Diaryl ether inhibitors of farnesyl-protein transferase

Article abstract of DOI:10.1016/S0960-894X(01)00162-7

Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The S_NAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of c

Full text of DOI:10.1016/S0960-894X(01)00162-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1257–1260
Diaryl Ether Inhibitors of Farnesyl-Protein Transferase
Suzanne C. MacTough,a,* S. Jane deSolms,a Anthony W. Shaw,a Marc T. Abrams,b
Terrence M. Ciccarone,a Joseph P. Davide,b Kelly A. Hamilton,b John H. Hutchinson,a
Kenneth S. Koblan,b Nancy E. Kohl,b Robert B. Lobell,b Ronald G. Robinsonb
and Samuel L. Grahama
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 15 December 2000; accepted 21 February 2001
Abstract—Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used
to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commer-
cially available phenols quickly identified compounds which proved active in cells. # 2001 Elsevier Science Ltd. All rights reserved.
The protein encoded by ras is an important part of the
signal transduction pathway in the life cycle of the cell.
Mutations in the ras gene can lead to uncontrolled cell
growth and have been reported in human tumors.1 A
crucial post-translational modification required for
activation of the Ras protein is farnesylation of the
C-terminus. Blockade of this activation step through
inhibition of farnesyl-protein transferase (FPTase) has
been accomplished by an assortment of synthetic com-
pounds as possible therapeutics in the treatment of
human cancer.2ꢀ4 In particular, piperazinones of the gen-
eral formula 1a have been shown to be potent inhibitors of
FPTase.5
data) with a low energy conformer of the biphenyl ether
2.9 This indicates that, when the imidazole and cyano-
phenyl groups are closely aligned, the biphenyl group of
2 is indeed able to reach the lipophilic pocket occupied
by the X-phenyl group of 1b.
In order to generate structural information about the
bound conformation of these inhibitors, transferred
NOE (trNOE) studies using the FPTase enzyme and
piperazinone 1b (IC50=475 nM) were performed.6 The
results support the folded conformation shown for these
compounds in the active site with the p-cyanophenyl
moiety positioned underneath the piperazinone ring.
Examination of a model based on the trNOE data indi-
cated that the lipophilic binding site occupied by the
X-phenyl group in 1b could be accessed from the cya-
nophenyl group rather than from the imidazole
group.7,8 In support of this proposal, Figure 1 shows an
overlay of 1b (in a conformation consistent with trNOE
To test this hypothesis, compound 2 was prepared as
outlined in Scheme 1. It was tested against both FPTase
and the closely related enzyme geranylgeranyl-protein
transferase-I (GGPTase-I). 2 inhibited the in vitro
farnesylation of Ras by FPTase (Table 1,
IC50=86ꢁ43 nM) and was more selective versus
GGPTase-I (IC50=12,000 nM). Related analogues
(Table 1) were synthesized using the same methodology
with the exception of compound 3, which was prepared
as shown in Scheme 2. Removal of the phenyl group to
give 5 led to a 10-fold loss in potency. The effect of
altering the spacing between the aryl groups was
explored. Extending the chain length by one or two
carbon atoms (6 and 7) improved potency against
FPTase 2- to 4-fold. Truncation to a two-atom linker
(4) resulted in slightly reduced activity. Further con-
traction to the diaryl ether provided compound 3 with
*Corresponding author. Fax: +1+215-652-7310; e-mail: sue_
mactough@merck.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00162-7

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S. C. MacTough et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1257–1260
potency comparable to 6 and only 3-fold less potent
than the initial lead compound 2. A somewhat different
pattern was observed for GGPTase-I activity, which
was minimally affected as the length of the linker
increased from one to four atoms but improved 10-fold
with the five-atom linker in 7.
The amenability of the synthetic route outlined in
Scheme 2 to rapid analogue synthesis led to the identi-
fication of compound 3 as a promising structure for
further exploration. An SNAr displacement reaction was
used to couple N-(4-cyano-3-fluorobenzyl) imidazole 8
with a wide variety of commercially available phenols.
Representative examples of compounds prepared in this
manner are shown in Table 2. Single chlorine substitu-
tion in any of the positions gave compounds (9, 11, and
13) that were 4-fold more potent than the unsubstituted
parent 3. Methoxy (14, 15, and 16) and phenyl (17, 18,
and 19) substitution exhibited a marked preference for
the meta-position. This finding was reinforced by the
loss in potency seen in going from 2-naphthyl (20) to 1-
naphthyl (21).
Figure 1. Stick overlay (2 in green).
Other meta-substituents in Table 2 show an increase in
their ability to inhibit the FPTase enzyme with an
increase in size, such as the 4- to 6-fold increase of the
halogens from F to Cl to Br (10, 11, and 12). The alkyne
(26) and its analogous, less potent nitrile (27), in con-
junction with the acetyl (28) and t-butyl (29), suggest
that there is a large space that can accommodate these
structures.
Scheme 1. Reagents and conditions: (a) CH3OH, HCl, rt, 5 h, 96%;
(b) HCl, NaNO2, THF, 0 ꢂC, 20 min; KI, 45 min, 14%; (c) Zn(CN)2,
Pd(Ph3P)4, DMF, 100 ꢂC, 1 h, 83%; (d) LiBH4, THF, reflux 22 h,
48%; (e) CBr4, Ph3P, DMF, CH2Cl2, rt, 1.5 h, 78%; (f) imidazole,
DMF, rt, 16 h, 64%; (g) 2-(1,10-biphenyl-4-yl)ethyl methanesulfonate,
Disubstituted phenoxy compounds (20–25) were among
the most potent. Interestingly, the 2,4-dichloro com-
pound 22 was 5-fold more potent against FPTase than
the mono-substituted analogues with concomitant
improvement in GGPTase-I activity. 2,3-Dimethoxy
analogue 24 was a potent FPTase inhibitor (IC50 1 nM)
but a very weak inhibitor of GGPTase-I (IC50 15 mM).
ꢂ
Cs2CO3, D MF, 80 C, 1.5 h, 63%.
Table 1. Activity of unsubstituted arylalkyl ethers
Compounds
X
n
FPTase IC50 (nM)a GGPTase-I IC50 (nM)b
2
3
4
5
6
7
Ph
H
H
H
H
H
2
0
1
2
3
4
86ꢁ43
230
1500
860
200
440
12,000ꢁ1000
3700ꢁ900 (2)
6800ꢁ900 (2)
7000ꢁ3100 (2)
3100ꢁ900 (2)
380ꢁ50 (3)
aConcentration of compound required to inhibit by 50% the rate of
incorporation of [3H]FPP into recombinant Ras-CVIM by human
FPTase.
bConcentration of compound required to inhibit by 50% the rate of
incorporation of [3H]GGPP into a biotinylated peptide corresponding
to the C-terminus of human Ki-Ras by human GGPTase-I. In both
FPTase and GGPTase-I, results from a single experiment are reported
unless noted otherwise in parentheses; values are estimated to be reli-
able within 2-fold. In the FPTase assay, two sets of dilution plates
were prepared and tested in all experiments.
Scheme 2. Synthesis of N-(4-cyano-3-fluorobenzyl)imidazole 8 and
SNAr displacement by phenol. Reactions and conditions: (a) KMnO4,
ꢂ
.
pyridine, H2O, reflux, 40 h, 94%; (b)ꢂBH3 THF, 5 C to rt, 24 h, 92%;
(c) Zn(CN)2, Pd(Ph3P)4, D MF, 95C, 18 h, 66%; (d) NBS, DMS,
CH2Cl2, ꢀ20 to 0 ꢂC to rt, 18 h, 65%; (e) imidazole, DMF, rt, 18 h,
ꢂ
51%; (f) phenol, Cs2CO3, D MF, 50 C, 3 h, 88%.

S. C. MacTough et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1257–1260
1259
The 3-phenyl substituted compound 18 is among the
more potent FPTase inhibitors. The biphenyl unit was
extended by insertion of several heteroatom linkers
(Table 3). Inhibitory potency against FPTase was gen-
erally independent of the electronic nature of the linker
as the amides 34 and 35, ketone 31, ether 32, and aniline
33 were equipotent. However, the methylene-linked
compound 30 was 16-fold less active. In the GGPTase-I
assay, the amides were the least well-tolerated among
the linking elements.
The attempt to discover novel structures by screening a
wide variety of commercially available phenols in this
library led to compound 36. The inclusion of the amide
group into the otherwise lipophilic seven-membered ring
of the large, racemic caprolactam 36 yielded one of the
most potent compounds, both in the FPTase and
GGPTase-I assays.
Table 2. Mono- and disubstituted phenyl ethers
The nucleophilic aromatic substitution reaction also
provided a facile synthesis of diaryl thioethers (Table 4).
The thioether 37 was significantly more potent than the
ether analogue 22. The sulfoxide 38 and sulfone 39 of
thioether 37 were also prepared and tested. As the oxi-
dation state of the sulfur increased, potency drastically
decreased in both the FPTase and GGPTase-I assays.
Compounds
Ar
FPTase
IC50 (nM)a
GGPTase-I
IC50 (nM)a
9
2-Cl-Ph
3-F-Ph
3-Cl-Ph
3-Br-Ph
4-Cl-Ph
2-OCH3-Ph
3-OCH3-Ph
4-OCH3-Ph
2-Ph-Ph
3-Ph-Ph
4-Ph-Ph
2-Naphthyl
1-Naphthyl
2,4-diCl-Ph
3,4-diCl-Ph
2,3-diOCH3-Ph
2,5-diOCH3-Ph
3-Ethynyl-Ph
3-Cyano-Ph
3-Acetyl-Ph
3-t-Butyl-Ph
41ꢁ12 (2)
1100ꢁ200 (2)
4400
The most potent of the library compounds were tested
further in a cell-based assay which measured displace-
ment of a radiolabeled inhibitor from FPTase in cells.10
Of the compounds tested, the racemic caprolactam 36
had the highest FPTase occupancy level in cells with an
IC50 of 4.9 nM (Table 5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
180
53ꢁ20 (2)
3200ꢁ600 (2)
38
38ꢁ18 (2)
2900
920ꢁ90 (3)
28,000
3500
340
40
290
110
9.6
3400
3500
740ꢁ80 (2)
520
1300
From the analysis of information provided by trNOE
experiments, a novel series of aryl ether inhibitors was
elucidated. Identification of a core structure (3) that
could be readily prepared by derivatization of the
1.1ꢁ0.1 (2)
520
3200
280
1200
15,000
18,000
2600
16,000
6100
520
150
8
28
1
100
Table 4. Effect of oxidation on thioethers
27
130ꢁ10 (2)
45
27ꢁ13 (2)
aSee corresponding notes in Table 1.
Compounds
Z
FPTase IC50 (nM)a GGPTase-I IC50 (nM)a
22
37
38S
39
O
S
8
<1
390
5400
280
23ꢁ12 (3)
Table 3. Heteroatom aryl linkers
¼O
2000ꢁ800 (2)
8800ꢁ2000 (2)
O¼S¼O
aSee corresponding notes in Table 1.
Table 5. Cell-based assay
Compounds
Z
FPTase
IC50 (nM)a
GGPTase-I
IC50 (nM)a
Compound
FPTase cell assay
IC50 (nM)a
30
31
32
33
34
35
CH2
C¼O
O
230
13
600
840
280
630
22
24
36
37
260
14
14ꢁ1.0 (2)
4.9ꢁ0.1 (2)
N
17
38ꢁ21 (2)
38
96ꢁ53 (3)
C(O)NH
HNC(O)
3300ꢁ1000 (3)
aConcentration of sample required to displace 50% of a radiolabeled
farnesyltransferase inhibitor (FTI) from FPTase in cultured v-Ha-ras-
transformed RAT1 cells.10
2000ꢁ30 (2)
aSee corresponding notes in Table 1.

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S. C. MacTough et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1257–1260
fluoro-cyano intermediate 8 via a one-step synthesis
expedited the discovery process. Overall, the meta-posi-
tion was recognized as the best site for optimization.
Commercial availability of a wide variety of substrates
broadened the number and scope of substrates that
could be investigated. Inhibitors were discovered that
were potent and selective against farnesyl-protein
transferase and showed a wide range of activity against
geranylgeranyl-protein transferase-I. Selected com-
pounds exhibited activity in cell culture. Further ela-
boration of these diaryl ethers will be reported in
subsequent publications.
Schaber, M. D.; Robinson, R. G.; Koblan, K. S.; Huber, H.
E.; Graham, S. L.; Hartman, G. D.; Huff, J. R.; Williams, T.
M. J. Am. Chem. Soc 2001, 123, 2107.
7. Distance restraints from trNOE data for 1b were used to
generate conformations using JG. Kearsley, S. K., Merck &
Co., Inc., unpublished.
8. These conformations were minimized within Macromodel
using the MMFF force field with a 4r distance-dependent
dielectric, and with trNOE distance constraints applied.
Mohamadi, F.; Richards, N. F. J.; Guida, W. C.; Liskamp,
R.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J.
Comput. Chem. 1990, 11, 440.
9. One hundred conformations of 2 were generated using JG.
The subsequent conformations were energy minimized with
Macromodel using the procedure outlined above. The result-
ing energy minimized conformations were superimposed using
SQ. Miller, M. D.; Sheridan, R. P.; Kearsley, S. K. J. Med.
Chem. 1999, 42, 1505.
10. Lobell, R. B.; Gibson, R., manuscript in preparation. To
assess the potency of the FTIs in cells, a competitive binding
assay was utilized to determine the concentration of the FTI
required to displace 50% of a radiolabeled FTI from FPTase
in cultured v-Ha-ras transformed RAT1 cells. This radio-
tracer, [125I] 4-{[5-({(2S)-4-(3-iodophenyl)-2-[2-(methylsulfonyl)-
ethyl]-5-oxopiperazin-1-yl}methyl)-1H-imidazol-1-yl]methyl}-
benzonitrile, has ꢃ50,000 high affinity binding sites (apparent
Kd of ꢃ1 nM) in the Rat1 cell line. The nonspecific binding
signal, determined by the addition of 1000-fold excess unla-
beled competitor FTI, is typically 5-fold lower than the spe-
cific binding signal. The assay provides comparable results
using a variety of cell lines, and is performed as follows. Cells
are seeded at 200,000 cells per well in 24-well tissue culture
plates and cultured for 16 h. The radiotracer (ꢃ300–1000 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 ꢂC, the cells are briefly rinsed with phosphate-buffered sal-
ine, removed from the culture plate by trypsinization, and then
subjected to 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.
Acknowledgements
The authors wish to thank J. Christopher Culberson for
the modeling studies.
References and Notes
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