Exploration of novel aryl binding sites of farnesyltransferase using molecular modeling and benzophenone-based farnesyltransferase inhibitors

Article abstract of DOI:10.1021/jm010873j

Most non-thiol CAAX-peptidomimetic farnesyltransferase inhibitors bear nitrogen-containing heterocycles in place of the terminal cysteine which are supposed to coordinate the enzyme-bound zinc. However, it has been shown that those nitrogen-containing heterocycles can be replaced by carbocyclic aromatic moieties which are unable to coordinate the zinc ion, a conclusion that resulted in the postulation of one or two hitherto unknown aryl binding sites. No indication has been given about the spatial location of these novel binding sites. Employing flexible docking of several non-thiol farnesyltransferase inhibitors known from the literature and some model compounds based on our benzophenone scaffold as well as performing GRID searches, we have identified two regions in the farnesyltransferase's active site which we suggest being the postulated aryl binding sites. One aryl binding region is located in close proximity to the zinc ion and is defined by the aromatic side chains of Tyr 300β, Trp 303β, Tyr 361β, and Tyr 365β. The second aryl binding site is defined by the side chains of Tyr 300β, Leu 295β, Lys 294β, Lys 353β, and Lys 356β. This second aryl binding site has been used for the design of a non-thiol farnesyltransferase inhibitor (9c) with an IC₅₀ of 35 nM.

Full text of DOI:10.1021/jm010873j

J . Med. Chem. 2001, 44, 3117-3124
3117
Exp lor a tion of Novel Ar yl Bin d in g Sites of F a r n esyltr a n sfer a se Usin g Molecu la r
Mod elin g a n d Ben zop h en on e-Ba sed F a r n esyltr a n sfer a se In h ibitor s
Markus Bo¨hm,# Andreas Mitsch,# Pia Wissner,# Isabel Sattler,§ and Martin Schlitzer*,#
Institut fu¨r Pharmazeutische Chemie, Philipps-Universita¨t Marburg, Marbacher Weg 6, D-35032 Marburg, Germany, and
Hans-Kno¨ll-Institut fu¨r Naturstoff-Forschung e. V., Beutenbergstrasse 11, D-07745 J ena, Germany
Received March 7, 2001
Most non-thiol CAAX-peptidomimetic farnesyltransferase inhibitors bear nitrogen-containing
heterocycles in place of the terminal cysteine which are supposed to coordinate the enzyme-
bound zinc. However, it has been shown that those nitrogen-containing heterocycles can be
replaced by carbocyclic aromatic moieties which are unable to coordinate the zinc ion, a
conclusion that resulted in the postulation of one or two hitherto unknown aryl binding sites.
No indication has been given about the spatial location of these novel binding sites. Employing
flexible docking of several non-thiol farnesyltransferase inhibitors known from the literature
and some model compounds based on our benzophenone scaffold as well as performing GRID
searches, we have identified two regions in the farnesyltransferase’s active site which we suggest
being the postulated aryl binding sites. One aryl binding region is located in close proximity to
the zinc ion and is defined by the aromatic side chains of Tyr 300â, Trp 303â, Tyr 361â, and
Tyr 365â. The second aryl binding site is defined by the side chains of Tyr 300â, Leu 295â, Lys
294â, Lys 353â, and Lys 356â. This second aryl binding site has been used for the design of a
non-thiol farnesyltransferase inhibitor (9c) with an IC50 of 35 nM.
Inhibition of farnesyltransferase has received consid-
erable interest in recent years as a strategy for the
development of novel potential cancer drugs.1-3 Farne-
syltransferase catalyzes the transfer of a farnesyl
residue from farnesylpyrophosphate to the thiol of a
cysteine side chain of protein bearing the CAAX-
tetrapeptide sequence (C: cysteine, A: aliphatic amino
acid, X: serine or methionine) at its C-terminus.4 The
rationale for using inhibitors of farnesyltransferase as
anticancer agents results from the observation that
farnesylation is a prerequisite for the transforming
activity of oncogenic Ras, found in approximately 30%
of all cancers in humans. However, there is accumulat-
ing evidence that prevention of Ras farnesylation may
not be the crucial cellular event responsible for the
antiproliferative effect of farnesyltransferase inhibitors.5
Focus has shifted to RhoB, another member of the class
of small GTPases which is involved in receptor traffick-
ing.6,7 Irrespective of the unresolved issue of mechanism
by which farnesyltransferase inhibitors exert their
antiproliferative effects, the efficacy of these compounds
and their low toxicity has been demonstrated,8 and
therefore, administration of such compounds is regarded
as a major strategy emerging in cancer therapy.
effects,10 and thus the development of farnesyltrans-
ferase inhibitors is clearly directed toward the so-called
non-thiol farnesyltransferase inhibitors. Most frequently
used replacements for cysteine are nitrogen-containing
heterocycles. The ring nitrogen is supposed to coordinate
to the enzyme-bound zinc similarly to the cysteine thiol
group.11 However, it has been shown that nitrogen
heterocycles can be replaced by aryl residues lacking
the ability to coordinate metal atoms without loosing
too much of their farnesyltransferase inhibitory activ-
ity.12,13 Therefore, the existence of at least one hitherto
unknown aryl binding region in the farnesyltrans-
ferase’s active site has been postulated.14,15 Until now,
no evidence has been provided for the spatial location
of this postulated aryl binding site in the farnesyltrans-
ferase’s active site.
We intended to address this issue using flexible
docking methods of both representative non-thiol far-
nesyltransferase inhibitors known from literature12-14,16
(Chart 1) as well as some model compounds based on
our benzophenone AAX-peptidomimetic scaffold.17,18 In
addition, we performed GRID analyses searching for
regions favorable for aryl binding. Using these molec-
ular modeling studies, we are able to provide a reason-
able suggestion about the location of the above-men-
tionedpostulatedarylbindingregionsinthefarnesyltransferase’s
active site.
Most inhibitors described in the literature are pepti-
domimetics resembling the CAAX-tetrapeptide recogni-
tion sequence of farnesylated proteins. The majority of
these CAAX-peptidomimetics exhibit a free thiol group1-3
which is believed to coordinate the enzyme-bound zinc
ion as shown for the native peptide substrate.9 However,
free thiols are associated with several adverse drug
Ch em istr y
The p-tolylacetylamino-5-aminobenzophenone scaffold
6 has been prepared, as described,19 in two steps from
commercially available starting material. Compound 6
was acylated by appropriate acyl chlorides in hot toluene
to yield the target compounds 7, 8a -f, and 9a -e
(Scheme 1).
* To whom correspondence should be addressed. Phone: +49 6421
2825825. Fax: +49 6421 2827052. E-mail: schlitze@mailer.uni-
marburg.de.
#
Universita¨t Marburg.
§ Hans-Kno¨ll-Institut fu¨r Naturstoff-Forschung.
10.1021/jm010873j CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/10/2001

3118 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Bo¨hm et al.
Ch a r t 1. Structures and Activities of Representative Non-Thiol Farnesyltransferase Inhibitors Described in the
Literature That Were Used for Flexible Docking
Sch em e 1a
In case of the model compounds 8b and 8e, the tolyl-
OC-NH-moiety was assumed to be isosteric to the
HOOC-CRH-NH- fragment of the CAAX-peptide18 and
was therefore used as the starting fragment. The
docking runs provided sets of solutions which were
inspected according to their suggested binding energy.
Obviously unreasonable solutions were excluded, for
instance, those where major parts of the inhibitors were
exposed merely to the solvent without showing specific
interactions within the active site.
a
(I) R-COCl, toluene or toluene/dioxane, reflux, 2 h.
GRID An a lysis
F lexible Dock in g
The program GRID22-25 allows the indentification of
regions of energetically favorable interactions between
a molecule and a probe. It can therefore be applied to
detect those regions in a protein’s active site which are
important for the binding of partial structures with
certain physicochemical properties. A three-dimensional
grid is placed in the active site large enough to encom-
pass all residues relevant for protein-ligand interaction.
At each grid node, interaction energies between a
specific probe and all atoms of the target (binding site
of farnesyltransferase) are calculated. Depending upon
which chemical information is desired, GRID allows
different types of probes to be used which are specific
for a certain atom or fragment. Here we utilized the
“C1)” probe (sp2 CH aromatic moiety) to detect favor-
able positions for aromatic binding. As some of our
highly potent benzophenone-based inhibitors contain a
nitro group, the “ON” probe can highlight those regions
favorable for oxygen atoms of a nitro group. In contrast,
ligands containing a bromine or chlorine substituent at
the same position as the nitro group show a reduced
inhibitory activity. Therefore, inspecting the interaction
energies derived from the “CL” (chlorine) probe, for
instance, can help to explain this loss of activity.
Flexible docking of both non-thiol farnesyltransferase
inhibitors 1-5 known from literature12-14,16 (Chart 1)
as well as the model compounds 8b and 8e (Chart 2)
was performed using the program FlexX.20 On the basis
of the coordinates of the published crystal structure9 of
a ternary complex of farnesyltransferase, a farnesylpy-
rophosphate analogue and N-acetyl-Cys-Val-Ile-sele-
noMetOH (PDB-code 1QBQ), we have calculated the
solvent accessible surface of the farnesyltransferase’s
active site using the program MOLCAD which is
implemented in the molecular modeling software pack-
age SYBYL.21 Due to our working hypothesis, the
postulated aryl binding sites should be located next to
the peptide binding region. Thus, we included the
farnesylpyrophosphate as part of the enzyme’s molec-
ular surface. The position of the HOOC-CHR-NH-moiety
of the CAAX-peptide as found in the crystal structure
was used as a starting fragment for the docking of
inhibitors 1-5 into the farnesyltransferase’s active site.
Subsequently, the docking program places the remain-
ing fragments of the inhibitors in a piece-wise fashion
into the active site searching for favorable hydrophobic
and H-bond interactions while avoiding steric overlaps.

Novel Aryl Binding Sites of Farnesyltransferase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3119
Ch a r t 2. Structures and Activities of Benzophenone-Based Farnesyltransferase Inhibitors
Resu lts a n d Discu ssion
To explore these postulated aryl binding sites, we next
prepared some model compounds varying the distance
between the phenyl residue and the AAX-peptidomi-
metic benzophenone scaffold by introducing spacer
groups with successively increasing numbers of meth-
ylene groups between the phenyl ring and the terminal
amide moiety (8b-f, Chart 2). Introduction of one
methylene group in 8a as in the phenylacetic acid
derivative 8b resulted in a 3-fold improvement in
activity. Elongation of the spacer by one additional
methylene unit (8c) decreased the farnesyltransferase
inhibitory activity. Compounds 8d and 8e incorporating
spacers of three and four methylene units, respectively,
display considerably enhanced inhibitory activities with
IC50 values in the submicromolar range. Further elon-
gation of the spacer resulted again in a reduction of
activity. These results are consistent with the existence
of two different aryl binding sites as it has been
postulated.15
As a first step to embark into the class of non-thiol
farnesyltransferase inhibitors based on our benzophe-
none scaffold, we replaced the terminal cysteinyl residue
in our lead structure 1018 by a nicotinoyl moiety (7). This
resulted in a significant loss of activity (10: IC50 ) 77
nM; 7: IC50 ) 22 µM). With respect to published
results,26 this reduction in activity was not unexpected.
The subsequent replacement of the pyridyl by a phenyl
ring improved activity (8a : IC50 ) 6 µM), confirming
that the ability to coordinate the enzyme-bound zinc is
not mandatory for farnesyltransferase inhibitory activ-
ity. This result is also consistent with observations
collected by other groups12,13 and led to the postulation
of one or two hitherto unknown aryl binding sites in
the farnesyltransferase’s active site.14,15 Considering the
improved activity of the carbocycle-containing compound
8a compared to the nicotinyl derivative 7 and, above
all, because making usage of a lipophilic moiety instead
of a zinc-coordinating group represents a novel concept
in farnesyltransferase inhibitor design, we decided to
focus our attention on this type of compounds instead
of addressing the effect of different heterocyles such as
imidazoles.
Since no indication has been given about the spatial
location of these novel aryl binding sites, we performed
some docking experiments using both representative
non-thiol farnesyltransferase inhibitors 1-5 known
from literature as well as our benzophenone-type inhibi-

3120 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Bo¨hm et al.
F igu r e 1. Results from docking runs of compounds 1 (red), 2
(yellow), and 8b (blue) placing the terminal aryl residue into
a lipophilic cleft next to the zinc (magenta). Hydrophobic
properties of the enzyme’s surface are indicated in brown,
hydrophilic in green to blue colors. Enzyme-bound zinc is
shown as a magenta sphere.
tors 8b and 8f. We set forward the working hypothesis
that the aryl binding sites should be located next to the
peptide binding region in the farnesyltransferase’s
active site. As most of the compounds seem to be unable
to adopt such a strongly bended conformation which
would be necessary to fit into the peptide binding site
and, at the same time, into the prenyl binding site, it
seems appropriate to assume that the farnesyltrans-
ferase-farnesylpyrophosphate complex is the target of
this type of inhibitor although no kinetic studies were
carried out to clearly indicate this assumption. There-
fore, farnesylpyrophosphate was integrated into the
binding site while computing the protein’s molecular
surface and performing the docking runs as well as the
GRID analyses.
Flexible docking of inhibitors 1, 2, and 8b exposing
their terminal aryl moiety in relatively short distance
to the center of the molecules resulted in all three cases
in sets of solutions showing the aryl residues placed into
a lipophilic binding cleft (Figure 1) in close proximity
to the enzyme-bound zinc. This binding site (“near”) is
defined by the aromatic amino acid side chains of Tyr
300â, Trp 303â, Tyr 361â, and Tyr 365â (Figure 3).
Hydrophobic interactions of the aryl moieties of 1, 2,
and 8b with respect to the first three neighboring amino
acid side chains are suggested by FlexX for all three
inhibitors. Compounds 3, 4, 5, and 8e are obviously too
large to fit into this binding cleft. In the case of
inhibitors 3, 4, and 8e, two alternative solutions are
proposed by FlexX. One solution shows the aryl residues
bended toward the “southern hemisphere” of farnesyl-
transferase’s active site (Figure 2a). Here a hydrophobic
interaction with the phenyl residue of Tyr 361â is found.
It should be noted that this interaction formed to the
opposite side of the Tyr 361â side chain is different from
that seen with the shorter molecules 1, 2, and 8b. In
addition, the N-benzyl substituent of compound 2 is
placed by FlexX into this region. However, no compa-
rable solution in which the lipophilic residue is placed
into this southern region was predicted for compound
5. In the second subset of reasonable solutions, the aryl
moieties of all four inhibitors 3, 4, 5, and 8e are placed
into an area apparently corresponding to a separate
binding cleft located east of the peptide binding region
F igu r e 2. (a) Representative solutions of docking runs of
compounds 3 (red), 4 (yellow), and 8e (blue) which orient their
terminal aryl residues into close proximity to the hydrophobic
surface in the southern hemisphere of the farnesyltransferase’s
active site. Hydrophobic properties of the enzyme’s surface are
indicated in brown, hydrophilic in green to blue colors.
Enzyme-bound zinc is shown as a magenta sphere. (b) Rep-
resentative solutions of docking runs of compounds 3 (red), 4
(yellow), 5 (cyan), and 8e (blue) resulting in an alternative
binding mode placing the terminal aryl residues into another
separate binding cleft located east of the farnesyltransferase’s
active site.
and being connected to it via a small channel (Figure
2b). This region (“far binding site”) is defined mainly
by Tyr 300â, Leu 295â, and three lysine side chains:
Lys 294â, Lys 353â, and Lys 356â (Figure 3). Again,
the interactions of these larger compounds occur op-
posite to the phenyl ring of Tyr 300â which appears to
be involved in interactions with 1, 2, and 8b. Although
it cannot be shown conclusively, we strongly favor the
latter, the eastern region, as the second (“far”) aryl
binding site. In this relatively narrow environment
interactions between the aryl moieties of the inhibitors
and the amino acid side chains should be more pro-
nounced compared to the southern location where the
aryl moiety is located on a rather flat molecular surface
and is therefore much more exposed to the aqueous
medium than in the eastern cleft. In addition to the
flexible docking, we performed GRID searches in the
farnesyltransferase’s active site and its neighboring
regions. Using the “C1)” probe allows to search for
locations favorable to interact with aromatic carbons.

Novel Aryl Binding Sites of Farnesyltransferase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3121
F igu r e 3. Location of the two suggested aryl binding sites
(near binding site: white; far binding site: yellow) with the
principal amino acid side chains highlighted (HFP: R-hy-
droxyfarnesylphosphat, FPP analogue used for the crystal
structure). Amino acids belonging to the near binding site are
labeled white, those belonging to the far binding site are
labeled yellow. Tyr300 belongs to both binding sites.
F igu r e 5. Results of the GRID analysis searching for regions
favorable for interaction with (a) nitro oxygen and (b) chlorine.
Energy levels: -5.0 kcal mol-1, blue; -6.0 kcal mol-1, red; -7.0
kcal mol-1, yellow; -8.0 kcal mol-1, white. Enzyme-bound zinc
is shown as a magenta sphere. The proposed far aryl binding
site strongly favors the binding of nitro over chlorine.
F igu r e 4. Results of the GRID analysis searching for regions
favorable for interaction with arylic moieties. The position of
the proposed near and far aryl binding sites is indicated in
white (near) and yellow (far), respectively. Energy levels: -2.25
configurated double bond forces the biaryl moiety into
the desired direction. To our disappointment, the first
biaryl derivative 9a was only weakly active (IC50 ) 10.3
µM). However, introduction of a nitro group into the
para position of the terminal phenyl (9b) resulted in a
more than 20-fold improvement in activity (IC50 ) 455
nM). Replacement of the central thiazole by a furan
(compound 9c) resulted in an additional 13-fold im-
provement in farnesyltransferase inhibitory activity
(IC50 ) 35 nM). Replacement of the nitro group by
bromine or chlorine (compounds 9d and 9e) led to a
significant reduction in activity (IC50 ) 925 nM and IC50
) 6.3 µM, respectively). GRID analysis using the “ON”
and the “CL” probe searching for regions favorable for
binding of nitro and chlorine, respectively, revealed that
the proposed far aryl binding site possesses good binding
properties for the terminal oxygen of nitro groups
(Figure 5a) while binding of chlorine is less favored in
this particular region (Figure 5b). These computational
results mirror nicely the farnesyltransferase inhibitor
activity observed in this first series of inhibitors de-
signed with our binding model in mind. This in turn
provides further argument for the correctness of our
binding model.
kcal mol-1, red; -2.75 kcal mol-1, yellow; -3.25 kcal mol-1
,
white. Enzyme-bound zinc is shown as a magenta sphere. As
can be seen from the yellow and white colored areas, aryl
binding is strongly favored in this regions.
As shown in Figure 4, regions favorable for aryl binding
are found exactly at the same positions where FlexX
has placed the aryl residues of our model compounds.
Hence, a second computational method confirmed the
results obtained by flexible docking, thus providing
further arguments for the reasonableness of our sug-
gestion concerning the location of the postulated aryl
binding sites.
In a second step we set out to design some novel non-
thiol farnesyltransferase inhibitors based on our ben-
zophenone scaffold which are better suited to fill the
novel aryl binding sites than the first model compounds
8a -f. We decided to first focus our attention on the far
aryl binding site. A biaryl structure consisting of a five-
membered and a six-membered aromatic ring should be
able to fill this far aryl binding site. As a linking
structure between this biaryl moiety and our benzophe-
none scaffold, an acrylic acid structure seemed to be
appropriate since it (1) ensures the correct distance
between this two partial structures and (2) the trans-

3122 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Bo¨hm et al.
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
4-p h en ylbu tyr yl a m id e (8d ): from 4-phenylbutuyryl chloride
(0.185 mg, 1.0 mmol) and 6 (0.344 g, 1.0 mmol). Purification:
chromatographed on silica gel (ethyl acetate:n-hexane 2:3) to
give a yellow solid: yield 0.407 g (83%); mp 117 °C; IR (KBr)
In conclusion, we suggest that the two regions in the
farnesyltransferase’s active site defined by the side
chains of Tyr 300â, Trp 303â, Tyr 361â, and Tyr 365â
and by the side chains of Tyr 300â, Leu 295â, Lys 294â,
Lys 353â, and Lys 356â, respectively, could be the
postulated aryl binding regions. These aryl binding sides
may be used for the rational design of non-thiol farne-
syltransferase inhibitors as demonstrated with com-
pound 9c. Our ongoing research is directed toward the
establishment of deeper structure-activity relationships
of this class of farnesyltransferase inhibitors.
ν 3430, 3290, 2945, 1700, 1645 cm-1 1H NMR (400 MHz,
;
CDCl3) δ 1.94 (m, 2H), 2.20 (m, 2H), 2.26 (s, 3H), 2.60 (m, 2H),
3.60 (s, 2H), 7.10 (m, 5H), 7.17 (m, 5H), 7.41 (m, 3H), 7.53 (m,
1H), 7.61 (m, 2H), 7.72 (m, 1H), 8.44 (m, 1H), 10.41 (s, 1H);
MS (EI) m/z 490 (100, M+), 358 (52). Anal. (C32H30N2O3) C, H,
N.
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
5-p h en ylva ler yl a m id e (8e): from 5-phenylvaleryl chloride
(0.200 g, 1.0 mmol) and 6 (0.344 g, 1.0 mmol). Purification:
chromatographed on silica gel (ethylactetate:n-hexan 2:3) to
give a yellow solid: yield 0.247 (49%); mp 126 °C; IR ν 3433,
3262, 2932, 1657, 1560, 1504 cm-1; 1H NMR (400 MHz, CDCl3)
δ 1.61 (m, 4H), 2.21 (m, 2H), 2.26 (s, 3H), 2.55 (m, 2H), 3.60
(s, 2H), 7.09 (m, 5H), 7.18 (m, 5H), 7.40 (m, 3H), 7.51 (m, 1H),
7.62 (m, 2H), 7.76 (m, 1H), 8.41 (m, 1H), 10.41 (s, 1H); MS
(EI) m/z 504 (50, M+), 212 (100), 105 (68). Anal. (C33H32N2O3)
C, H, N.
Exp er im en ta l Section
1H and 13C NMR spectra were recorded on a J EOL J MN-
GX-400 and a J EOL J MN-LA-500 spectrometer. Mass spectra
were obtained with a Vacuum Generators VG 7070 H using a
Vector 1 data acquisition system from Teknivent 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 (0.062-0.200 mm) from Merck.
Gen er a l P r oced u r e for P r ep a r a tion of 7 a n d 8a -f.
5-Amino-2-tolylacetylaminobenzophenone (6) was dissolved in
hot toluene (approximately 50 mL), and a solution of 1 equiv
of various carbonic acid chlorides in dioxane (approximately
10 mL) was added. The mixtures were heated under reflux
for 2 h. Solvent was removed in vacuo to give the crude
products.
N-[3-Ben zoyl-4-[(4-m eth ylp h en yl)a cetyla m in o]p h en yl]-
n icotin oyl a m id e (7): from nicotinoyl chloride (0.142 g, 1.0
mmol) and 6 (0.344 g, 1.0 mmol). Purification: chromato-
graphed on silica gel (ethyl acetate:n-hexane 3:2) to give a
yellow solid: yield 0.099 mg (22%); mp 198 °C; IR (KBr) ν 3400,
2925, 1675, 1635, 1595, 1555 cm-1; 1H NMR (400 MHz, CDCl3)
δ 2.26 (s, 3H), 3.60 (s, 2H), 7.08 (m, 2H), 7.15 (m, 2H), 7.30
(m, 1H), 7.41 (m, 2H), 7.51 (m, 1H), 7.58 (m, 1H), 7.64 (m,
2H), 7.91 (m, 1H), 8.14 (m, 1H), 8.47 (m, 1H), 8.65 (s, 1H),
8.97 (s, 1H), 10.44 (s, 1H); MS (EI) m/z 449 (75, M+), 317 (100).
Anal. (C28H23N3O3) C, H, N.
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
6-p h en ylh exa n oyl a m id e (8f): from 6-phenylhexanoyl chlo-
ride (0.190 g, 1.0 mmol) and 6 (0.344 g, 1.0 mmol). Purifica-
tion: recrystallized from toluene to give a yellow solid: yield
0.186 g (36%); mp 75 °C; IR (KBr) ν 3268, 2930, 1666, 1596,
1506 cm-1 1H NMR (500 MHz, CDCl3) δ 1.35 (m,2H), 1.62
;
(m, 2H), 1.68 (m, 2H), 2.26 (m, 2H), 2.33 (s, 3H), 2.58 (m, 2H),
3.69 (s, 2H), 7.14 (m, 5H), 7.24 (m, 5H), 7.48 (m, 3H), 7.58 (m,
1H), 7.69 (m, 2H), 7.83 (m, 1H), 8.50 (m, 1H), 10.47 (s, 1H);
MS (EI) m/z 518 (42, M+), 105 (100), 91 (99). Anal. (C34H34N2O3)
C, H, N.
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
3-(2-p h en yl-4-th ia zolyl)a cr ylic a cid a m id e (9a ): from 3-(2-
phenyl-4-thiazolyl)acrylic acid chloride (0.3 g, 1.2 mmol) and
6 (0.413 g, 1.2 mmol). Purification: recrystallized from toluene
to give a yellow solid: yield: 0.394 g (59%); mp 183 °C; IR
(KBr) ν 3345, 3270, 3075, 1680, 1660, 1655, 1595, 1550 cm-1
.
1H NMR (400 MHz, CDCl3) δ 2.32, (s, 3H), 3.69, (s, 2H), 6.99
(d, J ) 16 Hz, 1H), 7.14-7.16 (m, 2H), 7.23-7.25 (m, 2H),
7.31-7.32 (m, 1H), 7.39-7.48 (m, 5H), 7.54-7.61 (m, 3H),
7.69-7.71 (m, 2H), 7.84 (s, br, 1H), 7.90-7.93 (m, 2H), 8.03
(s, br, 1H), 8.51 (d, J ) 9 Hz, 1H), 10.52 (s, br, 1H); MS m/z
(%) 557 (96) [M+], 558 (37), 452 (21), 425 (21), 344 (27), 214
(100), 212 (24). Anal. (C34H27N3O3S): C, H, N, S.
N-[3-Ben zoyl-4-[(4-m eth ylp h en yl)a cetyla m in o]p h en yl]-
ben za m id e (8b): from benzoyl chloride (0.14 g, 1.0 mmol) and
6 (0.344 g, 1.0 mmol). Purification: recrystallized from toluene
to give a yellow solid: yield 0.363 g (81%); mp 212 °C; IR (KBr)
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
3-[2-(4-n itr op h en yl)-4-th ia zolyl]a cr ylic a cid a m id e (9b):
From 3-[2-(4-nitrophenyl)-4-thiazolyl]acrylic acid chloride (0.354
g, 1.2 mmol) and 6 (0.413 g, 1.2 mmol). Purification: MPLC
EtOAc:n-hexane 2:1 to give a yellow solid: yield 0.376 g (52%);
mp 243 °C; IR (KBr) ν 3335, 3085, 2920, 1685, 1660, 1635,
1595, 1550, 1515 cm-1; 1H NMR (500 MHz, CDCl3) δ 2.34, (s,
3H), 3.71, (s, 2H), 7.01 (d, J ) 16 Hz, 1H), 7.17-7.18 (m, 2H),
7.24-7.28 (m, 4H), 7.44-7.54 (m, 3H), 7.59-7.68 (m, 2H),
7.72-7.74 (m, 2H), 8.00 (s, br, 1H), 8.12-8.14 (m, 2H), 8.28-
8.32 (m, 2H), 8.57-8.59 (m, 1H), 10.54 (s, br, 1H); MS m/z (%)
602 (5) [M+], 345 (17), 344 (79), 259 (14), 239 (20), 238 (13),
213 (22), 212 (100), 211 (47), 142 (13), 105 (42), 44 (11), 40
(24). Anal. (C34H26N4O5S): C, H, N, S.
ν 3420, 1650, 1620, 1550, 1500 cm-1 1H NMR (400 MHz,
;
CDCl3) δ 2.34 (s, 3H), 3.66 (s, 2H), 7.14 (m, 2H), 7.22 (m, 2H),
7.40-7.50 (m, 5H), 7.57 (m, 1H), 7.62 (m, 1H), 7.70 (m, 2H),
7.78 (m, 2H), 7.90 (m, 1H), 7.98 (m, 1H), 8.55 (m, 1H), 10.52
(s, 1H); MS (EI) m/z 448 (96, M+), 316 (100). Anal. (C29H24N2O3)
C, H, N.
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
2-p h en yla ceta m id e (8b): from 2-phenyl acetyl chloride (0.2
mL, 1.5 mmol) and 6 (0.516 g, 1.5 mmol). Purification:
recrystallized from toluene to give a yellow solid: yield 0.472
g (68%); mp 134 °C; IR (KBr) ν 3290, 1700, 1670, 1630 cm-1
;
1H NMR (400 MHz, CDCl3) δ 2.30 (s, 3H), 3.61 (s, 2H), 3.64
(s, 2H), 7.13 (m, 2H), 7.20 (m, 2H), 7.25-7.45 (m, 6H), 7.57
(m, 1H), 7.43 (m, 2H), 7.55 (m, 1H), 7.65 (m, 2H), 7.87 (m,
1H), 8.43 (m, 1H), 10.44 (s, 1H); MS (EI) m/z 462 (100, M+),
330 (79). Anal. (C30H26N2O3) C, H, N.
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
3-[5-(4-n itr op h en yl)-2-fu r yl]a cr ylic a cid a m id e (9c). From
3-[5-(4-nitrophenyl)-2-furyl]acrylic acid chloride (0.333 g, 1.2
mmol) and 6 (0.413 g, 1.2 mmol). Purification: MPLC EtOAc:
n-hexane 1:1 to give a yellow solid: yield 0.311 g (48%); mp
221 °C; IR (KBr): ν 3260, 2920, 1665, 1630, 1595, 1550, 1510
cm-1; 1H NMR (500 MHz, CDCl3) δ 2.33, (s, 3H), 3.69, (s, 2H),
6.59 (d, J ) 15 Hz, 1H), 6.67-6.68 (m, 1H), 6.90-6.91 (m, 1H),
7.15-7.17 (m, 2H), 7.23-7.25 (m, 2H), 7.45-7.48 (m, 3H),
7.56-7.59 (m, 1H), 7.61-7.64 (m, 1H), 7.70-7.71 (m, 2H), 7.76
(d, J ) 8.8 Hz, 2H), 7.93 (s, br, 1H), 8.03 (s, br, 1H), 8.20 (d,
J ) 8.8 Hz, 2H), 8.51 (d, J ) 9 Hz 1H), 10.52 (s, br, 1H); MS
m/z (%) 585 (52) [M+], 583 (15), 453 (18), 345 (17), 344 (71),
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
3-p h en ylp r op ion yla m id e (8c): from 3-phenylpropinyl chlo-
ride (0.17 mL, 1.0 mmol) and 6 (0.344 g, 1.0 mmol). Purifica-
tion: recrystallized from toluene to give a yellow solid: yield
0.72 g (15%); mp 59 °C; IR (KBr) ν 3420, 3270, 1655, 1595,
1
1555, 1505 cm-1; H NMR (400 MHz, CDCl3) δ 2.32 (s, 3H),
2.56 (t, J ) 8 Hz, 2H), 2.96 (t, J ) 8 Hz, 2H), 3.66 (s, 2H), 7.14
(m, 5H), 7.22 (m, 5H), 7.35 (m, 1H), 7.47 (m, 2H), 7.58 (m,
1H), 7.66 (m, 2H), 7.75 (m, 1H), 8.44 (m, 1H), 10.47 (s, 1H);
MS (EI) m/z 476 (57, M+), 458 (100). Anal. (C31H28N2O3) C, H,
N.

Novel Aryl Binding Sites of Farnesyltransferase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3123
326 (18), 325 (20), 243 (15), 242 (100), 238 (15), 212 (80), 211
(29), 196 (22), 105 (39), 40 (11). Anal. (C35H27N3O6): C, H, N.
N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
3-[5-(4-b r om op h en yl)-2-fu r yl]a cr ylic a cid a m id e (9d ):
from 3-[5-(4-bromophenyl)-2-furyl]acrylic acid chloride (0.355
g, 1.2 mmol) and 6 (0.413 g, 1.2 mmol). Purification: MPLC
EtOAc:n-hexane 1:1 to give a yellow solid: yield 0.468 g (63%);
mp 237 °C; IR (KBr) ν 3300, 3045, 2360, 1685, 1660, 1635,
1595, 1550, 1505 cm-1; 1H NMR (500 MHz, CDCl3) δ 2.33, (s,
3H), 3.68, (s, 2H), 6.43 (d, J ) 15 Hz, 1H), 6.62-6.64 (m, 1H),
6.68-6.70 (m, 1H), 7.13-7.16 (m, 2H), 7.21-7.25 (m, 3H), 7.30
(s, br, 1H), 7.42-7.62 (m, 8H), 7.71-7.72 (m, 2H), 7.94 (s, br,
1H), 8.54 (d, J ) 9 Hz, 1H), 10.46 (s, br, 1H); MS m/z (%) 620
(34) [M+ + 2], 618 (26) [M+], 607 (26), 344 (44), 277 (100), 275
Dr. S. Grabley and PD Dr. R. Thiericke for generous
support and Ms. S. Egner for excellent technical as-
sistance. Financial support by the German Pharmaceu-
tical Society is gratefully acknowledged.
Refer en ces
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Target. J . Med. Chem. 1997, 40, 2971-2990.
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(3) Sebti, S. M.; Hamilton, A. D. New approaches to anticancer drug
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Discovery Today 1998, 3, 26-32.
(88), 265 (40), 212 (24), 105 (23), 40 (46). Anal. (C35H27
BrN2O4): C, H, Br, N.
-
(4) Zhang, F. L.; Casey, P. J . Protein Prenylation: Molecular
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N-[3-Ben zoyl-4-[(4-m eth ylph en yl)acetylam in o]ph en yl]-
3-[5-(4-ch lor oph en yl)-2-fu r yl]acr ylic acid am ide (9e): from
3-[5-(4-chlorophenyl)-2-furyl]acrylic acid chloride (0.321 g, 1.2
mmol) and 6 (0.413 g, 1.2 mmol). Purification: MPLC EtOAc:
n-hexane 1:1 to give a yellow solid: yield 0.493 g (71%); mp
240 °C; IR (KBr) ν 3320, 1685, 1660, 1635, 1595, 1555, 1505
(5) Cox, A. D.; Der, C. J . Farnesyltransferase inhibitors and cancer
treatment: targeting simply Ras? Biochim. Biophys. Acta 1997,
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(6) Du, W.; Lebowitz, P. F.; Prendergast, G. C. Cell Growth
Inhibition by Farnesyltransferase Inhibitors Is Mediated by Gain
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1
cm-1; H NMR (500 MHz, DMSO-D6) δ 2.26, (s, 3H), 3.38, (s,
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basis of cancer. Biochim. Biophys. Acta 1999, 1423, C19-C30.
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Generation Peptidomimetic Inhibitors of Protein Farnesyltrans-
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Containing Inhibitors of Protein Farnesyltransferase. Bioorg.
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Replacements in CAAX-Tetrapeptide Based Farnesyltransferase
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2H), 6.70 (d, J ) 15 Hz, 1H), 6.94-6.95 (m, 1H), 7.00-7.02
(m, 2H), 7.04-7.06 (m, 2H), 7.12-7.13 (m, 1H), 7.38 (d, J )
15 Hz, 1H), 7.48-7.55 (m, 4H), 7.62-7.65 (m, 2H), 7.69-7.70
(m, 2H), 7.77-7.79 (m, 3H), 7.88-7.90 (m, 1H), 10.03 (s, br,
1H), 10.30 (s, br, 1H); MS m/z (%) 576 (29) [M++2], 574 (72)
[M+], 345 (12), 344 (48), 233 (33), 232 (15), 231 (100), 212 (16).
Anal. (C35H27ClN2O4): C, H, Cl, N.
En zym e P r ep a r a tion . Yeast farnesyltransferase was used
as a fusion to glutathione S-transferase at the N-terminus of
the â-subunit. Farnesyltransferase was expressed in Escheri-
chia coli DH5R grown in LB media containing ampicillin and
chloramphenicol for coexpression of pGEX-DPR1 and pBC-
RAM2 for farnesyltransferase production.27 The enzyme was
purified by standard procedures with glutathione-agarose
beads for selective binding of the target protein.
F a r n esyltr a n sfer a se Assa y. The assay was conducted as
described elsewhere.28 Farnesylpyrophosphate (FPP) was ob-
tained as a solution of the ammonium salt in methanol-10 mM
aqueous NH4Cl (7:3) from Sigma-Aldrich. Dansyl-GlyCys-
ValLeuSer (Ds-GCVLS) was custom synthesized by ZMBH,
Heidelberg, Germany. The assay mixture (100 µL volume)
contained 50 mM Tris/HCl pH 7.4, 5 mM MgCl2, 10 µM ZnCl2,
5 mM dithiothreitol (DTT), 7 µM Ds-GCVLS, 20 µM FPP, and
5 nmol (approximately) yeast GST-farnesyltransferase and 1%
of various concentrations of the test compounds dissolved in
dimethyl sulfoxide (DMSO). The progress of the enzyme
reaction was followed by monitoring the enhancement of the
fluorescence emission at 505 nm (excitation 340 nm). The
reaction was started by addition of the enzyme and run in a
Quartz cuvette thermostated at 30 °C. Fluorescence emission
was recorded with a Perkin-Elmer LS50B spectrometer. IC50
values (concentrations resulting in 50% inhibition) were
calculated from initial velocity of three independent measure-
ments of four to five different concentrations of inhibitor.
Molecu la r Mod elin g. All molecular modeling was carried
out using SYBYL21 version 6.6/6.7 running on
a Silicon
Graphics O2 (R10000). Flexible docking was performed using
FlexX20 version 1.7.6. The FlexX command MAPREF and the
perturbate mode of the PLACEBAS command were used.
Default parameters were employed except the MAX•ENERGY
value which was set to 10 kJ mol-1. GRID25 (version 19)
analyses were realized using a grid box with dimensions of 22
Å × 30 Å × 20 Å. A grid spacing of 0.5 Å was used to get a
detailed description of the binding region and to retrieve
smooth contour maps better for interpretation.
(18) Sakowski, J .; Bo¨hm, M.; Sattler, I.; Dahse, H.-M.; Schlitzer, M.
Synthesis, Molecular Modeling and Structure Activity Relation-
ship of Benzophenone-Based CAAX-Peptidomimetic Farnesyl-
transferase Inhibitors. Submitted.
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tors: the concept of benzophenone-based bisubstrate analogue
farnesyltransferase inhibitors. Eur. J . Med. Chem. 2000, 35,
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docking method using an incremental construction algorithm.
J . Mol. Biol. 1996, 261, 470-489.
Ack n ow led gm en t. The authors gratefully thank
Prof. Dr. G. Klebe who provided us with all facilities
needed for the modeling and also for helpful discussions.
The pGEX-DPR1 and pBC-RAM2 plasmids were kindly
provided by Prof. F. Tamanoi (UCLA). I.S. thanks Prof.

3124 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Bo¨hm et al.
(21) SYBYL molecular modeling software; Tripos Inc.: 1699 South
Hanley Rd, Suite 303, St. Louis, MO 63144.
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energetically favorable binding sites on biologically important
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(25) GRID, version 19; Molecular Discovery Ltd.: West Way House,
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(26) Augeri, D. J .; O’Conner, S. J .; J anowick, D.; Szczepankiewicz,
B.; Sullivan, G.; Larsen, J .; Kalvin, D.; Cohen, J .; Devine, E.;
Zhang, H.; Cherian, S.; Saeed, B.; Ng, S.-C.; Rosenberg, S. Potent
and Selective Non-Cysteine-Containing Inhibitors of Protein
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bonds. J . Med. Chem. 1993, 36, 140-147. Wade, R. C.; Goodford,
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J M010873J