Non-thiol farnesyltransferase inhibitors: Structure-activity relationships of aralkylsubsituted benzophenones

Article abstract of DOI:10.1002/1521-4184(200102)334:2<40::AID-ARDP40>3.0.CO;2-W

We describe a novel class of benzophenone-based farnesyltransferase inhibitors exploiting a novel aryl binding region in the farnesyltransferase's active site. The present study was mainly focussed on structural modifications of the trimethylene spacer of the 4-phenyl butyroyl residue of our lead structure (IC₅₀ = 530 nM). These modifications turned out to have little effect on activity as had the replacement of the terminal aryl by cyclohexyl (IC₅₀ = 440 nM vs. IC₅₀ = 530 nM).

Full text of DOI:10.1002/1521-4184(200102)334:2<40::AID-ARDP40>3.0.CO;2-W

40
Mitsch, Wißner, Sattler, and Schlitzer
Non-Thiol Farnesyltransferase Inhibitors: Structure-Activity
Relationships of Aralkylsubsituted Benzophenones
a)
a)
b)
a)
Andreas Mitsch , Pia Wißner , Isabel Sattler , and Martin Schlitzer *
a) Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
b) Hans-Knöll-Institut für Naturstoff-Forschung e. V., Beutenbergstraße 11, D-07745 Jena, Germany
Key Words: Farnesyltransferase inhibitors; peptidomimetics, stucture-activity relationship
nesyltransferase inhibitor based on the benzophenone scaf-
Summary
fold and c) the arylalkyl-substituted benzophenone 1d which
terminal phenyl residue should occupy the postulated aryl
binding region.
We describe a novel class of benzophenone-based farnesyltrans-
ferase inhibitors exploiting a novel aryl binding region in the
farnesyltransferase’s active site. The present study was mainly
focussed on structural modifications of the trimethylene spacer of
the 4-phenyl butyroyl residue of our lead structure (IC50
=
530 nM). These modifications turned out to have little effect on
activity as had the replacement of the terminal aryl by cyclohexyl
(IC50 = 440 nM vs. IC50 = 530 nM).
Introduction
One of the most important posttranslational modifications
[1]
of proteins is protein prenylation , which is the transfer of
either a farnesyl or a geranylgeranyl residue from the corre-
sponding pyrophosphates to the thiol of a cysteine side chain
of proteins carrying the C-terminal CAAX-sequence (C: cys-
teine, A: amino acid with aliphatic side chain, X: variable
amino acid). Due to the involvement of farnesylated proteins
in carcinogenesis, inhibition of farnesyltransferase has re-
[2–5]
ceived much interest in recent years
. Farnesyltransferase
inhibitors have been demonstrated to inhibit a wide range of
tumor cell lines in vitro and display synergistic effects with
[6–8]
other tumor therapeutics
. Farnesyltransferase inhibitors
are therefore regarded as a major emerging strategy in tumor
therapy.
Most farnesyltransferase inhibitors described to date are
structural analogs of the CAAX recognition sequence of the
farnesylated proteins, thus they carry a free thiol function.
However, a free thiol is an undesireable feature in potential
drugs because of its sensitivity towards oxidation and, more
importantly, as a potential source of severe adverse drug
[9]
effects . Development is therefore directed towards the so
[10]
Figure 1. See text for explanation.
called “non-thiol” farnesyltransferase inhibitors. We
and
[11–14]
others
have shown that the cysteine residue in CAAX-
peptidomimetic farnesyltransferase inhibitors can be re-
placed by aryl moieties, a finding that lead to the postulation
In this study, we describe the synthesis and farnesyltrans-
ferase inhibitory activity of some structural variants of se-
lected examples of our model compounds 1a–f.
of two hitherto unknown aryl binding sites in or near the
[13,14]
farnesyltransferase’s active site
. Using several model
compounds (1a–f) based on our benzophenone AAX-pep-
[15]
tidomimetic scaffold
and docking studies, we have pro-
Chemistry
vided evidence for the location of the postulated binding
sites . Figure 1 provides an schematic representation of
farnesyltransferase’s active site showing a) the physiological the 5-aminobenzophenone 2
[10]
The target compounds 3–5 were prepared by acylation of
[15]
by appropriate carboxylic
substrates (CAAX-tetrapeptide and farnesylpyrophosphate acids activated either as acid chlorides oras mixed anhydrides
[FPP]), b) a thiol-containing CAAX-peptidomimetic far- (Scheme 1). Glycine derivatives 4e and 4f were prepared
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0365-6233/01/0202/0040 $17.50 +.50/0

Non-Thiol Farnesyltransferase Inhibitors
41
H
R
N
H
O
R
X
H2N
R
N
I or II
O
O
+
O
O
O
NH
NH
NH
R:
2
O
O
O
or
O
Cl
O
X:
3, 4, 5
4a
4b
4c
4d
4e
1a
S
1b
1c
O
O
IV, V
O2N
O2N
4e
4f
O
H
III
2
N
O
NH
O
N
1d
1e
1f
H
O
IV, VI, VII
S
6
O
H
N
Scheme 1. (I) toluene/dioxane, reflux, 2 h; (II) DMF, –15 °C → rt, 4 h;
(III) a) N-Boc-Gly, IBCF, NMM, DMF, –15 °C; 5 min.; b) add 2, DMF to
a), DMF, –15 °C → rt, 4 h; (IV) HCl/dioxane, rt, 1 h; (V) Ph-COCl, tolu-
ene/dioxane, reflux, 2 h; (VI) Ph-CHO, NEM, toluene, reflux 8 h; (VII)
NaBH4, toluene, rt, overnight.
O
O
4f
H
N
3a
4g
4h
3b
3c
from the N-protected glycine amide 6. After acidic removal
of the Boc protective group, the resulting amino group was
acylated by benzoyl chloride to yield 4e or reductively alky-
lated to yield 4f.
3d
5a
Biological Evaluation
5c
The inhibitory activity of the compounds prepared on far-
nesyltransferase was determined using a fluorescence en-
hancement assay. This assay, established by Pompliano et al.
(Merck Inc.) as an easy to handle test system for the evalu-
ation of potential farnesyltransferase inhibitors, yields
5b
equivalent results to those assays employing ras and tritium-
[16]
labeled FPP as substrates
. The assay employed yeast
Figure 2
farnesyltransferase (FTase) fused to glutathione S-transferase
[17]
at the N-terminus of the β-subunit.
Yeast farnesyltrans-
Table 1. Farnesyltransferase inhibitory activity of compounds 1 and 3–5.
——————————————————————————————
ferase is a close homolog and functionally similar to the
human enzyme; it is widely used for the evaluation of far-
[17,18]
Compd. IC50 (µM)
Compd. IC50 (µM)
Compd. IC50 (µM)
nesyltransferase inhibitors.
Farnesylpyrophosphate
and the dansylated pentapeptide Ds-GlyCysValLeuSer were
used as substrates. Upon farnesylation of the cysteine thiol
the dansyl residue is placed in a lipophilic environment; the
resulting enhancement of fluorescence at 505 nm is used to
monitor the enzyme reaction.
——————————————————————————————
1a
1b
1c
1d
1e
1f
6.0 ± 0.4
3b
3c
3d
4a
4b
4c
4d
5.0 ± 0.1
4e
4f
24.8 ± 0.2
9.6 ± 0.4
26.6 ± 1.3
0.99 ± 0.05
5.6 ± 0.2
4.2 ± 0.1
7.0 ± 0.2
2.0 ± 0.5
0.59 ± 0.05
0.44 ± 0.04
3.7 ± 0.12
6.1 ± 0.23
0.66 ± 0.03
1.0 ± 0.06
6.9 ± 0.6
4g
4h
5a
5b
5c
0.53 ± 0.04
0.4 ± 0.005
1.1 ± 0.06
6.6 ± 0.3
Results and Discussion
3a
Compounds 1a–f were used as model compounds for aryl-
substituted non-thiol farnesyltransferase inhibitors to explore
the postulated novel aryl binding sites in the farnesyltrans-
——————————————————————————————
tory activity of the phenyl derivative 1a and the cyclohexyl
derivative 3a (1a: IC = 6.0 µM; 3a: IC = 6.6 µM). With
[10]
ferase’s active site . Common structural features of our
50
50
the homolog compound pair 1b/3b, a significant difference
model compounds are a terminal phenyl residue connected
via a polymethylene spacer of variable length (n = 0–5) and
an amide moiety to our AAX-peptidomimetic benzophenone
scaffold. Now we address the question of how structural
modifications ofour model compounds influence theirinhibi-
tory activity. In a first series we replaced the terminal phenyl
in activity is observed (1b: IC = 2.0 µM; 3b: IC = 5.0 µM)
50
50
with the cyclohexyl derivative being 2.5-fold less active than
the phenyl derivative 1b. A significant difference in activity
was also observed with the next pair of compounds (1c: IC
50
= 6.9 µM; 3c: IC = 0.59 µM). In contrast to the preceding
50
residue of 1 by a cyclohexyl residue. For compound 3a, little pair, the cyclohexyl derivative in this case is about tenfold
difference is observed between the farnesyltransferase inhibi- more active than the phenyl derivative. However, this trend
Arch. Pharm. Pharm. Med. Chem. 334, 40–44 (2001)

42
Mitsch, Wißner, Sattler, and Schlitzer
did not continue with the next pair of homologs. Here, the
cyclohexyl derivative 3d is only slightly more active than the
Experimental
1H- and 13C-NMR spectra were recorded on a Jeol JMN-GX-400 and a
Jeol JMN-LA-500 spectrometer. Mass spectra were obtained with a Vacuum
Generators VG 7070 H using a Vector 1 data aquisition system from
Teknivent or a AutoSpec mass spectrometer from Micromass. IR spectra
were recorded on a Nicolet 510P FT-IR-spectrometer. Microanalyses were
obtained from a CH analyzer according to Dr. Salzer from Labormatic and
from a Hewlett Packard CHN-analyser type 185. Column chromatography
was carried out using silica gel 60 (0.062–0.200 mm) from Merck. Medium
Pressure Liquid Chromatography (MPLC) was performed using a pump type
688 from Büchi and a column of 3.5 cm diameter and 45 cm length filled
with silica gel 60 (0.015–0.040 mm) from Merck.
phenyl derivative 1d (1d: IC = 0.53 µM; 3d: IC
0.44 µM).
=
50
50
With the next series of compounds, we varied the linking
trimethylene chain in the lead structure 1d. Replacement of
the central methylene group by sulfur (4a) or oxygen (4b)
resulted in a marked drop in activity, with compound 4a being
7-fold and compound 4b 12-fold less active than the lead.
Introduction of a nitro group into the para-position of the
phenyl residue of 1d and 4a, respectively, yielded inconclu-
sive results. While the nitro derivative 4c of the lead com-
pound 1d was slightly less active than the unsubstituted lead
General Procedure 1: Preparation of Carboxylic Acid Chlorides
(1d: IC = 0.53 µM; 4c: IC = 0.66 µM), the nitro group
containing thioether 4d was about 4-fold more active than the
50
50
The various carboxylic acids were dissolved in toluene and 0.1 ml SOCl2
per mmol acid was added. The mixture was heated under reflux for 2 h and
then, the solvent was evaporated in vacuo. The acid chlorides obtained were
used as described in General Procedure 2.
unsubstituted thioether 4a (4a: IC = 3.7 µM; 4d: IC
=
50
50
1.0 µM). Replacement of the two terminal methylene groups
of the linking chain in the lead structure 1d by an amide
moiety was of particular interest, allowing access to a wide
variety of structural modifications easily accessible from a
number of amino acids and various benzoic acid chlorides.
Unfortunately, the presence of an amide moiety in the linking
structure is deleterious for inhibitory activity (4e:
General Procedure 2: Reaction of Carboxylic Acid Chlorides with the
5-Aminobenzophenone 2
The various carboxylic acid chlorides were dissolved in toluene. Where
necessary, a small amount of dioxane was added to obtain a clear solution.
This solution was added to a solution of one equivalent of 2 in toluene and
the mixture was heated under reflux for 2 h. Then the solvent was evaporated
to yield the crude product. Further purification is described at the particular
compound.
IC = 24.8 µM). Three novel molecular features are intro-
50
duced into the linking structure by the amide moiety: 1) a
carbonyl group, 2) an amide N-H and 3) a trans-configuration
due to the restricted rotation around the CO-NH bond. We
sought for the molecular feature responsible for the marked
reduction in activity with the last three compounds. This is
obviously not due to the trans-conformation enforced by the
amide moiety because compound 4h, carrying a trans-con-
figurated C-C double bond, is only slightly less active than
General Procedure 3: Activation of Carboxylic Acids as Mixed Anhydrides
and Reaction with the 5-Aminobenzophenone 2
The various carboxylic acids were dissolved in dry DMF under an atmos-
phere of argon. 0.3 ml N-Ethyl-morpholine per mmol acid was added. The
temperature was reduced to –15 °C and 0.13 ml isobutyl chloroformate per
mmol acid was added. After 5 min, a solution of one equivalent of 2,
dissolved in dry DMF, was added and the resulting mixture was stirred for
4h while it was allowed to reach room temperature. The reaction mixture was
poured into brine to give a solid. Further purification is described at the
particular compound.
the lead compound 1d (1d: IC = 0.53 µM; 4h: IC
=
50
50
0.99 µM). In contrast, a carbonyl group in terminal position
of the linking substructure is sufficient to produce the ob-
served drop in inhibitory activity (4g: IC = 26.6 µM). As
50
seen with the hetero atoms sulfur and oxygen in the central
position of the linking chain, a nitrogen alone also produces
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]cyclohexane
Carboxylic Acid Amide 3a
a marked drop in activity (4f: IC = 9.6 µM). Altogether, the
50
structure-activity relationships in the different series of com-
pounds are not easy to interpret. No apparent correlation
between structural modifications and farnesyltransferase in-
hibitory activity can be delineated.
Finally, the phenyl residue in compound 1a was replaced
by more spacious aryl moieties (compounds 5a–c). These
variations showed no significant influence on the farnesyl-
transferase inhibitory activity.
In summary, none of the structural modifications employed
in this study produced an exploitable improvement in inhibi-
tory activity of our model compounds used to explore the
novel aryl binding sites of the farnesyltransferase.
From cyclohexanecarboxylic acid chloride (0.16 ml, 1.0 mmol) according
to general procedure 2. Purification: recrystallisation from toluene. Yield:
0.37 g (81%).– Mp 204 °C.– IR (KBr): ν = 3292, 3052, 2930, 2855, 1668,
1553, 1506 cm1.– 1H NMR (CDCl3): δ = 1.17 (m, 2H), 1.38 (m, 2H), 1.54
(m, 2H), 1.72 (m, 2H), 1.80 (m, 2H), 2.08 (m,1H), 2.26 (s, 3H), 3.60 (s, 2H),
7.14 (m, 5H), 7.46 (m, 4H), 7.62 (m, 2H), 7.77 (s, 1H), 8.43 (m, 1H), 10.40
(s, 1H).–13C NMR (CDCl3): δ = 21.1, 25.6, 29.6, 45.1, 46.4, 122.4, 124.2,
124.4, 125.1, 128.4, 129.3, 129.6, 130.0, 131.2, 132.6, 132.7, 136.1, 137.0,
138.1, 170.2, 174.3, 198.6.– EI-MS: m/z (%) 454 (83) M+, 322 (93), 212
(100), 105 (76). Anal. (C29H30N2O3) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]cyclohexylacetic
Acid Amide 3b
From cyclohexylacetic acid (0.21 g, 1.5 mmol) according to general
procedures 1 and 2. Purification: flash-chromatography EtOAc:n-hexane
2:3. Yield: 0.15 g (33%).– Mp 163 °C.– IR (KBr): ν = 3278, 2922, 2850,
1695, 1681, 1652, 1558, 1505 cm–1.– 1H NMR (CDCl3): δ = 0.89 (m, 2H),
1.20 (m, 2H), 1.64 (m, 6H), 1.76 (m, 1H), 2.07 (d, J=7 Hz, 2H), 2.26 (s, 3H),
3.60 (s, 2H), 7.15 (m, 5H), 7.46 (m, 4H), 7.64 (m, 2H), 7.79 (s, 1H), 8.43 (m,
1H), 10.40 (s, 1H).– 13C NMR (CDCl3): δ = 21.1, 26.0, 26.1, 33.2, 35.3, 45.1,
45.7, 122.4, 124.3, 125.1, 128.3, 129.3, 129.6, 130.0, 131.2, 132.4, 132.7,
136.2, 137.0, 138.1, 170.2, 170.7, 198.4.– EI-MS: m/z (%) 468 (100) M+,
336 (74), 212 (89). Anal. (C30H32N2O3) C, H, N.
Acknowledgement
The pGEX-DPR1 and pBC-RAM2 plasmids were kindly providedbyProf.
F. Tamanoi (UCLA). M. Schlitzer thanks Prof. W. Hanefeld for generous
support, Ms. K. Burk for technical assistance and Dr. M. T. Stubbs for
proofreading the manuscript. Financial support by the Deutsche Pharmazeu-
tische Gesellschaft is gratefully acknowledged. I. S. wishes to thank Prof.
Dr. S. Grabley and PD Dr. R. Thiericke for generous support and Ms. S.
Egner for technical assistance.
Arch. Pharm. Pharm. Med. Chem. 334, 40–44 (2001)

Non-Thiol Farnesyltransferase Inhibitors
43
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-3-cyclohexyl-
propionic Acid Amide 3c
(s, 2H), 7.15 (m, 2H), 7.22 (m, 2H), 7.47 (m, 5H), 7.61 (m, 1H), 7.70 (m,
2H), 7.82 (s, 1H), 8.10 (m, 2H), 8.31 (s, 1H), 8.50 (m, 1H), 10.47 (s, 1H).–
13C NMR (CDCl3): δ = 21.1, 35.8, 36.2, 45.0, 122.5, 123.9, 124.1, 124.4,
125.0, 128.4, 129.3, 129.6, 129.8, 130.0, 131.1, 131.8, 132.8, 136.7, 137.1,
137.9, 144.4, 147.2, 166.4, 170.4, 198.3.– EI-MS: m/z (%) 553 (0.5) M+, 554
(85), 421 (100). Anal. (C31H27N3O5S) C, H, N, S.
From 3-cyclohexylpropionic acid chloride (0.17 ml, 1.0 mmol) according
to general procedure 2. Purification: recrystallisation from toluene. Yield:
0.23 g (48%).– Mp 55 °C.– IR (KBr): ν = 3285, 2925, 2850, 1660, 1550,
1510 cm–1.– 1H NMR (CDCl3): δ = 1.14–1.25 (m, 4H), 1.55 (m, 2H), 1.67
(m, 6H), 2.27 (m, 2H), 2.34 (m, 4H), 3.66 (s, 2H), 7.14 (m, 5H), 7.46 (m,
4H), 7.64 (m, 2H), 7.78 (s, 1H), 8.43 (m, 1H), 10.40 (s, 1H).– EI-MS: m/z
(%) 482 (100) M+, 350 (57). Anal. (C31H34N2O3) C,H,N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-Nα-benzoylglycine
Amide 4e
2 was acylated with N-BOC-glycine according to general procedure 3
yielding 6. Compound 6 (0.5 g, 1 mmol) was stirred in a 4N solution of
HCl(g) in dioxane for 1h. After the volatiles were removed in vacuo, the
residue was dissolved in toluene and acylated with benzoylchloride (140 mg,
1 mmol) acording to procedure 2. Purification: recrystallisation from EtOAc.
Yield: 0.30 g (59%).– Mp 196 °C.– IR (KBr): ν = 3392, 1679, 1639, 1572,
1517 cm–1.– 1H NMR (DMSO-D6): δ = 2.26, (s, 3H), 3.37, (s, 2H), 4.05–
4.06, (m, 2H), 7.00–7.06 (m, 4H), 7.44–7.55 (m, 5H), 7.59–7.64 (m, 2H),
7.67–7.70 (m, 3H), 7.77–7.81 (m, 1H), 7.88–7.90 (m, 2H), 8.66–8.72 (m,
1H), 10.00 (s, br, 1H), 10.12 (s, br, 1H).– 13C NMR (DMSO-D6): δ = 20.4,
42.6, 43.6, 120.5, 122.3, 124.1, 127.2, 128.0, 128.1, 128.7, 128.9, 129.5,
130.7, 131.2, 131.8, 132.2, 132.5, 134.0, 135.0, 135.4, 137.2, 166.6, 167.8,
168.9, 195.0.– EI-MS: m/z (%) 505 (67) M+, 344 (85), 212 (100), 105 (40).
Anal. (C31H27N3O4) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-cyclohexylbutyr
ic Acid Amide 3d
From 4-cyclohexylbutyric acid (0.26 g, 1.5 mmol) according to general
procedures 1 and 2. Purification: flash-chromatography EtOAc:n-hexane 2:3
and recrystallisation from ethanol. Yield: 0.08 g (14%).– Mp 135 °C.– IR
(KBr): ν = 3317, 2923, 2849, 1619, 1553, 1506 cm–1.– 1H NMR (DMSO d6):
δ = 0.85 (m, 2H), 1.18 (m, 6H), 1.56–1.64 (m, 7H), 2.21 (m, 2H), 2.24 (s,
3H), 3.34 (s, 2H), 6.97–7.05 (m, 4H), 7.45–7.54 (m, 3H), 7.59–7.66 (m, 4H),
7.77 (m, 1H), 9.87 (s, 1H), 9.95 (s, 1H).– EI-MS: m/z (%) 496 (100) M+, 364
(70), 212 (91), 105 (100), 40 (61). Anal. (C32H36N2O3) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]benzylthioacetic
Acid Amide 4a
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-Nα-benzylglycine
Amide 4f
From S-benzylthioglycolic acid (0.27 g, 1.5 mmol) according to general
procedures 1 and 2. Purification: MPLC EtOAc:n-hexane 1:1 and recrystal-
lisation from cyclohexane/ethanol. Yield: 0.41 g (67%).– Mp 120 ºC.– IR
(KBr): ν = 3304, 1699, 1676, 1635, 1589, 1512 cm–1.– 1H NMR (CDCl3): δ
= 2.34 (s, 3H), 3.23 (s, 2H), 3.68, (s, 2H), 3.73 (s, 2H), 7.19 (m, 9H), 7.37
(m, 1H), 7.50 (m, 2H), 7.60 (m, 1H), 7.71 (m, 3H), 8.33 (s, 1H), 8.51 (m,
1H), 10.52 (s, 1H).– 13C NMR (CDCl3): δ = 21.1, 36.4, 37.6, 45.0, 122.2,
124.1, 124.3, 125.1, 127.6, 128.3, 128.8, 128.8, 129.3, 129.6, 130.0, 131.1,
131.7, 132.8, 136.6, 137.0, 138.0, 166.6, 170.2, 198.5.– EI-MS: m/z (%) =
508 (100) [M+], 386 (52), 254 (84), 91 (54). Anal. (C31H28N2O3S) C, H, N,
S.
Compound 6 (0.5 g, 1 mmol) was deprotected as described above. After
the volatiles were removed in vacuo, the residue was dissolved in toluene
and one equivalent of N-ethylmorpholine and benzaldehyde (1.0 ml,
1 mmol), respectively, were added. The mixture was heated under reflux for
8 h and stirred at room temperature overnight. NaBH4 (0.25 g, 6.5 mmol)
was added to the reaction mixture and stirring was continued. After filtration,
the solvent was removed and the crude product was purified on silica gel with
EtOAc.
Yield: 0.01 g (2%).– Mp 47 °C.–IR (KBr): ν = 3296, 2925, 2855, 1682,
1508 cm–1.– 1H NMR (CDCl3): δ = 2.27 (s, 3H), 3.31 (s, 2H), 3.61 (s, 2H),
3.74 (s, 2H), 7.10 (m, 2H), 7.17–7.26 (m, 8H), 7.42 (m, 3H), 7.52 (m, 1H),
7.64 (m, 2H), 7.85 (s, 1H), 8.48 (m, 1H), 9.14 (s, 1H), 10. 44 (s, 1H).– EI-MS:
m/z (%) 491 (7) M+, 344 (37), 212 (47), 105 (49), 91 (100). Anal.
(C31H29N3O3) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]benzyloxyacetic
Acid Amide 4b
From benzyloxyacetic acid chloride (0.19 g, 1.0 mmol) according to
general procedure 2. Purification: MPLC EtOAc:n-hexane 1:1 and recrystal-
lisation from n-hexane/acetone. Yield: 0.38 g (77%).– Mp 96 ºC.– IR (KBr):
ν = 3305, 3031, 2919, 1685, 1594, 1507 cm–1.– 1H NMR (CDCl3): δ = 2.34
(s, 3H), 3.69 (s, 2H), 4.04 (s, 2H), 4.62 (s, 2H), 7.17 (m, 2H), 7.24 (m, 2H),
7.35 (m, 5H), 7.49 (m, 2H), 7.54 (m, 1H), 7.60 (m, 1H), 7.71 (m, 2H), 7.89
(m 1H), 8.25 (s, 1H), 8.55 (m, 1H), 10.53 (s, 1H).– 13C-NMR (CDCl3): δ =
21.1, 45.1, 69.5, 73.9, 122.4, 124.2, 124.5, 125.3, 128.1, 128.4, 128.5, 128.8,
129.3, 129.6, 130.1, 131.2, 131.4, 132.8, 136.3, 136.7, 137.0, 138.1, 167.6,
170.3, 198.5.– EI-MS: m/z (%) 492 (100) [M+], 360 (65), 91(55). Anal.
(C31H28N2O4) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-3-benzoylpropion
ic Acid Amide 4g
From 3-benzoylpropionic acid (0.18 g, 1 mmol) according to general
procedure 3. Purification: recrystallisation from toluene. Yield 0.11 g
(21%).– Mp 150 °C.–IR (KBr): ν = 3357, 3060, 2922, 1685, 1665, 1597,
1554, 1508 cm–1.– 1H NMR (CDCl3): δ = 2.26 (s, 3H), 2.66 (t, J=7 Hz, 2H),
3.33 (t, J=7 Hz, 2H), 3.60 (s, 2H), 7.09 (m, 2H), 7.18 (m, 2H), 7.39 (m, 3H),
7.50 (m, 3H), 7.62 (m, 4H), 7.75 (s, 1H), 7.89 (m, 2H), 8.43 (m, 1H), 10.41
(s, 1H).– 13C NMR (CDCl3): δ = 21.1, 31.2, 34.0, 45.0, 122.2, 142.2, 125.1,
128.1, 128.3, 128.7, 129.3, 129.6, 130.0, 131.2, 132.5, 132.7, 133.5, 136.2,
136.9, 138.1, 170.2, 170.4, 198.6, 199.1.– EI-MS m/z (%) 504 (20) M+, 254
(41), 105 (100). Anal. (C32H28N2O4) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-(4-nitrophenyl)-
butyric Acid Amide 4c
From 4-(4-nitrophenyl)butyric acid (0.31 g, 1.5 mmol) according to gen-
eral procedures 1 and 2. Purification: recrystallisation from toluene. Yield:
0.33 g (62%).– Mp 103 °C.– IR (KBr): ν = 3271, 2924, 1667, 1598, 1549,
1513 cm–1.– 1H NMR (DMSO D6): δ = 1.90 (m, 2H), 2.23 (s, 3H), 2.29 (t,
J=8 Hz, 2H), 2.74 (t, J=8 Hz, 2H), 3.32 (s, 2H), 6.96–7.03 (m, 4H), 7.44–7.53
(m, 5H), 7.58–7.65 (m, 4H), 7.74–7.77 (m, 1H), 8.12 (m, 2H), 9.92 (s, 1H),
9.96 (s, 1H).– EI-MS m/z (%) 535 (25) M+, 403 (72), 212 (57), 105 (100).
Anal. (C32H27N3O5) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-phenyl-
3-butenoic Acid Amide 4h
From 4-phenyl-3-butenoic acid (0.24 g, 1.5 mmol) according to general
procedures 1 and 2. Purification: MPLC EtOAc:n-hexane 1:1. Yield: 0.38 g
(65%).– Mp 100 °C.– IR (KBr): ν = 3248, 2923, 1681, 1658, 1597, 1553,
1498 cm–1.– 1H NMR (CDCl3): δ = 2.32, (s, 3H), 3.18–3.20, (m, 2H), 3.66,
(s, 2H), 6.24–6.30 (m, 1H), 6.47–6.52 (m, 1H), 7.14–7.16 (m, 2H), 7.21–7.33
(m, 7H), 7.40–7.45 (m, 3H), 7.51–7.56 (m, 1H), 7.67–7.69 (m, 2H), 7.75 (s,
br, 1H), 7.92–7.93 (m, 1H), 8.45–8.46 (m, 1H), 10.49 (s,br, 1H).– 13C NMR
(CDCl3): δ = 21.1, 41.6, 45.0, 121.6, 122.4, 124.4, 124.7, 125.3, 126.3, 127.9,
128.3, 128.6, 129.2, 129.6, 130.0, 131.1, 132.4, 132.8, 135.1, 136.3, 136.4,
137.0, 138.0, 169.1, 170.4, 198.5.– EI-MS: m/z (%) 488 (100) M+, 489 (37),
383 (38), 356 (75), 105 (31). Anal. (C32H28N2O3) C, H, N.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-(4-nitrobenzyl)-
thioacetic Acid Amide 4d
From (4-nitrobenzyl)thioacetic acid (0.34 g, 1.5 mmol) according to gen-
eral procedures 1 and 2. Purification: MPLC EtOAc:n-hexane 1:1. Yield:
0.38 g (65%).– Mp 60 °C.– IR (KBr): ν = 3295, 1663, 1598, 1551, 1513
cm–1.– 1H NMR (CDCl3): δ = 2.34, (s, 3H), 3.16, (s, 2H), 3.68, (s, 2H), 3.82
Arch. Pharm. Pharm. Med. Chem. 334, 40–44 (2001)

44
Mitsch, Wißner, Sattler, and Schlitzer
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-1-naphthoic Acid
Amide 5a
References
[1] F. L. Zhang, P. J. Casey, Annu. Rev. Biochem. 1996, 65, 241–269.
[2] D. M. Leonard, J. Med. Chem. 1997, 40, 2971–2990.
From 1-naphthoic acid chloride (0.19 g, 1.0 mmol) according to general
procedure 2. Purification: MPLC EtOAc:n-hexane 1:1 and recrystallisation
from EtOH/acetone. Yield: 0.44 g (88%).– Mp 208 °C.– IR (KBr): ν = 3323,
1
3268, 1667, 1595, 1541, 1507 cm–1.– H NMR (DMSO-D6): δ = 2.26, (s,
[3] Y. Qian, S. M. Sebti, A. D. Hamilton, Biopolymers 1997, 43, 25–41.
[4] T. M. Williams, Exp. Opin. Therp. Patents 1999, 9, 1263–1280.
[5] M. Schlitzer, Pharm. i. u. Z. 1998, 27, 278–288.
3H), 3.37 (s, 2H), 6.99–7.06 (m, 4H), 7.47–7.51 (m, 2H), 7.56–7.65 (m, 5H),
7.70–7.75 (m, 3H), 7.89–7.91 (m, 1H), 7.98–8.06 (m, 3H), 8.18–8.20 (m,
1H), 10.05 (s, 1H), 10.61 (s, 1H).– 13C NMR (DMSO-D6): δ = 20.5, 42.2,
121.1, 122.9, 124.8, 125.0, 125.4, 126.2, 126.9, 128.0, 128.2, 128.9, 129.5,
130.1, 132.2, 132.5, 133.1, 134.3, 135.4, 135.5, 137.1, 167.2, 169.0, 195.0.–
EI-MS: m/z (%) 498 (100) M+, 499 (36), 366 (54), 155 (79). Anal.
(C33H26N2O3) C, H, N.
[6] A. Oliff, Biochim. Biophys. Acta 1999, 1423, C19–C30.
[7] J. S. Sebolt-Leopold, Emerging Drugs 1998, 3, 271–277.
[8] B. T. Hill, D. Perrin, A. Kruczynski, Crit. Rev. Oncol. Hematol. 2000,
33, 7–23.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-9-fluorene-
[9] Martindale The Extra Pharmacopeia, 31st ed. Reynolds, J. E. F. Ed.;
Royal Pharmaceutical Society of Great Britain: London, 1996; p. 821.
carboxylic Acid Amide 5b
From 9-fluorenecarboxylic acid (0.32 g, 1.5 mmol) according to general
procedures 1 and 2. Purification: recrystallisation from toluene. Yield: 0.48 g
(59%).– Mp 229 °C.– IR (KBr): ν = 3253, 3027, 1661, 1597, 1534 cm–1.–
1H NMR (DMSO d6): δ = 2.22 (s, 3H), 3.32 (s, 2H), 4.97 (s, 1H), 6.95–7.02
(m, 4H), 7.27–7.31 (m, 2H), 7.37–7.46 (m, 4H), 7.54–7.59 (m, 4H), 7.62–
7.65 (m, 2H), 7.68–7.69 (m, 1H), 7.80 (m, 1H), 7.83 (m, 2H), 9.99 (s, 1H),
10.68 (s, 1H).– EI-MS: m/z (%) 536(57) M+, 404 (45), 165 (100), 105 (95).
Anal. (C36H28N2O3) C, H, N.
[10] M. Schlitzer, M. Böhm, A. Mitsch, J. Sakowski, P. Wißner, I. Sattler
short communication presented at the annual meeting of the German
Pharmaceutical Society, Münster, 5. – 7.10.2000; Abstr.: Arch. Pharm.
Pharm. Med. Chem. 2000, 333, Suppl. 2, 12 (D 1.62).
[11] S. R. O’Connor, K. J. Barr, L. Wang, B. K. Sorensen, A. S. Tasker, H.
Sham, A.-C. Ng, J. Cohen, E. Devine, S. Cherian, B. Saeed, H. Zhang,
J. Y. Lee, R. Warner, S. Tahir, P. Kovar, P. Ewing, J. Alder, M. Mitten,
J. Leal, K. Marsh, J. Bauch, D. J. Hoffman, S. M. Sebti, S. H. Rosenberg,
J. Med. Chem. 1999, 42, 3701–3710.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-9-anthracene-
carboxylic Acid Amide 5c
[12] D. J. Augeri, D. Janowick, D. Kalvin, G. Sullivan, J. Larsen, D.
Dickman, H. Ding, J. Cohen, J. Lee, R. Warner, P. Kovar, S. Sherian,
B. Saeed, H. Zhang, S. Tahir, S.-C. Ng,H. Sham, S. H. Rosenberg,
Bioorg. Med. Chem. Lett. 1999, 9, 1069–1074.
From 9-anthracenecarboxylic acid (0.33 g, 1.5 mmol) according to general
procedures 1 and 2. Purification: flash-chromatography EtOAc:n-hexane
2:3. Yield: 0.08 g (10%).– Mp 228 °C.–IR (KBr): ν = 3424, 1718, 1689,
1595, 1504 cm–1.– 1H NMR (CDCl3): δ = 2.28 (s, 3H), 3.60 (s, 2H), 7.11 (m,
2H), 7.18 (m, 3H), 7.39–7.51 (m, 6H), 7.67 (m, 1H), 7.70–7.75 (m, 3H), 7.94
(m, 2H), 7.99 (m, 3H), 8.42 (s, 1H), 8.53 (m, 1H), 10.46 (s, 1H).– 13C NMR
(CDCl3): δ = 15.4, 39.5, 117.2, 118.7, 119.0, 119.2, 119.6, 120.0, 121.5,
122.5, 122.8, 123.0, 123.3, 123.7, 124.0, 124.4, 125.5, 125.7, 126.9, 127.1,
131.4, 132.5, 162.0, 164.6, 192.8.– EI-MS: m/z (%) 548 (28) M+, 205 (100),
177 (44). Anal. (C37H28N2O3) C, H, N.
[13] M. J. Breslin, J. deSolms, E. A. Giuliani, G. E. Stokker, S. L. Graham,
D. L. Pompliano, S. D. Mosser, K. A. Hamilton, J.H. Hutchinson,
Bioorg. Med. Chem. Lett. 1998, 8, 3311–3316.
[14] T. M. Ciccarone, S. C. MacTough, T. M. Williams, C. J. Dinsmore, T.
J. O’Neill, D. Shah, J. C. Culberson, K. S. Koblan, N. E. Kohl, J. B.
Gibbs, A. I. Oliff, S. L. Graham, G. D. Hartman, Bioorg. Med. Chem.
Lett. 1999, 9, 1991–1996.
Farnesyltransferase Assay
[15] M. Schlitzer, I. Sattler, H.-M. Dahse, Arch. Pharm. Pharm. Med. Chem.
1999, 332, 124–132.
The assay was carried out as described.[13] The assay mixture (100 µL
volume) contained 50 mM Tris/HCl pH 7.4, 5 mM MgCl2, 10 µM, ZnCl2, 5
mM DTT, 7 µM Ds-GCVLS, 20 µM FPP and 5 nmol GST-FTase and 1%
of various concentrations of the test compounds dissolved in DMSO. The
progress of the enzyme reaction was followed by the enhancement of the
fluorescence emission at 505 nm (excitation: 340 nm). Fluorescence emis-
sion was recorded with a Perkin Elmer LS50B spectrometer. IC50 values
(concentrations resulting in 50% inhibition) were calculated from initial
velocity of three independent measurements of four to five different concen-
trations of inhibitor.
[16] D. L. Pompliano, R. P. Gomez, N. J. Anthony, J. Am. Chem. Soc. 1992,
114, 7945–7946.
[17] K. Del Villar, H. Mitsuzawa, W. Yang, I. Sattler, F. Tamanoi, J. Biol.
Chem. 1997, 272, 680–687.
[18] C. L. Strickland, P. C. Weber, Curr. Opin. Drug Discovery Dev. 1999,
2, 475–483.
Received: September 1, 2000 [FP519]
Arch. Pharm. Pharm. Med. Chem. 334, 40–44 (2001)