Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
INHIBITORS OF PRENYL-PROTEIN TRANSFERASE
RELATED APPLICATION
The present patent application is a continuation-in-part
application of copending provisional application Serial No. fi0/057,080,
filed August 27,1997.
BACKGROUND OF THE INVENTION
The present invention relates to certain compounds that
are useful for the inhibition of prenyl-protein transferases and the
treatment of cancer. In particular, the invention relates to prenyl-
protein transferase inhibitors which are efficacious in vivo as inhibitors
of geranylgeranyl-protein transferase type I (GGTase-I) and that inhibit
the cellular processing of both the H-Ras protein and the K4B-Ras
protein.
Prenylation of proteins by prenyl-protein transferases
represents a class of post-translational modification (Glomset,
J. A., Gelb, M. H., and Farnsworth, C. C. (1990). Trends Biochem.
Sci. 15, 139-142; Maltese, W. A. (1990). FASEB J. 4, 3319-3328). This
modification typically is required for the membrane localization and
function of these proteins. Prenylated proteins share characteristic
C-terminal sequences including CAAX (C, Cys; A, an aliphatic
amino acid; X, another amino acid), XXCC, or XCXC. Three post-
translational processing steps have been described for proteins having
a C-terminal CAAX sequence: addition of either a 15 carbon (farnesyl)
or 20 carbon (geranylgeranyl) isoprenoid to the Cys residue, proteolytic
cleavage of the last 3 amino acids, and methylation of the new
C-terminal carboxylate (Cox, A. D. and Der, C. J. (1992a). Critical
Rev. Oncogenesis 3:365-400; Newman, C. M. H. and Magee, A. I.
(1993). Biochim. Biophys. Acta 1155:79-96). Some proteins may also
have a fourth modification: palmitoylation of one or two Cys residues
N-terminal to the farnesylated Cys. While some mammalian cell
proteins terminating in XCXC are carboxymethylated, it is not clear
whether carboxy methylation follows prenylation of proteins terminat-
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ing with a XXCC motif (Clarke, S. (1992). Annu. Rev. Biochem. 61, 355-
386). For all of the prenylated proteins, addition of the isoprenoid is the
first step and is required for the subsequent steps (Cox, A. D. and Der, C.
J. (1992a). Critical Rev. Oncogenesis 3:365-400; Cox, A. D. and Der, C. J.
(1992b) Current Opinion Cell Biol. 4:1008-1016).
Three enzymes have been described that catalyze protein
prenylation: farnesyl-protein transferase (FPTase), geranylgeranyl-
protein transferase type I (GGPTase-I), and geranylgeranyl-protein
transferase type-II (GGPTase-II, also called Rab GGPTase). These
IO enzymes are found in both yeast and mammalian cells (Clarke, 1992;
Schafer, W. R. and Rine, J. (1992) Annu. Rev. Genet. 30:209-237). Each
of these enzymes selectively uses farnesyl diphosphate or geranyl-
geranyl diphosphate as the isoprenoid donor and selectively recognizes
the protein substrate. FPTase farnesylates CaaX-containing proteins
that end with Ser, Met, Cys, Gln or Ala. For FPTase, CaaX tetrapeptides
comprise the minimum region required for interaction of the protein
substrate with the enzyme. The enzymological characterization of these
three enzymes has demonstrated that it is possible to selectively inhibit
one with little inhibitory effect on the others (Moores, S. L., Schaber, M.
D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall,
M. S., Pompliano, D. L., and Gibbs, J. B., J. Biol. Chem., 266:17438
(1991), U.S. Pat. No. 5,470,832).
The prenylation reactions have been shown genetically to
be essential for the function of a variety of proteins (Clarke, 1992; Cox
and Der, 1992a; Gibbs, J. B. (1991). Cell 65: 1-4; Newman and Magee,
1993; Schafer and Rine, 1992). This requirement often is demonstrated
by mutating the CaaX Cys acceptors so that the proteins can no longer be
prenylated. The resulting proteins are devoid of their central biological
activity. These studies provide a genetic "proof of principle" indicating
that inhibitors of prenylation can alter the physiological responses
regulated by prenylated proteins.
The Ras protein is part of a signaling pathway that links
cell surface growth factor receptors to nuclear signals initiating cellular
proliferation. Biological and biochemical studies of Ras action indicate
that Ras functions like a G-regulatory protein. In the inactive state,
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Ras is bound to GDP. Upon growth factor receptor activation, Ras is
induced to exchange GDP for GTP and undergoes a conformational
change. The GTP-bound form of Ras propagates the growth stimulatory
signal until the signal is terminated by the intrinsic GTPase activity of
Ras, which returns the protein to its inactive GDP bound form (D.R.
Lowy and D.M. Willumsen, Ann. Rev. Biochem. 62:851-891 (I993)).
Activation of Ras leads to activation of multiple intracellular signal
transduction pathways, including the MAP Kinase pathway and the
Rho/Rac pathway (Joneson et al., Science 271:810-812).
Mutated ran genes are found in many human cancers,
including colorectal carcinoma, exocrine pancreatic carcinoma, and
myeloid leukemias. The protein products of these genes are defective in
their GTPase activity and constitutively transmit a growth stimulatory
signal.
The Ras protein is one of several proteins that are known to
undergo post-translational modification. Farnesyl-protein transferase
utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group
of the Ras CAAX box with a farnesyl group (Reins et al., Cell, 62:81-88
(1990); Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et
al., Science, 249:1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci
USA, 87:7541-7545 (1990)).
Ras must be localized to the plasma membrane for both
normal and oncogenic functions. At least 3 post-translational modi-
fications are involved with Ras membrane localization, and all 3
modifications occur at the C-terminus of Ras. The Ras C-terminus
contains a sequence motif termed a "CAAX" or "Cys-Aaal-Aaa2-Xaa"
box (Cys is cysteine, Aaa is an aliphatic amino acid, the Xaa is any
amino acid) (Willumsen et al., Nature 310:583-586 (1984)). Depend-
ing on the specific sequence, this motif serves as a signal sequence for
the enzymes farnesyl-protein transferase or geranylgeranyl-protein
transferase, which catalyze the alkylation of the cysteine residue of the
CAAX motif with a C15 or C2p isoprenoid, respectively. (S. Clarke., Ann.
Rev. Biochem. 61:355-386 (1992); W.R. Schafer and J. Rine, Ann. Rev.
Genetics 30:209-237 (1992)). Direct inhibition of farnesyl-protein
transferase would be more specific and attended by fewer side effects
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than would occur with the required dose of a general inhibitor of
isoprene biosynthesis.
Other farnesylated proteins include the Ras-related GTP-
binding proteins such as RhoB, fungal mating factors, the nuclear
lamina, and the gamma subunit of transducin. James, et al., J. Biol.
Chem. 269, 14182 (1994) have identified a peroxisome associated protein
Pxf which is also farnesylated. James, et al., have also suggested that
there are farnesylated proteins of unknown structure and function in
addition to those listed above.
Inhibitors of farnesyl-protein transferase (FPTase) have
been described in two general classes. The first class includes analogs
of farnesyl diphosphate (FPP), while the second is related to protein
substrates (e.g., Ras) for the enzyme. The peptide derived inhibitors
that have been described are generally cysteine containing molecules
that are related to the CAAX motif that is the signal for protein
prenylation. (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al., PNAS,
88:732-736 (1991)). Such inhibitors may inhibit protein prenylation while
serving as alternate substrates for the farnesyl-protein transferase
enzyme, or may be purely competitive inhibitors (U.S. Patent 5,141,851,
University of Texas; N.E. Kohl et al., Science, 260:1934-1937 (1993);
Graham, et al., J. Med. Chem., 3?, 725 ( 1994)).
Mammalian cells express four types of Ras proteins
(H-, N-, K4A-, and K4B-Ras) among which K4B-Ras is the most
frequently mutated form of Ras in human cancers. The genes that
encode these proteins are abbreviated H-ras, N-ras , K4A-ras and K4B-
ras respectively. H-ras is an abbreviation for Harvey-ras. K4A-ras and
K4B-ras are abbreviations for the Kirsten splice variants of ras that
contain the 4A and 4B exons, respectively. Inhibition of farnesyl-protein
transferase has been shown to block the growth of H-ras-transformed
cells in soft agar and to modify other aspects of their transformed
phenotype. It has also been demonstrated that certain inhibitors of
farnesyl-protein transferase selectively block the processing of the H-Ras
oncoprotein intracellularly (N.E. Kohl et al., Science, 260:1934-1937 (1993)
and G.L. James et al., Science, 260:1937-1942 ( I993). Recently, it has been
shown that an inhibitor of farnesyl-protein transferase blocks the growth
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of H-ras-dependent tumors in nude mice (N.E. Kohl et al., Proc. Natl.
Acad. Sci U.S.A., 91:9141-9145 (1994) and induces regression of
mammary and salivary carcinomas in H-ras transgenic mice (N.E.
Kohl et al., Nature Medicine, 1:792-797 (1995).
Indirect inhibition of farnesyl-protein transferase in vivo
has been demonstrated with lovastatin (Merck & Co., Rahway, NJ)
and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al.,
Science 245:3?9 (1989)). These drugs inhibit HMG-CoA reductase,
the rate limiting enzyme for the production of polyisoprenoids includ-
ing farnesyl pyrophosphate. Inhibition of farnesyl pyrophosphate
biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane
localization in cultured cells.
It has been disclosed that the lysine-rich region and
terminal CVIM sequence of the C-terminus of K-RasB confer resist-
ante to inhibition of the cellular processing of that protein by certain
selective FPTase inhibitors. James, et al., J. Biol. Chem. 270, 6221 (1995)
Those FPTase inhibitors were effective in inhibiting the processing of H-
Ras proteins. James et al., suggested that prenylation of the K4B-Ras
protein by GGTase contributed to the resistance to the selective FPTase
inhibitors.
Several groups of scientists have recently disclosed
compounds that are non-selective FPTase,/GGTase inhibitors. (Nagasu
et al. Cancer Research, 55:5310-5314 (1995); PCT application
WO 95/25086).
It is the object of the instant invention to provide a
prenyl-protein transferase inhibitor which is efficacious in vivo as an
inhibitor of geranylgeranyl-protein transferase type I (GGTase-I), also
known as CAAX GGTase.
It is also the object of the present invention to provide a
compound which inhibits the cellular processing of both the
H-Ras protein and the K4B-Ras protein.
It is also the object of the present invention to provide a
compound which is efficacious in vivo as an inhibitor of the growth of
cancer cells characterized by a mutated K4B-Ras protein.
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A composition which comprises such an inhibitor
compound is used in the present invention to treat cancer.
SUMMARY OF THE INVENTION
The present invention comprises peptidomimetic
piperazinone-containing compounds which inhibit the prenyl-protein
transferases: farnesyl-protein transferase and geranylgeranyl-protein
transferase type I. Further contained in this invention are
chemotherapeutic compositions containing these prenyl transferase
inhibitors and methods for their production.
The compounds of this invention are illustrated by the
formula I:
(Rs)r N Rsa R2 O
V - A~(CRla2)~A2(CR~a2)n ~'~N N N-Z
(CR~b2)p X/ ~~ '/3
R R
DE'~'AIT ED DESCRIPTION OF THE INVENTION
The compounds of this invention are useful in the inhibition
of prenyl-protein transferases and the prenylation of the oncogene
protein Ras. In a first embodiment of this invention, the inhibitors of
prenyl-protein transferases are illustrated by the formula I:
(R8)r N Rsa R2 O
V - A~(CR~a2)nA2(CR~a2)n ~'~N N N-Z
(CRlb2)p X/ ~~ '/3
R R
wherein:
RIa is selected from: hydrogen or C1-Cg alkyl;
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Rxb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, R1~0-, -N(R1~)2 or C2-C6
alkenyl,
c) C1-Cg alkyl unsubstituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, R1~0-, or -N(Rl~)2;
R3 and R4 selected from H and CHg;
R2 is selected fromH; unsubstituted or substituted aryl, unsubstituted or
substituted heteroaryl,
N R6R~ ,
O
or C1_5 alkyl, unbranched or branched, unsubstituted or
substituted with one or more of:
1) aryl,
2) heterocycle,
3) OR6,
4) SR6a, S02R6a, or
NRsR~
O
and R2 and R3 are optionally attached to the same carbon atom;
R6 and R7 are independently selected from:
H; C1_4 alkyl, Cg_6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1_4 alkogy,
b) halogen,
c) perfluoro-C1_4 alkyl, or
d) aryl or heterocycle;
Rsa is selected from:
C1_4 alkyl or C3_6 cycloalkyl,
unsubstituted or substituted with:
a) C1_4 alkoxy,
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b) halogen, or
c) aryl or heterocycle;
R8 is independently selected from:
S a) hydrogen,
b) C1-Cg alkyl, C2-Cg alkenyl, C2-C6 alkynyl, C1-Cg
perfluoroalkyl, F, Cl, R100-, R10C(O)NR10-, CN, N02,
(R10)2N-C(NR10)-, R10C(O)-, -N(R10)2, or R110C(O)NR10_~
and
c) C1-Cg alkyl substituted by C1-Cg perfluoroalkyl, 8100-,
R10C(O)NR10_~ (R10)2N-C(NR10)-, R10C(O)-,
-N(R10)2, or R~lOC(O)NR10-;
R9a is hydrogen or methyl;
R10 is independently selected from hydrogen, C1-Cg alkyl, C1-Cg
perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
R11 is independently selected from C1-Cg alkyl and aryl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C=C-,
-C(O)-, -C(O)NR10_~ O~ _N(R10)-, or S(O)m
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolyl,
pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl,
quinolinyl, isoquinolinyl, and thienyl,
c) aryl,
d) C1-C2p alkyl wherein from 0 to 4 carbon atoms are replaced
with a heteroatom selected from O, S, and N, and
e) C2-C2p alkenyl, and
provided that V is not hydrogen if A1 is S(O)m and V is not hydrogen if
A1 is a bond, n is 0 and A2 is S(O)m;
_g_
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X is -CH2- or -C(=O)-;
Z is selected fram:
1) a unsubstituted or substituted group selected from aryl,
heteroaryl, arylmethyl, heteroarylmethyl, arylsulfonyl,
heteroarylsulfonyl, wherein the substituted group is
substituted with one or more of the following:
a) C1_4 alkyl, unsubstituted or substituted with:
C1-4 alkoxy, NR6R7, Cg_g cycloalkyl, unsubstituted or
substituted aryl, heterocycle, HO, -S(O)mR6a, or
-C(O)NRSR7,
b) aryl or heterocycle,
c) halogen,
d) OR6
e) NRSR7~
f) CN,
g) N02,
h) CFg;
i) -S(O)mR6a,
j) -C(O)NR6R7, or
k) Cg-Cg cycloalkyl; or
2) unsubstituted C1-C6 alkyl, substituted C1-Cg alkyl,
unsubstituted Cg-Cg cycloalkyl or substituted Cg-CS cycloalkyl,
wherein the substituted C1-C6 alkyl and substituted Cg-C6
cycloalkyl is substituted with one or two of the following:
a) C 1-4 alkogy,
b) NR6R7,
c) Cg_g cycloalkyl,
d) -NR6C(O)R7,
e) HO,
f) -S(O~R6a,
g) halogen, or
h) perfluoroalkyl;
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m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4; and
r is 0 to 5, provided that r is 0 when V is hydrogen;
provided that the substituent (R8)r- V - AI(CRIa2)nA2(CRIa2)n -
is not H;
and provided the compound is not selected from:
1-(3-Chlorophenyl)-4-[1-(4-cyanobenzyl)imidazolylmethyl]-2-
piperazinone
2(S)-n-Butyl-4-( I-naphthoyl)-1-[ 1-(2-naphthylmethyl)imidazol-5-
ylmethyl]-piperazine
2(S)-n-Butyl-1-[1-(4-cyanobenzyl)imidazol-5-ylmethyl]-4-( 1-
naphthoyl)piperazine
1-[1-(4-Bromobenzyl)imidazol-5-ylmethyl]-2(S)-n-butyl-4-(1-
naphthoyl)piperazine
1-{ [1-(4-cyanobenzyl)-1H-imidazol-5-yl] acetyl[-2(S)-n-
butyl-4-( 1-naphthoyl)piperazine
1-phenyl-4-[1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl]-piperazin-2-one
1-(3-trifluoromethylphenyl)-4-[1-(4-cyanobenzyl)-1H-imidazol-5-
ylmethyl]-piperazin-2-one
1-(3-bromophenyl)-4-[1-(4-cyanobenzyl)-1H-imidazol-5-ylmethyl]-
piperazin-2-one
5(S)-(2-[2,2,2-trifluoroethogy]ethyl)-1-(3-trifluoromethylphenyl)- 4-[1-(4-
cyanobenzyl)-4-imidazolylmethyl] -piperazin-2-one
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1-(5,6,7,8-tetrahydronaphthyl)-4-(1-(4-cyanobenzyl)-1H-imidazol-5-
ylmethyl]-piperazin-2-one
1-(2-methyl-3-chlorophenyl)-4-[1-(4-cyanobenzyl)-4-
imidazolylmethyl)]-piperazin-2-one
or the pharmaceutically acceptable salts thereof.
A preferred embodiment of the compounds of this invention
is illustrated by the following formula I-a:
H
N~N
/N N-Z
(CRlb2)p X ~~J
2
R
~R8)r 1-a
wherein:
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, cycloalkyl, R1~0-, -N(R1~)2 or C2-C6
alkenyl,
c) C1-Cg alkyl unsubstituted or substituted by aryl,
heterocycle, cycloalkyl, alkenyl, RlaO-, or -N(R16)2;
R2 is selected from H; unsubstituted or substituted aryl or C 1-5 alkyl,
unbranched or branched, unsubstituted or substituted with one or
more of:
1) aryl,
2) heteroaryl,
3) OR6, or
4) SRSa;
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R6 and R7 are independently selected from: C 1_4 alkyl, aryl, and
heteroaryl, unsubstituted or substituted with:
a) C1_4 alkoxy,
b) halogen,
c) perfluoro-C 1_4 alkyl, or
d) aryl or heteroaryl;
R6a is selected from:
C 1_4 alkyl, unsubstituted or substituted with:
a) C1_4 alkoxy, or
b) aryl or heteroaryl;
R8 is independently selected from:
a) hydrogen,
b) C1-C6 alkyl, C2-Cg alkenyl, C2-Cg alkynyl, C1-Cg
perfluoroalkyl, F, Cl, R100-, R10C(O)NR10-, CN, N02,
(R10)2N_C(~,10)-~ R10C(O)-, -N(R10)2~ or R110C(O)NR10-
and
c) C1-Cg alkyl substituted by C1-Cg perfluoroalkyl, 8100-,
R,lOC(O)NR,10_~ (R10)2N_C(NR10)-, R10C(O)-,
-N(R10)2, or R110C(O)NR10_;
R10 is independently selected from hydrogen, C 1-Cg alkyl, benzyl and
aryl;
Rll is independently selected from C1-Cg alkyl and aryl;
X is -CHI- or -C(=O)-;
Z is an unsubstituted or substituted group selected from aryl, arylmethyl
and arylsulfonyl, wherein the substituted group is substituted with
one or more of the following:
a) C1_4 alkyl, unsubstituted or substituted with:
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' C],-4 alkoxy, NR6R7, C3-g cycloalkyl, unsubstituted or
substituted aryl, heterocycle, HO, -S(O)mRSa,
or
-C(O)NRSR7
,
b) aryl or heterocycle,
c) halogen,
d) ORS
e) NR6R7~
f7 CN,
g) NO~,
h) CFg;
i) -S(O)mR6a,
j ) -C(O)NR6R7, or
k) Cg-Cg cycloalkyl;
m is 0, 1 or 2; and
p is 0, 1, 2, 3 or 4; and
r is O to 3;
and provided the compound is not selected from:
1-(3-Chlorophenyl)-4-[1-(4-cyanobenzyl)imidazolylmethyl]-2-
piperazinone
2(S)-n-Butyl-4-(1-naphthoyl)-1-[1-(2-naphthylmethyl)imidazol-5-
ylmethyl]-piperazine
2(S)-n-Butyl-1-[1-(4-cyanobenzyl)imidazol-5-ylmethyl]-4-( 1-
naphthoyl)piperazine
1-[1-(4-Bromobenzyl)imidazol-5-ylmethyl]-2(S)-n-butyl-4-( 1-
naphthoyl)piperazine
1-{ [1-(4-cyanobenzyl)-1H-imidazol-5-yl]acetyl}-2(S)-n-
butyl-4-( 1-naphthoyl)piperazine
1-phenyl-4-[1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl]-piperazin-2-one
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I-(3-trifluoromethylphenyl)-4-[1-(4-cyanobenzyl)-1H-imidazol-5-
ylmethyl]-piperazin-2-one
1-(3-bromophenyl)-4-[ 1-(4-cyanobenzyl)-1H-imidazol-5-ylmethyl] -
piperazin-2-one
5(S)-(2-[2,2,2-trifluoroethoxy]ethyl)-1-(3-trifluoromethylphenyl)- 4-[1-(4-
cyanobenzyl)-4-imidazolylmethyl]-piperazin-2-one
to
1-(5,6,7,8-tetrahydronaphthyl)-4-[1-(4-cyanobenzyl)-1H-imidazol-5-
ylmethyl]-piperazin-2-one
1-(2-methyl-3-chlorophenyl)-4-[1-(4-cyanobenzyl)-4-
15 imidazolylmethyl)]-piperazin-2-one
or the pharmaceutically acceptable salts thereof.
The preferred compounds of this invention are as follows:
20 1-(3-Trifluoromethoxyphenyl)-4-[1-(4-cyanobenzyl)imidazolylmethyl]-
2-piperazinone
OCF3
NC ~ ~ NON
O
'N
1-(2,5-Dimethylphenyl)-4-[1-(4-cyanobenzyl)imidazolylmethyl]-2-
piperazinone
Me
NC ~ ~ NON
N \
<N O Me
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1-(3-Methylphenyl)-4-[1-(4-cyanobenzyl)imidazolylmethyl]-2-
piperazinone
Me
NC ~ ~ NON
N
'N
1-(3-Iodophenyl)-4-[1-(4-cyanobenzyl)imidazolylmethyl]-2-
piperazinone
I
NC ~ ~ NON
N ~ O
'N
1-(3-Chlorophenyl)-4-[ 1-(3-methoxy-4-cyanobenzyl)imidazolylmethyl]-
2-piperazinone
CI
NC ~ ~ NON
N
<N
MeO
1-(3-Trifluoromethoxyphenyl)-4-[1-(3-methoxy-4-
cyanobenzyl)imidazolylmethyl]-2-piperazinone and
OCF3
NC ~ ~ NON
N
Me0 ~ O
N
(R)-5-[(Benzyloxy)methyl]-1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-
imidazolylmethyl]-2-piperazinone
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Ph-1
CI
NC ~ ~ N N
N'
<N O
or the pharmaceutically acceptable salts or optical isomers thereof.
Other specific examples of compounds of this invention are:
1-(3-Chlorophenyl)-4-[1-(2-lluoro-4-cyanobenzyl)-1H-imidazol-5-
ylmethyl] piperazin-2-one
4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-(3-
methylthiophenyl)piperazin-2-one
4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-(3,5-
dichlorophenyl)piperazin-2-one
1-(3-Chlorophenyl)-4-[[1-(4-cyanophenyl)-1-ethyl]-1H-imidazol-5-
ylmethyl}piperazin-2-one
1-(3-Chloro-4-fluorophenyl)-4-[1-(4-cyanobenzyl)-1H-imidazol-5-
ylmethyl] piperazin-2-one
4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-(3,5-
dimethylphenyl)piperazin-2-one
(S)-5-Benzyl-4-[3-(4-cyanobenzyl-1-imidazol-5-yl)prop-1-yl]-1-phenyl-2-
piperazinone
1-(3-Chlorophenyl)-4-[1-(4-nitrobenzyl)-1H-imidazol-5-
ylmethyl] piperazin-2-one
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4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-(3,5-
difluorophenyl)piperazin-2-one
4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-(3,4-
difluorophenyl)piperazin-2-one
or the pharmaceutically acceptable salts or optical isomers thereof.
The compounds of the instant invention direr from
previously disclosed piperazinone-containing compounds, (PCT Publ.
No. WO 97/30343 - October 3,1996; PCT Publ. No. WO 97/36593 - October 9,
1997; PCT Publ. No. WO 97/36592 - October 9, 1997) that were described as
selective inhibitors of farnesyl-protein transferase, in that the instant
compounds are dual inhibitors of farnesyl-protein transferase and
geranylgeranyl-protein transferase type I (GGTase-I). Preferably, the
compounds of the instant invention inhibit FPTase in vitro (Example 15)
at an ICso of less than 1 mM, inhibit GGTase-I in vitro (Example 16) at
an IC5o of less than 1 mM and inhibited the cellular processing
(farnesylation) of H-Ras (Example 1?) at an ICSO of less than 1 mM.
The compounds of the present invention may have
asymmetric centers and occur as racemates, racemic mixtures, and as
individual diastereomers, with all possible isomers, including optical
isomers, being included in the present invention. When any variable
(e.g. aryl, heterocycle, Rl, R2 etc.) occurs more than one time in any
constituent, its definition on each occurrence is independent at every
other occurrence. Also, combinations of substituents/or variables are
permissible only if such combinations result in stable compounds.
As used herein, "alkyl" is intended to include both branched
and straight-chain saturated aliphatic hydrocarbon groups having the
specified number of carbon atoms; "alkoxy" represents an alkyl group of
indicated number of carbon atoms attached through an oxygen bridge.
"Halogen" or "halo" as used herein means fluoro, chloro, bromo and
iodo.
As used herein, "aryl" is intended to mean any stable
monocyclic or bicyclic carbon ring of up to 7 members in each ring,
wherein at least one ring is aromatic. Examples of such aryl elements
_1~_.
CA 02301770 2000-02-24
WO 99/09985 PCT/US98/17696 _
include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl,
phenanthryl, anthryl or acenaphthyl.
The term heterocycle or heterocyclic, as used herein,
represents a stable 5- to 7-membered monocyclic or stable 8- to
11-membered bicyclic heterocyclic ring which is either saturated or
unsaturated, and which consists of carbon atoms and from one to four
heteroatoms selected from the group consisting of N, O, and S, and
including any bicyclic group in which any of the above-defined
heterocyclic rings is fused to a benzene ring. The heterocyclic ring
may be attached at any heteroatom or carbon atom which results in the
creation of a stable structure. Examples of such heterocyclic elements
include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl,
benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl,
benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl,
dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl,
dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,
isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl,
naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl,
2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl,
pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl,
pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl,
tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl,
thienofuryl, thienothienyl, and thienyl.
As used herein, "heteroaryl" is intended to mean any stable
monocyclic or bicyclic carbon ring of up to 7 members in each ring,
wherein at least one ring is aromatic and wherein from one to four
carbon atoms are replaced by heteroatoms selected from the group
consisting of N, O, and S. Examples of such heterocyclic elements
include, but are not limited to, benzimidazolyl, benzisoxazolyl,
benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl,
benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl,
dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl,
dihydrobenzothiopyranyl sulfone, furyl, imidazolyl, indolinyl, indolyl,
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WO 99/09985 PCT/US98/17696 _
isochromanyl, isoindolinyl, isoquinolinyl, isothiazolyl, naphthyridinyl,
oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl,
pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl,
tetrahydroquinolinyl, thiazolyl, thienofuryl, thienothienyl, and thienyl.
As used herein in the definition of R2 and R4, the term "the
substituted group" intended to mean a substituted C 1_g alkyl, substituted
C2_g alkenyl, substituted C2_g alkynyl, substituted aryl or substituted
heterocycle from which the substituent(s) R2 and R3 are selected:
As used herein in the definition of Rs, R6a, R7 and R7a,
the substituted C1_g alkyl, substituted C3_6 cycloalkyl, substituted aroyl,
substituted aryl, substituted heteroaroyl, substituted arylsulfonyl,
substituted heteroarylsulfonyl and substituted heterocycle include
moieties containing from 1 to 3 substituents in addition to the point of
attachment to the rest of the compound. Preferably, such substituents
are selected from the group which includes but is not limited to F, Cl, Br,
CFg, NH2, N(C1-Cg alkyl)2, N02, CN, (CI-Cg alkyl)O-, -OH,
(C1-Cg alkyl)S(O)m-, (C1-Cg alkyl)C(O)NH-, H2N-C(NH)-, (C1-Cg
alkyl)C(O)-, (C1-Cg alkyl)OC(O)-, N3,(C1-C6 alkyl)OC(O)NH-, phenyl,
pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl,
isothiazolyl and C1-C2p alkyl.
Lines drawn into the ring systems from substituents
(such as from R2, R3, R4 etc.) indicate that the indicated bond
may be attached to any of the substitutable ring carbon atoms.
Preferably, Rla and Rlb are independently selected from:
hydrogen, -N(Rl~)2, R10C(O)NR10- or unsubstituted or substituted
C 1-C6 alkyl wherein the substituent on the substituted C 1-Cg alkyl is
selected from unsubstituted or substituted phenyl, -N(R1~)2, R1~0-
and R1~C(O)NR10_,
Preferably, R2 is selected from:
H,
~RgR~
O
and an unsubstituted or substituted group, the group selected from C1_g
alkyl, C2_g alkenyl and C2_g alkynyl;
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WO 99!09985 PCTIUS98/17696 _
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle,
2) OR6,
3) SR6a, S02RSa,
Preferably, R4 is hydrogen.
Preferably, R6 and R7 are selected from: hydrogen,
unsubstituted or substituted C1-Cg alkyl, unsubstituted or substituted
aryl and unsubstituted or substituted Cg-Cg cycloalkyl.
Preferably, Rsa is unsubstituted or substituted C1-Cg.
Preferably, R9 is hydrogen.
Preferably, R10 is selected from H, C1-C6 alkyl and benzyl.
Preferably, A1 and AZ are independently selected from:
a bond, -C(O)NR10_~ _~,lOC(O)-, O, -N(R10)-, -S(O)2N(R10)- and
-N(R10)S(O)2-. Most preferably, A1 and A2 are a bond.
Preferably, V is selected from hydrogen, heterocycle and
aryl. More preferably, V is phenyl.
Preferably, Z is selected from unsubstituted or substituted
phenyl, unsubstituted or substituted naphthyl, unsubstituted or
substituted pyridyl, unsubstituted or substituted furanyl and
unsubstituted or substituted thienyl. More preferably, Z is unsubstituted
or substituted phenyl.
Preferably, n and r are independently 0, 1, or 2.
Preferably p is 1, 2 or 3.
Preferably, the moiety
(R8)~ N R9a
V - A1(CR~a2)nA2(CR~a2)n ~'~IV
(Cf~lb2)P
is selected from:
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WO 99109985 PCT/US98/17696
Rsa
=N I N
1
N / and N~ Rsa
CFi2-~- ~ - CH2_~_
(R$)r~~ 8
~R ~r
It is intended that the definition of any substituent or
variable (e.g., Rla, R9, n, etc.) at a particular location in a molecule
be independent of its definitions elsewhere in that molecule. Thus,
-N(Rl~)2 represents -NHH, -NHCHg, -NHC2H5, etc. It is understood
that substituents and substitution patterns on the compounds of the
instant invention can be selected by one of ordinary skill in the art to
provide compounds that are chemically stable and that can be readily
synthesized by techniques known in the art, as well as those methods
set forth below, from readily available starting materials.
The pharmaceutically acceptable salts of the compounds of
this invention include the conventional non-toxic salts of the compounds
of this invention as formed, e.g., from non-toxic inorganic or organic
acids. For example, such conventional non-toxic salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric and the like: and the salts
prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, malefic,
hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane
disulfonic, oxalic, isethionic, trifluoroacetic and the like.
The pharmaceutically acceptable salts of the compounds
of this invention can be synthesized from the compounds of this
invention which contain a basic moiety by conventional chemical
methods. Generally, the salts are prepared either by ion exchange
chromatography or by reacting the free base with stoichiometric
amounts or with an excess of the desired salt-forming inorganic or
organic acid in a suitable solvent or various combinations of solvents.
-21-
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wo moss rcTivs9sn~6~
Reactions used to generate the compounds of this invention
are prepared by employing reactions as shown in the Schemes 1-11,
in addition to other standard manipulations such as ester hydrolysis,
cleavage of protecting groups, etc., as may be known in the literature or
exemplified in the experimental procedures. Substituent R, as shown in
the Schemes, represents the substituents R2, R3, R4, and R5~ however
the point of attachment to the ring is illustrative only and is not meant
to be limiting.
These reactions may be employed in a linear sequence
to provide the compounds of the invention or they may be used to
synthesize fragments which are subsequently joined by the reductive
alkylation reactions described in the Schemes.
~y~o_psis o~ chemes 1-11:
The requisite intermediates are in some cases
commercially available, or can be prepared according to literature
procedures, for the most part.
Piperazin-5-ones can be prepared as shown in Scheme 1.
Thus, the protected suitably substituted amino acid IV can be converted
to the corresponding aldehyde V by first forming the amide and then
reducing it with T-AH. Reductive amination of Boc-protected amino
aldehydes V gives rise to compound VI. The intermediate VI can be
converted to a piperazinone by acylation with chloroacetyl chloride to
give VII, followed by base-induced cyclization to VIII. Deprotection,
followed by reductive alkylation with a protected imidazole carboxalde-
hyde leads to IX, which can be alkylated with an arylmethylhalide to
give the imidazolium salt X. Final removal of protecting groups by
either solvolysis with a lower alkyl alcohol, such as methanol, or
treatment with triethylsilane in methylene chloride in the presence of
trifluoroacetic acid gives the final product XI.
The intermediate VIII can be reductively alkylated with a
variety of aldehydes, such as XII. The aldehydes can be prepared by
standard procedures, such as that described by O. P. Goel, U. Krolls,
M. Stier and S. Kesten in Orgy n~ is Syntheses, 1988, 67, 69-75, from
the appropriate amino acid (Scheme 2). The reductive alkylation can
-22-
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WO 99/09985 PCTNS98/17696
be accomplished at pH 5-7 with a variety of reducing agents, such as
sodium triacetoxyborohydride or sodium cyanoborohydride in a solvent
such as dichloroethane, methanol or dimethylformamide. The product
XIII can be deprotected to give the final compounds XIV with trifluoro-
acetic acid in methylene chloride. The final product XIV is isolated
in the salt form, for example, as a trifluoroacetate, hydrochloride
or acetate salt, among others. The product diamine XIV can further
be selectively protected to obtain XV, which can subsequently be
reductively alkylated with a second aldehyde to obtain XVI. Removal
of the protecting group, and conversion to cyclized products such as the
dihydroimidazole XVII can be accomplished by literature procedures.
Alternatively, the imidazole acetic acid XVIII can be
converted to the acetate XIX by standard procedures, and XIX can be
first reacted with an alkyl halide, then treated with refluxing methanol
to provide the regiospecifically alkylated imidazole acetic acid ester XX
(Scheme 3). Hydrolysis and reaction with piperazinone VIII in the
presence of condensing reagents such as 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide (EDC) leads to acylated products such as XXI.
If the piperazinone VIII is reductively alkylated with an
aldehyde which also has a protected hydroxyl group, such as XXII
in Scheme 4, the protecting groups can be subsequently removed
to unmask the hydroxyl group (Schemes 4, 5). The alcohol can be
oxidized under standard conditions to e.g. an aldehyde, which can then
be reacted with a variety of organometallic reagents such as Grignard
reagents, to obtain secondary alcohols such as XXIV. In addition, the
fully deprotected amino alcohol XXV can be reductively alkylated (under
conditions described previously) with a variety of aldehydes to obtain
secondary amines, such as XXVI (Scheme 5), or tertiary amines.
The Boc protected amino alcohol XXIII can also be utilized
to synthesize 2-aziridinylmethylpiperazinones such as XXVII (Scheme
6). Treating XXIII with 1,1'-sulfonyldiimidazole and sodium hydride in
a solvent such as dimethylformamide led to the formation of aziridine
XXVII. The aziridine reacted in the presence of a nucleophile, such as
a thiol, in the presence of base to yield the ring-opened product XXVIII.
-23-
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WO 99/09985 PCTNS98/17696 _
In addition, the piperazinone VIII can be reacted with
aldehydes derived from amino acids such as O-alkylated tyrosines,
according to standard procedures, to obtain compounds such as ~
(Scheme 7). When R' is an aryl group, XXX can first be hydrogenated to
unmask the phenol, and the amine group deprotected with acid to
produce XXXI. Alternatively, the amine protecting group in ~ can be
removed, and O-alkylated phenolic amines such as III produced.
Scheme 8 illustrates the use of an optionally substituted
homoserine lactone XXXIII to prepare a Boc-protected piperazinone
XXXVII. Intermediate XXXXVII may be deprotected and reductively'
alkylated or acylated as illustrated in the previous Schemes.
Alternatively, the hydroxyl moiety of intermediate XX~~VII may be
mesylated and displaced by a suitable nucleophile, such as the sodium
salt of ethane thiol, to provide an intermediate XXXVIII. Intermediate
XXXVII may also be oxidized to provide the carboxylic acid on
intermediate IXL, which can be utilized form an ester or amide moiety.
N-Aralkyl-piperazin-5-ones can be prepared as shown in
Scheme S. Reductive amination of Boc-protected amino aldehydes V
(prepared from III as described previously) gives rise to compound XL.
This is then reacted with bromoacetyl bromide under Schotten-
Baumann conditions; ring closure is effected with a base such as
sodium hydride in a polar aprotic solvent such as dimethylformamide to
give XLI. The carbamate protecting group is removed under acidic
conditions such as trifluoroacetic acid in methylene chloride, or
hydrogen chloride gas in methanol or ethyl acetate, and the resulting
piperazine can then be carried on to final products as described in
Schemes I-7.
The isomeric piperazin-3-ones can be prepared as described
in Scheme 10. The imine formed from arylcarboxamides XLII and
2-aminoglycinal diethyl acetal (XLIII) can be reduced under a variety of
conditions, including sodium triacetoxyborohydride in dichloroethane,
to give the amine XLIV. Amino acids I can be coupled to amines XLIV
under standard conditions, and the resulting amide XLV when treated
with aqueous acid in tetrahydrofuran can cyclize to the unsaturated
XLVI. Catalytic hydrogenation under standard conditions gives the
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WO 99/09985 PCT/US98/17696 _
requisite intermediate XLVII, which is elaborated to final products as
described in Schemes 1-7.
Amino acids of the general formula IL which have a
sidechain not found in natural amino acids may be prepared by the
reactions illustrated in Scheme 11 starting with the readily prepared
imine XLVIII.
O Ra O Ra
OH O~ N N(CH3)OCH3
O N
H O CH3NHOCH3 ~ HCI H O
Iv EDC . HCI, HOBT
DMF, Et3N, pH 7
R R H
LAH, Et20 BocNH~CHO ArNH2 BocNH~N~Ar
NaBH(OAc)3
V CICH2CH2CI VI
R R
O
CI BocNH N-Ar NaH BocN N-Ar HCI
~CI
CI~4 DMF ~ EtOAc
EtOAc / H20
NaHC03 VII VIII
-25-
CA 02301770 2000-02-24
WO 99/09985 PGT/US98/17696 _
CHO
R ~ ~ R
HCI-HN N-Ar C(Ph)3 N N-Ar
/N ~ O
O NaBH(OAc)3 'N
VIII CICH2CH2CI (Ph)3C IX
pH 5-6
R
ArCH2X Ark ~ -Ar
CH3CN ~ ' ~ O
N ~ X
(Ph)3C
X
R
MeOH
or Ar'~ ~ -Ar
N
TFA, CH2CI ~ ~ O
(C2Hs)aSiH N
XI
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WO 99/09985 PCT/US98/17696
Boc NH XII
R
Boc NH CHO
HCI~H ~ -Ar
NaBH(OAc)3
Et3N , CICH2CH2Ci
VIII
R
CF3C02H
Boc NH N N-Ar -----
CH2CI2
O
NHBoc XIII
R
BOC20
NH2 N N-Ar ---
CH2C12
NH2 O
XIV
~ CHO
R w I ~-
BocNH_ r--N- N-Ar NaBH(OAc)3
Et3N , CICH2CH2CI
NH2
XV
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WO 99/09985 PCTNS98/17696
SCHEME 2 (continued)
R
CF3C02H, CH2CI2;
BocNH~N~ -Ar
NaHC03
NH O
XVI
R
NH2 N N-Ar ~ NC
NH O AgCN
~ ~
R
N N-Ar
NON O
XVI I
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WO 99109985 PCT/US98/17696
CH2C02H N CH2C02CH3
CH30H
HCI H ~ HCI
XIX
CH2C02CH3 1 ) ArCH2X CH3CN
C H CBr reflux
( 6 5)3
(C2H5)3N N 2) CH30H, reflux
DMF Tr
XIX
Ar~N CH2C02CH3 2.5N HClag '
55°C
N
Ar~N CH2C02H
N
XX
-29-
CA 02301770 2000-02-24
qrp 99/p9ggs PCT/US98/17696
R
Ar~N CH2C02H
HCI ~ H N N-Ar
N
O
XX
VIII
EDC ~ HCI
HOBt
DMF
Ar R
O
N~ -Ar
11 //
N O
XXI
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WO 99/09985 PCTIUS98/17696
SCHEME 4
R NaBH(OAc)3
Et3N , CICH2CH2CI
HCI HN N-Ar
Bn0
O
VIII BocNH CHO
XXI I
R
20% Pd(OH)2 H2
BnO~ ~ -Ar
CH30H
NHBoc O CH3C02H
R
CICOCOCI
HO~N~ -Ar r
DMSO CH2CI2
NHBoc O (C2H5)3N
XXIII
R R
1. R'MgX
O N N-Ar (C2H5)20 HO, /-'N' N Ar
2. TFA R'~-.--~NH2 ~O
H NHBoc O CH2CI2
XXIV
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SCHEME 5
R
CF3C02H
HO~ ~ -Ar
CH2CI2
NHBoc O
XXIII
R
R'CHO
HO~ ~ -Ar
NaBH(OAc)3
NH2 O CICH2CH2CI
XXV
R
HO~ ~ -Ar
~NH O
R'CH2
XXVI
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H H
N=1 ~ N
~N~ S.N J
HO~N~ -Ar
~NHBoc~O NaH, DMF 0°C
XXIII
N N-Ar R'SH R~S N, N-Ar
O ~C2Hs)sN NH2 O
H CH30H ~ XXVIII
XXVI I
~C~
HO / HO
\ ~ 1 ) Boc20, K2C03
THF-H20 \
H2N C02H 2) CH2N2, EtOAc BocNH C02CH3
HO
LiAIH4 \ I R'CH2X
THF Cs2C03
0-20°C BocNH CH20H DMF
R'CH O
R CH O / I pyridine ' S03 2
\ \
DMSO
BocNH CH OH ~C2H5)3N BocNH CHO
20°C
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SCHEME 7 (continued)
R'CH20. / . R
HCI ~ HN~N-Ar
BocNH CHO O
XXIX ~ VIII
NaBH(OAc)3
CICH2CH2CI
-- R
R'CH20 ~ -Ar
O
NHBoc
XXX HCI
ETOAc
1 ) 20% Pd(OH)~
CH30H, CH3C02H
-- R
2) HCI, EtOAc
R'CH20 ~ -Ar
NH2 O
R ' XXXII
HO ~ -Ar
NH2 O
XXXf
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WO 99/09985 PCT/US98117696 _
SCE
su ~~ 1, goc20, i-Pr2EtN su \~
O O
H2N O 2. DIBAL BocHN OH
HCI
XXXI11 OH
~/ sub
ArNH2 ~ N
NaBH(OAc)3 BocNH Ar
CICH2CH2C1 XXXIV
HO sub
O
CI
CI BocNH N-Ar
EtOAc
/ H20
NaHC03 CI O
XXXV
H ~~ ub
Cs2CO3
BocN N-Ar
DMF
O
XXXVI
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WO 99/09985 PCT/US98117696
SCHEME 8 (continued)
HC~~ ub
Bo/cN N-Ar
O 1. (COCI)2, Et3N
DMSO
1. MsCI, iPr2NEt XXXVI
2. NaCl02, t-BuOH
2. NaSEt, DMF 2-Me-2-butene
NaH2P04
Et ~ sub HO ~ ub
O
BocN N-Ar
Boc ~ -Ar
O O
XXXVI II IXL
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WO 99/09985 PCT/US98/17696
O Ra ArCH2NH2 _
~O~ N H NaBH(OAc)3
H p CICH2CH2CI
pH 6
V
O R 1 ) BrCH2COBr
~O~ N ~ NHCH2Ar EtOAc; H20, NaHC03
H 2) NaH, THF, DMF
XL
Ra
O ~ 1 ) TFA, CH2C12
y-- N N-~
~O ~ Ar
O
XLI
Ra
H ~ --~
Ar
O
-37-
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BCHEME 10
ArCHO + NH2CH2CH(OC2H5)2 NaBH(OAc)3
XLII XLIiI
O Ra
~O~ N OH
Ar CH NHCH CH(OC H )
2 2 2 52
i H O
XLIV EDC . HCI, HOST
DMF, Et3N, pH 7
O Ra ~Ar 6N HCI
~O~ N N~CH(OC2H5)2 THF
H O
XLV
Ra O
H2 10%Pd/C
-N N--~ CH30H
O U Ar
XLVI
Ra O
O
-N N~
O ~--J Ar
XLVII
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WO 99/09985 PCT/US98/17696 _
1. KOtBu, THF R2
~C02Et R2X >-C02Et
/~ N _ H2N
2. 5% aqueous HCI HCI
XLVIII
1. Boc20, NaHCO3 R2
--C02H
BocHN
2. LiAIH4, Et20
IL
The instant compounds are useful as pharmaceutical
agents for mammals, especially for humans. These compounds may be
administered to patients for use in the treatment of cancer. Examples
of the type of cancer which may be treated with the compounds of this
invention include, but are not limited to, colorectal carcinoma, exocrine
pancreatic carcinoma, myeloid leukemias and neurological tumors.
Such tumors may arise by mutations in the ras genes themselves,
mutations in the proteins that can regulate Ras activity (i.e.,
neurofibromin (NF-1), neu, src, abl, lck, fyn) or by other mechanisms.
The compounds of the instant invention inhibit prenyl-
protein transferase and the prenylation of the oncogene protein Ras.
The instant compounds may also inhibit tumor angiogenesis, thereby
affecting the growth of tumors (J. Rak et al. Cancer Research, 55:
4575-4580 (1995)). Such anti-angiogenesis properties of the instant
compounds may also be useful in the treatment of certain forms of
vision deficit related to retinal vascularization.
The compounds of this invention are also useful for
inhibiting other proliferative diseases, both benign and malignant,
wherein Ras proteins are aberrantly activated as a result of oncogenic
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WO 99/09985 PCT/US98/17696 _
mutation in other genes (i.e., the Ras gene itself is not activated by
mutation to an oncogenic form) with said inhibition being accomplished
by the administration of an effective amount of the compounds of the
invention to a mammal in need of such treatment. For example, a
component of NF-1 is a benign proliferative disorder.
The instant compounds may also be useful in the treatment
of certain viral infections, in particular in the treatment of hepatitis
delta and related viruses (J.S. Glenn et al. Science, 256:1331-1333 (1992).
The compounds of the instant invention are also useful in
the prevention of restenosis after percutaneous transluminal coronary
angioplasty by inhibiting neointimal formation (C. Indolfi et al. Nature
medicine, 1:541-545(1995).
The instant compounds may also be useful in the treatment
and prevention of polycystic kidney disease (D.L. Schaffner et al.
American Journal of Pathology, 142:1051-1060 ( 1993) and B. Cowley, Jr.
et aI.FASEB Journal, 2:A3160 (1988)).
The instant compounds may also be useful for the treatment
of fungal infections.
The instant compounds may also be useful as inhibitors of
proliferation of vascular smooth muscle cells and therefore useful in the
prevention and therapy of arteriosclerosis and diabetic vascular
pathologies.
The compounds of this invention may be administered
to mammals, preferably humans, either alone or, preferably, in
combination with pharmaceutically acceptable carriers, excipients or
diluents, in a pharmaceutical composition, according to standard
pharmaceutical practice. The compounds can be administered orally
or parenterally, including the intravenous, intramuscular,
intraperitoneal, subcutaneous, rectal and topical routes of
administration.
The pharmaceutical compositions containing the active
ingredient may be in a form suitable for oral use, for example, as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsions, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any
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method known to the art for the manufacture of pharmaceutical
compositions and such compositions may contain one or more agents
selected from the group consisting of sweetening agents, flavoring
agents, coloring agents and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets contain
the active ingredient in admixture with non-toxic pharmaceutically
acceptable excipients which are suitable for the manufacture of tablets.
These excipients may be for example, inert diluents, such as calcium
carbonate, sodium carbonate, lactose, calcium phosphate or sodium
phosphate; granulating and disintegrating agents, for example,
microcrystalline cellulose, sodium crosscarmellose, corn starch, or
alginic acid; binding agents, for example starch, gelatin, polyvinyl-
pyrrolidone or acacia, and lubricating agents, for example, magnesium
stearate, stearic acid or talc. The tablets may be uncoated or they may
be coated by known techniques to mask the unpleasant taste of the drug
or delay disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period. For example,
a water soluble taste masking material such as hydroxypropylmethyl-
cellulose or hydroxypropylcellulose, ar a time delay material such as
ethyl cellulose, cellulose acetate buryrate may be employed.
Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an inert
solid diluent, for example, calcium carbonate, calcium phosphate or
kaolin, or as soft gelatin capsules wherein the active ingredient is mixed
with water soluble carrier such as polyethyleneglycol or an oil medium,
for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-
cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and
gum acacia; dispersing or wetting agents may be a naturally-occurring
phosphatide, for example lecithin, or condensation products of an
alkylene oxide with fatty acids, for example polyoxyethylene stearate,
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or condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethylene-oxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty acids
and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived from
fatty acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or
more coloring agents, one or more flavoring agents, and one or more
sweetening agents, such as sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the
active ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The
oily suspensions may contain a thickening agent, for example beeswax,
hard paraffin or cetyl alcohol. Sweetening agents such as those set forth
above, and flavoring agents may be added to provide a palatable oral
preparation. These compositions may be preserved by the addition of
an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.
Dispersible powders and granules suitable for preparation
of an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent, suspending
agent and one or more preservatives. Suitable dispersing or wetting
agents and suspending agents are exemplified by those already
mentioned above. Additional excipients, for example sweetening,
flavoring and coloring agents, may also be present. These compositions
may be preserved by the addition of an anti-oxidant such as ascorbic
acid.
The pharmaceutical compositions of the invention may also
be in the form of an oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral oil, for
example liquid paraffin or mixtures of these. Suitable emulsifying
agents may be naturally-occurring phosphatides, for example soy bean
lecithin, and esters or partial esters derived from fatty acids and hexitol
anhydrides, for example sorbitan monooleate, and condensation
products of the said partial esters with ethylene oxide, for example
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polyoxyethylene sorbitan monooleate. The emulsions may also contain
sweetening, flavouring agents, preservatives and antioxidants.
Syrups and elixirs may be formulated with sweetening
agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative, flavoring
and coloring agents and antioxidant.
The pharmaceutical compositions may be in the form of a
sterile injectable aqueous solutions. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and isotonic
sodium chloride solution.
The sterile injectable preparation may also be a sterile
injectable oil-in-water microemulsion where the active ingredient is
dissolved in the oily phase. For example, the active ingredient may be
first dissolved in a mixture of soybean oil and lecithin. The oil solution
then introduced into a water and glycerol mixture and processed to form
a microemulation.
The injectable solutions or microemulaions may be
introduced into a patient's blood-stream by local bolus injection.
Alternatively, it may be advantageous to administer the solution or
microemulsion in such a way as to maintain a constant circulating
concentration of the instant compound. In order to maintain such a
constant concentration, a continuous intravenous delivery device may
be utilized. An example of such a device is the Deltec CARD-PLUSTM
model 5400 intravenous pump.
The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleagenous suspension for intramuscular
and subcutaneous administration. This suspension may be formulated
according to the known art using those suitable dispersing or wetting
agents and suspending agents which have been mentioned above. The
sterile injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally-acceptable diluent or solvent, for
example as a solution in 1,3-butane diol. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including synthetic
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mono- or diglycerides. In addition, fatty acids such as oleic acid find use
in the preparation of injectables.
Compounds of Formula A may also be administered in the
form of a suppositories for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable non-
irritating excipient which is solid at ordinary temperatures but liquid at
the rectal temperature and will therefore melt in the rectum to release
the drug. Such materials include cocoa butter, glycerinated gelatin,
hydrogenated vegetable oils, mixtures of polyethylene glycols of various
molecular weights and fatty acid esters of polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or
suspensions, etc., containing the compound of Formula A are employed.
(For purposes of this application, topical application shall include mouth
washes and gargles.)
The compounds for the present invention can be
administered in intranasal form via topical use of suitable intranasal
vehicles and delivery devices, or via transdermal routes, using those
forms of transdermal skin patches well known to those of ordinary skill
in the art. To be administered in the form of a transdermal delivery
system, the dosage administration will, of course, be continuous rather
than intermittent throughout the dosage regimen.
As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the specific
amounts, as well as any product which results, directly or indirectly,
from combination of the specific ingredients in the specified amounts.
When a compound according to this invention is
administered into a human subject, the daily dosage will normally be
determined by the prescribing physician with the dosage generally
varying according to the age, weight, sex and response of the individual
patient, as well as the severity of the patient's symptoms.
In one exemplary application, a suitable amount of
compound is administered to a mammal undergoing treatment for
cancer. Administration occurs in an amount between about 0.1 mg/kg
of body weight to about 60 mg/kg of body weight per day, preferably of
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between 0.5 mg/kg of body weight to about 40 mg/kg of body weight per
day. .
The compounds of the instant invention may also be
co-administered with other well known therapeutic agents that are
selected for their particular usefulness against the condition that is
being treated. For example, the compounds of the instant invention
may also be co-administered with other well known cancer
therapeutic agents that are selected for their particular usefulness
against the condition that is being treated. Included in such
combinations of therapeutic agents are combinations of the instant
farnesyl-protein tranaferase inhibitors and an antineoplastic agent.
It is also understood that such a combination of antineoplastic agent
and inhibitor of farnesyl-protein transferase may be used in
conjunction with other methods of treating cancer and/or tumors,
including radiation therapy and surgery.
Examples of an antineoplastic agent include, in general,
microtubule-stabilizing agents ( such as paclitaxel (also known as
Taxol~), docetaxel (also known as Taxotere~), epothilone A, epothilone
B, desoxyepothilone A, desoxyepothilone B or their derivatives);
microtubule-disruptor agents; alkylating agents, anti-metabolites;
epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;
procarbazine; mitoxantrone; platinum coordination complexes;
biological response modifiers and growth inhibitors; hormonal/anti-
hormonal therapeutic agents and haematopoietic growth factors.
Example classes of antineoplastic agents include, for
example, the anthracycline family of drugs, the vinca drugs, the
mitomycins, the bleomycins, the cytotoxic nucleosides, the taxanes,
the epothilones, discodermolide, the pteridine family of drugs, diynenes
and the podophyllotoxins. Particularly useful members of those classes
include, for example, doxorubicin, carminomycin,. daunorubicin,
aminopterin, methotrexate, methopterin, dichloro-methotrexate,
mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine,
gemcitabine, cytosine arabinoside, podophyllotoxin or podo-phyllotoxin
derivatives such as etoposide, etoposide phosphate or teniposide,
melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine,
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paclitaxel and the like. Other useful antineoplastic agents include
estramustine, cisplatin, carboplatin, cyclophosphamide, bleomycin,
tamoxifen, ifosamide, melphalan, hexamethyl melamine, thiotepa,
cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase,
camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide,
leuprolide, pyridobenzoindole derivatives, interferons and interleukins.
The preferred class of antineoplastic agents is the
taxanes and the preferred antineoplastic agent is paclitaxel.
Radiation therapy, including x-rays or gamma rays which
are delivered from either an externally applied beam or by implantation
of tiny radioactive sources, may also be used in combination with the
instant inhibitor of faruesyl-protein transferase alone to treat cancer.
Additionally, compounds of the instant invention may also
be useful as radiation sensitizers, as described in WO 97/3869?,
published on October 23, 1997, and herein incorporated by reference.
The instant compounds may also be useful in combination
with other inhibitors of parts of the signaling pathway that links cell
surface growth factor receptors to nuclear signals initiating cellular
proliferation. Thus, the instant compounds may be utilized in
combination with farnesyl pyrophosphate competitive inhibitors of
the activity of farnesyl-protein transferase or in combination with a
compound which has Raf antagonist activity. The instant compounds
may also be co-administered with compounds that are selective
inhibitors of geranylgeranyl protein transferase or farnesyl-protein
transferase.
In particular, the compounds disclosed in the following patents
and publications may be useful as farnesyl pyrophosphate-competitive.
inhibitor component of the instant composition: U.S. Ser. Nos. 08/254,228
and 08/435,047. Those patents and publications are incorporated herein
by reference.
In practicing methods of this invention, which comprise
administering, simultaneously or sequentially or in any order, two or
more of a protein substrate-competitive inhibitor and a farnesyl
pyrophosphate-competitive inhibitor, such administration can be orally
or parenterally, including intravenous, intramuacular, intraperitoneal,
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subcutaneous, rectal and topical routes of administration. It is
preferred that such administration be orally. It is more preferred that
such administration be orally and simultaneously. When the protein
substrate-competitive inhibitor and farnesyl pyrophosphate-competitive
inhibitor are administered sequentially, the administration of each can
be by the same method or by different methods.
The instant compounds may also be useful in combination
with an integrin antagonist for the treatment of cancer, as described in
U.S. Ser. No. 09/055,487, filed April 6, 1998, which is incorporated herein
by reference.
As used herein the term an integrin antagonist refers to
compounds which selectively antagonize, inhibit or counteract binding
of a physiological ligand to an integrin(s) that is involved in the
regulation of angiogenisis, or in the growth and invasiveness of tumor
1 S cells. In particular, the term refers to compounds which selectively
antagonize, inhibit or counteract binding of a physiological ligand to
the avb3 integrin, which selectively antagonize, inhibit or counteract
binding of a physiological ligand to the avb5 integrin, which antagonize,
inhibit or counteract binding of a physiological ligand to both the avb3
integrin and the avb5 integrin, or which antagonize, inhibit or
counteract the activity of the particular integrin(s) expressed on
capillary endothelial cells. The term also refers to antagonists of the
avb6, avb8, albl, a2bl, a5bl, a6b1 and a6b4 integrins. The term also
refers to antagonists of any combination of avb3, avb5, avb6, avb8, albl,
a2bl, a5bl, a6b1 and a6b4 integrins. The instant compounds may also be
useful with other agents that inhibit angiogenisis and thereby inhibit the
growth and invasiveness of tumor cells, including, but not limited to
angiostatin and endostatin.
Similarly, the instant compounds may be useful in
combination with agents that are elective in the treatment and
prevention of NF-1, restenosis, polycystic kidney disease, infections
of hepatitis delta and related viruses and fungal infections.
If formulated as a fixed dose, such combination products
employ the combinations of this invention within the dosage range
described below and the other pharmaceutically active agents) within
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its approved dosage range. Combinations of the instant invention may
alternatively be used sequentially with known pharmaceutically
acceptable agents) when a multiple combination formulation is
inappropriate.
EXAMPLES
Examples provided are intended to assist in a further
understanding of the invention. Particular materials employed, species
and conditions are intended to be further illustrative of the invention and
not limitative of the reasonable scope thereof.
EXAMPLES 1
1-(3-Chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-
~,perazinnnP dihydrn~l~loride (Con~.pound 1)
Step A: Preparation of 1-triphenylmethyl-4-(hydroxymethyl)-
imida~n]e
To a solution of 4-(hydroxymethyl)imidazole
hydrochloride (35.0 g, 260 mural) in 250 mL of dry DMF at room
temperature was added triethylamine (90.6 mL, 650 mmol). A white
solid precipitated from the solution. Chlorotriphenylmethane (76.1 g,
273 mmol) in 500 mL of DMF was added dropwise. The reaction
mixture was stirred for 20 hours, poured over ice, filtered, and
washed with ice water. The resulting product was slurried with cold
dioxane, filtered, and dried in vacuo to provide the titled product as a
white solid which was sufficiently pure for use in the next step.
~te~ B_: Preparation of 1-triphenylmethyl-4-(acetoxymethyl)-
imidazole
Alcohol from Step A (260 mmol, prepared above)
was suspended in 500 mL of pyridine. Acetic anhydride (74 mL,
780 mmol) was added dropwise, and the reaction was stirred for
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48 hours during which it became homogeneous. The solution was
poured into 2 L of EtOAc, washed with water (3 x 1 L), 5% aq. HCl
soln. (2 x 1 L), sat. aq. NaHC03, and brine, then dried (Na2S04),
filtered, and concentrated in vacuo to provide the crude product. The
acetate was isolated as a white powder which was sufficiently pure
for use in the next reaction.
Step C: Preparation of 1-(4-cyanobenzyl)-5-(acetoxymethyl)-
imidazole hydrobromide
A solution of the product from Step B (85.8 g, 225 mmol)
and a-bromo-p-tolunitrile (50.1 g, 232 mmol) in 500 mL of EtOAc was
stirred at 60°C for 20 hours, during which a pale yellow precipitate
formed. The reaction was cooled to room temperature and filtered
to provide the solid imidazolium bromide salt. The filtrate was
concentrated in vacuo to a volume 200 mL, reheated at 60°C for two
hours, cooled to room temperature, and filtered again. The filtrate
was concentrated in vacuo to a volume 100 mL, reheated at 60°C for
another two hours, cooled to room temperature, and concentrated in
vacuo to provide a pale yellow solid. All of the solid material was
combined, dissolved in 500 mL of methanol, and warmed to 60°C.
After two hours, the solution was reconcentrated in vacuo to provide
a white solid which was triturated with hexane to remove soluble
materials. Removal of residual solvents in vacuo provided the titled
product hydrobromide as a white solid which was used in the next
step without further purification.
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Stew D: Preparation of 1-(4-cyanobenzyl)-5-(hydroxymethyl)-
imidazole
To a solution of the acetate from Step C (50.4 g,
150 mmol) in 1.5 L of 3:1 THF/water at 0°C was added lithium
hydroxide monohydrate (18.9 g, 450 mmol). After one hour, the
reaction was concentrated in vacuo, diluted with EtOAc (3 L), and
washed with water, sat. aq. NaHC03 and brine. The solution was
then dried (Na2S04), filtered, and concentrated in vacuo to provide
the crude product as a pale yellow fluffy solid which was sufficiently
pure for use in the next step without further purification.
Step E: Preparation of 1-(4-cyanobenzyl)-5-
To a solution of the alcohol from Step D (2L5 g,
101 mmol) in 500 mL of DMSO at room temperature was added
triethylamine (56 mL, 402 mmol), then S03-pyridine complex (40.5 g,
254 mmol). After 45 minutes, the reaction was poured into 2.5 L of
EtOAc, washed with water (4 x 1 L) and brine, dried (Na2S04),
filtered, and concentrated in vacuo to provide the aldehyde as a white
powder which was sufficiently pure for use in the next step without
further purification.
Step F: Preparation of N-(3-chlorophenyl)ethylenediamine
hydrochloride
To a solution of 3-chloroaniline (30.0 mL, 284 mmol) in
500 mL of dichloromethane at 0°C was added dropwise a solution
of 4 N HCl in 1,4-dioxane (80 mL, 320 mmol HCl). The solution
was warmed to room temperature, then concentrated to dryness
in vacuo to provide a white powder. A mixture of this powder
with 2-oxazolidinone (24.6 g, 282 mmol) was heated under nitrogen
atmosphere at 160°C for 10 hours, during which the solids melted,
and gas evolution was observed. The reaction was allowed to cool,
forming the crude diamine hydrochloride salt as a pale brown solid.
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Step G: Preparation of N-(tert-butoxycarbonyl)-N'-(3-
chloronhenyl)ethylenediamine
The amine hydrochloride from Step F (ca. 282 mmol,
crude material prepared above) was taken up in 500 mL of THF
and 500 mL of sat. aq. NaHC03 soln., cooled to 0°C, and di-tert-
butylpyrocarbonate (61.6 g, 282 mmol) was added. After 30 h, the
reaction was poured into EtOAc, washed with water and brine, dried
(Na2S04), filtered, and concentrated in vacuo to provide the titled
carbamate as a brown oil which was used in the next step without
further purification.
Preparation of N-[2-(tert-butoxycarbamoyl)ethyl]-N-(3-
chlorophenyl)-2-chloroacetamide
A solution of the product from Step G (77 g, ca. 282
mmol) and triethylamine (67 mL, 480 mmol) in 500 mL of CH2Cl2
was cooled to 0°C. Chloroacetyl chloride (25.5 mL, 320 mmol) was
added dropwise, and the reaction was maintained at 0°C with
stirring. After 3 h, another portion of chloroacetyl chloride (3.0 mL)
was added dropwise. After 30 min, the reaction was poured into
EtOAc (2 L) and washed with water, sat. aq. NH4C1 soln, sat. ,
aq. NaHC03 soln., and brine. The solution was dried (Na2S04),
filtered, and concentrated in vacuo to provide the chloroacetamide
as a brown oil which was used in the next step without further
purification.
_ tee I: Preparation of 4-(tent-butoxycarbonyl)-1-(3-chlorophenyl)-
2-ninerazinone
To a solution of the chloroacetamide fram Step H (ca. 282
mmol) in 700 mL of dry DMF was added K2C03 (88 g, 0.64 mol). The
solution was heated in an oil bath at 70-75°C for 20 hrs., cooled to
room temperature, and concentrated in vacuo to remove ca. 500 mL of
DMF. The remaining material was poured into 33% EtOAc/hexane,
washed with water and brine, dried (Na2S04), filtered, and
concentrated in vacuo to provide the product as a brown oil. This
material was purified by silica gel chromatography (25-50%
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EtOAc/hexane) to yield pure product, along with a sample of product
(ca. 65% pure by HPLC) containing a less polar impurity.
Step J: P_~g~arat»n of 1-(3-chlorophenvl)-2-ninerazinone
S Through a solution of Boc-protected piperazinone
from Step I (17.19 g, 55.4 mmol) in 500 mL of EtOAc at -78°C was
bubbled anhydrous HCl gas. The saturated solution was warmed to
0°C, and stirred for 12 hours. Nitrogen gas was bubbled through the
reaction to remove excess HCI, and the mixture was warmed to room
temperature. The solution was concentrated in vacuo to provide the
hydrochloride as a white powder. This material was taken up in 300
mL of CH2C12 and treated with dilute aqueous NaHC03 solution.
The aqueous phase was extracted with CH2Cl2 (8 x 300 mL) until
tlc analysis indicated complete extraction. The combined organic
1 S mixture was dried (Na2S04), filtered, and concentrated in vacuo to
provide the titled free amine as a pale brown oil.
Preparation of 1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-
imidazolylmethyl]-2-piperazinone dihydrochloride
To a solution of the amine from Step J (55.4 mmol,
prepared above) in 200 mL of 1,2-dichloroethane at 0°C was added
4th powdered molecular sieves ( 10 g), followed by sodium triacetoxy-
borohydride (17.? g, 83.3 mmol). The imidazole carboxaldehyde from
Step E of Example 1 (11.9 g, 56.4 mmol) was added, and the reaction
2S was stirred at 0°C. After 26 hours, the reaction was poured into
EtOAc, washed with dilute aq. NaHC03, and the aqueous layer was
back-extracted with EtOAc. The combined organics were washed
with brine, dried (Na2S04), filtered, and concentrated in vacuo. The
resulting product was taken up in 500 mL of 5:1 benzene:CH2C12, and
propylamine (20 mL) was added. The mixture was stirred for 12
hours, then concentrated in vacuo to afford a pale yellow foam. This
material was purified by silica gel chromatography (2-7%
MeOH/CH2C12), and the resultant white foam was taken up in
CH2C12 and treated with 2.1 equivalents of 1 M HCl/ether solution.
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After concentrated in vacuo, the product dihydrochloride was isolated
as a white powder.
Examples 2 and 3 (Table 1) were prepared using the above protocol,
which describes the synthesis of the structurally related compound
Table 1 lists other compounds of the instant invention that were
prepared using the procedure described in Example 1. In Step F,
the appropriately substituted aniline was used in place of 3-
chloroaniline.
Table 1: 1-Aryl-4-[1-(4-cyanobenzyl)imidazolylmethyl]-2-
yinerazinones
iX
NC ~ ~ N N
N
O
N
FAB mass
spectrum CHN
Fxamnle X (M+1) Analysis
2 3-OCF3 456 C~H2p F3N502 ~ 2.OHCl ~ 0.60H20
calcd; C, 51.24; H, 4.34; N, 12.99.
found; C, 51.31; H, 4.33; N, 12.94.
3 2,5-(CH3)2 400 C24H25N50 ~ 2.OOHC1 ~ 0.65H20
calcd; C, 59.54; H, 5.89; N, 14.47
found; C, 59.54; H, 5.95; N, 14.12.
4 3-CH3 386 C23H23N50 ~ 2.OHC1 ~ 0.80H20
calcd; C, 58.43; H, 5.67; N, 14.81.
found; C, 58.67; H, 6.00; N, 14.23.
5 3-I 498 C22H20N50I~2.25HC1~0.90H20
calcd; C, 44.36; H, 4.07; N, 11.76.
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found; C, 44.37; H, 4.06; N, 11.42.
1-(3-chlorophenyl)-4-[1-(4-cyano-3-methoxybenzyl)-5-imidazolylmethyl]-2-
giperazinone dihydrochlo~cide
Step A: Preparation of Methyl 4-Amino-3-~~droxybenzoate
Through a solution of 4-amino-3-hydroxybenzoic acid (75
g, 0.49 mol) in 2.0 L of dry methanol at room temperature was bubbled
anhydrous HCl gas until the solution was saturated. The solution
was stirred for 48 hours, then concentrated in vacuo. The product
was partitioned between EtOAc and saturated aq. NaHC03 solution,
and the organic layer was washed with brine, dried (Na2S04), and
concentrated in vacuo to provide the titled compound.
Step B: Preparation of Meth l~ 3-Hydroxy-4-iodobenzoate
A cloudy, dark solution of the product from Step A (?9 g,
0.47 mol), 3N HCl (750 mL), and THF (250 mL) was cooled to 0°C. A
solution of NaN02 (35.9 g, 0.52 mol) in I15 mL of water was added
over ca. 5 minutes, and the solution was stirred for another 25
minutes. A solution of potassium iodide (312 g, 1.88 mol) in 235 mL
of water was added all at once, and the reaction was stirred for an
additional 15 minutes. The mixture was poured into EtOAc, shaken,
and the layers were separated. The organic phase was washed with
water and brine, dried (Na2S04), and concentrated in vacuo to
provide the crude product (148 g). Purification by column
chromatography through silica gel (0%-50% EtOAc/hexane) provided
the titled product.
Step C: Preparation of Methvl 4-Cvano-3-hy~oxvbenzoate
A mixture of the iodide product from Step B (101 g, 0.36
mol) and zinc(II)cyanide (30 g, 0.25 mol) in 400 mL of dry DMF was
degassed by bubbling argon through the solution for 20 minutes.
Tetrakis(triphenylphosphine)palladium (8.5 g, 7.2 mmol) was added,
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and the solution was heated to 80°C for 4 hours. The solution was
cooled to room temperature, then stirred for an additional 36 hours.
The reaction was poured into EtOAc/water, and the organic layer
was washed with brine (4x), dried (Na2S04), and concentrated in
S vacuo to provide the crude product. Purification by column
chromatography through silica gel (30%-50% EtOAc/hexane)
provided the titled product.
Step D: ~g~aration of Methyl 4-Cyano-3-methoxvbenzoate
Sodium hydride {9 g, 0.24 mol as 60% wt. disp. mineral
oil) was aded to a solution of the phenol from Step C (36.1 g, 204 mmol)
in 400 mL of dry DMF at room temperature. Iodomethane was added
(14 mL. 0.22 mol) was added, and the reaction was stirred for 2 hours.
The mixture was poured into EtOAc/water, and the organic layer
1 S was washed with water and brine (4x), dried (Na2S04), and
concentrated in vacuo to provide the titled.
Step E: naration ~f 4-Cvano-3-methoxvbenzyl Alcohol
To a solution of the ester from Step D (48.8 g, 255 mmol)
in 400 mL of dry THF under argon at room temperature was added
lithium borohydride (255 mL, 510 mmol, 2M THF) over 5 minutes.
After 1.5 hours, the reaction was warmed to reflux for 0.5 hours, then
cooled to room temperature. The solution was poured into EtOAc/1N
HCl soln. [CAUTION], and the layers were separated. The organic
2S layer was washed with water, sat Na2C03 soln. and brine (4x), dried
(Na2S04), and concentrated in vacuo to provide the titled product.
SteR F: ~enaration of ~-Cvano-3-methoxvbenzyl Bromide
A solution of the alcohol from Step E (35.5 g, 218 mmol)
in 500 mL of dry THF was cooled to 0°C. Triphenylphosphine was
added (85.7 g, 327 mmol), followed by carbontetrabromide (108.5 g, 327
mmol). The reaction was stirred at 0°C for 30 minutes, then at room
temperature for 21 hours. Silica gel was added (ca. 300 g), and the
suspension was concentrated in vacuo. The resulting solid was
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loaded onto a silica gel chromatography column. Purification by
flash chromatography (30%-50% EtOAc/hexane) provided the titled.
Step G: Preparation of 1-(4-cyano-3-methoxybenzyl)-5-
~~cetoxy~,eth-~~~, zole hydrobromide
The titled product was prepared by reacting the bromide
from Step F (21.7 g, 96 mmol) with the imidazole product from Step B
of Example 1 (34.9 g, 91 mmol) using the procedure outlined in Step C
of Example 1. The crude product was triturated with hexane to
provide the titled product hydrobromide.
St_e~: Preparation of ~1-(4-cyano-3-methoxybenzyl)-5-
(hydroxymethyl)-imidazole
The titled product was prepared by hydrolysis of the
acetate from Step G (19.43 g, 68.1 mmol) using the procedure outlined
in Step D of Example 1. The crude titled product was isolated in
modest yield (11 g, 66% yield). Concentration of the aqueous extracts
provided solid material (ca. 100 g) which contained a significant
quantity of the titled product , as judged by 1H NMR spectroscopy.
Step I: Preparation of 1-(4-cyano-3-methoxybenzyl)-5-
imida ,~lecarboxaldehyde
The titled product was prepared by oxidizing the alcohol
from Step H (11 g, 45 mmol) using the procedure outlined in Step E of
Example 1. The titled aldehyde was isolated as a white powder which
was sufficiently pure for use in the next step without further
purification.
Step J: Preparation of 1-(3-chlorophenyl)-4-[1-(4-cyano-3-
methoxybenzyl)-5-imidazolylmethyl]-2-piperazinone
d_i~vdrochloride
The titled product was prepared by reductive alkylation
of the aldehyde from Step I (859 mg, 3.56 mmol) and the amine
(hydrochloride) from Step K of Example 1 (800 mg, 3.24 mmol) using
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the procedure outlined in Step H of Example 1. Purification by flash
column chromatography through silica gel (50%-75% acetone
CH2C12) and conversion of the resulting white foam to its
dihydrochloride salt provided the titled product as a white powder.
FAB ms (m+1) 437.
Anal. Calc. for C23H23C1N502~2.OHC1~0.35CH2Ci2:
C, 51.97; H, 4.80; N, 12.98.
Found: C, 52.11; H, 4.80; N, 12.21.
~X,P~IPLE 7
1-(3-trifluoromethoxyphenyl)-4-[1-(4-cyano-3-methoxybenzyl)- 5-
imidazo lyl met 11-2-p~inerazinone dihydroc~loride
1-(3-trifluoromethoxy-phenyl)-2-piperazinone
hydrochloride was prepared from 3-trifluoromethoxyaniline using
Steps F-J of Example 1. This amine (1.75 g, 5.93 mmol) was coupled
to the aldehyde from Step I of Example 6 (1.57 g, 6.52 mmol) using the
procedure outlined in Step H of Example 1. Purification by flash
column chromatography through silica gel (60%-100% acetone
CH2Cl2) and conversion of the resulting white foam to its
dihydrochloride salt provided the titled product as a white powder.
FAB ms (m+1) 486.
Anal. Calc. for C24H23F3N503 ~ 2.OHC1 ~ 0.60H20:
C, 50.64; H, 4.46; N, 12.30.
Found: C, 50.69; H, 4.52; N, 12.13.
(R)-5-[(Benzyloxy)methyl]-1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)- 5-
imidazolvlmethyll-2-~'~erazinone dihvdrochloride
Steps A E: Preparation of (R)-5-[(benzyloxy)methyl]-1-(3-
chloroy envl)-2-~perazinone hydrochloride:
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The titled compound was prepared using an adaptation
of the following protocol, which describes the synthesis of the
structurally related compound 5(S)-n-butyl-1-(2,3-dimethylphenyl)-
2-piperazinone hydrochloride. In Step A, N-Boc-Ser(OBn)-OH was
used instead of 2(S)-(butoxycarbonylamino)hexanoic acid.
Step A: N-Methoxy-N-methyl 2(S)-(tert-butoxycarbonylamino)-
hexanamide
2(S)-(Butoxycarbonylamino)hexanoic acid (24.6 g, 0.106
mol), N,O-dimethylhydroxylamine hydrochloride ( 15.5 g, 0.15 mol),
EDC hydrochloride ( 22.3 g, 0.117 mol) and HOBT (14.3 g, 0.106 mol)
were stirred in dry, degassed DMF (300 mL) at 20°C under nitrogen.
N-Methylmorpholine was added to obtain pH 7. The reaction was
stirred overnight, the DMF distilled under high vacuum, and the
residue partitioned between ethyl acetate and 2% potassium hydrogen
sulfate. The organic phase was washed with saturated sodium
bicarbonate, water, and saturated brine, and dried with magnesium
sulfate. The solvent was removed in vacuo to give the title compound.
Step B: 2(S)-(tent-Butoxvc~~bony,~amino)hexanal
A mechanically stirred suspension of lithium
aluminum hydride (5.00 g, 0.131 mol) in ether (250 mL) was cooled
to -45°C under nitrogen. A solution of the product from Step A (28.3 g,
0.103 mol) in ether ( 125 mL) was added, maintaining the tempera-
ture below -35°C. When the addition was complete, the reaction
was warmed to 5°C, then retooled to -45°C. A solution of
potassium
hydrogen sulfate (27.3 g, 0.200 mol) in water was slowly added,
maintaining the temperature below -5°C. After quenching, the
reaction was stirred at room temperature for lh. The mixture was
filtered through Celite, the ether evaporated, and the remainder
partitioned between ethyl acetate and 2% potassium hydrogen sulfate.
After washing with saturated brine, drying over magnesium sulfate
and solvent removal, the title compound was obtained.
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Step C: N-(2,3-Dimethylphenyl)-2(S)-(tent-butoxycarbonylamino)-
]hexayamine
2,3-Dimethylaniline (8.32 mL, 68.3 mmol) was dissolved
in dichloroethane under nitrogen. Acetic acid was added to obtain pH
5, and sodium triacetoxyborohydride (17.2 g, 80.8 mmol) and crushed
molecular sieves (4 g) were added. A solution of the product from
Step B ( 13.3 g, 62.1 mmol) in dichloroethane (80 mL) was added slowly
dropwise at 20°C. The reaction was stirred overnight, then quenched
with saturated sodium bicarbonate solution. The aqueous layer was
removed, the organic phase washed with saturated brine and dried
over magnesium sulfate. Crystallization from hexane gave the title
compound.
Step D: 4-tent-Butoxycarbonyl-5(S)-n-butyl-1-(2,3-
dimethy~~ enyl)-2-~perazinone
A solution of the product from Step C (8.50 g, 26.5 mmol)
in ethyl acetate (250 mL) was vigorously stirred at 0°C with saturated
sodium bicarbonate (150 mL). Chloroacetyl chloride (2.33 mL, 29.1
mmol) was added, and the reaction stirred at ) 0°C for lh. The layers
were separated, and the ethyl acetate phase was washed with
saturated brine, and dried over magnesium sulfate. The crude
product was dissolved in DMF (300 mL) and cooled to 0°C under
nitrogen. Sodium hydride (1.79 g, 60% dispersion in oil, 44.9 mmol)
was added portionwise to maintain moderate hydrogen evolution.
After 30 min, an additional amount of sodium hydride was added (0.8
g). The reaction was stirred another 30 min, then quenched with
saturated ammonium chloride. The DMF was distilled in vacuo, and
the residue partitioned between ethyl acetate and water. The organic
phase was washed with water, saturated brine, and dried over
magnesium sulfate. The crude product was chromatographed on
silica gel with 20-30% ethyl acetate in hexane
to obtain the title compound. -
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A solution of the product from Step D (0.570 g, 1.58 mmol}
in ethyl acetate (50 mL) was cooled to -15°C under nitrogen. HCl gas
was bubbled through for 15 min, and the reaction solution warmed to
0°C for 2h. The solvent was removed in vacuo to provide the titled
product.
Sten FF: Preparation of (R)-5-[(Benzyloxy)methyl]-1-(3-
chlorophenyl)-4- [ 1-( 4-cyanobenzyl )-5-imidazolylmethyl] -
~~perazinone dihydrochloride
The titled product was prepared by reductive alkylation
of the aldehyde from Step E of Example 1 (181 mg, 0.858 mmol) and
(R)-5-[(benzyloxy)methyl]-1-(3-chlorophenyl)-2-piperazinone hydro-
chloride from the present Example (205 mg, 0.558 mmol) using the
procedure outlined in Step K of Example 1. Purification by flash
column chromatography through silica gel (acetone/CH2CI2) and
conversion to the dihydrochloride salt provided the titled product
as a white powder. FAB ms (m+1) 526.
Anal. Calc. for C34H28C1N502~2.15HC1~0.55H20:
C, 58.65; H, 5.13; N, 11.40.
Found: C, 58.fi3; H, 5.13; N, 11.18.
1-(3-Chlorophenyl)-4-[1-(4-cyano-3-(trifluoromethoxy)benzyl)-5-
imidazolylmethyll-2-~nerazinone di~vdrochloride
Step A: Preparation of 4-bromo-2-(trifluoromethoxy) benzonitrile
To a solution of 4-bromo-2-(trifluoromethoxy)iodobenzene (25
g, 68 mmol) and zinc(II) cyanide (4.0 g, 34 mmol} in 150 mL of degassed
dimethylformamide was added tetrakis(triphenylphosphine)palladium
(3.1 g, 4 mole %). The solution was stirred at 80 °C for one hour, then
cooled to room temperature. Additional portions of zinc(II) cyanide (800
mg) and tetrakis(triphenylphosphine)palladium (700 mg) were added,
and the solution was heated a t 80 °C for 3 hours. The mixture was
diluted with EtOAc and extracted with saturated NaHCOg solution and
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brine, dried over sodium sulfate, filtered, and concentrated in vacuo.
Purification by silica gel column chromatography (0-5% ether/hexane)
gave the titled product.
Step B: Preparation of methyl 4-cyano-3-(trifluoromethoxy)
benzoate
Through a solution of the product from Step A (3.82 g,
14.4 mmol), palladium(II) acetate (150 mg, 0.4 wt %), 1,3-bis(diphenyl-
phosphino)propane (300 mg), and triethylamine (3.5 mL) in 30 mL
of MeOH and 15 mL of DMSO was bubbled carbon monoxide gas for
6 hours. The reaction was heated to 80°C and stirred under a baloon
of carbon monoxide. After ca: 16 hours, another aditional portions of
palladium(II) acetate (100 mg) and 1,3-bis(diphenylphosphino)propane
(200 mg) were added, and the solution was stirred for an additional 20
hours. The mixture was diluted with EtOAc and extracted with water
and brine, dried over sodium sulfate, filtered, and concentrated in
vacuo. Purification by silica gel column chromatography (20%
EtOAc/hexane) gave the titled product.
Step C: Preparation of 4-cyano-3-(trifluoromethoxy) benzyl
~,~ol
To a solution of the product from Step B (4.29 g, 17.5 mmol)
in 100 mL of methanol at 0 °C was added sodium borohydride (1.3 g, 35
mmol). The solution was allowed to warm to room temperature over 2
hours. An additional portion of sodium borohydried was added (500 mg),
and the solution stirred for 30 minutes. The mixture was diluted with
EtOAc and extracted with saturated NaHC03 solution and brine, dried
over sodium sulfate, filtered, and concentrated in vacuo to give the titled
product.
Sten D: Preparation of 1-[4-cyano-3-(trifluoromethoxy)benzyl]-~
~aceto ~e ~y~-imidazole
To a solution of the product from Step C ( 1.5 g, 6.9 mmol)
and the product from Step B of Example 1 (2.6 g, 6.9 mmol) in 10 mL of
dichloromethane at -78 °C was added diisopropylethylamine (2.4 mL, 14
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mmol), followed by slow addition of trifluoromethanesulfonic anhydride
(1.26 mL, 7.5 mmol). The solution was stirred for 15 minutes, then
allowed to warm to room temperature. After 2 hours, methanol was
added (10 mL), and the solution was stirred for 48 hours. The reaction
was concentrated in vacuo, diluted with EtOAc and extracted with
saturated NaHC03 solution and brine, dried over sodium sulfate,
filtered, and concentrated in vacuo. Purification by silica gel column
chroiriatography (5-10% MeOH/EtOAc) gave the titled product.
Step E: Preparation of 1-[4-cyano-3-(trifluoromethoxy)benzyl]-5
vdroxymethyl)-imidazole
The titled compound was prepared from the product of Step
D (1.94 g, 5.72 mmol) using the procedure described in Step D of Example
1. This provided the titled product.
Stp~F: Preparation of 1-[4-cyano-3-(trifluoromethoxy)benzyl]
iJmi dazole-5-carboxaldehyde
The titled compound was prepared from the product of Step
E (1.31 g, 4.41 mmol) using the procedure described in Step E of Example
1. This provided the titled product.
Step Preparation of 1-(3-Chlorophenyl)-4-[1-(4-cyano-3-
(trifluoromethoxy)benzyl)-5-imidazolylmethyl]-2-
~i~eraz~ one di vdrochloric~e
The titled compound was prepared from the product of Step
F {264 mg, 0.89 mmol) and the product of Step J of Example 1 using the
procedure described in Step K of Example 1. Purification by silica gel
column chromatography (50-65% acetone/dichloro methane) and
conversion to the dihydrochloride salt using excess ethereal HCl solution
gave the titled product as a white powder.
FAB ms (m+1) 490.1.
Anal. Calc. for C23H1gC1F3N502 ~ 2.00 HCl ~ 1.0 H20:
C, 4?.56; H, 3.99; N, 12.06.
Found: C, 47.58; H, 4.02; N, 11.91.
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4-[1-(4-Cyano-3-(trifluoromethoxy)benzyl)-5-imidazolylmethyl]-1-
~t~fluorometho~v)nhenyll-2-pinerazinone di~ydrochloride
Stew A: Preparation of 1-[3-(trifluoromethoxy)phenyl]-2-
~pera~innnP hv~drochloride
The titled compound was prepared from 3-
(trifluoromethoxy)aniline using the procedures described in Steps F-J of
Example 1.
Step B: Preparation of 4-[1-(4-cyano-3-(trifluoromethoxy)benzyl)-
5-imidazolylmethyl]-1-[3-(trifluoromethoxy)phenyl]-2-
p~nerazinong dihydrochloride
The titled compound was prepared from the product of
Step A and the product of Step F of Example 9 using the procedure
described in Step K of Example 1. Purification by silica gel column
chromatography (50-65% acetoneldichloro methane) and conversion
to the dihydrochloride salt using excess ethereal HCl solution gave
the titled product as a white powder.
FAB ms (m+1) 540.2.
Anal. Calc. for C24H1gFSN502 ~2.00 HCI ~ 1.15 H20 ~0.50 CH2Cl2:
C, 44.61; H, 3.71; N, 10.62.
Found: C, 44.63; H, 3.70; N, 10.56.
EXAMPLE 11
1-(3-Chlorophenyl)-4-[1-(4-cyano-3-fluorobenzyl)-5-imidazolylmethyl]-
2-rioerazinone dihxdrochloride
~,te~A_: ~~aration of 4-cyano-3-fluorotoluene
To a degassed solution of 4-bromo-3-fluorotoluene (50.0 g,
264 mmol) in 500 mL of DMF was added Zn(CN)2 (18.6 g, 159 mmol) and
Pd(PPh3)4 (6.1 g, 5.3 mmol). The reaction was stirred at 80°C for 6
hours,
then cooled to room temperature. The solution was poured into EtOAc,
washed with water, sat. aq. NaHC03, and brine, then dried (Na2S04),
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filtered, and concentrated in vacuo to provide the crude product.
Purification by silica gel chromatography (0-5% EtOAc/hexane) provided
the titled product.
Steg B: red ration of 4-cyano-3-fluorobenzylbromide
To a solution of the product from Step A (22.2 g, 165 mmol)
in 220 mL of carbontetrachloride was added N-bromosuccinimide (29.2 g,
164 mmol) and benzoylperoxide ( l.lg). The reaction was heated to reflux
for 30 minutes, then cooled to room temperature. The solution was
concentrated in vacuo to one-third the original volume, poured into
EtOAc, washed with water, sat. aq. NaHC03, and brine, then dried
(Na2S04), filtered, and concentrated in vacuo to provide the crude
product. Analysis by 1H NMR indicated only partial conversion, so the
crude material was resubjected to the same reaction conditions for 2.5
hours, using 18 g (102 mmol) of N-bromosuccinimide. After workup, the
crude material was purified by silica gel chromatography (0-10%
EtOAc/hexane) to provide the desired product.
Ste~C: Preparation of 1-(4-cyano-3-fluorobenzyl)-5-(acetoxymethyl)-
imidazole hydrobromide
A solution of the product from Step B (20.67 g, 96.14 mmol)
and the product from Step B of Example 1 (36.72 g, 96.14 mmol) in 250
mL of EtOAc was stirred at 60 °C for 20 hours, during which a white
precipitate formed. The reaction was cooled to room temperature and
filtered to provide the solid imidazolium bromide salt. The filtrate was
concentrated in vacuo to a volume of 100 mL, reheated at 60 °C for two
hours, cooled to room temperature, and filtered again. The filtrate was
concentrated in vacuo to a volume 40 mL, reheated at 60 °C for another
two hours, cooled to room temperature, and concentrated in vacuo to
provide a pale yellow solid. All of the solid material was combined,
dissolved in 300 mL of methanol, and warmed to 60 °C. After two hours,
the solution was reconcentrated in vacuo to provide a white solid which
was triturated with hexane to remove soluble materials. Removal of
residual solvents in vacuo provided the titled product hydrobromide as a
white solid which was used in the next step without further purification.
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Step D: Preparation of 1-(4-cyano-3-fluorobenzyl)-5-
~ydr~o , ~nethy~)imidazole
To a solution of the product from Step C (31.87 g, 89.77
mmol) in 300 mL of 2:1 THF/water at 0°C was added lithium hydroxide
monohydrate (7.53 g, 179 mmol). After two hours, the reaction was
concentrated in vacuo to a 100 mL volume, stored at 0°C for 30 minutes,
then filtered and washed with 700 mL of cold water to provide a brown
solid. This material was dried in vacuo next to P2O5 to provide the titled
product as a pale brown powder which was sufficiently pure for use in
the next step without further purification.
Preparation of 1-(4-cyano-3-fluorobenzyl)-5-
To a solution of the alcohol from Step D (2.31 g, 10.0 mmol)
in 20 mL of DMSO at 0 °C was added triethylamine (5.6 mL, 40 mmol),
then S03-pyridine complex (3.89 g, 25 mmol). After 30 minutes, the
reaction was poured into EtOAc, washed with water and brine, dried
(Na2S04), filtered, and concentrated in vacuo to provide the aldehyde as
a pale yellow powder which was sufficiently pure for use in the next step
without further purification.
~g~"~ Preparation of 1-(3-Chlorophenyl)-4-[1-(4-cyano-3
fluorobenzyl)-5-imidazolylmethyl]- 2-piperazinone
~y~rochlori~e
The titled compound was prepared from the product of
Step A and the product of Step F of Example 9 using the procedure
described in Step K of Example 1. Purification by silica gel column
chromatography (50-609'o acetone/dichioromethane) and conversion to
the dihydrochloride salt using excess ethereal HCl solution gave the
titled product as a white powder.
FAB ms (m+1) 424.2.
Anal. Calc. for C22H19C1FN5O2 ~2.00 HCl ~1.15 H20:
C, 51.05; H, 4.54; N, 13.53.
Found: C, 51.08; H, 4.62; N,13.44.
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1-(3-Chlorophenyl)-4-[1-(4-cyano-3-(methylthio)benzyl)-5-
imida~ lmet ~ 1~l- 2-~i_Derazinone dihvdrochloride
To a solution of the Example 11 product (52 mg, 0.12
mmol) in 1 mL of DMF was added sodium thiomethoxide (17 mg, 0.24
mmol). After ca. 16 hours, the reaction was diluted with EtOAc and
extracted with saturated NaHC03 solution and brine, dried over
sodium sulfate, filtered, and concentrated in vacuo. Purification by
silica gel preparative thin-layer chromatography (2 x 0.5 mm, 10%
CHCh/methanol) and conversion to the dihydrochloride salt using
excess ethereal HCl solution gave the titled product as a white
powder.
HPLC: 100% purity at 220 nm; retention time = 8.24 min; 5-95%
gradient: acetonitrile/0.1% TFA-water over 15 min.
Anal. Calc. for C23H22C1N50S ~2.00 HCl ~0.15 H20 ~0.30 CH2C12:
C, 50.59; H, 4.54; N, 12.66.
Found: C, 51.21; H, 5.08; N, 11.88.
1-(3-Chlorophenyl)-4-[1-(4-cyano-3-(phenoxy)benzyl)-5-
~m~~AZoI l~meth~yll- 2-~perazinone dihydrochloride
To a solution of the Example 11 product (50 mg,
0.12 mmol) in 1 mL of DMSO was added phenol (33 mg, 0.35
mmol), followed by cesium carbonate (114 mg, 0.35 mmol). After
ca. 16 hours, the reaction was diluted with EtOAc and extracted
with water and brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. Purification by silica gel preparative thin-
layer chromatography (2 x 0.5 mm, 90:10:1 CHCh/methanol/NH40H)
and conversion to the dihydrochloride salt using excess ethereal HCl
solution gave the titled product as a white powder.
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FAB ms (m+1) 498.2.
Anal. Calc. for C28H24C1N502 ~2.00 HCl ~0.50 H20 ~0.10 CH2C12:
C, 57.35; H, 4.66; N, 11.90.
Found: C, 57.37; H, 4.67; N, 11.13.
EXAMPLE 14
The following compounds were also prepared by procedures
analogous to those described in Examples 1-13:
1-(3-Chlorophenyl)-4-[1-(2-fluoro-4-cyanobenzyl)-1H-imidazol-5-
ylmethyl]piperazin-2-one dihydrochloride
Anal. C22H19C1FN5O ~ 2 HCl ~ 1H20
Calc: C, 51.33; H, 4.50; N, 13.60
Found: C, 51.41; H, 4.49; N, 13.16
4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-( 3-
methylthiophenyl)piperazin-2-one dihydrochloride
Anal. CZgH23NbOS ~ 2.5 HCl
Calc: C, 54.33; H, 5.06; N, 13.77
Found: C, 54.37; H, 4.75; N, 13.13
4-[1-(4-Cyanabenzyl)-1H-imidazol-5-ylmethyl]-1-(3,5-
dichlorophenyl)piperazin-2-one dihydrochloride
Anal. C~H19C12N50 ~ 2.55 HCl ~ 1 H20
Calc: C, 47.92; H, 4.31; N, 12.70
Found: C, 47.95; H, 4.31; N, 12.65
1-(3-Chlorophenyl)-4-{ [1-(4-cyanophenyl)-1-ethyl]-1H-imidazol-5-
ylmethyl}piperazin-2-one dihydrochloride
Anal. C29H~C1N80 ~ 2 HCl ~ 1.3 H20
Calc: C, 53.51; H, 5.19; N, 13.57
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Found: C, 53.59; H, 5.39; N, 13.44
1-(3-Chloro-4-fluorophenyl)-4-(1-(4-cyanobenzyl)-1H-imidazol-5-
ylmethyl]piperazin-2-one dihydrochloride
Anal. C~H,eCIFNbO ~ 2 HCl
Calc: C, 53.19; H, 4.26; N, 14.10
Found: C, 52.84; H, 4.37; N, 13.76
4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-(3,5-
dimethylphenyl)piperazin-2-one dihydrochloride
Anal. C24Hz5N5O ~ 2 HCl 0.1 H2O
Calc: C, 60.79; H, 5.78; N, 14.77
Found: C, 60.79; H, 6.32; N, 14.34
(S)-5-Benzyl-4-[3-(4-cyanobenzyl-1-imidazol-5-yl)prop-1-yl]-1-phenyl-2-
piperazinone dihydrochloride
FAB ms m/e 490 (m+1).
Anal. Cg1H31N5O ~ 2 HCl 1.45 H20
Calc: C, 63.25; H, 6.15; N, 11.90
Found: C, 63.22; H, 5.98; N,11.64
1-(3-Chlorophenyl)-4-[1-(4-nitrobenzyl)-1H-imidazol-5-
ylmethyl] piperazin-2-one
Anal. CZ1H20C'1NSO3 ~ 0.15 H2O
Calc: C, 58.85; H, 4.77; N, 16.34
Found: C, 58.82; H, 4.55; N, 16.35
4-[1-(4-Cyanobenzyl)-1H-imidazol-5-ylmethyl]-1-(3,5-
difluorophenyl)piperazin-2-one dihydrochloride
Anal. C~HI9FzN50 ~ 2 HCl ~ 0.25 EtOAc
Calc: C, 54.98; H, 4.61; N, 13.94
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Found: C, 54.72; H, 4.68; N, 13.80
4-[1-(4-Cyanobenzyi)-1H-imidazol-5-ylmethyl]-1-(3,4
difluorophenyl)piperazin-2-.one ditrifluoroacetic acid salt
Anal. C22H19F21V50 ~ 2 TFA ~ 0.35 H20
Calc: C, 48.66; H, 3.41; N,10.91
Found: C, 48.29; H, 3.44; N, 11.30
E~~AMPLE l~
In vitro in ibition of ras farnesyl transferase
Transferase Assays. Isoprenyl-protein transferase
activity assays are carried out at 30 °C unless noted otherwise. A
typical reaction contains (in a final volume of 50 mL): [3H]farnesyl
diphosphate, Ras protein , 50 mM HEPES, pH 7.5, 5 mM MgCl2, 5 mM
dithiothreitol, 10 mM ZnCl2, 0.1% polyethyleneglycol (PEG) (15,000-20,000
mw) and isoprenyl-protein transferase. The FPTase employed in the
assay is prepared by recombinant expression as described in Omer,
C.A., Kral, A.M., Diehl, R.E., Prendergast, G.C., Powers, S., Allen,
C.M., Gibbs, J.B. and Kohl, N.E. (1993) Biochemistry 32:5167-5176. After
thermally pre-equilibrating the assay mixture in the absence of enzyme,
reactions are initiated by the addition of isoprenyl-protein transferase
and stopped at timed intervals (typically 15 min) by the addition of 1 M
HCl in ethanol (1 mL). The quenched reactions are allowed to stand for
15 m (to complete the precipitation process). After adding 2 mL of 100%
ethanol, the reactions are vacuum-filtered through Whatman GF/C
filters. Filters are washed four times with 2 mL aliquots of 100010
ethanol, mixed with scintillation fluid ( 10 mL) and then counted in a
Beckman LS3801 scintillation counter.
For inhibition studies, assays are run as described above,
except inhibitors are prepared as concentrated solutions in 100%
dimethyl sulfoxide and then diluted 20-fold into the enzyme assay
mixture. Substrate concentrations for inhibitor IC50 determinations are
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as follows: FTase, 650 nM Ras-CVLS (SE~.ID.NO.: 1), 100 nM farnesyl
diphoaphate.
The compounds of the instant invention described in the
above Examples 1-14 were tested for inhibitory activity against human
FPTase by the assay described above and were found to have IC50 of
<_ 50 mM.
IO ModifiedIn vitro GGTase inhibition assay
The modified geranylgeranyl-protein transferase inhibition
assay is carried out at room temperature. A typical reaction contains (in
a final volume of 50 mL): (3H]geranylgeranyl diphosphate, biotinylated
Ras peptide, 50 mM HEPES, pH 7.5, a modulating anion (for example 10
mM glycerophosphate or 5mM ATP), 5 mM MgCl2, 10 mM ZnCl2, 0.1%
PEG ( 15,000-20,000 mw), 2 mM dithiothreitol, and geranylgeranyl-
protein transferase type I(GGTase). The GGTase-type I enzyme
employed in the assay is prepared as described in U.S. Pat. No. 5,470,832,
incorporated by reference. The Ras peptide is derived from the K4B-Ras
protein and has the following sequence: biotinyl-GKKKKKKSKTKCVIM
(single amino acid code) (SEQ.ID.NO.: 2). Reactions are initiated by the
addition of GGTase and stopped at timed intervals (typically 15 min) by
the addition of 200 mL of a 3 mg/mL suspension of streptavidin SPA
beads (Scintillation Proximity Assay beads, Amersham) in 0.2 M sodium
phosphate, pH 4, containing 50 mM EDTA, and 0.5% BSA. The
quenched reactions are allowed to stand for 2 hours before analysis on a
Packard TopCount scintillation counter.
For inhibition studies, assays are run as described above,
except inhibitors are prepared as concentrated solutions in 100%
dimethyl sulfoxide and then diluted 25-fold into the enzyme assay
mixture. IC50 values are determined with Ras peptide near KM
concentrations. Enzyme and substrate concentrations for inhibitor
IC50 determinations are as followa: 75 pM GGTase-I, 1.6
mM Ras peptide, 100 nM geranylgeranyl diphosphate.
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EXAMPLE 17
Cell-basedin vitro ras fa~ylation assay
The cell line used in this assay is a v-ras line derived
from either Ratl or NIH3T3 cells, which expressed viral Ha-ras p21.
The assay is performed essentially as described in DeClue, J.E. ~t ~1.,
dancer Research 51:712-717, (1991). Cells in 10 cm dishes at 50-?5%
confluency are treated with the test compound (final concentration of
solvent, methanol or dimethyl sulfoxide, is 0.1%). After 4 hours at
37°C,
the cells are labeled in 3 ml methionine-free DMEM supple-mented with
10% regular DMEM, 2% fetal bovine serum and 400 mCi[35S)methionine
(1000 Ci/mmol). After an additional 20 hours, the cells are lysed in 1 ml
lysis buffer (1% NP40/20 mM HEPES, pH 7.5/5 mM MgCl~lmM DTT/10
mg/ml aprotinen/2 mg/ml leupeptin/2 mg/ml antipain/0.5~ mM PMSF)
and the Iysates cleared by centrifugation at 100,000 x g for 45 min.
Aliquots of lysates containing equal numbers of acid-precipitable counts
are bought to 1 ml with IP buffer (lysis buffer lacking DTT) and immuno-
precipitated with the ras-specific monoclonal antibody Y13-259 (Forth,
M.E. ~ ~1_., J. Virol. 43:294-304, (1982)). Following a 2 hour antibody
incubation at 4°C, 200 ml of a 25% suspension of protein A-Sepharose
coated with rabbit anti rat IgG is added for 45 min. The immuno-
precipitates are washed four times with IP buffer (20 nM HEPES, pH
7.5/1 mM EDTA/1% Triton X-100Ø5oh deoxycholate/0.1%/SDS/0.1 M
NaCl) boiled in SDS-PAGE sample buffer and loaded on 13% acrylamide
gels. When the dye front reached the bottom, the gel is fixed, soaked in
Enlightening, dried and autoradiographed. The intensities of the bands
corresponding to farnesylated and nonfarnesylated ras proteins are
compared to determine the percent inhibition of farnesyl transfer to
protein.
Cg,~l-basedin vitro ~owth inhibition assay
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To determine the biological consequences of FPTase
inhibition, the effect of the compounds of the instant invention on the
anchorage-independent growth of Rat1 cells transformed with either
a v-ras, v-raf, or v-mos oncogene is tested. Cells transformed by v-Raf
and v-Mos maybe included in the analysis to evaluate the specificity of
instant compounds for Ras-induced cell transformation.
Rat 1 cells transformed with either v-ras, v-raf, or v-mos
are seeded at a density of 1 x 104 cells per plate (35 mm in diameter)
in a 0.3% top agarose layer in medium A (Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum) over a bottom
agarose layer (0.6%). Both layers contain 0.1% methanol or an
appropriate concentration of the instant compound (dissolved in
methanol at 1000 times the final concentration used in the assay).
The cells are fed twice weekly with 0.5 ml of medium A containing
0.1% methanol or the concentration of the instant compound.
Photomicrographs are taken 16 days after the cultures are seeded
and comparisons are made.
Construction of SEAP re~rter Dlasmid ~DSE100
The SEAP reporter plasmid, pDSE100 was constructed by
ligating a restriction fragment containing the SEAP coding sequence
into the plasmid pCMV-RE-AKI. The SEAP gene is derived from the
plasmid pSEAP2-Basic (Clontech, Palo Alto, CA). The plasmid pCMV
RE-AKI was constructed by Deborah Jones (Merck) and contains 5
35
sequential copies of the 'dyad symmetry response element' cloned
upstream of a 'CAT-TATA' sequence derived from the cytomegalovirus
immediate early promoter. The plasmid also contains a bovine growth
hormone poly-A sequence.
The plasmid, pDSE100 was constructed as follows. A
restriction fragment encoding the SEAP coding sequence was cut out
of the plasmid pSEAP2-Basic using the restriction enzymes EcoRl and
HpaI. The ends of the linear DNA fragments were filled in with the
HIenow fragment of E. coli DNA Polymerase I. The 'blunt ended' DNA
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containing the SEAP gene was isolated by electrophoresing the digest in
an agarose gel and cutting out the 1694 base pair fragment. The vector
plasmid pCMV-RE-AKI was linearized with the restriction enzyme Bgl-
II and the ends filled in with Klenow DNA Polymerase I. The SEAP
S DNA fragment was blunt end ligated into the pCMV-RE-AKI vector and
the ligation products were transformed into DH5-alpha E. coli cells
(Gibco-BRL). Tranaformants were screened for the proper insert and
then mapped for restriction fragment orientation. Properly oriented
recombinant constructs were sequenced across the cloning junctions to
verify the correct sequence. The resulting plasmid contains the SEAP
coding sequence downstream of the DSE and CAT-TATA promoter
elements and upstream of the BGH poly-A sequence.
Alternative Construction of SEAP reporter plasmid.,nDS_E101
The SEAP repotrer plasmid, pDSE101 is also constructed by
ligating a restriction fragment containing the SEAP coding sequence
into the plasmid pCMV-RE-AKI. The SEAP gene is derived from
plasmid pGEM7zf1-)/SEAP.
The plasmid pDSE101 was constructed as follows: A
restriction fragment containing part of the SEAP gene coding sequence
was cut out of the plasmid pGEM?zf(-)/SEAP using the restriction
enzymes Apa I and KpnI. The ends of the linear DNA fragments were
chewed back with the Klenow fragment of E. coli DNA Polymerase I.
The "blunt ended" DNA containing the truncated SEAP gene was
isolated by electrophoresing the digest in an agarose gel and cutting out
the 1910 base pair fragment. This 1910 base pair fragment was ligated
into the plasmid pCMV-RE-AKI which had been cut with Bgl-II and
filled in with E. coli Klenow fragment DNA polymerase. Recombinant
plasmids were screened for insert orientation and sequenced through
the ligated junctions. The plasmid pCMV-RE-AKI is derived from
plasmid pCMVIE-AKI-DHFR (Whang , Y., Silberklang, M., Morgan,
A., Munahi, S., Lenny, A.B., Ellis, R.W., and Kieff, E. (1987) J. Virol.,
61, 1796-1807) by removing an EcoRI fragment containing the DHFR and
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Neomycin markers. Five copies of the fos promoter serum response
element were inserted as described previously (Jones, R.E., Defeo-Jones,
D., McAvoy, E.M., Vuocolo, G.A., Wegrzyn, R.J., Haskell, K.M. and
Oliff, A. (1991) Oncogene, 6, ?45-751) to create plasmid pCMV-RE-AKI.
The plasmid pGEM7zf(-)/SEAP was constructed as follows. The SEAP
gene was PCRed, in two segments from a human placenta cDNA library
(Clontech) using the following oligos.
Sense strand N-terminal SEAP : 5'
GAGAGGGAATTCGGGCCCTTCCTGCAT
GCTGCTGCTGCTGCTGCTGCTGGGC 3' (SEQ.ID.N0.:3)
Antisense strand N-terminal SEAP: 5'
GAGAGAGCTCGAGGTTAACCCGGGT
GCGCGGCGTCGGTGGT 3' (SEQ.ID.N0.:4)
Sense strand C-terminal SEAP: 5'
GAGAGAGTCTAGAGTTAACCCGTGGTCC
CCGCGTTGCTTCCT 3' (SEQ.ID.N0.:5)
Antisense strand C-terminal SEAP: 5'
GAAGAGGAAGCTTGGTACCGCCACTG
GGCTGTAGGTGGTGGCT 3' (SEQ.ID.N0.:6)
The N-terminal oligos (SEQ.ID.NO.: 4 and SEQ.ID.NO.: 5) were used
to generate a 1560 by N-terminal PCR product that contained EcoRI
and HpaI restriction sites at the ends. The Antisense N-terminal oligo
(SEQ.ID.NO.: 4) introduces an internal translation STOP codon within
the SEAP gene along with the HpaI site. The C-terminal oligos
(SEQ.ID.NO.: 5 and SEQ.ID.NO.: 6) were used to amplify a 412 by C-
terminal PCR product containing HpaI and HindIII restriction sites.
The sense strand C-terminal oligo (SEQ.ID.NO.: 5) introduces the
internal STOP codon as well as the HpaI site. Next, the N-terminal
amplicon was digested with EcoRI and HpaI while the C-terminal
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amplicon was digested with HpaI and HindIII. The two fragments
comprising each end of the SEAP gene were isolated by electrophoresing
the digest in an agarose gel and isolating the 1560 and 412 base pair
fragments. These two fragments were then co-ligated into the vector
pGEM7zft-) (Promega) which had been restriction digested with EcoRI
and HindIII and isolated on an agarose gel. The resulting clone,
pGEM7zf(-)/SEAP contains the coding sequence for the SEAP gene from
amino acids.
P 1 P
An expression plasmid constitutively expressing the SEAP
protein was created by placing the sequence encoding a truncated SEAP
gene downstream of the cytomegalovirus (CMV) IE-1 promoter. The
expression plasmid also includes the CMV intron A region 5' to the
SEAP gene as well as the 3' untranslated region of the bovine growth
hormone gene 3' to the SEAP gene.
The plasmid pCMVIE-AKI-DHFR (Whang et al, 1987)
containing the CMV immediate early promoter was cut with EcoRI
generating two fragments. The vector fragment was isolated by agarose
electrophoresis and religated. The resulting plasmid is named pCMV-
AKI. Next, the cytomegalovirus intron A nucleotide sequence was
inserted downstream of the CMV IE1 promter in pCMV-AKI. The
intron A sequence was isolated from a genomic clone bank and
subcloned into pBR322 to generate plasmid pl6T-286. The intron A
sequence was mutated at nucleotide 1856 (nucleotide numbering as in
Chapman, B.S., Thayer, R.M., Vincent, K.A. and Haigwood, N.L.,
Nuc.Acids Res. 19, 3979-3986) to remove a SacI restriction site using site
directed mutagenesis. The mutated intron A sequence was PCRed from
the plasmid pl6T-287 using the following oligos.
Sense strand: 5' GGCAGAGCTCGTTTAGTGAACCGTCAG 3'
(SEQ.ID.NO.: 7)
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Antisense strand: 5' GAGAGATCTCAAGGACGGTGACTGCAG 3'
(SEQ.ID.NO.: 8)
These two oligos generate a 991 base pair fragment with
a SacI site incorporated by the sense oligo and a Bgl-II fragment
incorporated by the antisense oligo. The PCR fragment is trimmed with
SacI and Bgl-II and isolated on an agarose gel. The vector pCMV-AKI
is cut with SacI and Bgl-II and the larger vector fragment isolated by
agarose gel electrophoresis. The two gel isolated fragments are ligated
at their respective SacI and Bgl-II sites to create plasmid pCMV-AHI-
InA.
The DNA sequence encoding the truncated SEAP gene is
inserted into the pCMV-AHI-InA plasmid at the Bgl-II site of the vector.
The SEAP gene is cut out of plasmid pGEM7zf(-)/SEAP (described above)
using EcoRI and HindIII. The fragment is filled in with HIenow DNA
polymerise and the 1970 base pair fragment isolated from the vector
fragment by agarose gel electrophoresis. The pCMV-AKI-InA vector is
prepared by digesting with Bgl-II and filling in the ends with HIenow
DNA polymerise. The final construct is generated by blunt end Iigating
the SEAP fragment into the pCMV-AKI-InA vector. Transformants
were screened for the proper insert and then mapped for restriction
fragment orientation. Properly oriented recombinant constructs were
sequenced across the cloning junctions to verify the correct sequence.
The resulting plasmid, named pCMV-SEAP, contains a modified SEAP
sequence downstream of the cytomegalovirus immediately early
promoter IE-1 and intron A sequence and upstream of the bovine growth
hormone poly-A sequence. The plasmid expresses SEAP in a
constitutive manner when transfected into mammalian cells.
~loni of a ms's 3~lated viral-H-ras ex~~ression lap amid
A DNA fragment containing viral-H-ras can be PCRed from
plasmid "H-1" (Ellis R. et al. J. Virol. 36, 408, 1980) or "HB-11 (deposited
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in the ATCC under Budapest Treaty on August 27, 1997, and designated
ATCC 209,218) using the following oligos.
Se se strand:
5'TCTCCTCGAGGCCACCATGGGGAGTAGCAAGAGCAAGCCTAA
GGACCCCAGCCAGCGCCGGATGACAGAATACAAGCTTGTGGTG
G 3'. (SEQ.ID.NO.: 9)
Antisense:
5'CACATCTAGATCAGGACAGCACAGACTTGCAGC 3'.
(SEQ.ID.NO.: 10)
A sequence encoding the first 15 aminoacids of the v-src
gene, containing a myristylation site, is incorporated into the sense
strand oligo. The sense strand oligo also optimizes the 'Kozak'
translation initiation sequence immediately 5' to the ATG start site.
To prevent prenylation at the viral-ras C-terminus, cysteine 186 would
be mutated to a serine by substituting a G residue for a C residue in the
C-terminal antisense oligo. The PCR primer oligos introduce an XhoI
site at the 5' end and a XbaI site at the 3'end. The XhoI-XbaI fragment
can be ligated into the mammalian expression plasmid pCI (Promega)
cut with Xhol and XbaI. This results in a plasmid in which the
recombinant myr-viral-H-ras gene is constitutively transcribed from
the CMV promoter of the pCI vector.
loning of a viral-~i-ras-CULL ex~re~,g~on plasmid
A viral-H-ras clone with a C-terminal sequence encoding
the amino acids CVLL can be cloned from the plasmid "H-1" (Ellis R.
et al. J. Virol. 36, 408, 1980) or "HB-11 (deposited in the ATCC under
Budapest Treaty on August 27, 1997, and designated ATCC 209,218) by
PCR using the following oligos.
Sense strand:
5'TCTCCTCGAGGCCACCATGACAGAATACAAGCTTGTGGTGG-3'
(SEQ.ID:NO.: 11)
Antisense strand:
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5'CACTCTAGACTGGTGTCAGAGCAGCACACACTTGCAGC-3'
(SEQ.ID.NO.: 12)
The sense strand oligo optimizes the 'Kozak' sequence and
adds an XhoI site. The antisense strand mutates serine 189 to leucine
and adds an Xbal site. The PCR fragment can be trimmed with XhoI
and XbaI and ligated into the XhoI-XbaI cut vector pCI (Promega). This
results in a plasmid in which the mutated viral-H-ras-CULL gene
is constitutively transcribed from the CMV promoter of the pCI vector.
1C oni_ of c-H-ras-Leu61 expression ulasmid
The human c-H-ras gene can be PCRed from a human
cerebral cortex cDNA library (Clontech) using the following
1 S oligonucleotide primers.
Sense strand:
5'-GAGAGAATTCGCCACCATGACGGAATATAAGCTGGTGG-3'
(SEQ.ID.NO.: 13)
Antisense strand:
5'-GAGAGTCGACGCGTCAGGAGAGCACACACTTGC-3'
(SEQ.ID.NO.: 14)
The primers will amplify a c-H-ras encoding DNA
fragment with the primers contributing an optimized 'Kozak'
translation start sequence, an EcoRI site at the N-terminus and a Sal I
stite at the
C-terminal end. After trimming the ends of the PCR product with
EcoRI and Sal I, the c-H-ras fragment can be ligated ligated into an
EcoRI -Sal I cut mutagenesis vector pAlter-1 (Promega). Mutation of
glutamine-61 to a leucine can be accomplished using the
manufacturer's protocols and the following oligonucleotide:
5'-CCGCCGGCCTGGAGGAGTACAG-3' (SEQ.ID.NO.: 15)
After selection and sequencing for the correct nucleotide
substitution, the mutated c-H-ras-Leu61 can be excised from the pAlter-1
vector, using EcoRI and Sal I, and be directly ligated into the vector pCI
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(Promega) which has been digested with EcoRI and Sal I. The new
recombinant plasmid will constitutiveiy transcribe c-H-ras-Leu61 from
the CMV promoter of the pCI vector.
to i of a c-N-ras-Val-12 ex ren scion nlasmid
The human c-N-ras gene can be PCRed from a human
cerebral cortex cDNA library (Clontech) using the following
oligonucleotide primers.
dense strand:
5'-GAGAGAATTCGCCACCATGACTGAGTACAAACTGGTGG-3'
(SEQ.ID.NO.: 16)
Antisense strand:
5'-GAGAGTCGACTTGTTACATCACCACACATGGC-3' (SEQ.ID.NO.:
17)
The primers will amplify a c-N-ras encoding DNA
fragment with the primers contributing an optimized 'Kozak'
translation start sequence, an EcoRI site at the N-terminus and a Sal I
stite at the C-terminal end. After trimming the ends of the PCR product
with EcoRI and Sal I, the c-N-ras fragment can be ligated into an EcoRI
-Sal I cut mutagenesis vector pAlter-1 (Promega). Mutation of glycine-12
to a valine can be accomplished using the manufacturer's protocols and
the following oligonucleotide:
5'-GTTGGAGCAGTTGGTGTTGGG-3' (SEQ.ID.NO.: 18)
After selection and sequencing for the correct nucleotide
substitution, the mutated c-N-ras-Val-12 can be excised from the pAlter-
1 vector, using EcoRI and Sal I, and be directly iigated into the vector
pCI (Promega) which has been digested with EcoRI and Sal I. The new
recombinant plasmid will constitutively transcribe c-N-ras-Val-12 from
the CMV promoter of the pCI vector.
Cloning of a ~-K-ras-Val-12 ex~~ression lp asmid
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The human c-K-ras gene can be PCRed fram a human
cerebral cortex cDNA library (Clontech) using the following
oligonucleotide primers.
Sense sand:
5'-GAGAGGTACCGCCACCATGACTGAATATAAACTTGTGG-3'
(SEQ.ID.NO.: 19)
Antisens~ strand:
5'-CTCTGTCGACGTATTTACATAATTACACACTTTGTC-3'
(SEQ.ID.NO.: 20)
The primers will amplify a c-K-ras encoding DNA
fragment with the primers contributing an optimized 'Kozak'
translation start sequence, a KpnI site at the N-terminus and a Sal I
stite at the C-terminal end. After trimming the ends of the PCR product
with Kpn I and Sal I, the c-K-ras fragment can be ligated into a Kpnl -
Sal I cut mutagenesis vector pAlter-1 (Promega). Mutation of cysteine-
12 to a valine can be accomplished using the manufacturer's protocols
and the following oligonucleotide:
5'-GTAGTTGGAGCTGTTGGCGTAGGC-3' (SEQ.ID.NO.: 21)
After selection and sequencing for the correct nucleotide
substitution, the mutated c-K-ras-Val-12 can be excised from the pAlter-
1 vector, using KpnI and Sal I, and be directly ligated into the vector
pCI (Promega) which has been digested with KpnI and Sal I. The new
recombinant plasmid will constitutively transcribe c-K-ras-Val-12 from
the CMV promoter of the pCI vector.
Human C33A cells (human epitheial carcenoma - ATTC
collection) are seeded in IOcm tissue culture plates in DMEM + 10% fetal
calf serum + 1X Pen/Strep + 1X glutamine + 1X NEAR. Cells are grown
at 37oC in a 5% C02 atmosphere until they reach 50 -80% of confluency.
The transient transfection is performed by the CaP04
method (Sambrook et al., 1989). Thus, expression plasmids for H-ras,
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N-ras, K-ras, Myr-ras or H-ras-CVLL are co-precipitated with the DSE-
SEAP reporter construct. For lOcm plates 600m1 of CaCl2 -DNA solution
is added dropwise while vortexing to 600m1 of 2X HBS buffer to give l.2ml
of precipitate solution (see recipes below). This is allowed to sit at room
temperature for 20 to 30 minutes. While the precipitate is forming, the
media on the C33A cells is replaced with DMEM (minus phenol red;
Gibco cat. # 31053-028)+ 0.5% charcoal stripped calf serum + 1X
(Pen/Strep, Glutamine and nonessential aminoacids). The CaP04-DNA
precipitate is added dropwise to the cells and the plate rocked gently to
distribute. DNA uptake is allowed
to proceed for 5-6 hrs at 37oC under a 5% C02 atmosphere.
Following the DNA incubation period, the cells are washed
with PBS and trypsinized with lml of 0.05% trypsin. The 1 ml of
trypsinized cells is diluted into lOml of phenol red free DMEM + 0.2%
1 S charcoal stripped calf serum + 1X (Pen/Strep, Glutamine and NEAR ).
Transfected cells are plated in a 96 well microtiter plate (100m1/well)
to which drug, diluted in media, has already been added in a volume of
100m1. The final volume per well is 200m1 with each drug concentration
repeated in triplicate over a range of half log steps.
Incubation of cells and drugs is for 36 hrs at 37oC
under C02. At the end of the incubation period, cells are examined
microscopically for evidence of cell distress. Next, 100m1 of media
containing the secreted alkaline phosphatase is removed from each well
and transferred to a microtube array for heat treatment at 65oC for 1 hr
to inactivate endogenous alkaline phosphatases (but not the heat stable
secreted phosphatase).
The heat treated media is assayed for alkaline phosphatase
by a luminescence assay using the luminescence reagent CSPD~
(Tropix, Bedford, Mass. ). A volume of 50 ml media is combined with 200
ml of CSPD cocktail and incubated for 60 minutes at room temperature.
Luminesence is monitored using an ML2200 microplate luminometer
(Dynatech). Luminescence reflects the level of activation of the fos
reporter construct stimulated by the transiently expressed protein.
DATA-CaPO~p_recioitate for lOcm.~late o cells
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Ras expression plasmid (lmg/ml) lOml
DSE-SEAP Plasmid (lmg/ml) 2m1
Sheared Calf Thymus DNA (lmg/ml) 8m1
2M CaCl2 74m1
dH20 506m1
2X~'.BS Buffer
280mM NaCI
lOmM KCl
l.5mM Na2HP04 2H20
l2mM dextrose
50mM HEPES
Final pH = 7.05
Assay Buffer 20m1
Emerald ReagentT"' (Tropix) 2.5m1
100mM homoarginine 2.5m1
CSPD Reagent~ (Tropix) l.Om1
essay Buffer
Add 0.05M Na2C03 to 0.05M NaHCOg to obtain pH 9.5.
Make 1mM in MgCl2
EXAMPLE 20
The processing assays employed are modifications of that
described by DeClue et al (Cancer Research 51, 712-717, 1991].
PSN-1 (human pancreatic carcinoma) or viral-K4B-ras-
transformed Ratl cells are used for analysis of protein processing.
Subconfluent cells in 100 mm dishes are fed with 3.5 ml of media
(methionine-free RPMI supplemented with 2% fetal bovine serum or
cysteine-free/methionine-free DMEM supplemented with 0.035 ml of 200
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mM glutamine (Gibco), 2% fetal bovine serum, respectively) containing
the desired concentration of test compound, lovastatin or solvent alone.
Cells treated with lovastatin (5-10 ~.M), a compound that blocks Ras
processing in cells by inhibiting a rate-limiting step in the isoprenoid
biosynthetic pathway, serve as a positive control. Test compounds are
prepared as 1000x concentrated solutions in DMSO to yield a final solvent
concentration of 0.1%. Following incubation at 3?oC for two hours 204
~,Ci/ml [35S]Pro-Mix (Amersham, cell labeling grade) is added.
After introducing the label amino acid mixture, the cells
are incubated at 37oC for an additional period of time (typically 6 to
24 hours). The media is then removed and the cells are washed once
with cold PBS. The cells are scraped into 1 ml of cold PBS, collected by
centrifugation (10,000 x g for 10 sec at room temperature), and lysed by
vortexing in 1 ml of lysis buffer (1% Nonidet P-40, 20 mM HEPES, pH 7.5,
150 mM NaCI, 1 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT,
10 ~.g/ml AEBSF, 10 ~.g/ml aprotinin, 2 ~g/ml leupeptin and 2 ~tg/ml
antipain). The lysate is then centrifuged at 15,000 x g for 10 min at 4oC
and the supernatant saved.
For immunoprecipitation of Ki4B-Ras, samples of lysate
supernatant containing equal amounts of protein are utilized. Protein
concentration is determined by the Bradford method utilizing bovine
serum albumin as a standard. The appropriate volume of lysate is
brought to I ml with lysis buffer lacking DTT and 8 ~g of the pan Ras
monoclonal antibody, Y13-259, added. The protein/antibody mixture is
incubated on ice at 4oC for 24 hours. The immune complex is collected
on pansorbin (Calbiochem) coated with rabbit antiserum to rat IgG
(Cappel) by tumbling at 4oC for 45 minutes. The pellet is washed 3
times with 1 ml of lysis buffer lacking DTT and protease inhibitors and
resuspended in 100 ml elution buffer ( IO mM Tris pH 7.4, 1% SDS). The
Ras is eluted from the beads by heating at 95°C for 5 minutes,
after
which the beads are pelleted by brief centrifugation ( 15,000 x g for 30 sec.
at room temperature).
The supernatant is added to 1 ml of Dilution Buffer 0.1%
Triton X-100, 5 mM EDTA, 50 mM NaCI, 10 mM Tris pH 7.4) with 2 mg
Kirsten-ras specific monoclonal antibody, c-K-ras Ab-1 (Calbiochem).
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The second protein/antibody mixture is incubated on ice at 4oC for 1-2
hours. The immune complex is collected on pansorbin (Calbiochem)
coated with rabbit antiserum to rat IgG (Cappel) by tumbling at 4oC for
45 minutes. The pellet is washed 3 times with 1 ml of lysis buffer lacking
DTT and protease inhibitors and resuspended in Laemmli sample
buffer. The Ras is eluted from the beads by heating at 95°C for 5
minutes,
after which the beads are pelleted by brief centrifugation. The
supernatant is subjected to SDS-PAGE on a 12% acrylamide gel (bis-
acrylamide:acrylamide, I:100), and the Ras visualized by fluorography.
Rapl rn oces i~n~ inhibition assav
Protocol A:
Cells are labeled, incubated and lysed as described in
Example 20.
For immunoprecipitation of Rapl, samples of lysate
supernatant containing equal amounts of protein are utilized. Protein
concentration is determined by the bradford method utilizing bovine
serum albumin as a standard. The appropriate volume of lysate is
brought to 1 ml with lysis buffer lacking DTT and 2 ~,g of the Rapl
antibody, Rap1/Krevl (121) (Santa Cruz Biotech), is added. The
protein/antibody mixture is incubated on ice at 4oC for 1 hour. The
immune complex is collected on pansorbin (Calbiochem) by tumbling
at 4oC for 45 minutes. The pellet is washed 3 times with 1 ml of lysis
buffer lacking DTT and protease inhibitors and resuspended in 100 ml
elution buffer (IO mM Tris pH 7.4, 1% SDS). The Rapl is eluted from the
beads by heating at 95°C for 5 minutes, after which the beads are
pelleted
by brief centrifugation ( 15,000 x g for 30 sec, at room temperature).
The supernatant is added to 1 ml of Dilution Buffer (0.1%
Triton X-100, 5 mM EDTA, 50 mM NaCI, 10 mM Tris pH 7.4) with 2 mg
Rapl antibody, Rapl/Krevl (121) (Santa Cruz Biotech). The second
protein/antibody mixture is incubated on ice at 4oC for 1-2 hours. The
immune complex is collected on pansorbin (Calbiochem) by tumbling
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at 4oC for 45 minutes. The pellet is washed 3 times with 1 ml of lysis
buffer lacking DTT and protease inhibitors and resuspended in Laemmli
sample buffer. The Rapt is eluted from the beads by heating at 95°C for
5
minutes, after which the beads are pelleted by brief centrifugation. The
supernatant is subjected to SDS-PAGE on a 12% acrylamide gel (bis-
acrylamide:acrylamide, 1:100), and the Rapl visualized by fluorography.
Protocol B:
PSN-1 cells are passaged every 3-4 days in l0cm plates,
splitting near-confluent plates 1:20 and 1:40. The day before the assay is
set up, 5x 106 cells are plated on l5cm plates to ensure the same stage
of confluency in each assay. The media for these cells is RPM1 1640
(Gibco), with 15% fetal bovine serum and lx Pen/Strep antibiotic mix.
The day of the assay, cells are collected from the IScm plates
by trypsinization and diluted to 400,000 cells/ml in media. 0.5m1 of these
diluted cells are added to each well of 24-well plates, for a final cell
number of 200,000 per well. The cells are then grown at 37°C overnight.
The compounds to be assayed are diluted in DMSO in
1/2-log dilutions. The range of final concentrations to be assayed is
generally 0.1-100~.M. Four concentrations per compound is typical. The
compounds are diluted so that each concentration is IOOOx of the final
concentration (i.e., for a 10~,M data point, a lOmM stock of the compound
is needed).
2~,L of each 1000x compound stock is diluted into lml media
to produce a 2X stock of compound. A vehicle control solution (2~,L
DMSO to lml media), is utilized. 0.5 ml of the 2X stocks of compound are
added to the cells.
After 24 hours, the media is aspirated from the assay
plates. Each well is rinsed with lml PBS, and the PBS is aspirated.
180~.L SDS-PAGE sample buffer (Novex) containing 5% 2-mercapto-
ethanol is added to each well. The plates are heated to 100°C for 5
minutes using a heat block containing an adapter for assay plates.
The plates are placed on ice. After 10 minutes, 20N.L of an RNAse/
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DNase mix is added per well. This mix is lmg/ml DNaseI (Worthington
Enzymes), 0.25mg/ml Rnase A (Worthington Enzymes), 0.5M Tris-HCl
pH8.0 and 50mM MgCl2. The plate is left on ice for 10 minutes. Samples
are then either loaded on the gel, or stored at -70°C until use.
Each assay plate (usually 3 compounds, each in 4-
point titrations, plus controls) requires one 15-well 14% Novex gel. 25,1
of each sample is loaded onto the gel. The gel is run at l5mA for about
3.5 hours. It is important to run the gel far enough so that there will be
adequate separation between 2lkd (Rapl) and 29kd (Rab6).
The gels are then transferred to Novex pre-cut PVDF
membranes for 1.5 hours at 30V (constant voltage). Immediately after
transferring, the membranes are blocked overnight in 20m1 Western
blocking buffer (2% nonfat dry milk in Western wash buffer (PBS +
0.1°Jo
Tween-20). If blocked over the weekend, 0.02% sodium azide is added.
The membranes are blocked at 4°C with slow rocking.
The blocking solution is discarded and 20m1 fresh
blocking solution containing the anti Rapla antibody (Santa Cruz
Biochemical SC1482) at 1:1000 (diluted in Western blocking buffer) and
the anti Rab6 antibody (Santa Cruz Biochemical SC310) at 1:5000 (diluted
in Western blocking buffer) are added. The membranes are incubated at
room temperature for 1 hour with mild rocking. The blocking solution is
then discarded and the membrane is washed 3 times with Western wash
buffer for 15 minutes per wash. 20m1 blocking solution containing 1:1000
(diluted in Western blocking buffer) each of two alkaline phosphatase
conjugated antibodies (Alkaline phosphatase conjugated Anti-goat IgG
and Alkaline phosphatase conjugated anti-rabbit IgG [Santa Cruz
Biochemical)) is then added. The membrane is incubated for one hour
and washed 3x as above.
About 2m1 per gel of the Amersham ECF detection
reagent is placed on an overhead transparency (ECF) and the PVDF
membranes are placed face-down onto the detection reagent. This is
incubated for one minute, then the membrane is placed onto a fresh
transparency sheet.
The developed transparency sheet is scanned on a
phosphorimager and the Rapla Minimum Inhibitory Concentration is
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determined from the lowest concentration of compound that produces a
detectable Rapla Western signal. The Rapla antibody used recognizes
only unprenylated/unprocessed Rapla, so that the pretence of a
detectable Rapla Western signal is indicative of inhibition of Rapla
prenylation.
~n vivo tumor errowth inhibition assay (nude mouse)
In vivo efficacy as an inhibitor of the growth of cancer cells
may be confirmed by several protocols well known in the art. Examples
of such in vivo efficacy studies are described by N. E. Kohl et al. (Nature
Medicine, 1:792-797 (1995)) and N. E. Kohl et al. (Proc. Nat. Acad. Sci.
U.S.A., 91:9141-9145 (1994)).
Rodent fibroblasts transformed with oncogenically mutated
human Ha-ras or Ki-ras (106 cellslanimal in 1 ml of DMEM salts) are
injected subcutaneously into the left flank of $-12 week old female nude
mice (Harlan) on day 0. The mice in each oncogene group are randomly
assigned to a vehicle, compound or combination treatment group.
Animals are dosed subcutaneously starting on day 1 and daily for the
duration of the experiment. Alternatively, the farnesyl-protein
transferase inhibitor may be administered by a continuous infusion
pump. Compound, compound combination or vehicle is delivered in a
total volume of 0.1 ml. Tumors are excised and weighed when all of the
vehicle-treated animals exhibited lesions of 0.5 - 1.0 cm in diameter,
typically 11-15 days after the cells were injected. The average weight of
the tumors in each treatment group for each cell line is calculated.
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SEQUENCE LISTING
<110> Merck & Co., Inc.
Dinsmore, Christopher J.
Hutchinson, John H.
Williams, theresa M.
<120> INHIBITORS OF PRENYL-PROTEIN TRANSFERASE
<130> 20040Y
<150> 60/057,080
<151> 1997-08-27
<160> 21
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 4
<212> PRT
<213> Artificial Sequence
<400> 1
Cys Val Leu Ser
1
<210> 2
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 2
Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met
1 5 10 15
<210> 3
<211> 52
<212> DNA
<213> Artificial Sequence
<400> 3
gagagggaat tcgggccctt cctgcatgct gctgctgctg ctgctgctgg gc 52
<210> 4
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 4
gagagagctc gaggttaacc cgggtgcgcg gcgtcggtgg t 41
1
CA 02301770 2000-02-24
WO 99/09985 PCT/US98/17696
<210> 5
<211> 42
<212> DNA
<213> Artificial Sequence
<400> 5
gagagagtct agagttaacc cgtggtcccc gcgttgcttc ct 42
<210> 6
<211> 43
<212> DNA
<213> Artificial Sequence
<400> 6
gaagaggaag cttggtaccg ccactgggct gtaggtggtg get 43
<210> 7
<211> 27
<212> DNA
<2i3> Artificial Sequence
<400> 7
ggcagagctc gtttagtgaa ccgtcag 27
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 8
gagagatctc aaggacggtg actgcag 27
<210> g
<211> 86
<212> DNA
<213> Artificial Sequence
<400> 9
tctcctcgag gccaccatgg ggagtagcaa gagcaagcct aaggacccca gccagcgccg 60
gatgacagaa tacaagcttg tggtgg 86
<210> 10
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 10
cecatctaga tcaggacagc acagacttgc agc 33
<210> 11
<211> 41
<212> DNA
2
CA 02301770 2000-02-24
WO 99109985 PCT/US98/17696
<213> Artificial Sequence
<400> 11
tctcctcgag gccaccatga cagaatacaa gcttgtggtg g 41
<210> 12
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 12
cactctagac tggtgtcaga gcagcacaca cttgcagc 38
<210> 13
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 13
gagagaattc gccaccatga cggaatataa gctggtgg 38
<210> 14
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 14
gagagtcgac gcgtcaggag agcacacact tgc 33
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 15
ccgccggcct ggaggagtac ag 22
<210> 16
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 16
gagagaattc gccaccatga ctgagtacaa actggtgg 38
<210> 17
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 17
gagagtcgac ttgttacatc accacacatg gc 32
3
CA 02301770 2000-02-24
WO 99/09985 PCT/US98/17696 -
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 18
gttggagcag ttggtgttgg g 21
<210> 19
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 19
gagaggtacc gccaccatga ctgaatataa acttgtgg 38
<210> 20
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 20
ctctgtcgac gtatttacat aattacacac tttgtc 36
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 21
gtagttggag ctgttggcgt aggc 24
4
