C-terminal modifications of histidyl-N-benzylglycinamides to give improved inhibition of Ras farnesyltransferase, cellular activity, and anticancer activity in mice.

Full text of DOI:10.1021/jm970470c

© Copyright 1997 by the American Chemical Society
Volume 40, Number 21
October 10, 1997
Communications to the Editor
Ch a r t 1. Structures of PD83176 and PD152440 (5a )
C-Ter m in a l Mod ifica tion s of
Histid yl-N-ben zylglycin a m id es To Give
Im p r oved In h ibition of Ra s
F a r n esyltr a n sfer a se, Cellu la r Activity,
a n d An tica n cer Activity in Mice
Dennis J . McNamara,*,† Ellen Dobrusin,†
Daniele M. Leonard,† Kevon R. Shuler,†
J ames S. Kaltenbronn,† J ohn Quin III,† Scott Bur,†
Charles E. Thomas,† Annette M. Doherty,†
J effrey D. Scholten,‡ Karen K. Zimmerman,‡
Barbara S. Gibbs, Richard C. Gowan,
Michael P. Latash, Wilbur R. Leopold,§
Sally A. Przybranowski,§ and
J udith S. Sebolt-Leopold
Parke-Davis Pharmaceutical Research, Division of
Warner-Lambert Company, 2800 Plymouth Road,
Ann Arbor, Michigan 48105
Received J uly 18, 1997
To accomplish its critical role in transmitting the
signal for cell growth, the Ras protein (H, N, K-4A, and
K-4B) must be attached to the inside of the cell
membrane.1 This is achieved in large part by lipophilic
modifications to the four amino acids at its C-terminus,
termed the CAAX box, where C is cysteine, A is any
amino acid, and X is serine or methionine.2 The first
and most important of these modifications is the at-
tachment of a farnesyl group, a 15-carbon isoprenoid
unit, to the sulfur of cysteine. This reaction is catalyzed
by the enzyme farnesyltransferase (FT) and utilizes
farnesylpyrophosphate (FPP) as the farnesyl donor.
Mutations in Ras are frequently encountered in
cancer: 30% of all human cancers contain a Ras
mutation, including 90% of pancreatic, 50% of colon, and
30% of lung cancers.3 Since mutated Ras must be
farnesylated in order to transform cells to a malignant
state,4 inhibition of FT is an attractive target for
anticancer activity. In fact, several groups have re-
ported anticancer activity in mice utilizing this
strategy.5-9
We have previously reported that PD83176 (Chart 1),
a protected pentapeptide discovered by screening our
compound library, inhibited the isolated rat FT enzyme
with an IC50 ) 0.076 µM.10 However, it did not inhibit
Ras farnesylation in cells, probably due to an inability
to enter cells. By truncating the C-terminus of PD83176
at the serine R-carbon and transposing, in peptoid
fashion, the benzylated side chain of the tyrosine to its
R-nitrogen, PD152440 (5a ) (Chart 1) was designed.11
While less active than PD83176 against the isolated FT
enzyme with an IC50 ) 0.32 µM, 5a did inhibit Ras
farnesylation in cells at 1 µM. We here report on
modifications to the C-terminal amide of 5a , which
result in dramatically improved enzymatic and cellular
activities, culminating in anticancer activity in mice.
Ch em istr y. The synthesis of 5a is illustrated in
Scheme 1. Glycine methyl ester hydrochloride was
reductively aminated with 4-(benzyloxy)benzaldehyde
using sodium triacetoxyborohydride to give the N-
benzylglycine 1 in 40% yield. Acylation with CbzHis-
* To whom correspondence should be addressed. Tel: 313-996-7372.
Fax: 313-996-7879. E-mail: mcnamad@aa.wl.com.
† Department of Chemistry.
‡ Department of Biochemistry.
§ Department of Cancer Research.
Department of Cell Biology.
S0022-2623(97)00470-6 CCC: $14.00 © 1997 American Chemical Society

3320 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Sch em e 1. Synthesis of 5a a
Communications to the Editor
a
Reagents: (a) HCl‚H2NCH2CO2CH3, Na(OAc)3BH, CH2Cl2 (40%); (b) CbzHis(Trt), PyBOP, DIEA, CH2Cl2 (55%); (c) LiOH, THF, H2O
(89%); (d) HCl‚H2NCH2CH2OBn, HBTU, DIEA, CH2Cl2; (e) TFA, CH2Cl2 (54%, two steps).
Ta ble 1. Inhibitory Activities against the Isolated FT Enzyme and in Transformed Cells
compd
R1
R2
FT IC50 (µM)a
0.076
0.32 ( 0.07 (6)
0.062 ( 0.007 (9)
4
0.003 ( 0.001 (4)
0.041 ( 0.012 (5)
0.27
0.008 ( 0.001 (5)
0.005
0.016
0.002
Ras farnesylation MED (µM)b
colony formation IC50 (µM)c
PD83176
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
see Chart 1
CH2CH2OBn
CH2CH2Ph
CH2CH2Ph
>100
1
NTd
14.3 ( 3.6 (4)
4.3 ( 0.8 (7)
NT
0.71 ( 0.23 (3)
6.3
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.1
NT
0.05
0.2
CH(Me)CH2Ph (R)
CH(Me)CH2Ph (S)
C(Me)2CH2Ph
CH2CH(Me)Ph
CH2CH(Me)Ph (R)
CH2CH(Me)Ph (S)
CH2CH(CN)Ph
CH2CH(Et)Ph
CH2CH(n-Pr)Ph
CH2C(Me)2Ph
CH2C(Et)2Ph
1
NT
0.05
0.05
0.2
0.05
0.05
0.2
0.05
0.2
0.02
0.02
0.2
0.31 ( 0.06 (4)
0.30
0.57
0.19 ( 0.06 (3)
0.40 ( 0.08 (3)
>1
0.18 ( 0.04 (8)
>1
0.18
0.005 ( 0.001 (3)
0.025
0.004 ( 0.001 (3)
0.032
0.007
CH2C(CH2)2Ph
CH2C(CH2)3Ph
CH2C(CH2)4Ph
0.006
0.017
0.088 ( 0.022 (6)
0.33
a
Concentration of compound required to inhibit by 50% the incorporation of [3H]FPP into a biotinylated hexapeptide from the C-terminus
of KB-Ras by Sf9-affinity-purified rat FT. Values are averages of the number of determinations in parentheses ( SEM or, where unnoted,
b
of two determinations. Minimum effective dose (MED) of compound required to detect unfarnesylated Ras in H-Ras-F cells as determined
by Western transfer techniques. Values are confirmed data from at least two determinations. c Concentration of compound required to
inhibit by 50% the number of colonies of H-Ras-F cells when grown on soft agar. Values are averages of the number of determinations in
d
parentheses ( SEM or, where unnoted, of two determinations. Not tested.
(Trt)12 gave 2 in 55% yield. The methyl ester was
hydrolyzed to the acid 3 (88% yield), which was coupled
to 2-(benzyloxy)ethylamine hydrochloride13 to give the
protected intermediate 4a . Detritylation of the imida-
zole furnished the target 5a in 54% yield over two steps.
Compounds 5b-q (Table 1) were synthesized analo-
gously by coupling the required phenethylamines to 3.
R esu lt s a n d Discu ssion . The compounds in this
series were first tested for inhibition of the isolated rat
FT enzyme (Table 1).14 The cellular activity of these
compounds was then investigated, first by measuring
the inhibition of Ras farnesylation in H-Ras-trans-
formed NIH3T3 (H-Ras-F) cells15 and then by measur-
ing the inhibition of colony formation of these same cells
when grown in soft agar. This latter assay is especially
relevant to anticancer activity since colony formation
in soft agar is well correlated with tumorogenicity in
the nude mouse.16 In general there was very good
agreement between these three assays for this series
of compounds.
The greater activity of PD83176 relative to 5a against
the FT enzyme suggested that the activity of 5a could
be enhanced by substituting the C-terminal amide.
Initial efforts to simplify the scaffold on which to carry
out structure-activity studies led to the unsubstituted
phenethylamine 5b, which displayed a 5-fold improve-
ment in activity against FT, with a corresponding
improvement in cellular activity (Table 1). Methylation

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3321
of the C-terminal amide nitrogen (5c) markedly de-
creased activity against the enzyme. Substitution on
the R-carbon of the phenethylamine with a (R)-methyl
group (5d ) resulted in a 20-fold improvement in activity
against FT relative to 5b, with improved cellular
activity. (S)-Methyl substitution (5e) did not result in
a significant improvement in activity, and dimethyl
substitution (5f) resulted in decreased activity. Methyl
substitution on the â-carbon (5g), especially in the
R-configuration (5h vs 5i), resulted in a 10-fold im-
provement in activity against FT relative to 5b, with
improved cellular activity. Other small substituents,
as with the cyano (5j) and ethyl (5k ) groups, maintained
this enzymatic and cellular activity, while the larger
n-propyl substituent (5l) did not. Dimethyl substitution
(5m ) resulted in increased activity, whereas diethyl
substitution (5n ) resulted in decreased activity. Cyclo-
propyl (5o) and cyclobutyl (5p ) substitution also resulted
in increased activity, whereas cyclopentyl (5q) resulted
in decreased activity. In general, small substituents on
the R and â positions of the phenethylamine scaffold
resulted in compounds with dramatically improved
activity against the isolated FT enzyme and in cells.
F igu r e 1. Mean tumor burden of mice inoculated subcutane-
ously with H-Ras-F cells on day 0 and treated intraperitoneally
with 5m at 150 mg/kg/day (0) or 100 mg/kg/day (4) or vehicle
control (×) once a day for 14 consecutive days after tumor
implantation.
Compound 5m , which inhibited the isolated FT
enzyme with an IC50 ) 0.004 µM, inhibited Ras farne-
sylation in transformed cells at 0.05 µM, and inhibited
the colony formation of transformed cells in soft agar
with an IC50 ) 0.18 µM, was one of the most active
compounds in this series. Investigation of its inhibition
of FT using steady state kinetic analysis revealed it to
be competitive with the FPP substrate (data not shown),
with a Ki ) 0.74 ( 0.04 nM.
Adrienne D. Cox (University of North Carolina) for the
H-Ras-F cells.
Su p p or tin g In for m a tion Ava ila ble: Details for the
synthesis of 5a -q; assays for inhibition of the FT enzyme, the
GGT1 enzyme, cellular Ras farnesylation, and colony forma-
tion; and testing of 5m in mice (10 pages). Ordering informa-
tion is given on any current masthead page.
A further requirement for these FT inhibitors as
potential anticancer drugs is selectivity for the FT
enzyme compared to the closely related geranylgera-
nyltransferase-1 (GGT1) enzyme. GGT1 catalyzes the
transfer of a geranylgeranyl group, a 20-carbon iso-
prenoid unit, to proteins containing a CAAX box, where
X is leucine.2 Since many more proteins in the body
are geranylgeranylated than farnesylated, we believed
that selectivity for FT was important. Our lead com-
pound in this series, 5a , displayed only 2-fold selectivity
for FT (IC50 ) 0.32 µM) over GGT1 (IC50 ) 0.6 µM).
However 5m was a very selective inhibitor (4500-fold),
with an IC50 of 0.004 µM against FT and an IC50 of 18
µM against GGT1.
Finally, we evaluated 5m for anticancer activity in
mice (Figure 1). Athymic mice were implanted subcu-
taneously with H-Ras-F cells and treated intraperito-
neally with 5m , once a day for 14 consecutive days after
tumor implantation. When administered at 150 mg/kg/
day, 5m inhibited tumor growth by 88% relative to
untreated controls when assessed on day 15. At 100
mg/kg/day, 5m inhibited tumor growth by 53%.
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