Non-thiol farnesyltransferase inhibitors: FTase-inhibition and cellular activity of benzophenone-based bisubstrate analogue farnesyltransferase inhibitors

Article abstract of DOI:10.1002/ardp.200300758

Some 5-acylaminoacylamino-benzophenone derivatives were designed as bisubstrate analogue farnesyltransferase inhibitors. These compounds turned out to be only weakly active against farnesyltransferase, but displayed an antiproliferative effect rendering them suitable for further development as a novel type of cytostatic agents.

Full text of DOI:10.1002/ardp.200300758

242 Mitsch et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
Andreas Mitscha,
Silke Bergemannb,
Ronald Gustb,
Non-thiol Farnesyltransferase Inhibitors:
FTase-Inhibition and Cellular Activity of
Benzophenone-based Bisubstrate Analogue
Farnesyltransferase Inhibitors
Isabel Sattlerc,
Martin Schlitzerd
Some 5-acylaminoacylamino-benzophenone derivatives were designed as bisub-
strate analogue farnesyltransferase inhibitors. These compounds turned out to be
only weakly active against farnesyltransferase, but displayed an antiproliferative ef-
fect rendering them suitable for further development as a novel type of cytostatic
agents.
a
Institut für Pharmazeutische
Chemie, Philipps-Universität
Marburg, Marburg, Germany
Institut für Pharmazie,
Freie Universität Berlin,
Berlin, Germany
b
Keywords:Non-thiol farnesyltransferase inhibitors;Structure/Activity relationships;
Bisubstrate analogs; MCF-7 breast cancer cells; Antiproliferative agents
c
Hans-Knöll-Institut für
Naturstoff-Forschung e.V.,
Jena, Germany
Department für Pharmazie –
Received: January 6, 2003; Accepted: March 4, 2003 [FP758]
DOI 10.1002/ardp.200300758
d
Zentrum für
Pharmaforschung,
Ludwig-Maximilians-
Universität München,
München, Germany
free thiols are associated with several adverse drug ef-
fects [13] and, therefore, the development of farnesyl-
transferase inhibitors is clearly directed towards the so-
called non-thiol farnesyltransferase inhibitors.
Introduction
Inhibition of farnesyltransferase has become a major
strategy for the development of novel potential anti-can-
cer drugs [1–8].Farnesyltransferase catalyzes the trans-
fer of a farnesyl residue from farnesylpyrophosphate to
the thiol of a cysteine side chain of proteins bearing the
CAAX-tetrapeptide sequence (C: cysteine, A: aliphatic
amino acid, X:serine or methionine) C-terminally [9, 10].
Farnesylation is a prerequisite for the transforming activ-
ity of oncogenic Ras, which is found in approximately
30% of all cancers in humans. However, there is accu-
mulating evidence that prevention of Ras farnesylation
may not be the crucial cellular event responsible for the
antiproliferative effect of farnesyltransferase inhibitors
[5, 8]. Disregarding the unresolved mechanism of action
of farnesyltransferase inhibitors, the efficacy of these
compounds and their low toxicity has been demonstrat-
ed [5, 8, 11].
We have described a series of benzophenone-based
bisubstrate analogue farnesyltransferase inhibitors (e.g.
1) (Figure 1), compounds displaying molecular features
of both, the peptidic and the prenylic substrate [14]. In
this type of compounds, the 2-acylaminobenzophenone
moiety represents the peptidomimetic and the fatty acid
the farnesylmimetic substructure. Here, we have re-
placedthealiphaticfarnesyl surrogatebyaromaticacryl-
ic acid derivatives. In an earlier study, we identified such
aromatic acrylic acids as farnesyl mimetics [15]. The
compounds were evaluated for their farnesyltransferase
inhibitory activity and their effect on the growth of MCF-7
breast cancer cells. In addition, two compounds were
evaluated in the NCIs cancer cell line screen.
Most inhibitors described in literature are peptidomimet-
ics resembling the CAAX-tetrapeptide recognition se-
quence of farnesylated proteins. The majority of these
CAAX-peptidomimetics exhibits a free thiol group [1-8]
which coordinates the enzyme-bound zinc ion as dem-
onstrated for the native peptide substrate [12]. However,
Chemistry
The compounds were prepared from the appropriate 2-
acylamino-5-aminobenzophenones [16] (10) either by
acylation of the 5-amino function by the appropriate N-
acylamino acid [17, 18] activated as mixed anhydride or
by acylation of 2-(tolylacetyl)amino-5-aminobenzophe-
none [16] (10b) by the appropriate N-Boc protected ami-
no acid and subsequent removal of the protective group
followed by acylation with 4-phenylcinnamic acid chlo-
ride (Scheme 1).
Correspondence: Martin Schlitzer, Department für Pharma-
zie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Uni-
versität München, Butenandtstraße 5–13, D-81377 München,
Germany. Phone: +49 89 2180-77804, Fax: +49 89 2180-79992,
e-mail: martin.schlitzer@cup.uni-muenchen.de
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/04–05/0242 $ 17.50+.50/0

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
Non-thiol Farnesyltransferase Inhibitors 243
Figure 1. Stuctures of inhibitors 1–9.
Scheme 1. (I) N-4-benzyloxycinnamoyl-β-alanine or N-4-phenylcinnamoy-β-alanine or N-4-phenylcinnamoylpiperi-
dine-3-carboxylic acid, isobutyl chloroformate, N-methylmorpholine, DMF, –15°C, 5 min., then add 10, DMF, –15°C to
RT, overnight;(II) N-Boc-glycine or N-Boc-N-methyl-glycine or N-Boc-phenylalanine or N-Boc-proline, isobutyl chloro-
formate, N-methylmorpholine, DMF, –15°C, 5 min., then add 10b, DMF, –15°C to RT, overnight;(III) HCl 4 M in dioxane,
RT, 1 h; (IV) 4-phenylcinnammic acid chloride, N-methylmorpholine, DCM, 0°C → RT, 4 h.

244 Mitsch et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
Farnesyltransferase inhibition assay
Cell culture assays
The inhibitory activity was determined using the fluores-
cence enhancement assay as described by Pompliano
[20]. The assay employs yeast farnesyltransferase
(FTase) fused to Glutathione S-transferase at the N-ter-
minus of the β-subunit [21].Farnesylpyrophosphate and
the dansylated pentapeptide Ds-GlyCysValLeuSer were
used as substrates. Upon farnesylation of the cysteine
thiol, the dansyl residue is placed into a lipophilic envi-
ronment.The resulting enhancement of fluorescence at
505 nm is used to monitor the enzyme reaction.
The influence of the FTase inhibitors on the proliferation
of breast cancer cells was evaluated on the MCF-7 cell
line. MCF-7 cells were treated with the compounds 2–9
in three concentrations (1, 5, 10 µM) 48 h after seeding.
In this experiment, the drug- (and vehicle (DMSO)) con-
taining culture media were left unchanged throughout
the incubation period of about 244 h. The results are
summarized in Figure 2. In these plots of T/Ccorr vs. time
of incubation, time zero indicates the time of drug addi-
tion. T/Ccorr was calculated according to Equation 1
Figure 2. Activity of compounds 2–9 against MCF-7 breast cancer cells.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
Non-thiol Farnesyltransferase Inhibitors 245
(Experimental). In addition, compounds 4 and 5 have
been assayed against the full panel of cancer cell lines of
the NCI [22].
placement of β-alanine by glycine (5) resulted in a de-
creased antiproliferative activity when assayed in a con-
centration of 5 µM, while the activity remained nearly
constant in the higher concentration of 10 µM. Virtually
inactive compounds (7–9) were obtained when
β-alanine was replaced by phenylalanine, proline, or pip-
eridine-3-carboxylic acid.The use of N-methylglycine in
the central position resulted in compound 6.The time ac-
tivity curve of 6 presented in Figure 2 reveals high cyto-
cidal effects in the 5 µM and 10 µM concentration after
100–150 h of incubation (10 µM, τ = –46% after 148 h;5
µM τ = –25% after 128 h).However, the cells develop re-
sistance against 6 because the cytocidal effects are lost
towards the end of the test.On the other hand, 3, 4,and 5
preserved their strong antiproliferative potency during
the time of incubation, so 4 and 5 were selected for fur-
ther evaluation against 60 different cancer cell lines in
the NCI’s drug screen (Table 2).
Results and discussion
In contrast to the benzophenone-based bisubstrate ana-
logue farnesyltransferase inhibitors (e.g. 1) described
earlier [14], compounds 2–9, carrying an aromatic far-
nesylmimetic instead of an aliphatic one, exhibited only
weak farnesyltransferase inhibitory activity.The IC50-val-
ues are ranging between 10 and 36 µM (Table 1). Al-
though the effects are weak, some structure/activity re-
lationships (SAR) can be delineated. Compound 3, car-
rying a tolylacetic acid at the 2-amino group of the ben-
zophenone scaffold, was more active than the phenyl-
acetic acid carrying inhibitor 2.The same SAR has also
been observed with benzophenone-CAAX-peptidomi-
metics[16].Inaddition, deviationfromβ-alanineaslinker
between the peptidomimetic and the farnesylmimetic
substructures resulted in a decreased activity in most
cases, a result, whichhasalsobeen obtainedwithbisub-
strate analogues of different structure [18].
GI50-values are predominantly in the low micromolar
range with little differences between the two com-
pounds. No superiority of one inhibitor over the other is
thereby visible.A total growth inhibition (TGI) is generally
observed at similar concentrations while those required
reducing the cell number by 50% (LC50) are in some cas-
es significantly higher.
Although these compounds have initially been designed
as bisubstrate analogue farnesyltransferase inhibitors
and some parallels being between antiproliferative and
farnesyltransferase inhibitory activity, there are two facts
arguing strongly for an antiproliferative mechanism dif-
ferent from farnesyltransferase inhibition. First, antipro-
liferative activity is generally observed at lower concen-
trations than farnesyltransferase inhibition and second,
compound 5 displays considerable antiproliferative ac-
tivity while its farnesyltransferase inhibition (IC50 = 36
µM) is the weakest of the whole series.
Table 1. Farnesyltransferase inhibitory activity of com-
pounds 1–9.
Compd.
IC50 (µM)
Compd.
IC50 (µM)
1 [14]
0.4 0.033
6
7
8
9
10
32
25
6
9
7
2
3
4
5
22
14
9.3
8
5
4
30 11
36 10
Therefore, we consider these compounds as a new type
of antiproliferative agents with a mechanism yet to be de-
termined. Early structure activity relationships can be
deduced from this series, which suggest that a further
development may be worthwhile.
However, some of these compounds displayed concen-
tration-dependent cytotoxic–orat higher concentrations
– even cytocidal effects against MCF-7 breast cancer
cells.The compounds 4 and 6 were more active than the
antitumor drug cisplatin (see Figure 2).
Again, a clear dependency of the antiproliferative effect
on the structure of the compounds was visible. In analo-
gy to the activity enhancing effect of the methyl group at
the arylacetic acid substructure with respect to farnesyl-
transferase inhibition this residue also enhanced anti-
proliferative activity (Figure 2;3 vs.2).Direct comparison
of the inhibitors 3 and 4 revealed some superiority of the
4-phenylcinnamoyl-substituted compound 4 over the 4-
benzyloxycinnamoyl derivative 3. Therefore, this study
was continued with phenylcinnamoyl derivatives.The re-
Acknowledgements
The Developmental Therapeutics Program, DCTD, NCI,
NIH, Bethesda, MD, USA is gratefully acknowledged for
the in vitro screening data. The pGEX-DPR1 and pBC-
RAM2 plasmids were kindly provided by Prof.F.Tamanoi
(UCLA). Financial support by the Deutsche Phar-
mazeutische Gesellschaft is gratefully acknowledged.
I. S. wishes to thank Prof. Dr. S. Grabley for generous
support and Ms. S. Egner for technical assistance.

246 Mitsch et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
Table 2. Activity of compounds 4 and 5 in NCI’s drug screen. GI50: 50% growth inhibition;TGI: total growth inhibition
(0% growth); LC50: 50% reduction of cell number.
Compd. 5
GI50
Compd. 4
TGI
Panel/Cell Line
TGI
LC50
GI50
LC50
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
4.78E-06
7.44E-06
3.33E-06
3.50E-06
1.04E-05
1.84E-06
1.71E-05
2.07E-05
1.03E-05
1.53E-05
2.92E-05
4.9cE-06
4.94E-05
4.82E-05
3.21E-05
3.92E-05
8.22E-05
1.67E-05
2.16E-06
1.69E-05
1.11E-05
4.17E-06
2.40E-06
8.84E-06
5.44E-05
>1.00E-04
9.16E-06
1.47E-05
9.71E-05
>1.00E-04
>1.00E-04
>1.00E-04
8.02E-05
Non-Small Cell Lung
Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
2.03E-06
3.47E-06
2.56E-06
4.17E-06
2.79E-06
2.06E-06
5.21E-06
2.86E-06
5.13E-06
5.37E-06
1.58E-05
6.95E-06
1.54E-05
8.39E-06
4.71E-06
2.46E-05
1.11E-05
1.84E-05
2.09E-05
4.71E-05
2.67E-05
4.45E-05
3.49E-05
1.21E-05
>1.00E-04
3.72E-05
4.73E-05
1.77E-06
1.32E-06
2.11E-06
4.32E-06
3.58E-06
4.15E-06
1.16E-05
9.71E-06
8.18E-06
2.42E-06
1.85E-06
3.47E-06
1.88E-06
6.15E-06
4.20E-06
1.77E-05
4.60E-06
2.42E-05
9.50E-06
>1.00E-04
1.70E-05
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
6.93E-06
1.68E-06
1.37E-06
3.69E-06
1.58E-06
3.57E-06
4.75E-06
3.20E-05
4.98E-06
2.65E-06
1.32E-05
2.99E-06
1.45E-05
1.95E-05
>1.00E-04
1.84E-05
5.15E-06
3.63E-05
5.65E-06
4.49E-05
8.73E-05
1.75E-06
2.37E-08
1.12E-06
2.71E-06
5.06E-06
2.93E-06
1.57E-06
4.53E-06
1.61E-06
2.96E-06
9.53E-06
2.03E-05
1.46E-05
3.44E-06
1.39E-05
8.20E-06
7.82E-06
4.04E-05
6.63E-05
>1.00E-04
7.50E-06
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
2.46E-06
2.22E-06
2.70E-06
4.57E-06
7.51E-06
4.86E-06
8.85E-06
2.03E-05
3.29E-05
1.19E-05
3.69E-05
7.08E-05
2.62E-06
1.36E-06
1.68E-06
3.06E-06
1.93E-06
1.85E-06
9.47E-06
3.05E-06
3.67E-06
1.60E-05
6.59E-06
4.35E-06
7.41E-05
6.84E-06
8.03E-06
8.55E-05
3.26E-05
1.07E-05
2.77E-06
8.67E-06
3.24E-05
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
3.69E-06
2.99E-06
4.81E-06
1.39E-05
1.17E-05
1.69E-06
6.33E-06
1.31E-05
1.34E-05
1.23E-05
1.73E-05
3.53E-05
6.66E-05
6.32E-06
2.27E-05
3.08E-05
3.88E-05
7.73E-05
5.31E-05
8.99E-05
>1.00E-04
2.60E-05
6.35E-05
7.27E-05
2.03E-06
3.39E-06
2.57E-06
1.89E-06
3.04E-06
2.10E-06
2.05E-06
2.17E-06
4.84E-06
1.15E-05
7.30E-06
5.79E-06
9.00E-06
5.14E-06
4.71E-06
4.87E-06
1.45E-05
6.06E-05
3.13E-05
2.25E-05
3.95E-05
1.60E-05
1.23E-05
1.30E-05

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
Non-thiol Farnesyltransferase Inhibitors 247
Table 2. (continued)
Compd. 5
Compd. 4
Panel/Cell Line
GI50
TGI
LC50
GI50
TGI
LC50
Ovarian Cancer
IGROV1
8.25E-06
4.97E-06
2.17E-05
1.91E-05
5.11E-05
4.93E-05
9.75E-07
1.88E-06
1.48E-06
2.09E-06
8.72E-07
1.85E-06
2.16E-06
4.32E-06
3.41E-06
5.77E-06
2.21E-06
4.36E-06
4.72E-06
9.89E-06
7.82E-06
2.79E-05
5.12E-06
1.07E-05
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
4.48E-06
1.59E-06
1.07E-05
2.31E-05
3.27E-06
2.74E-05
>1.00E-04
6.73E-06
6.98E-05
Renal Cancer
786-0
A498
2.27E-06
9.89E-07
4.79E-06
4.89E-06
2.38E-06
2.74E-06
2.08E-06
1.18E-05
5.47E-06
8.73E-06
1.62E-05
2.04E-05
6.60E-06
9.93E-06
5.95E-06
2.47E-05
1.88E-05
3.27E-05
4.17E-05
6.05E-05
2.36E-05
3.69E-05
2.56E-05
5.18E-05
1.60E-06
2.31E-06
3.08E-06
3.29E-06
1.46E-06
1.92E-06
2.78E-06
3.67E-06
3.34E-06
5.31E-06
1.09E-05
1.13E-05
3.52E-06
5.51E-06
8.72E-06
1.37E-05
6.97E-06
1.74E-05
4.17E-05
7.09E-05
8.50E-06
2.62E-05
>1.00E-04
3.71E-05
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
DU-145
1.98E-06
2.49E-06
4.07E-06
7.54E-06
8.37E-06
3.59E-05
1.46E-06
3.93E-06
3.48E-06
>1.00E-04
8.25E-06
>1.00E-04
Breast Cancer
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
BT-549
1.81E-06
9.71E-06
2.62E-06
4.71E-06
3.67E-06
4.74E-06
1.09E-05
1.76E-06
3.69E-06
2.98E-05
9.21E-06
2.11E-05
1.61E-05
2.09E-05
2.40E-05
3.89E-06
7.51E-06
9.02E-05
3.40E-05
9.11E-05
4.76E-05
7.99E-05
5.27E-05
8.62E-06
1.72E-06
5.41E-06
1.87E-06
1.73E-06
3.27E-06
2.50E-06
1.46E-06
2.85E-06
4.92E-06
>1.00E-04
5.64E-06
3.74E-06
1.08E-05
6.61E-06
3.11E-06
1.80E-05
3.09E-05
>1.00E-04
2.45E-05
8.07E-06
4.32E-05
2.53E-05
6.64E-06
>1.00E-04
T-47D
tained with a Leitz microscope (Leitz, Wetzlar, Germany) and
are uncorrected. Column chromatography was carried out us-
ing silica gel 60 (0.062–0.200 mm) from Merck (Darmstadt,
Germany).
Experimental
General
1H-NMR spectra were recorded on a Jeol JMN-GX-400 and a
Jeol JMN-LA-500 spectrometer (Jeol USA Inc., Peabody, MA,
USA). Mass spectra were obtained with a Vacuum Generators
VG 7070 H (VacuumGenerators, Manchester, UK) using aVec-
tor 1 data acquisition system from Teknivent (Teknivent Corp.,
Maryland Heights, MO, USA) or an AutoSpec mass spectrome-
ter from Micromass (Micromass, Manchester, UK). IR spectra
were recorded on a Nicolet 510P FT-IR-spectrometer (Thermo
Nicolet Corp., Madison, WI, USA). Microanalyses were ob-
tained from a CH analyzer according to Dr. Salzer from Labor-
matic and from a Hewlett Packard CHN-analyzer type 185
(Hewlett Packard, Palo Alto, CA, USA).Melting points were ob-
General procedure A:amide formation using acids activated as
mixed anhydrides.
The appropriate acid was dissolved in a sufficient amount of
dry DMF in a flame dried flask under Ar atmosphere.After addi-
tion of N-methylmorpholine [NMM] (0.25 ml per mmol acid) the
solution was cooled to –15°C and isobutyl chloroformate
(0.13 ml per mmol acid) was added. A solution of the amine
component (1 equivalent) in dry DMF was added after 5 min.In
case the amine component was employed as a hydrochloride,
additional NMM (0.25 mL per mmol) was added. The mixture

248 Mitsch et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
was allowed to warm up to room temperature overnight, and
then poured into brine (400–800 mL). In case of solid precipi-
tate formation, this was collected by suction and thoroughly
washed with water. Otherwise, the aqueous mixture was ex-
tracted with ethyl acetate (3 × 100 mL) and the combined or-
ganic extracts were washed successively with 2 N citric acid,
sat. NaHCO3-solution and brine, and dried with MgSO4. The
residue obtained after removal of the solvent was purified by
recrystallisation or flash chromatography.
MS (EI): m/z 223 (100), 266 (75), 212 (64), 207 (63), 178 (54),
379 (51), 621 (18) [M+].Anal.calcd.for C40H35N3O4:C, 77.27;H,
5.67; N, 6.76; found: C, 76.95; H, 5.67; N, 6.48.
N-[[3-Benzoyl-4-(p-tolylacetylamino)phenylcarbamoyl]-
methyl]-3-biphenyl-4-yl-acrylamide (5)
From 4-phenylcinnamic acid (224 mg, 1 mmol) according to
general procedure B. Yield: 476 mg (78%). Mp 223°C. – IR
(KBr): ν = 3406, 3276, 3059, 2925, 1695, 1654, 1608, 1560,
1509 cm–1. 1H NMR (DMSO-d6): δ = 2.23 (s, 3H), 3.34 (s, 2H),
3.99 (s, 2H), 6.77 (d, J = 16 Hz, 1H), 6.97 (m, 2H), 7.02 (m,
2H), 7.20 (m, 1H), 7.34–7.37 (m, 2H), 7.44–7.49 (m, 4H),
7.55–7.71 (m, 9H), 7.75 (m, 2H), 8.32 (m, 1H), 9.99 (s, 1H),
10.10 (s, 1H).– MS (EI):m/z 325 (100), 212 (54), 207 (44), 458
(32), 344 (25), 607 (8) [M+]. Anal. calcd for C39H33N3O4: C,
77.08; H, 5.47; N, 6.92; found: C, 77.36; H, 5.75; N, 6.66.
General procedure B: amide formation using acids chlorides
Carboxylic acids were dissolved in dichloromethane and
0.2 mL oxalychloride per mmol acid was added. The mixture
was stirred at room temperature for 2 h and the volatiles were
evaporated in vacuo.The residue obtained was dissolved in tol-
uene or dioxane (approx. 10 mL) and added to a solution of the
appropriate aromatic amine in hot toluene (approx.50 mL).The
mixtures were heated under reflux for 2 h. Then, the solvent
was removed in vacuo to give the crude products.
N-[[3-Benzoyl-4-(p-tolylacetylamino)phenylcarbamoyl]-
methyl]-3-biphenyl-4-yl-N-methylacrylamide (6)
From 11b (773 mg, 1.5 mmol) according to general procedure
C.Yield: 457 mg (49%). Mp 202°C. – IR (KBr): ν = 3277, 3056,
General procedure C: N-Boc-deprotection and acylation using
acid chlorides
1
3032, 2923, 1689, 1646, 1599, 1559, 1508 cm–1. – H NMR
(CDCl3):δ 2.32 (s, 3H), 3.31 (s, 3H), 3.67 (s, 2H), 4.17 (s, 2H),
6.88 (d, J = 16 Hz, 1H), 7.15 (m, 2H), 7.25 (m, 2H), 7.37 (m,
1H), 7.42–7.49 (m, 4H), 7.53–7.62 (m, 8H), 7.66–7.73 (m,
3H), 7.85 (m, 1H), 8.47 (m, 1H), 9.07 (s, 1H), 10.48 (s, 1H). –
MS (EI): m/z 212 (100), 344 (86), 207 (59), 278 (55), 309 (52),
621 (2) [M+]. Anal. calcd. for C40H35N3O4: C, 77.27; H, 5.67; N,
6.76; found: C, 77.48; H, 5.53; N, 6.72.
The appropriate Boc-protected derivative 11 was stirred in a
4 N solution of HCl(g) in dioxane for 1 h.After the volatiles were
removed in vacuo, the residue 12 was used without further puri-
fication for the reaction with phenylcinnamic acid chloride ac-
cording to general procedure B.
N-[2-[3-Benzoyl-4-(phenylacetylamino)phenylcarbamoyl]ethyl]-
3-(4-benzyloxyphenyl)-acrylamide (2)
(S)-N-[1-[3-Benzoyl-4-(p-tolylacetylamino)phenylcarbamoyl]-
2-phenylethyl]-3-biphenyl-4-yl-acrylamide (7)
From 3-[3-(4-benzyloxyphenyl)acryloylamino]propionic acid
(440 mg, 1.35 mmol) according to general procedure A.Yield:
800 mg (93%). Mp 138°C. – IR (KBr): ν = 3304, 3064, 3032,
From 11 c (591 mg, 1 mmol) according to general procedure C.
Yield: 420 mg (60%). Mp 211°C. – IR (KBr): ν = 3260, 3018,
1
2962, 1654, 1601, 1560, 1509 cm–1. – H NMR (CDCl3): δ =
1
2890, 1649, 1620, 1514 cm–1. – H NMR (CDCl3): δ 2.23 (s,
2.54 (t, J = 6 Hz, 2H), 3.55 (t, J = 6 Hz, 2H), 3.64 (s, 2H), 4.99
(s, 2H), 6.13 (d, J = 16 Hz, 1H), 6.43 (m, 1H), 6.85 (m, 2H),
7.28–7.41 (m, 16H), 7.45 (m, 1H), 7.59 (m, 2H), 7.75 (m, 1H),
8.43 (m, 1H), 8.58 (s, 1H), 10.45 (s, 1H). – MS (EI): m/z 91
(100), 366 (27), 253 (27), 311 (22), 212 (17), 266 (15), 637 (0.2)
[M+]. Anal. calcd. for C40H35N3O5: C, 75.34; H, 5.53; N, 6.59;
found: C, 75.13; H, 5.70; N, 6.22.
3H), 3.00 (m, 2H), 3.60 (s, 2H), 4.91 (dd, J = 7/14 Hz, 1H),
6.37 (d, J = 16 Hz, 1H), 7.06–7.13 (m, 7H), 7.16–7.30 (m, 7H),
7.35 (m, 5H), 7.39–7.47 (m, 7H), 7.77 (s, 1H), 8.43 (m, 1H),
8.82 (s, 1H), 10.05 (s, 1H). – MS (EI): m/z 476 (100), 362 (91),
167 (85), 679 (76), 494 (73), 262 (63), 697 (9) [M+].Anal.calcd.
for C46H39N3O4: C, 79.17; H, 5.63; N, 6.02; found: C, 78.94; H,
5.58; N, 6.20.
N-[2-[3-Benzoyl-4-(p-tolylacetylamino)phenylcarbamoyl]-
ethyl]-3-(4-benzyloxy-phenyl)acrylamide (3)
(S)-N-[3-Benzoyl-4-(p-tolyl-acetylamino)phenyl]-1-(3-bi-
phenyl-4-yl-acryloyl)-pyrrolidine-2-carboxylic acid amide (8)
From 3-[3-(4-benzyloxyphenyl)acryloylamino]propionic acid
(327 mg, 1 mmol) according to general procedure A. Yield:
443 mg (68%). Mp 137°C. – IR (KBr): ν = 3296, 3061, 2923,
1656, 1602, 1539, 1510, 1251, 1174 cm–1.– 1H NMR (CDCl3):δ
= 2.26 (s, 3H), 2.54(m, 2H), 3.58(m, 2H), 3.60(s, 2H), 5.00 (s,
2H), 6.13 (d, J = 15 Hz, 1H), 6.26 (m, 1H), 6.86 (m, 2H), 7.10
(m, 2H), 7.16 (m, 2H), 7.21–7.43 (m, 11H), 7.59 (m, 3H), 7.75
(s, 1H), 8.29 (s, 1H), 8.43 (m, 1H), 10.39 (s, 1H). – MS (EI):
m/z 91 (100), 105 (14), 253 (12), 649 (0.7) [M+]. Anal. calcd. for
C41H37N3O5:C, 75.56;H, 5.72;N, 6.45;found:C, 75.35;H, 5.59;
N, 6.39.
From 11d (537 mg, 1 mmol) according to general procedure C.
Yield: 101 mg (16%). Mp 135 C. – IR (KBr): ν = 3277, 2876,
1
1700, 1647, 1594, 1560, 1507 cm–1. – H NMR (CDCl3): δ =
1.77 (m, 1H), 2.00 (m, 1H), 2.09 (m, 1H), 2.26 (s, 3H), 2.52 (m,
1H), 3.57 (m, 1H), 3.59 (s, 2H), 3.72 (m, 1H), 4.81 (m, 1H),
6.70 (d, J = 16 Hz, 1H), 7.09 (m, 2H), 7.15–7.20 (m, 2H), 7.31
(m, 1H), 7.39 (m, 4H), 7.49–7.57 (m, 8H), 7.63 (m, 2H), 7.69
(m, 1H), 7.79 (m, 1H), 8.41 (m, 1H), 9.94 (s, 1H), 10.33 (s,
1H). – MS (EI):m/z 207 (100), 344 (24), 304 (24), 647 (5) [M+].
Anal.calcd.for C42H37N3O4:C, 77.88;H, 5.76;N, 6.49;found:C,
77.95; H, 5.74; N, 6.38.
N-[2-[3-Benzoyl-4-(p-tolylacetylamino)phenylcarbamoyl]-
ethyl]-3-biphenyl-4-yl-acrylamide (4)
(RS)-N-[3-Benzoyl-4-(p-tolyl-acetylamino)phenyl]-1-(3-bi-
phenyl-4-yl-acryloyl)-piperidine-3-carboxylic acid amide (9)
From3-(3-biphenyl-4-yl-acryloylamino)propionic acid (307 mg,
1 mmol) according to general procedure A. Yield: 385 mg
(62%). Mp 181°C. – IR (KBr): ν = 3270, 3030, 2921, 1653,
1612, 1537, 1510 cm–1.– 1H NMR (CDCl3:δ = 2.25 (s, 3H), 2.54
(t, J = 6 Hz, 2H), 3.57 (s, 2H), 3.58 (t, J = 6 Hz, 2H), 6.30 (d, J =
16 Hz, 1H), 6.55 (m, 1H), 7.08 (m, 2H), 7.14 (m, 2H), 7.28 (m,
1H), 7.30–7.44 (m, 6H), 7.45–7.51 (m, 6H), 7.55–7.61 (m,
3H), 7.77 (m, 1H), 8.40 (m, 1H), 8.60 (s, 1H), 10.38 (s, 1H). –
From 10b (334 mg, 1 mmol) according to general procedure B.
Yield: 330 mg (50%). Mp 149°C. – IR (KBr): ν = 3430, 3277,
1
3058, 2938, 2860, 1653, 1640, 1560, 1507 cm–1. – H NMR
(CDCl3): δ = 1.56 (m, 2H), 1.81 (m, 1H), 2.21 (m, 1H), 2.26 (s,
3H), 2.56 (m, 1H), 3.54 (m, 1H), 3.61 (s, 2H), 3.63 (m, 1H),
3.72 (m, 1H), 4.03 (m, 1H), 6.77 (d, J = 16 Hz, 1H), 7.08 (m,
3H), 7.17 (m, 2H), 7.30 (m, 1H), 7.37–7.43 (m, 7H), 7.47 (m,

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
Non-thiol Farnesyltransferase Inhibitors 249
2H), 7.49–7.55 (m, 2H), 7.65 (m, 3H), 7.91 (m, 1H), 8.45 (m,
1H), 9.23 (s, 1H), 10.44 (s, 1H).– MS (EI):m/z 149 (100), 212
(79), 105 (78), 344 (73), 244 (55), 661 (16) [M+].Anal.calcd.for
C43H39N3O4:C, 78.04;H, 5.94;N, 6.35;found:C, 77.70;H, 5.75;
N, 6.41.
GCVLS, 20 µM FPP and 5 nmol (approx.) yeast GST-farnesyl-
transferase and 1% of various concentrations of the test com-
pounds dissolved in dimethylsulfoxide (DMSO). The progress
of the enzyme reaction was followed by monitoring the en-
hancement of the fluorescence emission at 505 nm (excitation
340 nm). The reaction was started by addition of the enzyme
and run in a Quartz cuvette thermostated at 30°C. Fluores-
cence emission was recorded with a Perkin Elmer LS50B spec-
trometer (Perkin Elmer, Shelton, CT, USA). IC50 values (con-
centrations resulting in 50% inhibition) were calculated from in-
itial velocity of three independent measurements of four to five
different concentrations of the respective inhibitor.
N-[[3-Benzoyl-4-(p-tolyl-acetylamino)phenylcarbamoyl]-
methyl]-N-methylcarbamic acid tert-butyl ester (11b)
From Boc-sarcosine (378 mg, 2 mmol) according to general
procedure A.Yield:592 mg (57%).– 1H NMR (CDCl3):δ 1.37 (s,
9H), 2.26 (s, 3H), 2.89 (s, 3H), 3.61 (s, 2H), 3.82 (s, 2H), 7.09
(m, 2H), 7.17 (m, 2H), 7.40 (m, 2H), 7.44 (m, 1H), 7.51 (m,
1H), 7.58–7.63 (m, 3H), 7.74 (s, 1H), 8.44 (m, 1H), 10.43 (s,
1H).
Cytotoxicity studies on the MCF-7 cell line
N-[1-[3-Benzoyl-4-(p-tolylacetylamino)phenylcarbamoyl]-
2-phenylethyl]carbamic acid tert- butylester (11c)
The human MCF-7 breast cancer cell line was obtained from
the American Type Culture Collection (ATCC, Rockville, Md.;
USA). Cell line banking and quality control were performed ac-
cording to the seed stock concept reviewed by Hay [23]. The
MCF-7 cells were maintained in L-glutamine containing Eagle’s
MEM (Sigma, München, Germany), supplemented with
NaHCO3 (2.2 g/L) sodium pyruvate (110 mg/L), gentamycin
(50 mg/L; Sebio Walchsing, Germany), and 10% fetal calf se-
rum (FCS; Gibco Eggenheim, Germany) using 75 cm2 culture
flasks (Falcon Plastics 3023) in a water-saturated atmosphere
(95% air/5% CO2) at 37°C.The cell line was weekly passaged
after treatment with trypsin (0.05%) / ethylenediamine-
tetraacetic acid (0.02%; EDTA; Boehringer, Mannheim, Ger-
many). Mycoplasma contamination was routinely monitored,
and only mycoplasma-free cultures were used.
From Boc-phenylalanine (530 mg, 2 mmol) according to gener-
al procedure A.Yield:763 mg (65%).– 1H NMR (CDCl3):δ 1.35
(s, 9H), 2.33 (s, 3H), 3.05 (d, J = 7 Hz, 2H), 3.66 (s, 2H), 3.69
(t, J = 7 Hz, 1H), 7.14–7.25 (m, 7H), 7.34 (m, 1H), 7.46 (m,
3H), 7.58 (m, 1H), 7.66 (m, 4H), 7.92 (s, 1H), 8.43 (m, 1H),
10.48 (s, 1H).
N-[3-Benzoyl-4-(p-tolylacetylamino)phenyl]-Nα-tert.-butyl-
oxycarbonylprolineamide (11d)
From Boc-proline (430 mg, 2 mmol) accorrding to general pro-
cedure A.Yield: 688 mg (64%). – 1H NMR (CDCl3): δ 1.36 (s,
9H), 1.60 (m, 1H), 1.83 (m, 1H), 2.26 (s, 3H), 2.28 (m, 1H),
2.48 (m, 1H), 3.30 (m, 1H), 3.60 (s, 2H), 3.81 (m, 1H), 4.36 (m,
1H), 7.10 (m, 3H), 7.18 (m, 2H), 7.39 (m, 2H), 7.49 (m, 2H),
7.62 (m, 2H), 7.70 (m, 1H), 8.42 (s, 1H), 10.38 (s, 1H).
In vitro chemosensitivity assay
1-(3-Biphenyl-4-yl-acryloyl)-piperidine-3-carboxylic acid
The in vitro testing for antitumor activity was carried out on ex-
ponentially dividing human breast cancer cells according to a
previously published microtiter assay [24, 25].Briefly, in 96-well
microtiter plates (Costar), 100 µL of a cell suspension at 500
cells/ml culture medium were plated into each well and incubat-
ed at 37°C for 2–3 d in a humidified atmosphere (5% CO2). By
addition of an adequate volume of a stock solution of the re-
spective compound (solvent: DMSO) to the medium the de-
sired test concentration was obtained (max. content of DMSO
in the medium: 1 ppm). For each test concentration and for the
control, which contained the corresponding amount of DMSO,
16 wells were used. After the proper incubation time the medi-
um was removed, the cells were fixed with a glutardialdehyde
solution and stored at 4°C. Cell biomass was determined by a
crystal violet staining technique [24, 25]. The effectiveness of
the complexes is expressed as correctedT/C values according
to the following equations:
4-Phenylcinnamic acid (450 mg, 2 mmol) was converted to the
acid chloride following general procedure B and added, dis-
solved in acetone (20 mL), to the solution of nipecotic acid
(260 mg, 2 mmol) in water (40 mL) and K2CO3 (860 mg).After
1 h at 0°C and 1 h at room temperature, ice water was added
and the pH was adjusted to 1 by addition of conc. HCl.The re-
sulting solid was washed with water and dried. Yield: 380 mg
(57%). – 1H NMR (DMSO-d6): δ 1.40 (m, 1H), 1.65 (m, 2H),
1.95 (m1H), 3.10 (m, 1H), 3.48 (m, 1H), 3.93(m, 1H), 4.07 (m,
1H), 4.45 (m, 1H), 7.24 (d, J = 16 Hz, 1H), 7.34 (m, 1H), 7.43
(m, 2H), 7.47 (d, J = 16 Hz, 1H), 7.66 (m, 4H), 7.74 (m, 2H),
12.23 (s, 1H).
Enzyme preparation
Yeast farnesyltransferase was used as a fusion protein to Glu-
tathione S-transferase at the N-terminus of the b-subunit. Far-
nesyltransferase was expressed in Escherichia coli DH5α
grown in LB media containing ampicillin and chloramphenicol
for co-expression of pGEX-DPR1 and pBC-RAM2 for farnesyl-
transferase production [21].The enzyme was purified by stand-
ard procedures with glutathione-agarose beads for selective
binding of the target protein.
Cytotoxic effect: T/Ccorr [%] = [(T-Co)/(C-Co)] × 100
Eq. (1)
where T (test) and C (control) are the optical densities at
578 nm of the crystal violet extract of the cell lawn in the wells
(i.e. the chromatin-bound crystal violet extracted with ethanol
70%), and Co is the density of the cell extract immediately be-
fore treatment.
Farnesyltransferase assay
The assay was conducted as described [20]. Farnesylpyro-
phosphate (FPP) was obtained as a solution of the ammonium
salt in methanol-10 mM aqueous NH4Cl (7:3) from Sigma-
Aldrich (Sigma-Aldrich Deutschland, Taufkirchen, Germany).
Dansyl-GlyCysValLeuSer (Ds-GCVLS) was custom synthe-
sized by ZMBH, Heidelberg, Germany. The assay mixture
(100 mL volume) contained 50 mM Tris/HCl pH 7.4, 5 mM
MgCl2, 10 µM ZnCl2, 5 mM dithiothreitol (DTT), 7 µM Ds-
Cytocidal effect: τ [%] = [(T-Co)/Co] × 100
Eq. (2)
For the automatic estimation of the optical density of the crystal
violet extract in the wells a Microplate EL 309 Autoreader (BIO-
TEK Instruments, Winooski, VT, USA) was used.

250 Mitsch et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 242–250
[14] M.Schlitzer, M.Böhm, I.Sattler, Bioorg.Med.Chem.2002,
10, 615.
References
[1] D. M. Leonard, J. Med. Chem. 1997, 40, 2971.
[15] M. Schlitzer, I. Sattler, Bioorg. Med. Chem. 1999, 7, 2391.
[2] Y.S.Qian, M.Sebti, A.D.Hamilton, Biopolymers 1997, 43,
[16] J.Sakowski, M.Böhm, I.Sattler, H.-M.Dahse, M.Schlitzer,
25.
J. Med. Chem. 2001, 44, 2886.
[3] T. M. Williams, Exp. Opin. Therp. Patents 1998, 8, 553.
[4] T. M. Williams Exp. Opin. Therp. Patents 1999, 9, 1263.
[5] I. M. Bell, Exp. Opin. Therp. Patents 2000, 10, 1813.
[17] M.Schlitzer, M.Böhm, I.Sattler, H.-M.Dahse Bioorg.Med.
Chem. 2000, 8, 1991.
[18] M.Schlitzer, I.Sattler, Pharm.Pharmacol.Commun.2000,
5, 117.
[6] T. M. Williams, C. J. Dinsmore, Adv. Med. Chem. 1999, 4,
[19] M. Fieser, L. F. Fieser, E .Toromanoff, Y. Hirata, H. Hey-
mann, M.Tefft, S. Bhattacharya, J. Am. Chem. Soc. 1956,
78, 2825.
273.
[7] A. Wittinghofer, H. Waldmann, Angew. Chem. 2000, 112,
4360; Angew. Chem. Int. Ed. 2000, 39, 4192.
[20] D. L. Pompliano, R. P. Gomez, N. J. Anthony J. Am. Chem.
Soc. 1992, 114, 7945.
[8] E.K.Rowinsky, A. Patnaik, Emerging Drugs 2000, 5, 161.
[9] F. L. Zhang, P. J. Casey, Annu. Rev. Biochem. 1996, 65,
[21] K. Del Villar, H. Mitsuzawa, W.Yang, I. Sattler, F.Tamanoi,
241.
J. Biol. Chem. 1997, 272, 680.
[10] H.-W. Fu, P. J. Casey, Rec. Prog. Hormon Res. 1999, 54,
[22] M. R. Boyd, K. D. Paull, Drug Dev. Rev. 1995, 34, 91.
[23] R. Hay, J. Anal. Biochem. 1988, 171, 225.
315.
[11] A. Oliff, Biochim. Biophys. Acta 1999, 1423, C19.
[24] G.Bernhardt, H.Reile, H.Birnböck,T.Spruß, H.Schönen-
[12] C. L. Strickland,;W.T.Windsor, R. Syto, L.Wang, R. Bond,
R.Wu, J. Schwartz, H.V. Le, L. S. Beese, P. C.Weber, Bio-
chemistry 1998, 37, 16601.
berger, J. Cancer Res. Clin. Oncol. 1992, 118, 35.
[25] H.Reile, H.Birnböck, G.Bernhardt,T.Spruß, H.Schönen-
berger, Anal. Biochem. 1990, 187, 262.
[13] Martindale The Extra Pharmacopeia, 31st ed. (Ed.: J. E. F.
Reynolds), Royal Pharmaceutical Society of Great Brit-
ain, London, 1996; p. 821.
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