Oxo-piperazine derivatives of N-arylpiperazinones as inhibitors of farnesyltransferase

Article abstract of DOI:10.1016/S0960-894X(00)00710-1

The evaluation of SAR associated with the insertion of carbonyl groups at various positions of N-arylpiperazinone farnesyltransferase inhibitors is described herein. 1-Aryl-2,3-diketopiperazine derivatives exhibited the best balance of potency and pharmac

Full text of DOI:10.1016/S0960-894X(00)00710-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 537±540
Oxo-piperazine Derivatives of N-Arylpiperazinones as
Inhibitors of Farnesyltransferase
Christopher J. Dinsmore,a,* Je€rey M. Bergman,a Donna D. Wei,a C. Blair Zartman,a
Joseph P. Davide,b Ian B. Greenberg,b Dongming Liu,b Timothy J. O'Neill,b
Jackson B. Gibbs,b Kenneth S. Koblan,b Nancy E. Kohl,b Robert B. Lobell,b
I-Wu Chen,c Debra A. McLoughlin,c Timothy V. Olah,c Samuel L. Graham,a
George D. Hartmana and Theresa M. Williamsa
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
Received 6 October 2000; accepted 7 December 2000
AbstractÐThe evaluation of SAR associated with the insertion of carbonyl groups at various positions of N-arylpiperazinone far-
nesyltransferase inhibitors is described herein. 1-Aryl-2,3-diketopiperazine derivatives exhibited the best balance of potency and
pharmacokinetic pro®le relative to the parent 1-aryl-2-piperazinones. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
FTIs, the use of conformational constraints in conjunction
with carboxyl deletion and cysteinyl group replacement
provided low molecular weight piperazinone FTIs with
potent in vitro and in vivo activity.5 Thus, 1-aryl-2-
piperazinone FTIs (e.g., 1) exhibit high levels of potency
and antiproliferative activity, and have good oral bioa-
vailability (Fig. 1).5,6
Farnesyltransferase (FTase) is a zinc metalloenzyme
that catalyzes the S-alkylation of a cysteine residue in
the C-terminal tetrapeptide sequence of proteins using
farnesylpyrophosphate (FPP), thus enabling their parti-
cipation in signal transduction during cell proliferation.1
One of the protein substrates for FTase, K-Ras, has
been implicated in the growth of 20±30% of all human
tumors.2 In these tumors, mutated Ras loses its normal
GTPase function, becomes constitutively bound to
GTP, and transmits growth signals independent of
extracellular growth factors to downstream mitogenic
e€ectors. Strategies for regulating unimpeded oncogenic
ras signalling have focused on FTase, inhibitors of
which have signi®cant potential as cancer chemother-
apeutic agents.3 FTase 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 currently undergoing human
clinical trials both as single agents and in combination
with other anti-cancer agents.4
Figure 1. 1-Aryl-2-piperazinone FTI 1 and oxo-piperazine derivatives 2.
We chose to investigate SAR of compounds closely
related to 1. The in vivo duration of 1 is limited by
rapid metabolic transformation via N-dealkylations
and C5-side-chain substituent hydroxylations.6 In
attempts to improve the overall pharmacokinetic pro®le
of compounds related to 1, we investigated oxo-piper-
azines 2 with the hope of blocking or attenuating meta-
bolism, and altering compound polarity. Herein, we
describe features that are consistent with maintaining
A wide array of FTIs that mimic the C-terminal Ca1a2X
tetrapeptide of Ras (e.g., CVIM) has been described.4a,e
In a previous approach to pharmacologically improved
*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 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00710-1

538
C. J. Dinsmore et al. / Bioorg. Med. Chem. Lett. 11 (2001) 537±540
Scheme 1. Reagents and conditions: (a) Gly-OMe, Na(AcO)3BH, 4 A
MS, DCE, 56%; (b) Boc-Gly-OH, EDC, HOBt, Et3N, DMF, 81%; (c)
HCl, EtOAc, 0 ꢀC, 100%; (d) Et3N, DCM, 62%; (e) Ph3Bi, Cu(OAc)2,
Et3N, DCM, 34%.
high enzyme activity and cell-potency, and that provide
improvements in pharmacokinetic half-life.7
Scheme 2. Reagents and conditions: (a) 2-oxazolidinone, MeO(CH2)2-
O(CH2)2OH, 160 ꢀC; (b) Boc2O, NaHCO3, THF±H2O, 80% for 2
steps; (c) N-Boc-norleucinal, Na(AcO)3BH, AcOH, 4 A MS, DCE,
63%; (d) BrCH2CO2Me, K2CO3, DMF, 60 ꢀC, 45% from 8, 58%
from 10; (e) HCl, EtOAc, 0 ꢀC, 100%; (f) ClCOCO2Me, NaHCO3,
EtOAc±H2O, 0 ꢀC, 90% from 8, 58% from 10; (g) Na(AcO)3BH, 4 A
MS, DCE, 0 ꢀC to rt, 45% from 11, 48% from 12, 18% from 13, 18%
from 14.
Chemistry
The 1-aryl-2-piperazinones 19, 22, and 25 (cf 2; Z=O)
were synthesized by analogy to previously described
methods.5,6,8 The 1-aryl-2,5-diketopiperazine 6 (cf. 2; X,
Z=O) was prepared as described in Scheme 1. Aldehyde 35
was reductively alkylated with glycine methyl ester, then
acylated with N-Boc-glycine to provide 4. Deprotection
and cyclization gave diketopiperazine 5, which was
subjected to Cu(OAc)2-mediated N-arylation9 to give 6.
The piperazine carboxamides 23 and 24 (cf 2; W=O
and W, Z=O, respectively) were prepared by sodium
chlorite oxidation10 of aldehyde 3, followed by EDC-
mediated amide coupling to 1-(3-chlorophenyl)piper-
azine and 1-(3-chlorophenyl)-2-piperazinone,8 respec-
tively.
premature cyclization was exacerbated by the Thorpe±
Ingold e€ect.13
Structure±Activity Relationships
An initial survey of N-[1-(4-cyanobenzyl)imidazolyl-5-
methyl]-2-piperazinone analogues (Table 1) revealed
that the position of oxo-groups within the framework
signi®cantly impacts the enzyme activities. Relative to
the parent piperazinone 19 (FTase IC50 5 nM), the more
polar 2,3-diketopiperazine 20 was only 3-fold less active
versus FTase, and at least several fold less active versus
The 1-aryl-3-piperazinones 15 and 17 (cf. 2; Y=O) and
the 1-aryl-2,3-diketopiperazines 16, 18, 20, and 26 (cf. 2;
Y, Z=O) were prepared using a tandem reductive alkyl-
ation and cyclization process recently developed for
convergent syntheses of unsymmetrical 2,3-diketopiper-
azines (Scheme 2).11 For example, intermediate 8, prepared
by aminoethylation12 and protection of 3-chloroaniline
7, was alkylated with methyl bromoacetate to give the
salt 11 after treatment with HCl. This was subjected to a
tandem reductive alkylation and cyclization reaction
with aldehyde 3 to a€ord the desired piperazinone 15.
Likewise, 8 was acylated with methyl oxalyl chloride to
provide the salt 12 after deprotection, and this was
converted to 2,3-diketopiperazine 16. The substituted
analogues 17 and 18 were constructed by reductive
alkylation of 2,3-dimethylaniline 9 with N-Boc-norleucinal
to give 10, followed by the same reaction sequences
described above. The reactions of substrates 11 and 12
with aldehyde 3 are less ecient than similar reactions
using nonbasic aldehydes (cf. 45% yield using 3 vs 61±
74% using substituted benzaldehydes).11 Greater pro-
portions of unalkylated piperazinone or diketopiprazine
byproducts were formed. This may be due, in part, to
the basicity of the imidazole moiety, which may accel-
erate premature cyclization relative to reductive alkyla-
tion. The reactions of the substituted analogues 13 and
14 were less ecient still (18% yield), probably because
the
related
enzyme
geranylgeranyltransferase-I
(GGTase-I). The isomeric 2,5-diketopiperazine 6, with
similar physical properties, was substantially less active
(FTase IC50 950 nM). Taken with the known increase in
Table 1. FTase inhibition, GGTase-I inhibition, and physical prop-
erty data for parent piperazinone 19 and diketopiperazines 20 and 6
Compd
X
Y
Z
FTase IC50
(nM)a
GGTase-I IC50
(mM)b
Log Pc
19
20
6
H2 H2
O
O
O
5
16
950
8.7
>20
nd
1.32
0.46
0.55
H2
O
O
H2
aConcentration of compound required to reduce the human FTase-
catalyzed incorporation of [3H]FPP into recombinant Ras-CVIM by
50% (ref 14a).
bConcentration of compound required to reduce the human GGTase-
I-catalyzed incorporation of [3H]GGPP into biotinylated peptide cor-
responding to the C-terminus of human Ki-Ras by 50% (ref 14b).
cLog of the octanol±water partition coecient.

C. J. Dinsmore et al. / Bioorg. Med. Chem. Lett. 11 (2001) 537±540
539
potency induced by the hydrophobic n-butyl group in 1
and related compounds,5,6 this indicates the presence of
a hydrophobic binding pocket in the FTase active site
that is intolerant to the presence of a carbonyl group at
the piperazine C5 position.
best in vitro activity relative to 22, and also exhibited
the lowest log P in the series. That the activities of these
compounds in vitro and in cell culture roughly correlate,
and that general cell cytotoxicity is only observed at
much higher concentrations (>25 mM) than are
required for growth inhibition, suggest that the intra-
cellular mechanism is due to inhibition of FTase. Taken
together, the results suggest that converting the amino-
group in 1-aryl-2-piperazinones to an sp2-hybridized
amide decreases potency by an amount dependent on
the placement of the carbonyl group.
Additional studies (Table 2) focused on compounds
lacking the o€ending C5 carbonyl group (X=O in 2).
The unoxygenated piperazine 21 (IC50=250 nM), was
100-fold less potent than the parent 1-aryl-2-piper-
azinone 22 (IC50=2 nM), and was the least active in the
series. This reduction in potency was accompanied by
ꢁ10-fold increase in the concentration required to inhi-
bit the anchorage-independent growth of Harvey-ras
transformed cells in soft agar. The 1-aryl-3-piperazinone
15 was 50-fold less active than 22, while another iso-
meric compound 23 was 10-fold less active. However,
the 2,3-diketopiperazine 16 (IC50=11 nM) exhibited the
Closer derivatives of 1-aryl-2-piperazinone compound 1
were investigated with the expectation that the biological
activity of oxo-piperazine analogues could be improved
by the addition of the lipophilic alkyl chain to the
piperazine ring (Table 3). Although 20-fold improve-
ment in activity was observed in the 1-aryl-2-piper-
azinone series upon C5-alkylation (25, IC50=11 nM vs
1, IC50=0.44 nM), only 7-fold bene®t was a€orded in the
2,3-diketopiperazines (26, IC50=20 nM vs 18, IC50=3
nM). Surprisingly, the 1-aryl-3-piperazinone analogue 17
was as potent as 18 in vitro, an unexpected result in light of
the 10-fold di€erence between non-alkylated analogues 15
(IC50=100 nM) and 16 (IC50=11 nM). Interestingly,
despite their equivalent activity in vitro, a >100-fold
di€erence in cell potency between 17 and 18 was
observed both in soft agar and on poly(HEMA) coated
microtiter plates, with 18 achieving the best activity
overall (H-ras poly-HEMA IC50=15 nM).
Table 2. FTase inhibition, cell growth inhibition, cytotoxicity, and
physical property data for compounds 15±16 and 21±24
Compd
W
Y
Z
FTase
H-ras
Cytotoxic Log Pd
endpoint
IC50 (nM)a soft agar
IC90 (mM)b IC20 (mM)c
21
22
15
23
24
16
H2 H2 H2
H2 H2
250
2
100
21
33
11
1.0 25
0.1 >100
>3.4
2.11
2.51
2.79
nd
In pharmacokinetic analyses, the compounds were
administered orally to two dogs as a mixture with up to
eleven other compounds, each at 1 mg/kg, with com-
pound 22 included as a reference in each combination
dose (Table 4).18 To monitor the data for potential
artifacts due to drug±drug interactions using this pro-
tocol, pharmacokinetic pro®les relative to the internal
standard 22 were assessed and were considered mean-
ingful since the data for 22 were not signi®cantly altered
from single administration data. Relative to the corre-
sponding 1-aryl-2-piperazinones, the 1-aryl-3-piper-
O
H2
H2
O
O
1
0.1±1
>100
>100
>100
>100
H2 H2
O
H2
H2
O
O
O
1
1
1.24
aSee footnote a in Table 1.
bConcentration required to achieve 90% reduction of anchorage-
independent growth of RAT1 v-Ha-ras transformed cells in soft agar
relative to vehicle-treated control (ref 15).
cHighest nontoxic concentration (ꢁ80% cell survival) for cultured
RAT1 cells as assessed by viability staining with MTT (ref 16).
dSee footnote c in Table 1.
Table 3. FTase inhibition, cell growth inhibition, cytotoxicity, and physical property data for compounds 1, 17±18, and 25±26
Compd
R
Y
Z
FTase
IC50 (nM)a
H-ras soft agar
IC90 (mM)c
H-ras poly(HEMA)
IC50 (nM)d
Cytotoxic endpoint
IC20 (mM)e
Log Pf
25
26
1
17
18
H
H
n-Bu
n-Bu
n-Bu
H2
O
H2
O
O
O
O
H2
O
11
20
0.44b
1.3b
3
1
>1
0.1±1
1±10
0.1±1
83
nd
28
2,510
15
10
10
25-50
>100
10
2.00
1.11
>3.7
>3.5
3.30
O
aSee footnote a in Table 1.
bEnzyme concentration=10 pM instead of 1 nM in assay.
cSee footnote b in Table 2.
dConcentration required to inhibit by 50% the anchorage-independent proliferation of RAT1 v-Ha-ras transformed cells on poly-(HEMA)-coated
plates relative to vehicle-treated control (ref 17).
eSee footnote c in Table 2.
fSee footnote c in Table 1.

540
C. J. Dinsmore et al. / Bioorg. Med. Chem. Lett. 11 (2001) 537±540
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Table 4. Pharmacokinetic data in dogs following combination dosing
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1-Aryl-3-piperazinones
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0.96
1.2
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1-Aryl-2,3-diketopiperazines
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12.50
4.43
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Conclusion
The manipulation of the oxo group position within the
1-aryl-2-piperazinone framework results in the modulation
of FTase enzyme activity, polarity, and pharmacokinetic
pro®les in dogs following oral administration. In the case of
1-aryl-3-piperazinones, a C5-alkyl substituent is bene®cial
to in vitro FTase inhibition activity, but activity in cell
culture (e.g., for 17) is poor. In comparison, the 2,3-
diketopiperazines are in some cases as potent as the
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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 analy-
tical support, R. Robinson and E. S. Walsh for technical
assistance, and J. R. Hu€ and A. I. Oli€ for their support
of this work.
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