Synthesis and evaluation of homofarnesoyl-substituted CAAX-peptidomimetics as farnesyltransferase inhibitors and antiproliferative agents

Article abstract of DOI:10.1016/S0968-0896(99)00165-0

Several CAAX-peptidomimetics were linked to homofarnesoic acid via a β-alanyl spacer with the intention to obtain a novel type of bisubstrate analogue farnesyltransferase inhibitors. However, the compounds were found to be only weakly active in the farnesyltransferase inhibition assay. Nevertheless, they displayed antiproliferative activity against different tumor cell lines in the low micromolar range. Replacement of the β-alanine moiety by aspartic acid-1-methyl ester resulted in a compound which inhibited the farnesyltransferase with an IC₅₀ of 860 nM. The corresponding free acid showed a eightfold loss in activity (IC₅₀ = 6.9 μM).

Full text of DOI:10.1016/S0968-0896(99)00165-0

Bioorganic & Medicinal Chemistry 7 (1999) 2037±2045
Synthesis and Evaluation of Homofarnesoyl-Substituted
CAAX-Peptidomimetics as Farnesyltransferase Inhibitors
and Antiproliferative Agents
Martin Schlitzer, a,* Isabel Sattler b and Hans-Martin Dahse b
a
Institut fur Pharmazeutische Chemie, Philipps-Universitat Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
È
È
Hans-Knoll-Institut fur Natursto€-Forschung e.V., Beutenbergstraûe 11, D-07745 Jena, Germany
b
È
È
Received 8 March 1999; accepted 3 May 1999
AbstractÐSeveral CAAX-peptidomimetics were linked to homofarnesoic acid via a b-alanyl spacer with the intention to obtain a
novel type of bisubstrate analogue farnesyltransferase inhibitors. However, the compounds were found to be only weakly active in
the farnesyltransferase inhibition assay. Nevertheless, they displayed antiproliferative activity against di€erent tumor cell lines in
the low micromolar range. Replacement of the b-alanine moiety by aspartic acid-1-methyl ester resulted in a compound which
inhibited the farnesyltransferase with an IC50 of 860 nM. The corresponding free acid showed a eightfold loss in activity (IC50=6.9
mM). # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
Inhibition of the farnesylation reaction reverses the
transformation caused by oncogenic ras. Therefore,
inhibition of the farnesyltransferase was identi®ed as a
promising target in cancer therapy. Intense e€orts of
several groups have resulted in a large number of dif-
ferent FTase inhibitors.1±8 Most groups used the CAAX
recognition motif as a template for their inhibitors. In
comparison to CAAX mimetics obtained by this
approach, stable non-substrate analogues of the second
substrate farnesylpyrophosphate are much less com-
mon.1±8 Since the ras farnesylation is a bisubstrate
reaction and therefore two binding domains for two
di€erent substrates have to exist in the enzyme in close
proximity, an interesting approach is the development
of bisubstrate analogues: molecules that display struc-
tural features of both substrates, the CAAX tetrapep-
tide and the farnesyl pyrophosphate. One example of
these bisubstrate analogues is 1 which is 100-fold more
active (IC50=0.033 mM) than its parent tetrapeptide
CVLS (IC50=4 mM).2 Representative examples of the
few published bisubstrate analogues (1±4)9±11 are shown
in Chart 1.
Ras proteins are members of the family of small guanyl
nucleotide binding proteins. They serve as molecular
switches in the signal transduction cascade which con-
trols cell di€erentiation and proliferation. Point muta-
tions in the ras oncogene yield ras proteins locked in
their GTP-bound active state. 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 oncogenic functions, ras proteins must undergo a
series of post-translational modi®cations. First, a 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
(FTase) and farnesylated at the cysteine thiol function.
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 reversibly converted into its meth-
yl ester. From these post-translational modi®cation
events only the farnesylation is obligatory for ras activity
(see reviews1±8).
Inhibitors 1±4 contain an unmodi®ed AAX tripeptide
motif and the `original' farnesyl residue. These bisub-
strate analogues di€er only in the nature of the linker
connecting the two parts of the molecule. We intended
to explore whether it is possible to develop novel bisub-
strate analogue inhibitors of farnesyltransferase by
replacing the peptidic structures by non-peptidic CAAX
Key words: Antiproliferative agents; bisubstrate analogues; farnesyl-
transferase inhibitors; peptidomimetics; ras.
* Corresponding author.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00165-0

2038
M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
surrogates. From six CAAX and CAA-peptidomimetics
16
ing methyl esters 26a and 27a. The aspartic acid deriva-
tive 39 was prepared by the addition of N-Z-aspartic
acid-1-methyl ester to 32. After hydrogenolytic removal
of the N-protective group, acylation with homo-
farnesoic acid to 41a was carried out as described
above. The synthesis was completed by the saponi®ca-
tion of the methyl ester yielding the target compound
41b (Scheme 3).
previously described12
we used the AAX- and AA-
mimetic substructures, respectively, for our envisioned
bisubstrate analogues. These were linked via b-alanine
with homofarnesoic acid which functioned as the
lipophilic part of our target structures. We chose
homofarnesoic acid because in a series of farnesylpyr-
ophosphate-based inhibitors of farnesyltransferase the
homofarnesoyl derivative turned out to be a more active
antagonist of FPP than the corresponding farnesoyl
derivative.17
Farnesyltransferase inhibition assay
The inhibitory activity of the potential synthetic inhibi-
tors was determined using the ¯uorescence assay as
described by Pompliano.12 The assay employed yeast
farnesyltransferase (FTase) fused to Glutathione S-
transferase at the N-terminus of the b-subunit.20 Farne-
sylpyrophosphate and the dansylated pentapeptide
DsGlyCysValLeuSer 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.
Chemistry
The amino acid 2,3-dimethylanilides 6 and 12 have been
prepared from 2,3-dimethylaniline and the appropriate
N-protected amino acid (5 and 11). Either acidic or
hydrogenolytic removal of the N-protective group and
subsequent acylation with N-Boc-b-alanine, activated as
mixed anhydride, yielded compounds 8 and 14, respec-
tively. The N-Boc protective groups were removed with
HCl in dioxane and the resulting hydrochlorides 9 and
15 were coupled with homofarnesoic acid using PyBOP
methodology (Scheme 1).
Antiproliferation assay
Homofarnesoic acid was prepared as described17 in two
steps from farnesyl bromide. The nitrobenzamides 18,
29 and nitrobenzenesulfonamides 19, 30, respectively,
were prepared by reaction of the appropriate nitro-
benzoic acid chloride and nitrobenzenesulfonyl chloride
with 2,3-dimethylaniline 28 and methionine methyl ester
hydrochloride 17, respectively. Reduction of the nitro
group using tin(II)chloride dihydrate in boiling ethyl
acetate18 yielded the corresponding aminobenzene deri-
vatives 20, 21, 31 and 32. Addition of N-Boc-b-alanine,
acidic removal of the protective group and subsequent
acylation with homofarnesoic acid as described above
completed the synthesis (Scheme 2). In case of the
methionine derivatives 26b and 27b, the ®nal step of the
synthesis was the alkaline hydrolysis of the correspond-
The target compounds were tested against cell lines K-
562 (chronic myeloid leukaemic cell line),21 THP-1
(acute monocytic leukaemic cell line),22 and HL-60
(acute myeloid leukaemic cell line)23 for their anti-
proliferative e€ects. The cells were incubated with dif-
ferent concentrations of the test compounds and an
automatic volumetric cell analysis was carried out dur-
ing the logarithmic phase of cell proliferation.
Results and Discussion
The results of the farnesyltansferase inhibiton assay are
displayed in Table 1; unfortunately, the Nꢀ-homo-
farnesoyl-b-alanine amides were only weakly active.
Most of the compounds inhibited the enzyme less than
50% at 100 mM, the highest concentration assayed. In
case of the methionine derivatives the presence of the
Figure 1. Farnesylation of ras proteins.
Chart 1. Literature known bisubstrate analogues.

M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
2039
carboxyl group either as its methyl ester as in 26a and
27a or as a sodium salt as in 26b and 27b made no sig-
ni®cant di€erence in activity. Only the isoleucine deri-
vative 10 displayed a somewhat higher activity. b-
alanine was chosen as the linker connecting the lipohilic
and the peptidomimetic substructures of our target
molecules because the distance between its carbonyl
carbon and its nitrogen is the same as the distance
between the carbonyl carbon and the thiol sulfur in
cysteine. However, the introduction of a methoxy-
carbonyl group into the linking structure by replace-
ment of the b-alanine by aspartic acid-1-methyl ester
(compound 41a) resulted in a dramatic enhancement in
the farnesyltransferase inhibitory activity. One could
speculate that the side chain ester carbonyl group exerts
a much stronger interaction with the crucial zinc atom
in the farnesyltransferases catalytic center than the
amide moiety of the other compounds. This interaction
obviously adds a considerable amount of binding a-
nity which might be responsible for the improved inhi-
bitory activity. Surprisingly, saponi®cation of 41a to the
corresponding free acid 41b led to a eightfold reduction
of the inhibitory activity. The compounds were applied
to an antiproliferation assay using three di€erent tumor
cell lines. Because compounds carrying free carboxyl
functions usually show poor membrane penetration,
Scheme 1. I. (a) isobutyl chloroformate, NMM, DMF, 15ꢀC, 5 min; (b) 2,3-dimethylaniline, DMF, 15ꢀC ! rt, overnight; II. 4 N HCl/dioxane,
rt, 2 h; III. (a) N-Boc-b-alanine, isobutyl chloroformate, NMM, DMF, 15ꢀC, 5 min; (b) add 7, NMM, DMF to (a), 15ꢀC ! rt, overnight; IV. 4
N HCl/dioxane, rt, 2 h; V. homofarnesoic acid, PyBOP, DIPEA, DMF, rt, overnight; VI. (a) isobutyl chloroformate, NMM, DMF, 15ꢀC, 5 min;
(b) 2,3-dimethylaniline, DMF, 15ꢀC ! rt, overnight; VII. Pd/C, H2, EtOH, rt, 2 h; VIII. (a) N-Boc-b-alanine, isobutyl chloroformate, NMM,
DMF, 15ꢀC, 5 min; (b) add 13, NMM, DMF to (a), 15ꢀC ! rt, overnight; IX. 4 N HCl/dioxane, rt, 2 h; X. homofarnesoic acid, PyBOP,
DIPEA, DMF, rt, overnight.
Table 1. Farnesyltransferase inhibitory and antiproliferative activity of homofarnesoyl substituted CAA(X) mimetics
Compound
FTase inhibition
Antiproliferative activity [IC50 (mM)]
HL-60
THP-1
K-562
10
16
77% at 100 mM
0% at 100 mM
35% at 100 mM
0% at 100 mM
21% at 100 mM
5% at 100 mM
31.4
74.2
14.7
nda
6.5
nd
11.2
5.3
>93
>96
13.3
nd
22.2
nd
14.7
8.5
>78
nd
55.2
>96
27.1
nd
12.2
nd
15.8
6.9
>78
nd
26a
26b
27a
27b
37
38
41a
41b
16% at 100 mM
22% at 100 mM
IC50=860‹52 nM
IC50=6.9‹0.9 mM
60.7
nd
a
nd: not determined.

2040
M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
Scheme 2. I. 4-Nitrobenzoic acid chloride or 4-nitrobenzenesulfonic acid chloride, NMM, CH2Cl2, 0ꢀC ! rt, overnight; II. SnCl2ÂH2O, EtOAc,
re¯ux, 2 h; III. (a) N-Boc-b-alanine, isobutyl chloroformate, NMM, DMF, 15ꢀC, 5 min; (b) add 20 or 21, DMF to (a), 15ꢀC ! rt, overnight; IV.
4 N HCl/dioxane, rt, 2 h; V. homofarnesoic acid, PyBOP, DIPEA, DMF, rt, overnight; VI. 1 N NaOH, THF/MeOH, rt; VII. 3-nitrobenzoic acid
chloride or 3-nitrobenzenesulfonic acid chloride, NMM, CH2Cl2, 0ꢀC ! rt, overnight; VIII. SnCl2Â2H2O, EtOAc, re¯ux, 2 h; IX. (a) N-Boc-b-
alanine, isobutyl chloroformate, NMM, DMF, 15ꢀC, 5 min; (b) add 31 or 32, DMF to (a), 15ꢀC ! rt, overnight; X. 4 N HCl/dioxane, rt, 2 h;
XI. homofarnesoic acid, PyBOP, DIPEA, DMF, rt, overnight.
Scheme 3. I. (a) N-Z-aspartic acid-1-methyl ester, isobutyl chloroformate, NMM, DMF, 15ꢀC, 5 min; (b) add 32, DMF to (a), 15ꢀC ! rt,
overnight; II. H2, Pd/C, EtOAc, rt; III. homofarnesoic acid, PyBOP, DIPEA, DMF, rt, overnight; IV. (a) 1 N LiOH, DME, rt; (b) HCl.

M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
2041
compounds 26b, 27b and 41b were excluded from this
assay. The isoleucine derivative 10 and the proline deri-
vative 16 showed no signi®cant e€ect in the anti-
proliferation assay. In contrast, the benzoic acid
derivatives 26a and 37 and the benzenesulfonic acid
derivatives 27a and 38 showed a strong antiproliferative
activity against all three cell lines. Highest activity was
found with the sulfonamide 38, which inhibited the
proliferation of all cell lines in low micromolar doses.
Replacement of the sulfonamide moiety of 38 by an
amide as in 37 resulted in a decrease of activity with the
sulfonamide 38 being approximately twice as active as
the corresponding amide 37. With the methionine deri-
vatives 26a and 27a, the e€ect of the replacement of the
amide by a sulfonamide is not that obvious. While the
sulfonamide derivative 27a is roughly twice as active
against the HL-60 and the K-562 cells as the amide
derivative 26a, this ratio is reversed for the THP-1 cells.
Interestingly, the presence of a carbonyl group in the
linker substructure as in 41a which resulted in a sig-
ni®cant increase in the farnesyltransferase inhibition
had a detrimental e€ect on the antiproliferative activity.
Since compounds 26a, 27a, 37 and 38 display con-
siderable antiproliferative activity but do not inhibit
farnesyltransferase, and in contrast, 41a is a good far-
nesyltransferase inhibitor but does not have an anti-
proliferative e€ect, it can be concluded that ras
inhibition is most likely not responsible for the observed
antiproliferative activity. The mechanism by which the
observed activity is exerted, is open for speculation. It
has been shown, that another farnesylated molecule, S-
farnesylthioacetic acid, induces apoptosis in HL-60 cell.
The authors suggest that this may be caused by an
interference of S-farnesylthioacetic acid with a speci®c
recognition of isoprenylated protein moieties.24 There is
accumulating evidence that isoprenoid moieties of cer-
tain proteins could play an important role in protein±
protein interactions.25±30 In fact, an isoprenoid acceptor
structure has been identi®ed in rho-GDI, the guanosine
diphosphate dissociation inhibitor of rho proteins.31
Inhibition of such interactions has been demonstrated
with lipids32 and prenylated molecules.33,34 Because of
their chemical structure, our molecules might well be
able to interrupt isoprenoid-mediated proteinÐprotein
interactions.
Investigations addressing structure±activity relationships
and the mode of action of these agents are under way.
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
FT±IR-spectrometer. Microanalyses were obtained
from a CH analyzer according to Dr. Salzer from
Labormatic and from a Hewlett Packard CHN-analyzer
type 185. Column chromatography was carried out
using silica gel 60 (0.062Ð0.200 mm) from Merck.
General protocol for the preparation of nitrobenzamides
18, 29 and nitrobenzenesulfonamides 19 and 30. To a
solution of 2,3-dimethylaniline or methionine methyl
ester hydrochloride in a sucient amount of dry
CH2Cl2 and N-methylmorpholine (0.24 mL per mmol
amine) 1 equiv of the appropriate nitrobenzoic acid or
nitrobenzenesulfonamide, respectively was added at 0ꢀC.
Stirring was continued overnight. The reaction mixture
was diluted with CH2Cl2 and washed successively with 2
N citric acid, sat. NaHCO3-solution and brine and dried
with MgSO4. The product obtained after the removal of
the solvent was used without further puri®cation or
characterization.
General protocol for the reduction of nitrobenzene deri-
vatives 18, 19, 29 and 30 to the corresponding amines 20,
21, 31 and 32. To a solution of the nitro derivative in
EtOAc (approx 5 mL per mmol) SnCl2ÂH2O (1.125 g
per mmol nitro compound) was added. The soln. 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 EtOAc (3Â100±200 mL) and the com-
bined organic extracts were thoroughly washed with
brine and dried over MgSO4. The products obtained
after the removal of the solvent were used without fur-
ther puri®cation or characterization.
In summary, with compounds 41a,b we have obtained a
new lead structure for bisubstrate analogue farnesyl-
transferase inhibitors. This compounds are di€erent
from known bisubstrate analogues in having the pepti-
dic moiety replaced by a peptidomimetic structure. Our
ongoing research is directed to further improvement of
the inhibitory activity of this novel class of farnesyl-
transferase inhibitors. Special attention will be paid to
the replacement of the homofarnesoyl residue by other
non-prenylic lipophilic structures. In addition, the pep-
tidomimetic moiety will be replaced by building blocks
which structural relation to the farnesyltransferase sub-
strate is even less pronounced. Very recently, we repor-
ted the ®rst results of this strategy.35,36
General protocol for the coupling of N-protected amino
acids with various amines using mixed anhydride
activation
Protocol 1. The N-protected amino acid 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 15ꢀC and isobutyl chlor-
oformate (0.13 mL per mmol acid) was added. A solu-
tion of the amine component (1 equiv) in dry DMF was
added after 5 min. When the amine component was
employed as a hydrochloride, additional NMM (0.25
mL per mmol) was added. The mixture was allowed to
warm up to room temperature overnight and then
Furthermore, a novel potent class of antiproliferative
agents as exempli®ed by compound 38 was discovered.

2042
M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
poured into brine (400±800 mL). In case a solid pre-
cipitate was formed, this was collected by suction and
thoroughly washed with water. Otherwise the aqueous
mixture was extracted with EtOAc (3Â100 mL) and the
combined organic extracts were washed successively
with 2 N citric acid, satd NaHCO3-soln and brine and
dried with MgSO4. The residue obtained after removal
of the solvent was puri®ed by recrystallization or ¯ash
chromatography.
2.6 mmol) was dissolved in EtOH and stirred after
addition of Pd/C (10%) under an atmosphere of H2 for
2 h at room temperature. The solution was ®ltered
through Celite and silica gel. The residue obtained after
removal of the solvent was coupled with N-Boc-b-ala-
nine following protocol 1. Compound 14 was puri®ed
by ¯ash chromatography (EtOAc). Yield: 993 mg
(98%); mp 65ꢀC. IR (KBr) n 3305, 2975, 1700, 1640
1
cm 1. H NMR (CDCl3) d 1.43 (s, 9H), 1.70 (m, 1H),
1.85 (m, 1H), 2.05 (m, 1H), 2.15 (s, 3H), 2.28 (s, 3H),
2.56 (t, J=6 Hz, 2H), 2.60±2.64 (m, 1H), 3.41±3.48 (m,
3H), 3.52 (m, 1H), 4.82 (d, J=8 Hz, 1H), 5.28 (s, 1H),
6.95±6.97 (m, 1H), 7.06±7.09 (m, 1H), 7.66±7.68 (m,
1H), 9.03 (s, 1H); 13C NMR (CDCl3) d 13.7, 20.6, 25.0,
26.8, 28.4, 34.8, 36.0, 47.5, 60.3, 79.3, 120.9, 125.7,
126.6, 127.5, 135.9, 137.1, 155.9, 168.9, 172.5. MS m/z
(%) 389 (3) [M+], 142 (25), 70 (100). Anal. calcd for
C21H31N3O4 (389.49): C, 64.76; H, 8.02; N, 10.79;
found: C, 64.39; H, 8.09; N, 10.67.
General protocol for the N-Boc-deprotection
Protocol 2. The N-Boc derivatives were dissolved in 4 N
HCl in dioxane (10 mL per mmol) and stirred for 2 h at
room temperature. After addition of diethylether the
volatiles were distilled in vacuo into a ¯ask immersed in
liquid N2. The solid residue was used without further
puri®cation.
General protocol for the coupling of homofarnesoic acid
with ꢀ-alanine amides using PyBOP methology
N-[4-(3-tert-Butyloxycarbonylaminopropionyl)aminobenz-
oyl]methionine methyl ester 22. Compound 20 (530 mg,
1.88 mmol) was coupled with N-Boc-b-alanine follow-
Protocol 3. One equivalent of homofarnesoic acid, the
b-alanine amide and PyBOP (520 mg per mmol homo-
farnesoic acid), respectively, were dissolved in dry DMF
in a ¯ame dried ¯ask. After addition of diisopropy-
lethylamine (0.65 mL per mmol homofarnesoic acid) the
mixture was stirred at room temperature overnight.
Then the mixture was poured into brine (400±800 mL).
The aqueous mixture was extracted with EtOAc (3Â100
mL) and the combined organic extracts were washed
successively with 2 N citric acid, satd NaHCO3-solution
and brine and dried with MgSO4. The residue obtained
after removal of the solvent was puri®ed by ¯ash chro-
matography.
ing protocol 1. Yield: 546 mg (64%); mp 148ꢀC
1
(EtOAc). IR (KBr) n 3310, 2980, 1750, 1635, 1600 cm
.
1H NMR (DMSO-d6) d 1.38 (s, 9H), 2.06 (m, 5H), 2.49±
2.59 (m, 4H), 3.23 (m, 2H), 3.65 (s, 3H), 4.57 (m, 1H),
6.82 (s, 1H), 7.66±7.68 (m, 2H), 7.82±7.85 (m, 2H), 8.58
(d, J=8 Hz, 1H), 10.12 (s, 1H); 13C NMR (DMSO-d6) d
14.5, 28.2, 29.9, 30.1, 36.3, 36.8, 51.6, 51.8, 118.1, 127.9,
128.3, 142.0, 155.5, 166.1, 169.8, 172.4. MS m/z (%) 453
(2) [M+], 305, (62), 279 (64), 217 (100), 120 (82). Anal.
calcd for C21H31N3O6S (453.55): C, 55.61; H, 6.89; N,
9.26; found: C, 55.78; H, 6.80; N, 9.05.
N-[4-(3-tert-Butyloxycarbonylaminopropionyl)aminophe-
nylsulfonyl]methionine methyl ester 23. Compound 21
(795 mg, 2.5 mmol) was coupled with N-Boc-b-alanine
following protocol 1. Yield: 860 mg (70%); mp 145ꢀC
(EtOAc/n-hexane). IR (KBr) n 3360, 3270, 2980, 1745,
N-tert-Butyloxy-N-(2,3-dimethylphenyl)isoleucine amide
6. Compound 6 was prepared from Boc-Ile-OH (5) fol-
lowing protocol 1.
1
N-Benzyloxy-N-(2,3-dimethylphenyl)proline amide 12.
Compound 12 was prepared from Z-Pro-OH (11) fol-
lowing protocol 1.
1680 cm 1. H NMR (CDCl3) d 1.48 (s, 9H), 1.86±1.95
(m, 1H), 2.00±2.05 (m, 1H), 2.06 (s, 3H), 2.56 (m, 2H),
2.65 (m, 2H), 3.51 (m, 2H), 3.55 (s, 3H), 4.07 (m, 1H),
5.19 (m, 1H), 5.52 (d, J=8 Hz, 1H), 7.70±7.72 (m, 2H),
7.76±7.78 (m, 2H) 8.64 (s, 1H); 13C NMR (CDCl3) d
15.3, 28.4, 29.6, 32.5, 36.3, 38.2, 52.7, 54.6, 80.1, 119.3,
128.4, 134.0, 142.3, 156.6, 170.1, 171.8. MS m/z (%) 313
(12), 164 (51), 162 (100). Anal. calcd for C20H31N3O7S2
(489.60): C, 49.06; H, 6.38; N, 8.58; found: C, 48.70; H,
6.15; N, 8.42.
N-(3-tert-Butyloxycarbonylaminopropionyl)-N-(2,3-di-
methylphenyl)isoleucine amide 8. Compound 6 (668 mg,
2 mmol) was deprotected following protocol 2 and cou-
pled with N-Boc-b-alanine following protocol 1. Yield:
668 mg (82%); mp 204ꢀC (EtOAc/MeOH). IR (KBr) n
1
3330, 3295, 2965, 2935, 1690, 1645 cm
.
1H NMR
(CDCl3) d 0.93 (t, J=7 Hz, 3H), 1.01 (d, J=6 Hz, 3H),
1.20 (m, 1H), 1.40 (s, 9H), 1.60 (m, 1H), 2.00 (m, 1H),
2.08 (s, 3H), 2.24 (s, 3H), 2.45 (m, 2H), 3.38 (m, 2H),
4.41 (m, 1H), 5.09 (s, 1H), 6.49 (m, 1H), 6.98 (m, 1H),
7.03 (m, 1H), 7.37 (m, 1H), 7.74 (s, 1H); 13C NMR
(CDCl3) d 11.2, 13.8, 15.7, 20.5, 25.1, 28.3, 36.3, 36.7,
58.5, 79.4, 122.1, 125.8, 127.6, 129.4, 134.8, 137.5, 156.0,
169.6, 171.9. MS m/z (%) 405 (1) [M+], 121 (100).
Anal. calcd for C22H35N3O4 (405.54): C, 65.16; H, 8.70;
found: C, 65.58; H, 9.08.
3 - [(3 - tert - Butyloxycarbonylaminopropionyl)amino] - N -
(2,3-dimethylphenyl)-benzamide 33. Compound 31 (480
mg, 2 mmol) was coupled with N-Boc-b-alanine follow-
ing protocol 1. Compound 33 was puri®ed by ¯ash
chromatography EtOAc. Yield: 428 mg (52%); mp
159ꢀC (toluene). IR (KBr) n 3295, 1710, 1685, 1650,
1
1590, 1430, 1280, 1170 cm 1. H NMR (DMSO-d6) d
1.35 (s, 9H), 2.09 (s, 3H), 2.27 (s, 3H), 2.48 (m, 2H), 3.23
(m, 2H), 6.80 (s, 1H), 7.06±7.13 (m, 3H), 7.42 (m, 1H),
7.64 (m, 1H), 7.81 (m, 1H), 8.11 (m, 1H), 9.84 (s, 1H),
10.06 (s, 1H). MS m/z (%) 411 (6) [M+], 311 (91), 283
(65), 217 (65), 191 (100). Anal. calcd for C23H29N3O4
N-(3-tert-Butyloxycarbonylaminopropionyl)-N-(2,3-di-
methylphenyl)proline amide 14. Compound 12 (915 mg,

M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
2043
(411.50): C, 67.13; H, 7.10; N, 10.21; found: C, 67.20; H,
7.05; N, 10.15.
¯ash chromatography (acetone:n-hexane, 2:1). Yield:
135 mg (22%); mp 54ꢀC. IR (KBr) n 3300, 2970, 1655,
1535 cm±1. H NMR (CDCl3) d 1.59 (s, 8H), 1.67 (s,
1
3-(tert-Butyloxycarbonylamino)-N-[3-(2,3-dimethylphenyl-
aminosulfonyl)phenyl]propionamide 34. Compound 32
(610 mg, 2.2 mmol) was coupled with N-Boc-b-alanine
following protocol 1. Compound 34 was puri®ed by
¯ash chromatography (EtOAc:n-hexane, 3:2). Yield:
340 mg (35%); mp 124ꢀC. IR (KBr) n 3330, 2975, 1685
cm 1. 1H NMR (DMSO-d6) d 1.37 (s, 9H), 1.96 (s, 3H),
2.17 (s, 3H), 2.49 (m, 2H), 3.21 (m, 2H), 5.47 (s, 1H),
6.70±6.79 (m, 2H), 6.91±7.01 (m, 2H), 7.21±7.29 (m,
1H), 7.41±7.46 (m, 1H), 7.76 (m, 1H), 8.01 (s, 1H), 9.49
(s, 1H), 10.16 (s, 1H); 13C NMR (DMSO-d6) d 15.6,
20.7, 28.8, 36.8, 37.2, 78.0, 117.3, 121.8, 123.1, 125.7,
126.3, 128.0. 130.0, 134.7, 138.1, 140.2, 141.7, 152.8,
156.0, 170.4. MS m/z (%) 447 (0.1) [M+], 373 (33), 276
(66), 120 (100). Anal. calcd for C22H29N3O5S (447.55):
C, 59.04; H, 6.53; N, 9.39; found: C, 59.07; H, 6.10; N,
9.72.
3H), 1.75 (s, 1H), 1.81±191 (m, 1H), 1.95±2.11 (m, 10H),
2.15 (s, 3H), 2.28 (s, 3H), 2.52±2.62 (m, 2H), 2.92 (d,
J=7 Hz, 2H), 3.52 (m, 1H), 3.54±3.62 (m, 3H), 4.77 (d,
J=7 Hz, 1H), 5.08 (m, 2H), 5.25 (m, 1H), 6.49 (m, 1H),
6.95±6.97 (m, 1H), 7.07 (m, 1H), 7.60±7.62 (m, 1H), 8.93
(s, 1H); 13C NMR (CDCl3) d 13.6, 16.0, 16.2, 17.7, 20.6,
23.4, 25.0, 25.6, 26.7, 26.9, 34.3, 35.0, 35.9, 39.6, 39.7,
47.6, 60.4, 116.3, 116.9, 121.0, 123.1, 124.2, 125.7, 126.8,
128.1, 131.3, 135.8, 137.1, 141.0, 169.0, 171.6, 172.4. MS
m/z (%) 521 (13) [M+], 452 (119, 219 (21), 70 (100).
HRMS calcd for C32H47N3O3 (521.74): 521.3617;
found: 521.3598.
(E,E)-N-{4-[3-(4,8,12-Trimethyl-1-oxo-3,7,11-tridecatri-
enyl)aminopropionyl]-aminobenzoyl}methionine methyl ester
26a. Compound 22 (545 mg, 1.2 mmol) was deprotected
following protocol 2 and coupled with homofarnesoic
acid following protocol 3. Compound 26 was puri®ed
by ¯ash chromatography (EtOAc). Yield: 50 mg (7%);
mp 110ꢀC. IR (KBr) n 3260, 3080, 2920, 1750, 1635
2-(2S)-(Benzyloxycarbonylamino)-3-[3-(2,3-dimethylphe-
nylaminosulfonyl)phenylcarbamoyl]propionic acid methyl
ester 39. Compound 32 (552 mg, 2 mmol) was coupled
with N-benzyloxycarbonyl-aspartic acid-1-methyl ester
(562 mg, 2 mmol) following protocol 1. Compound 39
was puri®ed by ¯ash chromatography (EtOAc:n-
1
cm 1. H NMR (CDCl3) d 1.52 (s, 8H), 1.60 (s, 3H),
1.89±2.10 (m, 13H), 2.19 (m, 1H), 2.50±2.58 (m, 4H),
2.90 (d, J=7 Hz, 2H), 3.50 (m, 2H), 3.71 (s, 3H), 4.84
(m, 1H), 5.00 (t, J=7 Hz, 2H), 5.20 (t, J=7 Hz, 1H),
6.51 (t, J=5 Hz, 1H), 7.01 (d, J=8 Hz, 1H), 7.59±7.62
(m, 2H), 7.68±7.71 (m, 2H), 9.18 (s, 1H); 13C NMR
(CDCl3) d 15.5, 16.0, 16.2, 17.6, 25.6, 26.3, 26.7, 30.1,
31.4, 35.5, 35.8, 37.0, 39.5, 39.6, 52.1, 52.6, 115.9, 119.1,
123.6, 124.2, 128.1, 128.7, 131.3, 135.5, 141.7, 141.8,
166.6, 170.1, 172.5, 172.6. MS m/z (%) 585 (37) [M+],
286 (33), 217 (31), 191 (33), 120 (100). HRMS calcd for
C32H47N3O5S (585.80): 585.3236; found: 585.3245.
hexane, 3:2). Yield: 605 mg (56%); mp 59ꢀC. H
1
NMR (CDCl3) d 1.97 (s, 3H), 2.18 (s, 3H), 2.97 (m, 1H),
3.14 (m, 1H), 3.74 (s, 3H), 4.66 (m, 1H), 5.08 (s, 2H),
6.10 (d, J=8 Hz, 1H), 6.78 (s, 1H), 6.92 (m, 2H), 6.98
(m, 1H), 7.29 (m, 6H), 7.86 (s, 2H), 8.26 (s, 1H). ESI±
MS m/z 540 [M+H]+, 562 [M+Na]+. ESI±HRMS
calcd for C27H30N3 O7S [M+H]+: 540.180448;
found: 540.181993; calcd for C27H29NaN3O7S
[M+Na]+: 562.162392; found: 562.162391.
(E,E)-N-{4-[3-(4,8,12-Trimethyl-1-oxo-3,7,11-tridecatri-
enyl)aminopropionyl]-aminobenzoyl}methionine, sodium salt
26b. Compound 26 (130 mg, 0.22 mmol) was dissolved
in a 1:1 mixture of THF and MeOH (15 mL) and stirred
after additon of 1 N NaOH (0.24 mL, 0.24 mmol) at
room temperature until TLC analysis indicated com-
pletion of the reaction. Then the solvents were removed
in vacuo. Yield: 128 mg (98%); mp 160ꢀC. IR (KBr) n
(E,E)-N-[3-(4,8,12-Trimethyl-1-oxo-3,7,11-tridecatrienyl)-
aminopropionyl]-N-(2,3-dimethylphenyl)isoleucine amide
10. Compound 8 (465 mg, 1.2 mmol) was deprotected
following protocol 2 and coupled with homofarnesoic
acid following protocol 3. Yield: 320 mg (50%); mp
148ꢀC (EtOAc:MeOH:n-pentane). IR (KBr) n 3275,
1
3065, 2965, 1655, 1640 cm 1. H NMR (DMSO-d6) d
1
0.85 (t, J=7 Hz, 3H), 0.92 (d, J=7 Hz, 3H), 1.18 (m,
2H), 1.54 (s, 8H), 1.62 (s, 3H), 1.65 (s, 1H), 1.80±1.90
(m, 2H), 1.91±2.04 (m, 7H), 2.04 (s, 3H), 2.23 (s, 3H),
2.32±2.37 (m, 2H), 2.77 (d, J=7 Hz, 2H), 3.22±3.38 (m,
2H), 4.36 (m, 1H), 5.07 (m, 2H), 5.23 (t, J=7 Hz, 1H),
6.99±7.08 (m, 3H), 7.65 (m, 1H), 7.99 (d, J=8 Hz,
1H), 9.40 (s, 1H); 13C NMR (DMSO-d6) d 10.9, 13.8,
15.4, 15.6, 16.0, 17.4, 19.9, 24.4, 25.9, 26.0, 26.1, 35.1,
35.4, 36.4, 37.9, 38.9, 57.3, 117.9, 123.5, 123.7, 123.8,
124.0, 124.9, 126.8, 131.1, 135.8, 136.7, 170.1, 170.5. MS
m/z (%) 537 (10) [M+], 304 (14), 121 (100). HRMS
calcd for C33H51N3O3 (537.79): 537.3930; found:
537.3927.
3300, 2920, 1605 cm 1. H NMR (DMSO-d6) d 1.56 (s,
8H), 1.62 (s, 3H), 1.90±1.96 (m, 4H), 2.01 (m, 9H), 2.50
(m, 4H), 2.80 (md, J=7 Hz, 2H), 3.30 (m, 2H), 4.00 (m,
1H), 5.06 (m, 2H), 5.24 (m, 1H), 7.68±7.74 (m, 4H),
10.32 (s, 1H). ESI-MS m/z (%) 572 [M+2H Na]+. ESI-
HRMS calcd for C31H46N3O5S: 572.315819; found:
572.313579.
(E,E)-N-{4-[3-(4,8,12-Trimethyl-1-oxo-3,7,11-tridecatrieny-
l)aminopropionyl]-aminophenylsulfonyl}methionine methyl
ester 27a. Compound 23 (400 mg, 0.8 mmol) was
deprotected following protocol 2 and coupled with
homofarnesoic acid following protocol 3. Compound 27
was puri®ed by ¯ash chromatography (EtOAc). Yield:
165 mg (33%); mp 90ꢀC. IR (KBr) n 3265, 2920, 1735,
1680, 1645 cm 1. 1H NMR (CDCl3) d 1.53 (s, 8H), 1.61
(s, 3H), 1.75 (s, 1H), 1.80±2.04 (m, 13H), 2.49 (m, 2H),
2.61 (t, J=6 Hz, 3H), 2.91 (d, J=7 Hz, 2H), 3.52 (m,
2H), 4.00 (m, 1H), 5.01 (t, J=7 Hz, 2H), 5.21 (t, J=7
(E,E)-N-[3-(4,8,12-Trimethyl-1-oxo-3,7,11-tridecatrienyl)-
aminopropionyl]-N-(2,3-dimethylphenyl)proline amide 16.
Compound 14 (466 mg, 1.2 mmol) was deprotected fol-
lowing protocol 2 and coupled with homofarnesoic acid
following protocol 3. Compound 16 was puri®ed by

2044
M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
Hz, 1H), 5.47 (d, J=9 Hz, 1H), 6.44 (t, J=6 Hz, 1H),
7.69 (m, 4H), 9.28 (s, 1H); 13C NMR (CDCl3) d 15.3,
16.0, 16.2, 17.7, 25.7, 26.3, 26.7, 29.6, 32.5, 35.6, 35.8,
37.5, 39.5, 39.7, 52.7, 54.6, 115.8, 119.3, 123.5, 124.2,
128.4, 131.4, 134.0, 135.7, 142.2, 142.6, 170.1, 171.9,
172.8. MS m/z (%) 621 (28) [M+], 552 (23), 260 (50),
162 (100). Anal. calcd for C31H47N3O6S2 (621.85): C,
59.88; H, 7.62; N, 6.76 ; found: C, 60.04; H, 7.61; N,
7.16.
1H); 13C NMR (CDCl3) d 13.9, 16.2, 17.7, 20.6, 25.7,
26.4, 26.7, 35.6, 35.8, 37.2, 39.6, 39.7, 115.9, 117.8,
120.6, 123.5, 123.6, 123.8, 124.2, 15.8, 128.5, 129.4,
131.4, 132.3, 133.9, 135.6, 138.0, 139.1, 140.3, 141.9,
170.2, 172.7. MS m/z (%) 579 (16) [M+], 330 (14), 119
(100). Anal. calcd for C33H45N3O4S (579.80): C, 68.36;
H, 7.82; N, 7.25; found: C, 68.50; H, 8.04; N, 7.95.
HRMS calcd for C33H45N3O4S: 579.313079; found:
579.310120.
(E,E)-N-{4-[3-(4,8,12-Trimethyl-1-oxo-3,7,11-tridecatri-
enyl) aminopropionyl] - aminophenylsulfonyl} methionine,
sodium salt 27b. Compound 27 (120 mg, 0.19 mmol)
was dissolved in a 1:1 mixture of THF and MeOH (15
mL) and stirred after additon of 1 N NaOH (0.2 mL,
0.2 mmol) at room termperature until TLC analysis
indicated completion of the reaction. Then, the solvents
were removed in vacuo. Yield: 118 mg (99%); mp
3-[3-(2,3-Dimethylphenylaminosulfonyl)phenylcarbamoyl]-
2-(2S)-(4,8,12-trimethyl-3,7,11-tridecatrienoylamino)pro-
pionic acid methyl ester 41a. Compound 39 (485 mg, 0.9
mmol) was dissolved in EtOAc (20 mL) and stirred after
addition of Pd/C (10%) under an atmosphere of H2 for
2 h at room temperature. The solution was ®ltered
through Celite and silica gel. The residue obtained after
removal of the solvent was coupled with homofarnesoic
acid (223 mg, 0.89 mmol) following protocol 3. Com-
pound 41a was puri®ed by ¯ash-chomatography
248ꢀC. IR (KBr) n 3310, 2920, 1660 cm 1. H NMR
1
(DMSO-d6) d 1.55 (s, 8H), 1.63 (s, 3H), 1.71 (m, 1H),
1.87±2.08 (m, 13H), 2.30 (m, 2H), 2.39±2.50 (m, 2H),
2.79 (d, J=7 Hz, 2H), 3.18 (m, 1H), 3.29 (m, 2H), 5.10
(m, 2H), 5.27 (m, 1H), 7.41 (s, 4H). ESI-MS m/z (%)
652 (100) [M H+2Na]+, 674 (27) [M 2H+3Na]+.
ESI-HRMS calcd for C30H45NaN3O6S2 [M+Na]+:
630.264750; found: 630.264572; calcd for C30H44Na2
N3O6S2 [M H+2Na]+: 652.246695; found: 652.247846.
(EtOAc:hexane, 3:2). Yield: 128 mg (22%); mp 41ꢀC.
1
IR (KBr) n 2925, 1750, 1720, 1545 cm
.
1H NMR
(DMSO-d6) d 1.57 (m, 8H), 1.66 (m, 5H), 1.92±2.03 (m,
9H), 2.04 (s, 3H), 2.20 (s, 3H), 2.98 (m, 2H), 3.04±3.15
(m, 2H), 3.76 (s, 3H), 4.79 (m, 1H), 5.08 (m, 2H), 5.30
(m, 1H), 6.89±7.03 (m, 4H), 7.10, (m, 1H) 7.30 (m, 2H),
7.93 (m, 1H), 8.91 (m, 1H). ESI-MS m/z 638 [M+H]+,
660 [M+Na]+. ESI-HRMS calcd for C35H48N3O6S
[M+H]+: 638.326384; found: 638.333211; calcd for
C35H47NaN3O6S [M+Na]+: 660.308328; found:
660.311051.
3-{[3-(4,8,12-Trimethyl-1-oxo-3,7,11-tridecatrienyl)amino-
propionyl]amino}-N-(2,3-dimethylphenyl)benzamide 37.
Compound 33 (575 mg, 1.4 mmmol) was deprotected
following protocol 2 and coupled with homofarnesoic
acid following protocol 3. Compound 37 was puri®ed
by ¯ash chromatography (EtOAc:n-hexane, 3:2). Yield:
68 mg (11%); mp 88ꢀC. IR (KBr) n 3390, 3300, 2920,
3-[3-(2,3-Dimethylphenylaminosulfonyl)phenylcarbamoyl]-
2-(2S)-(4,8,12-trimethyl-3,7,11-tridecatrienoylamino)pro-
pionic acid 41b. To a solution of 41 (120 mg, 0.19 mmol)
in DME (15 mL) 1 N LiOH-solution (15 mL) was
added and the mixture was stirred at room temperature
until the reaction was complete (TLC). Then, the vola-
tiles were removed in vacuo and the residue was dis-
solved in water (20 mL). This solution was extracted
with EtOAc. The organic phase was discarded. The
aqueous solution was adjusted to pH 1 with concd
HCl and extracted three times with EtOAc. The com-
bined organic extracts were dried with MgSO4 and
evaporated to dryness. Yield: 52 mg (47%); mp 64ꢀC.
1
1655 cm 1. H NMR (CDCl3) d 1.58 (s, 8H), 1.61 (s,
1H), 1.66 (s, 3H), 1.94±1.97 (m, 2H), 2.02±2.09 (m, 6H),
2.20 (s, 3H), 2.30 (s, 3H), 2.57 (t, J=6 Hz, 2H), 2.95 (d,
J=7 Hz, 2H), 3.55 (m, 2H), 5.07 (t, J=7 Hz, 2H), 5.27
(t, J=7 Hz, 1H), 6.34 (t, J=6 Hz, 1H), 7.06 (m, 1H),
7.12 (m, 1H), 7.40 (m, 1H), 7.51 (m, 1H), 7.59 (m, 1H),
7.76 (s, 1H), 7.81 (m, 1H), 8.10 (s, 1H), 8.76 (s, 1H); 13C
NMR (CDCl3) d 13.9, 16.0, 16.3, 17.7, 20.5, 25.7, 26.4,
26.8, 35.6, 35.9, 37.2, 39.6, 39.7, 116.1, 118.4, 122.5,
122.6, 123.0, 123.7, 124.3, 126.0, 127.9, 129.5, 130.1,
131.4, 135.3, 135.6, 137.7, 138.9, 141.7, 165.7, 170.2,
172.3. MS m/z (%) 544 (39) [M+], 543 (100), 120 (63).
HRMS calcd for C34H45N3O3 (543.75): 543.346093;
found: 543.349899.
1
IR (KBr) n 2925, 1720, 1545 cm 1. H NMR (DMSO-
d6) d 1.54 (m, 8H), 1.62 (m, 5H), 1.90±2.05 (m, 9H),
1.95 (s, 3H), 2.15 (s, 3H), 2.86 (m, 2H), 4.58 (m, 1H),
5.05 (m, 2H), 5.23 (m, 1H), 6.70 (m, 1H), 6.92 (m,
1H), 6.92 (m, 1H), 6.98 (m, 1H), 7.27 (m, 1H), 7.43
(m, 1H), 7.75 (m, 1H), 8.03 (m, 1H), 9.47 (s, 1H). ESI±
MS m/z 624 [M+H]+, 646 [M+Na]+. ESI-HRMS
calcd for C34H46N3O6S [M+H]+: 624.310734; found:
624.302916; calcd for C34H45NaN3O6S [M+Na]+:
646.292678; found: 646.292515.
N-{2-[3-(2,3-Dimethylphenylaminosulfonyl)phenylamino-
carbonyl]ethyl} - 4,8,12 - trimethyl - 3,7,11 - tridecatrienoic
amide 38. Compound 34 (340 mg, 0.76 mmol) was
deprotected following protocol 2 and coupled with
homofarnesoic acid following protocol 3. Compound 38
was puri®ed by ¯ash chromatography (EtOAc:n-hex-
ane, 3:2). Yield: 105 mg (24%); mp 105ꢀC. IR (KBr) n
3350, 2925, 1690, 1640, 1600 cm 1. 1H NMR (CDCl3) d
1.49 (s, 8H), 1.59 (s, 3H), 1.68 (s, 1H), 1.84±2.05 (m,
8H), 1.97 (s, 3H), 2.13 (s, 3H), 2.59 (t, J=6 Hz, 2H),
2.88 (d, J=7 Hz, 2H), 3.32 (m, 2H), 4.97±5.05 (m, 2H),
5.16 (m, 1H), 6.49 (t, J=6 Hz, 1H), 6.88±6.94 (m, 4H),
7.22±7.27 (m, 2H), 7.81 (m, 1H), 8.05 (s, 1H), 9.09 (s,
Farnesyltransferase assay. The assay was carried out as
described.19 The assay mixture (100 mL volume) con-
tained 50 mM Tris/HCl pH 7.4, 5 mM MgCl2, 10 mM,
ZnCl2, 5 mM DTT, 7 mM Ds-GCVLS, 20 mM FPP and
5 nmol GST-FTase20 and 1% of various concentrations
of the test compounds dissolved in DMSO. The pro-
gress of the enzyme reaction was followed by the

M. Schlitzer et al. / Bioorg. Med. Chem. 7 (1999) 2037±2045
2045
enhancement of the ¯uorescence emission at 460 nm
(excitation: 320 nm). The slight deviation from the
emission maximum (505 nm) was chosen for technical
reasons. Fluorescence emission was recorded with BMG
Polarstar microplate reader. IC50s were calculated from
initial velocity of three independent measurements of each
inhibitor concentration and expressed as mean‹SD.
8. Sattler, I.; Tamanoi, F. In Regulation of the RAS Signaling
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9. Bhide, R. S.; Patel, D. V.; Patel, M. M.; Robinson, S. P.;
Hunihan, L. W.; Gordon, E. M. Bioorg. Med. Chem. Lett.
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10. Patel, D. V.; Gordon, E. M.; Schmidt, R. S.; Weller, H. N.;
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1995, 38, 435.
11. Patel, D. V.; Young, M. G.; Robinson, S. P.; Hunihan, L.;
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Cells and culture conditions. Established suspended
human leukaemic cell lines K-562 cells (chronic myeloid
leukaemic cell line), HL-60 cells (acute myeloid leu-
kaemic cell line) and THP-1 cells (acute monocytic leu-
kaemic cell line) were cultured in RPMI 1640 medium
(Gibco, cat.-no. 15140-114), supplemented with 100 U/
mL penicillin, 100 mg/mL streptomycin and 10% FBS.
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 pre-
viously 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 sub-
stances in 0.1 mL medium. The cells were incubated for
72 h at 37ꢀC in a humidi®ed atmosphere and 5% CO2.
17. Patel, D. V.; Schmidt, R. J.; Biller, S. A.; Gordon, E. M.;
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The pGEX-DPR1 and pBC-RAM2 plasmides were
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