Non-peptidic, non-prenylic bisubstrate farnesyltransferase inhibitors. Effect of a carboxyl group at the central group on farnesyltransferase inhibitory activity

Article abstract of DOI:10.1211/146080800128735601

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

Full text of DOI:10.1211/146080800128735601

Pharm. Pharmacol. Commun. 2000, 6: 507±512
Received May 3, 2000
Accepted August 3, 2000
# 2000 Pharm. Pharmacol. Commun.
Non-Peptidic, Non-Prenylic Bisubstrate Farnesyltransferase
Inhibitors. Effect of a Carboxyl Group at the Central Group on
Farnesyltransferase Inhibitory Activity
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 recently described non-peptidic, non-prenylic bisubstrate analogues as novel farnesyl-
transferase inhibitors comprising three modulesÐa farnesyl-mimetic, a linker and an
AAX-peptidomimetic substructure. In this study, we replaced the originally used b-alanyl
linker with aminomalonic, aspartic and glutamic acid, respectively, to introduce a second
functional group capable of complexing the essential zinc ion, located in the active site of
farnesyltransferase.
Apart from aminomalonic acid, all moieties showed reduced inhibitory activity. Inter-
estingly, the benzyl esters of the aspartic and glutamic acid derivatives were more active
than the free acids.
The results provide further evidence for an additional lipophilic binding cleft in the
active site of farnesyltransferase.
Cancer is caused by a stepwise accumulation of
mutations that affect growth control, differentiation
and cell survival (McCormick 1999). Ras proteins
play a central role in the signal transduction cas-
cades that control these processes (Macara et al
1996; Gomez et al 1998). Mutated forms of Ras,
which are constitutively active, are found in
approximately 30% of all cancers in man. Several
post-transformational modi®cations occur before
Ras acquires its full biological activity. The critical
step is the transfer of a farnesyl 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). There-
fore, the inhibition of farnesyltransferase has
received considerable interest in the recent years as
a strategy for the development of novel potential
cancer therapeutics (Leonard 1997; Qian et al
1997; Sebti & Hamilton 1998). However, there
is increasing evidence that Ras may not be the
only substrate of farnesyltransferase involved in
oncogenesis (Cox & Der 1997; Du et al 1999).
Irrespective of the mechanisms by which farne-
syltransferase inhibitors exert their antiproliferative
effects, the ef®cacy of these compounds and their
low toxicity has been demonstrated (Oliff 1999),
and they are, therefore, regarded as a major new
approach in cancer
therapy.
We have previously described novel types of
bisubstrate analogue farnesyltransferase inhibitors
(1; Figure 2) (Schlitzer & Sattler 1999). These
inhibitors are different from the few known
bisubstrate analogues in that they do not have
peptidic or prenylic substructures. They are com-
posed of three modular building blocksÐan AAX-
peptidomimetic, a linker or central moiety and a
farnesylmimetic (Figure 2). We demonstrated that
the replacement of the amide moiety which con-
nects the farnesylmimetic to the linker by an
ethylene bridge considerably reduces the farnesyl-
transferase inhibitory activity (Schlitzer et al 2000).
We suggest that the carbonyl oxygen interacts with
the farnesyltransferase active site and that this
interaction is important for the binding af®nity.
This interaction might be enhanced by introducing
a carboxylic acid into the central moiety as a
second function capable of complexing the
Correspondence: M. Schlitzer, Institut fuÈr Pharmazeutische
È
Chemie, Philipps-Universitat Marburg, Marbacher Weg 6,
D-35032 Marburg, Germany.

508
MARTIN SCHLITZER AND ISABEL SATTLER
essential zinc ion, located in the active site of
farnesyltransferase. Therefore, we prepared an-
alogues of 1 in which the central b-alanyl moiety
was replaced by aminomalonic, aspartic and glu-
tamic acid, respectively.
for 2 h at room temperature. After addition of
diethylether the volatile compounds were distilled
in-vacuo into a ¯ask immersed in liquid N2. The
solid residue was used without further puri®cation.
General procedure for the saponi®cation of esters
4a, 6a and 8a (Procedure 3)
Material and Methods
To a solution of esters 4a, 6a and 8a, respectively,
in dimethoxyethane (15 mL), 1 M LiOH solution
(15 mL) was added and the mixture was stirred at
room temperature until the reaction was complete
(TLC). The volatile compounds were removed in-
vacuo and the residue was dissolved in water
(20 mL). This solution was extracted with ethyl
acetate. The organic phase was discarded. The
aqueous solution was adjusted to pH 1 with con-
centrated HCl and extracted three times with ethyl
acetate. The combined organic extracts were dried
with MgSO4 and evaporated to dryness.
Chemistry
1H NMR and 13C NMR spectra were recorded on
Jeol JMN-GX-400 and Jeol JMN-LA-500 spec-
trometers. Mass spectra were acquired 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 a Hewlett Packard CHN-analyser (type
185). Column chromatography was performed on
silica gel 60 (0Á062±0Á200 mm) from Merck. N-
(2-(3-(2,3-Dimethylphenylaminosulphonyl)-phenyl-
aminocarbonyl)ethyl)hexadecanoic acid amide
(1), 3-amino-N-(2,3-dimethyl-phenyl)benzenesul-
phonamide (2) (Schlitzer & Sattler 1999; Schlitzer
et al 1999) and N-Boc-2-aminomalonic acid
monoethyl ester (Kawai et al 1982) were prepared
as described elsewhere.
2-(tert-Butyloxycarbonylamino)-2-[[3-(2,3-
dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]acetic acid ethyl ester (3)
Compound 2 (1Á38g, 5Á0 mmol) was acylated with
N-Boc-2-aminomalonic acid monoethyl ester
(1Á24 g, 5Á0 mmol) according to Procedure 1. Yield
1
2Á41 g (95%). H NMR (400 MHz, CDCl3): d 0Á92
(3H, m, O-CH2-CH3), 1Á46 (9H, m, t-Bu-H), 1Á96
(3H, s, Ph-CH3), 2Á20 (3H, s, Ph-CH3), 4Á02 (1H, m,
malonyl-a-H), 4Á25 (2H, m, O-CH2-CH3), 6Á20 (1H,
s, NH), 6Á78 (1H, m, aryl-H), 6Á96±7Á01 (4H, m,
aryl-H), 7Á04 (1H, m, aryl-H), 7Á16 (1H, m, aryl-H).
General procedure for the acylation of amines
using 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
1
of N-methylmorpholine (0Á25 mL mmol ) the
solution was cooled to 15ꢀC and isobutyl chloro-
2-(Heptadecanoylamino)-2-[[3-(2,3-
dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]acetic acid ethyl ester (4a)
1
formate (0Á13 mL mmol ) was added. A solution
of the amine component (1 equiv.) in dry DMF was
added after 5 min. When a hydrochloride was used
as the amine component, additional N-methylmor-
Compound 3 (1010 mg, 2 mmol) was deprotected
according to Procedure 2 and acylated by hepta-
decanoic acid (541 mg, 2 mmol) according to Pro-
cedure 1. Puri®cation by ¯ash chromatography with
ethyl acetate±n-hexane (2 : 3) as eluent yielded
1260 mg (96%);1H NMR (400 MHz, d6-DMSO): d
0Á85 (6H, m, heptadecanoyl-CH3, O-CH2-CH3),
1Á22 (26H, m, heptadecanoyl-CH2), 1Á46 (2H, m,
heptadecanoyl-CH2), 1Á94 (3H, s, Ph-CH3), 2Á15
(3H, m, Ph-CH3), 2Á25 (2H, m, heptadecanoyl-
CH2), 3Á93 (1H, m, malonyl-a-H), 4Á15 (2H, m, O-
CH2-CH3), 6Á70 (1H, m, aryl-H), 6Á92 (1H, m, aryl-
H), 6Á98 (1H, m, aryl-H), 7Á26 (1H, m, aryl-H), 7Á42
(1H, m, aryl-H), 7Á78 (1H, m, aryl-H), 8Á00 (1H, m,
aryl-H), 9Á44 (1H, s, NH), 10Á04 (1H, s, NH); 13C
NMR (125 MHz, d6-DMSO): d 13Á9, 14Á0, 14Á1,
18Á7, 18Á8, 20Á2, 22Á2, 25Á1, 25Á2, 27Á4, 28Á8, 28Á9,
1
pholine (0Á25 mL mmol ) was added. The mixture
was left to warm to room temperature overnight
and then poured into brine (400±800 mL). The
aqueous mixture was extracted with ethyl acetate
(3 6 100 mL) and the combined organic extracts
were washed successively with 0Á67 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.
General procedure for the N-Boc-deprotection
(Procedure 2)
The N-Boc derivatives were dissolved in dioxane
1
(10 mL mmol ) supplied with 4 M HCl and stirred

BISUBSTRATE FARNESYLTRANSFERASE INHIBITORS
509
29Á0, 29Á1, 29Á2, 31Á4, 31Á4 and 34Á7 (alkyl-C), 56Á1
(malonyl-a-C), 61Á7 (O-CH2-CH3), 117Á0, 121Á0,
122Á6, 124Á9, 124Á4, 128Á2, 129Á5, 133Á8, 134Á7,
137Á6, 139Á9 and 141Á3 (aryl-C), 166Á6, 171Á8 and
172Á7 (CO).
hexadecanoyl-CH2), 2Á15 (3H, s, Ph-CH3), 2Á74
(1H, m, Asp-b-H), 2Á89 (1H, m, Asp-b-H), 4Á70
(1H, m, Asp-a-H), 5Á08 (2H, s, Bzl-CH2), 6Á70 (1H,
m, aryl-H), 6Á92 (1H, m, aryl-H), 6Á98 (1H, m, aryl-
H), 7Á22 (1H, m, aryl-H), 7Á28 (5H, m, aryl-H), 7Á41
(1H, m, aryl-H), 7Á74 (1H, m, aryl-H), 8Á03 (1H, m,
aryl-H), 8Á25 (1H, m, NH), 9Á04 (1H, s, NH), 10Á24
(1H, s, NH); 13C NMR (125 MHz, d6-DMSO): d
13Á8, 13Á9, 20Á0, 20Á1, 21Á9, 24Á4, 25Á1, 28Á4, 28Á5,
28Á6, 28Á7, 28Á8, 28Á85, 28Á9, 31Á1, 33Á6 and 34Á9
(alkyl-C), 48Á7 (Asp-a-C), 65Á9 (Bzl-CH2), 116Á0,
116Á8, 119Á9, 121Á1, 121Á8, 122Á4, 124Á5, 125Á2,
127Á4, 127Á7, 127Á9, 128Á1, 129Á3, 133Á6, 134Á5,
134Á6, 135Á8, 137Á3, 139Á4, 139Á9 and 141Á2 (aryl-
C), 168Á0, 170Á8 and 171Á9 (CO).
2-(Heptadecanoylamino)-2-[[3-(2,3-
dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]acetic acid (4b)
Compound 4a (210 mg, 0Á32 mmol) was depro-
tected according to Procedure 3. Yield 180 mg
(89%); mp 94ꢀC. IR: n ˆ 3340, 3285, 2920, 2850,
1
1
1765, 1695, 1545 cm ; H NMR (400 MHz, d6-
DMSO): d 0Á84 (3H, m, heptadecanoyl-CH3), 1Á25
(26H, m, heptadecanoyl-CH2), 1Á48 (2H, m, hep-
tadecanoyl-CH2), 1Á96 (3H, s, Ph-CH3), 2Á16 (3H,
m, Ph-CH3), 2Á27 (2H, m, heptadecanoyl-CH2),
4Á88 (1H, m, malonyl-a-H), 6Á71 (1H, m, aryl-H),
6Á93 (1H, m, aryl-H), 6Á97 (1H, m, aryl-H), 7Á27
(1H, m, aryl-H), 7Á43 (1H, m, aryl-H), 7Á77 (1H, m,
aryl-H), 8Á00 (1H, m, aryl-H), 8Á25 (1H, m, NH),
9Á40 (1H, s, NH); 13C NMR (100 MHz, d6-DMSO):
d 13Á6, 13Á8, 19Á8, 21Á8Á 24Á7, 28Á4, 28Á5, 28Á7, 31Á0,
34Á4 and 36Á3 (alkyl-C), 56Á0 (malonyl-a-C), 116Á8,
124Á5, 125Á1, 127Á8, 129Á1, 137Á2, 139Á6 and 141Á0
(aryl-C), 167Á9, 171Á4 and 172Á3 (CO); HR-ESIMS:
2(S)-2-(Hexadecanoylamino)-3-
[[3-(2,3-dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]propionic acid (6b)
Compound 6a (145 mg, 0Á2 mmol) was deprotected
according to Procedure 3. Yield 120 mg (95%); mp
128ꢀC. IR: n ˆ 3370, 3250, 2920, 2860, 1720,
1 1
1650 cm ; H NMR (400 MHz, d6-DMSO): d 0Á83
(3H, m, hexadecanoyl-CH3), 1Á22 (24H, m, hexa-
decanoyl-CH2), 1Á47 (2H, m, hexadecanoyl-CH2),
1Á96 (3H, s, Ph-CH3), 2Á06 (2H, m, hexadecanoyl-
CH2), 2Á15 (3H, s, Ph-CH3), 2Á70 (1H, m, Asp-b-
H), 2Á83 (1H, m, Asp-b-H), 4Á60 (1H, m, Asp-a-H),
6Á72 (1H, m, aryl-H), 6Á92 (1H, m, aryl-H), 6Á98
(1H, m, aryl-H), 7Á20 (1H, m, aryl-H), 7Á43 (1H, m,
aryl-H), 7Á75 (1H, m, aryl-H), 8Á03 (1H, m, aryl-H),
9Á00 (1H, s, NH), 9Á45 (1H, s, NH), 10Á17 (1H, s,
NH), 11Á97 (1H, s br, COOH); 13C NMR
(125 MHz, d6-DMSO): d 13Á8, 13Á9, 20Á0, 20Á1,
20Á6, 20Á9, 21Á9, 24Á4, 25Á1, 28Á4, 28Á5, 28Á6, 28Á7,
28Á75, 28Á8, 28Á9, 31Á1, 33Á5 and 35Á0 (alkyl-C),
48Á5, 116Á0, 116Á8, 124Á4, 125Á2, 127Á6, 129Á3,
137Á1, 139Á5 and 141Á1 (aryl-C), 168Á2, 170Á9 and
172Á2 (CO); HR-ESIMS: exact mass calculated
‡
exact mass calculated for C34H52N3O6S [M ‡ H] ,
630Á3577; found 630Á3574.
2(S)-2-(tert-Butyloxycarbonylamino)-3-
[[3-(2,3-dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]propionic acid benzyl ester (5)
Compound 2 (1Á66 g, 6Á0 mmol) was acylated with
N-Boc-L-aspartic acid-1-benzyl ester (1Á81 g,
6Á0 mmol) according to Procedure 1. Yield 2Á86 g
(95%). 1H NMR (500 MHz, CDCl3): d 1Á32 (9H, m,
t-Bu-H), 1Á90 (3H, s, Ph-CH3), 2Á11 (3H, s,
Ph-CH3), 2Á79±2Á88 (2H, m, Asp-b-H), 4Á56 (1H,
m, Asp-a-H), 5Á10 (2H, m, Bzl-CH2), 6Á81±6Á95
(3H, m, aryl-H), 7Á06±7Á13 (1H, m, aryl-H), 7Á18±
7Á26 (6H, m, aryl-H), 7Á84 (2H, m, aryl-H).
‡
for C34H52N3O6S [M ‡ H] , 630Á3577; found
630Á3580; elemental analysis calculated for
C34H51N3O6S (629Á87) C, 64Á84; H, 8Á16; N, 6Á67;
found C, 64Á48; H, 8Á43; N, 6Á63.
2(S)-2-(Hexadecanoylamino)-3-
[[3-(2,3-dimethylphenylaminosuphonyl)phenyl]-
aminocarbonyl]propionic acid benzyl ester (6a)
Compound 5 (871 mg, 1Á5 mmol) was deprotected
according to Procedure 2 and acylated by palmitic
acid (386 mg, 1Á5 mmol) according to Procedure 1.
Puri®cation by ¯ash chromatography with ethyl
acetate±n-hexane (3 : 2) as eluent yielded 1040 mg
2(S)-2-(tert-Butyloxycarbonylamino)-4-
[[3-(2,3-dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]butyric acid benzyl ester (7)
Compound 2 (552 mg, 2Á0 mmol) was acylated with
N-Boc-L-glutamic acid-1-benzyl ester (674 mg,
2Á0 mmol) according to Procedure 1. Yield 1Á03 g
(87%).1H NMR (500 MHz, CDCl3): d 1Á36 (9H, m,
t-Bu-H), 1Á92 (3H, s, Ph-CH3), 2Á15 (3H, s, Ph-
CH3), 2Á20 (2H, m, Glu-b-H), 2Á36 (2H, m, Glu-g-
H), 4Á30 (1H, m, Glu-a-H), 5Á10 (2H, m, Bzl-CH2),
1
(96%); H NMR (400 MHz, d6-DMSO): d 0Á83
(3H, m, hexadecanoyl-CH3), 1Á21 (24H, m,
hexadecanoyl-CH2), 1Á45 (2H, m, hexadecanoyl-
CH2), 1Á94 (3H, s, Ph-CH3), 2Á06 (2H, m,

510
MARTIN SCHLITZER AND ISABEL SATTLER
6Á82 (1H, m, aryl-H), 6Á88±7Á00 (5H, m, aryl-H),
7Á11 (1H, m, aryl-H), 7Á27 (5H, m, aryl-H).
containing ampicillin and chloramphenicol for the
co-expression of pGEX-DPR1 and pBC-RAM2
plasmids for farnesyltransferase 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.
2(S)-2-(Pentadecanoylamino)-4-
[[3-(2,3-dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]butyric acid benzyl ester (8a)
Compound 7 (300 mg, 0Á5 mmol) was deprotected
according to Procedure 2 and acylated by penta-
decanoic acid (121 mg, 0Á5 mmol) according to
Procedure 1. Puri®cation by ¯ash chromatography
with ethyl acetate±n-hexane (2 : 3) as eluent yiel-
Farnesyltransferase assay
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.
1
ded 260 mg (72%); H NMR (400 MHz, CDCl3): d
0Á81 (3H, m, pentadecanoyl-CH3), 1Á19 (22H, m,
pentadecanoyl-CH2), 1Á52 (2H, m, pentadecanoyl-
CH2), 1Á94 (3H, s, Ph-CH3), 2Á12 (3H, s, Ph-CH3),
2Á16 (2H, m, Glu-b-H), 2Á23 (2H, m, pentadeca-
noyl-CH2), 2Á37 (2H, m, Glu-g-H), 4Á58 (1H, m,
Glu-a-H), 5Á08 (2H, s, Bzl-CH2), 6Á40 (1H, m, NH),
6Á64 (1H, m, aryl-H), 6Á90 (3H, m, aryl-H), 7Á24
(6H, m, aryl-H), 7Á78 (1H, m, aryl-H), 8Á07 (1H, m,
aryl-H), 9Á21 (1H, s, NH); 13C NMR (100 MHz,
CDCl3): d 13Á9, 14Á1, 20Á6, 22Á7, 25Á6, 29Á2, 29Á3,
29Á4, 29Á5, 29Á6, 31Á9, 33Á4, 33Á9 and 36Á7 (alkyl-C),
51Á8 (Glu-a-C), 67Á7 (Bzl-CH2), 117Á9, 123Á4,
123Á8, 125Á9, 128Á4, 128Á5, 128Á7, 129Á4, 134Á0,
134Á8, 137Á9 and 140Á3 (aryl-C). 170Á9, 171Á7 and
174Á6 (CO).
2(S)-2-(Pentadecanoylamino)-4-
[[3-(2,3-dimethylphenylaminosulphonyl)phenyl]-
aminocarbonyl]butyric acid (8b)
Compound 8a (80 mg, 0Á11 mmol) was deprotected
according to Procedure 3. Yield 60 mg (87%); mp
170ꢀC. IR: n ˆ 3365, 3285, 2925, 2855, 1705,
1
1600, 1545 cm ; 1H NMR (400 MHz, d6-DMSO):
d 0Á84 (3H, m, pentadecanoyl-CH3), 1Á23 (22H, m,
pentadecanoyl-CH2), 1Á47 (2H, m, pentadecanoyl-
CH2), 1Á96 (3H, s, Ph-CH3), 2Á09 (2H, m, Glu-b-H),
2Á15 (3H, s, Ph-CH3), 2Á24 (2H, m, pentadecanoyl-
CH2), 2Á38 (2H, m, Glu-g-H), 4Á23 (1H, m, Glu-a-
H), 6Á71 (1H, m, aryl-H), 6Á92 (1H, m, aryl-H), 6Á98
(1H, m, aryl-H), 7Á27 (1H, m, aryl-H), 7Á42 (1H, m,
aryl-H), 7Á75 (1H, m, aryl-H), 7Á90 (1H, m, NH),
8Á01 (1H, m, aryl-H), 9Á40 (1H, s, NH), 10Á09 (1H,
s, NH); HR-ESIMS: exact mass calculated
‡
for C34H52N3O6S [M ‡ H] , 630Á3577; found
630Á3570.
Figure 1. Synthesis of compounds 4, 6 and 8. i. N-Boc-amino
acid mono ester, isobutyl chloroformate, NMM, DMF, 15ꢀC,
5 min, then 2, 15ꢀC, room temp., overnight; ii. 4 M HCl=diox-
ane, room temp., 2 h; (iii) H3C(CH2)n-COOH, isobutyl chloro-
formate, NMM, DMF, 15ꢀC, 5 min, then deprotected 3, 5 or
7, 15ꢀC, room temp., overnight; and iv. 1 M LiOH, DME,
room temp.
Enzyme preparation
Yeast farnesyltransferase was used as a fusion to
glutathione S-transferase at the N-terminus of the
b-subunit. Farnesyltransferase was expressed in
Escherichia coli DH5a grown in LB media

BISUBSTRATE FARNESYLTRANSFERASE INHIBITORS
511
Dansyl-GlyCysValLeuSer (Ds-GCVLS) was syn-
thesized by ZMBH, Heidelberg, Germany. The
assay mixture (100 mL) contained 50 mM Tris=HCl
pH 7Á4, 5 mM MgCl2, 10 mM, ZnCl2, 5 mM dithio-
threitol (DTT), 7 mM Ds-GCVLS, 20 mM FPP and
approximately 5 nmol yeast GST-farnesyltransfer-
ase and 1% of various concentrations of the test
compounds dissolved in DMSO. The progress of
the enzyme reaction was followed by monitoring
the enhancement of the ¯uorescence emission at
505 nm (excitation 340 nm). The reaction was
started by addition of the enzyme and run in a
Quartz cuvette at 30ꢀC. Fluorescence emission 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 ®ve
different concentrations of inhibitor.
Results and Discussion
The preparation of the target structures was
achieved as outlined in Figure 1. The inhibitory
activity against yeast farnesyltransferase (Del Vil-
lar et al 1997) was determined as described else-
where (Pompliano et al 1992). The structures and
IC50 values are shown in Figure 2. Only the
Figure 2. Structure and farnesyltransferase inhibitory activity (IC50) of compounds 1, 4, 6 and 8.

512
MARTIN SCHLITZER AND ISABEL SATTLER
Cox, A. D., Der, C. J. (1997) Farnesyltransferase inhibitors and
cancer treatment: targeting simply Ras? Biochim. Biophys.
Acta 1333: F51±F71
aminomalonic acid derivative 4b was approxi-
mately as active as the lead compound 1. The
aspartic and the glutamic acid derivatives 6b and
8b showed a marked decrease in farnesyltransfer-
ase inhibitory activity. This effect was much more
pronounced in the larger glutamic acid derivative
8b. None of the three additional carboxyl groups
were in the correct position to enhance the inter-
action of the inhibitors with the enzyme-bound
zinc. In contrast, the carboxy groups of compounds
6b and 8b reduced the af®nity of the inhibitors
towards the enzyme.
Interestingly, the benzylesters 6a and 8a were
more active than the free carboxylic acids. Again,
this effect was more pronounced with the larger
glutamic acid derivative 8a. Obviously, the pres-
ence of the bulky lipophilic benzyl residue attenu-
ates the detrimental effect of the carboxyl group on
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Acknowledgements
The pGEX-DPR1 and pBC-RAM2 plasmids were
kindly provided by Professor F. Tamanoi (UCLA).
M. Schlitzer wishes to thank Ms K. Burk for
technical assistance and the German Pharmaceu-
tical Society for ®nancial support. I. Sattler wishes
to thank Professor S. Grabley and Dr R. Thiericke
for generous support and Ms S. Egner for excellent
technical assistance.
È
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