A novel class of highly potent, selective, and non-peptidic inhibitor of ras farnesyltransferase (FTase)

Article abstract of DOI:10.1016/S0960-894X(01)00624-2

Design, synthesis and structure-activity relationship of a class of aryl pyrroles as farnesyltransferase inhibitors are described. In vitro and in vivo evaluation of a panel of these inhibitors led to identification of 2 (LB42908) as a highly potent (IC₅₀=0.9 nM against H-Ras and 2.4 nM against K-Ras) antitumor agent that is currently undergoing preclinical studies.

Full text of DOI:10.1016/S0960-894X(01)00624-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3069–3072
A Novel Class of Highly Potent, Selective, and Non-Peptidic
Inhibitor of Ras Farnesyltransferase (FTase)
Hyunil Lee, Jinho Lee, Sangkyun Lee, Youseung Shin, Wonhee Jung, Jong-Hyun Kim,
Kiwon Park, Kwihwa Kim, Heung Soo Cho, Seonggu Ro, Sunhwa Lee, ShinWu Jeong,
Taesaeng Choi, Hyun-Ho Chung and Jong Sung Koh*
Life Science R&D, LGCI, Science Town, Taejon 305-380, Republic of Korea
Received 2 July 2001; accepted 17 September 2001
Abstract—Design, synthesis and structure–activity relationship of a class of aryl pyrroles as farnesyltransferase inhibitors are
described. In vitro and in vivo evaluation of a panel of these inhibitors led to identification of 2 (LB42908) as a highly potent
(IC50=0.9 nM against H-Ras and 2.4 nM against K-Ras) antitumor agent that is currently undergoing preclinical studies. # 2001
Elsevier Science Ltd. All rights reserved.
Mutated versions of three human ras genes are fre-
quently found in many human cancers, most notably
cancers of the pancreas, colon, and lung, implying an
important role for aberrant Ras function in human
tumor growth.1À4 The frequency of mutations is not
uniformed with three human ras genes. Thus, although
mutated H-ras was the first identified, and consequently
has been the most heavily studied, mutations in K- and
N-ras are much more frequently seen in human tumors.
The p21 Ras oncogenic products are synthesized as
cytosolic proteins, which undergo post-translational
modifications for attachment of the normal as well as
mutated Ras proteins to the membrane.5,6 A key step in
a series of posttranslational modifications of the onco-
genic product Ras is farnesylation of the thiol group of
cystein located at the C-terminal CAAX sequence in
Ras proteins. The enzyme farnesyltransferase (FTase)
catalyzes this reaction.7,8 Inhibitors of farnesylation
(FTIs) would therefore have potential as anticancer
agents for tumors in which a ras gene is oncogenically
mutated.9À11 In fact, the efficacy of FTIs in murine
models has been demonstrated by their ability to inhibit
H-, K-, and N-ras-dependent tumor growth in nude
mice and to induce tumor regression in H- and N-ras
transgenic mice.12À14
have been reported in the literatures.11,15À18 Avenues
toward improving the biological properties of
a
peptidomimetic inhibitor have included the use of non-
peptide surrogates for the central AA portion, deletion
of carboxyl containing terminus, and replacement of
cystein moiety. Notable examples have featured the
substitution of 4-aminobenzoic acid for central hydro-
phobic dipeptide and the use of an imidazole ring as an
alternative to the cystein thiol group, which is believed
to be involved in an important binding interaction with
Zn++ of FTase.16,17 However, the peptidic nature of
these inhibitors was considered as a liability for clinical
application. Our efforts focused on the discovery of
more stable and druglike molecules, with the specific
aims of finding a surrogate for the thiol as well as non-
peptidic template to which the putative zinc ligand
could be attached. In our search for a suitable template,
we have investigated pyrroles (Fig. 1). The hypothetical
pharmacophore model of pyrrole series features an
aromatic binding interaction, the spacer for the central
Numerous structurally diverse classes of FTIs that
mimic tetrapeptide of Ras C-terminal CAAX motif
*Corresponding author. Tel.: +82-42-866-2114; fax: +82-42-863-
8010; e-mail: jskob@lgci.co.kr
Figure 1. Structures of lead compound 1 and the potent FTase inhi-
bitor 2 (LB42908).
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00624-2

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H. Lee et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3069–3072
hydrophobic dipeptide, an H-bond acceptor, and zinc
binding ligand. Initially, compound 1 was designed and
synthesized. Pyrrole 1 inhibited human FTase against
H-Ras with IC50 of 500 nM. We optimized this com-
pound through the chemical modification along the
pyrrole and imidazole rings.19 In vitro and in vivo evalua-
tion of a panel of these inhibitors has led to identification
of [1-{[1-(1,3-benzodioxol-5-ylmethyl)-1H-imidazol-5-yl]-
methyl}-4-(1-naphtyl)-1H-pyrrol-3-yl](4-methyl-1-piper-
azynyl)methanone (2, LB42908) as a highly potent
antitumor agent that is currently undergoing preclinical
studies. Herein, we report the structure–activity
relationship (SAR) that has been observed for with this
class.
The compounds of this series were first tested for inhi-
bition of the recombinant human FTase and ger-
anylgeranylproteintransferase I (GGTase I). The IC50
values of 2 and its related compounds are presented in
Table 1. Initial lead compound 1 inhibited FTase with
modest activity with IC50 value of 500 nM. The dra-
matic boost of inhibitory activity from 12 to 11 is clearly
noteworthy, indicating the hydrophobic aromatic sub-
stituent (R1) at C-3 of the pyrrole is very important to
the inhibitory potency of the imidazole-containing pyr-
role. Other findings on the SARs of this series are as
follows: the replacement of ethyl ester of pyrrole (R2) by
a either morpholinyl group or 4-methylpiperazinyl
amide group enhanced potency to a great extent; the
hydrogen part of the imidazole (R3) was favorably
replaced by methyl group; and noticeably, the replace-
ment of hydrogen (R3) by a benzyl group greatly
enhanced the inhibitory activity of FTase against K-Ras
(Table 1). The representative compound 2 exhibited a
good selectivity (15,000-fold) for FTase inhibition ver-
sus GGTase I inhibition.
The synthesis of 2 is illustrated in Scheme 1. 5-Hydroxy-
methyl imidazole 4 was prepared from piperonyl amine
and dihydroxyacetone dimer by a literature proce-
dure.20 Compound 4 was chlorinated with thionyl chlo-
ride to give the chloromethyl hydride compound 5 in a
quantitative yield. Pyrrole ester 7 was prepared from 1-
naphtaldehyde as a starting material by a literature
procedure.21 Compound 7 was converted to the acid 8
with potassium hydroxide. Acid 8 was converted to the
corresponding acid chloride with thionyl chloride; the
acid chloride was subsequently reacted with 4-
methylpiperazine to give pyrrole amide 9. Compound 9
was coupled with chloromethyl imidazole 5, to give the
representative compound 2. Compound 1 was synthe-
sized by coupling the trityl protected 5-chloromethyl
imidazole hydrochloride with pyrrole 7 and deprotec-
tion of the coupled product in the presence of tri-
fluoroacetic acid and triethysilylhydride. 10–16 (Table 1)
were synthesized analogously by coupling the corre-
sponding 5-chloromethyl imidazole hydrochloride to
the corresponding pyrroles.
To determine the inhibition of anchorage-independent
growth of human tumor cells, soft agar growth assays
were performed using standard procedures.13,23 A vari-
ety of human tumor cell lines containing activated H-ras
and K-ras were both inhibited in their ability to form
colonies in soft agar, an important finding in light of the
prevalence of cells harboring mutations in K-ras in
human carcinomas (Table 2). Finally, in vivo anti-
tumor activity of compound 2:2HCl was evaluated by
testing its ability to inhibit the growth and to regress the
size of tumors in nude mice.24 Compound 2:2HCl
showed significant dose-dependent inhibition of the
growth of tumors by both HCT116, human colon car-
cinoma (containing mutated K-ras) at 40 and 60 mg/kg
Scheme 1. Synthesis of 2: (a) (1) 3-dihydroxyacetonedimer, KSCN, AcOH/i-PrOH; (b) HNO3, NaNO2/H2O (75%, two steps yield); (c) SOCl2/
CHCl3 (90%); (d) triethylphosphonoacetate, DBU/CH3CN (96%); (e) tosylmethylisocyanide, t-BuOK/THF (63.5%); (f) KOH/EtOH, reflux (84%);
(g) (1) DMF (0.05 equiv), SOCl2/CH2Cl2; (2) 4-methylpiperazine (85%); (h) 5, NaH (2.5 equiv)/DMF, 0 ꢀC (80%).

H. Lee et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3069–3072
3071
Table 1. Inhibitory activity of 2 and related compound against human FTase and GGTase Ia
FTASE IC50 (nM)
GGTase I
IC50 (nM)
Compd
R1
R2
R3
H-Ras
K-Ras
1
Naph
Naph
Naph
Ph
Naph
Naph
Naph
Naph
Naph
OCH2CH2
Piperidine
Morpholine
Morpholine
Morpholine
H
H
H
500
720
30
ND
ND
1830
ND
100
2.1
ND
ND
ND
ND
>100,000
ND
10
11
12
13
14
15
16
2
H
CH3
Benzyl
Piperony
Piperonyl
Piperonyl
2000
3.0
Morpholine
Morpholine
1.5
0.9
1.1
0.9
2.0
3.5
2.4
12,000
10,000
16,000
N(CH3)CH2CH2OCH3
4-Methylpiperazine
IC50 values are the means of three experiments.
aThe farnesyltransferase and geranylgeranytransferase I inhibitory assays were performed as described in Ref 22.
Table 2. Inhibition of anchorage-independent growth of ras-trans-
formed and human tumor cell lines by compound 2:2HCl10,23
Inhibition of cell growth in soft agar (GI50 nM)
HT29
K-ras
HCT116
K-ras
A549
K-ras
EJ
H-ras
T24
H-ras
LB42908
4.5
17.6
1.2
0.56
0.45
Table 3. Inhibition (%) of tumor growth relative to control tumors
in tumor xenograft nude mice by compound 2:2HCl
Tumor model
20 mg/kg, bid
40 mg/kg, bid
60 mg/kg, bid
c
HCT116a
EJb
—
78d
43d
Regressiond
55d
Regressiond
aDetermined on day 21.
bDetermined on day 15.
c—not tested.
dp<0.001 compared with control tumors by Student’s t-test.
In summary, we have discovered a novel class of pyrrole
based FTase inhibitors that inhibit FTase in the nano-
molar range against H-Ras and K-Ras, and devoid of
problematic thiol functional groups. Compound 2
(LB42908) showed strong activity against human FTase
and very good selectivity for FTase over GGTase I. In
addition, LB42908 showed potent inhibitory effects in
an anchorage-independent soft agar assays with several
human tumor cell lines harboring mutations in K-ras.
LB42908 is orally active and shows statistically sig-
nificant dose dependent inhibition on tumor growth in
nude mouse xenograft models.
Figure 2. In vivo efficacy studies carried out in human tumor xeno-
graft models in athymic nude mice. Nude mice bearing HCT116 or EJ
tumor xenograft were prepared following the method described in
ref 24. Drug treatment was initiated after tumor volume reached
around 100 mm3. Mean tumor volume for each group was plotted vs
days of treatment. Standard errors of the mean (SES) are indicated as
error bars. Panel A shows compound 2-induced tumor growth inhibi-
tion in a HCT116, human colon carcinoma model. Panel B shows
compound 2-induced tumor growth inhibition and tumor regression in
an EJ, human bladder carcinoma model.
References and Notes
and EJ, human bladder carcinoma (containing mutated
H-ras) at 20, 40 and 60 mg/kg, when given to animals
orally twice daily for 21 days (Fig. 2 and Table 3). In
addition, compound 2:2HCl regressed, in a dose-depen-
dent manner, the size of tumors by EJ cells into 5.8 and
0.4% of each initial size at dose levels of 40 and 60 mg/kg,
respectively (Fig. 2, Panel B).
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[3H]-geranylgeranyl pyrophosphate (GGPP, 15 Ci/mmol,
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IC50: the concentration of compound required to reduce
the enzyme-catalyzed incorporation of [3H]FPP or [3H]GGPP
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24. In vivo efficacy studies were carried out in xenograft
models. Animals (athymic female nude mice; BALB/c
AnNCrj-nu/nu; Charles River Laboratories, Japan; 5–7 weeks
old) were subcutaneously inoculated with either HCT116 or
EJ tumor cells on the intrascapular region at least 1 week of
acclimation period after arrival from vendor. The size of
inoculum for both tumor cell lines was 107 cells per animal.
Once palpable, the size of tumor was measured in three
dimensions every three days and the volume was calculated by
using the equation, V=1/6ÂpÂLÂWÂH (V, volume; L,
length; W, width; H, height). When the tumor reached a
volume of around 100 mm3, animals were randomly divided
into control or treatment groups (8 animals/group) and it was
assigned as Day 0. Treatment with compound 2 at the dose
level of 20, 40 or 60 mg/kg was initiated on Day 1 and con-
tinued twice a day (bid) by gastric gavage for 21 days. Vehicle
control received 0.9% saline. Data from tumor with ulcer were
not included in the calculation of group mean. Student’s t-test
was used for statistical analysis.