N-arylpiperazinone inhibitors of farnesyltransferase: Discovery and biological activity [1]

Full text of DOI:10.1021/jm990254z

J . Med. Chem. 1999, 42, 3779-3784
3779
Communications to the Editor
includes the reactive cysteine thiol. The a1a2 dipeptide
adjacent to the prenylated cysteine residue is usually
aliphatic and hydrophobic in nature, and in Ras pro-
teins, X is either serine or methionine. Analogues of the
Ca1a2X motif modified in a variety of ways are very
efficient inhibitors of FTase in vitro.
N-Ar ylp ip er a zin on e In h ibitor s of
F a r n esyltr a n sfer a se: Discover y a n d
Biologica l Activity
Theresa M. Williams,* J effrey M. Bergman,
Karen Brashear, Michael J . Breslin,
FTIs based on the Ca1a2X tetrapeptide generally
require the thiol and carboxylic acid functional groups
of the natural protein FTase substrates for high enzyme
affinity. However, it was desirable to re-engineer the
peptide FTIs to achieve high levels of inhibition in the
absence of these groups. Studies in vivo show that the
highly polar carboxylic acid impedes cell penetration,6
while a thiol-containing drug has potential antigenic
properties. An important consideration in the FTI drug
design process was the overall minimization of peptide
character, to maximize the chance of obtaining a drug
with suitable pharmacokinetic properties. An earlier
report describes our first attempt in this area, wherein
truncation and conformational constraint of Ca1a2X
peptides gave low-molecular-weight inhibitors such as
1.7 These compounds maintained high levels of FTase
inhibition in vitro (IC50 1 nM) and in vivo in the absence
of the carboxylate.
Christopher J . Dinsmore, J ohn H. Hutchinson,
Suzanne C. MacTough, Craig A. Stump,
Donna D. Wei, C. Blair Zartman,
Michael J . Bogusky, J . Christopher Culberson,
Carolyn Buser-Doepner,† J oseph Davide,†
Ian B. Greenberg,† Kelly A. Hamilton,†
Kenneth S. Koblan,† Nancy E. Kohl,† Dongming Liu,†
Robert B. Lobell,† Scott D. Mosser,†
Timothy J . O’Neill,† Elaine Rands,†
Michael D. Schaber,† Francine Wilson,†
Edith Senderak,∇ Sherri L. Motzel,
J ackson B. Gibbs,† Samuel L. Graham,
David C. Heimbrook,† George D. Hartman,
Allen I. Oliff,†,§ and J oel R. Huff
Departments of Medicinal Chemistry, Cancer Research,
Molecular Systems, Laboratory Animal Resources,
and Biometrics Research, Merck Research Laboratories,
West Point, Pennsylvania 19486
Received May 24, 1999
In tr od u ction . Ras is a small GTP-binding protein
intimately involved in cell signaling pathways and cell
growth. Mutations arising in the Ras protein that
impair GTP hydrolysis can lead to unregulated cell
growth and cellular transformation. A mutated ras gene
(ras oncogene) is often found in human tumors and is
estimated to occur in 30-50% of colon and 90% of
pancreatic cancers.1
In 1989, it was discovered that farnesylation of a
cysteine residue near the protein’s C-terminus was
required for its biological activity.2 Interfering with Ras
function by blocking the enzyme responsible for its
farnesylation thus became a pharmacological target to
suppress ras-dependent tumor growth. Since then, the
efficacy of farnesyltransferase inhibitors (FTIs) in mu-
rine models of cancer has been demonstrated by their
ability to inhibit H-, K-, and N-ras-dependent tumor
growth in nude mice3 and induce tumor regression in
H- and N-ras transgenic mice.4
FTIs have been obtained from a variety of sources,
including natural product, proprietary sample collection,
and combinatorial library screening, as well as rational
design based upon the structure of farnesyl protein
transferase (FTase) substrates.2,5 The C-terminal tet-
rapeptide of the Ras substrate constitutes a “Ca1a2X”
box, a characteristic signal sequence for prenylation that
Additional studies of Ca1a2X-based inhibitors indicate
that imidazole is a very good cysteine surrogate, perhaps
by serving as an alternative ligand for the FTase active
site zinc ion.8 In an important discovery, attaching a
benzyl group to the imidazole significantly improves
potency relative to the unsubstituted imidazole.9 It
seems likely that the added phenyl group takes advan-
tage of a novel, high-affinity aryl-binding site, originally
discovered in a separate investigation of Ca1a2X cysteine
replacements.10 Potency on the order of the original
Ca1a2X analogues was achieved with a variety of
substituted benzylimidazole cysteine surrogates.9
In contrast to the Ca1a2X series, imidazole by itself
is not a good cysteine surrogate in compounds such as
1. The imidazole analogue 2a experiences a 7000-fold
loss in potency relative to aminothiol 1. Fortunately,
however, the addition of a benzyl substituent to the
imidazole ring boosts potency 10-fold in the 1,5-isomer
2a (IC50 495 nM). As previously observed in the tet-
rapeptide series, this effect is highly dependent on the
regiochemistry of imidazole alkylation (e.g. the 1,4-
† Department of Cancer Research.
‡ Department of Molecular Systems.
∇ Department of Biometrics Research.
Department of Laboratory Animal Resources.
§ Current address: DuPont Pharmaceuticals Co., Rte 141 and Henry
Clay Rd, Wilmington, DE 19880.
substituted imidazole isomer of 2a has an IC50
100 000 nM). Substituents on the benzyl ring that
>
10.1021/jm990254z CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/03/1999

3780 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Ta ble 1. Influence of Imidazole Substitution on Activity
Communications to the Editor
aligned with the a1 substituent of 3 (Figure 1). In this
model, the acyl group in 2c occupies a region of space
similar to the lone amide carbonyl group necessary for
potency in Ca1a2X analogues. In the overlay, the cy-
anobenzyl substituent in 2c did not correlate to any part
of 3. From inspection of models, it is evident that a slight
rearrangement of functional groups to give N-arylpip-
erazinone 4a similarly overlays the enzyme-bound
conformation of 3 (Figure 2). On the basis of this
observation, piperazinone 4a was synthesized and sub-
sequently determined to be our first subnanomolar FTI
without thiol or carboxylic acid functional groups. We
are currently establishing structure-activity relation-
ships (SAR) within this series, with respect to both
intrinsic FTase potency and ability to block proliferation
of ras-transformed cells, and wish to report our pre-
liminary results.
entry
R
FTase IC50 (nM)a
2a
2b
2c
H
7300 ( 2100
495 ( 65
21 ( 1
(n ) 2)
(n ) 2)
(n ) 2)
PhCH2
(4-CN)PhCH2
a
Concentration of compound required to inhibit 50% of FTase-
catalyzed incorporation of [3H]FPP into recombinant human K-Ras
protein. Assay results were obtained using the protocol described
in ref 6.
enhanced potency in the Ca1a2X series were also screened
in the present context. The most dramatic increase in
activity relative to 2a results from introducing a p-cyano
group, giving benzylimidazole analogue 2c (IC50 21 nM)
(Table 1). Other substitutions on the benzyl ring are
less effective or detrimental to activity, suggesting
an as-yet unknown specific interaction for the cyano
group.
Ch em istr y. The synthesis of 4a proceeds as described
in Scheme 1 and is illustrative of the general procedure.
The Weinreb amide of N-Boc-L-norleucine is reduced
to the aldehyde, which undergoes reductive alkylation
with 2,3-dimethylaniline in the presence of sodium
triacetoxyborohydride to produce N-arylated ethylene-
diamine 6. Chloroacetylation followed by base-induced
cyclization furnishes the protected piperazinone 8.
The regioselective alkylation of 4-hydroxymethylimida-
zole utilizes chemistry developed by Anthony et al. for
the synthesis of regiochemically defined substituted
imidazoleacetic acid esters.9 Thus, the less hindered
nitrogen of 4-hydroxymethylimidazole reacts with trityl
chloride to give exclusively the trityl-protected 1,4-
substituted isomer. Fully protected trityl acetate 9 is
alkylated with 4-cyanobenzyl bromide to form the
imidazolium salt, which undergoes solvolysis in reflux-
ing methanol to imidazole hydrobromide 10. Acetate
hydrolysis and oxidation produces aldehyde 12, which
A computer-generated overlay using both electrostatic
and steric parameters11 matches conformations of 2c
with the conformation of FTase-bound CIFM analogue
3.12 The best-fit superpositions have the putative zinc
ligand imidazole of 2c overlaid with the aminothiol in
3, the N-aryl substituent of 2c positioned near the
hydrophobic a2 substituent of 3, and the n-butyl group
F igu r e 1. Computer-generated overlay of piperazine 2c and CIFM analogue 3.

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3781
F igu r e 2. Computer-generated overlay of CIFM analogue 3 and piperazinone 4a .
Sch em e 1. General Synthesis of Piperazinone FTIsa
Sch em e 2. Synthesis of Imidazole Intermediatea
a
(a) Ph3CCl, (C2H5)3N, DMF, 20 °C, 1 h, 94%; (b) (CH3CO)2O,
pyr, 20 °C, 5 h, 97%; (c) i. ArCH2Br, CH3CO2C2H5, 55 °C, 24 h, ii.
CH3OH, 55 °C; (d) LiOH, THF, H2O, 0 °C, 1 h, 66%; (e) SO3-pyr,
(C2H5)3N, DMSO, 20 °C, 2 h, 80%.
a
4d , 4e). Intrinsic FTase potency increases with the
length of the C-5 alkyl group; a C-5 butyl or ethylsufo-
nylmethyl substituent (4c, 4f) increases potency 20-
100-fold relative to the methyl-substituted analogue 4d .
Consistent with the model aligning the C-5 substituent
with the a1 side chain in Ca1a2X analogues, the most
active compounds contain hydrophobic groups at this
position. Moderately polar substituents such as sulfones
are also tolerated (4f).
To determine the ability of the compounds to act
intracellularly, compounds were assayed for their ability
to inhibit 50% of the anchorage-independent growth of
cultured H-ras-transformed RAT1 cells on poly(HEMA)-
coated microtiter plates (Table 3). In this assay, 4b and
4f have IC50 values between 1 and 10 nM. These
concentrations are well below the 10 µM to >100 µM
concentrations that are compatible with survival of 80-
90% of normal RAT1 cells, as determined by viable
(a) CH3NHOCH3, EDC, HOBT, (C2H5)3N, DMF, 95%; (b)
LiAlH4, (C2H5)2, 45-0 °C, 100%; (c) ArNH2, NaBH(OAc)3, DCE,
pH 6, 20 °C, 63%; (d) ClCH2COCl, aq NaHCO3, CH3CO2C2H5, 0
°C; (e) Cs2CO3, DMF, 0-20 °C, 96%; (f) HCl, CH3CO2C2H5; (g)
RCHO, NaBH(OAc)3, DCE, pH 6, 20 °C, 61%.
is reductively alkylated with the deprotected form
of piperazinone 8 to give the desired imidazole isomer
4a .
Resu lts a n d Discu ssion . The initial compound
prepared in the piperazinone series, 4a , inhibits FTase
with an IC50 of 0.4 nM (Table 2). Structure-activity
studies show that substitution of the N-aryl ring with
hydrophobic groups in either the ortho- or meta-posi-
tions is well-tolerated. Small hydrophobic groups in the
meta-position (4b, 4c) in particular give highly potent
inhibitors (IC50 e 0.1 nM). Variation of the piperazinone
C-5 substituent demonstrates that the S stereochemis-
try is preferred over the R by a factor of 10 (compare

3782 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Communications to the Editor
Ta ble 2. Influence of N-Aryl and Piperazinone Substitution on Activity
entry
Ar
R
FTase IC50 (nM)a
0.44 ( 0.5
<0.15 ( 0.07
0.11 ( 0.02
9 ( 1
80 ( 0
0.18 ( 0.11
GGTase IC50 (nM)b
4a
4b
4c
4d
4e
4f
2,3-Me2Ph
3-CF3OPh
3-ClPh
3-ClPh
3-ClPh
(S)-n-C4H9
(S)-n-C4H9
(S)-n-C4H9
(S)-CH3
(n ) 2)
(n ) 3)
(n ) 3)
(n ) 2)
(n ) 2)
(n ) 3)
10000
12000
NT
(n ) 1)
(n ) 1)
NT
NT
(R)-CH3
c
3-ClPh
(R)-C2H5SO2CH2
>100000
(n ) 1)
a
Concentration of compound required to inhibit 50% of FTase-catalyzed incorporation of [3H]FPP into recombinant human K-Ras
b
protein. Assay results were obtained using the protocol described in ref 6. Concentration of compound required to inhibit 50% of GGTase
I-catalyzed incorporation of [3H]GGPP into recombinant human RhoA protein. Assay results were obtained using the protocol described
in ref 6. c Compound 4f has the same absolute stereochemistry as compounds 4a -d , but group priority rules lead to the “R” designation.
Ta ble 3. Effect of Piperazinone FTIs on Cell Growth
poly(HEMA) IC50 (nM)a
MCF7
soft agar IC90 (nM)b
H-ras K-ras
100-1000e (n ) 2) 2500-10000 (n ) 2)
3-30 (n ) 2) 30-100 (n ) 2)
30 (n ) 2) >100
(n ) 1)f 1000
cytotoxic endpoint
entry
H-ras
DLD1
NT
IC20 (µM)c
4a
4b
4f
28 ( 16 (n ) 4)
2 ( 1
8 ( 4
NTd
25-50 (n ) 4)
(n ) 28) 4 ( 1 (n ) 2)
660 ( 8
(n ) 2)
10
(n ) 5)
(n ) 1)
(n ) 9) 5 ( 1 (n ) 2) 1500 ( 200 (n ) 2)
a
Concentration of compound required to inhibit 50% of cell growth on poly(HEMA)-coated microtiter plates. The assay is based on
b
conditions reported in ref 19 and is a measure of anchorage-independent cell growth. The concentration of compound required to inhibit
the anchorage-independent growth by 90% of either human H- or K-ras-transformed RAT1 cells suspended in soft agar, as compared to
vehicle-treated control. Assay conditions are described in ref 20. c Highest concentration of compound compatible with 80% cell survival
d
in cultured RAT1 cells, as assessed by viable staining with MTT. Assay conditions are described in ref 21. Not tested. e Determined in
viral H-ras-transformed RAT1 cells. f A second determination in viral H-ras-transformed RAT1 cells gave an identical result.
Ta ble 4. Selective FTIs Inhibit Both H-ras and K-ras Tumor
staining. To account for the differential effect of FTIs
on normal versus tumor tissue, there is evidence that
FTIs can induce apoptosis selectively in transformed
cells under certain conditions.13
Growth in Nude Micea
entry
cell line
dose
tumor growth inhibition (%)
4b
4b
4b
4f
4f
4f
H-ras/Rat1
H-ras/Rat1
K-ras/Rat1
H-ras/Rat1
H-ras/Rat1
K-ras/Rat1
40b
10b
80b
14c
1.4c
14c
100
68
64
100
73
60
(P < 0.01)d
(P < 0.01)
(P < 0.05)
(P < 0.01)
(P < 0.01)
(P < 0.01)
In another measure of anchorage-independent growth
inhibition, compounds 4a , 4b, and 4f were assayed for
their ability to block colony formation of H- and K-ras-
transformed RAT1 cells suspended in soft agar. One of
the most active compounds in this assay, 4b, inhibits
90% of H-Ras colony formation (IC90) at concentrations
between 3 and 30 nM, well below the 10 000 nM
concentration that ellicits 10-20% cytotoxicity in nor-
mal RAT1 cells (Table 3). While H-Ras is an exclusive
substrate for FTase, one complicating feature of Ras
biology relates to the ability of K-Ras to be a substrate
for either FTase or GGTase I.14 Since both prenylated
forms of the protein are capable of transforming
cells, it is reasonable to assume that both FTase and
GGTase I need to be suppressed to observe optimal
effects on K-ras-mediated cell growth.15 Although com-
pounds 4a , 4b, and 4f are poor inhibitors of GGTase I
(IC50 values greater than 10 000 nM, Table 2), they
block 90% of colony formation in K-ras-transformed
RAT1 cell lines at concentrations not toxic to normal
RAT1 cells (Table 3). Compared to cells expressing
H-Ras, however, approximately 10-30-fold higher con-
centrations of these compounds are needed to inhibit
K-ras-transformed cell proliferation to the same extent.
For example, FTI 4b blocks K-Ras colony formation with
an IC90 of 30-100 nM, a 3-10-fold increase relative to
its IC90 in H-ras-transformed cells. Overall, it is not
well-understood why selective FTIs interfere with pro-
liferation of K-ras-transformed cells. One possibility is
a
Female nude mice were subcutaneously injected with either
H- or K-ras-transformed RAT1 cells. After 24 h animals were
treated with either vehicle or FTI (n ) 8-10). Compound was
administered either by subcutaneous injection or by surgically
implanted Alzet osmotic pumps delivering 5 mL/h vehicle (50%
DMSO/water) or compound over a period of approximately 14 days.
b
Administered by subcutaneous injection. c Administered by con-
d
tinuous infusion. Statistical significance of tumor growth inhibi-
tion, relative to vehicle-treated control mice.
that other (non-Ras) farnesylated proteins are respon-
sible for the observed effects on growth in transformed
cells.16
In vivo studies in mice corroborate the activity
observed in cell culture.17 In nude mice bearing either
H- or K-ras-dependent tumors, treatment with 4b or 4f
for approximately 2 weeks produces statistically sig-
nificant tumor growth inhibition relative to vehicle-
treated controls. Compounds are administered either
subcutaneously once per day (4b) or by continuous
subcutaneous (sc) infusion via implanted Alzet minipump
(4f). In mice implanted with H-ras-transformed cells,
100% of tumor growth is blocked with either 40 mg/kg/
day 4b or 14 mg/kg/day 4f (Table 4). Approximately
8-10-fold higher doses are required to block the same
degree of K-ras tumor growth compared to H-ras. A 68%
reduction in tumor size occurs upon treatment of H-ras

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3783
T. J .; Shaber, M. D.; Gibbs, J . B.; Oliff, A. Inhibition of
Farnesyltransferase Induces Regression of Mammary and Sali-
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tumors with 10 mg/kg/day 4b, but 80 mg/kg/day 4b is
required to produce a 64% reduction in K-ras tumors.
Similarly for 4f, 1.4 mg/kg/day gives 73% reduction in
H-ras tumors, but 14 mg/kg/day is needed for 60%
inhibition of K-ras tumor growth.
Human tumor cell lines exhibit varying degrees of
sensitivity to FTIs 4b and 4f. In the MCF7 cell line, 4b
and 4f have IC50 values of 4 and 5 nM, respectively, in
the monolayer anchorage-independent growth assay
(Table 3). With the DLD1 cell line, 4b and 4f have IC50
values of 660 and 1500 nM, respectively. As noted in
earlier reports, the growth-inhibiting effect of FTIs on
cultured human tumor cell lines is largely independent
of their ras mutational status,18 and one of the most
sensitive cell lines, MCF7, is known to possess the wild-
type ras. It is possible that this and similar cell lines
contain activating mutations in proteins that function
upstream of Ras in the MAP/K signal transduction
pathway.
Con clu sion . Beginning with inhibitors that were
derived from the FTase Ras substrate Ca1a2X tetrapep-
tide, a series of modifications led to non-thiol, non-
carboxylate piperazinone FTIs that achieve potent levels
of inhibition. Significant aspects of inhibitor design en
route to 2c include conformational constraint via a
piperazine ring, optimization of the piperazine N-acyl
hydrophobic substituent, replacement of cysteine with
imidazole, and optimization of the imidazole substituent
to take advantage of a new high-affinity FTase binding
site. Modeling studies based on the FTase-bound con-
formation of a tetrapeptide analogue provided the
inspiration to pursue piperazinone 4a and analogues.
These compounds are subnanomolar FTIs that potently
inhibit the anchorage-independent growth of both H-
and K-ras-transformed cells and inhibit tumor growth
in a mouse tumor model. The overall profile of piper-
azinone FTIs, including their ability to suppress the
anchorage-independent growth of human tumor cell
lines at reasonable concentrations, encourages us to
continue to optimize their properties in the hope of
obtaining a clinically useful anticancer agent.
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Ack n ow led gm en t. The authors would like to thank
Graham M. Smith, Kenneth D. Anderson, Patrice A.
Ciecko, and Matthew M. Zrada for elemental analyses
and Harri J . Ramjit, Arthur B. Coddington, and Charles
W. Ross for mass spectra for the reported compounds.
We are also indebted to A. Burkhardt, D. Chen, Ronald
E. Diehl, Astrid M. Kral, Charles A. Omer, and Patricia
J . Miller for their assistance in carrying out mouse
experiments. Finally, we are grateful to J oy M. Hartzell
for preparation of this manuscript.
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