Non-peptidic, non-prenylic bisubstrate farnesyltransferase inhibitors, 4. Effect on farnesyltransferase inhibitory activity of conformational restrictions in the central group

Article abstract of DOI:10.1211/146080800128735746

We have recently described non-peptidic, non-prenylic bisubstrate analogues as novel farnesyltransferase inhibitors comprising three modules-a farnesylmimetic, a linker and an AAX-peptidomimetic substructure. In this study we replaced the originally used

Full text of DOI:10.1211/146080800128735746

Pharm. Pharmacol. Commun. 2000, 5: 117±124
Received January 5, 2000
Accepted January 19, 2000
# 2000 Pharm. Pharmacol. Commun.
Non-peptidic, Non-prenylic Bisubstrate Farnesyltransferase
Inhibitors, 4. Effect on Farnesyltransferase Inhibitory Activity
of Conformational Restrictions in the Central Group
MARTIN SCHLITZER AND ISABEL SATTLER*
È
Institut fur Pharmazeutische Chemie, Philipps-Universitat Marburg, Marbacher Weg 6, D-35032 Marburg
and *Hans-Knoll-Institut fur Naturstoff-Forschung e. V., Beutenbergstraûe 11, D-07745, Jena, Germany
È
È
È
Abstract
We have recently described non-peptidic, non-prenylic bisubstrate analogues as novel
farnesyltransferase inhibitors comprising three modules±a farnesylmimetic, a linker and an
AAX-peptidomimetic substructure. In this study we replaced the originally used b-alanyl
linker by several aliphatic and cyclic amino acids to investigate the effects on inhibitory
potential of the stereochemistry of this central group. Whereas replacement of b-alanine by
glycine did not affect inhibitory activity, all other modi®cations resulted in reduced
activity. This result, which will be helpful for further development, shows that the
bioactive conformation is none of those ®xed by the rigid linkers.
Cancer is caused by stepwise accumulation of
mutations that affect growth control, differentiation
and cell survival (McCormick 1999). Ras proteins
play a central role in the signal transduction cascades
controlling these processes (Macara et al 1996;
Gomez et al 1998). Mutated forms of Ras, which are
constitutively active, are found in 30% (approx.) of
all cancers in man. Several post-transformational
modi®cations occur before Ras acquires its full bio-
logical activity. The crucial step is transfer of a far-
nesyl residue from farnesylpyrophosphate to the thiol
of a cysteine side-chain of the C-terminal CAAX-
tetrapeptide sequence (C: cysteine, A: aliphatic
amino acid, X: serine or methionine) catalysed by the
enzyme farnesyltransferase (Zhang & Casey 1996).
Therefore, inhibition of farnesyltransferase has
received considerable interest in recent years as a
strategy for the development of novel potential can-
cer therapeutics (Leonard 1997; Quian et al 1997;
Sebti & Hamilton 1998). However, there is accu-
mulating evidence that Ras might not be the only
substrate of farnesyltransferase involved in onco-
genesis (Cox & Der 1997; Du et al 1999).
these compounds and their low toxicity have been
demonstrated and they are, therefore, regarded as a
major emerging strategy in cancer therapy.
We have previously described novel types of
bisubstrate analogue farnesyltransferase inhibitor
(1; Figure 1) (Schlitzer & Sattler 1999a). These
inhibitors are different from the few known
bisubstrate analogues in having no peptidic or
prenylic substructures. They are composed of three
modular building blocks±a AAX-peptidomimetic,
a linker or central group and a farnesylmimetic
(Figure 1). We have shown that replacement of the
amide group which connects the farnesylmimetic to
the linker by an ethylene bridge considerably
reduces the inhibitory activity of farnesyltrans-
ferase (Schlitzer & Sattler unpublished results).
We suggested that the carbonyl oxygen interacts
with the active site of the farnesyltransferase and
that this is important for the binding af®nity.
Because the position of this carbonyl group relative
to the peptidomimetic substructure is governed by
the length and conformational ¯exibility of the
linking substructure, we have prepared derivatives
of 1 in and that this b-alanyl linker is replaced by
other building blocks. It was hoped the use of rigid
cyclic structures would provide information about
the bioactive conformation of 1 (N-{2-[3-(2,3-
dimethyl}phenylaminosulphonyl)phenylaminocarb-
onyl]ethyl}hexadecanoic acid amide).
Irrespective of the unresolved issue of the
mechanism by which farnesyltransferase inhibitors
exert their antiproliferative effects, the ef®cacy of
Correspondence: M. Schlitzer, Institut fuÈr Pharmazeutische
È
Chemie, Philipps-Universitat Marburg,
Marbacher Weg 6, D-35032 Marburg, Germany.

118
MARTIN SCHLITZER AND ISABEL SATTLER
Figure 1. The modular composition of bisubstrate analogue farnesyltransferase inhibitor 1.
Material and Methods
General procedure for N-boc-deprotection
(Procedure 2)
Chemistry
N-Boc derivatives and HCl gas 4M HCl were dis-
solved in dioxane (10 mL mmolÀ1) and the solution
was stirred for 2 h at room temperature. After
addition of diethyl ether the volatile compounds
were distilled in-vacuo into a ¯ask immersed in
liquid N2. The solid residue was used without fur-
ther puri®cation.
H and C NMR spectra were recorded on Jeol
JMN-GX-400 and JMN-LA-500 spectrometers.
Mass spectra were acquired with a Vacuum Gen-
erators VG 7070H spectrometer and Vector 1 data
acquisition system from Tecnivent, or with an
AutoSpec mass spectrometer from Micromass. IR
spectra were recorded on a Nicolet 510P FTIR
spectrometer. Microanalysis was performed with a
CH analyser according to Dr Salzer from Labor-
matic, and with a Hewlett±Packard type 185 CHN
analyser. Column chromatography was performed
on silica gel 60 (0.062±0.200 mm) from Merck.
Compound 2 Schlitzer & Sattler 1999; was pre-
pared as described elsewhere (3-amino-N-(2,3-
dimethylphenylbenzene sulphonanide) Schlitzer et
al 1999).
General procedure for the preparation of the
N-acyl amino acids (Procedure 3)
The amino acid and K2CO3 (430 mg (mmol amino
acid)À1) were dissolved in water (40 mL) and
cooled to 0ꢀC. A solution of the appropriate acyl
chloride (1 equiv.) in acetone (40 mL) was added
dropwise. Stirring was continued for 1 h at 0ꢀC and
1 h at room temperature. Most of the acetone was
then removed by rotary evaporation and the
resulting aqueous solution was acidi®ed with conc.
hydrochloric acid to pH 1. The precipitate was
collected, washed with water and dried. The pro-
ducts were used without further puri®cation.
General procedure for acylation of amines by
mixed anhydride activation (Procedure 1)
The appropriate carboxylic acid was dissolved in
dry dimethylformamide (DMF) in a ¯ame-dried
¯ask under an atmosphere of argon. After addition
of N-methylmorpholine (0.25 mL (mmol acid)À1)
the solution was cooled to À15ꢀC and isobutyl
chloroformate (0.13 mL (mmol acid)À1) was added.
A solution of the amine component (1 equiv.) in
dry DMF was added after 5 min. When a hydro-
chloride was used as the amine component, addi-
tional N-methylmorpholine (0.25 mL mmolÀ1) was
added. The mixture was left to warm to room
temperature overnight and then poured into brine
(400±800 mL). If a solid precipitate formed, this
was collected by vacuum ®ltration and thoroughly
washed with water. Otherwise, the aqueous mixture
was extracted with ethyl acetate (36100 mL) and
the combined organic extracts were washed
successively with 0.67 M citric acid, saturated
NaHCO3 solution and brine and dried with MgSO4.
The residue obtained after removal of the solvent
was puri®ed by ¯ash chromatography.
2-(tert-Butyloxycarbonylamino)-N-[3-(2,3-dimethyl-
phenylaminosulphonyl)phenyl]aceticacidamide(11).
Compound 2 (1.66 g, 6.0 mmol) was acylated
with N-boc-glycine (1.05g, 6.0 mmol) according to
procedure 1. Yield 1.67g (65%). 1H NMR (500 MHz,
CDCl3): d1.43(9H, m), 1.97(3H,s), 2.17(3H,s), 3.94
(2H, m), 6.85±7.10 (5H, m), 7.29(2H, m), 8.05 (1H,
‡
m), 8.80 (1H, m); ESIMS: m=z ˆ 434 [M ‡ H] ; HR-
ESIMS: exact mass calculated for C21H27N3O5S
[M‡ H] 434.1749; found 434.1706.
‡
N-{[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]methyl}heptadecanoicacidamide(3).
Compound 11 (866 mg, 2 mmol) was deprotected
according to procedure 2 and acylated with heptade-
canoic acid (541 mg, 2 mmol) according to procedure
1. Puri®cation by ¯ash chromatography with ethyl
acetate±n-hexane (3 : 2) as eluent yielded 1060 mg

BISUBSTRATE FARNESYLTRANSFERASE INHIBITORS
119
(91%), mp 64ꢀC. IR: n ˆ 2920, 2850, 1715,
1645 cmÀ1; 1H NMR (500MHz, d6-DMSO): d 0.84
(3H,m),1.22(26H,m),1.48(2H,m),1.95(3H,s),2.15
(5H,m),3.84(2H,d,J ˆ 6Hz),6.70(1H,m),6.92(1H,
m), 6.98 (1H, m), 7.28 (1H, m), 7.45 (1H, m), 7.77
(1H, m), 7.99 (1H, m), 8.02 (1H, m), 9.47 (1H, s),
10.15 (1H, s); 13C NMR (125 MHz, d6-DMSO): d
14.1, 14.3, 20.3, 22.3, 24.8, 25.4, 28.8, 28.9, 28.95,
29.0, 29.1, 29.2, 29.3, 31.5, 34.0, 35.5, 43.3, 117.4,
121.5, 125.0, 125.6, 132.6, 134.0, 134.8, 137.7,
mg, 1.5 mmol) according to procedure 1. Yield
520 mg (78%). 1H NMR (500 MHz, CDCl3): d
1.38 (9H, m), 1.94 (3H, s), 2.19 (3H, s), 2.98
(3H, s), 3.91 (2H, s), 6.82±6.95 (5H, m), 7.86
(1H, s).
N-{[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]methyl}-N-methylheptadecanoic acid
amide (5). Compound 13 (520 mg, 1.17 mmol) was
deprotected according to procedure 2 and acylated
with heptadecanoic acid (316 mg, 1.17 mmol)
according to procedure 1. Puri®cation by ¯ash
chromatography with ethyl acetate±n-hexane
(3:2) then ethyl acetate as eluents yielded 320 mg
(46%), mp 58ꢀC. IR: n ˆ 2925, 2855, 1690,
1630 cmÀ1; 1H NMR (500 MHz, d6-DMSO): d
0.80 (3H, m), 1.20 (26H, m), 1.55 (2H, m), 2.01
(3H, s), 2.16 (3H, s), 2.35 (2H, m), 3.15 (3H, s),
4.10 (2H, s), 6.64 (1H, m), 6.92 (1H, m), 6.80 (2H,
m), 6.92 (1H, m), 7.15 (2H, m), 7.68 (1H, m), 7.76
(1H, m), 9.08 (1H, s); 13C NMR (125 MHz, d6-
DMSO): d 14.1, 20.6, 22.7, 24.8, 29.3, 29.4, 29.5,
29.6, 29.65, 29.7, 31.9, 33.3, 54.0, 118.0, 122.9,
123.5, 124.1, 125.8, 128.5, 129.3, 132.8, 134.0,
138.0, 138.5, 140.4, 168.0, 175.5; EIMS:
‡
139.5, 168.6, 172.0; EIMS: m=z ˆ 585 [M] ; HR-
‡
EIMS: exact mass calculated for C33H51N3O4S [M]
585.3600; found 585.3601.
4-(tert-Butyloxycarbonylamino)-N-[3-(2,3-dimethyl-
phenylaminosulphonyl)phenyl]butyric acid amide
(12). Compound 2 (690 mg, 2.5 mmol) was acy-
lated with N-boc-4-aminobutyric acid according to
procedure 1. Yield 875 mg (76%). 1H NMR
(400 MHz, CDCl3): d 1.42 (9H, m), 1.83 (2H, m),
1.98 (3H, s), 2.19(3H, s), 2.38 (2H, m), 3.19(2H,
m), 6.78 (1H, m), 6.96 (1H, m), 7.03 (1H, m), 7.31
(1H, m), 7.52 (1H, m), 7.81 (1H, m), 8.10 (1H, m);
‡
EIMS: m=z ˆ 461 [M] ; HR-EIMS: exact mass
‡
calculated for C23H31N3O5S [M]
found 461.1980.
461.1984;
‡
m=z ˆ 599 [M] ; elemental analysis calculated
N-{3-[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]propyl}pentadecanoic acid amide
(4). Compound 12 (507 mg, 1.1 mmol) was depro-
tected according to procedure 2 and acylated with
pentadecanoic acid (266 mg, 1.1 mmol) according
to procedure 1. Puri®cation by ¯ash chromatogra-
phy with ethyl acetate±n-hexane (3 : 2) as eluent
yielded 200 mg (31%), mp 45ꢀC. IR: n ˆ 2925,
for C34H53N3O4S (599.88) C, 68.08; H, 8.91; N,
7.00; found: C, 68.00; H, 8.75; N, 6.88.
N-Methyl-N-palmitoyl-b-alanine (14). N-Methyl-b-
alanine nitrile (0.94 mL, 10 mmol) was heated
under re¯ux for 2 h in a mixture of ethanol (25
mL), water (5 mL) and KOH (3.2 g). After evapora-
tion of the solvents the remaining residue was
dissolved in water (35 mL) and acylated with pal-
mitoyl chloride according to procedure 3 to yield
2.22 g (65%), mp 64ꢀC. IR: n ˆ 2920, 2850, 1710,
1605 cmÀ1; 1H NMR (500 MHz, d6-DMSO): d 0.83
(3H, m), 1.22 (24H, m), 1.46 (2H, m), 2.17 (2H, m),
2.37 (2H, m), 2.92 (3H, s), 3.43 (2H, s); EIMS:
1
2855, 1645, 1545 cmÀ1; H NMR (500 MHz, d6-
DMSO): d 0.83 (3H, m), 1.22 (22H, m), 1.46 (2H,
m), 1.68 (2H, m), 1.95 (3H, s), 2.02 (2H, m), 2.15
(3H, s), 2.28 (2H, m), 3.05 (2H, m), 6.71 (1H, m),
6.92 (1H, m), 6.97 (1H, m), 7.26 (1H, m), 7.41 (1H,
m), 7.64 (1H, m), 7.77 (1H, m), 8.00 (1H, m), 9.42
(1H, s), 10.06 (1H, s); 13C NMR (125 MHz, d6-
DMSO): d 13.6, 13.8, 19.8, 21.8, 24.9, 25.0, 28.3,
28.4, 28.5, 28.6, 28.7, 31.0, 33.7, 35.3, 37.8, 116.8,
120.7, 122.3, 124.4, 125.1, 127.7, 129.1, 133.4,
134.5, 137.2, 139.5, 141.1, 171.1, 171.9; EIMS:
‡
m=z ˆ 341 [M] .
N-{2-[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]ethyl}-N-methylhexadecanoic acid
amide (6). Compound 2 (414 mg, 1.5 mmol) was
acylated with 14 (512 mg, 1.5 mmol) according to
procedure 1. Puri®cation by ¯ash chromatography
with ethyl acetate±n-hexane (3 : 2) as eluent
yielded 284 mg (32%), mp 57ꢀC. IR: n ˆ 2925,
‡
m=z ˆ 585 [M] ; HR-EIMS: exact mass calculated
‡
for C33H51N3O4S [M] 585.3600; found 585.3609;
elemental analysis calculated for C33H51N3O4S
(585) C, 67.66; H, 8.77; N, 7.17; found: C, 67.76;
H, 8.90; N, 7.29.
1
2850, 1705, 1630 cmÀ1; H NMR (500 MHz, d6-
DMSO): d 0.80 (3H, m), 1.21 (24H, m), 1.50 (2H,
m), 1.95 (3H, s), 2.14 (3H, s), 2.24 (2H, m), 2.65
(2H, m), 2.99 (3H, s), 3.67 (2H, m), 6.68 (1H, m),
6.90 (3H, m), 7.25 (2H, m), 7.74 (1H, m), 8.12 (1H,
s), 9.57 (1H, s); 13C NMR (125 MHz, d6-DMSO): d
2-(N-tert-Butyloxycarbonyl-N-methyl-amino)-N-[3-
(2,3-dimethylphenylaminosulphonyl)phenyl]-acetic
acid amide (13). Compound 2 (414 mg 1.5 mmol)
was acylated with N-boc-N-methylglycine (284

120
MARTIN SCHLITZER AND ISABEL SATTLER
13.9, 14.1, 20.6, 22.7, 25.0, 29.1, 29.2, 29.3, 29.4,
29.5, 29.6, 29.7, 31.9, 33.6, 36.1, 36.4, 44.3, 118.1,
122.7. 123.2, 123.9, 125.9, 128.3, 129.3, 131.8,
134.1, 137.9, 139.3, 140.3, 169.4, 175.0; EIMS:
(300 mL) and the pH was adjusted to 7±8 by
addition of saturated NaHCO3 solution The mixture
was extracted with ethyl acetate (36200 mL). The
combined extracts were washed with brine, dried
with MgSO4 and evaporated to yield 16 as a yellow
foam. Yield 1.7 g (91%). IR: n ˆ 3360, 1660,
‡
m=z ˆ 599 [M] ; elemental analysis calculated for
C34H53N3O4S (599.88) C, 68.08; H, 8.91; N, 7.00;
found: C, 68.21; H, 8.81; N, 7.33.
1
1550 cmÀ1; H NMR (500 MHz, CDCl3): d 1.88
(3H, s), 2.12 (3H, s), 6.53 (1H, m), 6.84 (1H, m),
6.91 (1H, m), 6.93 (1H, m), 7.10 (1H, m), 7.18 (1H,
m), 7.25 (1H, m), 7.32 (1H, m), 7.54 (2H, m), 7.62
(1H, m), 7.77 (1H, s), 8.00 (2H, m), 8.29(1H, s).
N-Heptadecanoylproline (15). Proline (575 mg,
5 mmol) was acylated with heptadecanoyl chloride
(1.44 g, 5 mmol) according to procedure 3 to yield
1
1.7 g (93%). H NMR (500 MHz, CDCl3): d 0.88
(3H, m), 1.21 (26H, m), 1.65 (2H, m), 2.02 (2H, s),
2.33 (2H, m), 2.46 (2H, m), 3.45 (1H, M), 3.56 (1H,
m), 4.58 (1H, m).
N-{2-[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]phenyl}hexadecanoic acid amide
(8). Palmitoyl chloride (0.76 mL) was added to a
solution of 16 (1.0 g, 2.5 mmol) and N-methylmor-
pholine (0.6 mL) in dichloromethane (30 mL).
After stirring overnight at room temperature the
mixture was diluted with dichloromethane and
washed with 1 M hydrochloric acid, saturated
NaHCO3 solution and water and dried with MgSO4.
The residue obtained after evaporation of the sol-
vent was puri®ed by ¯ash chromatography with
ethyl acetate±n-hexane (2 : 3) as eluent to yield
1.12 g (71%), mp 105ꢀC. IR: n ˆ 3235, 2920,
N,N-{1-[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]tetramethylene}-heptadecanoic acid
amide (7). Compound 2 (552 mg, 2.0 mmol) was
acylated with 15 (734 mg, 2.0 mmol) according to
procedure 1. Puri®cation by ¯ash chromatography
with ethyl acetate±n-hexane (2 : 3) as eluent
yielded 360 mg (29%), mp 45ꢀC. IR: n ˆ 2925,
1
2855, 1680, 1620 cmÀ1; H NMR (500 MHz, d6-
DMSO): d 0.83 (3H, m), 1.22 (26H, m), 1.46 (2H,
m), 1.87 (2H, m), 1.96 (3H, s), 2.10 (2H, m), 2.16
(3H, s), 2.25 (2H, m), 3.49(1H, m), 3.54 (1H, m),
4.38 (1H, m), 6.70 (1H, m), 6.92 (3H, m), 6.98 (1H,
m), 7.38 (1H, m), 7.44 (1H, m), 7.78 (1H, m), 8.00
(1H, m), 9.43 (1H, s), 10.14 (1H, s); 13C NMR
(125 MHz, d6-DMSO): d 14.2, 14.4, 20.4, 22.3,
24.5, 24.7, 28.9, 29.0, 29.1, 29.2, 29.26, 29.3,
29.7, 31.6, 34.0, 47.2, 60.3, 117.5, 121.4, 123.0,
125.0, 125.6, 128.4, 129.7, 134.1, 135.0, 137.8,
1
2850, 1660, 1585 cmÀ1; H NMR (500 MHz, d6-
DMSO): d 0.84 (3H, m), 1.21 (24H, m), 1.54 (2H,
m), 1.99 (3H, s), 2.14 (3H, s), 2.27 (2H, m), 6.77
(1H, m), 6.93 (1H, m), 6.97 (1H, m), 7.19 (1H, m),
7.38 (1H, m), 7.49(2H, m), 7.73 (1H, m), 7.91 (1H,
m), 8.10 (1H, m), 8.16 (1H, m); 13C NMR
(125 MHz, d6-DMSO): d 13.6, 13.8, 19.8, 21.8,
24.3, 24.7, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 32.0,
33.5, 37.2, 118.2, 121.6. 121.7, 122.8, 123.8, 124.4,
125.1, 127.7, 128.5, 129.0, 131.5, 133.4, 134.5,
137.2, 137.6, 139.8, 141.1, 166.9, 174.1; EIMS:
‡
141.6, 171.3, 171.5; EIMS: m=z ˆ 625 [M] ; ele-
mental analysis calculated for C36H55N3O4S
(625.92) C, 69.08; H, 8.86; N, 6.71; found: C,
69.05; H, 8.60; N, 6.80.
‡
m=z ˆ 633 [M] ; elemental analysis calculated for
C37H51N3O4S (633.90) C, 70.11; H, 8.11; N, 6.63;
found: C, 70.43; H, 9.18; N, 6.29.
N-[3-(2,3-Dimethylphenylaminosulphonyl)phenyl]-
2-aminobenzoic acid amide (16). 2-Nitrobenzoyl
chloride (930 mg, 5 mmol) was added to a solution
of 2 (1.38 g, 5 mmol) in a mixture of toluene
(100 mL) and dioxane (20 mL) and heated to re¯ux.
After 2 h the solvents were evaporated to furnish a
yellow foam. Yield 2.1 g (95%). IR: n ˆ 1665,
N-Palmitoylpiperidine-3-carboxylic acid (17). Piper-
idine-3-carboxylic acid (1.29g, 10 mmol) was acy-
lated with palmitoyl chloride (3.0 mL, 10 mmol)
1
according to procedure 3 to yield 3.0 g (86%). H
NMR (400 MHz, d6-DMSO): d 0.84 (3H, m), 1.23
(24H, m), 1.45 (2H, m), 1.55 (1H, m), 1.65 (1H, m),
1.90 (1H, m), 2.28 (2H, m), 2.41 (1H, m), 2.69 (1H,
m), 3.18 (1H, m), 3.28 (1H, m), 3.72 (1H, m), 3.81
(1H, m).
1
1530, 1350 cmÀ1; H NMR (500 MHz, CDCl3): d
1.88 (3H, s), 2.12 (3H, s), 6.53 (1H, m), 6.84 (1H,
m), 6.91 (1H, m), 6.93 (1H, m), 7.10 (1H, m), 7.18
(1H, m), 7.25 (1H, m), 7.32 (1H, m), 7.54 (2H, m),
7.62 (1H, m), 7.77 (1H, s), 8.00 (2H, m), 8.29
(1H, s).
Tin(II)chloride dihydrate (5.34 g) was added to a
solution of this foam in ethyl acetate (70 mL). After
2 h under re¯ux the mixture was poured into water
N,N-{2-[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]pentamethylene}hexadecanoic acid
amide (9). Compound 2 (414 mg, 1.5 mmol) was
acylated with 17 (479mg, 1.5 mmol) according to
procedure 1. Puri®cation by ¯ash chromatography
with ethyl acetate±n-hexane (2 : 3) as eluent

BISUBSTRATE FARNESYLTRANSFERASE INHIBITORS
121
yielded 290 mg (31%), mp 104ꢀC. IR: n ˆ 2925,
(400 MHz, d6-DMSO): d 0.84 (3H, m), 1.23 (22H,
m), 1.59(2H, m), 2.31 (2H, m), 7.37 (1H, m), 7.57
(1H, m), 7.80 (1H, m), 8.18 (1H, m), 9.83 (1H, s)
1
1700, 1605 cmÀ1; H NMR (500 MHz, CDCl3): d
0.85 (3H, m), 1.23 (24H, m), 1.54 (3H, m), 1.66
(1H, m), 1.90 (1H, m), 1.98 (3H, s), 2.07 (1H, m),
2.18 (3H, s), 2.31 (2H, m), 2.60 (1H, m), 3.43 (1H,
m), 3.52 (1H, m), 3.75 (1H, m), 3.97 (1H, m), 6.76
(1H, m), 6.95 (3H, m), 7.29 (2H, m), 7.81 (1H, m),
8.12 (1H, s), 9.14 (1H, s); 13C NMR (125 MHz,
CDCl3): d 13.9, 14.1, 14.2, 20.6, 22.7, 24.6, 25.4,
26.9, 29.3, 29.4, 29.5, 29.6, 29.65, 29.7, 31.9, 33.3,
43.4, 44.0, 46.8, 118.1, 119.9, 122.6, 123.4, 123.9,
125.8, 128.3, 129.3, 132.1, 134.0, 137.9, 139.2,
N-{3-[3-(2,3-Dimethylphenylaminosulphonyl)phenyl-
aminocarbonyl]phenyl}pentadecanoic acid amide
(10). Compound 2 (552 mg, 2.0 mmol) was acyl-
ated with 18 (722 mg, 2.0 mmol) according to
procedure 1. Puri®cation by ¯ash chromatography
with ethyl acetate±n-hexane (2 : 3) as eluent
yielded 582 mg (47%), mp 176ꢀC. IR: n ˆ 2925,
2850, 1650 cmÀ1; 1H NMR (500 MHz, d6-DMSO):
d 0.84 (3H, m), 1.23 (22H, m), 1.60 (2H, m), 1.99
(3H, s), 2.17 (3H, s), 2.31 (2H, m), 6.75 (1H, m),
6.95 (1H, m), 7.36 (1H, m), 7.43 (1H, m), 7.51 (1H,
m), 7.60 (1H, m), 7.80 (1H, m), 7.96 (1H, m), 8.10
(1H, m), 8.23 (1H, m), 9.48 (1H, s), 9.97 (1H, s),
10.44 (1H, s); 13C NMR (125 MHz, d6-DMSO): d
14.0, 14.1, 20.1, 22.1, 25.1, 28.7, 28.8, 28.9, 29.0,
29.1, 31.3, 36.4, 118.3, 118.8, 119.8, 121.7, 123.0,
123.8, 124.8, 125.4, 128.1, 128.7, 129.4, 133.9,
134.7, 135.2, 137.6, 139.6, 139.8, 141.3, 165.9,
‡
140.3, 171.1, 173.0; EIMS: m=z ˆ 625 [M] ; HR-
EIMS: exact mass calculated for C36H55N3O4S
[M] 625.3913; found 625.3926; elemental analy-
‡
sis calculated for C36H55N3O4S (625.92) C, 69.08;
H, 8.86; N, 6.71; found: C, 69.29; H, 8.75; N, 6.89.
3-Pentadecanoylaminobenzoic acid (18). 3-Amino-
benzoic acid (1.37 g, 10 mmol) was acylated with
pentadecanoyl chloride (2.6 g, 10 mmol) according
1
to procedure 3 to yield 3.13 g (87%). H NMR
Figure 2. Synthesis of compounds 3±10: (i) N-Boc-amino acid, isobutyl chloroformate, N-methylmorpholine, dimethylforma-
mide, À15ꢀC, 5 min, then 2, À15ꢀC!room temperature, overnight; (ii) 4 M HCl in dioxane, room temperature, 2 h; (iii) RCOOH,
isobutyl chloroformate, N-methylmorpholine, dimethylformamide, À15ꢀC, 5 min, then deprotected 11, 12 or 13, À15ꢀC ! room
temperature, overnight; (iv) isobutyl chloroformate, N-methylmorpholine, dimethylformamide, À15ꢀC, 5 min, then 2,
À15ꢀC!room temperature, overnight; (v) 2-nitrobenzoic acid chloride, N-methylmorpholine, dimethylformamide, 0ꢀC ! room
temperature, overnight; (vi) SnCl2Á2H2O, ethyl acetate, re¯ux, 2 h; (vii) palmitoyl chloride, N-methylmorpholine, dichloromethane,
0ꢀC!room temperature, overnight.

122
MARTIN SCHLITZER AND ISABEL SATTLER
Figure 3. Structures and farnesyltransferase inhibitory activity of compounds 3±10.
‡
171.6; EIMS: m=z ˆ 619[M] ; elemental analysis
calculated for C36H49N3O4S (619.87) C, 69.76; H,
7.97; N, 6.78; found: C, 69.89; H, 8.03; N, 6.70.
minus of the subunit. FTase was expressed in
Escherichia coli DH5a grown in LB media
containing ampicillin and chloramphenicol for
co-expression of pGEX-DPR1 and pBC-RAM2 for
FTase production (Del Villar et al 1997). The
enzyme was puri®ed by standard procedures with
glutathione±agarose beads for selective binding of
the target protein.
Enzyme preparation
Yeast farnesyltransferase (FTase) was used as a
fusion to glutathione S-transferase at the N-ter-

BISUBSTRATE FARNESYLTRANSFERASE INHIBITORS
123
Farnesyltransferase assay
the glycine by proline prevents any rotation around
the Ca±N bond present in glycine. This obviously
forces the system into a conformation different
from the bioactive conformation, because activity
is reduced to less than half. Compounds 8 and 9
represent the syn and anti conformations, respec-
tively, of b-alanine. Both compounds are sig-
ni®cantly less active than the b-alanyl derivative 1.
The more pronounced drop in activity of 8 is not
entirely unexpected, because 8 represents the syn
conformation which is very unlikely to be adopted
by 1. In addition to their ®xing the conformation,
the cyclic linkers have a second effect which must
be considered. Because they are more spacious than
the acyclic linkers the loss of activity could also be
attributed to simple steric hindrance, although there
is evidence (Ciccarone et al 1999) that the stereo-
chemistry of that part of the farnesyltransferases
active site can accommodate the cyclic systems
used in this study. In conclusion, this study will be
helpful for further development because it shows
that none of the conformations ®xed by the dif-
ferent linkers represents the bioactive conformation
of our bisubstrate analogue farnesyltransferase
inhibitors.
The assay was conducted as described elsewhere
(Pompliano et al 1992). FPP was obtained as a
solution of the ammonium salt in methanol±10 mM
aqueous NH4Cl (7 : 3) from Sigma±Aldrich. Dan-
syl-Gly-Cys-Val-leu-Ser (Ds-GCVLS) was custom
synthesized by ZMBH, Heidelberg, Germany. The
assay mixture (100 L) contained 50 mM Tris±HCl
pH 7.4, 5 mM MgCl2, 10 mM, ZnCl2, 5 mM DTT
(dithiothreitol), 7 mM Ds-GCVLS, 20 mM FPP,
5 nmol (approx.) yeast GST-Ftase, and 1% of
different concentrations of the test compounds
dissolved in dimethylsulphoxide (DMSO). The
progress of the enzyme reaction was followed by
monitoring the enhancement of ¯uorescence emis-
sion at 505 nm (excitation 340 nm). The reaction
was started by addition of the enzyme and run in a
quartz cuvette thermostatted at 30ꢀC. Fluorescence
emission was recorded with a Perkin±Elmer
LS50B spectrometer. IC50 values (concentrations
resulting in 50% inhibition) were calculated from
the initial velocity of three independent measure-
ments of four or ®ve different concentrations of
inhibitor.
Results and Discussion
Acknowledgements
The preparation of the target structures was
accomplished as outlined in Figure 2. The inhibi-
tory activity against yeast farnesyltransferase (Del
Villar et al 1997) was determined as described
elsewhere (Pompliano et al 1992). The structures
and results are shown in Figure 3. The length of the
original b-alanyl linker was either reduced or
increased by one methylene unit. Whereas the
shorter glycine derivative 3 was as active (approx.)
as the lead structure 1, the longer g-aminobutyric
acid derivative 4 was half as active as 1 as an
inhibitor of farnesyltransferase. Therefore, glycine
and b-alanine were selected as linkers to determine
how an additional substituent on the amide nitrogen
would in¯uence the inhibitory potency of the
compounds. In both instances (5, 6) N-methylation
resulted in a considerable drop in activity. A pos-
sible interpretation that the free NH function is
necessary for activity and acts, for instance, as a
hydrogen-bond donor, does not hold true because
compounds 7 and 9, also lacking a free NH func-
tion, are signi®cantly more active than 5 or 6. The
distinct reduction in activity observed with 5 and 6
can be more readily attributed to a conformational
effect than to the loss of a hydrogen-bond donor.
With the use of rigid ring systems possible con-
formations of the linkers glycine, b-alanine and g-
aminobutyric acid can be frozen. Replacement of
The pGEX-DPR1 and pBC-RAM2 plasmids were
kindly provided by Professor F. Tamanoi (UCLA).
We thank K. Burk and S. Egner for technical
assistance, the German Pharmaceutical Society for
®nancial support and Professor Dr S. Grabley and
PD Dr R. Thiericke for generous support.
References
Ciccarone, T. M., MacTough, S. C., Williams, T. M., Dings-
more, C. J., O'Neill, T. J., Shah, D., Culberson, J. C.,
Koblan, K. S., Kohl, N. E., Gibbs, J. B., Oliff, A. I., Graham,
S. L., Hartman, G. D. (1999) Non-thiol 3-aminomethylben-
zamide inhibitors of farnesyl-protein transferase. Bioorg.
Med. Chem. Lett. 9: 1991±1996
Cox, A. D., Der, C. J. (1997) Farnesyltransferase inhibitors and
cancer treatment: targeting simply Ras? Biochim. Biophys.
Acta 1333: F51±F71
Del Villar, K., Mitsuzawa, H., Yang, W., Sattler, I., Tamanoi,
F. (1997) Amino acid substitutions that convert the protein
substrate speci®city of farneslytransferase to that of gera-
nylgeranyltransferase type I. J. Biol. Chem. 272: 680±687
Du, W., Lebowitz, P. F., Prendergast, G. C. (1999) Cell growth
inhibition by farnesyltransferase inhibitors is mediated by
gain of geranylgeranylated RhoB. Mol. Cell. Biol. 19:
1831±1840
Gomez, J., Martinez, A. C., Gonzalez, A., Rebollo, A. (1998)
Dual role of Ras and Rho proteins: at the cutting edge of life
and death. Immunol. Cell Biol. 76: 125±134

124
MARTIN SCHLITZER AND ISABEL SATTLER
Leonard, D. M. (1997) Ras farnesyltransferase: a new thera-
peutic target. J. Med. Chem. 40: 2971±2990
Macara, I. G., Lounsbury, K. M., Richards, S. A., McKiernan,
C., Bar-Sari, D. (1996) The Ras superfamily of GTPases.
FASEB J. 10: 625±630
McCormick, K. G. (1999) Signalling networks that cause
cancer. Trends Biochem. Sci. 24: M53±M56
Pompliano, D. L., Gomez, R. P., Anthony, N. J. (1992)
Intramolecular ¯uorescence enhancement: a continuous
assay of Ras farnesyl:protein transferase. J. Am. Chem.
Soc. 114: 7945±7946
Schlitzer, M., Sattler, I. (1999a) Design, synthesis and evalua-
tion of novel modular bisubstrate analogue inhibitors of
farnesyltransferase. Angew. Chem. Int. Engl. Ed. 38: 2032±
2034
Schlitzer, M., Sattler, I., Dahse, H.-M. (1999) Different amino
acid replacements in CAAX-tetrapeptide-based farnesyl-
transferase inhibitors. Arch. Pharm. Pharm. Med. Chem.
332: 124±132
Sebti, S. M., Hamilton, A. D. (1998) New approaches to
anticancer drug design based on the inhibition of farnesyl-
transferase. Drug Discov. Today 3: 26±32
Qian, Y., Sebti, S. M., Hamilton, A. D. (1997) Farnesyltrans-
ferase as a target for anticancer drug design. Biopolymers:
43: 25±41
Zhang, F. L., Casey, P. J. (1996) Protein prenylation: mole-
cular mechanism and functional consequences. Annu. Rev.
Biochem 65: 241±269