Synthesis and biological evaluation of cycloalkylidene carboxylic acids as novel effectors of Ras/Raf interaction

Article abstract of DOI:10.1021/jm011101q

The protooncogenes Ras and Raf play important roles in signal transduction pathways regulated by mitogen-activated protein kinases. Mutations of Ras that arrest the protein in its active state are frequently implicated in tumor formation. We used Ras and Raf proteins in the yeast two-hybrid system to search for natural or synthesized substances capable of modulating Ras/Raf interaction by specifically binding to one of the interacting partners. We found that cycloalkylidene carboxylic acids enhanced Ras/Raf interaction by acting on the cysteine-rich domain of Raf. Several analogues of the active substance 2-cyclohexylidene propanoic acid were synthesized and the importance of the semicyclic double bond in the stabilization of Ras/Raf interaction was demonstrated. Variation of the size and the substituents of the cyclic system as well as the length of the carboxylic acid resulted in enhanced Ras/Raf interaction.

Full text of DOI:10.1021/jm011101q

J . Med. Chem. 2002, 45, 1535-1542
1535
Syn th esis a n d Biologica l Eva lu a tion of Cycloa lk ylid en e Ca r boxylic Acid s a s
Novel Effector s of Ra s/Ra f In ter a ction
Anke Friese,†,‡ Katja Hell-Momeni,‡,§ Ilse Zu¨ndorf,† Thomas Winckler,*,† Theodor Dingermann,† and
Gerd Dannhardt§
Institut fu¨r Pharmazeutische Biologie, Universita¨t Frankfurt/ M. (Biozentrum), D-60439 Frankfurt am Main, Germany, and
Institut fu¨r Pharmazie, J ohannes Gutenberg-Universita¨t, D-55099 Mainz, Germany
Received November 13, 2001
The protooncogenes Ras and Raf play important roles in signal transduction pathways regulated
by mitogen-activated protein kinases. Mutations of Ras that arrest the protein in its active
state are frequently implicated in tumor formation. We used Ras and Raf proteins in the yeast
two-hybrid system to search for natural or synthesized substances capable of modulating Ras/
Raf interaction by specifically binding to one of the interacting partners. We found that
cycloalkylidene carboxylic acids enhanced Ras/Raf interaction by acting on the cysteine-rich
domain of Raf. Several analogues of the active substance 2-cyclohexylidene propanoic acid were
synthesized and the importance of the semicyclic double bond in the stabilization of Ras/Raf
interaction was demonstrated. Variation of the size and the substituents of the cyclic system
as well as the length of the carboxylic acid resulted in enhanced Ras/Raf interaction.
In tr od u ction
which causes its translocation to the plasma mem-
brane.6 The structures of both Ras binding sites in Raf,
spanning residues 51-131 and 139-184, have been
solved.7,8 Conformational changes in Raf upon binding
of Ras induce the exposure of the kinase domain (CR3)
and initiate a phosphorylation/activation cascade, which
activates the mitogen-activated protein kinases MEK
and ERK.9 ERK phosphorylates numerous substrates
including nuclear transcription factors, which serve to
activate the expression of genes involved in cell prolif-
eration and differentiation. They also activate p90RSK,
which plays an important role in apoptosis.10 The dual
role of Ras in the regulation of proliferation and apop-
tosis is still controversially discussed.11
Presently, only a few substances, e.g., sulindac sul-
fide12 or several peptides13-15 are known to directly
influence Ras/Raf interaction. In this paper, we describe
the synthesis and analysis of structure-activity rela-
tionships of cycloalkylidene carboxylic acids in Ras/Raf
interaction. 2-Cyclohexylidene propanoic acid (com-
pound 5) was originally developed as an analogue of
valproic acid in order to verify its antiepileptic po-
tency.16,17 It represents the first compound of this
structure class that showed a significant effect on the
Ras/Raf interaction. Using compound 5 as a lead, we
synthesized derivatives by varying the length of the
carboxylic acid as well as the size of the cyclic system.
Furthermore, substituents were introduced and varied
by their size and place (compounds 6-12) and the
carboxylic group was once interchanged by an aldehyde
function.
The small guanosine 5′-triphosphate (GTP) binding
protein p21 Ras and the serine/threonine protein kinase
Raf-1 play a pivotal, regulatory role in oncogenic,
mitogenic, and developmental signaling pathways. The
ras genes were originally found as the transforming
agents of the Harvey and Kirsten murine sarcoma
viruses.1 Identification of cellular ras homologues, the
presence of mutated alleles in human tumors, and the
strong conservation of ras genes in eukaryotes empha-
size their importance in cell growth control. The Ras
protein functions as a molecular switch, which cycles
between a GDP-bound inactive state and a GTP-bound
active state. Activation occurs transiently in response
to an array of extracellular signals triggered by growth
factors, cytokines, hormones, or neurotransmitters.2
Single amino acid substitutions of Gly-12, Gly-13, or
Gln-61 of the Ras protein create mutant proteins that
are locked in the active, GTP-bound conformation, thus
leading to constitutive, dysregulated activation of Ras
function.3 Mutated Ras proteins were detected in as
many as 30% of human tumors with the highest
incidence in ductal tumors of the exocrine pancreas,4
suggesting that drugs interacting with Ras to disturb
its interaction with downstream effectors may be a
promising tumor therapy.
Activated Ras utilizes a multitude of functionally
diverse effectors. One of the best characterized effectors
of Ras is the serine/threonine kinases of the Raf family.
The Raf family consists of at least three isoforms:
c-Raf-1 (herein referred to as Raf), B-Raf, and A-Raf.5
The proteins share homologous structural segments and
designated conserved regions (CRs) CR1, CR2, and CR3.
Ras binds to two sites in the regulatory region of Raf,
Ch em istr y
The cycloalkylidene alkanes evaluated in this study
are shown in Figure 2. The preparation of the cyclo-
heptylidene carboxylic acids was based on the Wittig-
Horner-Emmons reaction18 as shown in Scheme 1. The
corresponding ketone was treated with carbethoxyalky-
lphosphonate obtained by means of the Michaelis-
* To whom correspondence should be addressed. Tel.: +49-69-798-
29648. Fax: +49-69-798-29662. E-mail: winckler@em.uni-frankfurt.de.
† Universita¨t Frankfurt/M. (Biozentrum).
‡ These authors contributed equally to this work.
§ J ohannes Gutenberg-Universita¨t.
10.1021/jm011101q CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/28/2002

1536 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Friese et al.
Sch em e 1a
a
Reagent: (a) RCHBrCO2Et; (b) NaH/DME; (c) cycloheptanone; (d) H2O; (e) NaOH, EtOH.
Sch em e 2a
a
Reagent: (a) NaH; (b) NaH, R1CHBrCO2H; (c) NaH; (d) cycloalkanone, EtOH, HCl.
F igu r e 1. Compound 5 (2-cyclohexylidenpropanic acid) en-
hances Ras/Raf interaction. The basal â-galactosidase activity
achieved with the tester strains BD-Ras(G12V)1-166 and AD-
Raf1-275 (compare to Figure 4) in the presence of solvent (black
squares) and the control strain (open circles) was set equal to
1. Relative enzyme activities were calculated as the ratio of
the activity in the presence of compound divided by the effect
of pure solvent. Experiments are presented ( standard devia-
tion (SD) calculated from five individual experiments.
F igu r e 2. Synthesized compounds sorted according to the size
of the cycloalkylidene ring. First row, cycloheptylidenes; second
row, cyclohexylidenes; third row, cyclopentylidenes.
Arbuzov reaction19 of triethyl phosphite and 2-bromocar-
boxylic acid. The release of the free acids occurred easily
by hydrolysis of the resultant ester. To ensure the
semicyclic position of the double bond, the products were
characterized by 13C nuclear magnetic resonance (NMR)
addition of first the bromo-carboxylic acid and then
cycloalkanone, led to a good yield of the five- and six-
membered cycloalkylidenic acids existing as pure semi-
cyclic isomers.
1
as well as H NMR.
Because of isomerization of both the cyclohexylidenic
and the cyclopentylidenic derivatives, another way to
obtain compounds 4-14 had to be pursued. The method
of Britelli20 allowed us to synthesize the desired acids
in an one-vessel synthesis as shown in Scheme 2.
Addition of diethyl phosphite to a suspension of 3 equiv
of sodium hydride in 1,2-dimethoxyethane, followed by
The applied cycloalkanones were commercially avail-
able except for the adduct of compound 12, which was
synthesized as outlined in Scheme 3. The Grignard
reagent of 4-bromoanisole was treated with 1,4-cyclo-
hexanedionemonoethylenketale to give the 4-hydroxy-
cyclohexanone derivative. Treatment of the latter with
trifluoroacetic acid21 led to the corresponding cyclohex-

Cycloalkylidene Carboxylic Acids as Effectors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1537
Sch em e 3a
Compound 24 was prepared by the treatment of
diethyl formylmethylphosphonate with an equimolar
amount of cyclohexylamine. According to its acid sen-
sibility, distillation should be carried out in the presence
of anhydrous potassium carbonate. The IR spectra
indicated that the compound existed predominantly in
an enamino form. For completion of the formylolefina-
tion, the carbanion was generated in a convenient
manner by treatment of a tetrahydrofuran (THF) solu-
tion of 24 with an equimolar amount of sodium hydride
in the cold. After reaction of the carbanion with a slight
minority of the cycloheptanone, the amine was leniently
hydrolyzed with diluted oxalic acid in order to avoid
isomerization.
a
Reagent: (a) Mg/THF; (b) 1,4-cyclohexanedionmonoethyl-
eneketale; (c) CF3COOH; (d) Pd/C, H2.
Sch em e 4a
Resu lts a n d Discu ssion
The yeast two-hybrid system takes advantage of the
modular construction of the yeast GAL4 transcriptional
activator. DNA binding (BD) and transactivating (AD)
domains of GAL4 can be dissociated and expressed
separately from two different plasmids.27,28 The plasmid-
borne BD and AD domains of GAL4 can only induce
transcription of reporter genes such as the bacterial
â-galactosidase if proteins expressed as genetic fusions
with BD and AD are capable of interacting to reconsti-
tute GAL4. When Ras and Raf are fused to BD and AD,
respectively, compounds that bind specifically to either
Ras or Raf may influence Ras/Raf interaction and hence
reconstitution of active GAL4, which in turn results in
altered â-galactosidase reporter gene expression.
The assay applied to this study was performed with
BD-Ras and AD-Raf proteins. We made use of microtiter
plates in a high-throughput design in order to allow an
extended and fast screening of natural and synthesized
compounds. A tester strain was used, which expressed
the catalytic domain of the constitutively active
H-RasG12V (amino acids 1-166) fused to BD and the
regulatory domain of Raf (amino acids 1-275) fused to
AD. To demonstrate the specificity of effects of the tested
compounds on â-galactosidase reporter activity, a con-
trol strain was used that expressed genetic fusions of
AD and BD with the tumor suppressor protein p53 and
the large T-antigen of the Simian virus 40, which are
known to interact strongly.29,30 Interference of test
compounds with Ras and Raf was considered as specific
only if dose-dependent changes of â-galactosidase re-
porter activity in the tester strain but not in the control
strain were obtained.
a
Reagent: (a) Zn/I2, CH3CH2CHBrCO2Et; (b) NaOH, EtOH; (c)
acetic anhydride; (d) steam distillation.
enone, which could be hydrated by the means of
hydrogen catalyzed by palladium on charcoal.
Efforts to achieve butyric acids by the Britelli reaction
were unsuccessful. Compound 4 was therefore derived
from the Reformatzky22 reaction as shown in Scheme
4. In a first step, the ethyl 2-(1-hydroxycyclohexyl)-
butanoate was obtained.23 To prevent formation of the
endocyclic derivative, hydrolysis of the ester had to occur
before the dehydratization step. To separate the desired
semicyclic derivative from its endocyclic isomer, steam
distillation was carried out.
To examine the relevance of the functional group, the
carboxylic group was exchanged for an aldehyde func-
tion as demonstrated by compound 3. As discussed
above, the direct conversion of the cycloalkanones into
the R,â-unsaturated aldehydes could not be achieved
due to unwanted condensation reactions.24,25 For the
formylolefination of cycloheptanone, we used a method
developed by Nagata and Hyase,26 which is based on
the Wittig reaction (Scheme 5). Because the diethyl
2-(cyclohexylimino)ethylphosphonate (24) was consid-
ered to be less electrophilic than the parent carbonyl
compound, considerable self-condensation was not ex-
pected using this reaction.
Sch em e 5a
a
Reagent: (a) cycloheptanone; (b) H3O+.

1538 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Friese et al.
stabilize Ras/Raf interactions could be obtained (data
not shown). The cytotoxicity of compounds 1-7, 13, and
14 was calculated as EC50 values and was well above
300 µM, which is at least 6-fold above the concentration
used in the experiments (50 µM). Elongation of the
substituent in position 4′ from an ethyl group in
compound 8 to a propyl group in compound 9 resulted
in increased cytotoxicity (EC50 values of 125 and 74 µM,
respectively). Incubation of the yeast cells with com-
pound 10, which had an isobutylic substituent, revealed
a similar cytotoxicity as compound 9 (EC50 values of 81
µM). The correlation between larger substituents and
growth inhibition was additionally found by comparing
compounds 11 and 12, which differ by a methoxy group
(EC50 values of 143 and 130 µM).
F igu r e 3. Cycloalkylidene carboxylic acids affect Ras/Raf
interaction. The experiments were performed with 50 µM of
the indicated compounds. Reporter gene activity (hatched
columns) and control strain activity (black columns) obtained
with pure solvent was set equal to 1 (column “s”). Experiments
are presented ( SD calculated from three individual experi-
ments.
To identify domains in Ras and Raf required for the
stabilizing effects of the synthetic compounds, several
truncated derivatives of Ras and Raf were expressed as
AD and BD fusions, respectively (summarized in Figure
4A). In the experiments described above, amino acids
1-166 of H-Ras that fused to BD, named BD-Ras-
(G12V)1-166, were active in interacting with Raf. Un-
fortunately, it was impossible to achieve Ras/Raf inter-
action with any Ras derivative shorter than amino acids
1-166, probably because the loss of correct protein
structure or amino acids is critical for Raf binding.
Hence, the BD-Ras(G12V)1-166 protein was used in this
analysis. Yeast cells expressing wild-type Ras in fusion
with BD (BD-Ras1-166) had a significantly lower â-ga-
lactosidase activity than cells expressing the constitu-
tively active Ras mutant RasG12V (Figure 4B). Cycloalky-
lidene carboxylic acids increased the interaction of both
the mutant Ras(G12V)1-166 and the wild-type RasG12V
(Figure 4B). The minimal requirement of the Raf protein
to interact with Ras is the Ras binding domain (RBD),
which is located in CR1 at amino acid positions 51-
131 of the Raf protein (Figure 4A). Interaction of AD-
Raf51-131 with Ras was not enhanced by compound 5
(Figure 4B, column 2), suggesting that the compound
did not stabilize Ras/Raf interaction by binding to the
RBD of the Raf protein. We found that elimination of
CR2 and CR3 from AD-Raf fusions still allowed effects
of compound 5 on Ras/Raf interaction. On the other
hand, the cysteine-rich domain of Raf (amino acids 131-
194) seemed to be required to exert effects of compound
5 (Figure 4B). These data argue that the cycloalkylidene
carboxylic acids bind to the zinc fingerlike motif, which
is located between amino acids 131 and 194 of Raf.
Preliminary data support that the cycloalkylidene
carboxylic acids described in this paper may be able to
activate Ras/Raf-dependent pathways in mammalian
cell culture cells (data not presented). Hence, cycloalky-
lidene carboxylic acids may represent an interesting
new class of Ras/Raf effectors, which may be useful tools
in basic research of Ras/Raf-regulated pathways and
may also serve as lead structures in the development
of novel drugs.
In a random screening of natural and synthesized
substances as well as plant and fungal extracts, we
found that 2-cyclohexylidenpropanic acid (5) mediated
a significant stabilization of Ras/Raf binding (Figure 1).
An EC50 value for the specific enhancement of Ras/Raf
interaction by compound 5 was calculated as 64 µM.
To obtain more potent effectors of the Ras/Raf inter-
action, various cycloalkylidene carboxylic acids were
synthesized (Figure 2) and tested for effects on Ras/Raf
interaction in yeast two-hybrid screens. Structure-
activity relationships could be derived from these ex-
periments, which are summarized in Figure 3. The
compounds can be divided into three groups by their
effects on Ras/Raf interaction as compared to compound
5: (i) Modifications led to moderate changes of reporter
gene activity; substitution in position 3′ in compound 6
had almost no effect on Ras/Raf interaction (expressed
as changes in â-galactosidase activity). A methyl group
in position 4′ (compound 7) resulted in a slightly
increased â-galactosidase activity. (ii) Modifications
enhanced the reporter activity; enlargement of the ring
to cycloheptylidene in compounds 1 and 2 led to
increased â-galactosidase activities, indicating stabiliza-
tion of Ras/Raf interaction. The best results were seen
by treating the cells with compound 1, which differed
from 2 in the length of the acid chain. (iii) Modifications
resulted in decreased reporter activity; replacement of
the acid group in compound 2 by an aldehyde group in
compound 3 reduced the observed effects. The acid
group thus seemed to be of critical importance. Elonga-
tion of the propanoic acid in compound 5 to butanoic
acid in compound 4 showed only slight changes of
reporter activity. Decreasing the size of the ring system
to cyclopentylidenic acids in compounds 13 and 14
resulted in a complete loss of the Ras/Raf stabilizing
effect. To test the influence of semicyclic location of the
double bond, compound 5 was also tested as a mixture
of both the semicyclic and the endocyclic isomer in a
ratio of 1:1 (compound 5*). A significant decrease of
activity was observed (not shown).
Exp er im en ta l Section
Because of the fact that substituents in position 4′ as
well as enlargement of the ring system to cycloheptyl-
idenes led to more potent effectors, compounds 8-12
were synthesized and tested. These new cyclohexylidene
carboxylic acids exhibited unexpected cytotoxic effects,
such that no data on the potential of these drugs to
P la sm id s. The yeast two-hybrid RasG12V(1-166) construct
was generated by polymerase chain reaction (PCR) using the
DNA template pPC97RasG12Vverk. 31 The Ras carboxy terminus
containing the CaaX motif was deleted to prevent association
with the plasma membrane. Wild-type Ras was generated by
site specific mutation32,33 of RasG12V. All Ras constructs were

Cycloalkylidene Carboxylic Acids as Effectors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1539
µL of buffer Z (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM
KCl, and 1 mM MgSO4) and frozen as pellets for 15 min at
-80 °C. After resuspension in 90 µL of buffer Z, the cell
densities were measured at 630 nm. Cells were lysed by
addition of 4 µL of 0.1% sodium dodecyl sulfate and 6 µL of
CHCl3 and incubated for 10 min at 30 °C. â-Galactosidase
activity was tested by adding 100 µL of 0.2 mg/mL chlorophe-
nolred-â-D-galactopyranoside (CPRG), and the amount of
reaction product was measured time dependently in an
enzyme-linked immunosorbent assay reader at A550/630. En-
zyme activity was calculated as Miller units37 and presented
as the ratio of â-galactosidase activity obtained in the presence
of compound vs pure solvent. One Miller unit defines the
hydrolization of 1 µmol of CPRG to chlorophenol red and
D-galactose per minute and cell.
Gen er a l P r oced u r es. NMR spectra were recorded on
Bruker AC 300 or AM 400 MHz spectrometers with chemical
shifts reported as δ values in parts per million downfield from
tetramethylsilane as the internal standard. Splitting patterns
are designated as follows: s, singlet; d, double; t, triplet; q,
quartet; m, multiplet. IR spectra were recorded on a Beckmann
IR 42200, Perkin-Elmer 1310 Infrared Spectrophotometer or
Shimadzu Infrared Spectrophotometer IR-470. Mass spectra
were obtained on a Varian MAT 311A (70 eV) and a Varian
CH 7a (90 eV). Elemental analyses were performed at the
department of Organic Chemistry at the J ohannes Gutenberg-
University, Mainz. Melting points were determined with a Dr.
Tottoli melting point apparatus and are uncorrected. Flash
column chromatography was carried out with silica gel 60
(Merck).
R ea gen t s. All reagents were purchased from Aldrich
Chemical Co. and used without further purification except
anhydrous THF, which was distilled over potassium metal
under nitrogen or glyme, distilled over calcium hydride.
F igu r e 4. Evaluation of Raf domains required for effects by
cycloalkylidene carboxylic acids on Ras/Raf interaction. (A)
Schematic presentation of Ras and Raf proteins used in this
study. Ras is a 189 amino acid full-length protein, which
carries a carboxy-terminal farnesylation motif (CaaX motif).
We expressed genetic fusions of GAL4 BD with amino acids
1-166 of mutant H-Ras(G12V) or wild-type Ras. Full-length
Raf is 648 amino acids long. We expressed several truncated
versions of Raf as indicated by the numbers. The Ras-
interacting domain RBD spans amino acids 51-131. The
cysteine-rich, zinc fingerlike domain CRD is located between
positions 131 and 194. (B) Effects of 2-cyclohexylidenpropanic
acid 5 (100 µM) on reporter gene activity in the presence of
various truncated Raf mutants. Columns 1-6 represent
experiments performed with BD-Ras(G12V)1-166 as the Raf
binding partner, whereas wild-type Ras (BD-Ras1-166) was used
in experiments presented in columns 7 and 8. The following
Raf derivatives were used as Ras binding partners: column
Com p ou n d s of F ir st Sta ge Tr ieth yl P h osp h on op r op i-
on a te (15).19 A total of 40 g (221 mmol) of ethyl 2-bromopro-
pionate and 36.7 g (221 mmol) of triethyl phosphite were
heated at 155-160 °C for about 10 h during which bromoet-
hane formed. Bromoethane was removed continuously by the
help of a distillation bridge. The mixture was distilled under
reduced pressure to give 29.2 g (55.6%) of a colorless liquid;
bp 90 °C (1.6 × 10-1 mbar).
Tr ieth yl P h osp h on oa ceta te (16).19 Ethyl 2-bromoacetate
was used in the manner described for 15; 4.8%; 156 °C (18
mbar).
Eth yl 2-Cycloh ep tylid en p r op a n oa t (17).18 A 13.09 g (55
mmol) amount of 15 was added dropwise at 20 °C to a slurry
of NaH (1.2 g, 50 mmol) in 100 mL of dry glyme. The reaction
mixture was stirred for 1 h at room temperature until gas
evolution had ceased. Cycloheptanone was added dropwise at
such a rate that the temperature was maintained below 30
°C. After an additional stirring of 15 min, during which time
a viscous semisolid appeared, the mixture was taken up in a
large excess of water. The aqueous solution was extracted with
ether, and the organic layer was dried over magnesium sulfate
and evaporated. The crude product was purified by flash
chromatography (petroleum ether/ethyl acetate; 9.5:0.5; Rf
0.58) to give 2.1 g (18%) of a colorless liquid. IR 2900, 1700,
1, AD-Raf1-131; column 2, AD-Raf51-131; column 3, AD-Raf1-194
;
column 4, AD-Raf51-194; column 5, AD-Raf1-275; column 6, AD-
Raf51-275; column 7, AD-Raf1-194; column 8, AD-Raf1-275. Re-
porter gene activity obtained with pure solvent was set equal
to 1 (not shown in the graph). Experiments are presented (
SD calculated from three individual experiments.
1610 cm-1 1H NMR (CDCl3): δ 4.14 (dq, J ) 7.39 Hz, 2H,
.
cloned as EcoR I/Sal I DNA fragments into the pBD-GAL4Cam
vector (Stratagene). The Raf regulatory domain (amino acids
1-275) and truncated Raf mutants were PCR amplified from
the template pPC86HCRC2Raf 30 and cloned either with EcoR
I/Sal I (for DNA fragments coding for amino acids 1-131 and
51-131 of Raf) or EcoR I/Xba I (for DNA fragments coding
for amino acids 1-194, 51-194, 1-275, and 51-275 of Raf)
into the pAD-GAL4 vector (Stratagene).
O-CH2), 2.48 (t, J ) 7.75 Hz, 2H, CH2-CdC), 2.47 (t, J )
7.75 Hz, 2H, CH2-CdC), 1.73-1.55 (m, 4H, CH2-C-C-CH2),
1.68 (s, 3H, CH3-C-CO), 1.50-1.45 (m, 2H, CH2), 1.34-1.29
(m, 2H, CH2), 1.26 (dt, J ) 7.15 Hz, 3H, CH3-CO). 13C NMR
(DMSO): δ 169.37 (C-1), 149.76 (C-1′), 122.69 (C-2); 59.84
(C-1′′), 30.19, 28.77 (C-2′; C-7′), 28.42; 27.67 (C-3′; C-6′), 25.94,
24.00 (C-4′; C-5′), 15.33 (C-3), 14.42 (C-2′′). MS m/z 196, 181,
168, 151, 123, 108, 102. Anal. (C12H20O2) C, H.
Yeast Two-Hybr id System . Competent Y190 yeast cells34,35
were cotransformed with 1 µg of each of the two-hybrid vectors
according to Klebe36 and grown on synthetic medium lacking
leucine, tryptophane, and histidine. Single colonies were
picked and inoculated overnight at 30 °C. In microtiter plates,
140 µL per well liquid culture diluted to A600 0.2 were shaken
with 10 µL of probe dissolved in 50% dimethyl sulfoxide
(DMSO) for 12 h at 30 °C. Cells were washed twice with 150
Eth yl 2-Cycloh ep tylid en a ceta te (18).18 Preparation was
as described for 15 by use of triethyl phosphonoacetate (12.32
g, 55 mmol). The crude product was distilled giving 6.53 g
(71.7%) of a colorless liquid; bp 80 °C (4 × 10-2 mbar). IR 3020,
1
2900, 1700, 1610, 830 cm-1. H NMR (CDCl3): δ 5.65 (s, 1H,
H-CdC), 4.12 (q, J ) 7.15 Hz, 2H, O-CH2), 2.86 (t, J ) 6.08
Hz, 2H, CH2-CdC); 2.35 (t, J ) 5.72 Hz, 2H, CH2-CdC),

1540 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Friese et al.
1.74-1.44 (m, 8H, (CH2)4), 1.24 (t, J ) 7.15 Hz, 3H, CH3-C-
O). 13C NMR (DMSO): δ 172.27 (C-1), 166.67 (C-1′), 115.64
(C-2), 59.35 (C-1′′), 39.02, 32.09, 29.86, 29.02, 28.06, 26.88 (C-
2′; C-3′; C-4′; C-5′; C-6′; C-7′), 14.28 (C-2′′). MS m/z 182, 153,
137, 136, 109, 96, 82, 68, 56. Anal. (C11H18O2) C, H.
2-(1-Hyd r oxycycloh exyl)bu ta n oic Acid (23).23 A 15.4 g
(72 mmol) amount of 22 was treated with a solution of 3 g of
NaOH in 60 mL of ethanol (50%). After the solution was
refluxed for 4 h, the resulting solution was reduced to its half
and diluted with the same amount of water. The solution was
extracted with ether, and the aqueous layer was acidified with
HCl. After it was extracted with ether, the organic layer was
washed with water and dried over sodium sulfate, and the
solvent was evaporated. The resulting crystals were recrystal-
lized with a mixture of ether and petroleum ether; 5.2 g
(38.5%); mp 77 °C. IR 3400, 2900, 2550, 1680, 1430, 1310, 1265,
1180, 1120, 940 cm-1. 1H NMR (DMSO): δ 11.99 (s, 1H, CO2H),
2.37 (dt, J ) 6.78 Hz, 1H, CH-C-O), 2.17 (s, 1H, OH), 1.62-
1.30 (m, 10H, C6H10), 1.34 (m, 1H, CH2-C-C-O), 1.24 (m,
1H, CH2-C-C-O), 0.96 (t, J ) 7.54 Hz, 3H, CH3). 13C NMR
(DMSO): δ 176.04 (C-1), 70.79 (C-1′), 59.41 (C-2), 35.40, 33.70
(C-2′; C-6′), 25.85 (C-4′), 21.54 (C-3′; C-5′), 19.59 (C-3), 12.98
(C-4). MS m/z 186, 169, 143, 125, 112, 102, 99, 81. Anal.
(C10H18O3) C, H.
Dieth yl F or m ylm eth ylp h osp h on a te.26 A total of 71 g
(474 mmol) of triethyl phosphite and 94 g (474 mmol) of
2-bromo-1,1-diethoxyethane were refluxed for 3.5 h during
which bromoethane formed. Bromoethane was continuously
removed by the help of a distillation apparatus. After distil-
lation of the formed diethyl (2,2-diethoxyethyl)phosphonate (bp
128-130 °C, 2 mbar), the latter was given into its 3.5-fold
amount of refluxing HCl (2%) and extracted with ether for
about 3 days. Evaporation of the ether led to 33.91 g (75%) of
a colorless liquid.
4-H yd r oxy-4-(4-m e t h oxyp h e n yl)cycloh e xa n on e t h -
ylen k eta le (19). A 3.45 g (144 mmol) amount of magnesium
splinters was added to a solution of 25.5 g (136 mmol)
p-bromoanisole in dry THF (180 mL). The mixture was stirred
heavily and heated carefully until the reaction started. The
reaction was maintained until all magnesium had disappeared.
After the mixture was cooled to room temperature, 1,4-
cyclohexandionethylenketale (20.3 g, 123 mmol) in 180 mL of
dry THF was added dropwise and the resulting solution was
refluxed for 24 h. The reaction was stopped by adding 200 mL
of saturated NH4Cl and extracted three times with 100 mL
portions of ether. The organic layers were washed with NaOH
(10%, 40 mL) and water (100 mL) and dried over magnesium
sulfate. The solvent was evaporated, and the resulting product
1
was recrystallized from ether; 26.5 g (77.2%); mp 122 °C. H
NMR (CDCl3): δ 7.43 (d, J ) 8.82 Hz, 2H, AA′), 6.86 (d, J )
8.82 Hz, 2H, BB′), 3.96 (dd, J ) 2.63 Hz, 4H, O-(CH2)2-O),
3.79 (s, 3H, O-CH3), 2.19-2.01 (m, 4H, CH2-C-CH2), 1.86-
1.62 (m, 4H, OH, CH2-C-CH2). MS m/z 265, 204, 189, 165,
160, 150, 135, 101.
4-(4-Meth oxyp h en yl)cycloh exen e-1-on e (20). A 2.6 g
(100 mmol) amount of 19 was treated with 100 mL of
trifluoracetic acid and stirred at room temperature for 30 min.
The mixture was then given into a solution of saturated
NaHCO3 (50 mL) and extracted with chloroform. The organic
layer was washed with brine and dried over magnesium
sulfate, and the solvent was evaporated. Purification of the
crude product by flash chromatography (n-hexane/ethyl acetate
3:1, Rf 0.32) led to 8.3 g (41%) of orange crystals; mp 52 °C.
1H NMR (CDCl3): δ 7.33 (d, J ) 8.83 Hz, 2H, AA′), 6.88 (d, J
) 8.82 Hz, 2H, BB′), 5.99 (t, J ) 3.94 Hz, 1H, CdCH), 3.04
(m, 2H, CH-CH2), 2.86 (t, J ) 6.91 Hz, 2H, CH2), 2.63 (t, J )
6.91 Hz, 2H, CH2). 13C NMR (CDCl3): δ 210.35 (C-1), 159.08
(C-4′), 137.12 (C-4), 133.32 (C-1′), 126.37 (C-2′; C-6′), 119.30
(C-3), 113.85 (C-3′; C-5′), 55.37 (C-1′′), 39.97, 38.76 (C-5; C-6),
28.01 (C-2). MS m/z 202, 185, 173, 160, 147, 145, 129, 115, 77.
Dieth yl 2-(Cycloh exylim in o)eth ylp h osp h on a te (24).26
A solution of diethyl formylmethylphosphonate (30 g, 168
mmol) in methanol was cooled to 0 °C and treated under an
argon atmosphere with freshly distillated cyclohexylamine.
After the mixture was stirred for 10 min at room temperature,
the solvent was evaporated and the crude product was distil-
lated twice over K2CO3 to give 21.3 g (48.52%) of a colorless
liquid; bp 151 °C (0.04 mbar). IR 3300, 1430, 1290, 1210 cm-1
.
N -(2-C y c lo h e p t y li d e n e t h y li d e n e )-N -c y c lo h e x y l-
a m in e (25).26 Under an argon atmosphere and cooling in an
ice bath, a solution of 24 (10 g, 38 mmol) in 150 mL of dry
THF was given dropwise to a suspension of NaH (0.9 g, 38
mmol) in dry THF (10 mL). A solution of 3.6 g of cyclohep-
tanone (32 mmol) in dry THF (10%) was added slowly, and
the mixture was stirred for 2 h during which TLC was
conducted. The reaction mixture was given onto ice water,
extracted with ether, washed with saturated brine, and dried
over magnesium sulfate. Evaporation of the ether led to 3.1 g
(47.6%) of a colorless liquid.
Test Com p ou n d s. Gen er a l P r oced u r e for Com p ou n d s
1 a n d 2. A 44 mmol amount of the ester was added to 4.1 g of
NaOH dissolved in a mixture of 40 mL of ethanol and 30 mL
of water. The solution was refluxed for 3 h. After the solution
was cooled to room temperature, the ethanol was evaporated
and the residual was acidified with 8% HCl. The resulting
cloudy liquid was cooled in ice, which resulted in a white
precipitate. The acid was filtered and recrystallized in 20%
ethanol.
4-(4-Meth oxyp h en yl)cycloh exa n on e (21). A 800 mg
amount of Pd/C was given to a solution of 20 (8.3 g, 41 mmol)
in ethyl acetate (180 mL). The suspension was put under
hydrogen atmosphere and stirred at room temperature for
about 3 h (thin-layer chromatography (TLC) control). After the
catalyst was filtered and the solvent was evaporated, the crude
product (white crystals) was purified by flash chromatography
(n-hexane/ethyl acetate 3:1, Rf 0.32); 8.3 g (41%); mp 74 °C.
1H NMR (CDCl3): δ 7.16 (d, J ) 8.58 Hz, 2H, AA′), 6.87 (d, J
) 8.82 Hz, 2H, BB′), 3.79 (s, 3H, CH3), 3.04-2.92 (m, 1H, CH-
Ar), 2.53-2.46 (m, 4H, CH2-C-CH2), 2.26-2.15 (m, 2H, CH2),
1.99-1.83 (m, 2H, CH2). 13C NMR (CDCl3): δ 211.42 (C-1),
158.30 (C-4′), 136.97 (C-1′), 127.61 (C-2′; C-6′), 113.83 (C-3′;
C-5′), 55.31 (C-1′′), 41.85 (C-4), 41.46 (C-2; C-6), 34.26 (C-3;
C-5). MS m/z 204, 147, 134, 121, 119, 91, 77.
Eth yl 2-(1-Hyd r oxycycloh exyl)bu ta n oa te (22).23 In a
dry three neck flask, supplied with a drying tube filled with
calcium chloride, 12 g of zinc powder and a few crystals of
iodine were treated with 20 mL of a mixture of cyclohexanone
(16.6 g, 170 mmol), ethyl 2-bromobutanoic acid (31.5 g, 160
mmol), and glyme (25 mL). The suspension was stirred heavily
and heated on an oil bath (100-105 °C). As soon as the
reaction started, the rest of the solution was added dropwise
and the reaction was maintained for an additional hour. After
the mixture was cooled to room temperature, a mixture of ice
and 15 mL of concentrated sulfuric acid was added and the
solution was extracted several times with toluene. The organic
layer was washed with ice-cold solution of soda (10%) and then
with ice water. After the mixture was dried over sodium
sulfate, the toluene was evaporated and the resulting ester
was distillated under reduced pressure; 15.4 g (44.8%); bp 74
°C (10-2 mbar).
2-Cycloh ep tylid en p r op a n oic Acid (1). 20.73%; mp 66 °C.
1H NMR (DMSO): δ 12.07 (s, 1H, CO2H), 2.48 (t, J ) 6.40
Hz, 2H, CH2-CdC), 2.26 (t, J ) 5.65 Hz, 2H, CH2-CdC), 1.74
(s, 3H, CH3), 1.49 (m, 8H, (CH2)4). MS m/z 168, 159, 123, 110,
66. Anal. (C10H16O2) C, H.
2-Cycloh ep tylid en a cetic Acid (2).16 43.65%; mp 56 °C.
IR 3020, 2900, 1700, 1610, 830 cm-1. 1H NMR (CDCl3): δ 5.65
(s, 1H, H-CdC), 4.12 (q, J ) 7.15 Hz, 2H, O-CH2), 2.86 (t, J
) 6.08 Hz, 2H, CH2-CdC), 2.35 (t, J ) 5.72 Hz, 2H, CH2-
CdC), 1.74-1.44 (m, 8H, (CH2)4), 1.24 (t, J ) 7.15 Hz, 3H,
CH3-C-O). 13C NMR (DMSO): δ 167.51 (C-1), 165.57 (C-1′),
116.53 (C-2), 38.44, 31.51 (C-2′; C-7′), 29.59; 28.63 (C-5′; C-4′),
27.78, 26.49 (C-6′; C-3′). MS m/z 155, 137, 127, 112, 110, 101,
95, 80, 68. Anal. (C9H14O2) C, H.
2-Cycloh ep tylid en a ceta ld eh yd e (3). Compound 25 was
dissolved in the ratio of 1:40 in a mixture of CH2Cl2/THF
(4:1). An amount totaling 150 parts of 1% oxalic acid was

Cycloalkylidene Carboxylic Acids as Effectors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1541
added, and the mixture was refluxed for several hours. After
the mixture was cooled to room temperature, the solvent was
evaporated and the crude product was distillated by means of
bulb to bulb distillation; 888 mg (42.28%); bp 103 °C, 19 mbar.
(C-2′; C-6′), 31.11, 30.07 (C-3′, C-5′), 28.92 (C-7′), 15.31 (C-3),
11.70 (C-8′). MS m/z 182, 108, 79, 81, 67, 56. Anal. (C11H18O2)
C, H.
2-(4-P r op ylcycloh exylid en )p r op a n oic Acid (9). 59.55%;
IR 2820, 1725, 1425, 1385, 1270 cm-1 1H NMR (CDCl3): δ
.
1
mp 46 °C. IR 2900, 2600, 1670, 1620 cm-1. H NMR (DMSO):
9.80 (d, J ) 8.08 Hz, 1H, CHO), 5.57 (d, J ) 7.84 Hz, 1H, Cd
CH), 2.65 (t, J ) 5.89 Hz, 2H, CH2-CdC), 2.42 (t, J ) 5.52
Hz, 2H, CH2-CdC), 1.76-1.59 (m, 8H, (CH2)4). Anal. (C9H14O)
C, H.
δ 12.18 (s, 1H, CO2H), 2.77 (m, 2H, CH2-CdC), 2.53 (m, 2H,
CH2-CdC), 1.88-1.70 (m, 4H, CH2-C-CH2), 1.75 (s, 3H,
CH3-C-CO), 1.43 (m, 1H, CH), 1.29 (m, 2H, CH2-C-
Cyclohexyl), 1.14 (m, 2H, CH2-Cyclohexyl), 0.84 (t, J ) 7.16
Hz, 3H, CH3). 13C NMR (DMSO): δ 171.42 (C-1), 145.96
(C-1′), 120.47 (C-2), 38.62 (C-7′), 36.74 (C-4′), 34.20, 33.75
(C-2′; C-6′), 31.12, 30.13 (C-3′; C-5′), 19.92 (C-8′), 15.36 (C-9′),
14.49 (C-3). MS m/z 196, 162, 153, 122, 111, 100, 81, 79, 56,
44. Anal. (C12H20O2) C, H.
2-Cycloh exylid en bu ta n oic Acid (4).23 A 5.2 g (29 mmol)
amount of 23 in 6.0 mL of acetic anhydride were heated at
150 °C for 3 h. After the mixture was cooled to room
temperature, the solution was given into a big amount of water
and subjected to steam distillation. The distillate was cooled
to 0 °C during which the acid crystallized. The crystals were
washed and dried under vacuum; 404 mg (8.1%); mp 49 °C.
2-[4-(ter t-Bu t yl)cycloh exylid en ]p r op a n oic Acid (10).
1
48.32%; mp 114 °C. IR 2910, 2600, 1680, 1630 cm-1. H NMR
IR 2900, 2600, 1650, 1600, 1430, 1280, 1230, 1200, 930 cm-1
.
(DMSO): δ 12.14 (s, 1H, CO2H), 2.87 (m, 2H, CH2-CdC),
1.88-1.65 (m, 4H, CH2-C-CH2), 1.75 (s, 3H, CH3-C-CO),
1.20 (m, 1H, CH), 0.99 (m, 2H, CH2-CdC), 0.80 (s, 9H,
C(CH3)3). 13C NMR (DMSO): δ 171.08 (C-1), 146.04 (C-1′),
120.20 (C-2), 47.55 (C-4′), 32.41, 31.57, 30.67, 28.66, 28.26
(C-2′; C-3′; C-5′; C-6′; C-1′′), 27.66 (3 × CH3), 15.35 (C-3). MS
m/z 211, 210, 166, 195, 154, 139, 136, 125, 57. Anal. (C13H22O2)
C, H.
1H NMR (DMSO): δ 12.14 (s, 1H, CO2H), 2.33 (t, J ) 5.46
Hz, 2H, CH2-CdC), 2.21 (t, J ) 7.54 Hz, 2H, CH2-C-C-O),
2.15 (t, J ) 6.03 Hz, 2H, CH2-CdC), 1.51 (m, 6H, CH2-CH2-
CH2), 0.91 (t, J ) 7.54 Hz, 3H, CH3). 13C NMR (DMSO): δ
176.10 (C-1), 151.87 (C-1′), 125.73 (C-2), 32.74; 31.72, 28.50,
28.50, 26.57, 22.75 (C-2′; C-3′; C-4′; C-5′; C-6′; C-3), 11.37 (C-
4). MS m/z 169, 155, 151, 124, 108, 95, 81, 68. Anal. (C10H16O2)
C, H.
2-(4-P h en ylcycloh exyliden )pr opan oic Acid (11). 69.35%;
mp 146 °C. IR 3000, 2900, 2530, 1670, 1640, 1600, 790 cm-1
.
Gen er a l P r oced u r e for Com p ou n d s 5-14.20 To a sus-
pension of 31 mmol of NaH in 100 mL of dry glyme (argon
atmosphere), 4.0 mL (31 mmol) of diethyl phosphite was slowly
added. After the gas development had ceased, 31 mmol of the
2-bromocarboxylic acid, dissolved in 30 mL of dry glyme, was
added carefully and the solution was stirred until no more gas
development occurred. Afterward, 31 mmol of the cyclo-
alkanone was added dropwise and the mixture was stirred for
an additional hour. The reaction was stopped by adding 5 mL
of ethanol, and the mixture was tilted into a big excess of ice-
cold water. After it was extracted with ether, the aqueous layer
was brought to pH 4 with concentrated HCl and extracted once
again with ether. The organic layer was dried over magnesium
sulfate, and the solvent was evaporated. The resulting acid
was purified by recrystallization or flash chromatography.
2-Cycloh exylid en p r op a n oic Acid (5).20 41.72%; mp 78
°C. IR 3400, 2900, 1670, 1600 cm-1. 1H NMR (CDCl3): δ 11.70
(s, 1H, CO2H), 2.59 (t, J ) 5.86 Hz, 2H, CH2-CdC), 2.25 (t, J
) 5.86 Hz, 2H, CH2-CdC), 1.88 (s, 3H, CH3), 1.68-1.50 (m,
6H, CH2-CH2-CH2). 13C NMR (DMSO): δ 175.99 (C-1),
152.89 (C-1′), 118.60 (C-2), 32.38, 32.06 (C-2′; C-6′), 27.33,
27.83 (C-3′; C-5′), 26.39 (C-4′), 15.06 (C-3). MS m/z 154, 136,
111, 109, 81. Anal. (C9H14O2) C, H.
1H NMR (DMSO): δ 12.25 (s, 1H, CO2H), 7.28-7.01 (m, 5H,
Ar), 3.19 (m, 2H, CH2-CdC), 2.76 (m, 2H, CH2-CdC), 1.93
(m, 4H, CH2-C(Ar)-CH2), 1.81 (s, 3H, CH3), 1.45 (m, 1H, CH).
13C NMR (DMSO): δ 171.45 (C-1), 146.58 (C-1′), 144.73
(C-1′′), 128.62 (C-3′′; C-5′′), 126.99 (C-2′′; C-6′′), 126.27 (C-4′′),
121.15 (C-2), 43.79 (C-4′), 35.24, 34.84 (C-2′; C-6′), 31.62, 30.57
(C-3′; C-5′), 15.49 (C-3). MS m/z 230, 161, 122, 111, 100, 91,
79, 77, 44. Anal. (C15H18O2) C, H.
2-[4-(4-Meth oxyph en yl)cycloh exyliden ]pr opan oic Acid
(12). Adduct 21 was used to give 12; 21.43%; mp 138 °C. IR
3000, 2900, 2560, 1680, 1660, 1610, 1510, 1280, 1240, 1180,
1030, 810 cm-1. 1H NMR (CDCl3): δ 12.27 (s, 1H, CO2H), 7.12
(d, J ) 8.58 Hz, 2H, AA′), 6.83 (d, J ) 8.82 Hz, 2H, BB′), 3.78
(s, 3H, CH3), 3.50 (m, 1H, CH-Ar), 2.76 (m, 2H, CH2-CdC),
2.10-1.95 (m, 4H, CH2-C-CH2), 1.95 (s, 3H, CH3), 1.63-1.48
(m, 2H, CH2-CdCH). 13C NMR (DMSO): δ 171.48 (C-1),
157.81 (C-4′′), 144.76 (C-1′), 138.58 (C-1′′), 127.84 (C-2′′; C-6′′),
121.07 (C-2), 114.02 (C-3′′; C-5′′), 55.26 (C-1′′′), 42.92 (C-4′),
35.49, 35.09 (C-2′; C-6′), 31.66, 30.58 (C-3′; C-5′), 15.49 (C-3).
MS m/z 260, 215, 186, 147, 134, 126, 107. Anal. (C16H20O2) C,
H.
2-Cyclop en tylid en p r op a n oic Acid (13).23 21.53%; mp
100 °C. IR 2960, 2600, 1720, 1660, 1620, 1430, 1290, 730, 600,
2-(3-Meth ylcycloh exylid en )p r op a n oic Acid (6). 10.56%;
1
440 cm-1. H NMR (CDCl3): δ 11.43 (s, 1H, CO2H), 2.76 (t, J
mp 76 °C. IR 2840, 2600, 1650, 1430, 1380, 1270, 1210, 1100
) 6.40 Hz, 2H, CH2-CdC), 2.39 (t, J ) 6.40 Hz, 2H, CH2-
CdC), 1.86 (s, 1H, CH3), 1.70 (m, 4H, CH2-CH2). 13C NMR
(DMSO): δ 173.93 (C-1), 164.33 (C-1′), 118.01 (C-2), 34.66 (C-
2′; C-5′), 27.13, 25.52 (C-3′; C-4′); 15.83 (C-3). MS m/z 140, 95,
79, 67. Anal. (C8H12O2) C, H.
1
cm-1. H NMR (DMSO): δ 12.15 (s, 1H, CO2H), 2.48 (m, 4H,
2 × CH2-CdC), 1.76 (s, 3H, CH3-C-C-O), 1.86-0.99 (m, 5H,
CH2-CH2-CH), 0.99 (dd, J ) 6.40 Hz, 3H, CH3). 13C NMR
(DMSO): δ 171.50 (C-1), 145.17 (C-1′), 120.80, 34.58, 31.45
(C-2′; C-6′), 33.72 (C-3′), 30.25 (C-4′), 26.88 (C-5′), 22.37 (C-
7′), 15.35 (C-3). MS m/z 168, 150, 135, 123, 112, 111, 100, 95.
Anal. (C10H16O2) C, H.
2-Cyclop en tylid en a cetic Acid (14).16 3.5%; mp 51 °C. IR
3180, 3080, 2960, 1680, 1630, 860 cm-1 13C NMR (CDCl3): δ
.
168.08 (C-1), 167.65 (C-1′), 112.44 (C-2), 35.54, 32.44 (C-2′;
C-5′), 26.19, 25.29 (C-3′; C-4′). MS m/z 126, 125, 108, 97, 81,
80, 79, 71, 68, 67, 52. Anal. (C7H10O2) C, H.
2-(4-Meth ylcycloh exylid en )p r op a n oic Acid (7). 8.5%;
mp 54 °C. IR 2840, 2550, 1650, 1610, 1430, 1380, 1270, 920
1
cm-1. H NMR (DMSO): δ 12.17 (s, 1H, CO2H), 2.98 (m, 1H,
CH2-CdC), 2.52 (m, 1H, CH2-CdC), 1.86-1.69 (m, 4H, CH2-
C-CH2), 1.75 (s, 3H, CH3-C-C-O), 1.56 (m, 1H, CH), 0.96
(m, 2H, CH2-CdC), 0.85 (d, J ) 6.40 Hz, 3H, CH3). 13C NMR
(DMSO): δ 171.45 (C-1), 145.48 (C-1′), 120.64 (C-2), 36.20,
35.73 (C-2′; C-6′), 32.12 (C-4′), 31.15, 30.09 (C-3′; C-5′), 21.96
(C-7′), 15.40 (C-3). MS m/z 168, 150, 139, 135, 125, 123, 111,
108, 100, 94. Anal. (C10H16O2) C, H.
Ack n ow led gm en t. We are grateful to C. Block and
A. Wittinghofer for providing yeast two-hybrid plasmids.
This work was supported by grants from the Deutsche
Forschungsgesellschaft.
Refer en ces
2-(4-Eth ylcycloh exylid en )p r op a n oic Acid (8). 14.39%;
mp 41 °C. IR 2900, 2500, 1650, 1600, 1270, 1220 cm-1. 1H NMR
(DMSO): δ 12.16 (s, 1H, CO2H), 3.03 (m, 2H, CH2-CdC), 2.53
(m, 2H, CH2-CdC), 1.82 (m, 4H, CH2-C-CH2), 1.75 (s, 3H,
CH3-C-CO), 1.32 (m, 1H, CH), 1.18 (m, 2H, CH2-Cyclohexyl),
0.84 (t, J ) 7.16 Hz, 3H, CH3). 13C NMR (DMSO): δ 171.42
(C-1), 145.89 (C-1′), 120.50 (C-2), 38.74 (C-4′), 33.80, 33.35
(1) Ellis, R. W.; DeFeo, D.; Shih, T. Y.; Gonda, M. A.; Young, H. A.
Tsuchida, N.; Lowy, D. R.; Scolnick, E. M. The p21 src genes of
Harvey and Kirsten sarcoma viruses originate from divergent
members of a family of normal vertebrate genes. Nature 1981,
292, 506-511.
(2) Campbell, S. L.; Khosravi-Far, R.; Rossman, K. L.; Clark, G. J .;
Der, C. J . Increasing complexity of Ras signaling. Oncogene 1998,
17, 1395-1413.

1542 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Friese et al.
(3) Bos, J . L. p21Ras: an oncoprotein functioning in growth factor-
induced signal transduction. Eur. J . Cancer 1995, 31A, 1051-
1054.
(21) Kaiho, T.; San-nohe, K.; Kajiya, S.; Suzuki, T.; Otsuka, K.; Ito,
T.; Kamiya, J .; Maruyama, M. Cardiotonic agents. 1-Methyl-7-
(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinones and related
compounds. Synthesis and Activity. J . Med. Chem. 1989, 32,
351-357.
(22) Reformatsky, S. Neue Synthese zweiatomiger einbasischer Sa¨u-
ren aus den Ketonen. Ber. Dtsch. Chem. Ges. 1887, 20, 1210-
1211.
(4) Bos, J . L. Ras oncogenes in human cancer: a review. Cancer
Res. 1989, 49, 9 (17), 4682-4689.
(5) Yurvey, A.; Wennogle, L. P. The RAF family: an expanding
network of posttranslational controls and protein-protein in-
teractions. Cell Res. 1998, 8, 81-98.
(6) Morrison, D. K.; Cutler, R. E. The complexity of Raf-1 regulation.
Curr. Opin. Cell Biol. 1997, 9 (2), 174-179.
(23) Maillard, J .; Benard, M.; Morin, R. Acides cycloalphatiques doue´s
d′activite´ chole´re´tique. Bull. Soc. Chim. Fr. 1958, 244-248.
(24) Carnduff, J . Recent Advances in Aldehyd Synthesis. Quart. Rev.
1966, 20, 169-189.
(7) Nassar, N.; Horn, G.; Herrmann, C.; Scherer, A.; McCormick,
F.; Wittinghofer, A. The 2.2A crystal structure of the Ras-binding
domain of the serine/threonine kinase c-Raf-1 in complex with
Rap1A and a GTP analogue. Nature 1995, 375, 554-560.
(8) Mott, H. R.; Carpenter, J . W.; Zhong, S.; Ghosh, S.; Bell, R. M.;
Campbell, S. L. The solution structure of the Raf-1 cysteine-
rich domain: a novel Ras and phospholipid binding site. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93 (16), 8312-8317.
(9) Marais, R.; Marshall, C. J . Control of the ERK MAP kinase
cascade by Ras and Raf. Cancer Surv. 1996, 27, 101-125.
(10) Frodin, M.; Gammeltoft, S. Role and regulation of 90 kDa
ribosomal S6 kinase (RSK) in signal transduction. Mol. Cell.
Endocrinol. 1999, 151 (1-2), 65-77.
(25) Buddrus, J . Wittig Reaktion mit A¨ thylenoxid. Angew. Chem.
1968, 80, 535-536.
(26) Nagata, W.; Hayase, Y. Formylolefination of carbonyl com-
pounds. J . Chem. Soc. 1969, 460-466.
(27) Fields, S.; Song, O. A novel genetic system to detect protein-
protein interaction. Nature 1989, 340, 245-246.
(28) Chien, C.; Bartel, P.; Sternglanz, R.; Fields, S. The two-hybrid
system: A method to identify and clone genes for proteins that
interact with a protein of interest. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 9578-9582.
(29) Iwabuchi, K.; Li, B.; Bartel, P.; Fields, S. Use of the two-hybrid
system to identify the domain of p53 involved in oligomerization.
Oncogene 1993, 8, 1693-1696.
(30) Li, B.; Fields, S. Identification of mutations in p53 that affect
its binding to SV40 T antigen by using the yeast two-hybrid
system. FASEB J . 1993, 7, 957-963.
(31) J aitner, B. K.; Becker, J .; Linnemann, T.; Herrmann, C.;
Wittinghofer, A.; Block, C. Discrimination of amino acids
mediating Ras binding from noninteracting residues affecting
Raf activation by double mutant analysis. J . Biol. Chem. 1997,
272 (47), 29927-29933.
(32) Kunkel, T. A. Rapid and efficient site-specific mutagenesis
without phenotypic selection. Proc. Natl. Acad. Sci. U.S.A. 1985,
82, 488-492.
(33) Kunkel, T. A.; Roberts, J . D.; Zakour, R. A. Rapid and efficient
site-specific mutagenesis without phenotypic selection. Methods
Enzymol. 1987, 14, 367-382.
(34) Harper, J . W.; Adami, G. R.; Wei, N.; Keyomarsi, K.; Elledge, S.
J . The p21 Cdk-interacting protein Cip1 is a potent inhibitor of
G1 cyclin-dependent kinases. Cell 1993, 75 (4), 805-816.
(35) Flick, J . Two systems of glucose repression of the GAL1 promotor
in Saccharomyces cerevisiae. Mol. Cell. Biol. 1990, 10 (9), 4757-
4769.
(36) Klebe, R. J .; Harriss, J . V.; Sharp, Z. D.; Douglas, M. G. A general
method for polyethylene-glycol-induced genetic transformation
of bacteria and yeast. Gene 1983, 25, 333-341.
(37) Miller, J . H. Experiments in Molecular Genetics; Cold Spring
Harbor Laboratory: Cold Spring Harbor, NY, 1972.
(11) Downward, J . Ras signaling and apoptosis. Curr. Opin. Genet.
Dev. 1998, 8, 49-54.
(12) Herrmann, C.; Block, C.; Geisen, C.; Haas, K.; Weber, C.; Winde,
G.; Moroy, T.; Muller, O. Sulindac sulfide inhibits Ras signaling.
Oncogene 1998, 17 (14), 1769-1776.
(13) Clark, G. J .; Drugan, J . K.; Terrel, R. S.; Bradham, C.; Der, C.
J .; Ball, R. M.; Campbel, S. Peptides containing a consensus Ras
binding sequence from Raf-1 and the GTPase activating protein
NF1 inhibit Ras function. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 1577-1581.
(14) Barnard, D.; Diaz, B.; Hettich, L.; Chuang, E.; Zhang, X. F.;
Avruch, J .; Marshall, M. Identification of the sites of interaction
between c-Raf-1 and Ras-GTP. Oncogene 1995, 10 (7), 1283-
1290.
(15) Barnard, D.; Sun, H.; Baker, L.; Marshall, M. S. In vitro
inhibition of Ras-Raf association by short peptides. Biochem.
Biophys. Res. Commun. 1998, 247, 176-180.
(16) Palaty, J .; Abbott, F. S. Structure-activity relationships of
unsaturated analogues of valproic acid. J . Med. Chem. 1995, 38,
3398-3408.
(17) Lo¨scher, W. Pharmacological, toxicological and neurochemical
effects of ∆2(E)-valproate in animals. Pharm. Weekbl. Sci. Ed.
1992, 14, 139-143.
(18) Wadsworth, W. S., J r.; Emmons, W. D. The utility of phospho-
nates carbanions in olefin synthesis. J . Am. Chem. Soc. 1961,
83, 1733-1738.
(19) Arbusov, B. A. Michaelis-Arbusow- und Perkow-Reaktion. Pure
Appl. Chem. 1964, 9, 307-335.
(20) Britelli, D. R. Phosphite-mediated in situ carboxyvinylation: A
new general acrylic acid synthesis. J . Org. Chem. 1981, 46,
2514-2520.
J M011101Q