Different amino acid replacements in CAAX-tetrapeptide based peptidomimetic farnesyltransferase inhibitors

Article abstract of DOI:10.1002/(SICI)1521-4184(19994)332:4<124::AID-ARDP124>3.0.CO;2-G

In a series of CAAX-tetrapeptide based farnesyltransferase inhibitors it has been shown that the central AA-dipeptide can be replaced by tranexamic acid, 4-aminobenzenesulfonic acid, and 3-amino-N-(2,3- dimethylphenyl)benzenesulfonamide, respectively, yielding inhibitors active in the low micromolar range. Lipophilic derivatives of these compounds showed moderate antiproliferative activity against different tumor cell lines. A promising class of peptidomimetic farnesyltransferase inhibitors was discovered through the replacement of the terminal AAX motif of the CAAX- tetrapeptide by 2-acylamino-5-aminobenzophenones.

Full text of DOI:10.1002/(SICI)1521-4184(19994)332:4<124::AID-ARDP124>3.0.CO;2-G

124
Schlitzer, Sattler, and Dahse
Different Amino Acid Replacements in CAAX-Tetrapeptide Based
Peptidomimetic Farnesyltransferase Inhibitors
a)
b)
b)
Martin Schlitzer *, Isabel Sattler , and Hans-Martin Dahse
a) Institut für Parmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
b) Hans-Knöll-Institut für Naturstoff-Forschung e. V., Beutenbergstraße 11, D-07745 Jena, Germany
Key Words: ras; farnesyltransferase inhibitors; peptidomimetics
Summary
In a series of CAAX-tetrapeptide based farnesyltransferase inhibi-
tors it has been shown that the central AA-dipeptide can be
replaced by tranexamic acid, 4-aminobenzenesulfonic acid, and
3-amino-N-(2,3-dimethylphenyl)benzenesulfonamide, respec-
tively, yielding inhibitors active in the low micromolar range.
Lipophilic derivatives of these compounds showed moderate anti-
proliferative activity against different tumor cell lines. Apromising
class of peptidomimetic farnesyltransferase inhibitors was discov-
ered through the replacement of the terminal AAX motif of the
CAAX-tetrapeptide by 2-acylamino-5-aminobenzophenones.
Introduction
Interference with the post-translational modification of ras
proteins has been identified as a promising target in the
development of novel cancer therapeutics. Ras proteins relay
growth-stimulating signals from membrane-bound tyrosine
kinase receptors to cytosolic serine/threonine kinases and are
thus involved in the regulation of cell differentiation and
proliferation. In approximately 30% of all human cancers,
including 90% of pancreatic, 50% of colon, and 50% of
thyroid tumors, mutated ras proteins are found. These onco-
gene products have lost their ability to return from their active
into their inactive state by hydrolysis of the protein-bound
GTP. Post-translational modification is obligatory for the
activity of normal as well as mutated ras. It has been shown
that inhibition of the first modification step reverses the cell
box (C: cysteine, A: aliphatic amino acid, X: methionine or
serine) which is recognized by the enzyme that catalyzes this
[1–7]
modification, the farnesyltransferase (FTase) (Fig. 1)
.
A number of potent farnesyltransferase inhibitors have been
developed by modification of the CAAX recognition se-
quence. In any case, these modifications yielded non-sub-
strate competitive inhibitors. In contrast, only the unmodified
CAAX-tetrapeptides act as alternative substrates. A success-
fully applied strategy was the replacement of the central AA
motif by different non-peptidic linkers like 4-aminobenzoic
[13]
transformation caused by oncogenic ras without disturbing
acid (1) . Early structure-activity studies suggested that the
[1–7]
normal cell function
. The underlying mechanism of this
X amino acid is essential for the interaction of an inhibitor
with the farnesyltransferase. However, it was possible to
substitute this partial structure by substituted phenyl residues
[6,8–
selectivity is a topic of intense research and discussion
12]
.
The first modification step of the ras proteins is the transfer
of a farnesyl residue from farnesylpyrophosphate to the thiol
of a cysteine side chain near the C-terminus of the ras pro-
[14]
[15]
as in 2 oreven design cysteinyl derivatives like 3 which
are potent inhibitors in spite of lacking any structural equiva-
teins. This cysteine is part of the C-terminal so-called CAAX- lent of the C-terminal X amino acid (Fig. 2).
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/0404/0124 $17.50 +.50/0

Peptidomimetic Farnesyltransferase Inhibitors
125
In the course of our studies on the design of novel farnesyl-
The 5-chlorobenzophenone derivatives 31a,b,d–g were
transferase inhibitors we wished to explore the potency of prepared by acylation of 2-amino-5-chlorobenzophenone
several structurally different AA or AAX replacements in (30) with appropriate acyl chlorides in refluxing toluene. 31c
CAAX-tetrapeptide based farnesyltransferase inhibitors. was prepared by nucleophilic displacement from 31b and
Compound thus identified would then be used as partial 5-mercapto-1-methyltetrazole sodium salt. 31h was obtained
structures of other types of farnesyltransferase inhibitors or from 31g by ester saponification. The cysteine amide 36 was
as a starting point for further development.
prepared by acylation of 2-amino-5-nitrobenzophenone (32)
with phenylacetic acid chloride, successive reduction of the
nitro group and coupling with N-Boc,S-Trt-cysteine. Acidic
removal of the protective groups yielded the target compound
36 (Scheme 3).
Chemistry
Nitrobenzenesulfonamides 6 and 13 have been prepared
from the appropriate nitrobenzenesulfonyl chlorides and
methionine methyl ester hydrochloride 4 and 2,3-dimethyl-
Biological Evaluation
The inhibitory activity of the prepared compounds on the
farnesyltransferase was determined using the fluorescence
enhancement assay. This assay has been established by Pom-
pliano et al. (Merck Inc.) as an easy to handle test system for
the evaluation of potential farnesyltransferase inhibitors
yielding equivalent results as those assays employing ras and
tritium-labeled FPP as substrates
yeast farnesyltransferase (FTase) fused to Glutathione S-
transferase at the N-terminus of the β-subunit
farnesyltransferase is a close homolog and functionally simi-
lar to the human enzyme; it is widely used for the evaluation
of farnesyltransferase inhibitors. Farnesylpyrophosphate and
the dansylated pentapeptide Ds-GlyCysValLeuSer were used
as substrates. Upon farnesylation of the cysteine thiol the
dansyl residue is placed in a lipophilic environment; the
resulting enhancement of fluorescence at 505 nm is used to
monitor the enzyme reaction.
aniline 12, respectively. Then, the nitro group was reduced
[16]
by tin(II) chloride in refluxing ethyl acetate
. The resulting
aminobenzenesulfonamides 7 and 14 were acylated by N-
Boc-S-Trt-cysteine, activated as mixed anhydride. Removal
of the various protective groups yielded the desired CAAX
mimetics 11 and 17 (Scheme 1).
[18]
. The assay employed
For the preparation of 24 and 29, N-Boc-tranexamic acid
[17]
[19]
(18)
was activated as a mixed anhydride and coupled to
. Yeast
methionine methyl ester hydrochloride 4 and 2,3-dimethyl-
aniline 12, respectively. After acidic removal of the Boc
protective group, the synthesis was continued as described
above (Scheme 2).
The N,S-bis-Cbz derivatives 8, 15, 21, and 27 were pre-
pared following the route outlined in Schemes 1 and 2 using
N,S-bis-Cbz-cysteine instead of N-Boc,S-Trt-cysteine for the
reaction with 7, 14, 20, and 26, respectively.
Arch. Pharm. Pharm. Med. Chem. 332, 124–132 (1999)

126
Schlitzer, Sattler, and Dahse
Arch. Pharm. Pharm. Med. Chem. 332, 124–132 (1999)

Peptidomimetic Farnesyltransferase Inhibitors
127
free those compounds able to inhibit the farnesyltrans-
[23]
ferase
. Therefore, the O-methyl, N,S-bis-Cbz derivatives
8, 15, 21, and 27 of our CAAX analogs 11, 17, 24, and 29,
respectively, were prepared and studied on three different
tumor cell lines (Table 1). In the antiproliferation assay, low
to moderate activity was recorded for the tranexamic acid
derivatives. The sulfonamide derivatives 8 and 15 displayed
a slightly higher antiproliferative activity against all three
tumor cell lines than the tranexamic acid derivatives 21 and
27. Both sulfonamides are approximately twice as active
against the ras dependent cell lines HL-60 and THP-1 than
against the ras independent cell line K-562.
Selected compounds were tested against cell lines K-562
[20]
(chronic myeloid cell line) , THP-1 (acute monocytic
[22]
leukaemic cell line) , and HL-60 (acute myeloid leukaemic
[22]
cell line)
for their antiproliferative effects. The cells were
incubated with different concentrations ofthe test compounds
and an automatic volumetric cell analysis was carried out
during the logarithmic phase of cell proliferation.
Results and Discussion
In a first series of compounds, we used 4-aminobenzenesul-
fonic acid and 3-amino-N-(2,3-dimethylphenyl)benzenesul-
fonamide as AA replacements, respectively; either in a
CAAX- or in a CAA mimicking structure. While the corre-
sponding amide scaffolds have been successfully used by
other groups, nothing was known about the effect of exchang-
ing the amide moiety against a sulfonamide group. In the case
of the 4-amino derivative 11, the replacement of the peptide
bond by a sulfonamide moiety resulted in a marked loss of
[13]
activity (IC (11) = 2.7 µM vs. 50 nM for 1 ). In 11 the
50
sulfonamide group occupies the position between the AA
mimetic substructure and the X amino acid. This position
seems to be much more sensitive against structural changes
than the situation inside the AA mimetic substructure. The
two CAA mimetics with the sulfonamide (17) or the amide
linkage (3), located inside the AA mimetic substructure dis-
played inhibitory activities in a similar order of magnitude
[15]
(IC (17) = 2.3 µM; IC (3) < 50 µM
).
50
50
In a different approach towards farnesyltransferase inhibi-
tor design we wanted to explore the potency of 2-aminoben-
zophenone as a replacement of the central AA dipeptide in
the CAAX motif. In addition, lipophilic or acidic moieties
were added to the amino group to mimic the side chain or the
carboxylic group of the X amino acid thereby yielding a non-
peptide mimetic of the terminal AAX sequence of ras pro-
teins. In a preliminary investigation on this class of com-
pounds we evaluated a 8-compound library of 5-chloro-
Replacement of the central AA dipeptide of the CAAX
motif by tranexamic acid resulted in an inhibitor of moderate
activity (IC (24) = 5.6 µM). Additional replacement of the
50
terminal methionine by a 2,3-dimethylphenyl residue led to
a reduction in activity (IC (29) = 38.3 µM).
50
While free –SH, –NH , and –COOH functions are required
2
for inhibition of the enzyme, poor membrane penetration of
compounds is caused by these functionalities which renders
a cell culture assay of these compounds useless. To facilitate
membrane penetration into a cell a prodrug strategy is nesses-
sary. Functionalization of the amino and the mercapto group
as their N,S-bis-benzyloxycarbonyl (Cbz) derivatives and
masking of the carboxy group as its methyl ester has been
shown in similar cases to yield derivatives which are lipo-
philic enough to cross the membrane. Inside the cell the
benzophenone derivatives (31a–h) which were available
[24]
from a different study
. Inhibitory activity was observed
with three (31a, d, and h) out of eight compounds at a
concentration of 100 µM (Table 2). Although this concentra-
tion was rather high, structure activity relationships within
this series could clearly be delineated. For example, enlarge-
protective groups are cleaved by intracellular esterases to set ment of the lipophilic acyl side chain from phenyl (31a) to
Arch. Pharm. Pharm. Med. Chem. 332, 124–132 (1999)

128
Schlitzer, Sattler, and Dahse
N-(4-Nitrobenzenesulfonyl)methionine Methyl Ester 6
naphthyl (31d) improved the inhibitory activity while the
introduction of α-substituents (31b,c) had a detrimental ef-
fect. We further explored this class of compounds by replac-
ing the 5-chlorine in 31a by a cysteinylamino moiety, thus
transforming the AAX mimetics, as which the 5-chloroben-
zophenone derivatives 31 could be regarded, into true CAAX
peptidomimetics (36). This change resulted in a dramatic
Yield 3.1 g (89%).– 1H NMR (CDCl3): δ = 1.87–1.97 (m, 1H), 2.04–2.09
(m, 1H), 2.05 (s, 3H), 2.47–2.57 (m, 2H), 3.57 (s, 3H), 4.14–4.20 (m, 1H),
5.56 (d, J = 9 Hz, 1H), 8.02–8.06 (m, 2H), 8.32–8.36 (m, 2H).
3-Nitro-N-(2,3-dimethylphenyl)benzenesulfonamide 13
Yield 2.9 g (95%).– 1H NMR (CDCl3): δ = 1.93 (s, 3H), 2.16 (s, 3H), 6.42
(s, 1H), 6.88–7.00 (m, 3H), 7.57–7.61 (m, 1H), 7.92–7.94 (m, 1H), 8.32–8.35
(m, 1H), 8.52–8.53 (m, 1H).
enhancement of the inhibitory activity (IC = 654 nM (36)),
50
thereby confirming the usefulness of the 2-acylaminobenzo-
phenone scaffold as an AAX replacement.
General Protocol for Reduction of Nitrobenzenesulfonamides 6 and 13 to the
Corresponding Amines 7 and 14
Conclusion
To a solution of the nitro derivative in EtOAc (approx. 5 ml per mmol)
SnCl2•2H2O (1.125 g per mmol nitro compound) was added. Then the
solution was refluxed for 2 h. The cooled solution was diluted with water and
the pH was adjusted to 7–8 by addition of sat. NaHCO3 solution The aqueous
phase was extracted with EtOAc (3 × 100–200 ml) and the combined organic
extracts were thoroughly washed with brine and dried over MgSO4. The
products obtained after the removal of the solvent were used without further
purification.
In summary, tranexamic acid can be used as an AA substi-
tute in CAAX-peptidomimetics, resulting in moderately ac-
tive inhibitors of farnesyltransferase. Essentially the same
holds for the replacement of the amide linkage by a sulfon-
amide moiety in several CAAX peptidomimetics. These
compounds have been used as building blocks in other types
[25,26]
of farnesyltransferase inhibitors
. Lipophilic modifica-
tion of the peptidomimetics yielded compounds which dis-
played a moderate antiproliferative effect on tumor cells. In
the case of the sulfonamides 8 and 15 this antiproliferative
effect was more pronounced on ras-dependent than on ras-in-
dependent tumor cells. The replacement of the terminal AAX
tripeptide of the CAAX motif by N-acylbenzophenones
yielded compound 36 as a lead structure of a promising novel
class of peptidomimetic farnesyltransferase inhibitors. Our
ongoing research is directed towards the investigation of
structure-activity-relationships of the this novel inhibitors
and the improvement of their inhibitory potency.
3-Amino-N-(2,3-dimethylphenyl)benzenesulfonamide 17
Yield 2.1 g (80%).– 1H NMR (CDCl3): δ = 1.91 (s, 3H), 2.15 (s, 3H), 3.75
(s, 2H), 6.19 (s, 1H), 6.70–6.73 (m, 1H), 6.91–7.00 (m, 5H), 7.08–7.12 (m,
1H).
General Protocol for the Coupling of N,S-Protected Cysteines with Various
Amines Using Mixed Anhydride Activation (Protocol 1)
Under an argon atmosphere Boc-Cys(Trt)-OH or Cbz-Cys(Cbz)-OH was
dissolved in a sufficient amount of dry DMF in a flame dried flask. After
addition of N-methylmorpholine [NMM] (0.25 ml per mmol acid) the solu-
tion was cooled to –15 °C and isobutyl chloroformate (0.13 ml per mmol
acid) was added. After 5 min a solution of the amine component (1 equiva-
lent) in dry DMF was added. When the amine component was employed as
a hydrochloride, an additional equivalent of 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). If a solid precipitate was formed,
this was collected by suction and thoroughly washed with water. Otherwise,
the aqueous mixture was extracted with EtOAc (3 × 100 ml) and the
combined organic extracts were washed successively with 2N citric acid, sat.
NaHCO3 solution, and brine, and dried with MgSO4. The residue obtained
after removal of the solvent was purified by recrystallization or flash chro-
matography.
Acknowledgement
The pGEX-DPR1 and pBC-RAM2 plasmids were kindly provided by
Prof. F. Tamanoi (UCLA). M.S. wishes to thank Prof. W. Hanefeld for his
generous support. I.S. wishes to thank Prof. Dr. S. Grabley and
PD Dr. R. Thiericke for their generous support and S. Egner for technical
assistance.
Experimental
General Protocol for the O-Deprotection of Methionine Methyl Esters
(Protocol 2)
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 a AutoSpec mass spectrometer from Micromass. IR spectra
were recorded on a Nicolet 510P FT-IR-spectrometer. Microanalyses were
obtained with a CH analyzer according to Dr. Salzer from Labormatic and
from a Hewlett Packard CHN analyser type 185. Column chromatography
was carried out using silica gel 60 (0.062–200 mm) from Merck.
The methyl esters were dissolved in a 1:1 mixture of THF and methanol
(50 ml per mmol) and stirred after addition of 1 equivalent of 1N NaOH at
rt until the reaction was complete (TLC). The solvents were evaporated and
the solid residue was used without further purification for the following
N,S-deprotection step.
General Protocol for the N-Boc-S-Trt-Cysteine N,S-Deprotection
(Protocol 3)
General Protocol for the Preparation of Nitrobenzenesulfonamides 6 and 13
The anilide (12 or 22) or the product from the preceding O-deprotection
step (6 or 18) was dissolved in dry CH2Cl2 (6 ml per mmol). Upon addition
of trifluoroacetic acid (3 ml per mmol) the color of the solution turned brown.
Triethylsilane was added until the solution became colorless. Stirring was
continued for 1 h. After removal of the volatiles in vacuo the solid residue
was washed several times with n-hexane to remove most of the triphenyl-
methane. Then the solid was dissolved in a minimum amount of EtOAc and
an excess of diethyl ether, saturated with HCl(g) was added. The precipitate
was collected and washed with dry diethyl ether.
To a solution of 2,3-dimethylaniline 12 or methionine methyl ester hydro-
chloride 4 in a sufficient amount of dry CH2Cl2 and N-methylmorpholine
[NMM] (0.24 ml per mmol amine) one equivalent of the appropriate ni-
trobenzenesulfonyl chloride was added at 0 °C. Stirring was continued
overnight. The reaction mixture was diluted with CH2Cl2 and washed suc-
cessively with 2N citric acid, sat. NaHCO3-solution, and brine, and dried
with MgSO4. The product obtained after the removal of the solvent were used
without further purification.
Arch. Pharm. Pharm. Med. Chem. 332, 124–132 (1999)

Peptidomimetic Farnesyltransferase Inhibitors
129
N-[4-(N-tert-Butyloxycarbonyl-S-tritylcysteinylamino)phenylsulfonyl]-
N-[4-(N-tert-Butyloxycarbonyl-S-tritylcysteinylaminomethyl)-
methionine Methyl Ester 9
trans-cyclohexylcarbonyl]methionine Methyl Ester 22
From 7 (0.371 g, 1.17 mmol) according to protocol 1. Purification: flash-
chromatography EtOAc:n-hexane 3:2. Yield 0.504 g (56%).– Mp 74 °C. IR
From 20 (0.530 g, 1.32 mmol) according to protocol 1. Purification: flash-
chromatography EtOAc:n-hexane 3:2. Yield 0.7 g (71%).– Mp >200°C.– IR
(KBr): ν = 3315, 2975, 1750, 1695, 1650 cm–1.– 1H NMR (CDCl3): δ = 0.94
(m, 2H), 1.36–1.45 (m, 11H), 1.69 (s, 2H), 1.79 (m, 2H), 1.89 (m, 2H), 1.96
(m, 1H), 2.08 (s, 2H), 2.15 (m, 1H), 2.45–2.52 (m, 3H), 2.73 (m, 1H), 3.05
(m, 2H), 3.75 (m, 3H), 3.81 (s, 1H), 4.70 (m, 1H), 4.80 (s, 1H), 6.10 (s, 1H),
6.17 (d, J = 8 Hz, 1H), 7.19–7.31 (m, 9H), 7.39–7.42 (m, 6H).– 13C NMR
(CDCl3): δ = 15.5, 28.2, 28.7, 29.0, 29.7, 30.0, 31.6, 33.6, 37.2, 45.1, 51.3,
52.5, 53.6, 67.2, 80.3, 126.9, 128.0, 129.5, 144.4, 170.4, 172.6, 175.4.– Anal.
(C41H53N3O6S) C, H.
1
(KBr): ν = 3400, 1690, 1595 cm–1.– H NMR (CDCl3): δ = 1.45 (s, 9H),
1.83–1.92 (m, 1H), 1.97–2.04 (m, 1H), 2.05 (s, 3H), 2.54 (m, 2H), 2.73 (m,
1H), 2.81 (m, 1H), 3.52 (s, 3H), 3.87 (m, 1H), 4.02 (m, 1H), 4.75 (d, J = 7
Hz, 1H), 5.26 (d, J =9 Hz, 1H), 7.20–7.31 (m, 9H), 7.41–7.44 (m, 6H),
7.57–7.62 (m, 2H), 7.73–7.76 (m, 2H).– 13C NMR (CDCl3): δ = 15.4, 27.9,
28.2, 29.6, 32.6, 52.7, 54.6, 67.6, 81.2, 119.4, 127.0, 127.9, 128.1, 128.5,
129.5, 140.3, 141.7, 144.3, 169.2, 171.8. ESI-MS: m/z (%) 786 (100)
[M+Na]+, 802 (44) [M+K]+, 1550 (45) [2M+Na]+. ESI-HRMS calcd for
[M+Na]+ C39H45NaN3O7S3: 786.231736; found: 786.231001.
N-(4-Cysteinylaminomethyl-trans-cyclohexylcarbonyl)methionine
N-[4-(Cysteinylamino)phenylsulfonyl]methionine Hydrochloride 11
Hydrochloride 24
From 9 (0.222 g, 0.29 mmol) according to protocols 2 and 3. Yield 0.068 g
(53%). Mp 120 °C.– IR (KBr): ν = 3395, 1685, 1590 cm–1.– 1H NMR
([D6]DMSO): δ = 1.71–1.85 (m, 2H), 1.93 (s, 3H), 2.45 (m, 2H), 3.10 (m,
1H), 3.20 (m, 1H), 3.85 (m, 1H), 4.40 (m, 1H), 7.55 (m, 2H), 7.86 (m, 2H).
ESI-MS: m/z (%) 408 (97) [M+H]+, 430 (54) [M+Na]+, 452 (24) [M-
H+2Na]+. ESI-HRMS calcdfor [M+H]+C14H22N3O5S3: 408.072162; found:
408.073979.
From 22 (0.152 g, 0.2 mmol) according to protocols 2 and 3. Yield 0.052 g
(61%).– Mp 60 °C (dec.).– IR (KBr): ν = 3410, 2920, 1660, 1600 cm–1.– 1H
NMR ([D6]DMSO): δ = 0.91 (m, 2H), 1.28–1.49 (m, 4H), 1.72–1.87 (m, 4H),
1.91–1.98 (m, 1H), 2.03 (s, 3H), 2.12–2.16 (m, 1H), 2.50 (m, 2H), 2.89–3.07
(m, 2H), 3.35–3.40 (m, 2H), 3.98 (m, 1H), 4.28 (m, 1H), 7.97 (d, J = 8 Hz,
1H), 8.40 (s, 2H), 8.61 (t, J = 6 Hz, 1H).– ESI-MS: m/z (%) 392 (100)
[M+H]+, 414 (35) [M+Na]+, 773 (25) [2M+H]+.– ESI-HRMS calcd for
[M+H]+ C16H29N3O4S2: 392.167775; found: 392.163182.
3-(N-tert-Butyloxycarbonyl-S-tritylcysteinylamino)-N-(2,3-dimethyl-
phenyl)-benzenesulfonamide 16
4-(N-tert-Butyloxycarbonylaminomethyl)-N-(2,3-dimethylphenyl)-
trans-cyclohexylcarboxamide 25
From 14 (0.552 g, 2 mmol) according to protocol 1. Purification: flash-
chromatography EtOAc:n-hexane 3:2. Yield 0.685 g (47%).– Mp 101 °C. IR
(KBr): ν = 3385, 2975, 1690 cm–1.– 1H NMR (CDCl3): δ = 1.35 (s, 9H), 1.88
(s, 3H), 2.09 (s, 3H), 2.53–2.54 (m, 1H), 2.64–2.69 (m, 1H), 4.76 (m, 1H),
6.44 (s, 1H), 6.82–6.88 (m, 3H), 7.09–7.25 (m, 12H), 7.31–7.33 (m, 5H),
7.65–7.69 (m, 2H), 8.35 (s, 1H).– 13C NMR (CDCl3): δ = 13.9, 20.5, 28.2,
33.2, 54.6, 67.5, 81.2, 118.1, 122.9, 123.4, 123.9, 125.9, 127.0, 128.1, 128.5,
129.5, 132.1, 134.0, 141.3, 144.3, 169.1. ESI-MS: m/z (%) 722 (2) [M+H]+,
744 (45) [M+Na]+, 1466 (30) [2M+Na]+. ESI-HRMS calcd for [M+Na]+
C41H43NaN3O5S2: 744.254185; found: 744.256178.
From 18 (1.03 g, 4 mmol) and 12 (0.49 ml, 4 mmol) following protocol 1.
Purification: recrystallization from toluene/dioxane. Yield 1.3 g (94%).– 1H
NMR (CDCl3): δ = 0.94–1.06 (m, 2H), 1.43 (m, 11H), 1.52–1.62 (m, 3H),
1.86–1.89 (m, 2H), 2.03–2.08 (m, 2H), 2.12 (s, 3H), 2.16–2.25 (m, 1H), 2.99
(m, 2H), 4.58 (s, 1H), 6.97–7.00 (m, 2H), 7.08 (m, 1H), 7.45–7.47 (m, 1H).
4-Aminomethyl-N-(2,3-dimethylphenyl)-trans-cyclohexylcarboxamide
Hydrochloride 26
25 (0.720 g. 2 mmol) was dissolved in 4N HCl in dioxane (20 ml) and
stirred for 2 h at rt. Then, the solution was diluted with diethyl ether and the
precipitate was collected and washed with diethyl ether. Yield 0.380 mg
(64%). The product was used without further characterization for the next
step.
3-Cysteinylamino-N-(2,3-dimethylphenyl)benzenesulfonamide Hydrochlo-
ride 17
From 16 (0.292 g, 0.4 mmol) according to protocol 3. Yield 0.124 g
(75%).– Mp 129 °C. IR (KBr): ν = 3185, 3060, 2975, 1700, 1660 cm–1. 1H
NMR ([D6]DMSO): δ = 1.96 (s, 3H), 2.16 (s, 3H), 2.90 (m, 1H), 3.08 (m,
1H), 4.25 (s, 1H), 6.71 (m, 1H), 6.95 (m, 1H), 7.01 (m, 1H), 7.37 (m, 1H),
7.53 (m, 1H), 7.85 (m, 1H), 8.01 (m, 1H), 8.54 (s, 2H), 9.66 (s, 1H), 11.23
(s, 1H).– 13C NMR ([D6]DMSO): δ = 14.2, 20.2, 52.3, 117.6, 122.4, 123.3,
124.8, 125.5, 128.2, 129.7, 133.9, 134.6, 137.6, 138.6, 141.5, 166.0.– ESI-
MS: m/z (%) 380 (100) [M+H]+, 759 (55) [2M+H]+. ESI-HRMS calcd for
[M+H]+ C17H22N3O3S2: 380.110260; found: 380.112706; calcd for
[M+Na]+ C17H21NaN3O3S2: 402.092205; found: 402.094161.
4-(N-tert-Butyloxycarbonyl-S-tritylcysteinylaminomethyl)-N-(2,3-dimethyl-
phenyl)-trans-cyclohexylcarboxamide 28
From 26 (0.380 g, 1.28 mmol) according to protocol 1. Yield 0.606 g
(85%).– Mp 137 °C (toluene).– IR (KBr): ν = 3285, 2925, 1655 cm–1.– 1H
NMR (CDCl3): δ = 0.91–0.97 (m, 2H), 1.41 (s, 9H), 1.47–1.58 (m, 3H), 1.83
(m, 2H), 2.01 (m, 2H), 2.10 (s, 3H), 2.19 (m, 1H), 2.28 (s, 3H), 2.48–2.52
(m, 1H), 2.72 (m, 1H), 3.07 (m, 2H), 3.82 (m, 1H), 6.13 (s, 1H), 6.98–7.05
(m, 2H), 7.07 (m, 1H), 7.16–7.30 (m, 9 H), 7.40–7.44 (m, 6H).– 13C NMR
(CDCl3): δ = 13.7, 20.5, 28.2, 29.2, 29.7, 33.7, 37.3, 45.2, 46.0, 53.6, 67.2,
80.3, 122.2, 125.8, 126.9, 127.3, 128.0, 128.2, 129.0, 129.2, 129.5, 135.2,
137.3, 144.4, 170.4, 173.9.– ESI-MS: m/z (%) 728 (100) [M+Na]+, 744 (17)
[M+K]+, 1434 (32) [2M+Na]+. ESI-HRMS calcd for [M+Na]+
C43H51NaN3O4S: 728.349799; found: 728.348756.
N-[4-(N-tert-Butyloxycarbonylaminomethyl)-trans-cyclohexylcarbonyl]-
methionine Methyl Ester 19
From 18 and 4 following protocol 1. Yield: 1.75 g (87%).– Mp 134 °C
(EtOAc).– IR (KBr): ν = 3350, 2935, 1740, 1685, 1650 cm–1.– 1H NMR
(CDCl3): δ = 0.98 (m, 2H), 1.43 (s, 9H), 1.45–1.55 (m, 2H), 1.84 (m, 2H),
1.90–2.05 (m, 5H), 2.09 (s, 3H), 2.10–2.20 (m, 1H), 2.49 (m, 2H), 3.00 (m,
2H), 3.75 (s, 3H), 4.71 (m, 1H), 6.16 (m, 1H).– MS: m/z (%) 402 (6) [M+],
272 (100), 162 (55), 95 (75).– EI-HRMS calcd for C19H34N2O5S:
402.218844; found: 402.222839.
4-Cysteinylaminomethyl-N-(2,3-dimethylphenyl)-trans-cyclohexylcarbox-
amide Hydrochloride 29
From 28 (0.310 g, 0.44 mmol) according to protocol 3. Yield 0.175 g
(100%).– Mp 100 °C.– IR (KBr): ν = 3260, 2960, 1655, 1605 cm–1.– 1H
NMR ([D6]DMSO): δ = 0.98 (m, 2H), 1.42 (m, 2H), 1.86 (m, 2H), 2.02 (s,
3H), 2.22 (s, 3H), 2.35 (m, 1H), 2.82 (m, 1H), 2.96 (m, 2H), 3.05 (m, 1H),
3.98 (m, 1H), 7.00 (m, 3H), 8.35 (s, 1H), 8.58 (s, 2H), 9.21 (s, 1H).– 13C
NMR ([D6]DMSO): δ = 14.0, 20.2, 28.9, 29.7, 36.9, 44.2, 45.3, 51.2, 123.9,
125.1, 126.9, 131.5, 136.4, 136.8, 166.7, 174.2.– ESI-MS: m/z (%) 364 (100)
[M+H]+, 386 (7) [M+Na]+, 727 (22) [2M+H]+. ESI-HRMS calcd for [M+H]+
N-(4-Aminomethyl-trans-cyclohexylcarbonyl)methionine Methyl Ester
Hydrochloride 20
19 (0.800 g, 2 mmol) was dissolved in 4N HCl in dioxane (20 ml) and
stirred for 2 h at rt. Then the solution was diluted with diethyl ether and the
precipitate was collected and washed with diethyl ether. Yield 0.530 g (66%).
The product was used without further characterization for the next step.
Arch. Pharm. Pharm. Med. Chem. 332, 124–132 (1999)

130
Schlitzer, Sattler, and Dahse
C19H30N3O2S: 364.205874; found: 364.205350; calcd for [M+Na]+
C19H29NaN3O2S: 386.197819; found: 386.191135.
N-(2-Benzoyl-4-chlorphenyl)-2-phenylacetamide 31a
From 30 (0.693 g, 3 mmol) und phenylacetic acid chloride (0.45 ml,
3.3 mmol) according to protocol 4. Yield 0.91 g (87%).– Mp 94 °C. IR (KBr):
ν = 3290, 1690, 1640, 1595 cm–1.– 1H NMR (CDCl3): δ = 3.73 (s, 2H),
7.28–7.38 (m, 5H), 7.45–7.51 (m, 4H), 7.59–7.66 (m, 3H), 8.58 (m, 1H),
10.58 (s, 1H).– 13C NMR (CDCl3): δ = 45.5, 123.1, 127.5, 128.5, 129.0,
129.5, 129.9, 132.5, 132.9, 133.8, 134.0, 137.8, 138.7, 170.1, 198.0. MS: m/z
(%) 349 (20) [M+], 258 (45), 231 (86), 91 (100). Anal. (C21H16ClNO2): C,
H, N.
N-[4-(N,S-bis-Benzyloxycarbonylcysteinylamino)phenylsulfonyl]-
methionine Methyl Ester 8
From 7 (0.320 g, 1.0 mmol) according to protocol 1. Purification: flash-
chromatography EtOAc:n-hexane 3:2. Yield 0.068 g (10%). Mp 134 °C. IR
(KBr): ν = 3380, 3315, 1715, 1680, 1650, 1595, 1340, 1275, 1155 cm–1.– 1H
NMR (CDCl3): δ = 1.90 (m, 1H), 2.01–2.06 (m, 1H), 2.06 (s, 3H), 2.54 (m,
2H), 3.26 (dd, J = 15, 8 Hz, 1H), 3.40 (dd, J = 15, 5 Hz, 1H), 3.54 (s, 3H),
4.06 (m, 1H), 4.65 (m, 1H), 5.15 (s, 2H), 5.22 (s, 2H), 5.78 (d, J = 8 Hz, 1H),
7.30–7.36 (m, 10H), 7.57–7.60 (m, 2H), 7.75–7.78 (m, 2H).– 13C NMR
(CDCl3): δ = 15.4, 29.7, 32.6, 32.9, 52.8, 54.7, 56.3, 67.8, 70.0, 119.7, 127.0,
128.1, 128.4, 128.5, 128.6, 128.7, 128.8, 134.6, 134.8, 135.7, 141.4, 168.4,
171.7, 171.8.– ESI-MS: m/z (%) 712 (100) [M+Na]+, 728 (88) [M+K]+.
ESI-HRMS calcd for [M+Na]+ C31H35NaN3O9S3: 712.143315; found:
712.142578.
N-(2-Benzoyl-4-chlorophenyl)-2-chloro-2-phenylacetamide 31b
From 30 (2.3 g, 10 mmol) and α-chlorophenylacetic acid chloride
(1.75 ml, 10 mmol) according to protocol 4. Yield 3.7 g (96%).– Mp 140 °C.
IR (KBr): ν = 3260, 3060, 2950, 1680, 1640, 1575 cm–1. 1H NMR (CDCl3):
δ = 5.50 (s, 1H), 7.34–7.40 (m, 4H), 7.49–7.56 (m, 5H), 7.61–7.67 (m, 1H),
7.70–7.74 (m, 2H), 8.57 (m, 1H), 11.66 (s, 1H).– 13C NMR (CDCl3): δ =
62.2, 123.0, 125.4, 127.9, 128.4, 128.7, 129.1, 129.3, 130.0, 132.9, 133.1,
134.0, 136.6, 137.7, 138.1, 167.0, 198.1. MS: m/z (%) 385 (5) [M+], 260 (34),
258 (100), 180 (43), 91 (30).– Anal. (C21H15Cl2NO2) C, H, N, Cl.
3-(N,S-bis-Benzyloxycarbonylcysteinylamino)-N-(2,3-dimethylphenyl)-
benzenesulfonamide 15
N-(2-Benzoyl-4-chlorphenyl)-2-(1-methyltetrazol-5-ylmercapto)-2-phenyl-
acetamide 31c
From 14 (0.4 g, 1.46 mmol) according to protocol 1. Purification: flash-
chromatography EtOAc:n-hexane 3:2. Oil. Yield 0.150 g (15%).– 1H NMR
(CDCl3): δ = 1.99 (s, 3H), 2.19 (s, 3H), 3.26 (dd, J = 14, 7 Hz, 1H), 3.39 (dd,
J = 14, 4 Hz, 1H), 4.57 (m, 1H), 5.14 (s, 2H), 5.20 (d, J = 12 Hz, 1H), 5.22
(d, J = 12 Hz, 1H), 5.81 (d, J = 8 Hz, 1H), 6.48 (s, 1H), 6.95–6.97 (m, 3H),
7.32–7.37 (m, 13H), 7.90 (s, 1H).– 13C NMR (CDCl3): δ = 13.9, 20.6, 29.7,
56.2, 67.8, 69.9, 118.3, 123.3, 123.5, 124.2, 126.0, 128.1, 128.4, 128.6, 128.7,
129.6, 132.1, 133.9, 124.6, 135.8, 138.0, 140.5, 168.4, 171.6.– ESI-MS: m/z
(%) 670 (100) [M+Na]+, 686 (30) [M+K]+. ESI-HRMS calcd for [M+Na]+
C33H33NaN3O7S2: 670.165764; found: 670.164937.
31b (1.15 g, 3 mmol) and 5-mercapto-1-methyltetrazole sodium salt
(0.540 g, 3.4 mmol) were refluxed in methanol/dioxane (1:1, 50 ml) for 2 h.
The residue obtained after removal of the solvent was stirred with EtOAc.
Yield 1.0 g (72%).– Mp 108 °C.– IR (KBr): ν = 3250, 3065, 2950, 1690,
1625, 1595 cm–1.– 1H NMR (CDCl3): δ = 3.88 (s, 3H), 5.81 (s, 1H),
7.33–7.39 (m, 3H), 7.46–7.49 (m, 3H), 7.50–7.51 (m, 1H), 7.54–7.57 (m,
2H), 7.60–7.64 (m, 3H), 8.52 (m, 1H), 11.21 (s, 1H).– 13C NMR (CDCl3): δ
= 33.6, 58.3, 123.0, 125.0, 128.2, 128.4, 128.5, 129.4, 129.8, 132.7, 133.0,
133.9, 134.3, 137.5, 138.1, 152.5, 166.7, 198.1.– MS: m/z (%) 463 (1) [M+],
231 (32), 206 (100), 105 (56). Anal. (C23H18ClN5O2S) C, H, N, Cl.
N-{4-[N,S-bis-Benzyloxycarbonylcysteinyl]aminomethyl)-trans-cyclohexyl-
carbonyl}methionine Methyl Ester 21
N-(2-Benzoyl-4-chlorphenyl)-2-(2-naphthyl)acetamide 31d
From 30 (0.693 g, 3 mmol) and 2-naphthylacetic acid chloride (0.612 g,
3.0 mmol) according to protocol 4. Yield 1.0 g (83%).– Mp 88 °C (ether/pe-
troleum ether).–IR (KBr): ν = 3200, 3055, 1705, 1640, 1595 cm–1.– 1H NMR
(CDCl3): δ = 3.90 (s, 2H), 7.44–7.50 (m, 7H), 7.56–7.64 (m, 3H), 7.80–7.86
(m, 4H), 8.58 (d, J = 9 Hz, 1H), 10.65 (s, 1H).– 13C NMR (CDCl3): δ = 45.7,
123.2, 125.0, 126.0, 126.3, 127.3, 127.5, 127.7, 127.8, 128.4, 128.6, 128.8,
129.9, 131.6. 132.5, 132.7, 133.0, 133.7, 133.8, 137.8, 138.8, 170.1, 197.9.–
MS: m/z (%) 399 (9) [M+], 231 (32), 168 (100), 141 (82). Anal.
(C25H18ClNO2) C, H, N, Cl.
From 20 (0.508 g, 1.5 mmol) according to protocol 1. Yield 0.89 g (88%).–
Mp 170 °C (toluene). IR (KBr): ν = 3295, 3035, 1715, 1690, 1650, 1540
cm–1.– 1H NMR (CDCl3): δ = 0.97 (m, 2H), 1.48 (m, 2H), 1.63 (m, 1H), 1.78
(m, 2H), 1.90–2.05 (m, 4H), 2.10 (s, 3H), 2.10–2.20 (m, 1H), 2.50 (m, 2H),
3.10 (m, 2H), 3.22 (m, 1H), 3.32 (m, 1H), 3.77 (s, 1H), 4.39 (m, 1H), 4.71
(m, 1H), 5.24 (m, 2H), 5.68 (m, 1H), 6.16 (d, J = 8 Hz, 1H), 6.33 (s, 1H),
7.36 (s, 10H).– 13C NMR (CDCl3): δ = 15.6, 28.8, 29.0, 29.8, 30.1, 31.8,
33.2, 37.2, 45.2, 45.5, 51.4, 52.5, 52.6, 55.6, 67.4, 69.7, 128.1, 128.4, 128.5,
128.6, 128.7, 134.8, 136.1, 169.7, 171.6, 172.7, 175.4. ESI-MS: m/z (%) 674
(7) [M+H]+, 696 (100) [M+Na]+, 712 (14) [M+K]+. Anal. (C33H43N3O8S2)
C, H, N.
N-(2-Benzoyl-4-chlorophenyl)--methylcinnamic acid amide 31e
From 30 (1.15 g, 5 mmol) and α-methylcinnamic acid chloride (0.9 g,
5 mmol) according to protocol 4. Yield 0.1 g (5%).– Mp 91 °C. IR (KBr): ν
= 3285, 3050, 1675, 1640, 1620, 1595 cm–1.– 1H NMR (CDCl3): δ = 2.26
(s, 3H), 7.29–7.34 (m, 1H), 7.39–7.41 (m, 4H), 7.49–7.54 (m, 2H), 7.55–7.58
(m, 2H), 7.60–7.65 (m, 2H), 7.69–7.73 (m, 2H), 8.80 (s, 1H), 11.36 (s, 1H).–
13C NMR (CDCl3): δ = 14.0, 123.1, 124.5, 127.2, 128.1, 128.3, 128.5, 129.5,
129.8, 132.0, 132.8, 132.9, 134.1, 135.9, 136.2, 138.0, 139.5, 167.9, 198.7.–
MS: m/z (%) 375 (8) [M+], 146 (100), 117 (54). Anal. (C23H18ClNO2) C, H,
N, Cl.
4-(N,S-bis-Benzyloxycarbonylcysteinylaminomethyl)-N-(2,3-dimethyl-
phenyl)-trans-cyclohexylcarboxamide 27
From 26 (0.350 g, 1.18 mmol) according to protocol 1. Yield 0.12 g
(16%).– Mp: 202 °C (toluene). IR (KBr): ν = 3280, 3035, 1715, 1690, 1640
cm–1.– 1H NMR (CDCl3): δ = 0.90–1.00 (m, 2H), 1.35–1.45 (m, 2H),
1.75–1.78 (m, 2H), 1.85–1.87 (m, 3H), 2.03 (s, 3H), 2.24 (s, 3H), 2.31 (m,
1H), 2.85 (m, 2H), 2.95 (m, 1H), 3.08 (m, 1H), 4.23 (m, 1H), 5.05 (s, 2H),
5.26 (s, 2H), 6.97–7.07 (m, 4H), 7.30–7.40 (m, 9H), 7.54 (m, 1H), 8.00 (m,
1H), 9.16 (s, 1H).– 13C-NMR (CDCl3): δ = 13.7, 18.7, 19.9, 27.5, 28.7, 29.4,
32.9, 36.7, 44.1, 44.8, 54.2, 65.5, 68.6, 123.6, 124.8, 126.6, 127.4, 127.6,
128.0, 128.1, 128.2, 128.3, 131.2, 135.1, 136.1, 136.5, 136.7, 169.1, 169.5,
173.8.– ESI-MS: m/z (%) 632 (55) [M+H]+. Anal. (C35H41N3O6S) C, H, N.
N-(2-Benzoyl-4-chlorphenyl)-3-cyclohexylpropionamide 31f
From 30 (2.3 g, 10 mmol) and 3-cyclohexylpropionic acid chloride (1.2 g,
10 mmol) according to protocol 4. Yield 3.4 g (94%).– Mp 104 °C (etha-
nol).– IR (KBr): ν = 3370, 3065, 2920, 1680, 1660, 1595 cm–1.– 1H NMR
(CDCl3): δ = 0.88–0.99 (m, 2H), 1.10–1.32 (m, 4H), 1.60–1.79 (m, 7H),
2.39–2.48 (m, 2H), 7.49–7.54 (m, 4H), 7.59–7.65 (m, 1H), 7.67–7.74 (m,
2H), 8.62 (m, 1H), 10.64 (s, 1H).– 13C NMR (CDCl3): δ = 26.3, 26.6, 32.8,
33.1, 36.0, 37.3, 123.1, 124.5, 127.1, 128.6, 129.9, 132.7, 133.0, 134.0, 138.0,
139.2, 172.7, 198.5.– MS: m/z (%) 369 (8) [M+], 231 (100), 230 (48). Anal.
(C22H24ClNO2) C, H, N, Cl.
General protocol for the acylation of 2-aminobenzophenones (Protocol 4)
The 5-substituted 2-aminobenzophenone was dissolved in hot toluene and
the appropriate acid chloride was added. After 2 h under reflux the major part
of the solvent was removed in vacuo. The remaining solution was kept at rt
until the product precipitated.
Arch. Pharm. Pharm. Med. Chem. 332, 124–132 (1999)

Peptidomimetic Farnesyltransferase Inhibitors
131
3-[N-(2-Benzoyl-4-chlorophenyl)carbamoyl]propionic Acid Methyl Ester
31g
5 mM DTT, 7 µM Ds-GCVLS, 20 µM FPP, and 5 nmol GST-FTase and 1%
of various concentrations of the test compounds dissolved in DMSO. The
progress of the enzyme reaction was followed by the enhancement of the
fluorescence emission at 505 nm (excitation: 340 nm). Fluorescence emis-
sion was recorded with BMG Polarstar microplate reader. IC50’s were
calculated from initial velocity of three independent masurements of each
inhibitor concentration and expressed as mean ± SD.
From 30 (2.3 g, 10 mmol) and succinic acid methyl ester chloride (1.5 ml,
12 mmol) according to protocol 4. Yield 2.8 g (81%).– Mp 82 °C (ether/pe-
troleum ether). IR (KBr): ν = 3350, 2950, 1745, 1700, 1640, 1600 cm–1.– 1H
NMR (CDCl3): δ = 2.73 (m, 4H), 3.67 (s, 3H), 7.47–7.52 (m, 4H), 7.59–7.64
(m, 1H), 7.67–7.71 (m, 2H), 8.56–8.60 (m, 1H), 10.70 (s, 1H).– 13C NMR
(CDCl3): δ = 29.0, 32.5, 52.0, 123.2, 124.7, 127.3, 128.6, 130.0, 133.0, 134.0,
137.9, 138.9, 170.5, 173.0, 198.4.– MS: m/z (%) 345 (27) [M+], 231 (100),
230 (65), 115 (62).– Anal (C18H16ClNO4): C, H, N, Cl.
Cells and Culture Conditions
Established suspended human leukaemic cell lines K-562 cells, a chronic
myeloid leukaemic cell line, HL-60 cells, an acute myeloid leukaemic cell
line and THP-1 cells, an acute monocytic leukaemic cell line were cultured
in RPMI 1640 medium (Gibco, cat.-no. 15140-114), supplemented with 100
U/ml penicillin, 100 µg/ml streptomycin and 10% FBS.
3-[N-(2-Benzoyl-4-chlorphenyl)carbamoyl]propionic acid 31h
From 31f (0.173 g, 0.5 mmol) according to protocol 2. The crude product
was dissolved in water, acidified with conc. hydrochloric acid and extracted
with EtOAc. The title compound was obtained after removal of the solvent.
Yield 0.167 g (91%).– Mp >200 °C.– IR (KBr): ν = 3440, 1685, 1630,
1595 cm–1.– 1H-NMR ([D6]DMSO): δ = 2.21 (m, 4H), 3.37 (m, 1H), 7.50
(m, 3H), 7.62 (m, 4H), 10.04 (s, 1H).– 13C-NMR ([D6]DMSO): δ = 28.5,
30.4, 125.5, 128.1, 129.0, 129.3, 131.3, 132.7, 134.9, 136.5, 170.0, 173.2,
198.1.– Anal. (C17H14ClNO4): C, H, N.
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 microplates (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 humidified atmosphere, and 5% CO2.
N-(2-Benzoyl-4-nitrophenyl)-2-phenylacetamide 33
From 32 (1.2 g, 5 mmol) and phenylacetic acid chloride (0.8 ml, 5 mmol)
according to protocol 4. Yield 1.7 g (94%).– 1H-NMR (CDCl3): δ = 3.79 (s,
2H), 7.37 (m, 2H), 7.52 (m, 3H), 7.64 (m, 3H), 8.15 (m, 1H), 8.25 (m, 1H),
8.41 (m, 1H), 8.46 (m, 1H), 8.98 (m, 1H), 11.08 (s, 1H).
Methods of Evaluation
After incubation suspension cultures of K-562, HL-60, and THP-1 cells
were analysed by the Cell Counter + Analyser System CASY 1 (SCHÄRFE,
Reutlingen, Germany). The principle of measurement and evaluation of data
is described in ref.[27]. The 0.2 ml content of each well in the microplate was
diluted 1:50 with CASYTON (SCHÄRFE). Every count/ml was automat-
ically calculated from the arithmetic mean of three successive counts of
0.4 ml each. The software for data evaluation (CASYSTAT, SCHÄRFE)
offers a fast graphical evaluation of the measurement parameters, for exam-
ple, as diagrams of cell diameter distribution, overlays of different curves,
and cell volume distributions.
The essential parameters for the estimation of growth inhibition (GI) and
for changes in diameter distribution curves are expressed as diagrams. The
GI50 value was defined as being where the concentration-response curve
intersected the 50% line, determined by means of the cell counts/ml, com-
pared to control.
N-(4-Amino-2-benzoylphenyl)-2-phenylacetamide 34
From 33 (1.7 g, 4.7 mmol) by tin(II)chloride reduction as described above.
Yield 1.38 g (89%).– 1H-NMR (CDCl3): δ = 3.53 (s, 2H), 3.61 (s, 2H), 6.68
(m, 1H), 6.79 (m, 1H), 7.18–7.30 (m, 5H), 7.36–7.40 (m, 2H), 7.51 (m, 1H),
7.62 (m, 2H), 8.21 (m, 1H), 10.05 (s, 1H).
N-[3-Benzoyl-4-(2-phenylacetylamino)phenyl]-Nα-tert-butyloxycarbonyl-
S-tritylcysteine Amide 35
From BocCys(Trt)OH (0.695 g, 1.5 mmol) and 34 (0.495 g, 1.5 mmol)
according to protocol 1. Purification: column chomatography EtOAc:n-hex-
ane 3:2. Yield 1.11 g (96%).– Mp 80 °C.– IR (KBr): ν = 3310, 3060, 2980,
1690, 1595 cm–1.– 1H-NMR (CDCl3): δ = 1.38 (s, 9H), 2.55 (m, 1H), 2.74
(m, 1H), 3.72 (s, 2H), 4.75 (m, 1H), 7.13–7.33 (m, 7H), 7.35–7.42 (m, 6H),
7.45 (m, 2H), 7.56 (m, 1H), 7.64 (m, 2H), 7.71 (m, 1H), 8.52 (m, 1H), 10.51
(m, 1H).– ESI–MS: m/z 776 (4) [M+H+], 798 (100) [M+Na+], 814 (40)
[M+K+]. Anal. (C48H45N3O5S): C, H, N. ESI-HRMS calcd for [M+Na+]
C48H45N3O5SNa: 798.297764; found: 798.293261.
References
[1] P. J. Casey, M. C. Seabra, J. Biol. Chem. 1996, 271, 5289–5292.
[2] D. M. Leonard, J. Med. Chem. 1997, 40, 2971–2990.
[3] S. L. Graham, Exp. Opin. Ther. Patents 1995, 5, 1269–1285.
N-[3-Benzoyl-4-(2-phenylacetylamino)phenyl]cysteinamide Hydrochloride
36
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