α-Cyanocinnamide derivatives: A new family of non-peptide, non-sulfhydryl inhibitors of ras farnesylation

Article abstract of DOI:10.1016/S0968-0896(99)00118-2

Farnesylation of Ras and other proteins is required for their membrane attachment and normal function. Here we report on the synthesis of α-cyanocinnamide derivatives, a new family of farnesyltransferase inhibitors. These compounds are nonpeptidic and do not contain sulfhydryl groups. The most potent compound is a pure competitive inhibitor with respect to the Ras protein and mixed competitive with respect to farnesyl diphosphate. Selectivity studies against geranylgeranyltransferase and biological activities of selected compounds are described. Copyright (C) 1999.

Full text of DOI:10.1016/S0968-0896(99)00118-2

Bioorganic & Medicinal Chemistry 7 (1999) 1727±1736
ꢀ-Cyanocinnamide Derivatives: A New Family of Non-Peptide,
Non-Sulfhydryl Inhibitors of Ras Farnesylation
Enrique Poradosu, a Aviv Gazit, b Hadas Reuveni a and Alexander Levitzki a,*
aDepartment of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
bDepartment of Organic Chemistry, The Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received 2 March 1998; accepted 2 April 1999
AbstractÐFarnesylation of Ras and other proteins is required for their membrane attachment and normal function. Here we report
on the synthesis of a-cyanocinnamide derivatives, a new family of farnesyltransferase inhibitors. These compounds are nonpeptidic
and do not contain sulfhydryl groups. The most potent compound is a pure competitive inhibitor with respect to the Ras protein
and mixed competitive with respect to farnesyl diphosphate. Selectivity studies against geranylgeranyltransferase and biological
activities of selected compounds are described. # 1999 Published by Elsevier Science Ltd. All rights reserved.
Introduction
hypothesized to be a potentially speci®c chemother-
apeutic strategy.
The Ras proteins are a class of plasma membrane asso-
ciated G-proteins that act as a molecular switch in nor-
mal and pathogenic mitogenic signalling pathways
across the cell membrane. Point mutations in the ras
oncogenes which lock the Ras switch in its active GTP-
bound state are found in 40% of all cancers.1,2 The Ras
protein is localized to the inner lea¯et of the plasma
membrane. Anchoring to the membrane is achieved
through a series of post-translational modi®cations
directed by its carboxy terminal CAAX motif (C-
cysteine, A-aliphatic, X-methionine or serine), which is
farnesylated at the cysteine residue by the protein Ras
farnesyl transferase (FT). After a subsequent proteolytic
removal of the three C-terminal amino acids the farne-
sylated cysteine residue is methyl esteri®ed.3,4 The initial
farnesylation is a prerequisite for all subsequent cova-
lent modi®cations.5
Although biological e€ects exerted by FT inhibitors
were correlated with their ability to abolish Ras mem-
brane anchorage, several lines of evidence suggest that
the biological e€ects of FT inhibitors are mediated by
the inhibition of farnesylation of proteins other than
Ras, like Rho B.11±13
Despite the open questions concerning the mechanism
of action of FT inhibitors, experimental data obtained
in the past four years has clearly demonstated their
biological activity. FT inhibitors cause reversal of Ras
induced transformation in intact cells,14±18 inhibition of
Ras tumor growth in nude mice19±22 and tumor regres-
sion in Ha-Ras transgenic animals,23 with minimal toxic
e€ects. These ®ndings strongly support the use of FT
inhibitors as potential anticancer drugs.
The biological role of Ras prenylation has been exten-
sively studied; the modi®cation of Ras with a speci®c
isoprenoid is required for hSOS promoted guanine
nucleotide exchange,6 and the activation of Raf-1,7
B-Raf,8 and ERK.9 Moreover, membrane localization
of Ras is essential for its normal function and the
transforming activity of its oncogenic version.10
Thus, interfering with the Ras pathway by inhibiting
Ras farnesylation and membrane localization was
Analysis of the crystal structure of FT shows that it
possesses a bound zinc ion within an open coordination
sphere that includes a water molecule, suggesting a cat-
alytic function as opposed to the closed spheres char-
acteristic of structural metal ions. The crystallographic
data also indicates that the sulfhydryl group in the
CAAX motif is localized in close proximity to the zinc
atom and adjacent to the farnesylpyrophosphate a
phosphate.24 Accordingly, in CAAX motif peptidic
analogues the cysteine residue confers improved potency
to the inhibitor.25±28 However, the sulfhydryl moiety has
been replaced with various degrees of success in various
* Corresponding author.
0968-0896/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00118-2

1728
E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
peptidic derivatives16,17,29±34 and is not present in some
non-peptide inhibitors.35±37
imidazole and related heterocyclic groups is shown in
Table 1. The most e€ective compound was the imidazole
substituted a-cyanocinnamide (3) with an IC50 of 11.5 mM.
In this study we report on the synthesis and evaluation
of a new class of non-peptide, non-sulfhydryl FT inhi-
bitors based on the a-cyano-cinnamide structure. These
compounds exhibit FT inhibition activity in vitro and in
intact cells. The structure±activity relationship of a-
cyano-cinnamide derivatives, selectivity studies of FT
versus geranyl±geranyl transferase type I (GGT I) inhi-
bition,38,39 and their biological activities are described.
Next we examined the optimal length of the alkyl linker
between the pharmacophore and the cinnamide ring
and the optimal position on that ring (Table 2). The
most e€ective analogues had linkers in position ortho
and meta with an optimal length of 3 and 4 methylenes.
These inhibitors showed an IC50 of 17.5 and 11.5 mM
for the imidazole derivatives (compounds 10 and 3,
respectively) and 36.6 and 33.3 mM for the benzimida-
zole analogues (compounds 5 and 2, respectively).
Results and Discussion
In order to further explore the binding pocket of the
inhibitors a phenyl (14) and benzyl (29) derivatives of
compound 10 were prepared. While the benzyl ring
improved the inhibitory activity twofold (IC50 of
10.3 mM) the phenyl ring has a deleterious e€ect com-
pared to the parental compound (IC50 of 162 mM).
Further examination of phenyl and benzyl amide deri-
vatives yielded two di€erent families in which the rings
were systematically substituted with chemically di€erent
groups (Table 3). The most potent compound from this
series was 26, which inhibited FT with an IC50 of
1.8 mM.
ꢀ-Cyano-cinnamide derivatives design
With the aim to design non-peptide FT inhibitors lacking
a sulfhydryl group we ®rst synthesized a series of CAAX
peptides in which the cysteine residue was replaced by
various groups with di€erent anities towards Zn++
(data not shown). A direct correlation was observed
between the chelating capability to the inhibitory activity
towards FT. The best inhibitor of this series was HVFM
with an IC50 of 12 mM, in agreement with published
results in which the cysteine residue in CAAX peptidic
analogues was replaced by histidine.29
Interestingly, substitutions of di€erent chemical nature
in the phenyl ring at position R2 like the polar carboxyl
in compound 13 (IC50 12.1 mM) or Cl in compound 12
(IC50 7 mM) gave analogues with similar inhibitory
activities (Table 3). Free rotation of the phenyl ring in
space suggests the possibility that these substituents are
oriented to opposite sites in the same pocket. A similar
result was obtained in CAAX non-peptidic analogues.42,43
Since tyrosine kinase inhibitors from the tyrphostin family
which are hydroxy derivatives of a-cyanocinnamide were
shown to be tyrosine mimics,40 we chose as a non-pep-
tidic sca€old the a-cyanocinnamide structure with the
assumption that it will potentially mimic the phenylala-
nine residue in the potent CVFM peptide inhibitor.
a-Cyanocinnamide derivatives were prepared by a
straight-forward synthesis (Fig. 1); phenolic aldehydes
were reacted with excess a,o-dibromoalkanes and the
monobromo product puri®ed by chromatography. The
various thio-analogues were prepared from these bromo
compounds and the corresponding mercapto-hetero-
cycles, which were then condensed in the Knoevenagel
reaction with aryl cyano-acetamides41 or benzyl cyano-
acetamides (prepared analogueously). Yields were not
optimized.
As a preliminary test of this hypothesis we prepared the
phenyl imidazole derivatives with the double substitution
Table 1. Imidazole and related substitutions of a-cyanocinnamide. In
vitro IC50s against FT are reported for each compound. Each result
represents results of two to four tests. Assays were conducted as
described in Experimental
The inhibitory activity of a series of compounds based
on the a-cyanocinnamide structure substituted with
Compound no.
AGR
109
R
FT IC50 (mM)
1
500
2
3
71
33.3
11.5
124
Figure 1. A general scheme for the synthesis of a-cyanocinnamide
derivatives.

E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
1729
Table 2. Optimization of linker positioning and length in benzimi-
dazole and imidazole derivatives of a-cyanocinnamide. In vitro IC50
against FT are reported for each compound. Each result represents
results of two to four tests. Assays were conducted as described in
Experimental
and the inhibitor is <1. The inhibition pattern of
CAAX alternative substrates was found to be pure
competitive with respect to Ras and non-competitive
with respect to FPP.44 Although the mechanism of
inhibition of compound 26 implies a di€erent space
orientation within the active site than CAAX alternative
substrates it indicates that the inhibitor binds to the
catalytic pocket as expected.
s
Inhibition of FT versus GGT I
Early studies showed that the closely FT related prenyl
transferase GGT I transfers a geranyl±geranyl group to
CAAX containing sequences where X is preferably leu-
cine or phenylalanine.45±47 Since geranyl-geranylation of
normal proteins is 5±10 times more common than far-
nesylation,25,48,49 the common assumption was that a
compound displaying a strong selectivity towards FT
will have the advantage of reduced side e€ects than a
more general prenylation inhibitor. However, the dis-
covery that in cells resistant to FT inhibitors the K- and
N-Ras isoforms become geranyl-geranylated by GGT
I,50,51 and the ability of geranyl-geranylation inhibitors
to block platelet-derived and epidermal growth factor
dependent tyrosine phosphorylation,52 and to block
cells at G0/G153,54 has aroused new interest in this more
ubiquitous prenyl transferase reaction.
Compound no.
AGR
n (ortho)
FT
IC50 (mM)
4
5
6
93
85
94
2
3
4
153.1
36.6
115.1
Compound no.
AGR
n (meta)
FT
IC50 (mM)
112.3
81.6
33.3
100
7
8
2
9
87
79
71
74
2
3
4
5
A selectivity study of some FT inhibitors described here
was performed by testing their inhibitory activity
towards GGT I (Table 4). Most of the compounds tes-
ted inhibit both enzymes in vitro at comparable con-
centrations, (0.2±5 selectivity factor), with the highest
factor of selectivity towards FT (selectivity factor=33)
observed for compound 26. Similar to other series of FT
inhibitors35,55,56 selectivity was achieved despite the lack
of a structural element clearly corresponding to the
speci®city determining X residue in the CAAX motif.
Compound no.
AGR
n (ortho)
FT
IC50 (mM)
17.5
10
128
3
Compound no.
AGR
n (meta)
FT
IC50 (mM)
11.5
3
124
4
3,4 and 3,5-chloro,carboxy. These compounds inhibited
the FT reaction with an IC50 of 50.6 and 40 mM (18 and
25, respectively) (Table 3). This negative result implies
that the geometry of the hydrophobic and carboxylic
subpockets within this cleft requires a much more ¯ex-
ible structure than the phenyl ring to reach both sites
simultaneously. Further work is in progress to examine
this hypothesis.
Biological results
Compounds 13, 26, and 29 were tested for their ability
to inhibit protein prenylation in intact cells. Com-
pounds 13 and 26 completely inhibit Ras farnesylation
at a concentration of 120 mM, Rap-1 geranyl-geranyla-
tion was not a€ected at the same concentrations
(Fig. 3). The e€ect of these compounds on cell growth
was assessed on a LIM1899 colon carcinoma cell line
expressing a mutant K-Ras(Gly12!Cys12), NIH3T3
and v-H-Ras transformed NIH3T3 cell lines (Table 5).
The IC50s obtained (70±180 mM) are in correlation with
the concentrations required for FT inhibition in whole
cells. Previously it was reported that in K-Ras trans-
formed cells resistance to FT inhibitors could be
attributed to K-Ras geranyl-geranylation,50 however,
LIM1899 cells which express a mutated K-Ras were
inhibited to the same extent as NIH3T3 cells trans-
formed with v-H-Ras and normal NIH3T3 cells (Table
5). Compound 26 (AGR129) also inhibited colony for-
mation in soft agar of v-H-Ras transformed NIH3T3
cells (Fig. 4). At 100 mM, the concentration required for
complete inhibition of FT in whole cells, a 75% inhibi-
tion of colony formation was observed.
Although compound 10, which is the core of the inhi-
bitors described, was designed to mimic the HVF por-
tion of the HVFM peptide, the molecular structure of
the FT inhibitors developed di€er markedly from the
CAAX motif. Therefore we studied the kinetics of the
FT inhibition by the most potent in vitro inhibitor, 26.
Inhibition modalities other than Ras competition would
indicate a binding site di€erent from the catalytic
pocket to which the inhibitors were targeted based on
theoretical cosiderations. Compound 26 is a pure com-
petitive inhibitor of FT with respect to the Ras protein
and mixed competitive with respect to farnesyl pyro-
phosphate, as shown by the double reciprocal plots (1/
v versus 1/[S]) and the slope replots (slope versus [I])
(Fig. 2). The intersection of the reciprocal plots below
the 1/FPP axis means that the constant a€ecting Ki and
KFPP resulting from the mutual in¯uence between FPP

1730
E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
Conclusions
Experimental
General chemical procedures
Imidazole derivatives of a-cyano-cinnamide constitute a
novel class of non-peptidic, non-sulfhydryl FT inhibi-
tors. The most potent inhibitors of this class inhibit FT
with IC50 values at the low micromolar range and are
targeted to the catalytic site of the FT enzyme. These
compounds inhibit both the farnesylation of Ras in
intact cells and the cell growth of cells harboring muta-
ted v-H-Ras and K-Ras(Gly12!Cys12).
All starting materials were purchased from Aldrich.
NMR spectra were recorded on a Bruker 300 pulsed FT
spectrometer. Chemical shifts are in ppm relative to
TMS internal standard. Mass spectra were recorded
with a MAT311 instrument. Combustion analyses for
all new compounds were within 0.4% of the theoretical
value. Work up means adding to water, extracting with
Table 3. In vitro FT inhibition by phenyl (A) and benzyl (B) derivatives of AGR128 (compound 10). In vitro IC50s against FT are reported for
each compound. Each result represents results of two to four tests. Assays were conducted as described in Experimental
(A)
Compound no.
AGR
R1
R2
R3
R4
FT
IC50 (mM)
11
12
13
14
15
16
17
18
19
139
141
142
144
154
158
168
169
178
H
H
H
H
H
H
H
H
H
CH3
C1
COOH
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
72.6
7
12.1
162
>300
16.3
58.4
50.5
>64
H
COOCH2CH3
COOH
COOCH3
COOH
H
Cl
COOCH3
COOCH3
20
180
H
H
13.6
21
22
23
24
25
182
189
190
196
197
pH
H
H
H
H
H
NH2
COOCH3
Cl
H
H
OH
H
H
26
>128
>128
>64
40
COOCH3
H
COOCH3
COOH
Cl
H
(B)
Compound no.
AGR
R1
R2
R3
FT
IC50 (mM)
1.8
26
27
28
29
30
31
32
129
130
137
143
146
147
148
H
H
Cl
H
H
H
OCH3
Cl
H
H
H
OCH3
Cl
H
Cl
H
12.8
4.3
10.6
21.5
23.6
20.5
H
OCH3
Cl
OCH3
H

E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
1731
Figure 2. Kinetics of inhibition of the FT reaction by AGR129. The activity of FT was determined as described in Experimental except that the
reaction was performed in the presence of 1, 3, 6, 10, and 18 mM of GST-Ras, and a ®xed concentration of 0.5 mM FPP; or in the presence of 0.1, 0.3,
0.5, 1, and 1.8 mM of FPP and a ®xed concentration of 5 mM GST-Ras. Initial reaction velocity (v) is expressed as pmol of farnesylated GST-Ras/h/
mg enzyme. Families of double reciprocal plots and their respective slope replots are shown. AGR129 concentrations were as indicated in the ®gure.
dichloromethane (or with ethyl acetate when indicated),
drying the organic phase and evaporating to dryness.
Table 4. A comparison of FT and GGT I in vitro inhibitory activity
by selected inhibitors. Selectivity factor is expressed as the ratio of the
GGT I to FT inhibitory activites
Compound 1. (a). Bromo aldehyde. 25 g, 0.2 M, 3-hy-
droxy benzaldehyde, 70 g, 0.32 M, 1,4-dibromo butane,
and 17 g KOH in 150 mL ethanol were re¯uxed 18 h.
Work up and chromatography (silica gel, 70±230 mesh,
elution with CH2Cl2) gave 10.3 g, 20% yield, white oil.
NMR (CDCl3) d 9.97 (1H, s, CHO), 7.40 (3H, m), 7.18
(1H, m), 4.06 (2H, t, J=6.0 Hz), 3.50 (2H, t, J=6.0 Hz),
2.0 (4H, m). (b) Benzothiazole aldehyde. 0.5 g, 2.2 mM,
of the above bromo aldehyde, 0.38 g, 2.3 mM, 2-mer-
capto benzothiazole, and 0.2 g KOH in 30 mL ethanol
were stirred 19 h at room temperature. Work up (HCl
and EtAc) and chromatography gave after trituration
with benzene±hexane 0.11 g, 37% yield, white solid, mp
104ꢀC. NMR (CDCl3) d 9.95 (1H, s, CHO), 7.60 (4H,
m), 7.40 (3H, m), 7.22 (1H, m), 4.03 (2H, t, J=6.0 Hz),
3.53 (2H, t, J=6.0 Hz), 2.10 (4H, m). (c) 170 mg,
0.5 mM, 1b, 50 mg, 0.6 mM, cyano acetamide, and
15 mg b-alanine in 15 mL ethanol were re¯uxed 4 h.
Compound no.
FT
IC50 (mM)
GGT I
IC50 (mM)
Selectivity
factor
3
11.5
7
12.1
16.3
26
53.6
9.2
42.6
7.2
6.7
7.5
59.3
19.5
15.8
19.3
4.7
1.3
3.5
0.4
0.3
0.2
33
1.5
3.7
1.8
12
13
16
21
25
26
27
28
29
40
1.8
12.8
4.3
10.6
Evaporation and recrystalization from benzene gave
125 mg, 61% yield, white solid, mp 175ꢀC. NMR
(DMSO-d6) d 8.22 (1H, s, vinyl), 7.65 (4H, m), 7.30 (4H,
m), 4.10 (2H, t, J=5.8 Hz), 3.68 (2H, t, J=5.8 Hz), 2.12
(4H, m).

1732
E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
m), 3.93 (2H, t, J=5.7 Hz), 3.05 (2H, t, J=5.7 Hz), 1.60
(4H, m). (b) 260 mg, 0.93 mM, 3a, 84 mg, 1 mM, cyano
acetamide, and 18 mg b-alanine in 20 mL ethanol were
re¯uxed 3 h. Work up and trituration with acetone±
hexane gave 150 mg, 47% yield, white solid, mp 162ꢀC.
NMR (acetone-d6) d 8.18 (1H, s, vinyl), 7.45 (3H, m),
7.12 (2H, s), 7.08 (1H, m), 3.90 (2H, t, J=5.8 Hz), 3.10
(2H, t, J=5.8 Hz), 1.70 (4H, m).
Compound 4. (a). Bromo aldehyde. 7 g, 57 mM, salicyl
aldehyde, 15 g, 80 mM, dibromo ethane, and 7 g KOH
in 30 mL water and 50 mL ethanol were re¯uxed 20 h.
Work up and chromatography gave 0.83 g, 6% yield,
white oil. NMR (CDCl3) d 9.95 (1H, s, CHO), 7.40 (4H,
m), 4.33 (2H, t, J=6.0 Hz), 3.62 (2H, t, J=6.0 Hz). (b)
0.8 g, 3.5 mM, 4a, 0.5 g, 3.3 mM, 2-mercapto benzimi-
dazole, and 0.2 g KOH in 30 mL ethanol were stirred at
room temperature 14 h. Work up and trituration in
CH2Cl2±hexane gave 0.45 g, 44% yield, oily solid. NMR
(acetone-d6) d 9.93 (1H, s, CHO), 7.53 (4H, m), 7.30 (4H,
m), 4.45 (2H, t, J=6.0 Hz), 3.72 (2H, t, J=6.0 Hz). (c)
400 mg, 1.3 mM, 4b, 130 mg, 1.5 mM, cyano acetamide,
and 18 mg b-alanine in 20 mL ethanol were re¯uxed 3 h.
Work up and trituration with acetone±hexane gave
120 mg, 25% yield, white solid, mp 212ꢀC. NMR (ace-
tone-d6) d 8.26 (1H, s, vinyl), 7.45 (4H, m), 7.30 (4H,
m), 4.40 (2H, t, J=5.8 Hz), 3.60 (2H, t, J=5.8 Hz).
Figure 3. E€ect of a-cyanocinnamide derivatives on post-translational
processing of Ras and Rap1A/K-rev. v-H-Ras transformed NIH3T3
cells were treated with the indicated compounds at a concentration of
120 mM for 48 h or vehicle alone (control). Cell extracts were separated
by SDS±Page (40 mg of protein/lane) and visualized by western blot. (A)
v-H-Ras; (B) Rap1A. P, prenylated proteins; U, unprenylated protein.
Compound 13=AGR142, compound 29=AGR142, compound
26=AGR129.
Compound 5. (a). Bromo aldehyde, 7 g, 57 mM, salicyl
aldehyde, 16 g, 79 mM, 1,3-dibromo propane, and 7 g
KOH in 30 mL water and 50 mL ethanol were re¯uxed
24 h. Work up and chromatography gave 1.3 g, 9%
yield, white oil. NMR (CDCl3) d 9.95 (1H, s, CHO),
7.83 (1H, m), 7.55 (1H, m), 7.06 (2H, m), 4.24 (2H, t,
J=6.0 Hz), 3.62 (2H, t, J=6.0 Hz), 2.40 (2H, quint,
J=6.0 Hz). (b) 0.45 g, 1.8 mM, 5a, 0.2 g, 1.5 mM, 2-
mercapto benzimidazole, and 0.2 g KOH in 30 mL eth-
anol were stirred at room temperature 30 h. Work up
and trituration in CH2Cl2±hexane gave 0.29g, 61%
yield, white solid, mp 168ꢀC. NMR (acetone-d6) d 9.95
(1H, s, CHO), 7.53 (4H, m), 7.30 (4H, m), 4.45 (2H, t,
J=6.0 Hz), 3.72 (2H, t, J=6.0 Hz), 2.30 (2H, quint.,
J=6.0 Hz). (c) 160 mg, 0.5 mM, 5b, 45 mg, 0.5 mM,
cyano acetamide and 5 mg b-alanine in 20 mL ethanol
were re¯uxed 3 h. Work up and chromatography gave
40 mg, 21% yield, white solid, mp 77ꢀC. NMR (acetone-
d6) d 8.24 (1H, s, vinyl), 7.48 (4H, m), 7.36 (4H, m), 4.40
(2H, t, J=5.8 Hz), 3.60 (2H, t, J=5.8 Hz), 2.30 (2H,
quint., J=6.0 Hz).
Table 5. Cell growth inhibition. Inhibitions are expressed as IC50s in
mM. (3T3=NIH3T3, vRas=v-H-Ras transformed NIH3T3, LIM=
LIM1899)
Compound no.
3T3
vRas
LIM
13
26
98
186
80
152
73
100
Compound 2. (a). 1.6 g, 6.2 mM, bromo aldehyde 1a, 1 g,
6.5 mM, 2-mercapto benzimidazole, and 1 g, 10 mM,
Et3N in 30 mL ethanol were stirred 26 h at room tem-
perature. Work up and recrystalization from benzene
gave 0.6 g, 30% yield, viscous solid. NMR (acetone-d6)
d 10.0 (1H, s, CHO), 7.50 (4H, m), 7.25 (4H, m), 4.18
(2H, t, J=6.0 Hz), 3.86 (2H, t, J=6.0 Hz), 2.2 (4H, m).
(b) 310 mg, 0.95 mM, 2a, 84 mg, 1 mM cyano acetamide,
and 15 mg b-alanine in 20 mL ethanol were re¯uxed 8 h.
Evaporation and trituration in acetone±hexane gave
200 mg light-yellow solid, 54% yield, mp 154ꢀC. NMR
(acetone-d6) d 8.28 (1H, s, vinyl), 7.63 (4H, m), 7.30
(4H, m), 4.15 (2H, t, J=6.0 Hz), 3.72 (2H, t, J=6.0 Hz),
2.10 (4H, m).
Compound 6. (a). Bromo aldehyde. 7 g, 57 mM, salicyl
aldehyde, 15 g, 70 mM, 1,4-dibromo butane, and 7 g
KOH in 30 mL water and 40 mL ethanol were re¯uxed
24 h. Work up and chromatography gave 0.76 g, 5%
yield, white oil. NMR (CDCl3) d 9.95 (1H, s, CHO),
7.42 (4H, m), 4.06 (2H, t, J=6.0 Hz), 3.52 (2H, t,
J=6.0 Hz), 2.0 (4H, m). (b) 0.73 g, 2.8 mM, 6a, 0.4 g,
2.7 mM, 2-mercapto benzimidazole, and 0.2 g KOH in
30 mL ethanol were stirred at room temperature 14 h.
Work up and trituration in CH2Cl2±hexane gave 0.6 g,
65% yield, white solid, mp 68ꢀC. NMR (acetone-d6) d
9.95 (1H, s, CHO), 7.53 (4H, m), 7.31 (4H, m), 4.44
(2H, t, J=6.0 Hz), 3.70 (2H, t, J=6.0 Hz), 2.30 (2H, m).
Compound 3. (a). 1.6 g, 6.2 mM, 1a, 0.67 g, 6.7 mM, 2-
mercapto imidazole, and 0.5 KOH in 30 mL ethanol
were stirred 20 h at ambient temperature. Work up and
chromatography gave after trituration from acetone±
hexane 540 mg, 32% yield, white solid, mp 165ꢀC.
NMR (CDCl3) d 11.17 (1H, br.s, NH), 9.93 (1H, s,
CHO), 7.4 (3H, m), 7.15 (2H, s, imidazole), 7.10 (1H,

E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
1733
Figure 4. Inhibition of colony formation in soft agar by AGR129 (compound 26). v-H-Ras transformed NIH3T3 cells were seeded on a 96-well
plate (3500 cells/well) in soft agar (see Materials and Methods). Serial dilutions of the inhibitor were applied in the growth medium from the top.
After 12 days the colonies were stained with MTT, counted in the microscope and photographed. (a) 0 mM, (b) 25 mM, (c) 50 mM, (d) 100 mM, (e)
166 mM, (f) number of colonies per well plotted against the concentration of the inhibitor.
(c) 400 mg, 1.2 mM, 6b, 130 mg, 1.5 mM, cyano acet-
amide, and 10 mg b-alanine in 20 mL ethanol were
re¯uxed 3 h. Work up and chromatography gave 120 mg,
25% yield, white solid, mp 138ꢀC. NMR (acetone-d6) d
8.28 (1H, s, vinyl), 7.48 (4H, m), 7.36 (4H, m), 4.40 (2H,
t, J=5.8 Hz), 3.60 (2H, t, J=5.8 Hz), 2.34 (2H, m)
CHO), 7.42 (3H, m), 7.20 (1H, m), 4.35 (2H, t,
J=6.0 Hz), 3.66 (2H, t, J=6.0 Hz). (b) 0.43 g, 2.0 mM,
7a, 0.25 g, 1.6 mM, 2-mercapto benzimidazole, and 0.2 g
KOH in 30 mL ethanol were stirred at room tempera-
ture 30 h. Work up and trituration in CH2Cl2±hexane
gave 0.115 g, 24% yield, white solid, mp 164ꢀC. NMR
(acetone-d6) d 9.95 (1H, s, CHO), 7.53 (3H, m), 7.31
(4H, m), 7.15 (1H, m), 4.44 (2H, t, J=6.0 Hz), 3.70
(2H, t, J=6.0 Hz). (c) 105 mg, 0.35 mM, 7b, 30 mg,
1.5 mM, cyano acetamide, and 5 mg b-alanine in 20 mL
ethanol were re¯uxed 6 h. Workup and chromatography
gave 40 mg, 31% yield, white solid, mp 121ꢀC. NMR
Compound 7. (a). Bromo aldehyde, 4.9 g, 40 mM, 3-hy-
droxy benzaldehyde, 12.2 g, 65 mM, 1,2-dibromo ethane,
and 5 g KOH in 40 mL water and 40 mL ethanol were
re¯uxed 24 h. Work up and chromatography gave
1.05 g, 11% yield, white oil. NMR (CDCl3) d 9.97 (1H, s,

1734
E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
(acetone-d6) d 8.28 (1H, s, vinyl), 7.50 (3H, m), 7.38
(4H, m), 7.20 (1H, m), 4.40 (2H, t, J=5.9 Hz), 3.61 (2H,
t, J=5.9 Hz).
(1H, m), 4.16 (2H, t, J=6.0 Hz) 3.28 (2H, t, J= 6.0 Hz),
2.19 (2H, quint., J=6.0 Hz).
Compounds 11±32. These compounds were prepared
from the imidazole aldehyde 5a and the appropriate aryl
or benzyl cyano acetamide. One illustrating example is
given to each followed by data to the analogues of its
group.
Compound 8. (a). Bromo aldehyde, 5.1 g, 42 mM, 3-hy-
droxy benzaldehyde, 14 g, 69 mM, 1,3-dibromo propane,
and 5 g KOH in 60 mL water and 50 mL ethanol were
re¯uxed 18 h. Work up and chromatography gave 4.3 g,
33% yield, white oil. NMR (CDCl3) d 9.97 (1H, s, CHO),
7.45 (3H, m), 7.18 (1H, m), 4.16 (2H, t, J=6.0 Hz), 3.62
(2H, t, J=6.0 Hz), 2.34 (2H, quint., J=6.0 Hz). (b)
1.5 g, 6.2 mM, 8a, 0.9 g, 6.0 mM, 2-mercapto benzimi-
dazole, and 0.2 g KOH in 30 mL ethanol were stirred at
room temperature 26 h. Work up and chromatography
gave 0.29 g, 24% yield, viscous oil. NMR (acetone-d6) d
9.95 (1H, s, CHO), 7.53 (3H, m), 7.30 (4H, m), 7.25
(1H, m), 4.43 (2H, t, J=6.0 Hz), 3.74 (2H, t, J=6.0 Hz),
2.30 (2H, quint., J=6.0 Hz). (c) 300 mg, 0.96 mM, 8b,
90 mg, 1.07 mM, cyano acetamide, and 5 mg b-alanine
in 20 mL ethanol were re¯uxed 3 h. Workup and tri-
turation in CH2Cl2±hexane gave 160 mg, 44% yield,
white solid, mp 132ꢀC. NMR (acetone-d6) d 8.20 (1H, s,
vinyl), 7.48 (3H, m), 7.36 (4H, m), 7.19 (1H, m), 4.40
(2H, t, J=5.8 Hz), 3.60 (2H, t, J=5.8 Hz), 2.30 (2H,
quint., J=5.8 Hz).
Aryl analogues 11±25
Compound 12. (a). 4 g, 31 mM, 3-Cl aniline, and 4.7 g,
47 mM, methyl cyanoacetate were heated at 120ꢀC
without solvent at open ¯ask, for 15 h, the cooled reac-
tion was chromatographed directly (silica gel, 70±230
mesh, elution with dichloromethane) to give 0.92 g, 15%
yield, white solid, mp 132ꢀC. NMR (acetone-d6) d 7.84
(1H, m), 7.40 (2H, m), 7.16 (1H, m), 3.85 (2H, s). (b)
41 mg, 0.156 mM, 5a, 31 mg, 0.16 mM, 12a, and 6 mg b-
alanine in 20 mL ethanol were re¯uxed 4 h. Evaporation
and trituration in CH2Cl2±hexane gave 60 mg, 87%
yield, light-yellow solid, mp 152ꢀC. NMR (acetone-d6) d
9.06 (1H, s, vinyl), 8.25 (1H, m), 7.82 (1H, m), 7.5±7.0
(7H, m), 7.10 (2H, s, imidazole), 4.19 (2H, t, J=6.0 Hz),
3.30 (2H, t, J=6.0 Hz), 2.22 (2H, quint., J=6.0 Hz). MS
m/e 438,440 (M+, 12, 4%), 312 (M-NHAr, 14), 193
(20), 127 (100).
Compound 9. (a). Bromo aldehyde, 4.9 g, 40 mM, 3-hy-
droxy benzaldehyde, 12.6 g, 55 mM, 1,5-dibromo pen-
tane, and 5 g KOH in 40 mL water and 40 mL ethanol
were re¯uxed 20 h. Workup and chromatography gave
2.7 g, 25% yield, white oil. NMR (CDCl3) d 9.97 (1H, s,
CHO), 7.40 (3H, m), 7.18 (1H, m), 4.03 (2H, t, J=
6.0 Hz), 3.44 (2H, t, J=6.0 Hz), 2.0 (4H, m), 1.7 (2H,
m). (b) 1.3 g, 5.0 mM, 9a, 0.6 g, 4.0 mM, 2-mercapto
benzimidazole, and 0.3 g KOH in 30 mL ethanol were
stirred at room temperature 23 h. Workup and chroma-
tography gave 0.5 g, 29% yield, viscous oil. NMR (ace-
tone-d6) d 9.95 (1H, s, CHO), 7.47 (3H, m), 7.30 (4H,
m), 7.20 (1H, m), 4.43 (2H, t, J=6.0 Hz), 3.54 (2H, t,
J=6.0 Hz), 2.20 (4H, m), 1.85 (2H, m). (c) 250 mg,
0.73 mM, 9b, 67 mg, 0.8 mM, cyano acetamide, and
5 mg b-alanine in 20 mL ethanol were re¯uxed 8 h.
Work up and trituration in CH2Cl2±hexane gave
195 mg, 65% yield, white solid, mp 82ꢀC. NMR (ace-
tone-d6) d 8.25 (1H, s, vinyl), 7.48 (3H, m), 7.36 (4H,
m), 7.19 (1H, m), 4.40 (2H, t, J=5.8 Hz), 3.60 (2H, t,
J=5.8 Hz), 2.20 (4H, m), 1.90 (2H, m).
N-Aryl cyano acetamides. Compound, yield (%), mp
(ꢀC). 11, 19, 127. 13, 25, 256. 14, 17, 193. 15, 17, 138. 16,
30, 254. 17, 25, 178. 18, 30,222. 19, 47, 132. 20, 6, 138.
21, 7, 158. 22, 7, 112. 23, 5, 127. 24, 30, 187. 25, 13, 236.
Compounds 11±25. Compound, yield (%), mp (ꢀC). 11,
44, 148. 13, 60, 214. 14, 90, 182. 15, 92, 117. 16, 55, 215.
17, 66, 158. 18, 44, 148. 19, 97, 173. 20, 95, 197. 21. 84,
159. 22, 43, 133. 23, 50, 145. 24, 38, 170. 25, 42, 192.
Benzyl analogues 26±32
Compound 29. (a). 15 mL, 0.14 M, benzyl amine, and
13 mL, 0.14 M, methyl cyanoacetate were heated at
120ꢀC without solvent at open ¯ask, for 16 h, the cooled
reaction was chromatographed directly (silica gel, 70±
230 mesh, elution with dichloromethane) to give 9.4 g,
38 yield, white solid, mp 132ꢀC. NMR (CDCl3) d 7.4
(5H, m), 4.47 (2H, d, J=6.0 Hz), 3.38 (2H, s). (b) 63 mg,
0.24 mM, 5a, 46 mg, 0.26 mM, 29a, and 6 mg b-alanine
in 20 mL ethanol were re¯uxed 4 h. Evaporation and
trituration in CH2Cl2±hexane gave 36 mg, 37% yield,
light-yellow solid, mp 132ꢀC. NMR (acetone-d6) d 9.00
(1H, s, vinyl), 8.15 (1H, m), 7.82 (1H, m), 7.5±7.0 (7H,
m), 7.12 (2H, s, imidazole), 4.52 (2H, d, J=5.8 Hz), 4.24
(2H, t, J=6.0 Hz), 3.32 (2H, t, J=6.0 Hz), 2.26 (2H,
quint., J=6.0 Hz).
Compound 10. (a). 0.8 g, 3.3 mM, 5a, 0.32 g, 3.2 mM, 2-
mercapto imidazole, and 0.2 KOH in 30 mL ethanol
were stirred 20 h at ambient temperature. Work up and
chromatography gave after trituration from acetone
hexane 356 mg, 43% yield, white solid, mp 88ꢀC. NMR
(CDCl3) d 10.4 (1H, s, CHO), 7.80 (1H, d, J=7.8 Hz),
7.54 (1H, t), 7.10 (2H, s, imidazole), 7.04 (1H, t, J=
7.5 Hz), 6.96 (1H, d, J=8.4 Hz), 4.22 (2H, t, J=6.0 Hz)
3.24 (2H, t, J=6.0 Hz), 2.22 (2H, quint., J=6.0 Hz). (b)
45 mg, 0.17 mM, 3a, 20 mg, 0.24 mM, cyano acetamide,
and 4 mg b-alanine in 20 mL ethanol were re¯uxed 4 h.
Workup and trituration with acetone±hexane gave 44 mg,
80% yield, white solid, mp 164ꢀC. NMR (acetone-d6) d
9.0 (1H, s, vinyl), 8.4 (1H, d, J=8.2 Hz), 7.50 (1H, t,
J=7.8 Hz), 7.10 (1H, m), 7.07 (2H, s, imidazole), 6.93
N-Benzyl cyano acetamides. Compound, yield (%), mp
(ꢀC). 26, 80, 96. 27, 90, 115. 28, 51, 98. 30, 96, 133. 31,
85, 126. 32, 81, 134.
Compounds 26±32. Compound, yield (%), mp (ꢀC). 26,
43, 172. 27, 57, 147. 28, 53, oil. 30, 47, 141. 31, 41, 89.
32, 58, 111.

E. Poradosu et al. / Bioorg. Med. Chem. 7 (1999) 1727±1736
1735
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