Aryloxy substituted N-arylpiperazinones as dual inhibitors of farnesyltransferase and geranylgeranyltransferase-I

Article abstract of DOI:10.1016/S0960-894X(01)00240-2

A series of aryloxy substituted piperazinones with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to have potent inhibitory activity in vitro and are promising agents for the inhibition of

Full text of DOI:10.1016/S0960-894X(01)00240-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1411–1415
Aryloxy Substituted N-Arylpiperazinones as Dual Inhibitors of
Farnesyltransferase and Geranylgeranyltransferase-I
Jeffrey M. Bergman,a,* Marc T. Abrams,b Joseph P. Davide,b Ian B. Greenberg,b
Ronald G. Robinson,b Carolyn A. Buser,b Hans E. Huber,b Kenneth S. Koblan,b
Nancy E. Kohl,b Robert B. Lobell,b Samuel L. Graham,a George D. Hartman,a
Theresa M. Williamsa and Christopher J. Dinsmorea
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
Received 31 January 2001; accepted 29 March 2001
Abstract—A series of aryloxy substituted piperazinones with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity
was prepared. These compounds were found to have potent inhibitory activity in vitro and are promising agents for the inhibition
of Ki-Ras signaling. # 2001 Published by Elsevier Science Ltd. All rights reserved.
Introduction
is inhibited by an FTI, geranylgeranyltransferase-I
(GGPTase-I), an analogous prenyltransferase, is able to
activate Ki-Ras through geranylgeranylation. Thus,
dual FPTase/GGPTase-I inhibitors should prevent pre-
nylation of Ki-Ras, and have significant potential as
cancer chemotherapeutic agents.
Oncogenically activated Ras protein has been impli-
cated in the growth of 20–30% of all human tumors.1
Transforming mutations eliminate the intrinsic GTPase
activity of Ras, resulting in constitutively activated Ras
and growth signaling independent of extracellular
growth factors, leading to uncontrolled proliferation.
Among the ras genes, Ki-ras is the most relevant target
for an anticancer agent since this accounts for approxi-
mately 90% of the altered Ras found in human cancers.
Strategies for controlling Ras mediated oncogenic cel-
lular proliferation have focused on preventing the pre-
nylation of Ras by inhibition of farnesyl-protein
transferase (FPTase), an enzyme which catalyzes the S-
alkylation of a cysteine residue in the C-terminal tetra-
peptide sequence of Ras.2 This post translational mod-
ification is required for Ras activation and its inhibition
in altered Ras should control proliferation. FPTase
inhibitors (FTIs) have been shown to selectively inhibit
ras-transformed cell growth in cell culture, to inhibit the
growth of ras-dependent tumors in mice, and are cur-
rently undergoing human clinical trials both as single
agents and in combination with other anti-cancer
agents.3 However, Ki-Ras prenylation in FTI treated
cells has been reported.4 When farnesylation of Ki-Ras
Inhibitor Design
A wide array of FPTase inhibitors that mimic the
Ca1a2X tetrapeptide C-terminus of Ras have been
described.3d,e Improvements in the biological prop-
erties of FTIs have been achieved through the use
of non-peptide structural replacements for the cen-
tral a1a2 portion, the deletion of the carboxyl-con-
taining terminus, and substitution of the cysteine
moiety with alternative non-thiol groups. Extensive
work in this area resulted in the identification of
piperazinone FTIs,5 such as 1 (Fig. 1), which underwent
clinical evaluation.3c,e This compound is a dual prenyl-
protein transferase inhibitor (FPTase IC50=2 nM,
GGPTase-I IC50=98 nM).6 Our desire to improve
GGPTase-I inhibitory activity prompted investigation
of piperazinone derivatives with substitution on the
cyanobenzyl ring. Through this work, it was discovered
that aryloxy substitution (e.g. 2) can have an enhancing
effect on GGPTase-I activity. Herein, we describe the
optimization of these compounds as dual prenyl-
transferase inhibitors.
*Corresponding author. Tel.: +1-215-652-8257; fax: +1-215-652-
7310; e-mail: christopher_dinsmore@merck.com
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00240-2

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J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1411–1415
Synthesis of Inhibitors
FPTase activity relatively unaffected (Table 1). Addition
of phenoxide to the 3-position of 1 gave 8a (FPTase
IC50=7 nM, GGPTase-I IC50=22 nM). This repre-
sented a 5-fold increase in affinity for GGPTase-I while
maintaining FPTase potency similar to 1. Substituting
thiophenoxy in this position (8b) reduced affinity for
FPTase. In some cases, substitution on the aromatic
ring had a significant effect on activities. The ortho-
chlorophenoxy derivative 8c had the most profound
effect, maintaining the high intrinsic FPTase potency of
1 while increasing GGPTase-I activity over 100-fold
(GGPTase-I IC50=0.7 nM). The meta and para chloro
analogues were equipotent to unsubstituted 8a in
GGPTase-I inhibition. Increasing the size of the phen-
oxy substituent had a deleterious effect on GGPTase-I
activity. Compounds 8g and 8h had reduced GGPTase-I
inhibitory activity relative to 1 but maintained high
potency versus FPTase.
The cyanofluorobenzyl imidazole aldehydes 6 were pre-
pared from the corresponding bromofluorotoluenes as
outlined in Scheme 1. For example, 1-(4-cyano-3-
fluorobenzyl)-5-imidazolecarboxaldehyde 6a was pre-
pared in five steps starting from 4-bromo-3-fluoro-
toluene. Cyanation with zinc cyanide was followed by
NBS bromination to give 4a. A protected imidazole was
alkylated with this benzyl bromide giving 5a. Deprotec-
tion of the hydroxyl group and subsequent oxidation
gave the aldehyde 6a.
Reductive amination of aldehydes 6a and 6b with the
piperazinone amine 77 was followed by treatment of the
product with an aryloxide and cesium carbonate in
DMF to give the desired aryloxypiperazinones 8 and 9
(Scheme 2). Compounds without the nitrile (e.g., 10)
were prepared by reductive alkylation of the appro-
priate aryl piperazinone 7 with aldehyde 6c, followed by
Ullmann coupling8 with phenol.
It was found that some compounds behave as slow tight
binders to GGPTase-I.6a,b In these cases, in vitro studies
in which IC50 values are measured without prior incu-
bation of the inhibitor and enzyme gave higher IC50
values then those with a 30 min incubation period. For
example, 8c and 8i exhibited ca. 30-fold differences in
IC50 (Table 1). Furthermore, inhibition of GGPTase-I
by 8c was determined to be competitive with ger-
anylgeranylpyrophosphate (GGPP) and dependent on
the presence of inorganic or organic phosphate ions.
Many different phosphates could satisfy this require-
ment, and ATP was chosen because it is effective at
physiological concentrations.6a,b,10
Structure–Activity Relationships
Aryloxy substitution on the cyanophenyl ring has a
profound effect on GGPTase-I activity while leaving
Previous studies have documented the importance the
cyano group plays in FPTase inhibition activity.11 It
was anticipated that reorienting or removing this group
could reduce FPTase activity while maintaining
GGPTase-I activity. In an attempt to produce a
GGPTase-I selective inhibitor, such modifications were
explored (Table 2). In compound 9a the orientation of
the nitrile and phenoxy groups was reversed. Unexpect-
edly, FPTase potency was maintained while GGPTase-I
potency suffered. In compounds 10a–c the cyano group
Figure 1. Design of improved FTI-GGTIs.
Scheme 1. Reagents and conditions: (a) Pd(PPh3)4, Zn(CN)2, DMF,
80 ꢀC, 96%; (b) NBS, CCl4, benzoyl peroxide, reflux, 43–50%; (c) 1-
trityl-4-(acetoxymethyl)imidazole, EtOAc, 60 ꢀC; MeOH, 79–93%; (d)
LiOH, THF, H2O, 68–83%; (e) pyridine–SO3 complex, DMSO, Et3N,
86–90%.
Scheme 2. Reagents and conditions: (a) Na(OAc)3BH, 4 A sieves,
DCE, 34–67%; (b) for 8 and 9; ArOH or ArSH, Cs2CO3, DMF, 31–
.
60%; (c) for 10; PhOH, CuBr DMS, NaH, pyridine, reflux, 9–21%.

J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1411–1415
1413
is deleted. In these cases, FPTase inhibitory potency
has been drastically reduced yet these compounds
remain moderately potent and selective GGPTase-I
inhibitors. This supports the idea that the phenoxy
group is important to GGPTase-I binding, and that
selective inhibitors can be prepared by manipulating
substituents.
vitro FPTase IC50s. It is postulated that the increased
lipophilicity of the aryloxy compounds impedes cell
penetration. When polar substituents were introduced
onto the aryloxy groups, cellular FPTase values
responded positively. Substitution with O-(2-hydroxy-
ethyl)resorcinol gave 8i (FPTase IC50=2.5 nM,
GGPTase-I IC50=1.3 nM, FPTasecell IC50=8 nM)
which was more than 10-fold more active in the cell
based assay than the unsubstituted phenyl compound 8a.
Replacement of the phenol in 8a with 3-hydroxypyridine
gave 8j, the latter being 6-fold more potent in cells.
In addition to the in vitro FPTase inhibition assay,
FPTase binding in cells was also determined (Table 1).
An unfortunate consequence of the added lipophilicity
of these compounds was their poor performance in the
cell based assay. For many analogues, cellular FPTase
values were off by 10-fold or more as compared to in
Inhibition of the geranygeranylation of Rap1a is used as
a measure of the ability of a GGTI to prevent prenylation
Table 1. FPTase and GGPTase-I inhibition data for aryloxy derivatives 8a–j
In vitro (IC50, nM)
In cell culture (nM)
Compd
Y
H
FPTasea
GGPTase-lb
FPTase (IC50
bindingd
)
Rap1a (MIC)
prenylatione
1
2
7
98
18
3
1000
8a
110
300–1000
8b
8c
8d
8e
8f
18
7
12
0.7 (20)c
20
255
41
nd
300
nd
4
93
19
32
1
15
350
311
48
nd
62
nd
8g
8h
8i
199
nd
4
(290)c
1.3 (35)c
52
nd
8
nd
2.5
11
3000
1000
8j
17
aConcentration of compound required to reduce the human FPTase-catalyzed incorporation of [3H]FPP into recombinant Ras-CVIM by 50%.9a
bConcentration of compound required to reduce the human GGPTase-I-catalyzed incorporation of [3H]GGPP into biotinylated peptide
corresponding to the C-terminus of human Ki-Ras by 50%. Assay run with 30 min preincubation of enzyme and inhibitor in the presence of 5 mM
ATP.6
cSame as footnote b without prior incubation of inhibitor and enzyme.
dConcentration of sample required to displace 50% of a radiolabeled farnesyltransferase inhibitor (FTI) from FPTase in cultured Ha-ras trans-
formed RAT1 cells.9b
eMinimal concentration of compound required to inhibit Rap1a processing in PSN-1 cells.6

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J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1411–1415
Table 2. FPTase and GGPTase-I inhibition for piperazinones 9a and
10a–c
Acknowledgements
The authors are grateful to K. D. Anderson, P. A.
Ciecko, A. B. Coddington, G. M. Smith, H. G. Ramjit,
C. W. Ross III, B.-L. Wan, and M. M. Zrada for ana-
lytical support, J. R. Huff, D. C. Heimbrook, and A. I.
Oliff for their support of this work, and M.A. Guttman
for manuscript assistance.
Compd
X
Y
R
FPTase
IC50 (nM)a
GGPTase-I
IC50 (nM)b
8a
9a
10a
10b
10c
CN
OPh
H
H
H
OPh
CN
OPh
OPh
OPh
3-Cl
3-Cl
3-Cl
4-Cl
7
9
18
809
65
136
155
References and Notes
1230
670
5770
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aSee footnote a in Table 1.
bSee footnote b in Table 1.
3. (a) Recent reviews: Oliff, A. Biochim. Biophys. Acta 1999
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Gibbs, J. B. J. Clin. Invest. 2000, 105, 9. (d) Dinsmore, C. J.
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M. J.; Dinsmore, C. J.; Hutchinson, J. H.; MacTough, S. C.;
Stump, C. A.; Wei, D. D.; Zartman, C. B.; Bogusky, M. J.;
Culberson, J. C.; Buser-Doepner, C.; Davide, J.; Greenberg,
I. B.; Hamilton, K. A.; Koblan, K. S.; Kohl, N. E.; Liu, D.;
Lobell, R. B.; Mosser, S. D.; O’Neill, T. J.; Rands, E.; Schaber,
M. D.; Wilson, F.; Senderak, E.; Motzel, S. L.; Gibbs, J. B.;
Graham, S. L.; Heimbrook, D. C.; Hartman, G. D.; Oliff, A. I.;
Huff, J. R. J. Med. Chem. 1999, 42, 3779.
6. (a) Huber, H. E.; Abrams, M.; Anthony, N.; Graham, S.;
Hartman, G.; Lobell, R.; Lumma, W.; Nahas, D.; Robinson,
R.; Sisko, J.; Heimbrook, D. C. Proc. Am. Assoc. Cancer Res.
2000, 41, Abstract 2838. (b) Huber, H. E.; Robinson, R.;
Watkins, A.; Nahas, D.; Abrams, M.; Buser, C.; Lobell, R.;
Patrick, D.; Anthony, N.; Dinsmore, C.; Graham, S.; Hart-
man, G.; Lumma, W.; Williams, T.; Heimbrook, D. C. J. Biol.
Chem., in press. (c) Williams, T. M. et al., in preparation.
7. Weissman, S. A.; Lewis, S.; Askin, D.; Volante, R. P.;
Reider, P. J. Tetrahedron Lett. 1998, 39, 7459.
in cells.6a,b The compounds in this series that were tes-
ted in the Rap1a processing assay were active with
MICs in the 0.3–3 mM range, confirming GGPTase-I
inhibition in cells. Interestingly, Rap1a inhibition was
not significantly greater than that for 1 in spite of these
compounds having greater in vitro potency towards
GGPTase-I. This is most likely due to their reduced cell
penetration in comparison to 1.
In anchorage-independent growth inhibition assays in
soft agar, 8a was less effective than 1 at blocking colony
formation of v-H-ras transformed RAT1 cells (IC90
8a=0.3 mM, IC90 1=0.1 mM), consistent with its
reduced FTase binding in cell culture (vide supra).
General cell cytotoxicity elicited by 8a is only observed
at ꢁ30-fold higher concentrations (ꢁ80% RAT1 cell
survival up to 10 mM as assessed by viability staining
with MTT). Interestingly, inhibition by 8a of K-ras
transformed cell colonies required only slightly higher
concentration (IC90=0.3–1 mM) than was required
for H-ras, resulting in a ratio of K-ras/H-ras IC90
which is lower than for previously characterized selective
N-arylpiperazinone FTIs5 (ratio ca. 1–3 vs 10–20). A
determination of whether this is the result of dual versus
selective prenyltransferase inhibition will require further
studies.
8. Boger, D. L.; Yohannes, D. J. Am. Chem. Soc. 1991, 113,
1427.
9. (a) Graham, S. L.; deSolms, S. J.; Giuliani, E. A.; Kohl,
N. E.; Mosser, S. D.; Oliff, A. I.; Pompliano, D. L.; Rands, E.;
Breslin, M. J.; Deanna, A. A.; Garsky, V. M.; Scholz, T. H.;
Gibbs, J. B.; Smith, R. L. J. Med. Chem. 1994, 37, 725. (b)
Lobell, R. B.; Gibson, R. et al., in preparation. The radiotracer
used in the assay is [125I] 4-{[5-({(2S)-4-(3-iodophenyl)-2-[2-
(methylsulfonyl)-ethyl]-5-oxopiperazin-1-yl}methyl)-1H-imi-
dazol-1-yl]methyl}benzonitrile, which has ꢁ50,000 high affi-
nity binding sites (apparent Kd of ꢁ1 nM) in the RAT1 cell
line. The non-specific binding signal, determined by the addi-
tion of 1000-fold excess unlabeled competitor FTI, is typically
5-fold lower than the specific binding signal. The assay pro-
vides comparable results using a variety of cell lines. Cells are
seeded at 200,000 cells per well in 24-well tissue culture plates
and cultured for 16 h. The radiotracer (ꢁ300–1000 Ci/mmol)
is diluted into culture media to a concentration of 1 nM, along
with the desired concentration of test FTI, and then added to
the cell monolayers. After a 4 h incubation at 37 ꢀC, the cells
are briefly rinsed with phosphate-buffered saline, removed
from the culture plate by trypsinization, and then subjected to
Conclusion
The inclusion of aryloxy substituents on the cyano-
benzyl portions of certain dual FPTase/GGPTase-I
inhibitors can substantially improve GGPTase-I inhi-
bitory potency while leaving the relatively high
intrinsic FPTase inhibitory potency unaffected. The
poor cell penetration seen in this series can be posi-
tively addressed by the inclusion of polar function-
ality within the aryloxy substituent. Further
modifications, leading to the deletion of the nitrile,
yield GGPTase-I selective compounds. The ability to
modulate the relative degree of FPTase and GGPTase-I
inhibition could prove important in the design of
antineoplasic drugs of the prenyl-protein transferase
inhibitor class.

J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1411–1415
1415
gamma counting using a CobraII1 gamma counter (Packard
Instrument Company). Dose–inhibition curves and IC50 values
are derived from a four-parameter curve-fitting equation using
SigmaPlot1 software.
10. For 8c, the slope of a plot of log(IC50) versus log[GGPP]
is 0.93 in the presence of 5 mM ATP, suggestive of a GGPP-
competitive inhibitor. In the absence of 5 mM ATP in the
assay, GGPTase-I IC50=140 nM.
11. (a) Breslin, M. J.; deSolms, S. J.; Giuliani, E. A.; Stok-
ker, G. E.; Graham, S. L.; Pompliano, D. L.; Mosser, S. D.;
Hamilton, K. A.; Hutchinson, J. H. Bioorg. Med. Chem. Lett.
1998, 8, 3311. (b) Anthony, N.; Gomez, R. P.; Schaber,
M. D.; Mosser, S. D.; Hamilton, K. A.; O’Neil, T. J.;
Koblan, K. S.; Graham, S. L.; Hartman, G. D.; Shah, D.;
Rands, E.; Kohl, N. E.; Gibbs, J. B.; Oliff, A. I. J. Med.
Chem. 1999, 42, 3356.