Design, synthesis and early structure-activity relationship of farnesyltransferase inhibitors which mimic both the peptidic and the prenylic substrate

Article abstract of DOI:10.1016/S0968-0896(00)00138-3

Inhibition of the farnesylation of ras proteins has been identified as a promising target in tumor therapy. Only a few farnesyltransferase inhibitors are bisubstrate analogues displaying features of both substrates, the farnesylpyrophosphate and the C-terminal CAAX-tetrapeptide sequence of the ras protein. These known bisubstrate analogues consist of an AAX-tripeptide and a farnesyl residue connected through various linkers. We have developed a class of novel compounds that mimic a bisubstrate inhibitor structure and that differ from the known ones by lacking peptidic or farnesylic substructures. Long chain fatty acids and aryl-substituted carboxylic acids were used as farnesyl surrogates. These structures were linked to isoleucine amide, benzoic acid amide, N-substituted aminobenzenesulfonamides and N(α)-aryl-substituted methionine derivatives, respectively, which function as AA- or AAX-mimetics. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00138-3

Bioorganic & Medicinal Chemistry 8 (2000) 1991±2006
Design, Synthesis and Early Structure±Activity Relationship of
Farnesyltransferase Inhibitors Which Mimic Both the Peptidic and
the Prenylic Substrate
Martin Schlitzer,a,* Markus Bohm,a Isabel Sattlerb and Hans-Martin Dahseb
aInstitut fur Pharmazeutische Chemie, Philipps-Universitat Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
È
È
bHans-Knoll-Institut fur Natursto€-Forschung e.V., Beutenbergstr. 11, D-07745 Jena, Germany
È
È
Received 14 June 1999; accepted 3 May 2000
AbstractÐInhibition of the farnesylation of ras proteins has been identi®ed as a promising target in tumor therapy. Only a few
farnesyltransferase inhibitors are bisubstrate analogues displaying features of both substrates, the farnesylpyrophosphate and the C-
terminal CAAX-tetrapeptide sequence of the ras protein. These known bisubstrate analogues consist of an AAX-tripeptide and a
farnesyl residue connected through various linkers. We have developed a class of novel compounds that mimic a bisubstrate inhibitor
structure and that di€er from the known ones by lacking peptidic or farnesylic substructures. Long chain fatty acids and aryl-sub-
stituted carboxylic acids were used as farnesyl surrogates. These structures were linked to isoleucine amide, benzoic acid amide, N-
substituted aminobenzenesulfonamides and Na-aryl-substituted methionine derivatives, respectively, which function as AA- or
AAX-mimetics. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
reversibly converted into its methyl ester. In some cases
additional palmitoylation occurs at a nearby downstream
cysteine. From these post-translational modi®cation events
only the farnesylation is obligatory for ras activity.1,2
Ras proteins are members of the family of small guanyl
nucleotide binding proteins. They serve as molecular
switches in the signal transduction cascade which controls
cell di€erentiation and proliferation. Point mutations in
the Ras oncogene yield ras proteins whose GTPase
activity can no longer be activated upon binding of
GTPase activating proteins (GAP's). These mutated ras
proteins are locked in their GTP-bound active state and
thereby are constantly relaying growth signals to the
nucleus. In approximately 30% of all human cancers,
including 90% of pancreatic, 50% of colon, and 50% of
thyroid tumors, these mutated ras proteins are found.
To perform both, their normal and their oncogene
activity, ras proteins must undergo a series of post-
translational modi®cations. First the C-terminal CAAX
amino acid sequence (C: cysteine, A: aliphatic amino acid,
X: methionine or serine) of the ras protein is recognized by
the enzyme farnesyltransferase (FFase) and farnesylated
at the cysteine side chain thiol. In this reaction, farnesyl-
pyrophosphate (FPP) is serving as the prenyl group
donor (Fig. 1). After proteolytic removal of the AAX
tripeptide, the resulting C-terminal S-farnesyl cysteine is
Inhibition of the farnesylation reaction reverses the trans-
formation caused by oncogenic ras. Therefore, inhibition
of farnesyltransferase was identi®ed as a promising target
in cancer therapy. Intense e€orts of several groups have
resulted in a large number of di€erent FTase inhibi-
tors.2 Most researchers utilized the CAAX recognition
motif as a template for their inhibitor development. In
comparison to CAAX-mimetics, stable non-substrate
analogues of the second substrate farnesylpyrophos-
phate are much less common.2
A third approach for inhibitor design are bisubstrate
analogues: molecules that contain structural elements of
both substrates, the CAAX-tetrapeptide and the farnesyl-
pyrophosphate. Such bisubstrate inhibitors are of parti-
cular interest because of two reasons: ®rst, bisubstrate
analogues are lacking the free thiol found in most of the
CAAX peptidomimetics. A free thiol is an undesireable
molecular feature in potential drugs both due to its inher-
ent sensitivity towards oxidation and more importantly,
as a source of serious adverse e€ects as seen with the
ACE inhibitor captoprile.3 Second, recent studies on the
mechanism of the farnesyltransferase reaction revealed
*Corresponding author. +49-6421-28-5825; fax: +49-6421-28-7052;
e-mail: schlitze@mailer.uni-marburg.de
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00138-3

1992
M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
Figure 1. Farnesylation of ras proteins.
that the FTase has an unusually high anity for its reac-
tion product, the farnesylated ras protein which only is
released from the enzyme upon binding of a further FPP
molecule.4 Because of the high anity of the farnesyl-
transferase to its reaction product, structural analogues
of the farnesylated ras protein should be particularly
usefull enzyme inhibitors.4 Molecules which are com-
posed of the C-terminal tetrapeptide of the ras protein
and the farnesyl residue can be considered as bisubstrate
analogues as well as analogues of the farnesylated ras
protein, as product analogues.
farnesyltransferase inhibitors by design, determination of
their biochemical mechanism has not been established.
Known bisubstrate analogues contain amidic structures
in their peptidic substructure which are sensitive toward
hydrolytic or enzymatic degradation and several allylic
positions in the farnesyl residue which are susceptible
towards oxidation. In contrast, our compound lack those
sensitive structures (Fig. 3).
In a previous study on a series of non-prenylic farnesyl-
pyrophosphate analogue FTase inhibitors, we had iden-
ti®ed the 4-benzyloxycinnamoyl moiety (8) as a useful
farnesyl surrogate.10 The structure of the natural product
FPP-analogue FTase inhibitor chaetomellic acid (6)
(Fig. 4) suggests that long chain alkyl residues may be
suitable substituents for the farnesyl residue.11 There-
fore, the palmitoyl (7) and the 4-benzyloxycinnamoyl (8)
moieties were fused with AA- and AAX-mimetic partial
structures of several CAAX-peptidomimetic FTase
inhibitors through a b-alanyl (9) linker. We used the 2,3-
dimethylphenylamides of isoleucine (10),12 3-amino-
benzoic acid (11)13 and 3-aminobenzenesulfonic acid
(12),14 respectively, as AA-mimetic substructures and N-
(4-aminobenzoyl)methionine methyl ester (13)15 and N-
(4-aminophenylsulfonyl)methionine methyl ester (14)14
as AAX-mimetic substructures. The b-alanyl moiety (9)
was chosen as a linker because the distance between its
carbonyl carbon and its nitrogen resembles the distance
between the carbonyl carbon and the thiol sulfur in
cysteine.
Only a few bisubstrate analogues (or product analogues)
have been published. Recently, the natural product pep-
ticinnamine E 1 has been shown to act as a bisubstrate
inhibitor.5 In addition to this sole molecule, few synthetic
bisubstrate inhibitors had been published. Representative
examples of which (2±5)6 8 are shown in Figure 2.
Inhibitor design
All synthetic bisubstrate analogues (e.g. 2±5) contain an
unmodi®ed AAX tripeptide motif and the farnesyl resi-
due. These inhibitors only signi®cantly di€er in the nature
of the linker connecting the tripeptide and the farnesyl
residue. We intended to develop a novel class of farnesyl-
transferase inhibitors designed to be bisubstrate analogues
which di€er from the known ones in lacking prenylic or
peptidic substructures. In a ®rst approach we replaced
the AAX-peptide by several peptidomimetics yielding
homofarnesosyl-substituted CAAX-peptidomimetics.9a
In a second step, we also replaced the homofarnesoyl
residue by non-prenylic lipophilic structures thereby
yielding novel type inhibitors of the farnesyltransferase.9b
These novel, fully synthetic compounds are con-
veniently composed of three modular building blocks:
(1) a farnesyl-mimetic (2) a linker and (3) an AAX- or
AA-peptidomimetic. While the compounds are intended
to be non-peptidic, non-prenylic bisubstrate analogue
Chemistry
For the preparation of the target structures 18±22 a
convergent strategy was chosen. The corresponding
amines of the peptidomimetic partial structures 10±14
and the N-Acyl-b-alanine derivatives 17 (Table 1) were
prepared independently and joined in the last step of the

M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
1993
Figure 2. Literature known bisubstrate analogues.
synthesis. The N-acyl-b-alanine derivatives 17a±o were
prepared by additition of the appropriate acid chlorides
15 to b-alanine 16 (Scheme 1) following a known pro-
cedure.16 The cinnamic acid chlorides used in this reac-
tion were prepared in two steps from commercially
available aldehydes and malonic acid.17 2-(3-Phen-
oxybenzyloxy) acetic acid was obtained from 2-chloro-
acetic acid and 3-phenoxybenzyl alcohol.18 The N-acyl-
b-alanine derivatives 17 were activated as mixed anhy-
drides using iso-butyl chloroformate and coupled to the
appropriate amines (26, 29, 32a±i, 37 and 38) to yield
the target structures 18±22 (Scheme 1). The amines
employed in this reaction were prepared as follows. N-
Boc-isoleucine (24) was activated as mixed anhydride
and reacted with 2,3-dimethylaniline to the correspond-
ing amide (25), which was then deprotected with
hydrochloric acid to the corresponding hydrochloride
26 prior to coupling with the N-acyl-b-alanines 17c and
17i. 3-Nitro-N-(2,3-dimethylphenyl)benzamide (28) was
prepared from 3-nitrobenzoyl chloride 27 and 2,3-
dimethylaniline and then reduced to the corresponding
3-amino derivative 29 using tin(II)chlonde in re¯uxing
ethyl acetate.19 N-Substituted 3-aminobenzenesulfon-
amides 32a±g were prepared in the same way from 3-
nitrobenzenesulfonyl chloride (30) and the appropriate
amines. The N-substituted methionine derivatives 37
and 38 were prepared in the same manner from 4-
nitrobenzoyl chloride (33) and 4-nitrobenzenesulfonyl
chloride (34), respectively, and methionine methyl ester
hydrochloride. (Scheme 2).
Farnesyltransferase inhibition assay
The inhibitory activity of the potential synthetic inhibi-
tors was determined using the ¯uorescence enhancement
assay as described by Pompliano.20 The assay employed
yeast farnesyltransferase (FTase) fused to Glutathione S-
transferase at the N-terminus of the b-subunit.21 Yeast
farnesyltransferase is a close homologue and functionally
similar to the human enzyme; it is widely used for the
evaluation of farnesyltransferase inhibitors. Farnesyl-
pyrophosphate and the dansylated pentapeptide Ds-Gly-
CysValLeuSer were used as substrates. Upon farnesylation
of the cysteine thiol the dansyl residue is placed in a
lipophilic environment which results in an enhancement
of ¯uorescence at 505 nm which is used to monitor the
enzyme reaction.
Antiproliferation assay
The target compounds were tested against cell lines K-562
(chronic myeloid leukaemic cell line),22 THP-l (acute

1994
M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
Figure 3. The novel non-peptidic, non-prenylic inhibitors are composed of three building blocks.
Figure 4. Chaetomellic acid.
monocytic leukaemic cell line)23 and HL-60 (acute myeloid
leukaemic cell line)24 for their antiproliferative e€ects.
The cells were incubated with di€erent concentrations
of the test compounds and an automatic volumetric cell
analysis was carried out during the logarithmic phase of
cell proliferation.
Results and Discussion
Scheme 1. (I) (a) potassium carbonate, acetone/water, 0 ꢀC, 1 h, then
In the ®rst two series of compounds, the palmitoyl (7) and
the 4-benzyloxycinnamoyl (8) moieties were used as non-
prenylic farnesyl surrogates and connected via the b-ala-
nyl (9) spacer to the AA- or AAX-mimetic substructures
10±14 of several CAAX-peptidomimetic FTase inhibi-
tors yielding compounds 18a±e and 19a±e (Chart 1,
Table 2). All but two of the compounds showed farne-
syltransferase inhibitory activity in the low micromolar
range. Comparison of the palmitoyl derivatives 18 to
the respective 4-benzyloxycinnamoyl derivatives 19
revealed in all cases a higher inhibitory activity of the
palmitoyl derivatives. Furthermore, the amide derivatives
were more active than the corresponding sulfonamides,
rt, 1 h, (b) concentrated HCl; (II) (a) i-BuOCOCl, NMM, DMF,
15 ꢀC, 5 min; (b) 26, NMM or 29 or 32a±i or 37 or 38, DMF,
15 ꢀC!rt, overnight.
for example, 18a was three times more active than 18b
or 18c being twice as active as 18d. While the 4-benzyl-
oxycinnamoyl derivatives of isoleucineamide (19e) and
N-benzenesulfonylmethionine methyl ester (19b) showed
a considerable lower inhibitory activity, all derivatives of
the remaining three peptidomimetic substructures dis-
played inhibitory activities in the same range of magnitude.
Therefore, lead structures for early investigation on

M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
1995
Scheme 2. (I) (a) i-BuOCOCl, NMM, DMF, 15 ꢀC, 5 min, (b) 2,3-dimethylaniline, DMF, 15 ꢀC!rt, overnight; (II) HCl/dioxane, rt, 2 h; (III)
2,3-dimethylaniline, NMM, dichloromethane, 0 ꢀC!rt, overnight; (IV) SnCl2Â2H2O, ethyl acetate, re¯ux, 2 h; (V) R-NH2, NMM, dichloro-
methane, 0 ꢀC!rt, overnight; (VI) methionine methyl ester hydrochloride, NMM, dichloromethane, 0 ꢀC!rt, overnight.
Table 1. N-Acyl-b-alanines 17
Table 2. Farnesyltransferase inhibitory activity of compounds 18a±e
and 19a±e
Compound
R-CO-
Compound
IC50ÆSD (mM)
Compound
IC50ÆSD (mM)
17a
17b
17c
17d
17e
17f
17g
17h
17i
Lauroyl (C12:0)
Myristoyl (C14:0
Palmitoyl (C16:0
Margarinoyl (C17:0
Stearoyl (C18:0
Arachidoyl (C20:0
Elaidoyl (C18:1 (9t)
Oleoyl (C18:1 (9c)
Linoleoyl (C18:2 (9c,12c)
)
)
18a
18b
18c
18d
18e
3.7Æ0.7
13.0Æ3.5
5.6Æ0.3
9.8Æ2.6
7.8Æ0.3
19a
19b
19c
19d
19e
7.0Æ1.3
52.8Æ4.8
8.1Æ0.3
)
)
14.0Æ0.6
29.2Æ1.6
)
)
)
)
the palmitoyl residue resulted in an approximately 3-
fold loss in inhibitory activity. The C-14 myristoyl
derivative 20b and the C-17 margarinoyl derivative 20c
displayed nearly the same activity as the C-16 palmitoyl
substituted lead structure 18d. In contrast, compound 20d,
carrying the stearyl moiety which is by two methylene
units longer than the palmitoyl residue, was 4-fold more
active then the palmitoyl derivative (IC50=2.5mM). With
this compound, we reached the peak in activity in this
series since the compound 20e, carrying the C-20 arachi-
doyl residue displayed the same activity as the lead
structure 18d. The C-18 chain was used to explore the
in¯uence of the presence and the con®guration of double
bonds in the alkyl chain (compounds 20f±h). In any
case, the presence of double bond in the alkyl chain
caused a moderate decrease in inhibitory activity. This
17j
4-Benzyloxycinnamoyl
4-(2-Phenylethenyl)cinnamoyl
4-Phenylcinnamoyl
(4-Benzyloxyphenyl)oxyacetyl
(3-Phenyloxybenzyl)oxyacetyl
1-Naphthylacetyl
17k
171
17m
17n
17o
structure±activity relationship were choosen from these
compounds.
With our ®rst series of compounds we intended to inves-
tigate the in¯uence of the nature of the long chain fatty
acid on the inhibitory potency of these compounds using
18d as a lead (Table 3). Usage of the four methylene units
shorter C-12 lauroyl moiety (compound 20a) instead of

1996
M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
Table 3. In¯uence of the nature of the long chain fatty acid on the
inhibitory potency
Compound
R-COÐ
IC50ÆSD (mM)
18d
20a
20b
20c
20d
20e
20f
Palmitoyl (C16:0
)
9.8Æ2.6
31.1Æ1.1
12.4Æ0.7
12.7Æ0.9
2.5Æ0.8
9.9Æ2.7
13.4Æ0.4
8.7Æ0.8
3.8Æ0.3
Lauroyl (C12:0
Myristoyl (C14:0
Margarinoyl (C17:0
Stearoyl (C18:0
Arachidoyl (C20:0
Elaidoyl (C18:1 (9t)
Oleoyl (C18:1 (9c)
Linoleoyl (C18:2 (9c,12c)
)
)
)
)
)
)
20g
20h
)
)
Table 4. In¯uence of the nature of the N-substituent on the inhibi-
tory potency
Compound
-R
IC50ÆSD (mM)
18d
21a
21b
21c
21d
21e
21f
2,3-Dimethylphenyl
1-Naphthyl
Benzyl
2,3-Dichlorobenzyl
1-Naphthylmethyl
2-Phenylethyl
2-(3-Lindolyl)ethyl
iso-Pentyl
9.8Æ2.6
4.3Æ0.1
15.0Æ0.2
24.0Æ4.0
10.7Æ1.4
9.7Æ0.4
8.1Æ0.5
11.2Æ1.7
7.9Æ1.0
21g
21h
n-Docecyl
compound 21a. The benzylamides 21b and 21c were
considerably less active than the lead 18d, while the 1-
naphthylmethyl derivative 21d displayed almost the same
activity as 18d. No signi®cant changes were observed
upon the replacement of the 2,3-dimethylphenyl residue
in 18d by phenethyl (21e), 3-indolylethyl (21f), iso-pentyl
(21g) or dodecanyl (21h).
Chart 1. Compounds 18a±c and 19a±e.
e€ect was most pronounced with the E-con®gurated
double bond, present in the elaidoyl derivative 20e,
whereas inversion of the E- into the Z-con®guration
(oleoyl derivative 20f) attenuated the double bond e€ect.
Introduction of a second Z-con®gurated double bond
resulted in a 2-fold more active compound (linoleoyl
derivative 20h) whose IC50 of 3.8 mM was still slightly
higher than that of 20d leaving the stearoyl derivative as
the most active in this series.
A more pronounced sensitivity towards changes in
molecular geometry was observed upon variation of the
aromatic farnesyl surrogate in compound 19d (Table 5).
Replacement of the oxymethylene bridge connecting the
two phenyl residues in 19d by an E-con®gurated vinyl
bridge in 22a yielded a strongly ¯uorescent compound.
Only at a concentration as low as 1 mM could a reliable
value (9%) of its farnesyltransferase inhibition be
obtained, indicating an IC50 well above 15 mM thereby
representing no improvement over the lead structure.
However, total omission of the bridge between the two
aromatic residues in the farnesyl surrogate as in the
biphenyl derivative 22b resulted in a 4-fold improvement
of the inhibitory activity. Replacement of the E-con-
®gurated vinyl bridge connecting the phenyl residue and
Compound 18d was also chosen as a lead for the investi-
gation of the in¯uence of the N-substituent of the sulfona-
mide group of the terminal portion of the peptidomimetic
substructure (Table 4). A 2-fold improvement of inhibi-
tory activity was observed upon the repacement of the
2,3-dimethylphenyl residue by a 1-naphthyl group as in

M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
Table 5. In¯uence of the nature of the arylic acyl substituent on the
inhibitory potency
1997
Compound
R-CO-
IC50ÆSD (mM)
19
4-Benzyloxycinnamoyl
4-(2-Phenylethenyl)cinnamoyl
4-Phenylcinnamoyl
(4-Benzyloxyphenyl)oxyacetyl
(3-Phenyloxybenzyl)oxyacetyl
1-Naphthylacetyl
14.0Æ0.6
9% inh. at 1 mMa
3.4Æ0.4
22a
22b
22c
22d
22e
215Æ25
126Æ4
65.8Æ5.8
aDue to high ¯uorescence emission of 22a higher inhibitor concentrations
could not be determined.
Figure 5. Combination of the N-benzoylmethionine peptidomimetic
substructure of 18a and the stearoyl residue of 20d.
Figure 6. (a) Compond 18a docked into the farnesyltransferase's
active site by FlexX. Colors: protein surface: cyan; zinc: magenta; N-
Ac-Cys-Val-Ile-selenoMetOH: green; FPP: yellow; 18a: carbon: white,
nitrogen: blue, oxygen: red. (b) Interactions between 18a and amino
acid side chains of the active site as calculated by FlexX. Colors:
nitrogen: blue, oxygen: red, carbon of amino acid side chains: white,
carbon of 18a: green blue.
the carbonyl group in 19d by a conformationally more
¯exible oxymethylene bridge (compound 22c) was detri-
mental to activity, thereby stating the importance of an
E-con®gurated double bond in this position of the mole-
cule. Almost inactive compounds were also obtained
with the use of a (3-phenyloxybenzyl)oxyacetyl (com-
pound 22d) and a l-naphthylacetyl (compound 22e)
residue as farnesyl surrogates.
placed 18a into the farnesyltransferase's active site in a
way that it occupies considerable portions of the peptide
as well as the farnesylpyrophosphate binding regions
thus let it look like a bisubstrate analogue as postulated.
Figure 6b shows interactions found by FlexX between
18a and amino acid side chains in the active site. Inter-
actions are calculated between the methionine side chain
and Tyr 131a. Like the carbonyl group of seleno-methio-
nine in the crystal structure the carbonyl moiety of
methionine of 18a shows a hydrogen bond to Gln 167a
and to the guanidino group of Arg 202b. This side chain
also forms a hydrogen bond to the carbonyl of the central
aminobenzoyl moiety. Another hydrogen bond is
formed between the hydroxy group of Tyr 361b and the
amide nitrogen in the 4-position of the aminobenzoyl
moiety. The docking program calculated an interaction
between the carbonyl oxygen of the amide moiety con-
necting the b-alanyl linker to the palmitoyl residue and
the zinc. The middle portion of the palmitoyl alkyl
chain is placed in 2.8 to 3.8 A distance to the side chains
of Lys 164a, Lys 294b and Arg 291b which form the
positively charged cleft binding the pyrophosphate por-
tion of FPP.25 For the terminal portion of the fatty acid
hydrophobic interactions are found towards Tyr 166a,
Tyr 200a and Tyr 251b which form a part of the prenyl
As described above, replacement of the palmitoyl residue
by a stearoyl moiety in the benzenesulfonamide derivative
18d resulted in a 4-fold improvement in activity. How-
ever, the corresponding N-benzoylmethionine derivative
23 (Fig. 5) carrying a stearoyl instead of a palmitoyl
showed the same activity (IC50=3.8 mM) as the lead 18a.
Using the coordinates of the published25 crystal structure
of a ternary complex of farnesyltransferase, a farnesyl-
pyrophosphate analogue and N-Ac-Cys-Val-lle-seleno-
MetOH, a molecular surface of the farnesyltransferase's
active site was calculated using the program molcad
which is implemented in the molecular modeling soft-
ware package sybyl.26 Compound 18a was docked into
the active site using the ¯exible docking program
FlexX.27 The C-terminal methionine of 18a was used as
starting fragment and placed at the same position as the
seleno-methionine of N-Ac-Cys-Val-Ile-selenoMetOH
in the crystal structure. Then, the program placed the
remainder of the molecule fragment by fragment into
the active site searching for favorable interactions
between the ligand and the amino acid side chains while
avoiding steric overlaps. As shown in Figure 6a, FlexX

1998
M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
Table 6. Antiproliferative activity (GI50 (mM)) of selected com-
pounds
2-(3-Phenoxybenzyloxy)acetic acid chloride 15n. 3-Phe-
noxybenzyl alcohol (1.74 mL, 10 mmol) and 2-chlor-
oacetic acid (480 mg, 10 mmol) were dissolved in dry
THF (60 mL) under Ar. After addition of NaH (880mg of
a 60% suspension in oil, 22mmol) the mixture was heated
to re¯ux for 12 h. Then, the mixture was diluted with
water, acidi®ed to pH 1 with concentrated hydrochloric
acid and extracted with ethyl acetate. An oil was
obtained after removal of the solvent. Yield 2.2 g, 86%.
1H NMR (500 MHz, CDC13) d 4.14 (s, 2H), 4.61 (s, 2H),
6.93±7.01 (m, 4H), 7.07±7.12 (m, 2H), 7.31±7.35 (m,
3H). This oil was dissolved in toluene (30 mL) and after
addition of thionyl chloride (1.72 mL) heated to 80 ꢀC
for 2 h. The residue obtained after removal of the vola-
tiles in vacuo was used for the preparation of 17n with-
out further characterization.
Compound
HL-60
THP-1
K-562
18d
20h
22b
9.0
9.0
9.0
17.6
13.6
9.0
10.8
14.9
9.8
binding cleft.25 Thus, several interactions calculated
between 18a and amino acid side chains of the peptide as
well as the prenylphosphate binding regions provide argu-
ments for our substances being bisubstrate analogues.
Selected compounds were assayed against three di€erent
tumor cell lines (Table 6). Antiproliferative e€ects were
observed in the low micromolar range, thus showing
that that type of compounds is able to penetrate the cell
membrane.
General procedure for the preparation of the N-acyl-ꢀ-
alanines 17a±o. b-Alanine and K2CO3 (430mg per mmol
b-alanine) were dissolved in water (40 mL) and cooled to
0 ꢀC. The appropriate acyl chloride (1 equiv) dissolved in
acetone (40 mL) was added dropwise. Stirring was con-
tinued for 1 h at 0 ꢀC and 1 h at room temperature. Then,
most of the acetone was removed on a rotary evaporator
and the resulting aqueous solution was acidi®ed with
concentrated hydrochloric acid to pH 1. The precipitate
was collected, washed with water and dried. The pro-
ducts were used without further puri®cation.
Conclusion
We have described inhibitors of farnesyltransferase
which mimic structural features of both the peptidic and
the prenylic substrate of the farnesyltransferase and pro-
vided some computational evidence that they could act as
bisubstrate analogues. The compounds are composed of a
peptidomimetic substructure and simple aromatic or
aliphatic lipophilic structures to mimic the farnesyl
moiety. Early structure±activity relationships have been
established which form the basis for future development of
this class of farnesyltransferase inhibitors. Our com-
pounds are much more readily accessible than known
bisubstrate inhibitors from inexpensive starting material.
A particular advantage is the lack of sensitive peptidic
of prenyl substructures.
3-(Tetradecanoylamino)propionic acid (17b). Prepared as
described above from myristoyl chloride (2.46 g, 10mmol)
and b-alanine (890mg, 10mmol). Yield: 2.72g (91)%. 1H
NMR (400 MHz, DMSO-d6) d 0.84 (t, J=7Hz, 3H), 1.23
(m, 20H), 1.45 (m, 2H), 2.00 (m, 2H), 2.48 (m, 2H), 3.21
(m, 2H).
3-(Heptadecanoylamino)propionic acid (17d). Prepared
as described above from heptadecanoic acid chloride
(1.44 g, 5 mmol) and b-alanine (445mg, 5 mmol). Yield:
1.42g (83%); H NMR (400MHz, DMSO-d6) d 0.85 (t,
J=7Hz, 3H), 1.25 (m, 26H), 1.47 (m, 2H), 2.01 (m, 2H),
2.47 (m, 2H), 3.23 (m, 2H).
Our compounds are not only interesting as intermediate
steps towards the development of potential anti-cancer
agents but also as tools for studying the structure of
enzyme-inhibitor interactions.1c,5
1
3-(9-(E)-Octadecenoylamino)propionic acid (17g). Pre-
pared as described above from elaidic acid chloride
(600 mg, 2 mmol) and b-alanine (178 mg, 2 mmol). Yield:
551 mg (78%). 1H NMR (400 MHz, DMSO-d6) d 0.84 (t,
J=7 Hz, 3H), 1.26 (m, 20H), 1.45 (m, 2H), 1.92 (m, 4H),
2.00 (t, J=7 Hz, 2H), 2.31 (t, J=7 Hz, 2H), 3.20 (m, 2H),
5.35 (m, 2H).
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 acquisition system
from Tecnivent or an AutoSpec mass spectrometer from
Micromass. IR spectra were recorded on a Nicolet 510P
FTIR-spectrometer. Microanalyses were obtained from
a CH analyzer according to Dr. Salzer from Labormatic
and from a Hewlett Packard CHN-analyzer type 185
and are within Æ0.4% of the theoretical values. Melting
points were obtained with a Leitz-microscope and are
uncorrected. Column chromatography was carried out
using silica gel 60 (0.062±0.200 mm) from Merck. The
following compounds used as starting materials have
been described in the literature: 15m,28 17a,29 17c,30
17e,31 17f,32 17h,31 17o,33 26,12 29,13 37,34 38,35 32a,13
32g,3632b,37 32i.38 4-Substituted cinnammic acids chlor-
ides 15j±l have been prepared as described.10
3-(9-(Z),13-(Z)-Octadecadienoylamino)propionic acid (17i).
Prepared as described above from linoleic acid chloride
(896mg, 3 mmol) and b-alanine (267mg, 3 mmol). Yield:
1
737 mg (70%). H NMR (400MHz, CDCl3) d 0.86 (t,
J=7Hz, 3H), 1.29 (m, 14H), 1.58 (m, 2H), 2.02 (m, 4H),
2.14 (m, 2H), 2.57 (m, 2H), 2.72 (m, 2H), 3.47 (m, 2H),
5.31 (m, 4H).
3-{[3-(4-Benzyloxyphenyl)acryloyl]amino}propanoic acid
(17j). Prepared as described above from 4-benzyloxy-
cinnamoyl chloride (1.64 g, 6 mmol) and b-alanine
(534mg, 6 mmol). Yield: 1.7 g (87%). 1H NMR (400MHz,

M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
1999
DMSO-d6) d 2.43 (t, 2H, J=7Hz), 3.36 (m, 2H), 5.14 (s,
2H), 6.48 (d, 1H), J=16 Hz), 7.02±7.04 (m, 2H), 7.31±7.55
(m, 8H), 8.03 (t, 1H, J=5.5 Hz).
N-(1-Naphthylmethyl)-3-nitrobenzenesulfonamide (31e).
Prepared as described above from 30 (2.21 g, 10 mmol)
and 1-naphthylmethyl amine (1.57 g, 10 mmol). Yield:
1
2.2 g (64%). H NMR (500 MHz, CDCl3) d 4.62 (m,
3-[3-[4-(2-Phenylethenyl)phenyl]acryloyl]aminopropionic
acid (17k). Prepared as described above from 4-(2-phe-
nylethenyl)cinnamic acid chloride (641 mg, 2.38 mmol)
and b-alanine (211 mg, 2.38 mmol). Yield: 658 mg (86%).
1H NMR (400 MHz, DMSO-d6) d 2.44 (t, J=7 Hz, 2H),
3.37 (m, 2H), 6.64 (d, J=16 Hz, 1H), 7.29 (m, 3H), 7.38
(m, 3H), 7.54 (m, 2H), 7.62 (m, 4H), 8.12 (m, 1H).
2H), 4.94 (s, 1H), 7.20±7.26 (m, 2H), 7.36±7.41 (m, 3H),
7.61 (m, 1H), 7.68 (m, 1H), 7.78 (m, 1H), 7.88 (m, 1H),
8.14 (m, 1H), 8.38 (s, 1H).
3-Nitro-N-(2-phenylethyl)benzenesulfonamide (31f). Pre-
pared as described above from 20 (1.1 g, 5 mmol) and 2-
phenylethyl amine (0.97 mL, 5 mmol). Yield: 930 mg
1
(61%). H NMR (400MHz, CDCl33) d 2.73 (q, J=7Hz,
3-[(3-Biphenylylacryloyl)amino]propanoic acid (17l). Pre-
pared as described above from 4-phenylcinnamoyl chlo-
ride (704mg, 3.16mmol) and b-alanine (280mg, 3.16
mmol). Yield: 660 mg (71%). 1H NMR (400MHz,
DMSO-d6) d 2.48 (m, 2H), 3.40 (m, 2H), 6.68 (d,
J=16 Hz, 1H), 7.37 (m, 1H), 7.48 (m, 3H), 7.64 (m, 2H),
7.69±7.74 (m, 4H), 8.16 (m, 1H), 12.25 (s, 1H).
2H), 3.24 (m, 2H), 4.58 (t, J=6Hz, 1H), 7.00 (m, 2H), 7.18
(m, 3H), 7.64 (m, 1H), 8.01 (m, 1H), 8.32 (m, 1H), 8.51
(m, 1€).
N-(3-Methylbutyl)-3-nitrobenzenesulfonamide (31h). Pre-
pared as described above from 30 (1.1 g, 5 mmol) and iso-
pentyl amine (0.58 mL, 5 mmol). Yield: 800 mg (59%).
1H NMR (400 MHz, CDCl3) d 0.78 (d, J=7 Hz, 6H),
1.31 (q, J=7 Hz, 2H), 1.53, (m, 1H), 2.97 (q, J=7 Hz,
2H), 4.63 (t, J=6 Hz, 1H), 7.69 (m, 1H), 8.14 (m, 1H),
8.37 (m, 1H), 8.64 (m, 1H).
3-{[(4-Benzyloxyphenyl)oxyacetyl]amino}propanoic acid
(17m). Prepared as described above from (4-benzyloxy-
phenyl)oxyacetic acid chloride (1.37 g, 4.94 mmol) and
1
b-alanine (440 mg, 4.94 mmol). Yield: 1.3 g (80%). H
NMR (400 MHz, DMSO-d6) d 2.42 (m, 2H), 3.33 (m,
2H), 4.36 (s, 2H), 5.03 (s, 2H), 6.84±6.95 (m, 4H), 7.26±
7.43 (m, 5H), 8.02 (s, 1H).
General procedure for the preparation of the 3-amino-
benzenesulfonamides 32. To a solution of the nitro com-
pound (31) in ethyl acetate SnCl2ÂH2O (1.125 g per
mmol nitro compound) was added. Then, the solution
was re¯uxed for 2 h. The cooled solution was diluted
with water and the pH was adjusted to 7±8 by addition
of satd NaHCO3-solution. The aqueous phase was
extracted with ethyl acetate (3Â100±200 mL) and the
combined organic extracts were thoroughly washed with
brine and dried over MgSO4. The products obtained
after removal of the solvent were used without further
puri®cation.
3-{[(3-Phenyloxybenzyl)oxyacetyl]amino}propanoic acid
(17n). This compound was prepared as described above
from 2-(3-phenoxybenzyloxy)acetic acid chloride (2.37 g,
8.6 mmol) and b-alanine (765 mg, 8.6 mmol). Yield:
1.52 g (54%); oil. 1H NMR (500 MHz, CDCl3) d 2.51 (t,
2H, J=6 Hz), 3.48 (q, 2H, J=6 Hz), 3.91 (s, 2H), 4.45
(s, 2H), 6.85±6.96 (m, 4H), 7.01±7.05 (m, 2H), 7.21±7.28
(m, 3H).
General procedure for the preparation of 3-nitrobenzene-
sulfonamides 31. To a solution of the amine in dry
dichloromethane and N-methylmorpholine [NMM]
(0.24mL per mmol amine) 3-nitrobenzenesulfonyl chlo-
ride (30) (220 mg, per mmol amine) was added at 0 ꢀC.
Stirring was continued overnight. The reaction mixture
was diluted with dichloromethane and washed succes-
sively with 2 N citric acid, satd NaHCO3-soln and brine
and dried with MgSO4. The products obtained after
removal of the solvent were used without further pur-
i®cation.
3-Amino-N-benzylbenzenesulfonamide (32c). Prepared as
described above from 31c (0.900g, 3.1 mmol). Yield:
420 mg (52%). H NMR (400MHz, CDC13) d 3.84 (s,
2H), 4.06 (d, J=6Hz, 2H), 4.56 (t, J=6Hz, 1H), 6.79 (m,
1H), 7.08 (m, 1H), 7.12±7.17 (m, 3H), 7.19±7.25 (m, 4H).
1
3-Amino-N-(2,3-dichlorobenzyl)benzenesulfonamide (32d).
Prepared as described above from 31d (670 mg,
1.86 mmol). Yield: 615 mg (quant). 1H NMR (500 MHz,
CDCl3) d 3.88 (s, 2H), 4.25 (d, J=6 Hz, 2H), 5.04 (t,
J=6 Hz, 1H), 6.77 (m, 1H), 7.06 (m, 1H), 7.10±7.14 (m,
2H), 7.18±7.24 (m, 2H), 7.34 (m, 1H).
Preparation of N-benzyl-3-nitrobenzenesulfonamide (31c).
Prepared as described above from 30 (1.1 g, 5 mmol)
and benzyl amine (0.55 mL, 5 mmol). Yield: 905 mg
(62%). H NMR (400 MHz, CDCl3) d 4.24 (d, J=6 Hz,
2H), 5.01 (t, J=6 Hz, 1H), 7.16 (m, 2H),7.22 (m, 3H),
7.66 (m, 1H), 8.12 (m, 1H), 8.36 (m, 1H), 8.59 (m, 1H).
3-Amino-N-(1-naphthylmethyl)benzenesulfonamide (32e).
Prepared as described above from 31e (2.2 g, 6.43mmol).
Yield: 1.73g (86%). 1H NMR (500 MHz, CDCl3):
d=3.84 (s, 2H), 4.48 (m, 2H), 6.80 (m, 1H), 7.10 (m, 1H),
7.18±7.31 (m, 4H), 7.42 (m, 2H), 7.72 (m, 1H), 7.77 (m,
1H), 7.84 (m, 1H).
1
N-(2,3-Dichlorobenzyl)-3-nitrobenzenesulfonamide (31d).
Prepared as described above from 30 (607mg, 2.75 mmol)
and 2,3-dichlorobenzyl amine (485mg, 2.75 mmol). Yield:
3-Amino-N-(2-phenylethyl)benzenesulfonamide (32f). Pre-
pared as described above from 31f (0.826 g, 2.7 mmol).
Yield: 524 mg (70%). 1H NMR (400MHz, CDCl3):
d=2.75 (q, J=7Hz, 2H), 3.20 (m, 2H), 3.87 (s, 2H), 4.31
(t, J=7Hz, 1H), 6.82 (m, 1H), 7.08 (m, 3H), 7.12 (m, 1H),
7.25 (m, 4H).
1
690 mg (70%). H NMR (500MHz, CDCl3) d 4.32 (d,
J=6 Hz, 2H), 5.28 (t, J=6 Hz, 1H), 7.04 (m, 1H), 7.18
(m, 1H), 7.22 (m, 1H), 7.56 (m, 1H), 7.96 (m, 1H), 8.25
(m, 1H), 8.40 (m, 1H).

2000
M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
3-Amino-N-(3-methylbutyl)benzenesulfonamide (32h). Pre-
pared as described above from 31h (0.79 g, 2.9 mmol).
Yield: 600 mg (85%). 1H NMR (400MHz, CDCl3): d 0.77
(d, J=7Hz, 6H), 1.33 (m, 2H), 1.51, (m, 1H), 2.94 (m,
2H), 3.88 (s, 2H), 4.47 (m, 1H), 6.83 (m, 1H), 7.14±7.25
(m, 3H).
N-{2-[3-(2,3-Dimethylphenylaminocarbonyl)phenylamino-
carbonyl]ethyl}hexadecanoic acid amide (18c). Prepared
as described above from 17c (327 mg, 1 mmol) and 29
(240mg, 1 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield: 451 mg (82%); mp
107 ꢀC. IR (KBr) n 3300, 2920, 2850, 1640cm 1. 1H NMR
(400 MHz, DMSO-d6) d 0.84 (m, 3H), 1.13±1.29 (m,
24H), 1.45 (m, 2H), 2.00 (m, 2H), 2.09 (s, 3H), 2.27 (s,
3H), 2.48 (m, 2H), 3.31 (m, 2H), 7.05±7.11 (m, 3H), 7.41
(m, 1H), 7.63 (m, 1H), 7.82 (m, 2H), 8.11 (m, 1H), 9.82 (s,
1H), 10.05 (s, 1H); 13C NMR (125MHz, DMSO-d6) d
13.9, 14.2, 20.1, 22.1, 25.2, 25.3, 28.7, 28.8, 28.9, 29.0, 31.3,
34.1, 34.8, 35.0, 35.4, 36.5, 118.8, 121.8, 121.9, 124.6,
125.2, 127.5, 128.6, 132.6, 134.3, 134.9, 135.3, 139.4, 165.4,
169.7, 172.2. MS (EI) m/z 549(M+), 126, 113. Anal.
calcd for C34H51N3O3: C, 74.28; H, 9.35; N, 7.64; found
C, 74.34; H, 9.53; N, 7.81.
General procedure for the preparation of the N-acyl-ꢀ-
alanine amides 18±22. The appropriate N-acyl-b-alanine
17 was dissolved in a sucient amount of dry DMF in a
¯ame dried ¯ask under an atmosphere of Ar. After
addition of N-methylmorpholine [NMM] (0.25 mL per
mmol acid) the solution was cooled to 15 ꢀC and iso-
butyl chloroformate (0.13 mL per mmol acid) was
added. A solution of the amine component (1 equiv) in
dry DMF was added after 5 min. When 26 was used as
the amine component, additional NMM (0.25 mL per
mmole) was added. The mixture was allowed to warm
up to room temperature overnight and then poured into
brine (400±800 mL). In case a solid precipitate was
formed, this was collected by suction and thoroughly
washed with water. Otherwise, the aqueous mixture
was extracted with EtOAc (3Â100 mL) and the com-
bined organic extracts were washed successively with
2N citric acid, satd NaHCO3-soln and brine and dried
with MgSO4. The residue obtained after removal of
the solvent was puri®ed by recrystallisation or ¯ash
chromatography.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}hexadecanoic acid amide (18d). Prepared
as described above from 17c (490mg, 1.5 mmol) and 32a
(414mg, 1.5 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield: 600 mg (68%); mp
145 ꢀC. IR (KBr) n 3350, 1695, 1640, 1320, 1160cm 1. 1H
NMR (500 MHz, DMSO-d6) d 0.84 (t, 3H, J=7Hz),
1.18±1.22 (m, 24H), 1.44 (m, 2H), 1.96 (s, 3H), 2.01 (t, 2H,
J=7Hz), 2.16 (s, 3H), 2.48 (m, 2H), 3.29 (m, 2H), 6.70 (m,
1H), 6.92 (m, 1H), 6.98 (m, 1H), 7.27 (m, 1H), 7.43 (m,
1H), 7.77 (m, 1H), 7.82 (m, 1H), 8.05 (s, 1H), 9.49 (s, 1H),
10.16 (s, 1H); 13C NMR (125MHz, DMSO-d6) d 13.8,
13.9, 20.0, 21.9, 25.2, 28.5, 28.7, 28.9, 31.1, 34.8, 35.2, 36.3,
116.8, 120.9, 122.3, 124.5, 125.2, 127.9, 129.2, 133.6, 134.5,
137.3, 139.5, 141.1, 169.8, 171.1. MS (EI) m/z 585 (M+),
310, 120. Anal. calcd for C33 H51N3O4S: C, 67.66; H, 8.77;
N, 7.17; found C, 67.29; H, 8.43; N, 7.38.
N-{4-[(3-Hexadecanoylamino)propanoylamino]benzoyl}-
methionine methyl ester (18a). Prepared as described
above from 17c (654 mg, 2 mmol) and 37 (564 mg,
2 mmol) and puri®ed by ¯ash chromatography (ethyl
acetate). Yield: 917 mg (78%); mp 141 ꢀC. IR (KBr) n
1
3295, 2920, 2850, 1750, 1660, 1640 cm
.
1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J=7 Hz, 3H), 1.15±1.19
(m, 24H), 1.46 (m, 2H), 1.97±2.08 (m, 7H), 2.52±2.58
(m, 4H), 3.32 (m, 2H), 4.64 (s, 3H), 4.65 (m, 1H), 7.65±
7.68 (m, 2H), 7.82±7.84 (m, 2H), 8.27 (s, 1H), 8.55 (m,
1H), 10.10 (s, 1H). 13C NMR (100 MHz, DMSO-d6) d
13.7, 14.4, 21.9, 25.2, 28.7, 28.9, 29.2, 29.8, 30.1, 31.1,
34.7, 34.8, 35.2, 36.4, 51.5, 51.7, 117.9, 127.8, 128.2,
141.8, 165.9, 169.7, 172.0, 172.3. MS (EI) m/z 591 (M+),
518, 517, 310. Anal. calcd for C32H53N305S: C, 64.94; H,
9.03; N, 7.10; found: C, 65.00; H, 9.01; N, 7.37.
Nꢁ-3-(Hexadecanoylamino)propanoyl-N-(2,3-dimethyl-
phenyl)isoleucine amide (18e). Prepared as described
above from 17c (491 mg, 1.5 mmol) and 26 (351 mg,
1.5 mmol) and recrystallized from dioxane. Yield: 395 mg
(48%); mp 201 ꢀC. IR (KBr) n 3275, 2920, 1640cm 1. 1H
NMR (500 MHz, DMSO-d6) d 0.84±0.91 (m, 9H), 1.22 (m,
25H), 1.46 (m, 3H), 2.00 (m, 3H), 2.04 (s, 3H), 2.23 (s,
3H), 2.50 (m, 2H), 3.56 (m, 2H), 4.35 (m, 1H), 7.00 (m,
3H), 7.08 (m, 2H). MS (EI) m/z 543, 525, 311, 310 (M+).
Anal. calcd for C33H57N3O3 C, 72.88; H, 10.56; N, 7.73;
found: C, 72.83; H, 10.33; N, 8.10.
N-{4-[(3-Hexadecanoylamino)propanoylamino]phenylsul-
fonyl}methionine methyl ester (18b). Prepared as descri-
bed above from 17c (392 mg, 1.2 mmol) and 38 (382 mg,
1.2 mmol) and puri®ed by ¯ash chromatography (ethyl
acetate). Yield: 32 mg (4%); mp 142 ꢀC. IR (KBr) n
3320, 2920, 1735, 1685, 1645 cm 1. 1H NMR (400 MHz,
DMSO-d6) d 0.84 (t, J=7 Hz, 3H), 1.14±1.26 (m, 24H),
1.44 (m, 2H), 1.69±1.84 (m, 2H), 1.92 (s, 3H), 2.00 (m,
2H), 2.25±2.41 (m, 2H), 2.51 (m, 2H), 3.29 (m, 2H), 3.91
(m, 1H), 7.64±7.67 (m, 2H), 7.72±7.75 (m, 2H), 7.83 (m,
1H), 8.16 (m, 1H), 10.23 (m, 1H); 13C NMR (100 MHz,
DMSO-d6) d 13.8, 14.3, 21.9, 25.2, 28.5, 28.7, 28.9, 29.0,
31.1, 31.3, 34.8, 35.2, 36.5, 51.7, 54.2, 118.5, 127.4,
134.6, 142.6, 170.0, 171.4, 172.2. MS (EI) m/z 627 (M+),
402, 310, 162, 93. Anal. calcd for C31H53N3O6S2: C,
59.30; H, 8.51; N, 6.69; found: C, 59.31; H, 8.48; N,
6.48.
N-{4-{3-[3-(4-Benzyloxyphenyl)acryloylamino]propanoyl-
amino}benzoyl}methionine methyl ester (19a). Prepared
as described above from 17j (325 mg, 1 mmol) and 37
(282 mg, 1 mmol). Yield: 310 mg (53%); mp 218 ꢀC. IR
1
(KBr) n 1745, 1660, 1635 cm 1. H NMR (400 MHz,
DMSO-d6) d 2.03±2.05 (m, 5H), 2.49±2.61 (m, 4H), 3.35
(m, 2H), 3.64 (s, 3H), 4.57 (m, 1H), 5.13 (s, 2H), 6.48 (d,
1H, J=16 Hz), 7.02±7.04 (m, 2H), 7.36±7.49 (m, 7H),
7.67±7.71 (m, 2H), 7.81±7.85 (m, 2H), 8.04 (m, 1H), 8.58
(d, 1H, J=8Hz), 10.24 (s, 1H); 13C NMR (100MHz,
DMSO-d6) d 14.5, 29.9, 30.1, 36.4, 38.9, 51.6, 51.8, 114.9,
115.1, 118.1, 119.9, 127.5, 127.9, 128.2, 128.3, 128.5, 128.9,
136.7, 138.0, 143.0, 159.2, 165.1, 166.0, 169.9, 172.4. MS
(EI) m/z 92, 91. Anal. calcd for C32H35N3O6S: C, 65.18;
H, 5.98; N, 7.13; found: C, 65.48; H, 5.72; N, 6.78.

M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
2001
N-{4-{3-[3-(4-Benzyloxyphenyl)acryloylamino]propanoyl-
amjno}phenylsulfonyl}methionine methyl ester (19b).
Prepared as described above from 17j (489 mg, 1.5
mmol) and 38 (477 mg, 1.5 mmol) and puri®ed by ¯ash
chromatography (ethyl acetate). Yield: 497 mg (53%);
mp 194 ꢀC. IR (KBr) n 3285, 1740, 1655, 1600 cm 1. 1H
NMR (400 MHz, DMSO-d6) d 1.68±1.82 (m, 2H), 1.92
(s, 3H), 2.25±2.27 (m, 2H), 2.52Ð2.54 (m, 2H), 3.35 (m,
2H), 3.42 (s, 3H), 3.90 (m, 1H), 5.12 (s, 2H), 6.46 (d,
J=16 Hz, 1H), 7.01±7.04 (m, 2H), 7.30±7.38 (m, 3H),
7.40±7.48 (m, 3H), 7.64±7.67 (m, 2H), 7.73±7.76 (m,
2H), 8.00 (m, 1H), 8.19 (m, 1H), 10.28 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d 14.3, 29.0, 31.3, 34.8, 35.3, 36.4,
51.8, 54.2, 69.2, 115.1, 118.5, 119.8, 127.4, 127.6, 127.8,
128.3, 128.9, 134.6, 136.7, 138.0, 142.4, 159.3, 165.1,
170.1, 170.3, 171.4. MS (EI) m/z 625 (M+), 325, 162, 147,
91. Anal. calcd for C31H35N3O7S2: C, 59.50; H, 5.64; N,
6.72; found: C, 59.45; H, 5.34; N, 6.44.
(m, 3H), 0.93 (m, 3H), 1.19 (m, 1H), 1.55 (m, 1H), 1.85
(m, 1H), 2.05 (s, 3H), 2.23 (s, 3H), 2.37 (t, 1H, J=7 Hz),
2.43 (t, 1H, J=7 Hz), 3.36 (m, 2H), 4.37 (m, 1H), 5.14
(s, 2H), 6.48 (d, 1H, J=16 Hz), 6.97±7.09 (m, 5H), 7.31±
7.88 (m, 8H), 8.00 (m, 1H), 8.06 (m, 1H), 9.42 (s, 1H);
13C NMR (100 MHz, DMSO-d6) d 10.9, 13.8, 15.4, 19.9,
24.4, 35.1, 35.4, 36.4, 57.3, 69.2, 115.0, 115.1, 119.9,
123.5, 124.9, 126.8, 127.5, 127.7, 128.3, 128.9, 131.2,
135.8, 136.7, 137.9, 159.2, 165.1, 170.2, 170.5; MS (ESI)
m/z 542 ([M+H]+), 564 ([M+Na]+). HRMS calcd for
C33H40N3O4: 542.3019; found: 542.3075. Anal. calcd for
C33H39N3O4: C, 73.17; H, 7.26; N, 7.76; found: C,
72.82; H, 7.57; N, 7.44.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl}ethyl}dodecanoic acid amide (20a). Prepared as
described above from 17a (407mg, 1.5 mmol) and 32a
(414mg, 1.5 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield: 300 mg (38%); mp
172 ꢀC. IR (KBr) n 3350, 3240, 1695, 1640, 1320,
3-(4-Benzyloxyphenyl)-N-{2-[3-(2,3-dimethylphenylamino-
carbonyl)phenylaminocarbonyl]ethyl}acrylamide
1
(19c).
1160 cm 1. H NMR (500 MHz, DMSO-d6) d 0.84 (t,
Prepared as described above from 17j (490 mg, 1.5
mmol) and 29 (360 mg, 1.5 mmol) and puri®ed by ¯ash
chromatography (ethyl acetate:n-hexane, 3:2). Yield:
3H, J=7 Hz), 1.19±1.26 (m, 16H), 1.45 (m, 2H), 1.96 (s,
3H), 2.02 (t, 2H, J=7 Hz), 2.16 (s, 3H), 2.49 (m, 2H),
3.30 (m, 2H), 6.71 (m, 1H), 6.95 (m, 1H), 7.00 (m, 1H),
7.28 (m, 1H), 7.44 (m, 1H), 7.78 (m, 1H), 7.83 (m, 1H),
8.06 (s, 1H), 9.50 (s, 1H), 10.17 (s, 1H); 13C NMR
(125 MHz, DMSO-d6) d 13.8, 13.9, 20.0, 22.0, 25.2, 28.5,
28.6, 28.7, 28.8, 28.85, 28.9, 31.2, 34.8, 35.3, 36.4, 116.8,
120.9, 122.4, 124.6, 125.2, 127.9, 129.3, 133.6, 134.5, 137.4,
139.6, 141.1, 169.8, 172.2. MS (EI) m/z 529 (M+), 254,
120. Anal. calcd for C29H43N3O4S: C, 65.75; H, 8.18; N,
7.93; found: C, 65.61; H, 7.80; N, 7.66.
630 mg (77%); mp 183 ꢀC. IR (KBr) n 3295, 1660, 1605,
1
1510 cm
.
1H NMR (500MHz, DMSO-d6) d 2.09 (s,
3H), 2.28 (s, 3H), 2.56 (t, J=7Hz, 2H), 3.47 (m, 2H), 5.13
(s, 2H), 6.50 (d, J=16 Hz, 1H), 7.02±7.04 (m, 2H), 7.06±
7.12 (m, 2H), 7.30±7.34 (m, 3H), 7.37±7.39 (m, 1H), 7.41±
7.44 (m, 2H), 7.47±7.49 (m, 2H), 7.64 (m, 1H), 7.83 (m,
1H), 8.10 (t, J=6 Hz, 1H), 8.14 (s, 1H), 9.85 (s, 1H), 10.12
(s, 1H); 13C NMR (125MHz, DMSO-d6) d 14.1, 20.0,
35.1, 36.4, 69.2, 115.1, 118.7, 119.8, 121.8, 124.4, 125.1,
127.4, 127.6, 127.8, 128.3, 128.5, 128.9, 132.5, 135.3, 136.7,
136.8, 138.1, 139.2, 159.0, 165.2, 165.3, 169.9. MS (EI) m/z
547 (M+), 249, 174. Anal. calcd for C34H33N3O4: C,
74.59; H, 6.03; N, 7.68; found: C, 74.65; H, 6.14; N, 7.70.
N-(2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}tetradecanoic acid amide (20b). Prepared
as described above from 17b (449 mg, 1.5 mmol) and 32a
(414mg, 1.5 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield 240 mg (29%); mp
1H
170 ꢀC. IR (KBr) n 3350, 1640, 1320, 1160 cm
.
1
3-(4-Benzyloxyphenyl)-N-{2-[3-(2,3-dimethylphenylamino-
sulfonyl)phenylaminocarbonyl]ethyl}acrylamide
(19d).
NMR (500 MHz, DMSO-d6) d 0.85 (t, 3H, J=7 Hz),
1.19±1.26 (m, 20H), 1.45 (m, 2H), 1.97 (s, 3H), 2.02 (t,
2H, J=7 Hz), 2.17 (s, 3H), 2.49 (m, 2H), 3.31 (m, 2H),
6.72 (m, 1H), 6.93 (m, 1H), 7.00 (m, 1H), 7.28 (m, 1H),
7.44 (m, 1H), 7.78 (m, 1H), 7.83 (m, 1H), 8.06 (s, 1H),
9.50 (s, 1H), 10.17 (s, 1H), 13C NMR (125 MHz, DMSO-
d6) d 13.8, 13.9, 20.0, 22.0, 25.2, 28.5, 28.6, 28.7, 28.8,
28.9, 31.2, 34.8, 35.3, 36.4, 116.8, 120.9, 122.3, 124.6,
125.2, 127.9, 129.3, 133.6, 134.6, 137.3, 139.6, 141.1, 169.8,
172.2. MS (EI) m/z 557 (M+), 282, 120. Anal. calcd for
C31H47N3O4S. C, 66.75, H, 8.49, N, 7.53, found: C, 66.41,
14, 8.15, N, 7.83.
Prepared as described above from 17j (488 mg,
1.5 mmol) and 32a (414 mg, 1.5 mmol) and puri®ed by
¯ash chromatography (ethyl acetate). Yield: 110 mg
(13%); mp 190 ꢀC. IR (KBr) n 3425, 1655, 1600, 1310,
1
1155 cm 1. H NMR (400 MHz, DMSO-d6) d 1.96 (s,
3H), 2.15 (s, 3H), 2.55 (t, 2H, J=7 Hz), 3.43 (m, 2H),
5.13 (s, 2H), 6.48 (d, 1H, J=16 Hz), 6.69±6.71 (m, 1H),
6.90±7.04 (m, 4H), 7.27±7.49 (m, 10H), 7.78±7.80 (m,
1H), 8.05±8.07 (m, 2H), 9.49 (s, 1H), 10.21 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) d 13.9, 20.0, 35.0, 36.3, 69.2,
116.9, 119.8, 121.0, 122.4, 124.6, 125.2, 127.5, 127.6, 127.7,
127.9, 128.3, 128.9, 129.3, 133.6, 124.5, 126.7, 137.4, 138.1,
139.5, 141.1, 159.2, 165.2, 169.8. MS (EI) m/z 565 (MI),
276, 120, 91. Anal. calcd for C33H33N3O5S: C, 67.91; H,
5.70; N, 7.20; found: C, 67.53; H, 5.66; N, 7.56.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}heptadecanoic acid amide (20c). Prepared
as described above from 17d (682 mg, 2 mmol) and 32a
(552 mg, 2 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield: 910 mg (76%); mp
143 ꢀC. IR (KBr) n 3315, 2920, 2850, 1650cm 1. 1H NMR
(400MHz, DMSO-d6) d 0.84 (t, J=7Hz, 3H), 1.19±1.26
(m, 26H), 1.45 (m, 2H), 1.97 (s, 3H), 2.02 (m, 2H), 2.16 (s,
3H), 2.48 (m, 2H), 3.30 (m, 2H), 6.73 (m, 1H), 6.92 (m,
1H), 6.97 (m, 1H), 7.29 (m, 1H), 7.42 (m, 1H), 7.76 (m,
2H), 8.04 (s, 1H), 10.10 (s, 1H); 13C NMR (125 MHz,
Nꢁ-{3-[3-(4-Benzyloxyphenyl)acryloylamino]propanoyl}-
N-(2,3-dimethylphenyl)isoleucine amide (19e). Prepared
as described above from 17j (488mg, 1.5 mmol) and 26
(405mg, 1.5 mmol) and puri®ed by ¯ash chromatography
(1. ethyl acetate:n-hexane, 3:2, 2. ethyl acetate:methanol,
3:1). Yield: 720 mg (92%); mp >250 ꢀC. IR (KBr) n
3415, 1650 cm 1. 1H NMR (400 MHz, DMSO-d6) d 0.84

2002
M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
DMSO-d6) d 13.6, 13.8, 19.8, 21.8, 25.0, 28.4, 28.6, 28.7,
31.0, 34.7, 35.2, 36.3, 116.8, 120.8, 122.3, 124.4, 125.1,
127.7, 129.1, 134.5, 137.2, 139.4, 169.7, 172.0. MS (EI)
m/z 599 (10, M+), 121 (50), 120 (100). HRMS calcd for
C34H53N3O4S: 599.3757; found: 599.3747. Anal. calcd
for C34H53N3O4S: C, 68.08; H, 8.91; N, 7.00; found: C,
68.13; H, 8.95; N, 6.97.
1.5 mmol) and 32a (414mg, 1.5 mmol) and puri®ed by
¯ash chromatography (ethyl acetate:n-hexane, 3:2). Yield:
390 mg (43%); mp 125 ꢀC. IR (KBr) n 3350, 1655, 1320,
1160 cm 1. 1H NMR (500MHz, DMSO-d6) d 0.84 (t, 3H,
J=7Hz), 1.21±1.29 (m, 21H), 1.45 (m, 2H), 1.95±2.01 (m,
6H), 2.02 (m, 2H), 2.17 (s, 3H), 2.48 (m, 2H), 3.31 (m, 2H),
5.30 (m, 2H), 6.73 (m, 1H), 6.92 (m, 1H), 6.98 (m, 1H),
7.28 (m, 1H), 7.42 (m, 1H), 7.68 (m, 1H), 7.77 (m, 1H),
8.04 (s, 1H), 9.36 (s, 1H), 10.07 (s, 1H); 13C NMR
(125 MHz, DMSO-d6) d 13.8, 13.9, 20.0, 21.9, 25.2, 26.5,
28.5, 28.7, 29.0, 31.1, 34.8, 35.2, 36.4, 116.8, 120.9, 122.4,
124.6, 125.2, 127.9, 129.3, 129.5, 133.6, 134.5, 137.3,
139.5, 141.9, 169.8, 172.1. MS (EI) m/z 611 (M+), 335,
120. Anal. calcd for C35H53N3O4S: C, 68.70; H, 8.73; N,
6.87; found: C, 68.47; H, 8.40; N, 6.80.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}octadecanoic acid amide (20d). Prepared
as described above from 17e (530mg, 1.5 mmol) and 32a
(414mg, 1.5 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield: 236 mg (45%); mp
123 ꢀC. IR (KBr) n 3345, 3250, 2920, 2850, 1695, 1655,
1
1640 cm
.
1H NMR (500MHz, DMSO-d6) d 0.84 (t,
J=7Hz, 3H), 1.18±1.27 (m, 28H), 1.44 (m, 2H), 1.96 (s,
3H), 2.01 (m, 2H), 2.16 (s, 3H), 2.48 (m, 2H), 3.30, m, 2H),
6.71 (m, 1H), 6.92 (m, 1H), 6.97 (m, 1H), 7.43 (m, 1H),
7.77 (m, 1H), 7.82 (m, 1H), 8.05 (m, 1H), 10.16 (s, 1H); 13C
NMR (125 MHz, DMSO-d6) d 13.8, 13.9, 20.0, 22.0, 25.2,
28.6, 28.7, 28.9, 31.2, 34.8, 35.2, 36.4, 116.8, 120.9, 122.3,
124.4, 125.2, 127.7, 129.2, 133.2, 134.6, 137.3, 139.5, 141.2,
169.8, 172.1. MS (EI) m/z 613 (M+), 121, 120. Anal. calcd
for C35H55N3O4S: C, 68.48; H, 9.03; N, 6.84; found: C,
68.42; H, 9.45; N, 6.50.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}-(Z,Z)-9,13-octadecadienoic acid amide
(20h). Prepared as described above from 17i (527mg,
1.5 mmol) and 32a (414mg, 1.5 mmol) and puri®ed by
¯ash chromatography (ethyl acetate:n-hexane, 3:2). Yield:
816 mg (89%); mp 107 ꢀC. IR (KBr) n 3345, 3240, 3065,
1
1695, 1655, 1640cm 1. H NMR (500MHz, CDCl3) d
0.86 (t, J=7Hz, 3H), 1.21±1.35 (m, 14H), 1.54 (m, 2H),
1.98 (s, 3H), 2.01 (m, 4H), 2.14 (m, 2H), 2.18 (s, 3H),
2.65 (t, J=6Hz, 2H), 2.73 (t, J=7Hz, 2H), 3.58 (m, 2H),
5.26±5.37 (m, 4H), 6.47 (m, 1H), 6.92±6.94 (m, 2H), 6.95±
6.98 (m, 1H), 7.07 (m, 1H), 7.25±7.31 (m, 2H), 7.84 (m,
1H), 8.11 (m, 1H), 9.12 (s, 1H); 13C NMR (125 MHz,
CDCl3) d 13.9, 14.1, 20.6, 22.6, 25.6, 25.7, 27.2, 29.1,
29.2, 29.3, 29.6, 31.5, 35.4, 36.7, 37.2, 117.9, 122.7, 123.7,
123.8, 125.8, 128.0, 128.5, 129.4, 130.0, 130.2, 132.0,
133.8, 138.0, 139.1, 140.4, 170.5, 174.3. MS (EI) m/z 609
(M+), 121, 120, 106. Anal. calcd for C35H51N3O4S: C,
68.93; H, 8.43; N, 6.89; found: C, 68.75; H, 8.39; N,
7.02.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}eicosanoic acid amide (20e). Prepared as
described above from 17f (600mg, 1.56mmol) and 32a
(440mg, 1.56mmol) and puri®ed by ¯ash chromato-
graphy (ethyl acetate:n-hexane, 3:2). Yield: 700 mg (70%);
mp 120 ꢀC. IR (KBr) n 3350, 3250, 2920, 2850, 1695,
1
1640cm 1. H NMR (500MHz, DMSO-d6) d 0.84 (t,
J=7Hz), 1.18±1.22 (m, 32H), 1.46 (m, 2H), 1.96 (s, 3H),
2.01 (t, J=7Hz, 2H), 2.16 (s, 3H), 2.48 (m, 2H), 3.29 (m,
2H), 6.70 (m, 1H), 6.92 (m, 1H), 6.99 (m, 1H), 7.28 (m,
1H), 7.43 (m, 1H), 7.77 (m, 1H), 7.81 (m, 1H), 8.05 (m,
1H), 10.16 (s, 1H); 13C NMR (125MHz, DMSO-d6) d
13.8, 13.9, 20.0, 22.0, 25.2, 28.6, 28.7, 28.9, 31.2, 34.8, 25.2,
36.3, 116.8, 120.9, 122.4, 124.6, 125.2, 127.9, 129.3, 133.3,
134.5, 137.3, 139.6, 169.5, 172.1. MS (EI) m/z 641 (M+),
578, 366, 121, 120. Anal. calcd for C37H59N3O4S: C,
69.23; H, 9.26; N, 6.55; found: C, 68.92; H, 9.54; N, 6.15.
N-{2-[3-(1-Naphthylaminosulfonyl)phenylaminocarbonyl-
jethyl}hexadecanoic acid amide (21a). Prepared as
described above from 17c (290 mg, 0.89 mmol) and 32b
(265 mg, 0.89 mmol) and puri®ed by ¯ash chromato-
graphy (ethyl acetate:n-hexane, 3:2). Yield: 362 mg
(67%); mp 51 ꢀC. IR (KBr) n 2920, 2850, 1660, 1650,
1
1640 cm 1. H NMR (400 MHz, DMSO-d6) d 0.84 (m,
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}-(E)-9-octadecenoic acid amide (20f). Pre-
pared as described above from 17g (530mg, 1.5 mmol)
and 32a (414mg, 1.5 mmol) and puri®ed by ¯ash chro-
matography (ethyl acetate:n-hexane, 3:2). Yield: 588 mg
(64%); mp 111 ꢀC. IR (KBr) n 3340, 3300, 3240, 2920,
2850, 1695, 1645cm 1. 1H NMR (500MHz, DMSO-d6) d
0.84 (t, J=7Hz, 3H), 1.18±1.26 (m, 20H), 1.44 (m, 2H),
1.91 (s, 4H), 1.96 (s, 3H), 2.01 (m, 2H), 2.16 (s, 3H), 2.47
(m, 2H), 3.30 (m, 2H), 5.33 (m, 2H), 6.70 (m, 1H), 6.92
(m, 1H), 6.98 (m, 1H), 7.27 (m, 1H), 7.34 (m, 1H), 7.77
(m, 1H), 7.82 (m, 1H), 8.05 (m, 1H), 9.49 (s, 1H), 10.17
(s, 1H). MS (EI) m/z 611 (M+), 121, 120. Anal. calcd
for C35H53N3O4S: C, 68.70; H, 8.73; N, 6.87; found: C,
68.88; H, 8.40; N, 6.45.
3H), 1.17±1.25 (m, 24H), 1.45 (m, 2H), 2.00 (m, 1H),
2.16 (m, 1H), 2.44 (m, 2H), 3.27 (m, 2H), 7.13 (m, 1H),
7.31±7.48 (m, 5H), 7.68±7.77 (m, 2H), 7.79 (m, 1H), 7.86
(m, 1H), 8.05 (m, 1H), 8.11 (s, 1H), 10.12 (s, 1H); 13C
NMR (125 MHz, DMSO-d6) d 14.5, 22.6, 25.0, 25.8,
29.1, 29.22, 29.26, 29.3, 29.5, 29.6, 31.8, 34.2, 35.9, 37.0,
117.6, 121.6, 123.1, 123.8, 126.0, 126.5, 126.7, 128.4,
129.9, 130.0, 134.3, 140.2, 170.2, 172.8, 175.0. MS (EI)
m/z 607 (M+), 352, 298, 142. Anal. calcd for C35
H49N3O4S: C, 69.16; H, 8.13; N, 6.91; found: C, 69.21;
H, 8.28; N, 6.91.
N-{2-[3-(Benzylaminosulfonyl)phenylaminocarbonyl]eth-
yl}hexadecanoic acid amide (21b). Prepared as described
above from 17c (523 mg, 1.6 mmol) and 32c (420 mg,
1.6 mmol) and puri®ed by ¯ash chromatography (ethyl
acetate:n-hexane, 3:2). Yield: 650 mg (71%); mp 103 ꢀC.
IR (KBr) n 3315, 2955, 2850, 1660, 1640 cm 1. 1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J=7 Hz, 3H), 1.19±1.23
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl} - (Z) - 9 - octadecenoic acid amide (20g).
Prepared as described above from 17h (530mg,

M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
2003
(m, 24H), 1.47 (m, 2H), 2.02 (t, J=7Hz, 2H), 2.49 (m, 2H),
3.31 (m, 2H), 3.96 (d, J=6Hz, 2H), 7.21±7.29 (m, 4H),
7.45±7.47 (m, 2H), 7.75 (m, 1H), 7.84 (m, 1H), 8.10 (m,
1H), 8.18 (s, 1H), 10.10 (s, 1H); 13C NMR (125MHz,
DMSO-d6) d 13.8, 21.9, 25.2, 28.5, 28.6, 28.7, 28.8, 31.2,
34.8, 35.2, 36.4, 46.0, 116.8, 120.7, 122.2, 127.0, 127.4,
128.1, 129.4, 137.6, 139.6, 141.1, 169.8, 172.2. MS (EI) m/z
571 (M+), 402, 311, 310. Anal. calcd for C32 H49N3O4S: C,
67.22; H, 8.64; N, 7.35; found: C, 66.86; H, 8.39; N, 7.41.
N-{2-{3-[2-[(3-Indoly)ethyl]aminosulfonyl]phenylamino-
carbonyl}ethyl}hexadecanoic acid amide (21f). Prepared
as described above from 17c (1079 mg, 3.3 mmol) and 32g
(1040mg, 3.3 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield: 1.8 g (87%); mp
105 ꢀC. IR (KBr) n 3395, 3315, 2920, 2850, 1655, 164.0
cm 1. 1H NMR (400 MHz, CDCl3) d 0.81 (t, J=7 Hz, 3H),
1.14±1.20 (m, 24H), 1.50 (m, 2H), 2.08 (m, 2H), 2.55 (m,
2H), 2.84 (m, 2H), 3.24 (m, 2H), 3.53 (m, 1H), 4.62 (m, 1H),
6.18 (m, 1H), 6.68 (m, 1H), 6.98 (m, 1H), 7.10 (m, 1H),
7.24±7.43 (m, 6H); 13C NMR (125 MHz, CDCl3) d 12.0,
22.7, 25.4, 25.7, 29.3, 29.5, 29.7, 31.9, 35.4, 36.8, 37.3, 43.2,
111.3, 111.4, 117.9, 118.5, 119.5, 122.3, 122.4, 122.7, 123.0,
129.6, 138.2, 139.4, 140.4, 170.1, 172.0. MS (EI) m/z 625
(M+), 624, 130. Anal. calcd for C35H52N3O4S: C, 67.27;
H, 8.39; N, 8.97; found: C, 67.05; H, 8.68; N, 8.63.
N-{2-[3-(2,3-Dichlorobenzylaminosulfonyl)phenylamino-
carbonyl]ethyl}hexadecanoic acid amide (21c). Prepared
as described above from 17c (608 mg, 1.86mmol) and
32d (615mg, 1.86mmol) and puri®ed by ¯ash chromato-
graphy (ethyl acetate:n-hexane, 3:2). Yield: 1.18g (99%);
1
mp 124 ꢀC. IR (KBr) n 3315, 2920, 2850, 1655, 1645cm
.
1H NMR (500MHz, DMSO-d6) d 0.81 (t, J=7 Hz, 3H),
1.15±1.20 (m, 24H), 1.43 (m, 2H), 2.00 (m, 2H), 2.48 (m,
2H), 3.30 (m, 2H), 4.09 (s, 2H), 7.27 (m, 1H), 7.36 (m,
1H), 7.41±7.48 (m, 3H), 7.72 (m, 2H), 8.14 (s, 1H), 8.18
(s, 1H), 10.11 (s, 1H); 13C NMR (125 MHz, DMSO-d6)
d 13.8, 21.9, 25.2, 28.6, 28.7, 28.8, 28.89, 28.9, 31.2, 34.9,
25.3, 44.2, 116.9, 120.7, 122.5, 127.8, 128.2, 129.3, 129.4,
130.1, 131.6, 137.6, 139.7, 140.9, 169.9, 172.2. MS (EI)
m/z 639 (10, M+), 330 (23), 310 (100), 174 (40). Anal.
calcd for C32H47C12N3O4S: C, 59.99; H, 7.39; N, 6.56;
found C, 60.06; H, 7.40; N, 6.55.
N-{2-{3-[(3-Methylbutyl)aminosulfonyl]phenylaminocar-
bonyl}ethyl}hexadecanoic acid amide (21g). Prepared as
described above from 17c (810 mg, 2.5 mmol) and 32h
(600 mg, 2.5 mmol) and puri®ed by ¯ash chromato-
graphy (ethyl acetate:n-hexane, 3:2). Yield: 900 mg
(65%); mp 103 ꢀC. IR (KBr) n 3315, 2920, 2850, 1655,
1
1640 cm 1. H NMR (400 MHz, DMSO-d6) d 0.77 (d,
J=7 Hz, 6H), .84 (m, 3H), 1.18±1.27 (m, 24H), 1.43 (m,
2H), 1.55 (m, 1H), 2.00 (m, 2H), 2.49 (m, 4H), 2.74 (m,
2H), 3.32 (m, 2H), 7.38±7.48 (m, 3H), 7.73 (m, 1H), 7.82
(m, 1H), 8.15 (m, 1H), 10.18 (m, 1H); 13C NMR
(125 MHz, DMSO-d6) d 13.8, 21.9, 22.0, 24.7, 25.2, 28.5,
28.6, 28.8, 28.9, 31.2, 34.8, 35.3, 36.4, 37.8, 40.7, 116.8,
120.6, 122.1, 129.4, 139.6, 141.0, 169.8, 172.2. MS (EI)
m/z 551 (M+), 310. Anal. calcd for C30H53N3O4S: C,
65.30; H, 9.68; N, 7.61; found: C, 65.62; H, 9.62; N,
8.01.
N-{2-[3-(1-Naphthylmethylaminosulfonyl)phenylamino-
carbonyl]ethyl}hexadecanoic acid amide (21d). Prepared
as described above from 17c (654 mg, 2 mmnol) and 32e
(624 mg, 2 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate:n-hexane, 3:2). Yield: 845 mg (68%); mp
126 ꢀC. IR (KBr) n 3315, 3050, 2920, 2850, 1660,
1
1640cm 1. H NMR (500MHz, DMSO-d6) d 0.85 (t,
J=7Hz, 3H), 1.19±1.27 (m, 24H), 1.47 (m, 2H), 2.04 (m,
2H), 2.51 (m, 2H), 3.34 (m, 2H), 4.42 (d, J=6Hz, 2H),
7.42 (m, 2H), 7.50±7.53 (m, 4H), 7.77±7.79 (m, 2H), 7.82
(m, 1H), 7.90 (m, 1H), 8.04±8.08 (m, 2H), 8.22 (s, 1H),
10.16 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 13.8,
21.9, 25.2, 28.5, 28.6, 28.7, 28.8, 28.87, 28.9, 31.2, 34.9,
35.3, 44.3, 117.1, 120.8, 122.4, 123.4, 125.6, 126.4, 128.0,
128.3, 129.4, 130.8, 132.6, 133.2, 139.6, 140.9, 169.8, 172.2.
MS (EI) m/z 619 (15, M+), 555 (20), 120 (100). Anal. calcd
for C36H51N3O4S: C, 69.53; H, 8.27; N, 6.76; found: C,
69.54; H, 8.15; N, 6.67.
N-{2-[3-(Dodecylaminosulfonyl)phenylaminocarbonyl]eth-
yl}hexadecanoic acid amide (21h). Prepared as described
above from 17c (654 mg, 2 mmol) and 32i (680 mg,
2 mmol) and puri®ed by ¯ash chromatography (ethyl
acetate:n-hexane, 3:2). Yield: 559 mg (43%); mp 84 ꢀC.
1
IR (KBr) n 3350, 2920, 2850, 1659, 1655, 1635cm 1. H
NMR (400MHz, DMSO-d6) d 0.84 (m, 6H), 1.13±1.45
(m, 44H), 1.58 (m, 2H), 2.30 (m, 2H), 2.48 (m, 2H), 2.72
(m, 2H), 3.24 (m, 2H), 7.40 (m, 1H), 7.47 (m, 2H), 7.73 (m,
1H), 8.13 (m, 1H), 10.10 (s, 1H); 13C NMR (125MHz,
DMSO-d6) d 13.8, 22.0, 24.9, 25.9, 28.4, 28.5, 28.9, 31.2,
36.3, 42.4, 116.7, 120.5, 122.0, 129.4, 139.7, 141.1, 171.5.
MS (EI) m/z 650 (M+), 423, 184. Anal. calcd for C37
H67N3O4S: C, 68.37; H, 10.39; N, 6.46; found: C, 68.27;
H, 10.75; N, 6.41.
N-{2-{3-[(2-Phenylethyl)aminosulfonyl]phenylaminocar-
bonyl}ethyl}hexadecanoic acid amide (21e). Prepared as
described above from 17c (490mg, 1.5 mmol) and 32f
(414mg, 1.5 mmol) and recrystallized from toluene. Yield:
700 mg (80%); mp 93 ꢀC. IR (KBr) n 3310, 2920, 2850,
3-[4-(2-Phenylethenyl)phenyl]-N-{2-[3-(2,3-dimethylphe-
nylaminosulfonyl)phenylaminocarbonyl]ethyl}acrylamide
(22a). Prepared as described above from 17k (320 mg,
1 mmol) and 32a (276 mg, 1 mmol) and puri®ed by ¯ash
1
1640 cm 1. H NMR (400 MHz, DMSO-d6) d 0.84 (m,
3H), 1.28±1.23 (m, 24H), 1.44 (m, 2H), 2.06 (m, 2H),
2.46 (m, 2H), 2.67 (m, 2H), 2.95 (m, 2H), 3.26 (m, 2H),
7.14±7.19 (m, 4H), 7.22±7.26 (m, 2H), 7.68 (m, 1H), 7.75
(m, 1H), 7.83 (m, 1H), 8.16 (m, 1H), 10.20 (m, 1H), 13C
NMR (125 MHz, DMSO-d6) d 13.8, 20.0, 25.2, 28.6, 28.7,
28.9, 30.6, 31.2, 33.9, 35.3, 44.6, 116.8, 120.7, 122.3, 126.1,
128.2, 128.5, 129.5, 138.6, 139.7, 140.9, 169.9, 172.2,
172.8. MS (EI) m/z 585 (M+), 494, 423, 239. Anal. calcd
for C33H51N3O4S: C, 67.66; H, 8.77; N, 7.17; found: C,
67.90; H, 8.91; N, 7.26.
chromatography (ethyl acetate). Yield 132 mg (22%);
1
mp >200 ꢀC. IR (KBr) n 3275, 2925, 1655, 1600 cm
.
1H NMR (500 MHz, DMSO-d6) d 1.96 (s, 3H), 2.15 (s,
3H), 2.56 (t, J=7Hz, 2H), 3.46 (m, 2H), 6.65 (m, 1H),
6.69 (m, 1H), 6.92 (m, 1H), 6.98 (m, 1H), 7.27±7.31 (m,
3H), 7.36±7.40 (m, 3H), 7.54 (m, 2H), 7.59±7.66 (m, 4H),
7.80 (m, 1H), 8.06 (s, 1H), 8.19 (m, 1H), 9.52 (s, 1H), 10.24
(s, 1H); 13C NMR (125MHz, DMSO-d6) d 14.7, 20.7,

2004
M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
35.6, 37.0, 117.2, 121.3, 122.3, 122.9, 125.1, 125.6, 128.0,
128.3, 129.1, 129.2, 129.5, 130.4, 130.8, 131.0, 134.5, 134.9,
135.4, 136.1, 137.5, 138.0, 138.8, 139.4, 140.2, 141.8, 165.7,
170.8. MS (EI) m/z 579 (M+), 276, 233, 120. Anal. calcd
for C34H33N3O4S: C, 70.44; H, 5.74; N, 7.25; found: C,
70.26; H, 5.34; N, 7.46.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}-1-naphthylacetamide (22e). Prepared as
described above from 17o (386 mg, 1.5 mmol) and 32a
(414 mg, 1.5 mmol) and recrystallized from toluene/diox-
ane/petroleum ether. Yield: 653 mg (85%); mp 110ꢀC. IR
(KBr) n 3415, 1655 cm 1. 1H NMR (400 MHz, DMSO-d6)
d 1.97 (s, 3H), 2.15 (s, 3H), 2.49 (m, 2H), 3.35 (m, 2H), 3.87
(s, 2H), 6.71±6.73 (m, 1H), 6.92±7.03 (m, 2H), 7.28±7.30
(m, 1H), 7.38±7.45 (m, 5H), 7.76±7.78 (m, 2H), 7.86±7.89
(m, 1H), 8.02±8.06 (m, 2H), 8.23 (m, 1H), 9.50 (s, 1H),
10.17 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d 13.9,
20.0, 35.0, 36.1, 115.1, 116.9, 119.8, 121.0, 122.4, 124.6,
125.2, 127.5, 127.6, 127.7, 127.9, 128.3, 128.9, 129.3, 133.6,
134.5, 136.7, 137.4, 138.1, 139.5, 141.1, 169.8, 170.0. MS
(EI) m/z 515 (M+), 168, 120. Anal. calcd for C29H29N3O4S:
C, 67.55; H, 5.67; N, 8.15; found C, 67.59; H, 5.72; N, 8.12.
3-Biphenylyl-N-{2-[3-(2,3-dimethylphenylaminosulfonyl)-
phenylaminocarbonyl]ethyl}acrylamide (22b). Prepared
as described above from 17l (443mg, 1.5 mmol) and 32a
(414mg, 1.5 mmol) and puri®ed by ¯ash chromatography
(ethyl acetate). Yield: 249 mg (30%); mp 230 ꢀC. IR (KBr)
1
n 3350, 1695, 1660, 1600cm
.
1H NMR (400MHz,
DMSO-d6) d 1.96 (s, 3H), 2.15 (s, 3H), 2.57 (t, J=6Hz,
2H), 3.47 (q, J=6 Hz, 2H), 6.66±6.71 (m, 2H), 6.90±6.98
(m, 2H), 7.26±7.32 (m, 1H), 7.34±7.38 (m, 1H), 7.42±7.44
(m; 4H), 7.61±7.63 (m, 2H), 7.69±7.71 (m, 4H), 7.79±7.81
(m, 1H), 8.06 m, 1H), 8.19 (d, J=6 Hz, 1H), 9.48 (s,
1H), 10.22 (s, 1H); 13C NMR (125MHz, DMSO-d6) d
14.0. 20.0, 34.7, 36.3, 116.5, 120.8, 121.8, 122.0, 124.1,
125.0, 126.2, 127.3, 127.9, 128.5, 129.2, 133.4, 133.7, 134.3,
137.1, 137.8, 138.9, 139.1, 140.6, 140.8, 165.0, 169.5. MS
(EI) m/z 553 (M+), 223, 222, 207. Anal. calcd for C32
H30N3O4S: C, 69.42; H, 5.64; N, 7.59; found: C, 69.55; H,
5.65; N, 7.97.
N-{4-[(3-Octadecanoylamlno)propanoylaminolbenzoyl}-
methionine methyl ester (23). Prepared as described
above from 17e (530 mg, 1.5 mmol) and 37 (423 mg,
2 mmol) and puri®ed by ¯ash chromatography (ethyl
acetate:n-hexane, 3:2). Yield: 150 mg (16%); mp 122 ꢀC.
1
IR (KBr) n 3290, 2920, 2850, 1745, 1630 cm
.
1H
NMR (400 MHz, DMSO-d6) d 0.79 (t, J=7 Hz, 3H),
1.15±1.20 (m, 28H), 1.43 (m, 2H), 1.95±2.04 (m, 7H),
2.50±2.56 (m, 4H), 3.28 (m, 2H), 3.59 (s, 3H), 4.53 (m,
1H), 7.60±7.63 (m, 2H), 7.76±7.78 (m, 2H), 8.25 (s, 1H),
8.50 (m, 1H), 10.06 (s, 1H). MS (EI): m/z 619 (M+),
545, 457, 338, 212, 120. HRMS calcd for C34H57N3O5S:
619.4019; found: 619.4059.
2-(4-Benzyloxyphenyloxy)-N-{2-[3-(2,3-dimethylphenyla-
minosulfonyl)phenylaminocarbonyl]ethyl}acetamide (22c).
Prepared as described above from 17m (628mg, 2 mmol)
and 32a (552mg, 2 mmol) and washed with ethyl acetate:
n-hexane, 3:2 and n-hexane. Yield: 450 mg (38%); mp
1
153 ꢀC. IR (KBr) n 3185, 1660, 1600, 1310, 1155 cm
.
Farnesyltransferase assay
1H NMR (500 MHz, DMSO-d6) d 1.98 (s, 3H), 2.17 (s,
3H), 2.55 (t, 2H, J=7 Hz), 3.44 (m, 2H), 4.38 (s, 2H),
5.00 (s, 2H), 6.73±6.75 (m, 1H), 6.87±6.95 (m, 5H), 6.98±
6.99 (m, 1H), 7.28±7.31 (m, 2H), 7.35±7.45 (m, 5H), 7.76±
7.78 (m, 1H), 7.97 (m, 1H), 8.06 (s, 1H), 9.39 (s, 1H), 10.12
(s, 1H); 13C NMR (100MHz, DMSO-d6) d 13.9, 20.0,
34.7, 36.1, 67.7, 69.6, 115.6, 115.7, 116.9, 121.0, 122.5,
124.6, 125.2, 127.4, 127.6, 128.0, 128.2, 129.3, 137.2, 137.5,
139.5, 141.1, 151.9, 152.8, 167.8, 169.8. MS (EI) m/z 587
(M+), 210, 120, 91. Anal. calcd for C32H33N3O6S: C,
65.40; H, 5.66; N, 7.15; found: C, 65.10; H, 5.60; N, 6.79.
The assay employed yeast farnesyltransferase (FTase)
fused to Glutathione S-transferase at the N-terminus of
the b-subunit. FTase was expressed in Escherichia coli
DH5a grown in LB media containing ampicillin and
additional chloramphenicol for co-expression of pGEX-
DPR1 and pBC-RAM2 for FTase production.21 The
enzyme was puri®ed by standard protocol using glu-
tathione-agarose beads for selective binding of the target
proteins. The assay was carried out as described.5,20 FPP
was obtained as ammonium salt solution in methanol:
10mM aqueous NH4Cl (7:3) from Sigma-Aldrich. Dan-
syl-GCVLS was custom synthesized by ZMBH, Heidel-
berg, Germany. The assay mixture (100mL volume)
contained 50mM Tris/HCl pH 7.4, 5 mM MgCl2, 10mM,
ZnCl2, 5 mM DTT, 7 mM Ds-GCVLS, 20 mM FPP and
approx. 5 nmol yeast GST-FTase21 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 ¯uorescence emission 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. IC50s were calculated from
initial velocity of three independent measurements of each
inhibitor concentration and expressed as meanÆSD.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl}-2-(3-phenyloxyphenyloxy)acetamide (22d).
Prepared as described above from 17n (600mg,
2.32mmol) and 32a (640mg, 2.32mmol) and puri®ed by
¯ash chromatography (1. ethyl acetate:n-hexane, 3:2; 2.
ethyl acetate). Yield: 410 mg (30%); mp 45ꢀC. IR (KBr) n
3300, 1660, 1250, 1155 cm 1. 1H NMR (500 MHz, DMSO-d6)
d 1.96 (s, 3H), 2.15 (s, 3H), 2.52 (t, 2H, J=7 Hz), 3.39 (m, 2H),
3.87 (s, 2H), 4.50 (s, 2H), 6.70 (m, 1H), 6.88±6.92 (m, 2H),
6.97±7.00 (m, 4H), 7.09±7.13 (m, 2H), 7.27±7.44 (m, 5H),
7.78 (m, 1H), 7.83 (m, 1H), 8.03 (s, 1H), 9.47 (s, 1H), 10.18
(s, 1H); 13C NMR (100MHz, DMSO-d6) d 13.9, 20.0, 34.5,
36.1, 69.2, 71.7, 116.9, 117.6, 118.5, 121.0, 122.4, 123.3,
124.6, 125.2, 128.0, 129.8, 129.9, 133.6, 134.4, 139.3, 139.9,
141.0, 156.6, 168.7, 170.0. MS (EI) m/z 587 (M+), 330, 183,
120. MS (ESI) m/z 588 [M+H]+, 610 [M+Na]+. HRMS
calcd for C32H34N3O6S: 588.2168; found: 588.2152.
Anal. calcd for C32H33N3O6S: C, 65.40; H, 5.66; N,
7.15; found: C, 65.48; H, 5.78; N, 6.77.
Cells and culture conditions
Established suspended human leukaemic cell lines K-
562 cells (chronic myeloid leukaemic cell line), HL-60

M. Schlitzer et al. / Bioorg. Med. Chem. 8 (2000) 1991±2006
2005
cells (acute myeloid leukaemic cell line) and THP-1 cells
(acute monocytic leukaemic cell line) were cultured in
RPMI 1640 medium (Gibco, cat.-no. 15140-114), supple-
mented with 100 U/mL penicillin, 100 mg/mL streptomycin
and 10% FBS.
9. (a) Schlitzer, M.; Sattler, I.; Dahse, H.-M. Bioorg. Med.
Chem. 1999, 7, 2037. (b) Schlitzer, M.; Sattler, I. Angew.
Chem., Int. Ed. 1999, 38, 2032.
10. Schlitzer, M.; Sattler, I. Bioorg. Med. Chem. 1999, 7, 2391.
11. Gibbs, J. B.; Pompliano, D. L.; Mosser, S. D.; Rands, E.;
Lingham, R. B.; Singh, S. B.; Scolnick, E. M.; Kohl, N. E.;
Oli€, A. J. Biol. Chem. 1993, 268, 7617.
12. deSolms, S. J.; Deana, A. A.; Giuliani, E. A.; Kohl, N. E.;
Mosser, S. D.; Oli€, A. I.; Pompliano, D. L.; Rands, E.;
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Med. Chem. 1995, 38, 3967.
13. Ciccarone, T. M.; Williams, T. M.; Dinsmore, C. J.;
Stokker, G. E.; Wai, J. S. (Merck & Co., Inc.) Inhibitors of
Farnesyl-Protein Transferase, WO 96/30014, 3 October 1996.
Chem. Abstr. 1996, 125, 328305g.
14. Schlitzer, M.; Sattler, I.; Dahse, H.-M. Arch. Pharm.
Pharm. Med. Chem. 1999, 332, 124.
15. Qian, Y.; Blaskovich, M. A.; Saleem, M.; Seong, C.-M.;
Wathen, S. P.; Hamilton, A. D.; Sebti, S. M. J. Biol. Chem.
1994, 269, 12410.
16. Uchida, M.; Tabusa, F.; Komatsu, M.; Morita, S.; Kanbe,
T.; Nakagawa, K. Chem. Pharm. Bull. 1985, 33, 3775.
17. Jones, G. The Knoevenagel Condensation. In Organic
Reactions; Cope, A. C., Ed.; John Wiley & Sons: New York,
1967; Vol. 15, pp 204±599.
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19. Bellamy, F. D.; Qu, K. Tetrahedron Lett. 1984, 25, 839.
20. Pompliano, D. L.; Gomez, R. P.; Anthony, N. J. J. Am.
Chem. Soc. 1992, 114, 7945.
21. (a) Mitsuzawa, H.; Esson, K.; Tamanoi, F. Proc. Natl.
Acad. Sci. USA 1995, 92, 1704. (b) Del Villar, K.; Mitsuzawa,
H.; Yang, W.; Sattler, L; Tamanoi, F. J. Biol. Chem., 1997,
272, 680. (c) Finegold, A. A.; Johnson, D. I.; Farnsworth, C.
C.; Geib, M. H.; Judd, S. R.; Glomset, J. A.; Tamanoi, F.
Proc. Natl. Acad. Sci. USA 1991, 88, 4448.
22. Lozzio, C. B.; Lozzio, B. B. Blood 1975, 45, 321.
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Test conditions
For each experiment, approximately 10,000 cells were
seeded with 0.1 mL culture medium, containing sodium
bicarbonate, but without HEPES, into 96-well micro-
plates (NUNC). The plates were previously prepared
with dilutions of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128,
1:256, 1:512 and 1:1024 of test substances in 0.1 mL
medium. The cells were incubated for 72 h at 37 ꢀC in a
humidi®ed atmosphere and 5% CO2.
Acknowledgements
The pGEX-DPR1 and pBC-RAM2 plasmides were
kindly provided by Prof. F. Tamanoi (UCLA). M.
Schlitzer thanks Ms. K. Burk and Mr. T. Merdan for
technical assistance and Prof. W. Hanefeld and Prof. G.
Klebe for generous support. I. Sattler wishes to thank
Prof. Dr. S. Grabley and PD Dr. R. Thiericke of gen-
erous support and Ms. S. Egner for excellent technical
assistance.
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