Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE INVENTION
INHIBITORS OF PRENYL-PROTEIN TRANSFERASE
BACKGROUND OF THE INVENTION
The Ras proteins (Ha-Ras, Ki4a-Ras, Ki4b-Ras and N-Ras) are part of
a signalling 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, 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, Ayayz. Rev. Biochem. 62:851-891 (1993)).
Mutated ras genes (Ha-ras, Ki4a-ras, Ki4b-ras and N-ras) 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.
Ras must be localized to the plasma membrane for both normal
and oncogenic functions. At least 3 post-translational modifications 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-
Aaa'-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)). Depending on
the
specific sequence, this motif serves as a signal sequence for the enzymes
farnesyl-
protein transferase or geranylgeranyl-protein transferase type I, which
catalyze the
alkylation of the cysteine residue of the CAAX motif with a C15 or C2o
isoprenoid,
respectively. (S. Clarke., AnyZ. Rev. Biochem. 61:355-386 (1992); W.R. Schafer
and
J. Rine, Aran. Rev. Gefzetics 30:209-237 (1992)). The term prenyl-protein
transferase
may be used to refer generally to farnesyl-protein transferase and
geranylgeranyl-
protein transferase type I. The Ras protein is one of several proteins that
are known
to undergo post-translational farnesylation. Other farnesylated proteins
include the
Ras-related GTP-binding proteins such as Rho, fungal mating factors, the
nuclear
lamins, and the gamma subunit of transducin. James, et al., J. Biol. Clzem.
269, 14182
(1994) have identified a peroxisome associated protein Pxf which is also
farnesylated.
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James, et al., have also suggested that there are farnesylated proteins of
unknown
structure and function in addition to those listed above.
Inhibition of farnesyl-protein transferase has been shown to block
the growth of 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 Ras
oncoprotein
intracellularly (N.E. Kohl et al., Science, 260:1934-1937 (1993) and G.L.
James et
al., Science, 260:1937-1942 (1993). Recently, it has been shown that an
inhibitor
of farnesyl-protein transferase blocks the growth of 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 ras transgenic mice
(N.E. Kohl et al., Nature MedicifZe, 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 245:379 (1989)).
These drugs
inhibit HMG-CoA reductase, the rate limiting enzyme for the production of poly-
isoprenoids including farnesyl pyrophosphate. Farnesyl-protein transferase
utilizes
farnesyl pyrophosphate to covalently modify the Cys thiol group of the Ras
CAAX
box with a farnesyl group (Reiss 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. Aead. Sci USA, 87:7541-7545 (1990)). Inhibition of
farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks
Ras membrane localization in cultured cells. However, direct inhibition of
farnesyl-
protein transferase would be more specific and attended by fewer side effects
than
would occur with the required dose of a general inhibitor of isoprene
biosynthesis.
Inhibitors of farnesyl-protein transferase (FPTase) have been described
in two general classes. The first are analogs of farnesyl diphosphate (FPP),
while
the second class of inhibitors is related to the 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., 37, 725 (1994)).
In
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general, deletion of the thiol from a CAAX derivative has been shown to
dramatically
reduce the inhibitory potency of the compound. However, the thiol group
potentially
places limitations on the therapeutic application of FPTase inhibitors with
respect to
pharmacokinetics, pharmacodynamics and toxicity. Therefore, a functional
replacement for the thiol is desirable.
It has recently been reported that farnesyl-protein transferase inhibitors
are inhibitors of proliferation of vascular smooth muscle cells and are
therefore useful
in the prevention and therapy of arteriosclerosis and diabetic disturbance of
blood
vessels (JP H7-112930).
It has recently been disclosed that certain tricyclic compounds which
optionally incorporate a piperidine moiety are inhibitors of FPTase (WO
95/10514,
WO 95/10515 and WO 95/10516). Imidazole-containing inhibitors of farnesyl
protein transferase have also been disclosed (WO 95/09001 and EP 0 675 112
A1).
It is, therefore, an object of this invention to develop compounds that
do not have a thiol moiety, and that will inhibit prenyl-protein transferase
and thus,
the post-translational prenylation of proteins. It is a further object of this
invention to
develop chemotherapeutic compositions containing the compounds of this
invention
and methods for producing the compounds of this invention.
SUMMARY OF THE INVENTION
The present invention comprises spirocyclic compounds which inhibit
prenyl-protein transferase. Further contained in this invention are
chemotherapeutic
compositions containing these prenyl-protein transferase inhibitors and
methods for
their production.
The compounds of this invention are illustrated by the formula I:
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~R3)W~ AWRid)2)1 ~~C~RIa)2)n
Y
A1
1 C)2)s
3 ~ is
A WR )2)n
C R1°
)2)s
~/---~ C R1 b A2 C R1 b
C C )2)p ~ ~ )2)p
(R4)r
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention are useful in the inhibition of
prenyl-protein transferase and the prenylation of the oncogene protein Ras. In
a first
embodiment, the compounds of the instant invention are illustrated by the
formula I:
~R3)W ~ ~ WRi d)2)1 ~~C~R1 a)2)n
Y
A1
1 ~)2)s
A3 ~C~R1 a)2)n
C Ri°
)2)s
V~- C R1 b A2 C R1 b
)2)p ~ ~ )2)p
(R4)r
-4-
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wherein
M is selected from N, O or S;
A1, A2 and A3 are independently selected from
1) a bond,
2) O,
3) C(O),
N(R10)2~
105) C(O)NR10,
~10C(O)~
S(O)m
g) OS(O)m
9) ~lOC(O)~g10~
1510) OC(O),
11) C(O)O
12) CH=CH,
13) C---C,
14) OC(O)NR10, or
2015) NR10C(O)O;
Rla~ Rlb~ Rlc
and R1d are
independently
selected from
1) H,
2) unsubstituted or substituted C1-C(
alkyl,
253) unsubstituted or substituted C2-Cg
alkenyl,
4) unsubstituted or substituted C2-Cg
alkynyl,
5) unsubstituted or substituted aryl,
6) unsubstituted or substituted C3-C
10 cycloalkyl,
7) unsubstituted or substituted heterocycle,
30g) -OR10,
R11S(O)m-
10) -N(R10)2,
11) R10C(O)-,
12) -C(O)NR6R~, .
3513) R10C(O)NR10_~
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14) (R10)2NC(O)NR10-
15) R100C(O)-,
16) R10C(O)O-, or
17) R110C(O)NR10-
R3 is independentlyselected from
1) H,
2) halo,
3) -CN,
104) -N02,
5) unsubstituted or substituted C1-C(
alkyl,
6) unsubstituted or substituted C2-Cg
alkenyl,
7) unsubstituted or substituted C2-Cg
alkynyl,
8) unsubstituted or substituted aryl,
159) , unsubstituted or substituted heterocycle,
10) C1-Cg perfluoroalkyl,
11) -OR10,
12) OCF3,
13) -CH2CF3,
2014) unsubstituted or substituted C3-C
10 cycloalkyl,
15) NR6R7,
16) -C(O)R10,
17) -OC(O)R10,
18) -O(C1-C6 alkyl)OR10_~
2519) -S(O)mRll,
20) -C(O)NR6R7,
21) -NR6C(O)R10,
22) -(C1-C( alkyl)OR10, or
23) -(C1-C6 alkyl)C(O)R10;
R4 is independently selected from
1) H,
2) unsubstituted or substituted C1-C( alkyl,
3) unsubstituted or substituted C2-Cg alkenyl,
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4) unsubstituted or substituted C2-Cg
alkynyl,
5) unsubstituted or substituted aryl,
6) unsubstituted or substituted heterocycle,
7) unsubstituted or substituted C3-C10
cycloalkyl,
8) unsubstituted or substituted C1-C6
perfluoroalkyl,
9) halo,
10) -OR 10,
11 ) CN,
12) -S(O)mRll~
1013) -C(O)NRER7,
14) -NR10C(O)R10~
15) N02,
16) -N(R10)2,
17) -C(O)R10,
1518) -OC(O)R10,
19) R10)2NC(O)NR10_, or
20) R100C(O)NR10_~
R5 is independentlyselected from
201) H,
2) unsubstituted or substituted C1-C6
alkyl,
3) unsubstituted or substituted C2-Cg
alkenyl,
4) unsubstituted or substituted C2-Cg
alkynyl,
5) unsubstituted or substituted C3-C10
cycloalkyl,
256) unsubstituted or substituted aryl,
7) unsubstituted or substituted heterocycle,
8) -S(O)mRll~
9) _C(O)NR6R7,
10) -S(O)2NRER7,
3011) -C(O)RE,
12) -C(O)ORE,
13) -N(R10)2,
14) CN,
15) halo,
_7_
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16) -OR10,
17) N02, or
18) R110C(O)NR10_;
R6 and R7 are independently selected from: H, C1-C( alkyl, C3_6 cycloalkyl,
heterocycle, aryl, aroyl, heteroaroyl, arylsulfonyl, heteroarylsulfonyl,
unsubstituted or
substituted with:
a) C1_6 alkoxy,
b) C1-C2p alkyl
c) aryl or heterocycle,
d) halogen,
e) HO,
17 _C(O)Rlla
g) -S02R11, or
h) N(R10)2; or
R6 and R7 may be joined in a ring;
R10 is independently selected from
1) H,
2) unsubstituted or substituted C1-C6 alkyl,
3) unsubstituted or substituted perfluoroalkyl,
4) unsubstituted or substituted aralkyl,
5) unsubstituted or substituted aryl, or
6) unsubstituted or substituted heterocycle;
R11 is independently selected from
1) unsubstituted or substituted C1-C( alkyl,
2) unsubstituted or substituted aralkyl,
3) unsubstituted or substituted aryl, or
4) unsubstituted or substituted heterocycle;
G is selected from H2 or O;
_g_
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V is selected from
1) heterocycle or
2) aryl;
W is a heterocycle;
Y is selected
from
1) a bond,
2) C1-Cg alkyl,
3) C2-Cg alkenyl,
4) C2-Cg alkynyl,
5) C3-C10 cycloalkyl,
6) aryl, or
7) heterocycle;
m is 0, 1 or 2;
n is 0, 1, 2, 3, 4, 5 or 6;
p is 0, 1, 2, 3, 4, 5 or 6;
q is 0, 1, 2, 3 or 4;
r is 0, 1, 2, 3 or 4;
s is 0, 1, 2, 3, 4, 5 or 6;
t is 0, 1, 2, 3, or 4;
w is 0, 1, 2, 3 or 4, provided Y is not
a bond;
x is 0, 1, 2 or 3; and
y is 0, 1, 2 or 3;
or a pharmaceutically
acceptable
salt or
stereoisomer
thereof.
In another embodiment, the compounds of the instant invention are
illustrated by formula A:
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(C(R1 d)2) Ai
x
~Y ~ 1 a
( )w ~ (C(R )2)n
A3
\(R )a
( )r (C(Ri b)2)pA2(C(Ri b)2)p
A
wherein
5 A1, A2 and A3 are independently selected from
1) a bond,
2) O,
3) C(O),
N(R10)2~
5) C(O)NR10,
~lOC(O)~
~10C(O)~10~
OC(O),
9) C(O)O
10) OC(O)NR10, or
11) NR10C(O)O;
Rla~ Rlb and Rld are independently selected from
1) H,
2) unsubstituted or substituted C 1-C( alkyl,
3) unsubstituted or substituted aryl,
4) unsubstituted or substituted C3-C10 cycloalkyl,
- 10 -
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5) -OR10,
_N(R10)2~
7) R10C(O)-
8) -C(O)NR6R~, or
9) R10C(O)NR10-
R3 is independentlyselected from
1) H,
2) halo,
103) -CN,
4) -N02,
5) unsubstituted or substituted C1-C6
alkyl,
6) unsubstituted or substituted aryl,
7) -OR10,
158) OCF3,
9) unsubstituted or substituted C3-C1p
cycloalkyl,
10) NR6R~,
11) _C(O)RlOa
12) -C(O)NR6R~,
2013) -NR6C(O)R10,
14) -(C1-C( alkyl)OR10, or
15) -(C1-C( alkyl)C(O)R10~
R4 is independentlyselected from
251) H,
2) unsubstituted or substituted C
1-C( alkyl,
3) unsubstituted or substituted C2-Cg
alkenyl,
4) unsubstituted or substituted C2-Cg
alkynyl,
5) unsubstituted or substituted aryl,
306) unsubstituted or substituted heterocycle,
7) unsubstituted or substituted C3-C
10 cycloalkyl,
8) halo,
9) -OR10,
10) CN,
- 11 -
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11) -C(O)NR6R~,
12) -NR10C(O)R10~ or
13) -N(R10)2;
R5 is independently
selected from
1) H,
2) unsubstituted or substituted C1-C(
alkyl,
3) unsubstituted or substituted C3-C10
cycloalkyl,
4) unsubstituted or substituted aryl,
5) -C(O)NR6R~,
6) -C(O)RE,
7) -C(O)ORE,
8) halo, or
9) -OR10;
RE and R~ are
independently
selected from:
H, C1-CE alkyl,
C3_E cycloalkyl,
heterocycle, aroyl, heteroaroyl, arylsulfonyl, heteroarylsulfonyl,
aryl, unsubstituted or
substituted
with:
a) C1_E alkoxy,
b) C1-C20 alkyl
c) aryl or heterocycle,
d) halogen,
e) HO,
~ _C(O)R11~
g) -S02R11, or
h) N(R10)2; or
RE and R~ may be joined in a ring;
R10 is independently selected from
1) H,
2) unsubstituted or substituted C1-CE alkyl,
3) unsubstituted or substituted perfluoroalkyl,
4) unsubstituted or substituted aralkyl,
- 12 -
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5) unsubstituted or substituted aryl, or
6) unsubstituted or substituted heterocycle;
R11 is independently selected from
1) unsubstituted or substituted C1-C( alkyl,
2) unsubstituted or substituted aralkyl,
3) unsubstituted or substituted aryl, or
4) unsubstituted or substituted heterocycle;
G is selected from H~ or O;
V is selected from
1) heterocycle or
2) aryl;
W is a heterocycle selected from imidazolyl, pyridyl;
Y is selected
from
1) a bond,
2) C3-C10 cycloalkyl,
3) aryl, or
4) heterocycle;
m is 0, 1 or 2;
n is 0, 1, 2, 3, 4, 5 or 6;
p is 0, l, 2, 3, 4, 5 or 6;
q is 0, 1, 2, 3 or 4;
r is 0, 1, 2, 3 or 4;
t is 0, 1, 2, 3, or 4;
w is 0, 1, 2, 3 or 4, provided Y is not
a bond;
x is 1 or 2; and
yis 1 or2;
or a pharmaceutically
acceptable
salt or
stereoisomer
thereof.
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In another embodiment, the compounds of the instant invention are
illustrated by formula B:
N
'C(R1 d~2W Ai
~C~R1 a)2O
As /~~N
\ N. ~~ Rs
r
~C~R1 b~2~pA2~~!~R1 b~2~p
wherein
A1 and A2 are independently selected from
1) a bond,
2) O,
3) C(O),
4) C(O)NR10~
5) NR10C(O),
6) OC(O), or
7) C(O)O;
A3 is selected from
1) a bond,
2) O,
3) C(O),
4) C(O)NR10,
or
5) NR10C(O);
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Rla~ Rlb and
Rld are independently
selected from
1) H,
2) unsubstituted or substituted C1-C6
alkyl,
3) unsubstituted or substituted aryl,
4) unsubstituted or substituted C3-C10
cycloalkyl,
5) -OR10
6) -N(R10)2, or
R10C(O)-~
R3 is independently
selected from
1) H,
2) halo,
3) -CN,
154) -NO~,
5) unsubstituted or substituted C1-C6
alkyl,
6) unsubstituted or substituted aryl,
7) unsubstituted or substituted heterocycle,
8) -OR10,
209) OCF3,
10) unsubstituted or substituted C3-C10
cycloalkyl,
11) NR6R~,
12) -C(O)R10~
13) -C(O)NR6R~, or
2514) -NR6C(O)R10;
R4 is independentlyselected from
1 ) H,
2) unsubstituted or substituted C1-C6
alkyl,
303) unsubstituted or substituted C2-Cg
alkenyl,
4) unsubstituted or substituted C~-C8
alkynyl,
5) unsubstituted or substituted aryl,
6) unsubstituted or substituted heterocycle,
7) unsubstituted or substituted C3-C10
cycloalkyl,
358) halo,
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9) -OR 10,
10) CN, or
11) -N(R10)2;
R5 is independently selected from
1) H,
2) unsubstituted or substituted C1-C6 alkyl,
3) unsubstituted or substituted C3-C10 cycloalkyl,
4) unsubstituted or substituted aryl,
5) -C(O)RE,
6) -C(O)ORE,
7) halo, or
8) -OR10;
RE and R~ are independently selected from: H, C1-CE alkyl, C3-E cycloalkyl,
heterocycle, aryl, aroyl, heteroaroyl, arylsulfonyl, heteroarylsulfonyl,
unsubstituted or
substituted with:
a) C 1 _E alkoxy,
b) C 1-C20 alkyl
c) aryl or heterocycle,
d) halogen,
e) HO,
f) -C(O)R11~
g) -S02R11, or
h) N(R10)2; or
RE and R~ may be joined in a ring;
R10 is independently selected from
1) H,
2) unsubstituted or substituted C 1-CE alkyl,
3) unsubstituted or substituted perfluoroalkyl,
4) unsubstituted or substituted aralkyl,
5) unsubstituted or substituted aryl, or
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6) unsubstituted or substituted heterocycle;
R11 is independently selected from
1) unsubstituted or substituted C1-Cg alkyl,
2) unsubstituted or substituted axalkyl,
3) unsubstituted or substituted aryl, or
4) unsubstituted or substituted heterocycle;
G is selected from H2 or O;
Y is selected from
1) a bond,
2) C3-C10 cycloalkyl,
3) aryl, or
4) heterocycle;
m is 0, 1 or 2;
n is 0, 1, 2, 3, 4, 5 or 6;
p is 0, l, 2, 3, 4, 5 or 6;
q is 0, 1, 2, 3 or 4;
r is 0, 1, 2, 3 or 4;
t is 0, 1, 2, 3, or 4;
w is 0, 1, 2, 3 or 4, provided Y
is not a bond;
x is 1 or 2; and
y is 1 or 2;
or a pharmaceutically acceptable salt or stereoisomer thereof.
Examples of the compounds of the instant invention are:
- 17 -
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H O
N N~
~N
N
O
NC
(17R, 20R)-19,20,21,22-tetrahydro-19-oxo-17H-15,17:18,20-diethano-6,10:12,16-
dimetheno-20,21-propano-16H-imidazo [3,4-h] [ 1,8,11,14]oxatriazacycloeicosine-
9-
carbonitrile;
H O
N N~
O
N~ N
O
NC
(R,R)-15,16,17,17a,19,20,22,23-octahydro-19,22-dioxo-5H,21H 18,20-ethano-
12,14-etheno-6,10-metheno-20,21-propanobenz[d]imidazo[4,3-l] [1,6,9,13]
oxatriazacyclononadecosine-9-carbonitrile;
O
Br N,~N~
~N
7 ,N
N
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(R)-15-bromo-19,20,21,22-tetrahydro-19-oxo-17H 18,20-ethano-6,10:12,16-
dimetheno-20,21-propano-16H imidazo[3,4-h][1,8,11,14]oxatriazacycloeicosine-9-
carbonitrile;
J
S (R)-19,20,22,23-tetrahydro-19,22-dioxo-5H,21H-18,20-ethano-12,14-etheno-6,10-
metheno-20,21-propanobenz[d]imidazo[4,3-l]
[1,6,9,13]oxatriazacyclononadecosine-
9-carbonitrile;
H O
N N
~N
~N
(R,R)-15,16,17,17a,19,20,21,22-octahydro-15-oxa-19-oxo-5H-18,20-ethano
12,14-etheno-6,10-metheno-20,21-propano-18H-Benz [d]imidazo [4,3-k] [
1,6,9,12]
oxatriazacyclooctadecosine-9-carbonitrile;
or a pharmaceutically acceptable salt, optical isomer or stereoisomer thereof.
The compounds of the present invention may have asymmetric centers,
chiral axes and chiral planes, and occur as racemates, racemic mixtures, and
as
individual diastereomers, with all possible isomers, including optical
isomers, being
included in the present invention. (See E.L. Eliel and S.H. Wilen
StereoclaemistYy
- 19 -
. O I
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of Carbofz Compounds (John Wiley and Sons, New York 1994), in particular pages
1119-1190) When any variable (e.g. aryl, heterocycle, Rla, R3 etc.) occurs
more than
one time in any constituent, its definition on each occurrence is independent
at every
other occurrence. Also, combinations of substituents and/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 1 to 6 carbon
atoms,
unless otherwise indicated; "alkoxy" represents an alkyl group having 1 to 6
carbon
atoms, unless otherwise indicated, attached through an oxygen bridge.
"Halogen"
or "halo" as used herein means fluoro, chloro, bromo and iodo.
As used herein, "cycloalkyl" is intended to include non-aromatic
hydrocarbon groups having having from 3 to 10 carbon atoms, unless otherwise
specified. Examples of such cycloalkyl groups include, but are not limited to,
cyclopropyl, cyclobutyl, cyclohexyl, cycloheptyl, cyclooctyl, admantyl and the
like.
1S Optionally, a carbon atom in the cycloalkyl may be replaced with a
heteroatom,
such as O, N or S.
If no number of carbon atoms is specified, the term "alkenyl" refers
to a non-aromatic hydrocarbon, straight, branched or cyclic, containing from 2
to 10
carbon atoms, unless otherwise indicated, and at least one carbon to carbon
double
bond. Preferably one carbon to carbon double bond is present, and up to four
non-aromatic carbon-carbon double bonds may be present. Thus, "C2-C8 alkenyl"
means an alkenyl radical having from 2 to 8 carbon atoms. Examples of such
alkenyl
groups include, but are not limited to, ethenyl, propenyl, butenyl and
cyclohexenyl.
As described above with respect to alkyl, the straight, branched or cyclic
portion of
the alkenyl group may contain double bonds and may be substituted if a
substituted
alkenyl group is indicated.
The term "alkynyl" refers to a hydrocarbon radical straight, branched
or cyclic, containing from 2 to 10 carbon atoms, unless otherwise indicated,
and
at least one carbon to carbon triple bond. Up to three carbon-carbon triple
bonds
may be present. Thus, "CZ-C8 alkynyl" means an alkynyl radical having from 2
to
8 carbon atoms. Examples of such alkynyl groups include, but are not limited
to,
ethynyl, propynyl and butynyl. As described above with respect to alkyl, the
straight,
branched or cyclic portion of the alkynyl group may contain triple bonds and
may be
substituted if a substituted alkynyl group is indicated.
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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 include, but are not limited to,
phenyl,
naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl,
acenaphthyl
and the like.
As used herein, "aralkyl" is intended to mean an aryl moiety, as
defined above, attached through a Cl-C6 alkyl linker, where alkyl is defined
above.
Examples of aralkyls include, but are not limited to, benzyl, naphthylmethyl,
phenylbutyl and the like.
The term heterocycle or heterocyclic, as used herein, represents
a stable 5- to 7-membered monocyclic or stable ~- 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.
The term heterocycle or heterocyclic includes heteroaryl moieties. Examples of
such heterocyclic elements include, but are not limited to, azepinyl,
benzimidazolyl,
benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl,
benzo-
thiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl,
dihydrobenzofuryl,
dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone,
1,3-dioxolanyl, 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, 2-
pyridinonyl pyrazinyl,
pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl,
quinazolinyl,
quinolinyl, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,
tetrahydroquinolinyl,
thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl,
thienofuryl,
thienothienyl, thienyl and triazolyl.
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,
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benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl,
dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone,
furyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl,
isoquinolinyl,
isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl,
pyridazinyl,
pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl,
tetrahydroisoquinolinyl,
tetrahydroquinolinyl, thiazolyl, thienofuryl, thienothienyl, thienyl and
triazolyl.
As used herein, "heterocyclylalkyl" is intended to mean a heterocyclic
moiety, as defined above, attached through a C1-C6 alkyl linker, where alkyl
is defined above. Examples of heterocyclylalkyls include, but are not limited
to, 2-pyridylmethyl, 2-imidazolylethyl, 2-quinolinylmethyl, 2-
imidazolylmethyl,
1-piperazineethyl, and the like.
As used herein, the terms "substituted alkyl", "substituted alkenyl"
"substituted alkynyl" and "substituted alkoxy", unless otherwise defined, are
intended
to include the branch or straight-chain alkyl group of the specified number of
carbon
atoms, wherein the carbon atoms may be substituted with F, Cl, Br, I, CF3,
OCF3,
CN, N3, N02, NH2~ N(C1-C( alkyl)2, oxo, OH, -O(C1-C6 alkyl), -C(O)H, S(O)0_2,
(C1-C6 alkyl)S(O)0_~-, CZ-C( alkenyl, C~-C( alkynyl, -(C1-C( alkyl)S(O)0-2
(C1-C( alkyl), C3-C20 cycloalkyl, -C(O)NH2, HC(O)NH-, (C1-C6 alkyl)C(O)NH-,
H~NC(O)NH-, (C1-C( alkyl)C(O)-, -O(C1-C( alkyl)CF3, (C1-C6 alkyl)OC(O)-,
(C1-C6 alkyl)O(C1-C6 alkyl)-, (C1-C( alkyl)C(O)~(C1-C( alkyl)-, (C1-C6 alkyl)
OC(O)NH-, aryl, heterocycle, aralkyl, heterocyclylalkyl, halo-aryl, halo-
aralkyl, halo-
heterocycle, halo-heterocyclylalkyl, cyano-aryl, cyano-aralkyl, cyano-
heterocycle and
cyano-heterocyclylalkyl.
As used herein, the terms "substituted aryl", "substituted heterocycle",
"substituted heteroaryl", "substituted cycloalkyl", "substituted benzyl",
"substituted
aralkyl" and "substituted heterocyclylalkyl", unless otherwise defined, are
intended to
include the cyclic group containing from 1 to 3 substitutents in addition to
the point
of attachment to the rest of the compound. Such substitutents are preferably
selected
from the group which includes but is not limited to F, Cl, Br, I, CF3, OCF3,
NH2,
N(C1-C6 allcyl)~, NO~, CN, N3, C1-C20 alkyl, C3-C~0 cycloalkyl, -OH, -O(C1-Cg
allcyl), S(O)0-~, (C1-C6 alkyl)S(O)0_~-, (C1-C6 alkyl)S(O)0_2(C1-C6 alkyl)-,
-C(O)NH2, HC(O)NH-, (C1-C6 alkyl)C(O)NH-, H2NC(O)NH-, (C1-C6 alkyl)C(O)-,
(C1-C6 alkyl)OC(O)-, (C1-C6 alkyl)O(C1-C6 alkyl)-, (C1-C6)C(O)~(C1-C6 alkyl)-,
(C1-C6 alkyl)OC(O)NH-, aryl, aralkyl, heterocycle, heterocyclylalkyl, halo-
aryl,
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halo-aralkyl, haloheterocycle, haloheterocyclylalkyl, cyano-aryl, cyano-
aralkyl,
cyano-heterocycle and cyanoheterocyclylalkyl.
Lines drawn into the ring systems from substituents (such as from A3,
R4, R5 etc.) indicate that the indicated bond may be attached to any of the
substitut-
able ring carbon or nitrogen atoms.
Preferably, Rla, R1b Rlc and Rld are independently selected from:
hydrogen or unsubstituted or substituted C1-C( alkyl.
Preferably, A1, A2 and A3 are independently selected from a bond, O
or C(O). Most preferably, A1 is a bond or C(O), A2 is a bond and A3 is a bond
or O.
Preferably, R3 is independently selected from H, halo, unsubstituted or
substituted C1-C6 alkyl or OR10. Most preferably, R3 is independently selected
from
H or halo.
Preferably, R4 is independently selected from H, halo, CN,
unsubstituted or substituted aryl or unsubstituted or substituted C1-C6 alkyl.
Most preferably, variable r is 1 to 3, and at least one R4 is CN.
Preferably R5 is independently selected from H, unsubstituted or
substituted C1-C( alkyl or unsubstituted or substituted aryl. Most preferably,
R5
is hydrogen.
Preferably, G is O.
Preferably, V is aryl. Most preferably, V is phenyl.
Most preferably, W is imidazolyl.
Preferably, n, p, s and t are independently selected from 0, 1 or 2.
Preferably, q is 0 or 1.
Preferably, w is 0, 1 or 2.
Preferably, x is 1.
Preferably, y is 1.
Preferably, the moiety
~C~RIa)2O
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is
C Rid
)2)t
(C(Ri a)2)n
More preferably, the moiety
id
~C(R )2)t
(C(Ria)2)n
is
C Rid
~ ( )2)t
(C(Ria)2)n
Preferably, the moiety
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C Ria
( ( )2)n
C R1°
~ )2)s W (R5)a
U~'-CRib A2CRib~
C ( )2)p ( ( )2)p
(R4)r
is selected from
is ( /Ria)2)n
(C(R )2)S
~C~RIb)2) A2(C(Rib)2)p ~_
\ p \N~N (Rs)a
C -
(R4)r
or
1c C 1a
(C(R )2)s ( \( )2)n
\ (C(Ri b)2)pA2(C(R1 b)2)p N~ (R5)
yJ a
C, N
( R4)
Most preferably, the moiety
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C R1a
)2)n
C R1c
)2)s W ~R5)a
v~- C Rib A2 C Rlb
)2)p ~ ~ )2)p
~R4)r
is
~R1~)2)s (CH2)1-4
C \ \N~N ~R )a
~R4)r
It is intended that the definition of any substituent or variable (e.g.,
5 Rla, R3, n, etc.) at a particular location in a molecule be independent of
its definitions
elsewhere in that molecule. Thus, -N(R10)~ represents -NHH, -NHCH3, -NHC~HS,
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.
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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
stoichio-
metric amounts or with an excess of the desired salt-forming inorganic or
organic
acid in a suitable solvent or various combinations of solvents.
Abbreviations which may be used in the description of the
chemistry and in the Examples that follow include:
Ac20 Acetic anhydride;
AcOH Acetic acid;
AIBN 2,2'-Azobisisobutyronitrile;
BINAP 2,2'-Bis(diphenylphosphino)-l,l' binaphthyl;
Bn Benzyl;
BOCBoc tert-Butoxycarbonyl;
CBz Carbobenzyloxy;
DBAD Di-tert-butyl azodicarboxylate;
DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene;
DCE 1,2-Dichloroethane;
DIEA N,N Diisopropylethylamine;
DMAP 4-Dimethylaminopyridine;
l~ME 1,2-Dimethoxyethane;
DMF N,N-Dimethylformamide;
DMSO Methyl sulfoxide;
DPPA Diphenylphosphoryl azide;
DTT Dithiothreitol;
EDC 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide-hydrochloride;
EDTA Ethylenediaminetetraacetic acid;
Et2 Diethyl ether;
Et3N Triethylamine;
EtOAc Ethyl acetate;
EtOH Ethanol;
FAB Fast atom bombardment;
HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic
acid;
HOAc Acetic acid;
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HOBT 1-Hydroxybenzotriazole hydrate;
HOOBT 3-Hydroxy-1,2,2-benzotriazin-4(3I~-one;
HPLC High-performance liquid chromatography;
KOtBu Potassium tart-butoxide;
LAH Lithium aluminum hydride;
LCMS Liquid Chromatography Mass Spectroscopy;
MCPBA m-Chloroperoxybenzoic acid;
Me Methyl;
MeOH Methanol;
1Q Ms Methanesulfonyl;
MsCI Methanesulfonyl chloride;
n-Bu n-butyl;
n-Bu3P Tri-n-butylphosphine;
NaHMDS Sodium bis(trimethylsilyl)amide;
NBS N-Bromosuccinimide;
Pd(PPh3)q. Palladium tetrakis(triphenylphosphine);
Pd2(dba) 2 Tris(dibenzylideneacetone)dipalladium
(0)
Ph phenyl;
PMSF a-Toluenesulfonyl chloride;
Py or pyr Pyridine; .
PYBOP Benzotriazol-1-yloxytripyrrolidinophosphonium
(or PyBOP) hexafluorophosphate;
t-Bu tart-Butyl;
TBAF Tetrabutylammonium fluoride;
RPLC Reverse Phase Liquid Chromatography;
RT Room Temperature;
TBSCI tart-Butyldimethylsilyl chloride;
TFA Trifluoroacetic acid;
THF Tetrahydrofuran;
TIPS Triisopropylsilyl;
TMS Tetramethylsilane; and
Tr Trityl.
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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 alkylation reactions described in the Schemes.
Sepsis of Schemes
Scheme 1 illustrates the synthesis of the key amine intermediate 5
in optically enriched form. The 6-hydroxy-1-indanone 1 is protected as the
silyl ether
2, which is reduced to the (S)-alcohol 3 using (R)-methyl oxazaborolidine as
shown.
Treatment of 3 with DPPA and DBU in toluene provides the azide 4 with clean
inversion of stereochemistry, and this is reduced using LAH to give the (S)-
amine 5.
In Scheme 2, the synthesis of (R)-2-allyl-N (tart-butoxycarbonyl)
proline 9 (described in Khalil, Subasinghe & Johnson, Tetrahedrof2 Lett., 37,
3441-
3444 (1996)) is summarized. This acid can be coupled to amine 5 to afford the
amide
10 under standard PyBOP conditions.
Scheme 2A illustrates how a pipecolinate analogue (9A) of the proline
derivative 9 may be synthesized. This intermediate 9A could be used in analogy
to 9
in the other Schemes to produce alternative spiro products. The Cbz protecting
group
in 9A may be removed as necessary under standard conditions that are known to
one
skilled in the art.
The imidazolecarboxaldehyde intermediate 14 can be synthesized as
shown in Scheme 3. Palladium-catalyzed cyanation, followed by bromination, of
toluene 11 affords the benzyl bromide 12, which can be used to alkylate the
trityl-
protected imidazole as shown. The synthesis of such protected imidazoles has
been
previously described in Williams et al., J. Med. Chern., 42, 3779-3784 (1999).
The
initial imidazolium ion formed in the alkylation is treated with methanol to
give the
product 13. Removal of the acetyl protecting group, followed by oxidation with
pyridine-sulfur trioxide complex, provided the desired compound 14.
Amide 10 can be converted to the alcohol 15 by the oxidative
cleavage-reduction sequence shown in Scheme 4. Mitsunobu cyclodehydration
conditions may be used to convert this alcohol to the spiropyrrolidine 16, and
the
BOC group is removed by treatment with TFA. The resulting amine 17 is
reductively
alkylated with aldehyde 14 under standard NaCNBH3 conditions to provide
compound 18, and this may be converted to the macrocycle 19 via one pot ether
cleavage and cyclization, mediated by TBAF.
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In Scheme 5, a simple deprotection-reprotection sequence is used to
convert the anisole 20 to silyl ether 22. This compound is then subjected to
borane
reduction, conversion of the resulting alcohol to the azide 24, and finally
reduction of
this azide to the amine 25. Coupling of this amine with the acid 9 yields the
proline
derivative 26. Subjection of amide 26 to an analogous sequence to that used
for
amide 10 in Scheme 4 leads to the macrocyclic product 31, as shown.
The indanone 1 (Scheme 1) may be replaced with similar compounds,
such as a-tetralones or 4-chromanones, to afford analogous macrocyclic
products via
the same route used for 1. A representative synthesis is shown in Scheme 6,
using
compound 32 as either an a-tetralone (X = CHZ) or 4-chromanone (X = O)
starting
material.
In Scheme 7, analogous chemistry is performed using the amino-
naphthalene 44 as a substitute for the indanyl amine 5. Amine 44 is realized
via
protection of 1-amino-7-hydroxynaphthalene 41 to give 43 followed by selective
removal of the BOC protecting group with TFA, as shown. The standard protocols
already describe lead to the spiro intermediate 48.
Alkylation of trityl-protected imidazole derivatives with benzyl
bromides like A, to yield 1,5-disubstituted imidazoles B (as detailed in
Scheme 8) has
been previously described by Anthony et al., J. Med. Chem., 42, 3356-3368
(1999).
Ester B may be saponified using lithium hydroxide to afford the desired
carboxylate
50, and this can be coupled with amines such as 49, as shown in Scheme 9.
Removal
of the silyl ether followed by macrocyclization affords the desired final
product S3.
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S CREME 1
H Ph
O TBDPSCI O Ph
s~W imidazole
DMF
/
/ H3g- Me
CH CI
OH 2 OTBDPS
H
DPPA, DBU LAH
toluene THF
DPS ~R DPS
3 4
~~ H2
BDPS
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SCHEME 2
f BuCHO H
~,, H penoane '~ O
40 C N
N C02H H O
H
(S)-Proline
6
/ /
1 ) LDA, TH F
2) allyl bromide N ~~'' O
-78°C to -40°C 6 M HCI (aq) N C02H
H O H
8
7
NH2
BOC20, Me4NOH ,
H20, CH3CN N C02H y
BOC g (R3)w OTBDPS
O BOC
PyBOP N
DIEA H
CH2C12
DPS 10
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SCHElV~ 2A
0 1 ) LDA, TH F O
N ~ 2) allyl bromide N
O
O O O~O 0
\ \
/
N OH
TFA ~ I
O OO
9A /
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S CHEME 3
i) Tr~N~N
CH3 i) Zn(CN)2 Br (Rs)a
Pd(PPh3)4 OAc
DMF
F ii) NBS, AIBN ~ /~ (R4)r CH3CN
(R4)r Br CC14 F ii) MeOH
11 CN 12
~N (R5)a
_N (R5)a
(i) LiOH N
TH F, H20
OAc (ii) Py~S03 (R4)r i ~ CHO
R r Et3N /
( ) ~ ~ F DMSO F
CN 14
CN 13
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SCHEME 4
O NOC 1 ) Os04, Na104 O NOC
NH '~ MeOH, H20 NH
2) NaBH4, EtOAc
.~ s ~ ~ OH
~R3~ 10 ~R )w 15
OTBDPS OTBDPS
BOC
O N
Bu3P, DBAD N TFA
THF ~ CH2C12
16
~Rs)~
OTBDPS
O
O HN H N N~ s
N 14 ~R )a
NaCNBH3 ~~N
MeOH 1$ N
17
3DPS /(R4)r
OTBDPS
~R3)w
O F CN
H
N N
TBAF
THF N
NI
\ ~Rs)a
- J
19 NC (R4)
r
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SCHEME 5
(R3)w (R3)w TBDPSCI
C02H BBr3 ~ \ C02H imidazole
CH2CI2 f / DMF
20 OMe OH 21
(R3)w (R3)w
\ C02H BH3 ~\ OH
THF I / DPPA, DBU
toluene
22 OTBDPS 23 OTBDPS
(R3)w
N3 THF ~ \ NH2
(R )w ' / ~ /
24 OTBDPS 25 OTBDPS
(R3) O BOC
N~
PyBOP \
DIEA I NH
CH2C12
OTBDPS ~ 2~
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SCHEME 5 (CONTINUED)
BOC
~R3)w O NOC 1 ) Os04, Na104 ~R3)w O I
N'
NH .~' MeOH, H20 ~I~ NH ,,
2 NaBH4, EtO/~c
OTBDPS ~ 26 OTBDPS OH 27
BOC
(Rs) O N
Bu3P, DBAD \\ TFA
THF I N~ CH2C12
28
OTBDPS
lR3)w O
N N~ ~R5)q
Rs) O 14 /
C w HN NaCNBH3 ~~N
N MeOH OTBDPS N
OTBDPS 29 R4 %
)r
F CN
O
TBAF N'~ N~
THF ~R3)w~
-~ y y N-
O / \~ ~R4)r
NC 31
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SCHEME 6
(R3)w (R3)w H PhPh
TBDPSCI ~- O
imidazole X O
X O DMF i ~B
32 ~ ~ 33 H3B- Me
OTBDPS CH2CI2
OH
X ~"'OH DPPA, DBU X N3
LAH
(R3)w toluene THF
3
(R )w
34 OTBDPS 35 OTBDPS
O BOC
9
X NH2 PyBOP X NH ~,N/
(R3)w _ DI EA _
CH2C12
(Ra)
36 OTBDPS OTBDPS 37
(Rs) O BOC
1 ) Os04, Na104 ~I w ,,N
MeOH, H20 X NH Bu3P, DBAD
2) NaBH4, EtOAc THF
OH 38
OTBDPS
O BOC O
N \ HN
X N
_ TFA
CH2C12 40
(R3)~ ~ 39 (R
OTBDPS PS
X represents O or CH2
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SCI3EME 7
NH2 / ~ NHBOC
(R3),nr~ BOC20 (R3)w~
41 OH 42 OH
NHBOC
TBDPSCI
imidazole (R3)w~ TFA
DMF ~ ~ CH2C12
43 OTBDPS
O BOC
9
NH2 PyBOP / \ NH ',N,
DIEA
(R )w~ CH2C12
3
44 (R )w 45
OTBDPS OTBDPS
(R3) O BOC
w
1 ) Os04, Na104 ~ ~ ~ NH ~~N
MeOH, H20 Bu3P, DBAD
2, NaBH4, EtOAc_ THF
OH 46
OTBDPS
O BOC
O HN
N ~ ~ N
U TFA
47 CH2C12 v
48
(Rs)~ (Rs)/
OTBDPS OTBDPS
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SCHEME 8
s
R
Br y) Tr~N~~N N ~ Ra
R2 R2
C02Me ~\ C02Me
CH3CN
_F ~F
CN (ii) MeOH CN
A g
~N
N
R2 Ra
LiOH \
THF, H20 I C02Li
F
CN
5
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SCHEME 9
O HN (Rs)a
~N
I O
PYBOP N
DIEA Li0 F
( 49 DMF I \
R
DPS 5~ ~ ~ CN
(R4)r
H O
N N NX(R5)a 4 TBAF
_ (R )r THF
O N
CN
(R3)~
OTBDPS 51
F
(R3)W H O
\ N N, ~ R5)a Cs2CO3
J (R4) DMF
O N -/ r
52 ~ ~ CN
OH F
H O
N N~
~~RS)a
~ O N
iN
(Ra)f -
53
NC (R )r
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In a preferred embodiment of the instant invention the compounds of
the invention are selective inhibitors of farnesyl-protein transferase. A
compound is
considered a selective inhibitor of farnesyl-protein transferase, for example,
when
its ifz vitro farnesyl-protein transferase inhibitory activity, as assessed by
the assay
described in Example 4, is at least 100 times greater than the in vitro
activity of
the same compound against geranylgeranyl-protein transferase-type I in the
assay
described in Example 5. Preferably, a selective compound exhibits at least
1000
times greater activity against one of the enzymatic activities when comparing
geranylgeranyl-protein transferase-type I inhibition and farnesyl-protein
transferase
inhibition.
It is also preferred that the selective inhibitor of farnesyl-protein
transferase is further characterized by:
a) an IC50 (a measure of in vitro inhibitory activity) for inhibition of the
prenylation of newly synthesized K-Ras protein more than about 100-fold
higher than the EC50 for the inhibition of the farnesylation of hDJ protein.
When measuring such ICSOs and ECSps the assays described in Example 9 may be
utilized.
It is also preferred that the selective inhibitor of farnesyl-protein
transferase is further characterized by:
b) an IC50 (a measurement of irz vitro inhibitory activity) for inhibition of
K4B-
Ras dependent activation of MAP kinases in cells at least 100-fold greater
than the EC50 for inhibition of the farnesylation of the protein hDJ in cells.
It is also preferred that the selective inhibitor of farnesyl-protein
transferase is further characterized by:
c) an IC50 (a measurement of ira vitro inhibitory activity) against H-Ras
dependent activation of MAP lcinases in cells at least 1000 fold lower than
the
inhibitory activity (IC50) against H-ras-CULL (SEQ.ID.NO.: 1) dependent
activation of MAP kinases in cells.
When measuring Ras dependent activation of MAP kinases in cells the assays
described in Example 8 may be utilized.
In another preferred embodiment of the instant invention the
compounds of the invention are dual inhibitors of farnesyl-protein transferase
and
geranylgeranyl-protein transferase type I. Such a dual inhibitor may be termed
a
Class II prenyl-protein transferase inhibitor and will exhibit certain
characteristics
when assessed in in vitro assays, which are dependent on the type of assay
employed.
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In a SEAP assay, such as described in Example 8, it is preferred that
the dual inhibitor compound has an ih vitro inhibitory activity (IC50) that is
less than
about l2p.M against K4B-Ras dependent activation of MAP kinases in cells.
The Class II prenyl-protein transferase inhibitor may also be
characterized by:
a) an IC50 (a measurement of iy2 vitro inhibitory activity) for inhibiting K4B-
Ras
dependent activation of MAP lcinases in cells between 0.1 and 100 times the
IC50 for inhibiting the farnesylation of the protein hDJ in cells; and
b) an IC50 (a measurement of ifa vitro inhibitory activity) for inhibiting K4B-
Ras
dependent activation of MAP kinases in cells greater than 5-fold lower than
the inhibitory activity (IC50) against expression of the SEAP protein in cells
transfected with the pCMV-SEAP plasmid that constitutively expresses the
SEAP protein.
The Class II prenyl-protein transferase inhibitor may also be
characterized by:
a) an IC50 (a measurement of iyz vitro inhibitory activity) against H-Ras
dependent activation of MAP kinases in cells greater than 2 fold lower but
less than 20,000 fold lower than the inhibitory activity (IC50) against H-ras-
CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and
b) an IC50 (a measurement of iy2 vitro inhibitory activity) against H-ras-CVLL
dependent activation of MAP kinases in cells greater than 5-fold lower than
the inhibitory activity (IC50) against expression of the SEAP protein in cells
transfected with the pCMV-SEAP plasmid that constitutively expresses the
SEAP protein.
The Class II prenyl-protein transferase inhibitor may also be
characterized by:
a) an ICSp (a measurement of ifa vitro inhibitory activity) against H-Ras
dependent activation of MAP kinases in cells greater than 10-fold lower but
less than 2,500 fold lower than the inhibitory activity (IC50) against H-ras-
CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and
b) an IC50 (a measurement of in vitro inhibitory activity) against H-ras-CVLL
dependent activation of MAP kinases in cells greater than 5 fold lower than
the inhibitory activity (IC50) against expression of the SEAP protein in cells
transfected with the pCMV-SEAP plasmid that constitutively expresses the
SEAP protein.
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A method for measuring the activity of the inhibitors of prenyl-protein
transferase, as well as the instant combination compositions, utilized in the
instant
methods against Ras dependent activation of MAP kinases in cells is described
in
Example ~.
In yet another embodiment, a compound of the instant invention may
be a more potent inhibitor of geranylgeranyl-protein transferase-type I than
it is an
inhibitor of farnesyl-protein transferase.
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 farnesyl-protein
transferase and the farnesylation 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, 55:4575-450 (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 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, the composition
is
useful in the treatment of neurofibromatosis, which 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).
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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 the instant invention may also be useful in the
prevention and treatment of endometriosis, uterine fibroids, dysfunctional
uterine
bleeding and endometrial hyperplasia.
In such methods of prevention and treatment as described herein,
the prenyl-protein transferase inhibitors 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
prenyl-protein transferase inhibitor may be useful in further combination with
drugs
known to supress the activity of the ovaries and slow the growth of the
endometrial
tissue. Such drugs include but are not limited to oral contraceptives,
progestins,
danazol and GnRH (gonadotropin-releasing hormone) agonists.
Administration of the prenyl-protein transferase inhibitor may also
be combined with surgical treatment of endometriosis (such as surgical removal
of
misplaced endometrial tissue) where appropriate.
The instant compounds may also be useful as inhibitors of corneal
inflammation. These compounds may improve the treatment of corneal opacity
which results from cauterization-induced corneal inflammation. The instant
compounds may also be useful in reducing corneal edema and neovascularization.
(I~. Sonoda et al., Invest. OphthalnZOl. Vis. Sci., 1998, vol. 39, p 2245-
2251).
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.
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Additionally, the compounds of the instant invention may be
administered to a mammal in need thereof using a gel extrusion mechanism (GEM)
device, such as that described in U.S. Serial No. 60/144,643, filed on July
20, 1999,
which is hereby incorporated by reference.
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.
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 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 maslc 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 hydroxypropyl-methylcellulose or hydroxypropyl-
cellulose, or 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 lcaolin, 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.
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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, 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
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from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and
condensation products of the said partial esters with ethylene oxide, for
example
polyoxyethylene sorbitan monooleate. The emulsions may also contain
sweetening,
flavoring 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 microemulsions 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 CADD-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 mono- or diglycerides.
In addition, fatty acids such as oleic acid find use in the preparation of
injectables.
Compounds of Formula I 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-irntating excipient which is
solid at
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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 I are employed. (For purposes of this
application, topical application shall include mouthwashes 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. Compounds of the present invention
may also be delivered as a suppository employing bases such as cocoa butter,
glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene
glycols
of various molecular weights and fatty acid esters of polyethylene glycol.
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 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 prenyl-protein transferase inhibitors and an
antineoplastic
agent. It is also understood that such a combination of antineoplastic agent
and
inhibitor of prenyl-protein transferase may be used in conjunction with other
methods
of treating cancer and/or tumors, including radiation therapy and surgery. It
is further
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understood that any of the therapeutic agents described herein may also be
used in
combination with a compound of the instant invention and an antineoplastic
agent.
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, for
example,
nitrogen mustards, ethyleneimine compounds, alkyl sulfonates and other
compounds
with an alkylating action such as nitrosoureas, cisplatin, and dacarbazine;
anti-metabolites, for example, folic acid, purine or pyrimidine antagonists;
epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;
procarbazine;
mitoxantrone; platinum coordination complexes; biological response modifiers
and
growth inhibitors; mitotic inhibitors, for example, vinca alkaloids and
derivatives of
podophyllotoxin; cytotoxic antibiotics; hormonal/anti-hormonal therapeutic
agents,
haematopoietic growth factors and antibodies (such as trastuzumab, also known
as
HerceptinTM).
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, 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, dactinomycin, mechlorethamine
(nitrogen
mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU),
procarbazine, mitomycin, cytarabine, etoposide, methotrexate, bleomycin,
chlorambucil, camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide,
leuprolide, pyridobenzoindole derivatives, interferons and interleukins.
Particular
examples of antineoplastic, or chemotherapeutic, agents are described, for
example,
by D. J. Stewart in "Nausea and Vomiting: Recent Research and Clinical
Advances",
Eds. J. Kucharczyk, et al., CRC Press Inc., Boca Raton, Florida, USA (1991),
pages
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177-203, especially page 188. See also, R. J. Gralla, et al., Cancer Treatment
Reports, 68(1), 163-172 (1984).
The preferred class of antineoplastic agents is the taxanes and the
preferred antineoplastic agent is paclitaxel.
The compounds of the instant invention may also be co-administered
with antisense oligonucleotides which are specifically hybridizable with RNA
or
DNA deriving from human ras gene. Such antisense oligonucleotides are
described
in U.S. Patent No. 5,576,208 and PCT Publication No. WO 99/22772. The instant
compounds are particularly useful when co-administered with the antisense
1p oligonucleotide comprising the amino acid sequence of SEQ.ID.NO: 2 of U.S.
Patent
No. 5,576,208.
Certain compounds of the instant invention may exhibit very low
plasma concentrations and significant inter-individual variation in the plasma
levels
of the compound. It is believed that very low plasma concentrations and high
inter
subject variability achieved following administration of certain prenyl-
protein
transferase inhibitors to mammals may be due to extensive metabolism by cyto-
chrome P450 enzymes prior to entry of drug into the systemic circulation.
Prenyl-
protein transferase inhibitors may be metabolized by cytochrome P450 enzyme
systems, such as CYP3A4, CYP2D6, CYP2C9, CYP2C19 or other cytochrome P450
isoform. If a compound of the instant invention demonstrates an affinity for
one or
more of the cytochrome P450 enzyme systems, another compound with a higher
affinity for the P450 enzymes) involved in metabolism should be administered
concomitantly. Examples of compounds that have a comparatively very high
affinity
for CYP3A4, CYP2D6, CYP2C9, CYP2C19 or other P450 isoform include, but
are not limited to, piperonyl butoxide, troleandomycin, erythromycin,
proadifen,
isoniazid, allylisopropylacetamide, ethinylestradiol, chloramphenicol, 2-
ethynyl-
naphthalene and the like. Such a high affinity compound, when employed in
combination with a compound of formula I, may reduce the inter-individual
variation
and increase the plasma concentration of a compound of formula I to a level
having
substantial therapeutic activity by inhibiting the metabolism of the compound
of
formula I. Additionally, inhibiting the metabolism of a compound of the
instant
invention prolongs the pharmacokinetic half life, and thus the pharmacodynamic
effect, of the compound.
A compound of the present invention may be employed in
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conjunction with antiemetic agents to treat nausea or emesis, including acute,
delayed,
late-phase, and anticipatory emesis, which may result from the use of a
compound of
the present invention, alone or with radiation therapy. For the prevention or
treatment
of emesis a compound of the present invention may be used in conjunction with
other
anti-emetic agents, especially neurokinin-1 receptor antagonists, 5HT3
receptor
antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron,
GABAB
receptor agonists, such as baclofen, or a corticosteroid such as Decadron
(dexa-
methasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such
as
disclosed in U.S.Patent Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375,
3,929,768,
3,996,359, 3,928,326 and 3,749,712. For the treatment or prevention of emesis,
conjunctive therapy with a neurokinin-1 receptor antagonist, a 5HT3 receptor
antagonist and a corticosteroid is preferred.
Neurokinin-1 receptor antagonists of use in conjunction with the
compounds of the present invention are fully described, for example, in U.S.
Patent
Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270,
5,494,926,
5,496,833, 5,637,699, 5,719,147; European Patent Publication Nos. EP 0 360
390,
0 394 989, 0 428 434, 0 429 366, 0 430 771, 0 436 334, 0 443 132, 0 482 539,
0 498 069, 0 499 313, 0 512 901, 0 512 902, 0 514 273, 0 514 274, 0 514 275,
0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528 495, 0 532 456,
0 533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152,
0 599 538, 0 610 793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655,
0 699 674, 0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959,
0 733 632 and 0 776 893; PCT International Patent Publication Nos. WO
90/05525,
90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585,
92/17449,
92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01159,
93/01165,
93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084, 93/14113,
93/18023,
93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402,
94/02461,
94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843,
94/08997,
94/10165, 94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663,
94/14767,
94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,
95/02595,
95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880,
95/14017,
95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575,
95/21819,
95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744,
96/05181,
96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643,
96/20197,
96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385,
96/37489,
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97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206,
97/19084,
97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268
931,
2 269 170, 2 269 590, 2 271774, 2 292 144, 2 293 168, 2 293 169, and 2 302
689.
The preparation of such compounds is fully described in the aforementioned
patents
and publications.
A particularly preferred neurokinin-1 receptor antagonist for use in
conjunction with the compounds of the present invention is 2-(R)-(1-(R)-(3,5-
bis
(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-
triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof,
which is
described in U.S. Patent No. 5,719,147.
For the treatment of cancer, it may be desirable to employ a
compound of the present invention in conjunction with another
pharmacologically
active agent(s). A compound of the present invention and the other
pharmacologic-
ally active agents) may be administered to a patient simultaneously,
sequentially
or in combination. For example, the present compound may be employed directly
in
combination with the other active agent(s), or it may be administered prior,
con-
current or subsequent to the administration of the other active agent(s). In
general,
the currently available dosage forms of the known therapeutic agents for use
in such
combinations will be suitable.
For example, a compound of the present invention may be presented
together with another therapeutic agent in a combined preparation, such as
with an
antiemetic agent for simultaneous, separate, or sequential use in the relief
of emesis
associated with employing a compound of the present invention and radiation
therapy.
Such combined preparations may be, for example, in the form of a twin pack. A
preferred combination comprises a compound of the present invention with
antiemetic
agents, as described above.
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 prenyl-protein
transferase alone to treat cancer.
Additionally, compounds of the instant invention may also be useful as
radiation sensitizers, as described in WO 97/38697, 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
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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.
In particular, if the compound of the instant invention is a selective
inhibitor of farnesyl-protein transferase, co-administration with a compounds)
that is
a selective inhibitor of geranylgeranyl protein transferase may provide an
improved
therapeutic effect.
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. Serial 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 oral or parenteral, including intravenous,
intramuscular,
intraperitoneal, subcutaneous, rectal and topical routes of administration. It
is
preferred that such administration be oral. It is more preferred that such
administra-
tion be oral and simultaneous. 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.
Serial No.
09/055,487, filed April 6, 1998, and WO 98/44797, published on October 15,
1998,
which are incorporated herein by reference.
As used herein the term "integrin antagonist" refers to a compound
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 cells. In particular, the term refers to compounds
which
selectively antagonize, inhibit or counteract binding of a physiological
ligand to
the av[33 integrin, which selectively antagonize, inhibit or counteract
binding of a
physiological ligand to the av(35 integrin, which antagonize, inhibit or
counteract
binding of a physiological ligand to both the ccv(33 integrin and the ccv(35
integrin,
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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
x1(31, x2(31, x5(31, cc6(31 and x6(34 integrins. The term also refers to
antagonists
of any combination of ocv(33 integrin, av(35 integrin, a1~31, x2(31, x5(31,
oc6(31 and
x6(34 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.
The instant compounds may also be useful in combination with an
inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) for
the treatment of cancer. Compounds which have inhibitory activity for HMG-CoA
reductase can be readily identified by using assays well-known in the art. For
example, see the assays described or cited in U.S. Patent No. 4,231,938 at
col. 6,
and WO 84/02131 at pages 30-33. The terms "HMG-CoA reductase inhibitor" and
"inhibitor of HMG-CoA reductase" have the same meaning when used herein.
Examples of HMG-CoA reductase inhibitors that may be used include
but are not limited to lovastatin (MEVACOR~; see US Patent No. 4,231,938,
4,294,926 and 4,319,039), simvastatin (ZOCOR~; see US Patent No. 4,444,784,
4,820,850 and 4,916,239), pravastatin (PRAVACHOL~; see US Patent Nos.
4,346,227, 4,537,859, 4,410,629; 5,030,447 and 5,180,589), fluvastatin
(LESCOL~;
see US Patent Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853,
5,290,946
and 5,356,896), atorvastatin (LIPITOR~; see US Patent Nos. 5,273,995,
4,681,893,
5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin and
BAYCHOL~; see US Patent No. 5,177,080). The structural formulas of these and
additional HMG-CoA reductase inhibitors that may be used in the instant
methods
are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs",
Cheynistry &
Ihdust~y, pp. 85-89 (5 February 1996) and US Patent Nos. 4,782,084 and
4,885,314.
The term HMG-CoA reductase inhibitor as used herein includes all
pharmaceutically
acceptable lactone and open-acid forms (i.e., where the lactone ring is opened
to form
the free acid) as well as salt and ester forms of compounds which have HMG-CoA
reductase inhibitory activity, and therefor the use of such salts, esters,
open-acid and
lactone forms is included within the scope of this invention. An illustration
of the
lactone portion and its corresponding open-acid form is shown below as
structures I
and II.
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HO O HO COOH
O OH
Lactone Open-Acid
I II
In HMG-CoA reductase inhibitors where an open-acid form can exist,
salt and ester forms may preferably be formed from the open-acid, and all such
forms
are included within the meaning of the term "HMG-CoA reductase inhibitor" as
used
herein. Preferably, the HMG-CoA reductase inhibitor is selected from
lovastatin and
simvastatin, and most preferably simvastatin. Herein, the term
"pharmaceutically
acceptable salts" with respect to the HMG-CoA reductase inhibitor shall mean
non-
toxic salts of the compounds employed in this invention which are generally
prepared
by reacting the free acid with a suitable organic or inorganic base,
particularly those
formed from cations such as sodium, potassium, aluminum, calcium, lithium,
magnesium, zinc and tetramethylammonium, as well as those salts formed from
amines such as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine,
ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine,
diethanolamine,
procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidine-1'-yl-methyl-
benzimidazole, diethylamine, piperazine, and tris(hydroxymethyl) aminomethane.
Further examples of salt forms of HMG-CoA reductase inhibitors may include,
but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate,
bisulfate,
bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride,
clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate,
fumarate,
gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,
hydrabamine,
hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,
lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate,
mucate,
napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate,
phosphate/
diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate,
tannate,
tartrate, teoclate, tosylate, triethiodide, and valerate.
Ester derivatives of the described HMG-CoA reductase inhibitor
compounds may act as prodrugs which, when absorbed into the bloodstream of a
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warm-blooded animal, may cleave in such a manner as to release the drug form
and
permit the drug to afford improved therapeutic efficacy.
Similarly, the instant compounds may be useful in combination
with agents that are effective 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 above and
the
other pharmaceutically active agents) within its approved dosage range.
Combina-
tions of the instant invention may alternatively be used sequentially with
known
pharmaceutically acceptable agents) when a multiple combination formulation is
inappropriate.
The instant compounds may also be useful in combination with
prodrugs of antineoplastic agents. In particular, the instant compounds may be
co-administered either concurrently or sequentially with a conjugate (termed a
"PSA conjugate") which comprises an oligopeptide, that is selectively cleaved
by
enzymatically active prostate specific antigen (PSA), and an antineoplastic
agent.
Such co-administration will be particularly useful in the treatment of
prostate cancer
or other cancers which are characterized by the presence of enzymatically
active
PSA in the immediate surrounding cancer cells, which is secreted by the cancer
cells.
Compounds which are PSA conjugates and are therefore useful in such
a co-administration, and methods of synthesis thereof, can be found in the
following
patents, pending patent applications and publications which are herein
incorporated
by references:
U.S. Patent No. 5,599,686, granted on February 4, 1997;
WO 96/00503 (January 11, 1996); U.S. Serial No. 08/404,833, filed on March 15,
1995; U.S. Serial No. 08!468,161, filed on June 6, 1995;
U.S. Patent No. 5,866,679, granted on February 2, 1999;
U.S. Patent No. 5,998,362, granted on December 7, 1999;
U.S. Patent No. 5,948,750, granted on September 7, 1999;
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WO 99/02175 (January 21, 1999); U.S. Serial No. 09/112,656, filed on July 9,
1998;
and
WO 99/28345 (June 10, 1999); U.S. Serial No. 09/193,365, filed on November 17,
1998.
Compounds which are described as prodrugs wherein the active
therapeutic agent is released by the action of enzymatically active PSA and
therefore
may be useful in such a co-administration, and methods of synthesis thereof,
can be
found in the following patents, pending patent applications and publications,
which
are herein incorporated by reference: WO 98/52966 (November 26, 1998).
All patents, publications and pending patent applications identified are
herein incorporated by reference.
The compounds of the instant invention are also useful as a component
in an assay to rapidly determine the presence and quantity of farnesyl-protein
trans-
ferase (FPTase) in a composition. Thus the composition to be tested may be
divided
and the two portions contacted with mixtures which comprise a known substrate
of FPTase (for example a tetrapeptide having a cysteine at the amine terminus)
and farnesyl pyrophosphate and, in one of the mixtures, a compound of the
instant
invention. After the assay mixtures are incubated for a sufficient period of
time,
well known in the art, to allow the FPTase to farnesylate the substrate, the
chemical
content of the assay mixtures may be determined by well known immuno-logical,
radiochemical or chromatographic techniques. Because the compounds of the
instant
invention are selective inhibitors of FPTase, absence or quantitative
reduction of
the amount of substrate in the assay mixture without the compound of the
instant
invention relative to the presence of the unchanged substrate in the assay
containing
the instant compound is indicative of the presence of FPTase in the
composition to
be tested.
It would be readily apparent to one of ordinary skill in the art that such
an assay as described above would be useful in identifying tissue samples
which
contain farnesyl-protein transferase and quantitating the enzyme. Thus, potent
inhibitor compounds of the instant invention may be used in an active site
titration
assay to determine the quantity of enzyme in the sample. A series of samples
composed of aliquots of a tissue extract containing an unknown amount of
farnesyl-
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protein transferase, an excess amount of a known substrate of FPTase (for
example a
tetrapeptide having a cysteine at the amine terminus) and farnesyl
pyrophosphate are
incubated for an appropriate period of time in the presence of varying
concentrations
of a compound of the instant invention. The concentration of a sufficiently
potent
inhibitor (i.e., one that has a Ki substantially smaller than the
concentration of enzyme
in the assay vessel) required to inhibit the enzymatic activity of the sample
by 50°Io
is approximately equal to half of the concentration of the enzyme in that
particular
sample.
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 are not intended to limit the
reasonable
scope thereof.
EXAMPLE 1
H
N N~
~N
N
Preparation of (17R, 20R)-19,20,21,22-tetrahydro-19-oxo-17H 15,17:1~,20-
diethano-6,10:12,16-dimetheno-20,21-propano-16H-imidazo[3,4-h] [ 1,x,11,14]
oxatriazac~cloeicosine-9-carbonitrile
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Step A: 4-(H~~ethyl)-1-(triphenylmethyl)imidazole
To a solution of 4-(hydroxymethyl)imidazole hydrochloride (35.0 g,
260 mmol) in dry DMF (250 mL) at room temperature was added triethylamine
(90.6
mL, 650 mmol). A white solid precipitated from the solution. Chlorotriphenyl-
methane (76.1 g, 273 mmol) in DMF (500 mL) 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 iya
vacuo to
provide the titled product.
Step B: 4-(Acetox~xl)-1-(triphen l~ethyl)imidazole
4-(Hydroxymethyl)-1-(triphenylmethyl)imidazole, as described above
in Step A, (88.5 g, 260 mmol) was suspended in pyridine (500 mL). Acetic
anhydride
(74 mL, 780 mmol) was added dropwise, and the reaction was stirred for 48
hours
during which it became homogeneous. The solution was poured into EtOAc, and
washed sequentially with water, 5% aqueous HCl solution, saturated aqueous
NaHC03 solution, and brine. The organic extracts were dried (Na2S04), and
concentrated iu vacuo to provide the titled ester.
Step C_ 4-Cyano-3-fluorotoluene
To a deoxygenated solution of 4-bromo-3-fluorotoluene (25.0 g,
132 mmol) in DMF (500 mL) was added Zn(CN)2 (10.1 g, 86 mmol) and Pd(PPh3)4
(15 g, 13 mmol). The reaction was stirred at 100°C for 18 hours, then
cooled to
room temperature. The solution was poured into toluene (1 L), washed with 30%
aqueous NH40H (2 x 1 L), then brine (800 mL), then dried (Na2S04), filtered,
and concentrated in vacuo to provide the crude product. Purification by silica
gel
chromatography, eluting with a gradient of hexane - 0% to 7% EtOAc, yielded
the
titled product.
Step D_ 4-Cyano-3-fluorobenzyl bromide
To a solution of 4-cyano-3-fluorotoluene, as described above
in Step C, (5.0 g, 37.0 mmol) in carbon tetrachloride (300 mL) was added N
bromosuccinimide (7.57 g, 42.6 mmol) and 2,2'-azobisisobutyronitrile (610 mg,
3.7
mmol). The reaction mixture was heated to reflux under argon for 24 hours,
then
cooled to room temperature, filtered, and concentrated under reduced pressure.
The
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residue was purified by silica gel chromatography, eluting with a gradient of
hexane -
4% to 7% EtOAc, to yield the titled product.
Step E: 5-(Acetoxymethyl)-1-(4-cyano-3-fluorobenzyl)imidazole
hydrobromide
A mixture of 4-(acetoxymethyl)-1-(triphenylmethyl)imidazole, as
described above in Step B, (19.7 g, 51.4 mmol) and 4-cyano-3-fluorobenzyl
bromide,
as described above in Step D, (11.0 g, 51.4 mmol) in dry CH3CN (140 mL) was
stirred at 50°C for 3 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 iu vacuo to a volume of 70 mL,
reheated
at 50°C for 2 hours, cooled to room temperature, and filtered again.
The solid
material was combined and dissolved in MeOH (500 mL), and the solution was
heated to reflux for 2 hours. The solution was concentrated irz vacuo to a
volume of
20 mL, then cold hexane - EtOAc (1:1, 500 mL) was added and the precipitate
was
collected and dried in vacuo.
Step F: 1-(4-Cyano-3-fluorobenzyl)-5-(hydroxymethyl)imidazole
To a solution of 5-(acetoxymethyl)-1-(4-cyano-3-fluorobenzyl)
imidazole, as described above in Step E, (19.8 g, 72.5 mmol) in 5:1 THF/water
(430
mL) at ambient temperature was added lithium hydroxide monohydrate (3.33 g,
79.4
mmol). After 4 hours, the solution was adjusted to pH 7 with 1.0 N
hydrochloric acid
and concentrated ira vacuo. The residue was concentrated from toluene iyi
vacuo (3 x
100 mL) to give the titled product.
Step G: 1-(4-Cyano-3-fluorobenzyl)-5-imidazolecarboxaldehyde
To a solution of 1-(4-cyano-3-fluorobenzyl)-5-(hydroxymethyl)
imidazole, as described above in Step F, (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 iu vacuo to provide the
titled
aldehyde.
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St_ ep H: 6-tart-Butyldiphenylsil~rloxy-1-indanone
6-Hydroxy-1-indanone was prepared according to the procedure
described by Nayak & Chakraborti, Tetrahedron Lett., 38, 8749-8752 (1997). A
mixture of 6-hydroxy-1-indanone (5.00 g, 33.8 mmol), tart-butyldiphenylsilyl
chloride (23.1 g, 84.2 mmol), and imidazole (6.90 g, 101.4 mmol) in degassed
DMF
(100 mL) was heated at 60°C for 18 h. The solvent was removed irz vacuo
and the
residue purified by silica gel chromatography, eluting with a gradient of
hexane - 10%
to 15% EtOAc, to yield the titled product.
St_ ep I: (S)-6-tart-But~phen~~y-1-indanol
To a stirred solution of borane-methyl sulfide (0.155 mL, 1.55 mmol)
in dry CHZCl2 (4 mL), under argon, was added (R)-methyl oxazaborolidine (1 M
in
toluene, 1.55 mL, 1.55 mmol). To the resulting solution was added a solution
of 6-
tert-butyldiphenylsilyloxy-1-indanone, as described above in Step H, (500 mg,
1.29
mmol) in CH2C12 (5.5 mL), dropwise. The reaction mixture was stirred at
ambient
temperature for 2 h, then added carefully to MeOH (20 mL). The solvent was
removed by distillation at 1 atm. This MeOH addition-distillation procedure
was
repeated twice more, then the residue was purified by silica gel
chromatography,
eluting with CH2C12, to yield the titled product.
St_ ep J: (R)-6-tent-But~phen lsil~y-1-indanyl azide
To a solution of (S)-6-tent-butyldiphenylsilyloxy-1-indanol, as
described above in Step I, (283 mg, 0.728 mmol) and diphenylphosphoryl azide
(240
mg, 0.874 mmol) in toluene (1.3 mL) at 0°C was added 1,8-
diazabicyclo[5.4.0]undec-
7-ene (0.12 mL, 0.80 mmol), dropwise. The mixture was allowed to slowly warm
to
ambient temperature and was stirred for 18 h then diluted with CHZCh (15 mL).
The
resulting mixture was washed with 0.5 N HCl (5 mL), then H20 (5 mL), then
brine (5
mL) then dried over Na2S04, filtered, and concentrated ih vacuo. The residue
was
purified by silica gel chromatography, eluting with a gradient of hexane - 0%
to 4%
EtOAc, to yield the titled product.
St. ep K: (R)-6-tart-But~phen~sil loxy-1-indanylamine
To a stirred solution of (R)-6-tart-butyldiphenylsilyloxy-1-indanyl
azide, as described above in Step J, (273 mg, 0.66 mmol) in THF (8 mL) at
0°C was
added LiAlH4. (1.0 M in THF, 0.79 mL, 0.79 mmol), dropwise. The reaction
mixture
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was allowed to slowly warm to ambient temperature and was stirred for 18 h.
EtOAc
(0.035 mL) was added, followed by H20 (0.035 mL), then 15% aqueous NaOH
(0.035 mL), then HBO (0.10 mL). The quenched mixture was stirred at ambient
temperature for 30 min, then filtered, and concentrated ih vacuo. The residue
was
purified by silica gel chromatography, eluting with a gradient of CH2C12 - 0%
to 6%
MeOH - 0% to 0.6% NH40H, to yield the titled product.
Step L: (R,R)-2-Allyl-1-(tart-butoxycarbonyl)-N [6-(tert-
but~rldiphen~~loxy)indan-1-~pyrrolidine-2-carboxamide
(R)-2-Allyl-N (tart-butoxycarbonyl)proline was prepared in analogy
with the procedure described by Khalil, Subasinghe & Johnson, Tetrahedron
Lett.,
37, 3441-3444 (1996). To (R)-2-allyl-N (tart-butoxycarbonyl)proline (587 mg,
2.30
mmol) in dry CH2C12 (5 mL), at 0°C, under argon were added PYBOP (1.32
g, 2.53
mmol), (R)-6-tart-butyldiphenylsilyloxy-1-indanylamine, as described above in
Step
K, (980 mg, 2.53 mmol), and N,N diisopropylethylamine (0.44 mL, 2.53 mmol).
The
reaction mixture was stirred for 1 h, then poured into CH2C12 (50 mL) and 10%
aqueous citric acid (20 mL), and the organic layer was extracted, dried over
Na2S04,
filtered, and concentrated ifa vacuo. The crude product was~purified by flash
column
chromatography on silica, eluting with a gradient of CH2C12 - 0% to 5% EtOAc
to
yield the desired product.
Step M: (R,R)-1-(tart-Butoxycarbonyl)-N-[6-(tart-butyldiphenylsilyloxy)indan-
1-, l~(2-h d~yeth~)pxrrolidine-2-carboxamide
To a stirred solution of (R,R)-2-allyl-1-(tent-butoxycarbonyl)-N [6-
(tart-butyldiphenylsilyloxy)indan-1-yl]pyrrolidine-2-carboxamide, as described
above in Step L, (410 mg, 0.656 mmol), in MeOH (20 mL) and H20 (5 mL) at
ambient temperature was added osmium tetroxide (2.5% solution in tart-BuOH,
0.6
mL, 0.048 mol) followed by sodium periodate (421 mg, 1.97 mmol). The resulting
mixture was stirred for 90 min, then poured into H20 and EtOAc and extracted
with
EtOAc (2 x 75 mL). The combined organic extracts were washed with H20, then
brine, then dried over Na2S04, filtered, and concentrated in vacuo. This crude
product was dissolved in EtOAc (20 mL), the solution was cooled to -
78°C and
NaBH4. (0.1 M in i-PrOH, 2.5 mL, 0.26 mmol) was added dropwise. The mixture
was allowed to warm slowly for 2 h, then was poured into brine (10 mL) and the
organic layer was extracted. The aqueous layer was extracted further with
EtOAc
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(10 xnL), then the combined organic extracts were dried over NaZS04, filtered,
and concentrated ih vacuo. The crude product was purified by flash column
chromatography on silica, eluting with a gradient of CH2C12 - 0% to 50% EtOAc
to yield the desired product.
Step N: (R,R)-tart-Butyl 7-[6-(pert-butyldiphenylsilyloxy)indan-1-yl]-6-oxo-
1,7-diazaspirof4.41nonane-1-carboxylate
To a solution of di-tart-butyl azodicarboxylate (40 mg, 0.176 mmol)
in THF (0.5 mL) was added tri-re-butylphosphine (36 mg, 0.176 mmol) and the
mixture was stood for 5 min, then added dropwise to a solution of (R,R)-1-
(tert-
butoxycarbonyl)-N-[6-(tart-butyldiphenylsilyloxy)indan-1-yl]-2-(2-
hydroxyethyl)
pyrrolidine-2-carboxamide, as described above in Step M, (85 mg, 0.135 mmol),
in
THF (1 mL) at 0°C. The reaction mixture was allowed to warm slowly to
ambient
temperature and was stirred for 18 h, then poured into saturated aqueous
NaHC03 and
extracted with CH2C12 (2 x 10 mL). The combined organic extracts were dried
over
Na2S04, filtered, and concentrated in vacuo. The crude product was purified by
flash
column chromatography on silica, eluting with a gradient of CH2Cl2 - 0% to 35%
EtOAc to yield the desired product.
Step O: (R,R)-7-[6-(tent-Butyldiphenylsilyloxy)indan-1-yl]-6-oxo-1,7-
diazaspirof4.41nonane trifluoroacetate
A solution of tart-butyl 7-[6-(tart-butyldiphenylsilyloxy)indan-1-yl]-6-
oxo-1,7-diazaspiro[4.4]nonane-1-carboxylate, as described above in Step N, (62
mg,
0.101 mmol) in CHZCl2 (1 mL) and trifluoroacetic acid (0.5 mL) was stood at
ambient
temperature for 20 min, then concentrated in vacuo to yield the titled salt.
Step P: (R,R)-4-[5-({7-[6-(tart-Butyldiphenylsilyloxy)indan-1-yl]-6-oxo-
1,7-diazaspiro[4.4]non-1-yl }methyl)-1H-imidazol-1-ylmethyl]-2-
fluorobenzonitrile
(R,R)-7-[6-(tart-Butyldiphenylsilyloxy)indan-1-yl]-6-oxo-1,7-
diazaspiro[4.4]nonane trifluoroacetate, as described above in Step O, (62 mg,
0.099
mmol) and 1-(4-cyano-3-fluorobenzyl)-5-imidazolecarboxaldehyde, as described
above in Step G, (23 mg, 0.099 mmol), were stirred in MeOH (1 mL) and N,N
diisopropylethylamine was added dropwise to adjust the mixture to ca. pH 5, as
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judged by wetted pH paper. The mixture was stirred for 1 h at ambient
temperature,
then NaCNBH3 (9 mg, 0.143 mmol) was added, the pH was adjusted to pH 5 with
AcOH, and stirring was continued for 18 h. The reaction was quenched with
saturated aqueous NaHC03 (6 mL) and extracted with CH~C12 (2 x 15 mL). The
combined organic extracts were dried over Na2S04, filtered, and concentrated
in
vacuo. The crude product was purified by flash column chromatography on
silica,
eluting with a gradient of CHCl3 - 0% to 3% MeOH - 0% to 0.3% NHq.OH, to yield
the titled product.
Ste : (17R, 20R)-19,20,21,22-Tetrahydro-19-oxo-17H-15,17:18,20-
diethano-6,10:12,16-dimetheno-20,21-propano-16H-imidazo
f3 4-hlfl,8,11,141oxatriazacycloeicosine-9-carbonitrile
To a stirred solution of (R,R)-4-[5-({7-[6-(tart-butyldiphenylsilyloxy)
indan-1-yl]-6-oxo-1,7-diazaspiro[4.4]non-1-yl}methyl)-1H imidazol-1-ylmethyl]-
2-
fluorobenzonitrile, as described above in Step P, (74 mg, 0.10 mmol) in THF (2
mL)
was added TBAF (1.0 M in THF, 0.11 mL, 0.11 mmol), dropwise, and the resulting
mixture was stirred at ambient temperature for 18 h. The reaction was quenched
with
saturated aqueous NaHC03 (10 mL) and extracted with CH2C12 (2 x 20 mL). The
combined organic extracts were dried over Na2S04, filtered, and concentrated
ih
vacuo. The crude product was purified by flash column chromatography on
silica,
eluting with a gradient of CH~,Cl2 - 0% to 5% MeOH - 0% to 0.5% NH4OH, to
yield
the titled product.
1H NMR (CDC13): S 7.62 (1H, d, J= 8.3 Hz), 7.61 (1H, s), 7.28 (1H, d, J= 8.1
Hz),
7.17 (1H, dd, J= 8.3, 2.4 Hz), 7.06 (1H, d, J= 1.0 Hz), 6.99 (1H, s), 6.83
(1H, dd,
J= 8.1, 1.2 Hz), 6.69 (1H, d, J= 2.2 Hz), 5.67 (1H, dd, J= 8.8, 4.9 Hz), 5.23
(1H,
d,J=16.4Hz),5.01(lH,d,J=16.1Hz),3.70(lH,d,J=14.7Hz),3.33(lH,d,
J= 14.9 Hz), 3.23 (1H, m), 3.09 (1H, td, J= 8.1, 2.9 Hz), 3.05-2.90 (3H, m),
2.80
(1H, td, J= 9.5. 2.9 Hz), 2.45 (1H, m), 2.20 (1H, dt, J= 12.2, 8.3 Hz), 2.09-
1.78
(6H, m).
ES MS: 466 (MH+)
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EXAMPLE 2
H O
N N~
N~ N
O
NC
Preparation of (R,R)-15,16,17,17a,19,20,22,23-octahydro-19,22-dioxo-5H,21H
18,20-ethano-12,14-etheno-6,10-metheno-20,21-propanobenz[d]imidazo[4,3-L]
f 1 6 9 131oxatriazacXclononadecosine-9-carbonitrile
Step A: MethXl (imidazol-4-yl)acetate hydrochloride
A solution of 4-imidazoleacetic acid hydrochloride (25 mmol) in
MeOH (100 mL) is saturated with HCl (g). Trimethyl orthoformate (100 mmol)
is added and the mixture stands at ambient temperature for 18 hours, then is
concentrated to dryness irz vacuo to afford the titled ester.
Step B: Meths f 1-(triphen 1y methyl~lH-imidazol-4-yllacetate
To a solution of methyl 4-imidazoleacetate hydrochloride, as described
above in Step A, (24.3 mmol) in dry DMF (50 mL) are added triethylamine (53.5
mmol), then triphenylmethyl bromide (26.7 mmol). The mixture is stirred at
ambient
temperature, then partitioned between H20 and EtOAc. The organic layer is
dried
over Na2S04, filtered and concentrated under reduced pressure. The residue is
purified by flash column chromatography on silica to yield the titled product.
Step C: Methxl f 1-(4-cyano-3-fluorobenzyl)-1H-imidazol-5-yllacetate
A mixture of methyl [1-(triphenylmethyl)-1H-imidazol-4-yl]acetate,
as described above in Step B, (1.40 mmol) and 4-cyano-3-fluorobenzyl bromide,
as described in Example 1, Step D, (1.40 mmol) in acetonitrile (3 mL) is
heated to
50°C for 2 h. The mixture is allowed to cool, and the solid is
collected by filtration.
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The acetonitrile filtrate is concentrated ih vacuo to a volume of
approximately 1 mL
and then reheated to 50°C for 2 h, cooled, and the solid is removed by
filtration. The
two crops of precipitated irnidazolium salts are combined in MeOH (30 mL) and
the solution is heated to reflux for 2 h, then concentrated in vacuo. The
residue is
partitioned between saturated aqueous NaHC03 and CHC13. The aqueous layer is
extracted further with CHCl3 (2 x). The combined organic extracts are dried
over
Na2SQ4, filtered, and concentrated in vacuo. The crude product is purified by
flash
column chromatography on silica to yield the titled product.
Step D: Lithium f 1 ~4-cyano-3-fluorobenz~)-1H-imidazol-5-yllacetate
Methyl [1-(4-cyanobenzyl)-1H-imidazol-5-yl]acetate, as described
above in Step C, (free base) (0.95 mmol) is dissolved in THF (5 mL) and H2O (1
mL).
Lithium hydroxide (0.95 mmol) is added and the resulting mixture is stirred at
ambient temperature until the reaction is complete, as judged by HPLC, then is
adjusted to pH 7 with 1.0 N aqueous HCl and is concentrated to dryness ifa
vacuo
to give the titled lithium salt.
Step E: (R,R)-7-[7-(tart-Butyldiphenylsilyloxy)-1,2,3,4-tetrahydronaphthalen-
1-,~1-6-oxo-1 7-diazaspirof4.41nonane trifluoroacetate
Following the procedures described in Example l, Steps H to O, but
using 7-hydroxy-3,4-dihydronaphthalen-1(2H)-one, in place of 6-hydroxy-1-
indanone
in Step H, the above-titled compound is obtained.
Std F: (R,R)-4-[5-(2-{7-[7-(tent-Butyldiphenylsilyloxy)-1,2,3,4-
tetrahydronaphthalen-1-yl]-6-oxo-1,7-diazaspiro[4.4]non-1-yl }-
2-oxoeth~)-1H-imidazol-1-,~meth~l-2-fluorobenzonitrile
To lithium [1-(4-cyano-3-fluorobenzyl)-1H imidazol-5-yl]acetate, as
described above in Step D, (2.0 mmol) in dry DMF (5 mL) are added PYBOP (2.2
mmol), (R,R)-7-[7-(tart-butyldiphenylsilyloxy)-1,2,3,4-tetrahydronaphthalen-1-
yl]-
6-oxo-1,7-diazaspiro[4.4]nonane trifluoroacetate, as described above in Step
E, (2.2
mmol), and N,N diisopropylethylamine (2.2 mmol). The reaction mixture is
stirred
until the reaction is complete as judged by HPLC, then poured into EtOAc and
10%
aqueous citric acid, and the organic layer is extracted, dried over Na2S04,
filtered,
and concentrated in vacuo. The crude product is purified by flash column
chromatography on silica to yield the desired product.
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Step G: (R,R)-2-Fluoro-4-(5-{2-[7-(7-hydroxy-1,2,3,4-tetrahydronaphthalen-
1-yl)-6-oxo-1,7-diazaspiro[4.4]non-1-yl]-2-oxoethyl }-1H-imidazol-1-
l~yl)benzonitrile
To a stirred solution of (R,R)-4-[5-(2-{7-[7-(tert-butyldiphenylsilyl-
oxy)-1,2,3,4-tetrahydronaphthalen-1-yl]-6-oxo-1,7-diazaspiro[4.4]non-1-yl }-2-
oxoethyl)-1H-imidazol-1-ylmethyl]-2-fluorobenzonitrile, as described above in
Step
F, (1.0 mmol) in THF (10 mL) is added TBAF (1.0 M in THF, 1.1 mmol), dropwise,
and the resulting mixture is stirred at ambient temperature until the reaction
is
complete as judged by HPLC. The reaction is quenched with saturated aqueous
NaHC03 and extracted with CH2C12 (2 x). The combined organic extracts are
dried
over Na2S04, filtered, and concentrated iya vacuo. The crude product is
purified by
flash column chromatography on silica to yield the titled product.
Step H: (R,R)-15,16,17,17a,19,20,22,23-Octahydro-19,22-dioxo-5H,21H
18,20-ethano-12,14-etheno-6,10-metheno-20,21-propanobenz [d]
imidazof4,3-ll f 1,6,9,131oxatriazacyclononadecosine-9-carbonitrile
A mixture of (R,R)-2-fluoro-4-(5-{2-[7-(7-hydroxy-1,2,3,4-
tetrahydronaphthalen-1-yl)-6-oxo-1,7-diazaspiro[4.4]non-1-yl]-2-oxoethyl }-1H-
imidazol-1-ylmethyl)benzonitrile, as described above in Step G, (0.31 mrnol)
and
CsZC03 (0.62 mmol) in dry, degassed DMF (40 mL) is stirred at 50°C
under argon
until the reaction is complete as judged by LCMS. The solvent is removed under
reduced pressure, and the residue is partitioned between saturated aqueous
NaHC03
and CHCl3. The aqueous layer is extracted further with CHC13 (2 x). The
combined
organic extracts are dried over Na2S0ø, filtered, and concentrated ira vacuo.
The
crude product is purified by flash column chromatography on silica to yield
the titled
product.
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EXAMPLE 3
O
Br N,~N~
~N
N
O
NC
Preparation of (R)-15-bromo-19,20,21,22-tetrahydro-19-oxo-17H-18,20-
ethano-6,10:12,16-dimetheno-20,21-propano-16H-imidazo [3,4-h] [ 1, 8,11,14]
oxatriazacycloeicosine-9-carbonitrile
Step A: 2-Bromo-5-h~ybenzoic acid
To a solution of 2-bromo-5-methoxybenzoic acid (60 mmol) in CH2C12
(500 mL) at -78°C is added BBr3 (1 M solution in CH2Cl2, 132 mmol)
dropwise. The
reaction mixture is allowed to warm slowly to ambient temperature and stirred
for 18
h. The reaction is quenched with 10°70 aqueous citric acid and
extracted with EtOAc
(3x). The combined organic extracts are dried over NaZS04, filtered, and
concentrated
in vacuo to yield the titled product.
Step B: 2-Bromo-5-(tent-but~phen~ylox~r)benzoic acid
A solution of 2-bromo-5-hydroxybenzoic acid, as described above
in Step A, (50 mmol), tent-butyldiphenylsilyl chloride (55 mmol), and
imidazole
(100 mmol) in dry DMF (50 mL) is heated to 65°C, under argon, until the
reaction is
complete, as judged by HPLC. The reaction is poured into Ha0 and extracted
with
EtOAc (3x). The combined organic extracts are dried over Na2S04, filtered, and
concentrated in vacuo. The crude product is purified by flash column
chromatography on silica to yield the titled product.
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Step C: 2-Bromo-5-(tart-butyldiphenylsilyloxy)benzyl alcohol
To a stirred solution of 2-bromo-5-(tart-butyldiphenylsilyloxy)benzoic
acid, as described above in Step B, (25 mmol) in dry THF (50 mL) at 0°C
is added
borane-THF (1 M solution in THF, 37.5 mmol) dropwise. The mixture is allowed
to
warm to ambient temperature slowly and stirring is continued until the
reaction is
complete, as judged by HPLC. The reaction is quenched carefully with H20 and
extracted with EtOAc (3x). The combined organic extracts are dried over
Na2S04,
filtered, and concentrated iya vacuo. The crude product is purified by flash
column
chromatography on silica to yield the titled product.
Step D: (R)-15-Bromo-19,20,21,22-tetrahydro-19-oxo-17H 18,20-ethano-
6,10:12,16-dimetheno-20,21-propano-16H-imidazo [3,4-la]
[1,8,11,141oxatriazacycloeicosine-9-carbonitrile
Following the procedures described in Example 1, Steps J to Q, but
using 2-bromo-5-(tart-butyldiphenylsilyloxy)benzyl alcohol, in place of (S)-6-
tert-
butyldiphenylsilyloxy-1-indanol in Step J, the above-titled compound is
obtained.
EXAMPLE 4
IfZ vitro inhibition 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 ~L): [3H]farnesyl diphosphate, Ras protein, 50 mM HEPES, pH 7.5,
5 mM MgCl2, 5 mM dithiothreitol, 10 ~,M 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 100% ethanol, mixed with scintillation fluid (10
mL) and
then counted in a Beckman LS3801 scintillation counter.
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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
ICso determinations are as follows: FTase, 650 nM Ras-CVLS (SEQ.ll~.NO.: 1),
100
nM farnesyl diphosphate.
The compounds of the instant invention are tested for inhibitory
activity against human FPTase by the assay described above.
EXAMPLE 5
Modified In 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 p.L): j3H]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 p,M 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. Patent 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 p.L
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. ICso values are determined with
Ras
peptide near KM concentrations. Enzyme and substrate concentrations for
inhibitor
ICso determinations are as follows: 75 pM GGTase-I, 1.6 p,M Ras peptide, 100
nM
geranylgeranyl diphosphate.
The compounds of the instant invention are tested for inhibitory
activity against human GGTase-type I by the assay described above.
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EXAMPLE 6
Cell-based in vitro ras farnesylation 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. et al., Cancer Research 51:712-717,
(1991).
Cells in 10 cm dishes at 50-75% 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 wCi[35S]methionine (1000
Ci/mmol). After an additional 20 hours, the cells are lysed in 1 ml lysis
buffer (1%
NP40120 mM HEPES, pH 7.5/5 mM MgCl2/1mM DTT/10 mg/ml aprotinen/2 mg/ml
leupeptin/2 mg/ml antipain/0.5 mM PMSF) and the lysates 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 (Furth,
M.E.
et al., J. Virol. 43:294-304, (1982)). Following a 2 hour antibody incubation
at 4°C,
200 p,1 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-100Ø5% 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
farnesyl-
ated and nonfarnesylated ras proteins are compared to determine the percent
inhibition of farnesyl transfer to protein.
EXAMPLE 7
Cell-based ira vitro growth inhibition assa
To determine the biological consequences of FPTase inhibition,
the effect of the compounds of the instant invention on the anchorage-
independent
growth of Ratl 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
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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
concentra-
tion of the instant compound. Photomicrographs are taken 16 days after the
cultures
are seeded and comparisons are made.
EXAMPLE 8
Construction of SEAP reporter plasmid pDSE100
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 (Mercle)
and contains 5 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 EcoR1 and HpaI. The ends of the linear DNA
fragments were filled in with the Klenow fragment of E. coli DNA Polymerase I.
The 'blunt ended' DNA 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 DNA fragment was
blunt end ligated into the pCMV-RE-AKI vector and the ligation products were
transformed into DH5-alpha E. colt cells (Gibco-BRL). 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 contains the SEAP coding sequence
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downstream of the DSE and CAT-TATA promoter elements and upstream of the
BGH poly-A sequence.
Alternative Construction of SEAP reporter plasmid, pDSE101
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 pGEM7zf(-)/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 pGEM7zf(-)/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 Polymerise 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 polymerise. Recombinant plasmids were screened for insert orienta-
tion and sequenced through the ligated junctions. The plasmid pCMV-RE-AKI is
derived from plasmid pCMVIE-AKI-DHFR (Whang , Y., Silberklang, M., Morgan,
A., Munshi, 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 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, 745-751) 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.:4)
Antisense strand N-terminal SEAP: 5' GAGAGAGCTCGAGGTTAACCCGGGT
GCGCGGCGTCGGTGGT 3' (SEQ.ID.N0.:5)
Sense strand C-terminal SEAP: 5' GAGAGAGTCTAGAGTTAACCCGTGGTCC
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CCGCGTTGCTTCCT 3' (SEQ.ID.N0.:6)
Antisense strand C-terminal SEAP: 5' GAAGAGGAAGCTTGGTACCGCCACTG
GGCTGTAGGTGGTGGCT 3' (SEQ.ID.N0.:7)
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.: 5)
introduces an internal translation STOP codon within the SEAP gene along with
the
HpaI site. The C-terminal oligos (SEQ.ID.NO.: 6 and SEQ.ID.NO.: 7) were used
to
amplify a 412 by C-ternninal PCR product containing HpaI and HindIII
restriction
sites. The sense strand C-terminal oligo (SEQ.ID.NO.: 6) 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 amplicon was digested with HpaI and
HindIII. The two fragments comprising each end of the SEAP gene were isolated
by electro-phoresing the digest in an agarose gel and isolating the 1560 and
412 base
pair fragments. These two fragments were then co-legated into the vector
pGEM7zf
(-) (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.
Construction of a constitutively expressin Sg EAP plasmid pCMV-SEAP-A
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 , Y., Silberklang, M.,
Morgan, A., Munshi, S., Lenny, A.B., Ellis, R.W., and I~ieff, E. (1987) J.
Virol.,
61:1796-1807) containing the CMV immediate early promoter was cut with EcoRI
generating two fragments. The vector fragment was isolated by agarose electro-
phoresis and relegated. The resulting plasmid is named pCMV-AKI. Next, the
cytomegalovirus intron A nucleotide sequence was inserted downstream of the
CMV IEl promter in pCMV-AI~I. The intron A sequence was isolated from a
genomic clone bank and subcloned into pBR322 to generate plasmid pl6T-286.
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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.: 8)
Antisense strand: 5' GAGAGATCTCAAGGACGGTGACTGCAG 3' (SEQ.ID.NO.:
9)
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 Sacl 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-AKI-
InA.
The DNA sequence encoding the truncated SEAP gene is inserted into
the pCMV-AKI-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 Klenow DNA polymerase 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
Klenow
DNA polymerase. The final construct is generated by blunt end ligating 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-A (deposited in the
ATCC under Budapest Treaty on August 27, 1998, and designated ATCC), 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.
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Alternative construction of a constitutively expressin S~plasmid pCMV-SEAP-B
An expression plasmid constitutively expressing the SEAP protein can
be created by placing the sequence encoding a truncated SEAP gene downstream
of
the cytomegalovirus (CMV) IE-1 promoter and upstream of the 3' unstranslated
region of the bovine growth hormone gene.
The plasmid pCMVIE-AKI-DHFR (Whang, Y., Silberklang, M.,
Morgan, A., Munshi, S., Lenny, A.B., Ellis, R.W., and Kieff, E. (1987) J.
Virol.,
61:1796-1807) containing the CMV immediate early promoter and bovine growth
hormone poly-A sequence can be cut with EcoRI generating two fragments. The
vector fragment can be isolated by agarose electrophoresis and relegated. The
resulting plasmid is named pCMV-AKI. The DNA sequence encoding the truncated
SEAP gene can be inserted into the pCMV-AKI plasmid at a unique Bgl-II in the
vector. The SEAP gene is cut out of plasmid pGEMzf(-)/SEAP (described above)
using EcoRI and HindIII. The fragments are filled in with Klenow DNA
polymerase
and the 1970 base pair fragment is isolated from the vector fragment by
agarose gel
electrophoresis. The pCMV-AKI vector is prepared by digesting with Bgl-II and
filling in the ends with Klenow DNA polymerase. The final construct is
generated
by blunt end legating the SEAP fragment into the vector and transforming the
legation
reaction into E. coli DHSa cells. Transformants can then be screened for the
proper
insert and mapped for restriction fragment orientation. Properly oriented
recombinant
constructs would be sequenced across the cloning junctions to verify the
correct
sequence. The resulting plasmid, named pCMV-SEAP-B contains a modified SEAP
sequence downstream of the cytomegalovirus immediate early promoter, IEl, and
upstream of a bovine growth hormone poly-A sequence. The plasmid would express
SEAP in a constitutive nammer when transfected into mammalian cells.
Cloning of a M~stylated viral-H-ras expression plasmid pSMS600
A DNA fragment containing viral-H-ras can be PCRed from plasmid
"HB-11 (deposited in the ATCC under Budapest Treaty on August 27, 1997, and
designated ATCC 209,218) using the following oligos.
Sense strand:
5'TCTCCTCGAGGCCACCATGGGGAGTAGCAAGAGCAAGCCTAAGGACCC
CAGCCAGCGCCGGATGACAGAATACAAGCTTGTGGTGG 3'. (SEQ.ID.NO.:
10)
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Antisense:
S' CACATCTAGATCAGGACAGCACAGACTTGCAGC 3' .
(SEQ.ID.NO.: 11)
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 XhoI and XbaI. This
results
in a plasmid, pSMS600, in which the recombinant myr-viral-H-ras gene is
constitutively transcribed from the CMV promoter of the pCI vector.
Cloning of a viral-H-ras-CVLL expression plasmid pSMS601
A viral-H-ras clone with a C-terminal sequence encoding the amino
acids CULL can be cloned from the plasmid "HB-11" by PCR using the following
oligos.
Sense strand:
5'TCTCCTCGAGGCCACCATGACAGAATACAAGCTTGTGGTGG-3'
(SEQ.ID.NO.: 12)
Antisense strand:
5'CACTCTAGACTGGTGTCAGAGCAGCACACACTTGCAGC-3' (SEQ.ID.NO.:
13)
The sense strand oligo optimizes the 'Kozak' sequence and adds an
XhoI site. The antisense strand mutates serine 189 to leucine and adds an XbaI
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, pSMS601, in which
the
mutated viral-H-ras-CVLL gene is constitutively transcribed from the CMV
promoter
of the pCI vector.
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Cloning of cellular-H-ras-Leu61 expression plasmid pSMS620
The human cellular-H-ras gene can be PCRed from a human cerebral
cortex cDNA library (Clontech) using the following oligonucleotide primers.
Sense strand:
5'-GAGAGAATTCGCCACCATGACGGAATATAAGCTGGTGG-3'
(SEQ.ID.NO.: 14)
Antisense strand:
5'-GAGAGTCGACGCGTCAGGAGAGCACACACTTGC-3' (SEQ.ID.NO.: 15)
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 site 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.m.NO.: 16)
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 (Promega) which has been
digested
with EcoRI and Sal I. The new recombinant plasmid, pSMS620, will
constitutively
transcribe c-H-ras-Leu61 from the CMV promoter of the pCI vector.
Cloning of a c-N-ras-Val-12 expression plasmid pSMS630
The human c-N-ras gene can be PCRed from a human cerebral cortex
cDNA library (Clontech) using the following oligonucleotide primers.
Sense strand:
5' -GAGAGAATTCGCCACCATGACTGAGTACAAACTGGTGG-3'
(SEQ.ID.N0.:17)
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Antisense strand:
5'-GAGAGTCGACTTGTTACATCACCACACATGGC-3' (SEQ.ID.NO.: 18)
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 site 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.: 19)
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 ligated into the vector pCI (Promega) which has been
digested
with EcoRI and Sal I. The new recombinant plasmid, pSMS630, will
constitutively
transcribe c-N-ras-Val-12 from the CMV promoter of the pCI vector.
Clonin~gLof a c-K4B-ras-Val-12 expression plasmid pSMS640
The human c-K4B-ras gene can be PCRed from a human cerebral
cortex cDNA library (Clontech) using the following oligo-nucleotide primers.
Sense strand:
5' -GAGAGGTACCGCCACCATGACTGAATATAAACTTGTGG-3'
(SEQ.ID.NO.: 20)
Antisense strand:
5'-CTCTGTCGACGTATT'TACATAATTACACACTTTGTC-3' (SEQ.ID.N0.:21)
The primers will amplify a c-K4B-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 site at the C-terminal end. After trimming the
ends of
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the PCR product with Kpn I and Sal I, the c-K4B-ras fragment can be ligated
into a
KpnI -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.N0.:22)
After selection and sequencing for the correct nucleotide substitution,
the mutated c-K4B-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-K4B-ras-Val-12 from the CMV promoter of the pCI vector.
Cloning of c-K-ras4A-Val-12 expression plasmid pSMS650
The human c-K4A-ras gene can be PCRed from a human cerebral
cortex cDNA library (Clontech) using the following oligo-nucleotide primers.
Sense strand:
5'-GAGAGGTACCGCCACCATGACTGAATATAAACTTGTGG-3'
(SEQ.lD.N0.:23)
Antisense strand:
5' -CTCTGTCGACAGATTACATTATAATGCATTTTTTAATTTTCACAC-3'
(SEQ.m.NO.: 24)
The primers will amplify a c-K4A-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-ras4A fragment can be ligated
into a
KpnI -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.N0.:25)
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After selection and sequencing for the correct nucleotide substitution,
the mutated c-K4A-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, pSMS650, will
constitutively transcribe c-K4A-ras-Val-12 from the CMV promoter of the pCI
vector.
SEAP assay
Human C33A cells (human epitheial carcenoma - ATTC collection)
are seeded in lOcm tissue culture plates in DMEM + 10% fetal calf serum + 1X
Pen/Strep + 1X glutamine + 1X NEAR. Cells are grown at 37°C in a
5% COZ
atmosphere until they reach 50-80% of confluency.
The transient transfection is performed by the CaPO4 method
(Sambroolc et al., 1989). Thus, expression plasmids for H-ras, N-ras, K-ras,
Myr-
ras or H-ras-CVLL are co-precipitated with the DSE-SEAP reporter construct. (A
ras expression plasmid is not included when the cell is transfected with the
pCMV-
SEAP plasmid.) For 10 cm plates 600 ~,1 of CaCl2-DNA solution is added
dropwise
while vortexing to 600 p1 of 2X HBS buffer to give 1.2 ml 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. No. 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 37°C under a 5% CO~
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 10 ml of phenol red free DMEM + 0.2% charcoal stripped calf serum + 1X
(Pen/Strep, Glutamine and NEAA ). Transfected cells are plated in a 96 well
micro-
titer plate (100 p,l/well) to which drug, diluted in media, has already been
added in a
volume of 100 p,1. The final volume per well is 200 p,1 with each drug
concentration
repeated in triplicate over a range of half-log steps.
Incubation of cells and drugs is for 36 hours at 37°C under CO2.
At
the end of the incubation period, cells are examined micro-scopically for
evidence of
cell distress. Next, 100 ~,1 of media containing the secreted alkaline
phosphatase is
removed from each well and transferred to a microtube array for heat treatment
at
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65°C for 1 hour 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 ~,1 media is combined with 200 ~1 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.
DNA-CaP04~recipitate for lOcm. plate of cells
Ras expression plasmid (1 ~,g/~l) 10 p,1
DSE-SEAP Plasmid (1 ~.gh,l) 2 p,1
Sheared Calf Thymus DNA (1 p.g/~,1) 8 ~1
2M CaCl2 74 ~,1
dH20 506 ~1
2X HBS Buffer
280mM NaCI
lOmM KCl
l.5mM Na2HP04 2H20
l2mM dextrose
50mM HEPE5
Final pH = 7.05
Luminesence Buffer (26m1)
Assay Buffer 20m1
Emerald ReagentTM (Tropix) 2.5m1
100mM homoarginine 2.5m1
CSPD Reagent~ (Tropix) 1.0m1
Assay Buffer
Add 0.05M Na2C03 to 0.05M NaHC03 to obtain pH 9.5.
Make 1mM in MgCl2
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EXAMPLE 9
The processing assays employed are modifications of that described by
DeClue et al [Cancer Research 51, 712-7I7, 1991].
K4B-Ras processing-inhibition assay
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 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
37°C for 2 hours 204 ,uCi/ml [35S]Pro-Mix (Amersham, cell labeling
grade) is added.
After introducing the label amino acid mixture, the cells are incubated
at 37°C 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 NaCl, 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
,ug/ml
antipain). The lysate is then centrifuged at 15,000 x g for 10 min at
4°C 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
appropri-
ate volume of lysate is brought to 1 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 4°C for 24 hours. The immune complex is collected
on pansorbin
(Calbiochem) coated with rabbit antiserum to rat IgG (Cappel) by tumbling at
4°C for
45 minutes. The pellet is washed 3 times with 1 ml of lysis buffer lacking DTT
and
protease inhibitors and resuspended in 100 ~,l elution buffer (10 mM Tris pH
7.4, 1%
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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 p,g Kirsten-ras specific
monoclonal antibody, c-K-ras Ab-I (Calbiochem). The second protein/antibody
mixture is incubated on ice at 4°C for 1-2 hours. The immune complex is
collected
on pansorbin (Calbiochem) coated with rabbit antiserum to rat IgG (Cappel) by
tumbling at 4°C 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, 1:100), and the Ras visualized
by
fluorography.
hDJ processing inhibition assay
PSN-1 cells are seeded in 24-well assay plates. Fox each compound
to be tested, the cells are treated with a minimum of seven concentrations in
half-log
steps. The final solvent (DMSO) concentration is 0.1%. A vehicle-only control
is
included on each assay plate. The cells are treated fox 24 hours at
37°C / 5% C02.
The growth media is then aspirated and the samples are washed
with PBS. The cells are lysed with SDS-PAGE sample buffer containing 5% 2-
mercaptoethanol and heated to 95°C for 5 minutes. After cooling on ice
for 10
minutes, a mixture of nucleases is added to reduce viscosity of the samples.
The plates are incubated on ice for another 10 minutes. The samples
are loaded onto pre-cast 8% acrylamide gels and electrophoresed at 15 mA/gel
for
3-4 hours. The samples are then transferred from the gels to PVDF membranes by
Western blotting.
The membranes are blocked for at least 1 hour in buffer containing 2%
nonfat dry milk. The membranes are then treated with a monoclonal antibody to
hDJ-
2 (Neomarkers Cat. # MS-225), washed, and treated with an alkaline phosphatase-
conjugated secondary antibody. The membranes are then treated with a
fluorescent
detection reagent and scanned on a phosphorimager.
For each sample, the percent of total signal corresponding to the
unprenylated species of hDJ (the slower-migrating species) is calculated by
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densitometry. Dose-response curves and ECSO values are generated using 4-
parameter
curve fits in SigmaPlot software.
EXAMPLE 10
Rap1 processing inhibition assay
Protocol A:
Cells are labeled, incubated and lysed as described in Example 9.
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
appropri-
ate 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 4°C far 1 hour. The immune
complex is
collected on pansorbin (Calbiochem) by tumbling at 4°C for 45 minutes.
The pellet
is washed 3 times with 1 ml of lysis buffer lacking DTT and protease
inhibitors and
resuspended in 100 ~,l elution buffer (10 mM Tris pH 7.4, 1% SDS). The Rap1 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 ~.g Rapl antibody,
Rapl/Krevl (121) (Santa Cruz Biotech). The second protein/antibody mixture is
incubated on ice at 4°C for 1-2 hours. The immune complex is collected
on pansorbin
(Calbiochem) by tumbling at 4°C 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 Rapl 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 Rap1 visualized by fluorography.
Protocol B:
PSN-1 cells are passaged every 3-4 days in l0cm plates, splitting
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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
15 cm
plates by trypsinization and diluted to 400,000 cells/ml in media. p.5ml of
these
diluted cells are added to each well of 24-well plates, for a final cell
number of
200,040 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 1000x of the final concentration (i.e., for a 10 ,uM
data point,
a 10 mM stock of the compound is needed).
2 ~,L of each 1000x compound stock is diluted into 1 ml media to
produce a 2X stock of compound. A vehicle control solution (2 ,uL 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 ,uL 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, 20 ~.L of an
RNAse/DNase
mix is added per well. This mix is lmg~ml DNaseI (Worthington Enzymes), 0.25
mg/ml Rnase A (Worthington Enzymes), 0.5 M Tris-HCI pH 8.0 and 50 mM 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 ,u1 of each sample is loaded
onto
the gel. The gel is run at 15 mA for about 3.5 hours. It is important to run
the gel
far enough so that there will be adequate separation between 21 kd (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% Tween-20). If blocked over the weekend, 0.02%
sodium azide is added. The membranes are blocked at 4°C with slow
rocking.
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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
Bio-
chemical 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 2 ml 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 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
precence of a detectable Rapla Western signal is indicative of inhibition of
Rapla
prenylation.
Protocol C:
This protocol allows the determination of an ECSo for inhibition of
processing of Rapla. The assay is run as described in Protocol B with the
following
modifications. 20 ~,1 of sample is run on pre-cast 10-20% gradient acrylamide
mini
gels (Novex Inc.) at 15 mA/gel for 2.5-3 hours. Prenylated and unprenylated
forms
of Rapla are detected by blotting with a polyclonal antibody (Rap1lKrev-1
Ab#121;
Santa Cruz Research Products #sc-65), followed by an alkaline phosphatase-
conjugated anti-rabbit IgG antibody. The percentage of unprenylated Rapla
relative
to the total amount of Rapla is determined by peak integration using
ImagequantTM
software (Molecular Dynamics). Unprenylated Rapla is distinguished from prenyl-
ated protein by virtue of the greater apparent molecular weight of the
prenylated
protein. Dose-response curves and ECSO values are generated using 4-parameter
curve fits in SigmaPlot software.
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EXAMPLE 11
Ira vivo tumor growth 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 cells/animal in 1 ml of DMEM salts) are injected
subcutaneously
into the left flank of 8-12 week old female nude mice (Harlan) on day 0. The
mice
in each oncogene group are randomly assigned to a vehicle or compound
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 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.
Bell, Ian M.
<120> INHIBITORS OF PRENYL-PROTEIN TRANSFERASE
<130> 20770
<150> US 601243,236
<151> 2000-10-25
<160> 25
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 4
<212> PRT
<213> Homosapien
<400> 1
Cys Val Leu Leu
1
<210> 2
<211> 4
<212> PRT
<213> Homosapien
<400> 2
Cys Val Leu Ser
1
<210> 3
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 3
Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met
1 5 10 15
<210> 4
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
-1-
CA 02426684 2003-04-16
WO 02/060868 PCT/USO1/50527
<223> completely synthesized
<400> 4
gagagggaat tcgggccctt cctgcatgctgctgctgctg ctgctgctgg gc 52
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 5
gagagagctc gaggttaacc cgggtgcgcggcgtcggtgg t 41
<210> 6
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 6
gagagagtct agagttaacc cgtggtccccgcgttgcttc ct 42
<210> 7
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 7
gaagaggaag cttggtaccg ccactgggctgtaggtggtg get 43
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 8
ggcagagctc gtttagtgaa ccgtcag 27
<210> 9
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
CA 02426684 2003-04-16
WO 02/060868 PCT/USO1/50527
<223> completely synthesized
<400> 9
gagagatctc aaggacggtg actgcag 27
<210> 10
<211> 86
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 10
tctcctcgag gccaccatgg ggagtagcaagagcaagcct aaggacccca gccagcgccg60
gatgacagaa tacaagcttg tggtgg 86
<210> 11
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 11
cacatctaga tcaggacagc acagacttgcagc 33
<210> 12
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 12
tctcctcgag gccaccatga cagaatacaagcttgtggtg g 41
<210> 13
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 13
cactctagac tggtgtcaga gcagcacacacttgcagc 38
<210> 14
<211> 38
<212> DNA
<213> Artificial Sequence
-3-
CA 02426684 2003-04-16
WO 02/060868 PCT/USO1/50527
<220>
<223> completely synthesized
<400> 14
gagagaattc gccaccatga cggaatataa gctggtgg 38
<210> 15
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 15
gagagtcgac gcgtcaggag agcacacact tgc 33
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 16
ccgccggcct ggaggagtac ag 22
<210> 167
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 17
gagagaattc gccaccatga ctgagtacaa actggtgg 38
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 18
gagagtcgac ttgttacatc accacacatg gc 32
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence
-4-
CA 02426684 2003-04-16
WO 02/060868 PCT/USO1/50527
<220>
<223> completely synthesized
<400> 19
gttggagcag ttggtgttgg g 21
<210> 20
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 20
gagaggtacc gccaccatga ctgaatataa acttgtgg 38
<210> 21
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 21
ctctgtcgac gtatttacat aattacacac tttgtc 36
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 22
gtagttggag ctgttggcgt aggc 24
<210> 23
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 23
gagaggtacc gccaccatga ctgaatataa acttgtgg 38
<210> 24
<211> 45
<212> DNA
<213> Artificial Sequence
-S-
CA 02426684 2003-04-16
WO 02/060868 PCT/USO1/50527
<220>
<223> completely synthesized
<400> 24
ctctgtcgac agattacatt ataatgcatt ttttaatttt cacac 45
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 25
gtagttggag ctgttggcgt aggc 24
-6-
