spectrometer. Chemical shifts are given in ppm relative
to TMS at zero using the solvent peak at 5.32 ppm
(CD2Cl2), 3.30 ppm (CD3OD) and 7.26 ppm (CDCl3),
respectively, as an internal standard.
Aldol condensation of methyloleate and methylpyruvate.
Methyl oleate (5.29 g, 10 mmol) was reacted with 12
mmol of methylpyruvate in a manner identical to the
reaction just described. Silica gel ®ltration gave 6.52 g
(82.3%) of 1:1 mixture of diastereomeric aldol products
as oil. A small portion of this mixture was chromato-
graphed on a ¯ash silica gel (200 cc) column packed in
hexane. The column was eluted with 5% EtOAc in hex-
ane to a€ord the ®rst diastereomer (5b), a mixture of
both and the second diastereomer (6b), all as oil. 5b: 1H
NMR (CDCl3, d): 5.34 (2H, m), 3.76 (3H, s), 3.68 (3H, s),
3.65 (1H, brs), 2.76 (1H, dd, J=10.2, 3.9 Hz), 2.0 (4H, m),
1.66 (2H, m), 1.42 (3H,s), 1.27 (20H, brm), 0.88 (3H, t,
J=6.8 Hz); HRESI-FTMS (m/z): 399.3093 [M+H] (calcd
1
for C23H43O5: 399.3111). 6b: H NMR (CDCl3, d): 5.34
(2H, m), 3.80 (3H, s), 3.72 (3H,s), 3.51 (1H, brs), 2.72 (1H,
dd, J=11.7, 3.3 Hz), 2.0 (4H, m), 1.84 (2H, m), 1.43 (3H,s),
1.27 (20H, m), 0.88 (3H, t, J=6.6 Hz). HRESI-FTMS (m/
z): 399.3096 [M+H] (calcd for C23H43O5: 399.3111).
Elimination reaction of methylpalmitate aldol products.
To a solution of 5a (160 mg, 0.43 mmol) in CH2Cl2
(1.5 mL) and pyridine (1.0 mL) was added 2,6-di-tert-
butyl-4-methylpyridine (200 mg, 0.97 mmol) followed
by p-toluenesulfonic anhydride (570 mg, 1.74 mmol)
and the solution was heated at 50 ꢀC under N2 over-
night. Progress of the reaction was monitored on TLC
(hexane:EtOAc, 85:15). The tosylate was less polar than
starting alcohol. A small portion of the tosylate was
puri®ed on a silica gel column using 2 to 5% EtOAc in
hexane to give 7a (1H NMR (CDCl3, d): 7.76 (2H, d,
J=8.4 Hz), 7.32 (2H, d, J=8.4 Hz), 3.83 (3H, s), 3.67
(3H, s), 2.95 (1H, dd, J=11.7, 3.0 Hz), 2.44 (3H, s), 1.81
(3H, s), 1.60 (2H, m), 1.24 (24H, m), 0.88 (3H, t, J=6.6
Hz), FABMS (m/z): 527 (M+H)). DBU (0.5 mL) was
added and the reaction mixture was heated at 130±
Aldol condensation of methylpalmitate and methylpyr-
uvate. n-Butyl lithium (1.6 M in hexane, 8.5 mL, 13.6
mmol) was added to a cooled ( 78 ꢀC) solution of dii-
sopropyl amine (2.2 mL, 15 mmol) in THF (10 mL).
The solution was stirred under N2 at 78 ꢀC for 10 min
followed by at 0 ꢀC for 10 min. After cooling the LDA
solution at 78 ꢀC a THF (20 mL) solution of methyl
palmitate (2.7 g, 10 mmol) was added via a syringe over
a 10-min period and stirring was continued for 10 min.
The reaction mixture was slowly allowed to warm to
0 ꢀC and stirred for 30 min resulting in to a faint yel-
low color. This enolate solution was added drop wise
(total addition time 20 min) via a dropping funnel to a
thoroughly cooled ( 78 ꢀC) and stirred solution of

S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
577
140 ꢀC for 3 h to give citraconic acid diester (HPLC,
Whatman ODS-3 (C-18), 4.6Â250 mm, CH3OH:H2O
(92:8), ¯ow rate 1.5 mL/min; tR=8.8 min, ꢁ50%), ita-
conic acid diester analogue (tR=9.4 min, 40%) and
mesaconic acid diester analogue (tR=9.8 min, ꢁ10%).
t, J=7.2 Hz), 2.0 (4H, m), 1.90 (1H, m), 1.66 (1H, m),
1.27 (20H, m), 0.88 (3H, t, J=6.8 Hz); HRESI-FTMS
(m/z): 381.3003 [M+H] (calcd for C23H41O4: 381.3005).
1
11b: H NMR (CDCl3, d): 5.34 (2H, m), 3.78 (3H, s),
3.77 (3H, s), 2.44 (2H, t, J=7.4 Hz), 2.00 (7H, brs and
m), 1.40 (2H, m), 1.27 (18H, m), 0.88 (3H, t, J=6.8 Hz);
HRESI-FTMS (m/z): 381.3012 [M+H] (calcd for
C23H41O4: 381.3005).
Similarly, the reaction of 6a (90 mg, 0.26 mmol) in
CH2Cl2 (1.5 mL), pyridine (1 mL), 2,6-di-tert-butyl-4-
methylpyridine (100 mg) with p-toluenesulfonic anhy-
dride (270 mg) at 50 ꢀC followed by heating with DBU
for 20 min gave major mesaconic acid diester with
minor amounts of citraconate and itaconate isomers.
Both reaction mixtures were cooled to room tempera-
ture, combined and worked up together, they were
poured on to EtOAc (200 mL) and washed sequentially
with 4 N aqueous HCl (3Â50 mL), water, 10% aq
NaHCO3 (3Â50 mL) followed by water. The EtOAc
extract was dried (Na2SO4), evaporated under reduced
pressure. The product mixture was chromatographed on
a ¯ash silica gel column (100 cc) packed in hexane and
eluted with 2% followed by 3% EtOAc to give mesaco-
nate 11a (25 mg), itaconate 10a (88 mg) and citraconate
9a (80 mg) in order of their elution, a combined yield of
81%. The mixtures of aldol products could be used for
Elimination reaction of methylmyristate aldol products
The aldol reaction of methylmyristate with methylpyr-
uvate gave aldol products 5c/6c, which were tosylated to
give 7c/8c, and subjected to elimination reaction just
described to give 9c, 10c and 11c. 9c: 1H NMR (CDCl3,
d): 3.76 (3H, s), 3.75 (3H, s), 2.32 (2H, t, J=7.2 Hz),
1.94 (3H, brs), 1.43 (2H, m), 1.25 (18H, m), 0.88 (3H, t,
J=6.6 Hz); HRESI-FTMS (m/z): 327.2553 [M+H]
1
(calcd for C19H35O4: 327.2535). 10c: H NMR (CDCl3,
d): 6.35 (1H, s), 5.74 (1H, s), 3.76 (3H, s), 3.67 (3H, s),
3.49 (1H, t, J=7.2 Hz), 1.86 (1H, m), 1.65 (1H, m), 1.24
(20H, m), 0.87 (3H, t, J=6.3 Hz); HRESI-FTMS (m/z):
327.2552 [M+H] (calcd for C19H35O4: 327.2535). 11c:
1H NMR (CDCl3, d): 3.78 (3H, s), 3.77 (3H, s), 2.44
(2H, t, J=8.5 Hz), 1.99 (3H, brs), 1.40 (2H, m), 1.25
(18H, m), 0.88 (3H, t, J=7.8 Hz); HRESI-FTMS (m/z):
327.2553 [M+H] (calcd for C19H35O4: 327.2535).
1
the elimination reaction without any problem. 9a: H
NMR (CDCl3, d): 3.75 (3H, s), 3.74 (3H, s), 2.32 (2H, t,
J=7.2 Hz), 1.94 (3H, brs), 1.43 (2H, m), 1.24 (22H, m),
0.86 (3H, t, J=6.6 Hz); HREIMS (m/z): 354.2741 (calcd
for C21H38O4: 354.2808). 10a: 1H NMR (CDCl3, d): 6.35
(1H, s), 5.74 (1H, s), 3.76 (3H, s), 3.67 (3H, s), 3.49 (1H, t,
J=7.2 Hz), 1.86 (1H, m), 1.65 (1H, m), 1.24 (24H, m),
0.87 (3H, t, J=6.3 Hz); EIMS (m/z): 354 (M)+. 11a: 1H
NMR (CDCl3, d): 3.78 (3H,s), 3.77 (3H, s), 2.43 (2H, t,
J=8.5 Hz), 1.99 (3H, brs), 1.40 (2H, m), 1.24 (22H, m),
0.87 (3H, t, J=7.8 Hz); EIMS (m/z): 354 (M)+.
Hydrolysis of dimethyl esters
Chaetomellic acid A anhydride (12a). A solution of
citraconate dimethyl ester 9a (40 mg) in THF (1 mL),
methanol (1 mL), and water (1 mL) was added 4 N
NaOH (0.5 mL) and the mixture was heated at re¯ux
overnight. The progress of the reaction was monitored
on HPLC (Whatman C-18, 4.6Â250 mm, CH3CN:H2O,
90:10, ¯ow rate 1.5 mL/min). After completion of the
reaction it was cooled to 0 ꢀC and acidi®ed with 4 N
HCl to pH 2. The product was extracted with CH2Cl2
(2Â25 mL), washed with water, dried (Na2SO4) and
evaporated to give colorless anhydride (tR=8.9 min)
which solidi®ed upon cooling. This product was iden-
tical with the natural product (HPLC, 1H NMR).
Elimination reaction of methyloleate aldol products. To a
solution of the diastereomeric mixture of 5b and 6b (2.2
g, 5.5 mmol) in CH2Cl2 (10 mL) and pyridine (5 mL)
was added 2,6-di-tert-butyl-4-methylpyridine (2.3 g, 11
mmol) followed by p-toluenesulfonic anhydride (5.4 g,
16.5 mmol). The solution was stirred at room tempera-
ture overnight under N2. The progress of the reaction
was examined by TLC (hexane:EtOAc, 85:15). After
completion of the reaction DBU (4 mL) was added and
CH2Cl2 was removed under vacuum and the mixture
was heated at 130±140 ꢀC for 6 h. The reaction mixture
was cooled to room temperature and poured on to
EtOAc (400 mL) and washed sequentially with 4 N aq
HCl (3Â100 mL), water, 10% aq NaHCO3 (3Â100 mL)
followed by water. The EtOAc extract was dried
(Na2SO4), evaporated under reduced pressure and the
product was chromatographed on a ¯ash silica gel col-
umn (300 cc) packed in hexane. Elution with 1, 2% fol-
lowed by 3% EtOAc in hexane gave ®rst mesaconate
(trans) diester 11b (70 mg), itaconate diester 10b (1.56 g)
and citraconate (cis) diester 9b (160 mg) all as oil. 9b: 1H
NMR (CDCl3, d): 5.34 (2H, m), 3.76 (3H, s), 3.74 (3H,
s), 2.32 (2H, t, J=7.5 Hz), 2.00 (4H, m), 1.94 (3H, brs),
1.57 (2H, m), 1.42 (2H, m), 1.31±1.24 (18H, m), 0.88
(3H, t, J=6.0 Hz); HREIMS (m/z): 380.2928 (calcd for
Chaetomellic acid B anhydride (12b). A solution of cis-
dimethyl ester analogue 9b (55 mg) hydrolyzed accord-
ingly and the product was extracted with EtOAc (3Â50
mL). The EtOAc solution was washed with water, dried
(Na2SO4) and evaporated to give colorless product anhy-
dride as an oil. This product was identical with natural
chaetomellic acid B anhydride (HPLC, 1H NMR).
Chaetomellic acid C anhydride (12c). Hydrolysis of 9c in
an analogous manner gave the anhydride, 1H NMR
(CDCl3, d): 2.45 (2H, t, J=7.5 Hz), 2.07 (3H, s), 1.57
(2H, m), 1.26 (18 H, m), 0.88 (3H, t, J=7 Hz); HRESI-
FTMS of diacid (m/z): 297.2072 [M±H] (calcd for
C17H29O4: 297.2066).
trans-Chaetomellic acid A (14a). Using conditions
described above the mesaconate (trans) diester 11a (4.4
mg) was re¯uxed overnight with 4 N NaOH in metha-
nol (0.5 mL), THF (0.5 mL) and water (0.5 mL). The
1
C23H40O4: 380.2966). 10b: H NMR (CDCl3, d): 6.36
(1H, s), 5.75 (1H, s), 3.77 (3H, s), 3.68 (3H, s), 3.50 (1H,

578
S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
mixture was cooled to room temperature and acidi®ed
to pH 2 by addition of 4 N HCl. The diacid was extracted
with ethyl acetate (3Â20 mL), washed with 20 mL water,
dried (Na2SO4) and evaporated under reduced pressure to
mL) was added 1 mL of 4 N NaOH. The solution was
stirred at room temperature for overnight followed by
heating at re¯ux for 18 h. The reaction was cooled to
room temperature, acidi®ed to pH 2 by addition of
concd HCl and extracted with ethyl acetate (3Â50 mL).
The organic layer was washed with water, dried
(Na2SO4) and evaporated under reduced pressure to
give 240 mg (91.6%) of chromatographically homo-
geneous diastereomeric diacid 15a as colorless powder.
1H NMR (CD3OD, d): 2.73 (1H, m), 1.82 (1H, m), 1.66
(1H, m), 1.44, 1.41 (3H, s), 1.29 (24H, m), 0.89 (3H, t,
J=7.2 Hz); HRESI-FTMS (m/z): 343.2499 [M±H]
(calcd for C19H35O5: 343.2485).
1
give 4 mg of colorless powder of 14a. H NMR (CDCl3,
d): 2.58 (2H, t, J=7.8 Hz), 2.14 (3H, s), 1.52 (2H, m),
1.26 (22H, m), 0.89 (3H, t, J=6.6 Hz); HRESI-FTMS
(m/z): 325.2385 [M±H] (calcd for C19H33O4: 325.2379).
trans-Chaetomellic acid B (14b). Using conditions
described above mesaconate (trans) diester analogue 11b
(45 mg) was re¯uxed overnight to give 21 mg of diacid
14b after puri®cation by HPLC (Whatman C-18, 22Â250
mm, 60 to 80% aq CH3CN containing 0.1% TFA, at a
1
¯ow rate of 10 mL/min) as a powder. 14b: H NMR
Hydrolysis of 5b/6b. To a solution of 540 mg of the
diastereomeric mixtures of 5b/6b in THF (6 mL) was
added a 2 mL aqueous solution of 230 mg of lithium
hydroxide. The solution was stirred overnight at room
temperature followed by heating at 80 ꢀC for 4 h. The
progress of the reaction was monitored by TLC. After
completion, the reaction mixture was allowed to cool
down to ambient temperature and acidi®ed to pH 2 by
addition of concentrated hydrochloric acid, poured on
to EtOAc (100 mL), washed with water (2Â100 mL),
and dried (Na2SO4). EtOAc was removed by con-
centration under reduced pressure and dried in a
vacuum desiccator to give 500 mg (99.6%) of chroma-
tographically homogeneous diacid 15b as a gum. 1H
NMR (CDCl3, d): 5.36 (2H, m), 2.87, 2.86 (1H, each t,
J=9.6 Hz), 2.03 (4H, m), 1.55, 1.49 (3H, s), 1.34-126
(22H, m), 0.90 (3H, t, J=7.2 Hz), HRESI-FTMS (m/z):
370.2719 (calcd for C21H38O5: 370.2757).
(CDCl3, d): 5.34 (2H, m), 2.58 (2H, t, J=7.8 Hz), 2.15
(3H, s), 2.00 (4H, m), 1.52 (2H, m), 1.32, 1.26 (18H, m),
0.88 (3H, t, J=6.6 Hz); ESIMS (m/z): 375 (M+Na).
trans-Chaetomellic acid C (14c). A similar hydrolysis of
11c gave diacid 14c in quantitative yield as a powder.
14c: 1H NMR (CDCl3+CD3OD, d): 2.38 (2H, t, J=8.5
Hz), 1.93 (3H, s), 1.33 (2H, m), 1.13 (18H, m), 0.76 (3H,
t, J=7 Hz); HRESI-FTMS (m/z): 297.2073 [M-H]
(calcd for C17H29O4: 297.2066).
Vinyl-chaetomellic acid A (13a). Vinyl diester 10a (50
mg) was hydrolyzed following the conditions described
for cis- diester. The reaction was analyzed by HPLC
(Whatman C-18 (4.6Â250 mm), CH3CN±H2O +0.08%
TFA, and ¯ow rate 1.5 mL/min). The reaction produced
a mixture of three products, 13a (72%, tR=7.7 min),
12a (20%, tR=12.9 min) and 14a (8%, tR=5.9 min) in
an over all yield of ꢁ80%. These acids were puri®ed by
HPLC (Whatman C-18, 22Â250 mm, gradient elution
with CH3CN±H2O+0.1% TFA, 65 to 75% CH3CN
over 60 min followed by 90% at a ¯ow rate of 10 mL/
min) to give 13a as an amorphous powder. 13a: 1H
NMR (CDCl3, d): 6.52 (1H, s), 5.82 (1H,s), 3.37 (1H, t,
J=7 Hz), 1.93 (1H, m), 1.73 (1H, m), 1.25 (24 H, m),
0.88 (3H, t, J=6.6 Hz); HRESI-FTMS (m/z): 325.2390
[M±H] (calcd for C19H33O4: 325.2379).
Chaetomellic acid B anhydride (12b). The diastereomeric
diacids 15b (460 mg) in 3 mL acetic anhydride was heated
at re¯ux for 2 h. The progress of the reaction was followed
up by HPLC (Zorbax RX C-8, 4.6Â250 mm, CH3CN±
H2O+0.1% TFA, 85/15, 1 mL/min). After completion
of the reaction, acetic anhydride was distilled o€ under
reduced pressure, product was ®ltered through a small
bed of silica gel and eluted with ethyl acetate to give 405
mg (97.6%) homogeneous chaetomellic acid B anhydride
(12b) as a gum.
Vinyl-chaetomellic acid B (13b). A similar hydrolysis of
50 mg of 10b and puri®cation by HPLC (Whatman C-
18, 22Â250 mm, gradient elution with CH3CN:H2O
(containing 1% TFA) 65:35 to 75:25 over 60 min fol-
lowed by 90:10 at a ¯ow rate 10 mL/min) gave 13b as
major product as a gum. 12b and 14b were also isolated
as minor products. 13b: 1H NMR (CDCl3, d): 6.51 (1H,
s), 5.81 (1H, s), 5.35 (2H, m), 3.36 (1H, m), 2.62 (1H,
m), 2.01 (4H, m), 1.75 (1H, m), 1.26 (20H, m), 0.88 (3H,
t, J=7 Hz), HRESI-FTMS (m/z): 351.2530 [M±H]
(calcd for C21H35O4: 351.2535).
Chaetomellic acid A anhydride (12a). The diastereomer-
ic mixture of diacids 15a (12.2 mg) was similarly heated
in 1 mL acetic anhydride for 2 h to give 10.9 mg
(>99%) of chaetomellic acid A anhydride 12a.
Isolation of chaetomellic acids D (1b) and E (2b). A 1.2
L regrowth of sterile mycelia grown on liquid media was
extracted with methyl ethyl ketone (1.2 L) by shaking at
room temperature for 30 min. Methyl ethyl ketone was
evaporated under reduced pressure and residual water
was removed via lyophilization. The mixture was chro-
matographed on a 2.0 L Sephadex LH 20 column and
eluted with methanol. The FPTase active fractions were
pooled to give 600 mg of gum. This material was sus-
pended in 5 mL 1:1 mixture of CH3OH:CH3CN and
®ltered. The ®ltrate was chromatographed, in three
equal aliquots, on a Whatman C-18 column (22Â250
mm). Gradient elution with 70 to 80% aq CH3CN con-
taining 0.2% TFA in 60 min at a ¯ow rate of 10 mL/
Vinyl-chaetomellic acid C (13c). Prepared from 10c as
1
an amorphous powder, H NMR (CDCl3, d): 6.54 (1H,
s), 5.83 (1H, s), 3.36 (1H, m), 1.95 (1H, m) 1.75 (1H, m),
1.26 (20H, m), 0.88 (3H, t, J=7 Hz), HRESI-FTMS (m/
z): 297.2076 [M±H] (calcd for C17H29O4: 297.2066).
Hydrolysis of 5a/6a. To a solution of a mixture of 5a/6a
(284 mg) in methanol (2 mL), THF (2 mL) and water (2

S. B. Singh et al. / Bioorg. Med. Chem. 8 (2000) 571±580
579
min gave two fractions eluting from 31 to 44 min and 45
to 58 min. The former fraction weighing 100 mg con-
tained polar chaetomellic acids D and E whereas the
latter fraction was major and possessed about 300 mg of
1:1 mixture of chaetomellic acids A and B. The polar
fraction containing compounds 1b and 2b was chroma-
tographed again on a similar HPLC column. Elution
with a gradient of 50 to 90% aq CH3CN +0.2% TFA
over 60 min gave 6.4 mg of pure chaetomellic acid D 1b
(elution time 46±47 min) and 2.8 mg of chaetomellic
acid E (elution time 49 min), both as gums. 1b: 1H
NMR (CDCl3, d): 4.62 (2H, s), 2.60 (2H, t, J=8 Hz),
1.63 (2H, m), 1.36±1.29 (22H, m), 0.91 (3H, t, J=7.5
Hz); HREIMS (m/z): 342.2370 (calcd for C19H34O5:
Bovine GGPTase I assay. Partially puri®ed geranylger-
anyl protein transferase type 1 (GGPTase-1) was pre-
pared from bovine brain as described by Moores et al.28
GGPTase-1 was assayed in a volume of 100 mL con-
taining 100 mM Hepes pH 7.4, 5 mM MgC12, 5 mM
dithiothreitol, 100 nM [3H]-geranylgeranyl diphosphate
(15 Ci/mmol, American Radiolabeled Chemicals), 650
nM Ras-CAIL and 1 mg/mL GGPTase at 37 ꢀC for 30
min. Assay tubes were processed as described above for
farnesyl transferase.
Acknowledgements
The authors are thankful to Dr. Z. Guan and Ms. D.
Zink for mass spectral analysis, Ms. R. Jenkins for
regrowth of the fermentation, Drs. M. Goetz, J. Liesch
for their support, and Drs. S. Graham and J. Gibbs for
helpful discussions during the course of this work. The
authors are also grateful to Drs. Gino Salituro and
Robert Schwartz for providing samples of C-8 acid.
1
342.2406). 2b: H NMR (CDCl3, d): 5.38 (2H, m), 4.62
(2H, s), 2.60 (2H, t, J=8 Hz), 2.04 (4H, m), 1.63 (2H, m),
1.36±1.29 (18H, m), 0.91 (3H, t, J=7.5 Hz); HREIMS
(m/z): 368.2535 (calcd for C21H36O5: 368.2562).
Recombinant human FPTase assay. Human FPTase was
prepared as described by Omer et al.26 FPTase assays
were performed as described8 and contained the follow-
ing: 2 nM FPTase, 50 nM [3H]-farnesyl diphosphate
(FPP) and 100 nM Ras-CVIM or 400 nM Ras-CVLS.
All substrates were used at concentrations that corre-
sponded to Km levels. Reactions were kept at at 31 ꢀC
for 60 min. Reactions were initiated with FPTase and
terminated with one mL of 1.0 N HCl in ethanol. Pre-
cipitates were collected onto ®ltermats using a TomTec
Mach II cell harvestor, washed with 100% ethanol,
dried and counted in a LKB b-plate counter. The assay
was linear with respect to both substrates, FPTase levels
and time; less than 10% of the [3H]-FPP was utilized
during the reaction period. Fermentation extracts or
pure compounds were dissolved in 100% DMSO and
diluted 20-fold into the assay to give a ®nal solvent
concentration of 5%.
References
1. Barbacid, M. Ann. Rev. Biochem. 1987, 56, 779.
2. Gibbs, J. B.; Oli€, A.; Kohl, N. E. Cell 1994, 77, 175.
3. Gibbs, J. B.; Oli€, A. Annu. Rev. Pharmacol. Toxicol. 1997,
37, 143.
4. Leonard, D. M. J. Med. Chem. 1997, 40, 2971.
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Gomez, R. P.; Lee, T.-J.; Smith, R. L.; Graham, S. L.; Hart-
man, G. D.; Gibbs, J. B.; Oli€, A. Proc. Natl Acad. Sci. USA
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6. Kohl, N. E.; Omer, C. A.; Conner, M. W.; Anthony, N. J.;
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O'Neill, T. J.; Rands, E.; Schaber, M. D.; Gibbs, J. B.; Oli€,
A. Nature Medicine 1995, 1, 792.
Yeast FPTase assay. Reaction mixtures for the deter-
mination of IC50 values with rScFPTase (pGP114 was
obtained from Dr. C. Dale Poulter of the University of
Utah, rScFPTase was expressed in Esterichia coli from
this plasmid and puri®ed as described27) contained the
following (in a total volume of 0.05 mL): sodium Hepes
bu€er, pH 7.5 (50 mM), biotinyl-YRASNRSCVLS (1.0
mM), rSc FTase (0.05 activity units), [3H]-farnesyl pyro-
phosphate (0.94 mM; 3 Ci/mmol), MgCl2 (1 mM),
ZnCl2 (50 mM), dodecylmaltoside (0.05%), dithiothreitol
(5 mM), DMSO (5%) and test compound. Reaction
mixtures were prepared in 96-well microtiter plates
(Micro¯uor W Flat Bottom plates; Dynex Technologies
Inc). Reaction was initiated by the addition of enzyme.
Incubation was for 15 min at 30 ꢀC. Reaction was ter-
minated by addition to each well of the following solu-
tion (in a total volume of 0.1 mL): phosphoric acid, pH
3.5 (80 mM), MgCl2 (600 mM), sodium azide (0.03%),
skim milk (1.25%), BSA (2.5%), and Strepavidin SPA
beads (0.75 mg; 2-fold molar excess over the peptide
concentration). The contents of the wells were mixed by
rotary shaking and the plates were allowed to stand at
room temperature for 2±24 h before counting. Radio-
activity was determined with a Packard TopCount.
7. Singh, S. B.; Zink, D. L.; Liesch, J. M.; Goetz, M. A.; Jen-
kins, R. G.; Nallin-Omstead, M.; Silverman, K. C.; Bills, G.
F.; Mosley, R. T.; Gibbs, J. B.; Albers-Schonberg, G.; Ling-
ham, R. B. Tetrahedron 1993, 49, 5917.
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