Synthesis and biological evaluation of 4-[(3-methyl-3H-imidazol-4-yl)-(2-phenylethynyl-benzyloxy)-methyl]-benzonitrile as novel farnesyltransferase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2003.07.006

Farnesyltransferase inhibitors (FTIs) have emerged as a novel class of anticancer agents. Analogues of the potent FTI, 4-[3-biphenyl-1-hydroxy-1-(3-methyl-3H-imidazol-4-yl)-prop-2-ynyl] -1-yl-benzonitrile, were synthesized and tested in vitro for their in

Full text of DOI:10.1016/j.bmcl.2003.07.006

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3821–3825
Synthesis and Biological Evaluation of
4-[(3-Methyl-3H-imidazol-4-yl)-(2-phenylethynyl-benzyloxy)-
methyl]-benzonitrile as Novel Farnesyltransferase Inhibitor
Nan-Horng Lin,* Le Wang, Jerry Cohen, Wen-Zhen Gu, David Frost,
Haiying Zhang, Saul Rosenberg and Hing Sham
Cancer Research, R-47B, Global Pharmaceutical Products Division, Abbott Laboratories,
100 Abbott Park Road, Abbott Park, IL 60064-3500, USA
Received 28 May 2003; accepted 24 July 2003
Abstract—Farnesyltransferase inhibitors (FTIs) have emerged as a novel class of anticancer agents. Analogues of the potent FTI, 4-
[3-biphenyl-1-hydroxy-1-(3-methyl-3H-imidazol-4-yl)-prop-2-ynyl]-1-yl-benzonitrile, were synthesized and tested in vitro for their
inhibitory activities. The most promising compound identified from this series is analogue 11 that possesses potent enzymatic and
cellular activities.
# 2003 Elsevier Ltd. All rights reserved.
GTP-bound Ras proteins those are responsible for
initiating an intracellular phosphorylation cascade, and
consequently play an important role in normal cellular
physiology and pathophysiology.1 Oncogenic Ras pro-
teins commonly found in human tumors1,2 are locked in
the activated GTP-bound state, which leads to a con-
tinuously activated phosphorylation cascade. An essen-
tial prerequisite for the function of the Ras protein is its
association with the plasma membrane. Ras proteins are
initially synthesized in the cytoplasmwhere they
undergo posttranslational farnesylation of the cysteine
unit of the so-called CAAX box (C, cysteine; A, any
aliphatic amino acid; X, serine or methionine) in the
pre-Ras protein by the enzyme protein farnesyltransfer-
ase (FT).3 Once the protein substrate is farnesylated, the
AAX tripeptide is cleaved and the new C-terminal
cysteine carboxylate is methylated. The processed pro-
teins become relocated to the cell membrane, a step that
is essential for their function. This association transmits
extracellular signals to the nucleus and leads to cell
proliferation4,5
have been widely investigated in the past decade.6ꢀ9
FTIs were originally developed with the aimof inhibit-
ing the posttranslational prenylation and oncogenic
activity of Ras. It has become apparent that inhibition
of Ras prenylation is not necessary for these compounds
to exhibit antitumor activity. Instead, inhibition of
Rho-B and possibly other cellular proteins might also
account for the efficacy against malignant tumors.10ꢀ13
Although, the mechanism of action of these agents is
still debated, FTIs have shown impressive efficacy in
preclinical models of human cancers.
Our initial discovery of compound 1 as a potent, non-
peptidic, non-sulhydryl, selective inhibitor of FT
prompted an investigation of SAR centered around this
compound. The goal of our research is to identify addi-
tional molecules within this class of FTIs that not only
have good in vitro potency but may also have different
physical properties (i.e., solubility and crystallinity). We
have previously discovered that modification of the
phenyl ring of the biaryl skeleton in 1 affected the inhi-
bitory potency of analogues.14 However, we have not
thoroughly examined the effect of the linker on the
potency. Since it has been demonstrated previously that
the ortho phenyl ring could accommodate a large group,
we replaced the acetylenic moiety with a methyl ether
linkage in 1, and introduced an acetylenic likage
between the two ortho phenyl rings as shown in the
The antitransforming properties of farnesyltransferase
inhibitors (FTIs), a novel class of cancer therapeutics,
*Corresponding author. Fax: +1-847-935-5466; e-mail: nanhorng.
lin@abbott.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.006

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N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3821–3825
Scheme 1. (i) n-BuLi, ꢀ78 ꢁC; (ii) TBAF, THF; (iii) NBS, AIBN, CCl4; (iv) NaH, THF; (v) Pd(PPh3)4, CuI, THF; (vi) Pt/C; H2; (vii) Pd/C; H2.
structure A. It was anticipitated that these modifications
would increase the solubility of FTIs and maintain the
potency.
Acetylenic, vinyl and alkyl analogues of methyl ether
FTIs were tested for their inhibitory activity against
both FTase and GGTase-1 enzymes. The effects of var-
iation of the phenyl group in the phenyl acetylene series
have been studied in detail (Table 1). The 4-NO2 ana-
logue (21) was found to be the most potent FTase inhib-
itor in this series. Both electron-donating and
withdrawing groups are tolerated at the C3- and C4-
position. Interestingly, introduction of the ethylene
group between the phenyl group and acetylene had
little effect on the potency (17 vs 11). To probe the
steric volume of acetylene pocket we replaced the
phenyl ring with a long aliphatic chain, but this had
little impact on the potency (14 vs 11). The same
phenomenon was also observed when the phenyl moeity
was replaced with a bulkyl t-butyl group (12 vs 11).
Only a 2-fold decrease in potency was observed when
a polar hydroxyl group was introduced to the ali-
phatic chain (14 vs 19). Replacement of the phenyl
group with a saturated cyclohexyl moiety (20) gave
similar potency as well. In contrast, the inhibitory
activity appeared to be affected by steric effects at the
C4 position. Analogue 16 that possesses a large phenyl
group at the C4-position is less potent than either the
unsubstituted phenyl analogue 11 or cyano substituted
phenyl analogue 18. Finally, introduction of a nitrogen
atominto the acetylenic phenyl ring resulted in
decreased potency in both the FTase and GGTase-1
assays (15 vs 11).
Acetylenic analogues were prepared using the reaction
sequences shown in Scheme 1. Formation of the sec-
ondary alcohol 3 was accomplished by formation of an
imidazole anion with n-butyl lithiumfollowed by
addition of the anion to the aldehyde 2. The TES group
was then removed with TBAF. Bromide 4 was prepared
fromthe corresponding toluene by bromination with N-
bromosuccinimide in carbon tetrachloride. Alkylation
of alcohol 3 with bromide 4 gave the core intermediate 5,
which was coupled with various acetylenes utilizing
Sonogashira reaction conditions to give the acetylene
analogues 6. The acetylene analogues were hydrogenated
to olefinic compounds 7 with hydrogen in the presence of
platinumon charcoal. Further reduction to saturated
analogues 8 was then accomplished by hydrogenation of
the olefins with palladiumon carbon in ethyl acetate.
With regard to activity against the GGTase, introduc-
tion of electron withdrawing groups such as nitro or
cyano resulted in an increase in potency. In contrast, a
large group at the C4 position caused a huge reduction
in potency. In general, the acetylene series showed good
selectivity with the exception of compounds 14, 18, and
21, which have approximately only 150-fold selectivity
in favor of FTase inhibition.
Biphenyl analogues were synthesized using a similar
route (Scheme 2). Thus, coupling of a substituted benzyl
bromide 9 (prepared by bromination of the corre-
sponding toluene with NBS) with alcohol 3 provided the
bromide 10 as a common intermediate. Suzuki reaction
of bromide 10 with various substituted boronic acids 11
gave the biphenyl analogues 12 in reasonable yields.

N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3821–3825
3823
Scheme 2. (i) NaH, THF; (ii) Pd(PPh3)4, CsF, DME.
The introduction of a NO2 or CN group at the C4-
position of the phenyl ring in the acetylene series was
found to result in a 116- to 360-fold decrease in Ras
processing potency compared with analogue 11. Intro-
duction of a hydroxyl group at the C3 position caused a
similar reduction in potency, and potency was further
lowered by replacement of the phenyl moiety with ali-
phatic groups (cf. 12 and 17). The only acceptable sub-
stituent in the Ras processing was found to be the
cyclohexyl moiety (20).
The selectivity of olefinic analogues against FTase was
also evaluated. It was found that replacement of the
acetylene in the phenyl acetylene moiety with an olefinic
group caused a 5-fold decrease in selectivity (22 vs 15).
In contrast to this result, no change in selectivity was
observed when the acetylene was replaced with an olefin
in the 4-nitrophenyl analogues (23 vs 21).
Replacement of acetylene with an olefin in the acetylene
series results in a 16-fold increase in Ras processing
potency (23 vs 21). The nitro analogue 23 was identified
as the most potent compound in the olefin series syn-
thesized so far.
The effects of changing the acetylene to an olefin on the
potency of inhibiting FTase (Table 2) have been studied
with only a few analogues. The 4-NO2 analogue 23 was
found to be the most potent compound to inhibit
FTase in this series. However, only a minor change
in potency was observed when the 4-nitrophenyl
group was replaced with either pyridine or a long ali-
phatic moiety.
We then turned our attention to the effect of the acet-
ylenic linker on the inhibitory potency (Table 3).
Removal of the acetylene moiety of the phenyl acetylene
analogue resulted in only minor change in potency in
the inhibition of both the FTase and GGTase-1
Table 1. FTase inhibition, GGTase-1 inhibition, and Ras processing data for acetylenic analogues 11–21
Compd
R
FTase
IC50 (nM)a
GGTase
IC50 (nM)b
Selectivity (GGT/FT)
Ras
EC50 (nM)c
11
12
13
14
15
16
17
18
19
20
21
Ph
t-Bu
3-OH–Ph
n-C6H12
2-Pyridine
4-Ph–Ph
–CH2–CH2–Ph
4-CN–Ph
0.95
0.62
1
0.9
2.9
10
1.4
0.63
1.7
0.87
0.55
1800
490
1300
160
1900
790
1300
178
5517
>1000
386
0.1
76.9
>0.1
>0.1
>0.1
>0.1
130
11.6
20.9
1.93
36
16,000
>10,000
540
124
1600
845
196
941
971
273
–(CH2)4–OH
1-OH-cyclohexyl
4-NO2–Ph
150
aConcentration of compound required to reduce the human FTase-catalyzed incorporation of [3H] FPP into recombinant Ras CVIM by 50%.
bConcentration of compound required to reduce the human GGTase-catalyzed incorporation of [3H] GGPP into biotinylated peptide corresponding
to the C-terminal of human K-Ras by 50%.
cCompound concentration needed to reduce 50% of farnesylation in NIH-3T3H-ras cell line.

3824
N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3821–3825
Table 2. FTase inhibition, GGTase-1 inhibition, and Ras processing data for olefinic analogues 22–24
Compd
R1
FTase
IC50 (nM)a
GGTase
IC50 (nM)b
Selectivity (GGT/FT)
Ras
EC50 (nM)c
22
23
24
2-Pyridine
4-NO2–Ph
–CH–CH–n-Pr
1.6
0.61
1.3
1800
110
220
1125
180
169
23.1
2.24
16.7
aConcentration of compound required to reduce the human FTase-catalyzed incorporation of [3H] FPP into recombinant Ras CVIM by 50%.
bConcentration of compound required to reduce the human GGTase-catalyzed incorporation of [3H] GGPP into biotinylated peptide corresponding
to the C-terminal of human K-Ras by 50%.
cCompound concentration needed to reduce 50% of farnesylation in NIH-3T3H-ras cell line.
Table 3. FTase inhibition, GGTase-1 inhibition, and Ras processing data for biphenyl analogues 25–28 and 22
Compd
R
A
FTase
IC50 (nM)a
GGTase
IC50 (nM)b
Selectivity (GGT/FT)
Ras
EC50 (nM)c
25
11
26
18
27
16
21
23
12
28
Ph
Ph
Bond
CꢂC
1.3
0.95
1.3
0.63
1.1
3500
1800
9000
124
2690
1900
6900
196
140
0.1
4-CN–Ph
4-CN–Ph
4-Ph–Ph
4-Ph–Ph
4-NO2–Ph
4-NO2Ph
t-Bu
Bond
<0.1
11.6
<0.1
>0.1
36
2.24
76.9
0.54
CꢂC
Bond
CꢂC
150
136
10
>10,000
150
110
>1000
273
180
CꢂC
0.55
0.61
0.62
0.95
CH¼CH
CꢂC
490
310
790
326
t-Bu
CH2CH2
aConcentration of compound required to reduce the human FTase-catalyzed incorporation of [3H] FPP into recombinant Ras CVIM by 50%.
bConcentration of compound required to reduce the human GGTase-catalyzed incorporation of [3H] GGPP into biotinylated peptide corresponding
to the C-terminal of human K-Ras by 50%.
cCompound concentration needed to reduce 50% of farnesylation in NIH-3T3H-ras cell line.
enzymes (25 vs 11). In contrast to this result, a 77-fold
decrease in potency was observed in the inhibition of
GGTase-1 when the acetylene was removed from the 4-
CN phenyl analogue (26 vs 18). However, little change
in the FTase potency was observed (Table 3). Reduction
of the acetylene to an olefin had little effect on the
inhibition of both enzymes (23 vs 21). The same trend
was observed when the triple bond was reduced to a
single bond (12 vs 28). In general, acetylenic analogues
were less potent inhibitors of Ras processing activity
than their corresponding alkyl, alkenyl or biphenyl
analogues.
well tolerated at the C4-position of the phenyl ring. In
general, compounds having either the phenyl or alkyl
structure as shown in Table 1 possess acceptable
inhibitory activities against the GGTase-1 enzyme,
while the 2-pyridyl analogues possess the weakest activity.
The selectivity for FTase inhibition over GGTase-1 is
highest in the 2-pyridyl series as shown in Table 1. This
study has also shown that the effect of the acetylene moi-
ety on potency is substrate dependent. However, only a
small effect is observed when the acetylene is reduced to
either a double or single bond. Compound 11 is the most
potent FTI identified fromthis study.
In summary, we have varied the substituent pattern at
two phenyl rings of the imidazole acetylenic alcohol
such as compound 1 and examined the resultant
effects on inhibitory activity. The inhibitory activity
(Tables 1–3) indicates that large substituents are not
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