Non-thiol farnesyltransferase inhibitors: The concept of benzophenone- based bisubstrate analogue farnesyltransferase inhibitors

Article abstract of DOI:10.1016/S0223-5234(00)00162-8

Replacement of the thiol in a benzophenone-based CAAX-peptidomimetic farnesyltransferase inhibitor by a carboxylic acid moiety resulted in a marked drop in inhibitory potency. Transformation of these carboxylic acid derivatives into bisubstrate analogues by addition of a lipophilic alkyl chain, which should be able to occupy considerable portions of the farnesyl binding region in the farnesyltransferase's active site, resulted in a regain of the inhibitory activity. The bisubstrate analogues represent new lead structures for non-thiol farnesyltransferase inhibitors. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00162-8

Eur. J. Med. Chem. 35 (2000) 721−726
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001628/FLA
Preliminary communication
Non-thiol farnesyltransferase inhibitors: the concept of benzophenone-based
bisubstrate analogue farnesyltransferase inhibitors
Martin Schlitzera*, Isabel Sattlerb
aInstitut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
bHans-Knöll-Institut für Naturstoff-Forschung e. V., Beutenbergstraße 11, D-07745, Jena, Germany
Received 3 January 2000; revised 28 February 2000; accepted 6 March 2000
Abstract – Replacement of the thiol in a benzophenone-based CAAX-peptidomimetic farnesyltransferase inhibitor by a carboxylic acid
moiety resulted in a marked drop in inhibitory potency. Transformation of these carboxylic acid derivatives into bisubstrate analogues by
addition of a lipophilic alkyl chain, which should be able to occupy considerable portions of the farnesyl binding region in the
farnesyltransferase’s active site, resulted in a regain of the inhibitory activity. These bisubstrate analogues represent new lead structures for
non-thiol farnesyltransferase inhibitors. © 2000 Éditions scientifiques et médicales Elsevier SAS
farnesyltransferase / non-thiol farnesyltransferase inhibitors / bisubstrate analogues / benzophenones
1. Introduction
their antiproliferative effects, these compounds have
demonstrated their efficacy combined with a surprisingly
low toxicity and therefore are regarded as a major
emerging strategy in cancer therapy.
Cancer is caused by a stepwise accumulation of muta-
tions that affect growth control, differentiation and cell
survival [1]. Ras proteins play a central role in the signal
transduction cascades controlling these processes [2, 3].
Mutated forms of Ras, which are constitutively active, are
found in approximately 30% of all human cancers.
Several post-transformational modifications take place
before Ras obtains its full biological activity. The crucial
step is the transfer of a farnesyl residue from farnesylpy-
rophosphate to the thiol of a cysteine side chain of the
C-terminal CAAX-tetrapeptide sequence catalysed by the
enzyme farnesyltransferase [4]. Therefore, inhibition of
farnesyltransferase has received considerable interest in
past years [5–7] as a strategy for the development of
novel potential cancer therapeutics. However, there is
accumulating evidence that Ras may not be the only
substrate of the farnesyltransferase involved in oncogen-
esis [8, 9]. Regardless of the unresolved issue of the
mechanism by which farnesyltransferase inhibitors exert
We have developed 2-acylaminobenzophenones as
novel types of CAAX-peptidomimetic farnesyltransferase
inhibitors [10]. Compared to a native CAAX-tetrapeptide
in our lead structure 1 (IC50 = 0.08 µM) the entire
terminal tripeptide has been replaced by a non-peptidic
structure (figure 1a, b). In this initial approach, the
terminal cysteine with its thiol crucial for complexing the
essential zinc has been preserved. However, thiols are an
undesired feature in potential drugs because of their
sensitivity towards oxidation. More importantly, free
thiols are a source of adverse drug effects as has been
seen with the angiotensin converting enzyme inhibitor
captoprile [11]. Therefore, current development is clearly
directed towards the so called non-thiol farnesyltrans-
ferase inhibitors.
With the angiotensin converting enzyme inhibitors the
thiol has successfully been replaced by a carboxylic acid
moiety. We intended to explore whether this strategy
might also work with our farnesyltransferase inhibitors
(figure 1c).
* Correspondence and reprints: schlitze@mailer.uni-marburg.de

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M. Schlitzer, I. Sattler / Eur. J. Med. Chem. 35 (2000) 721–726
Figure 1. Schematic representation of the farnesyltransferase’s active site. Presumable alignment of: a) the natural substrates FPP and
CAAX-tetrapeptide; b) the cysteinyl benzophenone inhibitor 1; c) a carboxylic acid benzophenone inhibitor; d) a bisubstrate
benzophenone inhibitor.

M. Schlitzer, I. Sattler / Eur. J. Med. Chem. 35 (2000) 721–726
723
Figure 2. Synthesis of compounds 5–11: (I) H3C–C6H4–COCl, toluene, reflux; (II) SnCl2·2H2O, EtOAc, reflux; (III) succinic or
glutaric acid anhydride, toluene/dioxane, reflux; (IV) MeOOC–(CH2)n–COCl, toluene/dioxane, reflux; (V) H3C–(CH2)m–NH2, PyBOP,
DIPEA, DMF, RT.
2. Chemistry
tathione S-transferase at the N-terminus of the ꢀ-subunit
[13]. Yeast farnesyltransferase is a close homologue and
functionally similar to the human enzyme; it is widely
used for the evaluation of farnesyltransferase 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 which, upon excitation
at 340 nm, results in an enhancement of fluorescence at
505 nm which is used to monitor the enzyme reaction.
Compound 4 was prepared according to a procedure
published previously [10]. It was acylated by appropriate
carboxylic acid chlorides and anhydrides, respectively, in
an inert solvent. The long chain alkyl amide derivatives
10 and 11 were prepared from the carboxylic acid
derivatives 6 and 8 using the PyBOP methodology (figure
2).
3. Pharmacology
4. Results and discussion
The inhibitory activity of the potential synthetic inhibi-
tors was determined using the fluorescence enhancement
assay as described by Pompliano [12]. The assay em-
ployed yeast farnesyltransferase (FTase) fused to glu-
The carboxylic acid derivatives 5–9 of our lead struc-
ture turned out to be only very weak inhibitors of the
farnesyltransferase (table I), thereby it made no differ-

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M. Schlitzer, I. Sattler / Eur. J. Med. Chem. 35 (2000) 721–726
Table I. Inhibitory activity of compounds 5–11.
non-thiol farnesyltransferase inhibitors whose potential is
currently exploited. The results will be reported in due
course.
Compound
n
m
R
IC50 ± SD (µM)
5
6
7
8
9
10
11
1
2
2
3
3
2
3
–
–
–
–
–
14
13
Me
H
Me
H
Me
–
–
130 ± 35
> 200
130 ± 33
120 ± 42
180 ± 45
7.8 ± 0.4
1.0 ± 0.1
6. Experimental
6.1. Chemistry
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 acquisition system from
Tecnivent or an AutoSpec mass spectrometer from Mi-
cromass. IR spectra were recorded on a Nicolet 510P
FT-IR-spectrometer. Microanalyses were obtained from a
CH analyser 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.
ence whether there was a free carboxyl group or the
corresponding methyl ester situated at the terminus of the
molecules. Obviously, we lost a lot of binding energy
upon the replacement of the thiol by a carboxyl group.
Addition of a lipophilic moiety to the terminal carboxyl
group, which presumably occupies considerable portions
of that region in the farnesyltransferases active site which
nomally harbours the farnesyl residue, should result in a
regain of at least some of the lost affinity (figure 1d). The
additional hydrophobic interactions would enhance the
overall binding energy. Thus, we would transform our
peptidomimetic inhibitors into the so-called bisubstrate
analogues, compounds which display molecular features
of both the peptidic and the prenylic substrate.
Farnesyltransferase has the uncommon feature of dis-
playing a high affinity to its reaction products, the
farnesylated proteins. After the transfer of the farnesyl
residue to the cysteine thiol, the farnesylated proteins
remain tightly bound to the enzyme. They are only
released in a displacement reaction upon binding of
another molecule of farnesylpyrophosphate [14]. Bisub-
strate inhibitors can also be regarded as analogues of
these farnesylated proteins, e.g. as product analogues and
therefore are an interesting class of compounds to study.
The two amide derivatives 10 and 11 inhibited farne-
syltransferase in the low micromolar range. In the case of
11, this represents a more than 100-fold improvement in
the inhibitory activity of the bisubstrate analogue over the
corresponding carboxylic acid non-thiol CAAX-
peptidomimetic 8.
6.1.1. General procedure for compounds 5–9
To a solution of 4 in hot toluene (50–80 mL) the
appropriate carboxylic acid chloride or anhydride dis-
solved in dioxane (10 mL) was added. The resulting
mixture was refluxed for 2 h. After cooling, most of the
solvent was removed in vacuo. The resulting solution was
kept at 8 °C until crystallization occurred.
6.1.1.1. N-(2-Benzoyl-4-nitrophenyl)-
2-(4-methylphenyl)acetic acid amide 3
To a solution of 2-amino-5-nitrobenzophenone (2)
(1.2 g, 5 mmol) in hot toluene (50 mL), 2-(4-
methylphenyl)acetic acid chloride (0.843 g, 5 mmol) was
added. The mixture was refluxed for 2 h. Then most of
the solvent was removed in vacuo. The product crystal-
lized upon cooling and was recrystallized from EtOH.
Yield: 1.75 g (93%). 1H-NMR (CDCl3): δ = 2.33 (s, 3H),
3.74 (s, 2H), 7.17 (m, 2H), 7.24 (m, 2H), 7.51 (m, 2H),
7.65 (m, 3H), 8.37 (m, 1H), 8.41 (m, 1H), 8.88 (d, J =
9 Hz, 1H), 11.05 (s, 1H).
6.1.1.2. N-(4-Amino-2-benzoylphenyl)-
2-(4-methylphenyl)acetic acid amide 4
To a solution of 3 (1.75 g, 4.7 mmol) in hot EtOAc
(80 mL) SnCl2·2H2O (5.35 g) was added. The mixture
was refluxed for 2 h and then poured into water (300 mL).
The pH of the resulting mixture was adjusted to pH 8 by
addition of sat. NaHCO3-solution. The mixture was
extracted with EtOAc (3 × 150 mL). The combined or-
ganic extracts were washed with brine, dried with MgSO4
and evaporated to dryness. The resulting solid was
recrystallized from EtOH. Yield: 1.053 g (65%). 1H-
5. Conclusion
We have demonstrated that the transformation of the
CAAX-peptidomimetic farnesyltransferase inhibitors car-
rying a carboxylic acid moiety in place of the original
thiol into bisubstrate inhibitors enhances their inhibitory
activity by two orders of magnitude. These bisubstrate
analogues represent a lead structure for a novel type of

M. Schlitzer, I. Sattler / Eur. J. Med. Chem. 35 (2000) 721–726
725
NMR (CDCl3): δ = 2.25 (s, 3H), 3.55 (s, 2H), 6.66 (d, J =
3 Hz, 1H), 6.78 (m, 1H), 7.07 (m, 2H), 7.13 (m, 2H), 7.38
(m, 2H), 7.51 (m, 1H), 7.61 (m, 2H), 8.18 (d, J = 9 Hz,
1H), 9.97 (s, 1H).
(37, M+), 321 (55), 294 (100), 212 (47), 105 (100). Anal.
C27H26N2O5 (C, H, N).
6.1.1.6. 3-[N-[3-[3-Benzoyl-4-
[[2-(4-methylphenyl)acetyl]amino]-
phenylamino]carbamoyl]-butyric acid 8
6.1.1.3. 3-[N-[3-[3-Benzoyl-4-[[2-
(4-methylphenyl)acetyl]amino]phenyl-
amino]carbamoyl]acetic acid methyl ester 5
From 4 (0.688 g, 2 mmol) and glutaric acid anhydride
(0.228 g, 2 mmol). Purification: recrystallization from
toluene. Yield: 0.740 g (81%). M.p. 124 °C. IR (KBr):
From 4 (0.688 g, 2 mmol) and malonic acid methyl-
ester chloride (0.24 mL, 2.2 mmol). Purification: recrys-
tallization from toluene. Yield: 0.46 g (50%). M.p. 104 °C.
IR (KBr): ν = 3 295, 2 955, 1 740, 1 690, 1 660,
1 560 cm–1. 1H-NMR (CDCl3): δ = 2.22 (s, 3H), 3.32 (s,
2H), 3.40 (s, 2H), 3.62 (s, 3H), 6.95 (m, 2H), 7.01 (m,
2H), 7.45 (m, 2H), 7.54 (m, 1H), 7.62 (m, 4H), 7.73 (m,
1H), 10.0 (s, 1H), 10.22 (s, 1H). 13C-NMR (CDCl3):
δ = 16.7, 38.4, 39.5, 48.0, 116.5, 118.4, 120.4, 124.3,
124.9, 125.0, 125.6, 127.1, 128.1, 128.3, 128.7, 131.1,
131.3, 133.6, 160.1, 164.0, 165.2, 191.1. Anal.
C26H24N2O5 (C, H, N).
ν = 3 285, 2 900–2 600, 1 735, 1 690, 1 660 cm–1. H-
1
NMR (DMSO-d6): δ = 1.76 (m, 2H), 2.22 (m, 5H), 2.28
(m, 2H), 3.31 (s, 2H), 6.95 (m, 2H), 7.01 (m, 2H), 7.45
(m, 2H), 7.51 (m, 1H), 7.62 (m, 4H), 7.75 (m, 1H), 9.91
(s, 1H), 9.93 (s, 1H), 11.91 (s, 1H). 13C-NMR (DMSO-
d6): δ = 20.2, 20.4, 32.8, 35.2, 42.5, 120.1, 122.0, 124.0,
128.0, 128.6, 128.7, 129.3, 131.3, 132.1, 132.4, 135.2,
135.3, 137.0, 168.8, 170.6, 173.7, 194.3. MS (EI): m/z
(%) = 458 (60, M+), 326 (93), 212 (93), 105 (100). Anal.
C27H26N2O5 (C, H, N).
6.1.1.7. 3-[N-[3-[3-Benzoyl-4-[[2-
(4-methylphenyl)acetyl]amino]phenyl-
amino]carbamoyl]-butyric acid methyl ester 9
6.1.1.4. 3-[N-[3-[3-Benzoyl-4-
[[2-(4-methylphenyl)acetyl]amino]-
From 4 (0.344 g, 1 mmol) and glutaric acid methyl
ester chloride (0.17 mL, 1 mmol). Purification: recrystal-
lization from toluene. Yield: 0.310 g (65%). M.p. 106 °C.
IR (KBr): ν = 3 300, 3 050, 2 950, 1 740, 1 660,
1 560 cm–1. 1H-NMR (CDCl3): δ = 1.90 (m, 2H), 2.25 (s,
3H), 2.27 (m, 4H), 3.56 (s, 3H), 3.59 (s, 2H), 7.08 (m,
2H), 7.15 (m, 2H), 7.40 (m, 3H), 7.50 (m, 1H), 7.61 (m,
3H), 7.78 (m, 1H), 8.38 (m, 1H), 10.38 (s, 1H). 13C-NMR
(CDCl3): δ = 20.5, 21.1, 32.9, 36.1, 45.0, 51.6, 122.4,
124.3, 124.4, 125.1, 128.3, 129.3, 129.6, 130.0, 131.1,
132.6, 132.7, 136.0, 137.0, 138.0, 170.3, 170.5, 173.7,
198.5. MS (EI): m/z (%) = 472 (50, M+), 340 (70), 212
(81), 105 (83), 59 (100). Anal. C28H28N2O5 (C, H, N).
phenylamino]carbamoyl]-propionic acid 6
From 4 (0.688 g, 2 mmol) and succinic acid anhydride
(0.200 g, 2 mmol). Purification: recrystallization from
toluene. Yield: 0.880 g (99%). M.p. 158 °C. IR (KBr):
1
ν = 3 330, 2 900–2 600, 1 725, 1 655, 1 560 cm–1. H-
NMR (DMSO-d6): δ = 2.19 (s, 3H), 2.43 (m, 4H), 3.51
(s, 2H), 6.93 (m, 2H), 6.98 (m, 2H), 7.42 (m, 2H), 7.50
(m, 1H), 7.59 (m, 4H), 7.70 (m, 1H), 9.90 (s, 1H), 9.93 (s,
1H). 13C-NMR (DMSO-d6): δ = 20.4, 28.6, 30.9, 42.1,
120.0, 121.9, 124.0, 128.0, 128.6, 128.7, 129.3, 131.3,
132.1, 132.4, 135.2, 135.3, 137.0, 168.8, 170.0, 173.4,
194.9. MS (EI): m/z (%) = 444 (5, M+), 321 (45), 294
(100), 105 (74). Anal. C26H24N2O5 (C, H, N).
6.1.1.8. 3-[N-[3-[3-Benzoyl-4-[[2-
(4-methylphenyl)acetyl]amino]phenylamino]-
6.1.1.5. 3-[N-[3-[3-Benzoyl-4-[[2-
(4-methylphenyl)acetyl]amino]phenyl-
carbamoyl]-propionic acid N-pentadecyl amide 10
To a solution of 6 (458 mg, 1 mmol), pentadecylamine
(228 mg, 1 mmol) and PyBOP (520 mg 1 mmol) in dry
DMF (20 mL) diisopropylethylamine (0.65 mL) was
added. The mixture was stirred at room temperature
overnight. Then the mixture was poured into brine
(400 mL). The aqueous mixture was extracted with EtOAc
(3 × 100 mL) and the combined organic extracts were
washed successively with 2 N citric acid, sat. NaHCO3-
soln. and brine and dried with MgSO4. The residue
obtained after removal of the solvent was purified by
flash chromatography on silica gel (EtOAc:n-Hexan 3:2).
Yield: 0.50 g (76%). M.p. 105 °C. IR (KBr): ν = 3 300,
3 060, 2 925, 2 850, 1 640, 1 540 cm–1. 1H-NMR (CDCl3):
amino]carbamoyl]-propionic acid methyl ester 7
From 4 (0.344 g, 1 mmol) and succinic acid methyl-
ester chloride (0.20 mL, 1 mmol). Purification: recrystal-
lization from toluene. Yield: 0.348 g (76%). M.p. 144 °C.
IR (KBr): ν = 3 375, 2 950, 2 930, 1 735, 1 715, 1 690,
1
1 635, 1 550 cm–1. H-NMR (CDCl3): δ = 2.33 (s, 3H),
2.57 (m, 2H), 2.69 (m, 2H), 3.65 (s, 3H), 3.66 (s, 2H),
7.15 (m, 2H), 7.22 (m, 2H), 7.48 (m, 3H), 7.57 (m, 1H),
7.67 (m, 2H), 7.75 (m, 1H), 7.80 (m, 1H), 8.46 (m, 1H),
10.44 (s, 1H). 13C-NMR (CDCl3): δ = 21.1, 29.1, 31.8,
45.0, 52.0, 122.4, 124.2, 124.4, 125.1, 128.3, 129.3,
129.6, 130.0, 131.2, 132.4, 132.7, 135.2, 136.1, 137.0,
138.0, 169.7, 170.3, 173.6, 198.5. MS (EI): m/z (%) = 458

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M. Schlitzer, I. Sattler / Eur. J. Med. Chem. 35 (2000) 721–726
δ = 0.80 (t, J = 7 Hz, 3H), 1.21 (m, 22H), 1.36 (m, 2H),
1.57 (m, 2H), 2.26 (s, 3H), 2.45 (m, 2H), 2.54 (m, 2H),
3.10 (m, 2H), 3.60 (s, 2H), 5.67 (m, 1H), 7.08 (m, 2H),
7.18 (m, 4H), 7.39 (m, 2H), 7.48 (m, 2H), 7.61 (m, 2H),
7.77 (m, 1H), 8.42 (m, 1H), 8.71(s, 1H), 10.41 (s, 1H).
13C-NMR (CDCl3): δ = 14.1, 21.1, 22.7, 26.9, 29.2, 29.3,
29.5, 29.56, 29.62, 29.65, 29.66, 29.67, 31.5, 31.9, 33.0,
39.8, 45.0, 122.3, 124.2, 125.1, 128.3, 129.3, 129.6,
130.0, 131.2, 132.6, 132.7, 136.1, 136.9, 138.1, 170.2,
170.6, 172.2, 198.6. MS (EI): m/z (%) = 653 (1, M+), 294
(100), 293 (32), 212 (27), 105 (25). Anal. C41H55N3O4
(C, H, N).
Germany. 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 approx.
5 nmol yeast GST-FTase [13] and 1% of various concen-
trations 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). The reaction was started by addition
of the enzyme and run in a quartz cuvette thermostatted at
30 °C. Fluorescence emission was recorded with a Perkin
Elmer LS50B spectrometer. IC50s were calculated from
initial velocity of three independent measurements of
typically 4–5 inhibitor concentrations and expressed as
mean ± SD.
6.1.1.9. 3-[N-[3-[3-Benzoyl-4-[[2-
(4-methylphenyl)acetyl]amino]phenylamino]-
carbamoyl]-butyric acid N-tetradecyl amide 11
As described above from 8 (0.460 g, 1.0 mmol) and
tetradecylamine (0.220 g, 1.0 mmol). Purification: flash-
chromatography on silica gel (EtOAc:n-Hexan 3:2). Yield:
0.325 g (50%). M.p. 109 °C. IR (KBr): ν = 3 300, 3 060,
2 925, 2 855, 1 655, 1 550 cm–1. 1H-NMR (CDCl3):
δ = 0.81 (t, J = 7 Hz, 3H), 1.18 (m, 20H), 1.39 (m, 2H),
1.59 (m, 2H), 1.90 (m, 2H), 2.18 (m, 2H), 2.27 (s, 3H),
2.32 (m, 2H), 3.13 (m, 2H), 3.60 (s, 2H), 5.55 (m, 1H),
7.10 (m, 2H), 7.18 (m, 4H), 7.41 (m, 2H), 7.51 (m, 2H),
7.64 (m, 2H), 7.83 (m, 1H), 8.43 (m, 1H), 10.42 (s, 1H).
13C-NMR (CDCl3): δ = 14.1, 21.1, 22.7, 26.9, 29.2, 29.3,
29.5, 29.56, 29.6, 31.9, 35.0, 36.3, 39.7, 45.0, 122.3,
124.3, 125.1, 128.3, 129.3, 129.6, 130.0, 132.7, 132.8,
138.1, 170.2, 171.1, 172.7, 198.7. Anal. C41H55N3O4 (C,
H, N).
Acknowledgements
The pGEX-DPR1 and pBC-RAM2 plasmids were
kindly provided by Prof. F. Tamanoi (UCLA). M. Schl-
itzer wishes to thank Ms. K. Burk for technical assistance
and the German Pharmaceutical Society for financial
support. I. Sattler wishes to thank Prof. Dr S. Grabley and
PD Dr R. Thiericke for generous support and Ms. S.
Egner for excellent technical assistance.
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