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, Ann. 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-
Aaal-Aaa2-Xaa" box (Cys is cysteine, Aaa is an aliphatic amino acid, the Xaa
is
any amino acid) (Willumsen et al., Nature 310:583-586 (1984)). 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 C20
isoprenoid,
respectively. (S. Clarke., Ann. Rev. Biochem. 61:355-386 (1992); W.R. Schafer
and
J. Rine, Ann. Rev. genetics 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. Chem.
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., Sciehce, 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. Sei 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 Medicine, 1:792-797 (1995).
Indirect inhibition of farnesyl-protein transferase in vivo has been
demonstrated with lovastatin (Merck & Co., Rahway, NJ) and compactin (Hancock
et al., ibid; Casey et al., ibid; Schafer et al., Science 245:379 (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. Acad. 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.5. 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 fused bicyclic compounds which
inhibit prenyl-protein transferase. Further contained in this invention are
chemo-
therapeutic compositions containing these prenyl-protein transferase
inhibitors and
methods for their production.
The compounds of this invention are illustrated by the formula I:
-3-
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(R3)m Y~
~C~R1 b)2)p
8
NR
~R1)s O~ 1a
~R )2)n
..
R2b
I N
R2a
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)m Y\
~~C~R1 b)2)p
8
NR
~Ri)s O~ 7a
~R )2)n
w ..
R2b
I N
R2a
wherein
-4-
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is selected from
i ~ ~ ~ ~/ M~ ~~
M / ~ M / or
M ~~ ,ss'w
Mis CorN;
R1 is independently
selected
from
a) H,
b) unsubstituted or substituted
C1-C( alkyl,
c) unsubstituted or substituted
C1-C( alkoxy,
d) unsubstituted or substituted
aryl,
e) unsubstituted or substituted
C2-Cg alkenyl,
f) unsubstituted or substituted
C2-Cg alkynyl,
g) unsubstituted or substituted
perfluoroalkyl,
h) halo,
i) OR10,
J) Rus(O)m~
k) R10C(O)NR10_~
1) -C(O)N(R10)2,
m) CN,
n) N02,
o) R10C(O)-
P) RlOpC(O)_~
q) N3,
r) _N(R10)2~ or
s) R110C(O)NR10_;
R1a and R1b are independently selected from:
a) H,
b) unsubstituted or substituted C1-C( alkyl,
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c) unsubstituted or substituted C1-C6 alkoxy,
d) unsubstituted or substituted aryl,
e) unsubstituted or substituted heterocycle,
f) unsubstituted or substituted aralkyl, or
g) -(CH2)n heterocycle;
R2a and R2b are independently selected from:
a) H,
b) unsubstituted or substituted C1-C6 alkyl,
c) unsubstituted or substituted aryl,
d) oxo,
e) unsubstituted or substituted heterocycle, or
f) unsubstituted or substituted C1-C6 alkoxy;
wherein the substituted group is substituted with from one to three
substituents
selected from:
1) unsubstituted or substituted C1-C6 alkyl,
2) unsubstituted or substituted C 1-C6 alkoxy,
3) halo,
4) unsubstituted or substituted aryl,
5) unsubstituted or substituted heterocycle,
6) unsubstituted or substituted C2-Cg alkenyl,
7) unsubstituted or substituted C2-Cg alkynyl,
8) unsubstituted or substituted perfluoroalkyl,
-OR10
10) R11S(O)m-,
11) R1~C(O)NR10_~
12) -C(O)N(R10)2,
13) CN,
14) N02,
15) -N(R10)2,
16) R10C(O)-,
17) R100C(O)-,
18) N3, or
19) R110C(O)NR10_;
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RZa and RZb may optionally be joined in a ring;
R3 is independently
selected
from:
a) H,
b) unsubstituted or substituted C1-C6
alkyl,
c) unsubstituted or substituted CZ-Cg
alkenyl,
d) unsubstituted or substituted CZ-Cg
alkynyl,
e) unsubstituted or substituted perfluoroalkyl,
10f) halo,
g) -OR10,
h) Rlls(O)m-~
i) R10C(p)NR10_~
j) -C(O)~10
15k) CN,
1) NO2,
m) R10C(O)-~
n) R100C(O)_~
o) N3~
20p) -N(R 10)2, or
Rl lpC(O)~10_;
Rg is selected from:
a) unsubstituted or substituted C1-C(
alkyl,
25b) unsubstituted or substituted C3-C10
cycloalkyl,
c) unsubstituted or substituted aryl,
d) unsubstituted or substituted CZ-Cg
alkenyl, or
e) unsubstituted or substituted CZ-Cg
alkynyl;
wherein
the substituted
group
is selected
from:
30 1) unsubstituted or substituted C1-C(
alkyl,
2) unsubstituted or substituted CZ-Cg
alkenyl,
3) unsubstituted or substituted CZ-Cg
alkynyl,
4) unsubstituted or substituted C1-C(
alkoxy,
5) CN,
35 6) unsubstituted or substituted aryl,
or
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7) unsubstituted or substituted heterocycle;
R10 is selected
from:
a) H,
b) unsubstituted or substituted C1-C6
alkyl,
c) unsubstituted or substituted C3-C10
cycloalkyl,
d) unsubstituted or substituted aryl,
e) unsubstituted or substituted heterocycle,
f) unsubstituted or substituted aralkyl,
or
g) unsubstituted or substituted heterocyclylalkyl;
R11 is selected from
a) unsubstituted or substituted C1-C6
alkyl,
b) unsubstituted or substituted C3-C10
cycloalkyl,
c) unsubstituted or substituted aryl,
d) unsubstituted or substituted heterocycle,
e) unsubstituted or substituted aralkyl,
or
f) unsubstituted or substituted heterocyclylalkyl;
Y is selected from:
a) heterocycle,
b) C3-C10 cycloalkyl,
c) C2-Cg alkenyl,
d) C2-Cg alkynyl,
e) C1-C( alkoxy,
f) CN,
g) C(O),
h) C(O)OR10,
i) -OR 10,
j) -N(R10)2, or
k) C(_~)~10
m is 0 to 4;
nis Oto4;
p is 1 to 4;
_g_
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sis Oto4;
or a pharmaceutically acceptable salt or stereoisomer thereof.
In a further embodiment of this invention, the inhibitors of prenyl-
protein transferase are illustrated by the formula A:
(R3)~
(C(Ri b)2)p
NR8
(R1)s O~ 1a
(C(R )2)n
\~ s
2b
N R
R2a
A
wherein
Mis CorN;
R1 is independently selected from
a) Ii,
b) unsubstituted or substituted C1-C( alkyl,
c) unsubstituted or substituted C1-C6 alkoxy,
d) unsubstituted or substituted
aryl,
e) unsubstituted or substituted
C2-Cg alkenyl,
f) unsubstituted or substituted
C2-Cg alkynyl,
g) unsubstituted or substituted
perfluoroalkyl,
h) halo,
i) OR10,
J) Rlls(O)m~
k) R10C(O)NR10_~
1) -C(O)N(R10)2,
_g_
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m) CN,
n) N02,
o) R10C(O)_~
p) RlOpC(O)_~
q) N3,
r) -N(R10)2, or
s) RllpC(O)~10_;
R1a is independently selected from:
a) H,
b) unsubstituted or substituted C1-C( alkyl,
c) unsubstituted or substituted C 1-C( alkoxy,
d) unsubstituted or substituted aryl, or
e) unsubstituted or substituted heterocycle;
R1b is independently
selected
from:
a) H,
b) unsubstituted or substituted
C1-C( alkyl,
c) unsubstituted or substituted
C1-C6 alkoxy,
d) unsubstituted or substituted
aryl,
e) unsubstituted or substituted
heterocycle,
f) unsubstituted or substituted
aralkyl, or
g) -(CH2)n heterocycle;
R2a and R2b are independently selected from:
a) H,
b) unsubstituted or substituted C 1-C( alkyl,
c) unsubstituted or substituted aryl,
d) oxo,
e) unsubstituted or substituted heterocycle, or
f) unsubstituted or substituted C1-C( alkoxy;
wherein the
substituted
group is
substituted
with from
one to three
substituents
selected
from:
1) unsubstituted or substituted C1-Cg alkyl,
2) unsubstituted or substituted C1-C( alkoxy,
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3) halo,
4) unsubstituted or substituted
aryl,
5) unsubstituted or substituted
heterocycle,
6) unsubstituted or substituted
C2-Cg alkenyl,
7) unsubstituted or substituted
C2-Cg alkynyl,
8) unsubstituted or substituted
perfluoroalkyl,
9) -OR10,
10) R11S(O)m-,
11) R10C(O)NR10-
12) -C(O)N(R10)2,
13) CN,
14) N02,
15) -N(R10)2,
16) R10C(O)-,
17) R100C(O)-,
18) N3, or
19) R110C(O)NR10_;
R2a and R2b may optionally be joined in a ring;
R3 is independently
selected
from:
a) H,
b) unsubstituted or substituted
C 1-C6 alkyl,
c) unsubstituted or substituted
C2-Cg alkenyl,
d) unsubstituted or substituted
C2-Cg alkynyl,
e) unsubstituted or substituted
perfluoroalkyl,
f) halo,
g) -OR 10,
h) R11S(O)m-~
i) R10C(O)NR10_~
j) -C(O)NR10,
k) CN,
1) N02,
m) R10C(O)_~
n) R100C(O)-,
- 11 -
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o) N3
p) -N~R10)2~ or
Rl lpC~p)NR10-;
R8 is selected from:
a) unsubstituted or substituted C1-C6 alkyl,
b) unsubstituted or substituted C3-C10 cycloalkyl,
c) unsubstituted or substituted aryl,
d) unsubstituted or substituted C2-Cg alkenyl, or
e) unsubstituted or substituted C2-Cg alkynyl;
wherein the substituted group is selected from:
1) unsubstituted or substituted C1-C6 alkyl,
2) unsubstituted or substituted C2-Cg alkenyl,
3) unsubstituted or substituted C2-Cg alkynyl,
4) unsubstituted or substituted C 1-C( alkoxy,
5) CN,
6) unsubstituted or substituted aryl, or
7) unsubstituted or substituted heterocycle;
R10 is selected from:
a) H,
b) unsubstituted or substituted C1-C( alkyl,
c) unsubstituted or substituted C3-C 10 cycloalkyl,
d) unsubstituted or substituted
aryl,
e) unsubstituted or substituted
heterocycle,
f) unsubstituted or substituted
aralkyl, or
g) unsubstituted or substituted
heterocyclylalkyl;
R 11 is selected from
a) unsubstituted or substituted C1-C6 alkyl,
b) unsubstituted or substituted C3-C10 cycloalkyl,
c) unsubstituted or substituted aryl,
d) unsubstituted or substituted heterocycle,
e) unsubstituted or substituted aralkyl, or
- 12 -
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f) unsubstituted or substituted heterocyclylalkyl;
Y is selected
from:
a) heterocycle,
b) C3-C10 cycloalkyl,
c) C2-Cg alkenyl,
d) C2-Cg alkynyl,
e) C1-C( alkoxy,
f) CN,
g) C(O),
h) C(O)OR10, .
i) -OR10,
J) -N(R10)2~ or
k) C(_~)~10;
mis Oto4;
nis Oorl;
p is 1 to 4;
sis Oto4;
or a pharmaceutically acceptable salt or stereoisomer thereof.
In another embodiment of the instant invention, the inhibitors of
prenyl-protein transferase are illustrated by the formula A:
(R3)m Y \
~C~Rlb~2~p
\NRs
O
(rigs\ \ ~ (Ria~2)n
~ ~ ''~ R
M ~ N
R2a
A
- 13 -
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wherein
M is C or N;
R1 is independently
selected
from
a) H,
b) unsubstituted or substituted
C1-C( alkyl,
c) unsubstituted or substituted
C1-C( alkoxy,
d) unsubstituted or substituted
aryl,
e) unsubstituted or substituted
perfluoroalkyl,
f) halo,
g) OR10
h) R10C(O)NR10_~
i) -C(O)N(R10)2,
j) CN,
k) R10C(O)-
1) R100C(O)_,
m) _N(R10)2, or
n) R110C(O)NR10_;
Rla is independently selected from:
a) H,
b) unsubstituted or substituted C1-C( alkyl, or
c) unsubstituted or substituted C1-C( alkoxy;
Rlb is independently
selected
from:
a) H,
b) unsubstituted or substituted
C1-C6 alkyl,
c) unsubstituted or substituted
C1-C( alkoxy,
d) unsubstituted or substituted
aryl,
e) unsubstituted or substituted
heterocycle,
f) unsubstituted or substituted
aralkyl, or
g) -(CH2)n heterocycle;
R2a, and R2b are independently selected from:
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a) H,
b) unsubstituted or substituted C1-C( alkyl,
c) unsubstituted or substituted aryl,
d) oxo,
e) unsubstituted or substituted heterocycle, or
f) unsubstituted or substituted C1-C( alkoxy;
wherein the
substituted
group is
substituted
with from
one to three
substituents
selected
from:
1) unsubstituted or substituted Cl-C( alkyl,
2) unsubstituted or substituted C1-C( alkoxy,
3) halo,
4) unsubstituted or substituted aryl,
5) unsubstituted or substituted heterocycle,
6) unsubstituted or substituted perfluoroalkyl,
7) -OR10,
g) R10C(O)~10_~
9) -C(O)N(R10)2,
10) CN,
11 ) N02, or
12) -N(R10)2;
R2a and R2b may optionally be joined in a ring;
R3 is independently
selected
from:
a) H,
b) unsubstituted or substituted
Cl-C( alkyl,
c) halo,
d) -OR 10,
e) Rlls(O)m-
f) RlOC(O)NR10_~
g) -C(O)~10
h) RIOC(O)-~
i) -N(R10)2, or
j) RllpC(O)NR10-;
- 15 -
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R8 is selected from:
a) unsubstituted or substituted C1-C6 alkyl,
b) unsubstituted or substituted C3-C10 cycloalkyl,
c) unsubstituted or substituted aryl,
d) unsubstituted or substituted C2-Cg alkenyl, or
e) unsubstituted or substituted C2-Cg alkynyl,
wherein the substituted group is selected from:
1) unsubstituted or substituted C1-C6 alkyl,
2) unsubstituted or substituted C2-Cg alkenyl,
3) unsubstituted or substituted C2-Cg alkynyl,
4) unsubstituted or substituted C1-C6 alkoxy,
5) CN,
6) unsubstituted or substituted aryl, or
7) unsubstituted or substituted heterocycle;
R10 is selected from:
a) Ha
b) unsubstituted or substituted C1-C6 alkyl,
c) unsubstituted or substituted C3-C 10 cycloalkyl,
d) unsubstituted or substituted aryl,
e) unsubstituted or substituted heterocycle,
f) unsubstituted or substituted aralkyl, or
g) unsubstituted or substituted heterocyclylalkyl;
R11 is selected from
a) unsubstituted or substituted C1-C6 alkyl,
b) unsubstituted or substituted C3-C10 cycloalkyl,
c) unsubstituted or substituted aryl,
d) unsubstituted or substituted heterocycle,
e) unsubstituted or substituted aralkyl, or
f) unsubstituted or substituted heterocyclylalkyl;
Y is selected from:
a) heterocycle,
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b) C3-C10 cycloalkyl,
c) C2-Cg alkenyl,
d) C~-Cg alkynyl,
e) C1-Cg alkoxy,
f) CN,
g) C(O),
h) C(O)OR10,
i) -OR10,
J) -N(R10)2~ or
k) C(=NH)NHR10;
m is 0 to 4;
n is 0 or l;
p is 1 to 4;
sis Oto4;
or a pharmaceutically acceptable salt or stereoisomer thereof.
In another embodiment of this invention, the inhibitors of prenyl-
protein transferase are illustrated by the formula B:
wherein
(R3)~ Y~---(CH2)p
a
NR
O
(R1)S (CH2)n
' N~ R2b
R2a
B
R1 is independently selected from
a) H,
- 17 -
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b) unsubstituted or substituted C1-Cg alkyl,
c) unsubstituted or substituted C1-C( alkoxy,
d) unsubstituted or substituted aryl,
e) unsubstituted or substituted perfluoroalkyl,
f) halo,
g) OR10
h) R10C(O)~10_~
i) -C(O)N(R10)2,
j) CN,
10k) R10C(O)-, or
1) _N(R 10)2;
R2a and
R2b are
independently
selected
from:
a) H,
15b) unsubstituted or substituted C 1-C6 alkyl,
c) unsubstituted or substituted aryl,
d) oxo,
e) unsubstituted or substituted heterocycle, or
f) unsubstituted or substituted C1-C( alkoxy;
20wherein
the substituted
group
is substituted
with from
one to
three
substituents
selec ted from:
1) unsubstituted or substituted C1-C( alkyl,
2) unsubstituted or substituted C1-C( alkoxy,
3) halo,
25 4) unsubstituted or substituted aryl,
5) unsubstituted or substituted heterocycle, .
6) unsubstituted or substituted perfluoroalkyl,
7) -OR10,
g) R10C(O)~10_~
30 9) -C(O)N(R 10)2, or
10) -N(R10)2;
R2a and R2b may optionally be joined in a ring;
- 18 -
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R3 is independently
selected
from:
a) H,
b) , unsubstituted or substituted C1-C(
alkyl,
c) halo,
d) -OR 10,
e) R10C(O)~10_~
f) _C(O)NR10~
g) RIOC(O)_~ or
h) _N(R10)2~
Rg is selected
from:
a) unsubstituted or substituted C1-C6
alkyl,
b) unsubstituted or substituted C3-C10
cycloalkyl,
c) unsubstituted or substituted aryl,
15d) unsubstituted or substituted C2-Cg
alkenyl, or
e) unsubstituted or substituted C2-Cg
alkynyl;
wherein
the substituted
group
is selected
from:
1) unsubstituted or substituted C1-Cg
alkyl,
2) unsubstituted or substituted C2-Cg
alkenyl,
3) unsubstituted or substituted C2-Cg
alkynyl,
4) unsubstituted or substituted C
1-C( alkoxy,
5) CN,
6) unsubstituted or substituted aryl,
or
7) unsubstituted or substituted heterocycle;
R10 is
selected
from:
a) H,
b) unsubstituted or substituted C1-C(
alkyl,
c) unsubstituted or substituted C3-C
10 cycloalkyl,
30d) unsubstituted or substituted aryl,
e) unsubstituted or substituted heterocycle,
f) unsubstituted or substituted aralkyl,
or
g) unsubstituted or substituted heterocyclylalkyl;
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RI I is selected from
a) unsubstituted or substituted C 1-Cg alkyl,
b) unsubstituted or substituted C3-Clp cycloalkyl,
c) unsubstituted or substituted aryl,
d) unsubstituted or substituted heterocycle,
e) unsubstituted or substituted aralkyl,
or
f) unsubstituted or substituted heterocyclylalkyl;
Y is selected from:
IO a) heterocycle, which is selected from pyridinyl, furanyl, pyrazolyl,
pyrimidinyl, pyrazinyl, imidazolyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl or thiofuranyl;
b) C3-C10 cycloalkyl,
c) C2-Cg alkenyl,
d) C2-Cg alkynyl,
e) C 1-Cg alkoxy,
f) CN,
g) C(O),
h) C(O)OR10,
i) -OR 10,
J) -N(R10)2~
or
k) C(=NH)NHR10;
mis Oto4;
n is 0 or 1;
p is 1 to 4;
sis Oto4;
or a pharmaceutically acceptable salt or stereoisomer thereof.
Examples of the compounds of the instant invention are:
N-isopropyl-2-(1-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide;
N-isopropyl-2-(1-methyl-2-o-tolyl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-
acetamide;
N-isopropyl-2-(7-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide;
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2-[1-(2-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ylmethyl)acetamide;
N-isopropyl-2-(2-phenyl-1H-indol-1-yl)-N-(pyridin-4-ylmethyl)acetamide;
N-isopropyl-2-(2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-pyridin-4-ylmethyl-
acetamide;
N-benzyl-N-isopropyl-2-(2-phenyl-1 H-indol-3-yl )acetamide;
N-ethyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)acetamide;
2-[ 1-(4-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ylmethyl)acetamide;
2-[ 1-(3-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ylmethyl)acetamide;
2-(2-phenyl-1H-indol-3-yl)-N,N-bis(pyridin-3-ylmethyl)acetamide;
2-(1-benzyl-1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-ylmethyl)acetamide;
2-[2-(4-bromophenyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ylmethyl)acetamide;
2-( 1-ethyl-2-phenyl-1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-
ylmethyl)acetamide;
N-isopropyl-2-(2-phenyl-1-propyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide;
2-(1-benzyl-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-
ylmethyl)
acetamide;
1-benzyl-4-{ [[(1-benzyl-2-oxo-2,3-dihydro-IH-indol-3-yl)acetyl]
(isopropyl)amino]
methyl }pyridinium;
N-cyclobutyl-2-(2-phenyl-I H-indol-3-yl)-N-pyridin-3-ylmethyl-acetamide;
N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-3-ylmethyl-acetamide;
2-[2-(2-chloro-phenyl)-1H-indol-3-yl]-N-isopropyl-N-pyridin-4-ylmethyl-
acetamide;
N,N-diallyl-2-(2-phenyl-1H-indol-3-yl)-acetamide;
N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-3-ylmethyl-acetamide;
(S)-N-sec-butyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-acetamide;
N-furan-3-ylmethyl-N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-acetamide;
N-(2-cyano-ethyl)-N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-acetamide;
N-cyclobutyl-2-(2-phenyl-1 H-indol-3-yl)-N-pyridin-4-ylmethyl-acetamide;
N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-acetamide;
2-[2-(3-chloro-phenyl)-1H-indol-3-yl]-N-isopropyl-N-pyridin-4-ylmethyl-
acetamide;
N-(2-cyano-ethyl)-N-cyclopropyl-2-(1-methyl-2-phenyl-1H-indol-3-yl)-acetamide;
N-isopropyl-2-(2-phenyl-IH-indol-3-yl)-N-pyrimidin-5-ylmethyl-acetamide;
N-cyclopropyl-N-(2-methyl-2H-pyrazol-3-ylmethyl)-2-(2-phenyl-1H-indol-3-yl)-
acetamide;
(+)-N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)propanamide;
N-(2-furylmethyl)-N-isopropyl-2-(2-phenyl-1H-indol-3-yl)acetamide;
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N-isopropyl-N-[(1-methyl-1H-pyrazol-5-yl)methyl]-2- (2-phenyl-1H-indol-3-yl)
acetamide;
N-isobutyl-N-methyl-2-(2-phenyl-1H-indol-3-yl)acetamide;
N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyrazin-2-ylmethyl)acetamide;
N-cyclopropyl-N-(3-furylmethyl)-2-(2-phenyl-1H-indol-3-yl)acetamide;
N-(2-cyanoethyl)-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-3-ylmethyl)acetamide;
N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyrazin-2-ylmethyl)acetamide;
N-isopropyl-N-[(1-methyl-1H-pyrazol-4-yl)methyl]-2-(2-phenyl-1H-indol-3-yI)
acetamide;
N-isopropyl-N-[(1-methyl-1H-pyrazol-3-yl)methyl]-2-(2-phenyl-1H-indol-3-yl)
acetamide;
N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)acetamide;
N-(2-cyanoethyl)-N-cyclopropyl-2-[2-(2-methylphenyl)-1H-indol-3-yl]acetamide;
N-isopentyl-N-methyl-2-(2-phenyl-1H-indol-3-yl)acetamide;
N-(2-cyanoethyl)-N-isopropyl-2-(2-phenyl-1H-indol-3-yl)acetamide;
N-cyclopropyl-N-[(1-methyl-1H-pyrazol-4-yl)methyl]-2-(2-phenyl-1H-indol-3-yl)
acetamide;
N-cyclopropyl-N-[(1-methyl-1H-pyrazol-3-yl)methyl]-2-(2-phenyl-1H-indol-3-yl)
acetamide;
N-cyclobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)acetamide;
N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)acetamide;
N-cyclopropyl-N-(2-furylmethyl)-2-(2-phenyl-1H-indol-3-yl)acetamide;
N-isopropyl-2-[2-(2-methoxyphenyl)-1H-indol-3-yl]-N-(pyridin-4-ylmethyl)
acetamide;
N-isobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)acetamide;
2-[2-(2-chlorophenyl)-1H-indol-3-yl]-N-(2-cyanoethyl)-N-cyclopropylacetamide;
N-(tert-butyl)-N-(2-cyanoethyl)-2-(2-phenyl-1H-indol-3-yl)acetamide;
N-isobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-3-ylmethyl)acetamide;
methyl N-cyclopropyl-N-[(2-phenyl-1H-indol-3-yl)acetyl]-beta-alaninate;
N-isopropyl-2-(6-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide;
N-isopropyl-2-(4-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)
acetamide;
N-isobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)acetamide;
N-cyclopentyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)acetamide;
N-[(1R)-1-methylpropyl]-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)
acetamide;
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N,N-bis(2-methoxyethyl)-2-(2-phenyl-1H-indol-3-yl)acetamide;
N-isoprop yl-2-(5-methyl-2-phenyl-1 H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide;
N-(2-cyanoethyl)-N-cyclopropyl-2-[2-(2-methoxyphenyl)-1H-indol-3-yl]acetamide;
N-c yclopentyl-2-(2-phenyl-1 H-indol-3-yl)-N-(pyridin-2-ylmethyl)acetamide;
N-cyclopentyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-3-ylmethyl)acetamide;
N-isopropyl-N-phenyl-2-(2-phenyl-1H-indol-3-yl)acetamide;
(2-phenyl-1H-indol-3-yl)acetyl N-[3-(dimethylamino)propyl]-N'-
ethylimidocarbamate;
N-cyclopropyl-N-((2-phenyl-1H-indol-3-yl)acetyl]-beta-alanine;
N-Isopropyl-2-(2-pyridin-3-yl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-acetamide;
N-Isopropyl-2-(2-pyridin-4-yl-1H-indol-3-yl)-N-pyridin-3-ylmethyl-acetamide;
N-Benzyl-N-isopropyl-2-(2-pyridin-4-yl-1H-indol-3-yl)-acetamide;
or a pharmaceutically acceptable salt or stereoisomer thereof.
Specific examples of the compounds of the instant invention are
illustrated by
N-isopropyl-2-(1-methyl-2-o-tolyl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-
acetamide;
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Me Me
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Me
Me~
N
O
~N
\
Me
N-isopropyl-2-(1-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide;
Me
Me~
N
O
-N
/ N Br
2-[1-(2-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ylmethyl)acetamide;
Me
Me~
N
0
~N
\ ~ ~ /
/ ~/
N-isopropyl-2-(2-phenyl-1H-indol-1-yl)-N-(pyridin-4-ylmethyl)acetamide;
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Me
Me~
N
O
-N
N-isopropyl-2-(2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-pyridin-4-ylmethyl-
acetamide;
N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-3-ylmethyl-acetamide;
N,N-diallyl-2-(2-phenyl-1H-indol-3-yl)-acetamide;
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O~ Me
~I \~N~..
N-furan-3-ylmethyl-N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-acetamide;
N
5.
N-(2-cyano-ethyl)-N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-acetamide;
or a pharmaceutically acceptable salt or stereoisorner 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
Stereochemistry of Carbon 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/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"
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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 IO carbon atoms, unless otherwise
specified. Examples of such cycloalkyl groups includes, but are not limited
to,
cyclopropyl, cyclobutyl, cyclohexyl, cycloheptyl, cyclooctyl, admantyl and the
like.
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 I0
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-Cg 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, "C~-Cg 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.
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 CI-C( 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
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a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic
heterocyclic ring which is either saturated or unsaturated, and which consists
of
carbon atoms and from one to four heteroatoms selected from the group
consisting
of N, O, and S, and including any bicyclic group in which any of the above-
defined
heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be
attached
at any heteroatom or carbon atom which results in the creation of a stable
structure.
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,
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 C 1-Cg alkyl linker, where alkyl
is defined above. Examples of heterocyclylalkyls include, but are not limited
to,
2-pyridylmethyl, 2-imidazolylethyl, 2-quinolinylmethyl, 2-imidazolylmethyl,
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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-Cg alkyl)2, oxo, OH, -O(C1-C( alkyl), -C(O)H, S(O)0_2,
(C1-C6 alkyl)S(O)0-2-, C2-C6 alkenyl, C2-C6 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-,
H2NC(O)NH-, (C1-C6 alkyl)C(O)-, -O(C1-C6 alkyl)CF3, (C1-C( alkyl)OC(O)-,
(C1-C( alkyl)O(C1-C( alkyl)-, (C1-C( alkyl)C(O)2(C1-C( alkyl)-, (C1-Cg 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 alkyl)2, N02, CN, N3, C1-C20 alkyl, C3-C20 cycloalkyl, -OH, -O(C1-C6
alkyl), S(O)0_2, (C1-C6 alkyl)S(O)0_2-, (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)2(C1-C6 alkyl)-,
(C1-C6 alkyl)OC(O)NH-, aryl, aralkyl, heterocycle, heterocyclylalkyl, halo-
aryl,
halo-aralkyl, haloheterocycle, haloheterocyclylalkyl, cyano-aryl, cyano-
aralkyl,
cyano-heterocycle and cyanoheterocyclylalkyl.
Lines drawn into the ring systems from substituents (such as from R1,
R2a, R3 etc.) indicate that the indicated bond may be attached to any of the
substitut-
able ring carbon or nitrogen atoms.
Preferably, R1 is independently selected from: hydrogen, OR10'
-N(R10)2, halo or unsubstituted or substituted C1-C6 alkyl.
Preferably, Rla and Rlb are independently selected from: hydrogen,
or unsubstituted or substituted C1-C( alkyl.
Preferably, R2a and R2b are independently selected from: hydrogen,
unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted aryl,
oxo or
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unsubstituted or substituted C1-C( alkoxy. Most preferably, R2a is
unsubstituted or
substituted aryl.
Preferably, R3 is independently selected from: hydrogen, unsubstituted
or substituted C1-C6 alkyl, unsubstituted or substituted aryl and OR10,
Preferably, Rg is selected from hydrogen, unsubstituted or substituted
C1_6 alkyl, unsubstituted~or substituted C2_g alkenyl, or unsubstituted or
substituted
C3-10 cycloalkyl.
Preferably, Y is selected from heterocycle, 03_10 cycloalkyl, C(O),
CN, or-OR10 . If Y is a heterocycle, preferably, the heterocycle is selected
from
pyridinyl, furanyl, pyrazolyl, pyrimidinyl, pyrazinyl, imidazolyl, oxazolyl,
thiazolyl,
triazolyl, tetrazolyl or thiofuranyl. If Y is a heterocycle, most preferably,
the
heterocycle is selected from pyridinyl, furanyl, pyrazolyl, pyrimidinyl, or
pyrazinyl.
Preferably, m is 0 or 1.
Preferably, n is 0, 1, or 2. Most preferably, n is 1.
Preferably p is 0, 1, or 2.
Preferably s is 0 or 1.
Preferably, the moiety
1
(R )s (C(Ri a)2)n
K
'R2b
'N
R2a
is selected from
1 is 1 C Ria
(R )s (C(R )2)n (R )s ~ ( )2)n
N
K ~ 2b
or K ~ ~ 2b
N R ~ R
R2a R2a
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More preferably,
(R1)S (R1)S
N
or
2b K ~ \ 2b
~~ N R ~~ R
R2a R2a
is selected from
~R1)s ..~ ~R1)s
\ \ \\ N
N\ 2b or
R ~ R2b
R2a R2a
It is intended that the definition of any substituent or variable (e.g.,
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, -NHC2H5,
etc. It is understood that substituents and substitution patterns on the
compounds
of the instant invention can be selected by one of ordinary skill in the art
to provide
compounds that are chemically stable and that can be readily synthesized by
techniques known in the art, as well as those methods set forth below, from
readily
available starting materials.
The pharmaceutically acceptable salts of the compounds of this
invention include the conventional non-toxic salts of the compounds of this
invention
as formed, e.g., from non-toxic inorganic or organic acids. For example, such
conventional non-toxic salts include those derived from inorganic acids such
as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the
like: and the
salts prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic,
lactic, malic, tartaric, citric, ascorbic, pamoic, malefic, hydroxymaleic,
phenylacetic,
glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,
toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and
the like.
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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;
AIBN 2,2'-Azobisisobutyronitrile;
BINAP 2,2'-Bis(diphenylphosphino)-1,1' binaphthyl;
Bn Benzyl;
BOC/Boc t-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;
DME 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;
HOBT 1-Hydroxybenzotriazole hydrate;
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HOOBT 3-Hydroxy-1,2,2-benzotriazin-4(3~-one;
HPLC High-performance liquid chromatography;
KOtBu Potassium t-butoxide;
LAH Lithium aluminum hydride;
MCPBA m-Chloroperoxybenzoic acid;
Me Methyl;
MeOH Methanol;
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) Tris(dibenzylideneacetone)dipalladinm
2 (0)
Ph phenyl;
PMSF a-Toluenesulfonyl chloride;
Py or pyr Pyridine;
PYBOP Benzotriazole-1-yl-oxy-trispyrrolidinophosphonium
(or PyBOP)hexafluorophosphate;
t Bu tart-Butyl;
TBAF Tetrabutylammoniumfluoride;
RPLC Reverse Phase Liquid Chromatography;
RT Room Temperature;
TBSCI tart-Butyldimethylsilyl chloride;
TFA Trifluoroacetic acid;
THF Tetrahydrofuran;
TIPS Triisopropylsilyl;
TMS Tetramethylsilane; and
Tr Trityl.
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.
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Synopsis of Schemes 1-6:
In the schemes below, the substituents and variables illustrated (such
as n, R2a, R8, Y, etc.) are defined in Formula I. Substituent R depicts the
possible
one to three substituents available when substituents R2a or R2b, of Formula
I, are
substituted.
The requisite intermediates are in some cases commercially available,
or can be prepared according to literature procedures, for the most part. In
Scheme
1, for example, the synthesis of 2-phenylindole-3-acetamides is described. To
a
solution of aldehyde 1 and amine 2 in methanol at 0°C is added sodium
borohydride.
I0 Reductive amination product 3 is then coupled with 2-phenylindole-3-acetic
acid (4)
to give the target amide 5. N-alkylation of amide 5 can also be effected by
treatment
with potassium tert-butoxide and an alkyl iodide, providing compound 6.
Preparation of indole-3-acetamides with various substituents attached
at the 2-position is illustrated in Scheme 2. Indole-3-acetonitrile (7) is
brominated to
give intermediate 8. Subsequent Suzuki coupling provides 2-substituted
compound 9.
Hydrolysis of the cyano group affords acid 10, which is then coupled with
amine 3 to
provide compound 11.
Scheme 3 depicts the preparation of indoles substituted at the 4, 5, 6,
and/or 7 positions. Acyl substituted acid 12 is coupled to amine 3 under
standard
conditions to provide amide 13. Formation of hydrazone 15 is followed by
Fischer
indolization to give indole 16. Using acids of type 12 substituted at the 2-
alkyl
position provides the corresponding alpha-substituted indoleacetamides.
Scheme 4 details the synthesis of N-arylmethyl substituted
indoleacetamides. Indole-3-acetic acid (17) is coupled to amine 3 to provide
amide
18. Subsequent alkylation with bromide 19 affords the N-alkylated product 20.
The isomeric indoles 24 can be prepared as described in Scheme 5.
Treatment of 2-substituted indole 22 with potassium hydroxide and methyl bromo-
acetate in wet DMSO effects alkylation and ester cleavage to provide acid 23.
Peptide coupling using amine 3 gives compound 24.
The synthesis of 2-substituted-azaindole-3-acetamides is outlined in
Scheme 6. Azaindole 27 is prepared from aminopyridine 25 and Weinreb amide 26
using the method of Hands et al. (Hands, D.; Bishop, B.; Cameron, M.; Edwards,
J.
S.; Cottrell, I. F.; Wright, S. H. B. Syntlaesis 1996, 877-882). A Mannich
reaction
with paraformadehyde as the amine component yields azagramine 28. 1h situ
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quaternization and cyanide displacement provides indolyl acetonitrile 29.
Basic
hydrolysis and peptide coupling as illustrated above then provides acetamide
31.
SCHEME 1
H
R NaBH4
O
Y + ~NH2 MeOH
1 2
Ra PyBOP
~N~ iPr2EtN
3
R8
~N~
KOtBu, R2bl
18-crown-6
Et20
R
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SCHEME 2
N
~C
(R1)s ( R2aB(OH)2
S O LiCI, Na2C03
N 2 - Pd(PPh3)4
toluene
7 H 8 H
N,
(R ' NaOH (F
R2a MeOH/H20
9 H 1~ H
0
R8
,N~ Y
H 3 Y (F
PyBOP
iPr2EtN R2a
DMF
11 H
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SCHEME 3
O Ra O Rs
R OH 3 \N'~Y R2a NAY
2a lvln ~ EDCHDMF lv)n
12 13
~R1)s
~R1)s. i \ \
14
HN.NH3+CI~ HN~N R$
toluene, reflux ~ NAY
n
R2a
15 O
s
N~
Y
ZrtCl2 ~Ri)s O ~ ~n
170°C \\ ~~ 2a
'?-R
H 16
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SCHEME 4
R8
OH a ~N~\Y
R
O 3 \N~\RA O
H _
\ EDC, DMF I ~ \
N ~ N
H 17 H 18
Ra
~N'~\
Br Y
jRF O
1s ~
DMF I / \> 20
NaH, N
SCHEME 5
OH
~Rt)s O ~R~)s O
\ R2a Me0' v Br ~ N
R2a
N KOH, wet DMSO
22 H ~ / 23
R8
Ra ~N_\Y
~N~Y O
H
PyBOP ~ N
iPr2EtN ~/ ~ R2a
DMF
~Ri)s 24
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SCHEME 6
O 1
~R~~\ Me R2a~N.OMe ~R )\ 2
Ra
26 Me
N
N NHBoc BuLi, THF; N H 2~
25 5.5 M HCI
~ Mel, MeOH;
~H
KCN, H20 _
paraformaldehyde
AcOH/dioxane
28 H
~Ri )s N(
\\ ~i
~R2a NaOH
N MeOH/H20
N
H 29
H au
Rs N ~Y
~N~ /
H Y lRi)SO
PyBOP ~ \
iPr EtN C~ \ R
DMF 'N N
H 31
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 in vitro farnesyl-protein transferase inhibitory activity, as assessed by
the assay
described in Example 67, 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 68. Preferably, a selective compound exhibits at least
1000
times greater activity against one of the enzymatic activities when comparing
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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 ICSps and ECSps the assays described in Example 72 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 in vitro inhibitory activity) for inhibition of
K4B-
Ras dependent activation of MAP kinases in cells at least 100-fold greater
than the ECSp 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 ire vitro inhibitory activity) against H-Ras
dependent activation of MAP kinases in cells at least 1000 fold lower than the
inhibitory activity (IC50) against H-ras-CVLL (SEQ.ll~.NO.: 1) dependent
activation of MAP kinases in cells.
When measuring Ras dependent activation of MAP kinases in cells the assays
described in Example 71 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.
In a SEAP assay, such as described in Example 71, it is preferred that
the dual inhibitor compound has an in 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 in vitro inhibitory activity) for inhibiting K4B-
Ras
dependent activation of MAP kinases in cells between 0.1 and 100 times the
IC50 for inhibiting the farnesylation of the protein hDJ in cells; and
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b) an IC50 (a measurement of in 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 ICSp (a measurement of in 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-CULL
(SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and
b) an ICSp (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 (ICSp) 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 in 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.
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 7I.
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.
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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, 55:4575-4580 (1995)). Such anti-angiogenesis
properties of
the instant compounds may also be useful in the treatment of certain forms of
vision
deficit related to retinal vascularization.
The compounds of this invention are also useful for inhibiting other
proliferative diseases, both benign and malignant, wherein Ras proteins are
aberrantly
activated as a result of oncogenic 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).
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.
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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.
(K. Sonoda et al., Invest. Ophthalmol. 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.
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. 601144,643, 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
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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
rnay be uncoated or they may be coated by known techniques to mask the
unpleasant
taste of the drug or delay disintegration and absorption in the
gastrointestinal tract and
thereby provide a sustained action over a longer period. For example, a water
soluble
taste masking material such as 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 kaolin, or as soft gelatin
capsules wherein the active ingredient is mixed with water soluble carrier
such
as polyethyleneglycol or an oil medium, for example peanut oil, liquid
paraffin,
or olive oil.
Aqueous suspensions contain the active material in admixture with
excipients suitable for the manufacture of aqueous suspensions. Such
excipients are
suspending agents, for example sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum
tragacanth and gum acacia; dispersing or wetting agents may be a naturally-
occurnng
phosphatide, for example lecithin, or condensation products of an alkylene
oxide
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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-occurnng
phosphatides, for example soy bean lecithin, and esters or partial esters
derived
from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and
condensation products of the said partial esters with ethylene oxide, for
example
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.
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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 microernulation.
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 cari
be
prepared by mixing the drug with a suitable non-irritating excipient which is
solid at
ordinary temperatures but liquid at the rectal temperature and will therefore
melt in
the rectum to release the drug. Such materials include cocoa butter,
glycerinated
gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of
various
molecular weights and fatty acid esters of polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or suspensions,
etc., containing the compound of Formula I are employed. (For purposes of this
application, topical application shall include mouthwashes and gargles.)
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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
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
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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, hexarnethyl 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
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.
48 -
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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
oligonucleotide comprising the amino acid sequence of SEQ.)D.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
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
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WO 02/28831 PCT/USO1/42389
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 neurolcinin-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,
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, 2 271 774, 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.
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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 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 97138697, published on October 23,
1997,
and herein incorporated by reference.
The instant compounds may also be useful in combination with
other inhibitors of parts of the signaling pathway that links cell surface
growth
factor receptors to nuclear signals initiating cellular proliferation. Thus,
the instant
compounds may be utilized in combination with farnesyl pyrophosphate
competitive
inhibitors of the activity of farnesyl-protein transferase or in combination
with a
compound.which has Raf antagonist activity. The instant compounds may also be
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WO 02/28831 PCT/USO1/42389
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 orally or parenterally, including intravenous,
intramuscular,
intraperitoneal, subcutaneous, rectal and topical routes of administration. It
is
preferred that such administration be orally. It is more preferred that such
administration be orally and simultaneously. When the protein substrate-
competitive
inhibitor and farnesyl pyrophosphate-competitive inhibitor are administered
sequentially, the administration of each can be by the same method or by
different
methods.
The instant compounds may also be useful in combination with
an integrin antagonist for the treatment of cancer, as described in U.S.
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 an integrin antagonist refers to compounds
which selectively antagonize, inhibit or counteract binding of a physiological
ligand
to an integrin(s) that is involved in the regulation of angiogenisis, or in
the growth
and invasiveness of tumor cells. In particular, the term refers to compounds
which
selectively antagonize, inhibit or counteract binding of a physiological
ligand to
the av~i3 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 av(33 integrin and the av(35
integrin,
or which antagonize, inhibit or counteract the activity of the particular
integrin(s)
expressed on capillary endothelial cells. The term also refers to antagonists
of the
a!(31, x2(31, a5(31, a6~31 and x6(34 integrins. The term also refers to
antagonists
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WO 02/28831 PCT/USO1/42389
of any combination of ocv(33 integrin, ocv(35 integrin, aril, x2(31, a5~31,
x6[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",
Chemistry &
Industry, 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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WO 02/28831 PCT/USO1/42389
HO O HO COOH
O OH
Lac lone Open-Acid
I I
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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WO 02/28831 PCT/USO1/42389
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.
Combinations 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); US Serial No. 08/404,833, filed on March 15,
1995; US 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;
WO 99/02175 (January 21, 1999); US Serial No. 09/112,656, filed on July 9,
1998;
and
WO 99128345 (June 10, 1999); US Serial No. 09/193,365, filed on November 17,
1998.
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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-
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°70
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WO 02/28831 PCT/USO1/42389
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
Preparation of N-isopropyl-2-(1-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide
Me
Me~
Me
Step A: Pre aration of N-(pyridin-4- l~meth~propan-2-amine
To a solution of pyridine-4-carboxaldehyde (0.53 g, 4.95mmo1) in
5 mL of MeOH was added isopropylamine (0.421 mL, 4.95 mmol). The resulting
solution was stirred at room temperature for 25 minutes, then cooled to
0°C. Solid
sodium borohydride (0.206 g, 5.44 mmol) was added, and the reaction was
stirred at
room temperature for 80 minutes. Saturated aqueous ammonium chloride was added
to the reaction, and the resulting mixture was stirred until gas evolution
ceased. The
mixture was partitioned between CH2C12 and saturated aqueous sodium
bicarbonate,
and the aqueous solution was extraced with CH2C12 (7 x). The combined organic
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CA 02424222 2003-03-31
WO 02/28831 PCT/USO1/42389
solutions were dried (Na2S04), filtered, and concentrated in vacuo to provide
a clear
oil. ES mass spectrum m/e 151 (M+1).
Step B: Preparation of N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-
4-ylmethyl)acetamide
A solution of N-(pyridin-4-ylmethyl)propan-2-amine (0.650 g) and
2-phenylindole-3-acetic acid (1.09 g, 4.33 mmol) in 10 mL of THF and 5 mL of
DMF was cooled to 0°C. EDC (0.913 g, 4.76 mmol) was added in one
portion.
The reaction was stirred at room temperature for 6 h, then partitioned between
EtOAc
and 1 N aqueous NaHS04. The aqueous solution was basified by addition of
sodium
carbonate, then extracted with EtOAc (4 x). The combined organic solutions
were
dried (Na2S04), filtered, and concentrated in vacuo. Flash chromatography
(linear
gradient, 10-40% MeOH/EtOAc) afforded the title compound as a white foam.
Exact mass calcd for C25H25N30: 384.2071; found: 384.2076 (FAB).
Step C: Preparation of N-isopropyl-2-(1-methyl-2-phenyl-1H-indol-3-yl)-N-
~pyridin-4-ylmethyl)acetamide
The indole described in Step B above (0.500 g, 0.261 mmol) was
added to a suspension of 18-crown-6 (0.007 g, 0.03 mmol) and KOtBu (0.044 g,
0.39 mmol) in 3 mL of Et20. The resulting orange suspension was stirred at
room
temperature for 15 minutes. Methyl iodide (0.024 mL, 0.39 mmol) was added via
syringe, and the reaction was stirred at room temperature for 3 hours 20
minutes.
The mixture was then paritioned between EtOAc and water. The aqueous solution
was extracted with EtOAc (2 x). The combined organic solutions were dried
(Na2S04), filtered, and concentrated in vacuo. Flash chromatography (linear
gradient, 99% CH2C12/0.9% MeOH/0.1% aqueous NH40H - 97% CH2C12/3.6%
MeOH/0.4% aqueous NH40H) afforded the title compound, which was isolated as
its
hydrochloride salt. ES mass spectrum m/e 398 (M+1).
EXAMPLE 2
Preparation of N-Isopropyl-2-(1-methyl-2-o-tolyl-1H-indol-3-yl)-N-pyridin-4-
lmeth~-acetamide
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CA 02424222 2003-03-31
WO 02/28831 PCT/USO1/42389
Me
Me~
N
O
-N
'N
Me Me
Step A: Preparation of (2-Bromo-1H-indol-3-yl)-acetonitrile
To a solution of indole-3-acetonitrile (13.0 g, 83.23 mmol) and silica
gel (13 g) in 130 ml of dry CH2C12 at 0°C was added NBS (14.8 g, 83.23
mmol).
The ice bath was then removed and the reaction stirred at room temperature for
16
hours. The solution was filtered, and the liquid filtrate was washed with 5%
aqueous
sodium metabisulfite (3 x), then dried (MgS04), filtered, and concentrated in
vacuo.
Flash chromatography (5%-30% EtOAc/hexane) provided the titled product.
St_ ep B: P. reparation of (2-o-Tolyl-1H-indol-3-yl)-acetonitrile
To a solution of (2-bromo-1H-indol-3-yl)-acetonitrile (200 mg, .851
mmol) and ortho-tolyl boronic acid (174 mg, 1.28 mmol) in 12 ml of toluene and
12
ml of ethanol was added 1 M aqueous Na2C03 (2.13 ml, 2.13 mmol), Pd(PPh3)4
(49.2 mg, .04 mmol), and LiCI (108.2 mg, 2.55 mmol). The reaction was heated
to
reflux for 3 hours then cooled to room temperature and stirred for 14 hours.
Upon
completion the reaction was concentrated in vacuo. Purification by silica gel
chromatography (0-20% EtOAc/hexane) provided the titled product.
Step C: Preparation of (2-o-Tolyl-1H-indol-3-yl)-acetic acid
To a solution of 2-ortho-tolyl-1H-indol-3-yl)-acetonitrile (0.196 g,
0.796 mmol) in 5 ml of MeOH was added 5 ml of 40% aqueous NaOH. The reaction
was heated to reflux for 16 hours, then cooled to room temperature. The
solution was
then acidified with 3 N aqueous HCl and washed with CH2C12 (3 x). The solution
was dried (MgS04), filtered, and concentrated in vacuo to provide the titled
product.
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Step D: Preparation of N-Isopropyl-N-pyridin-4-ylmethyl-2-(2-o-tolyl-1H-
indol-3-yl)-acetamide
To a solution of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid (0.100 g,
0.377 mmol) in 0.800 ml of DMF was N-(pyridin-4-ylrriethyl)propan-2-amine
(0.056
g, 0.38 mmol), DIEA (0.200 mL, 1.13 mmol), and PyBOP (0.216 g, 0.410 mmol).
The solution stirred at room temperature for 3 days. Upon completion of the
reaction,
the solution was purified by reverse phase chromatography to provide the
titled
product.
Step E: Preparation of N-Isopropyl-2-(1-methyl-2-o-tolyl-1H-indol-3-yl)-N-
pyridin-4- l~yl-acetamide
To a suspension of 18-crown-6 (0.002 g, 0.01 mmol) and KOtBu
(0.010 g, 0.09 mmol) in EtaO (1 mL) was added N-Isopropyl-N-pyridin-4-ylmethyl-
2-(2-o-tolyl-1H-indol-3-yl)-acetamide in one portion. After stirring at room
tempera-
ture for 15 minutes, iodomethane (0.013 g, 0.090 mmol) was added. The reaction
was stirred at room temperature for 24 hours and then quenched with water. The
mixture was poured into EtOAc, washed with water, dried (MgS04), filtered and
concentrated in vacuo. Purification by reverse phase HPLC yielded the titled
product.
ES mass spectrum m/e 412 (M+1).
EXAMPLE 3
Preparation of N-isopropyl-2-(7-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ly methyl)acetamide
Me
Me-\
N
O
-N
N
H
Me
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Step A: Preparation of N-isopropyl-4-oxo-4-phenyl-N-(pyridin-4-
ylmethyl)butanamide
To a solution of 4-oxo-4-phenylbutanoic acid (0.500 g, 2.81 mmol)
in 3.5 mL of DMF at room temperature were added N-(pyridin-4-ylmethyl)propan-
2-amine (0.464 g, 3.09 mmol) and EDC (0.591 g, 3.09 mmol). A white solid
precipi-
tated from the solution. The reaction mixture was stirred for 7 hours, then
partitioned
between EtOAc and saturated aqueous sodium bicarbonate. The aqueous solution
was extracted with EtOAc (2 x). The combined organic solutions were washed
with
1:1 saturated aqueous sodium chloride:water (1 x) and saturated aqueous sodium
chloride (1 x), then dried over sodium sulfate, filtered, and concentrated.
Flash
chromatography (linear gradient, 99% CH2C12/0.9% MeOH/0.1 % aqueous NH40H -
97% CH2C12/2.7% MeOH/0.3% aqueous NH40H) afforded the title compound.
Step B: Preparation of N-isopropyl-2-(7-methyl-2-phenyl-1H-indol-3-yl)-N-
(pyridin-4-ylmethyl)acetamide
The ketone prepared in Step A (0.051 g, 0.164 mmol) was dissolved
in 1 mL of toluene. Ortho-Tolylhydrazine hydrochloride (0.026 g, 0.164 mmol)
was added in one portion, and the resulting suspension was heated to
100°C. After
1 hour, the reaction was cooled to room temperature and concentrated in vaeuo.
The
unpurified hydrazone was combined with zinc chloride (0.112 g, 0.82 mmol), and
the mixture was heated to 170°C. After 5 minutes, the reaction was
cooled to room
temperature and diluted with acetone to give a brown solution. The solution
was
partitioned between EtOAc and water. The organic solution was washed with
saturated aqueous sodium chloride (1 x). The aqueous solutions were extracted
with EtOAc (2 x), and the combined organic solutions were washed with
saturated
aqueous sodium chloride (1 x), then dried over sodium sulfate, filtered, and
concentrated in vacuo. Purification by reversed phase HPLC provided the title
compound as its trifluoroacetate salt. ES mass spectrum m/e 398 (M+1).
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EXAMPLE 4
Preparation of 2-[1-(2-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ly methyl)acetamide
Me
Me~
N
O
~N
N Br
Ste~A: Preparation of 2-(1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-
1y methyl)acetamide
A solution of N-(pyridin-4-ylmethyl)propan-2-amine (4.93 g, 32.87
mmol) and indole-3-acetic acid (5.75 g, 32.81 mmol) in 48 mL of THF and 48 mL
of
DMF was cooled to 0°C. EDC (6.92 g, 36.10 mmol) was added in one
portion. The
reaction was stirred at room temperature for 15 hours, then partitioned
between
EtOAc and saturated aqueous sodium bicarbonate. The aqueous solution was
extracted with EtOAc (2 x). The combined organic solutions were washed with
saturated aqueous sodium chloride, dried (Na2S04), filtered, and concentrated
in
vacuo. Flash chromatography (linear gradient, 98% CH2C12/1.8% MeOH/0.2%
aqueous NH40H - 95% CH2C12/4.5% MeOH/0.5% aqueous NH40H) afforded a
pink foam. ES mass spectrum m/e 308 (M+1).
Sten B: Preparation of 2-[1-(2-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-
~pyridin-4-~methyl)acetamide
A 25 mL round-bottomed flask was charged with a 60% dispersion
of sodium hydride in mineral oil (0.033 g, 0.81 mmol). The suspension was
washed
with hexanes (2 mL), and the flask was charged with 2.5 mL of DMF and 0.250g
of the indole prepared in Step A. The suspension was stirred at room
temperature
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for 30 minutes to give a green solution. The solution was cooled to
0°C, and 2-
bromobenzyl bromide (0.203 g, 0.81 mmol) was added in one portion. After 1
hour, the reaction was quenched with saturated aqueous ammonium chloride, then
partitioned between EtOAc and saturated aqueous sodium bicarbonate. The
aqueous
solution was extracted with EtOAc (2 x). The combined organic solutions were
washed with saturated aqueous sodium chloride, dried (Na2SO4), filtered, and
concentrated in vacuo. Flash chromatography (linear gradient, 99% CH2Cl2/0.9%
MeOH/0.1% aqueous NHq.OH - 97% CH2C12/2.7% MeOH/0.3% aqueous NH40H)
afforded a white foam. Exact mass calcd for C26H26BrN30: 416.1332; found:
476.1323 (FAB).
EXAMPLE 5
Preparation of N-isopropyl-2-(2-phenyl-1H-indol-1-yl)-N-(pyridin-4-ylmethyl)
acetamide
Me
Me~
N
O
~N
\ N -
Step A: Preparation of (2-phenyl-1H-indol-1-yl)acetic acid
Freshly crushed KOH (2.69 g, 48.02 mmol) and DMSO were
combined and stirred at room temperature for 5 minutes. 2-phenylindole (2.00
g,
10.35 mmol) was added in one portion, and the resulting mixture was stirred
for 45
minutes. The reaction was cooled to 0°C, and methyl bromoacetate (1.96
mL, 20.70
mmol) was added via syringe. The reaction was stirred at room temperature for
8
hours 30 minutes, then poured into water. The mixture was extracted with EtOAc
(2 x) to remove residual starting material. The pH of the aqueous solution was
then
adjusted to 4 by addition of conc. aqueous HCI, and the solution was extracted
with
EtOAc (2 x). The organic extracts were dried (Na2S04), filtered, and
concentrated
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in vacuo to provide the title compound.
Step B: Preparation of N-isopropyl-2-(2-phenyl-1H-indol-1-yl)-N-(pyridin-4-
lmethyl)acetamide
To a solution of the acid from Step A above (0.050 g, 0.199 mmol) and
N-(pyridin-4-ylmethyl)propan-2-amine (0.033 g, 0.220 mmol) in 1 mL of DMF were
added diisopropylethylamine (0.069 mL, 0.400 mmol) and PYBOP (0.114g, 0.220
mmol). The reaction was stirred at room temperature for 15 hours, then
purified
directly by reverse phase HPLC (Clg, acetonitrile/water/TFA). Further
purification
by flash chromatography (linear gradient, 99% CH2C12/0.9% MeOH/0.1% aqueous
NH40H - 97% CH2Cl2/2.7% MeOH/0.3% aqueous NH40H) afforded the title
compound as a white foam. Exact mass calcd for C25H25N30: 384.2070; found:
384.2054 (FAB).
EXAMPLE 6
Preparation of N-Isopropyl-2-(2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-
pyridin-
4- l~yl-acetamide
Me
Me~
N
O
-N
N
N H
Step A: Preparation of 2-Phenyl-3-pyrrolidin-1-ylmethyl-1H-pyrrolo[2,3-b]
pyridine
A solution of 2-phenyl-1H-pyrrolo[2,3-b]pyridine (1.0 g, 5.15 mmol,
prepared according to: Hands, D.; Bishop, B.; Cameron, M.; Edwards, J. S.;
Cottrell,
I. F.; Wright, S. H. B. Synthesis 1996, 877-882.), pyrrolidine (2.2 g, 30.89
mmol), and
formaldehyde (37% by wt., 2.51g, 30.89 mmol) in HOAc/dioxane (2m1/8m1) was
stirred at room temperature for 72 hours. The solution was acidified with lml
of 3N
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HCl/lOml H20 and washed with ether. The water layer was then basified with
solid
K2CO3, and a white precipitate formed. The precipitate was washed with water
and
then with CH2C12 to yield the titled product.
Step B: Preparation of 2-Phen,1-~1H-pyrrolof2,3-blpyridin-3-yl)-acetonitrile
To a solution of 2-phenyl-3-pyrrolidin-1-ylmethyl-1H-pyrrolo[2,3-b]
pyridine in MeOH (7 ml) was added iodomethane (0.958 g, 6.75 mmol). The
reaction
stirred at room temperature for 3 hours. The MeOH was then removed in vacuo.
The
residue was dissolved in THF (10 ml) and treated with KCN (0.916 g, 14.06
mmol)
in water. The reaction stirred at reflux for 16 hours. Additional KCN (0.549
g, 8.44
mmol) was added, and the reaction continued to stir at reflux for 72 hours.
The
reaction was cooled to room temperature, filtered, and washed with water to
yield the
titled product.
Step C: Preparation of (2-Phenyl-1H-pyrrolof2,3-blpyridin-3-~l~-acetic acid
To a solution of 2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-acetonitrile
(0.703 g, 3.014 mmol) in 15 ml of MeOH was added 15 rnl of 40% aqueous NaOH.
The reaction was heated to reflux for 26 hours, then cooled to room
temperature. The
solution was acidified with 3 N HCl and washed with CH2C12 (3 x). The aqueous
solution was then washed with n-butanol (3 x). Both CH2C12 and n-butanol
solutions
were combined and concentrated in vacuo to yield the titled product.
Step D: Preparation of N-Isopropyl-2-(2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-
yl)-N-pyridin-4-ylmethyl-acetamide
To a solution of (2-Phenyl-1H-pyrrolo(2,3-b]pyridin-3-yl)-acetic acid
(0.200g, 0.793 mmol) in lml of DMSO was added N-(pyridin-4-ylmethyl)propan-2-
amine (0.119 g ,0.79 mmol), DIEA (0.41 ml, 2.38 mmol), and PYBOP (0.454 mg,
0.87 mmol). The solution stirred at room temperature for 3 days. Upon
completion
the reaction was directly purified by reverse phase HPLC to provide the titled
product. ES mass spectrum m/e 412 (M+1).
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EXAMPLE 7
Preparation of N-Benz 1-N-isoprop 1-~phenyl-1H-indol-3-yl)acetamide
Me
Me'\N
O
~%~N
H
The above-titled compound was prepared according to the procedure
of Example l, Step B, using benzylisopropylamine in place of N-(pyridin-4-
ylmethyl)
propan-2-amine.
EXAMPLE 8
Preparation of N-ethyl-2-(2-phenyl-1H-indol-3- l~-(pyridin-4-
ylmethyl)acetamide
N
O
~N
~%~N
The above-titled compound was prepared according to the procedure
of Example 1, Step B, using 4-(ethylaminomethyl)pyridine in place of N-
(pyridin-4-
ylmethyl)propan-2-amine.
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EXAMPLE 9
Preparation of 2-[1-(4-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ly methyl)acetamide
Me
Me~
N
O
-N
N
w
Br
The above-titled compound was prepared according to the procedures
of Example 4, Step B, using 4-bromobenzyl bromide in place of 2- bromobenzyl
bromide. Exact mass calcd for C26H26BrN30: 476.1332; found: 476.1312 (FAB).
EXAMPLE 10
Preparation of 2-[1-(3-bromobenzyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
1y methyl)acetamide
Me
Me~
N
O
-N
\
N Br
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The above-titled compound was prepared according to the procedures
of Example 4, Step B, using 3-bromobenzyl bromide in place of 2- bromobenzyl
bromide. Exact mass calcd for C2(H2(BrN30: 476.1332; found: 476.1312 (FAB).
EXAMPLE 11
Preparation of 2-(2-phenyl-1H-indol-3-yl)-N.N-bis ,pyridin-3-
lmethyl~)acetamide
N
/ v
N
\N
O
hi
The above-titled compound was prepared according to the procedure
of Example 1, Step B, using 3,3'-dipicolylamine in place of N-(pyridin-4-
ylmethyl)
propan-2-amine.
EXAMPLE 12
Preparation of 2-(1-benzyl-1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-ylmethyl)
acetamide
Me
Me~
N
0
-N
N
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The above-titled compound was prepared according to the procedures
of Example 4, Step B, using benzyl bromide in place of 2- bromobenzyl bromide.
Exact mass calcd for C26H27N30: 398.2227; found: 398.2201 (FAB).
EXAMPLE 13
Preparation of 2-[2-(4-bromophenyl)-1H-indol-3-yl]-N-isopropyl-N-(pyridin-4-
ylmethYl)acetamide
Me
Me~
N
O
-N
/ Br
N
H
The above-titled compound was prepared according to the
procedures of Example 3 using 3-(4-bromobenzoyl) propionic acid in place of
4-oxo-4-phenylbutanoic acid in Step A and phenylhydrazine hydrochloride in
placef of ortho-tolylhydrazine hydrochloride in Step B. ES mass spectrum m/e
462
(M+1).
EXAMPLE 14
Preparation of 2-(1-ethyl-2-phenyl-1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-
ylmethXl)acetamide
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Me
Me~
N
O
-N
'N
Me4
The above-titled compound was prepared according to the procedure
of Example 1, Step C using ethyl iodide in place of methyl iodide. Exact mass
calcd
for C27H29N30: 412.2383; found: 412.2368 (FAB).
EXAMPLE 15
Preparation of N-isopropyl-2-(2-phenyl-1-propyl-1H-indol-3-yl)-N-(pyridin-4-
, 1y methyl)acetamide
Me
Me~
N
O
~N
N
Me
The above-titled compound was prepared according to the procedure
of Example 1, Step C using n-propyl iodide in place of methyl iodide.
Exact mass calcd for C2gH31N30: 426.2540; found: 426.2537 (FAB).
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EXAMPLE 16
Preaparation of 2-(1-benzyl-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-isopropyl-N
(pyridin-4-ylmethyl) acetamide and 1-benzyl-4-{ [[(1-benzyl-2-oxo-2,3-dihydro
1H-indol-3-~rl)acet 1y 1 fisoprop~rl)aminolmeth~lpyridinium trifluoroacetate
Me
Me-\
N
O /
~N
~O
N
O
Me
Me~ O~CF3
N
o /
N+ I
\ ~ \
N
To a solution of 2-(1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-ylmethyl)
acetamide (0.050 g, 0.163 mmol) in 2 mL of DMSO was added solid KOH. The
reaction was stirred for 15 minutes at room temperature, then benzyl bromide
(0.031
g, 0.180 mmol) was added via syringe. After 5 hours stirring at room
temperature,
the reaction was acidified with conc. HCl and purified directly by reverse
phase
HPLC to give:
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(a) 2-(1-benzyl-2-oxo-2,3-dihydro-1H-indol-3-yl)-N-isopropyl-N-(pyridin-4-
ylmethyl)-acetamide as its trifluoroacetate salt.
ES mass spectrum m/e 414 (M+1); and
(b) 1-benzyl-4-{ [[(1-benzyl-2-oxo-2,3-dihydro-1H-indol-3-yl)acetyl]
(isopropyl)-
amino]methyl}pyridinium trifluoroacetate.
ES mass spectrum m/e 414 (M+1).
EXAMPLE 17
Preparation of N-Cyclobutyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-3-ylmethyl-
acetamide '
N'
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-3-carboxaldehyde in place of
pyridine-
4-carboxaldehyde and cyclobutylamine in place of isopropylamine.
ES mass spectrum mle 396 (M+1).
EXAMPLE 18
Preparation of N-Cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-3-ylmethyl-
acetamide
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N'
I-I
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-3-carboxaldehyde in place of
pyridine-
s 4-carboxaldehyde and cyclopropylamine in place of isopropylamine. Exact mass
calcd for C25H23N30: 381.1914; found: 382.1909 (FAB).
EXAMPLE 19
Preparation of 2-[2-(2-Chloro-phenyl)-1H-indol-3-yl]-N-isopropyl-N-pyridin-4-
~rlmethyl-acetamide
Me
Me~
N
O
~N
CI
The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 2-chlorophenyl boronic acid in place of ortho-
tolyl
boronic acid. ES mass spectrum m/e 418 (M+1).
EXAMPLE 20
Preparation of N,N-Diallyl-2-(2-phenyl-1H-indol-3-yl)-acetamide
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H
The above-titled compound was prepared according to the procedure
of Example 2, Step D, using diallylamine in place of N-(pyridin-4-
ylmethyl)propan-
2-amine and 2-phenylindole-3-acetic acid in place of (2-ortho-tolyl-1H-indol-3-
yl)-
acetic acid. ES mass spectrum m/e 331 (M+1).
EXAMPLE 21
Preparation of N-Isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-3-ylmethyl-
acetamide
Me
Me~
N
O ~ \N
N
H
I5
Step A: Preparation of N-(pyridin-3- 1y methxl)propan-2-amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using pyridine-3-carboxaldehyde in place of pyridine-4-
carboxaldehyde.
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Step B: Preparation of N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-3-
l~yl-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D, using N-(pyridin-3-ylmethyl)propan-2-amine in place of N-
(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of
(2-ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 384 (M+1).
EXAMPLE 22
Preparation of (S)-N-sec-Butyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-
acetamide
IVIC
N/~N~ Me
Step A: Preparation of (S)-N-(pyridin-3- l~methyl)sec-butyl-2-amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using (S)-N-sec-butylamine in place of isopropylamine.
Step B: Preparation of (S)-N-sec-Butyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-
4- l~methyl-acetamide
The above-titled compound was prepared according to the
procedure of Example 2, Step D using (S)-N-(pyridin-3-ylmethyl)sec-butyl-2-
amine,
as described above in Step A, in place of N-(pyridin-4-ylmethyl)propan-2-amine
and
2-phenylindole-3-acetic acid in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic
acid.
ES mass spectrum mle 398 (M+1).
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EXAMPLE 23
Preparation of N-Furan-3-ylmethyl-N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-
acetamide
~e
0
N / Me
O
N
H
Step A: Preparation of N-furan-3-ylmethyl-N-isopropylamine
The above-titled compound was prepared according to the procedure
of Example l, Step A using 3-furaldehyde in place of pyridine-4-
carboxaldehyde.
Step B: Preparation of N-furan-3-ylmethyl-N-isopropyl-2-(2-phenyl-1H-indol-
3-yl)-acetamide
The above-titled compound was prepared according to the
procedure of Example 2, Step D using N-furan-3-ylmethyl-N-isopropylamine, as
described above in Step A, in place of N-(pyridin-4-ylmethyl)propan-2-amine
and
2-phenylindole-3-acetic acid in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic
acid.
Exact mass calcd for C24H~4N202: 373.1911; found: 373.1928 (FAB).
EXAMPLE 24
Preparation of N-(2-Cyano-ethyl)-N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-
acetamide
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N
H
The above-titled compound was prepared according to the
procedure of Example 2, Step.D using 1-(cyclopropylamino)-propionitrile in
place
of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in
place
of (2-orth~-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 344 (M+1).
EXAMPLE 25
Preparation of N-Cyclobutyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-
acetamide
N
O
-N
N
H
The above-titled compound was prepared according to the procedures
of Example l, Steps A and B, using cyclobutylamine in place of isopropylamine.
ES mass spectrum m/e 396 (M+1).
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EXAMPLE 26
Preparation of N-Cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyridin-4-ylmethyl-
acetamide
N
O
-N
/ N
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using cyclopropylamine in place of
isopropylamine.
Exact mass calcd for C25H23N302: 382.1914; found: 382.1893 (FAB).
EXAMPLE 27
Preparation of 2-[2-(3-Chloro-phenyl)-1H-indol-3-yl]-N-isopropyl-N-pyridin-4-
ylmethyl-acetamide
Me
Me~
N
O
~N
N
CI
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The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 3-chlorophenyl boronic acid in place of ortho-
tolyl
boronic acid. ES mass spectrum m/e 418 (M+1).
EXAMPLE 28
Preparation of N-(2-Cyano-ethyl)-N-cyclopropyl-2-(1-methyl-2-phenyl-1H-indol-3-
yl)-acetamide
N\~
N
O
N
Me
The above-titled compound was prepared according to the procedure
of Example l, Step C using N-(2-Cyano-ethyl)-N-cyclopropyl-2-(2-phenyl-1H-
indol-
3-yI)-acetamide (Example 24) in place of N-isopropyl-2-(2-phenyl-1H-indol-3-
yl)-N-
(pyridin-4-ylmethyl)acetamide. ES mass spectrum m/e 358 (M+1).
EXAMPLE 29
Preparation of N-Isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyrimidin-5-ylmethyl-
acetamide
Me
Me~
N ~N
N
N
H
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Step A: Preparation of N-pyrimidine-5-ylmethyl-N-isopropylamine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using pyrimidine-5-carboxaldehyde in place of pyridine-4-
carboxaldehyde.
Step B: Preparation of N-Isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-pyrimidin-
5-, l~hyl-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-pyrimidine-5-ylmethyl-N-isopropylamine in place
of
N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of
(2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd for C24H24N40:
385.2023; found: 385.2040 (FAB).
EXAMPLE 30
Preparation of N-Cyclopropyl-N-(2-methyl-2H-pyrazol-3-ylmethyl)-2-(2-phenyl-1H-
indol-3-yl)-acetamide
Me
i
.N
N ~ N
O
\ -
N
Step A: Preparation of N-Cyclopropyl-N-(2-methyl-2H-pyrazol-3-
l~xl)amine
The above-titled compound was prepared according to the procedure
of Example l, Step A using 1-methyl-1H-pyrazole-5-carboxaldehyde in place of
pyridine-4-carboxaldehyde and cyclopropylamine in place of isopropylamine.
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Step B: Preparation of N-Cyclopropyl-N-(2-methyl-2H-pyrazol-3-ylmethyl)-2-
(2-phenyl-1H-indol-3-yl)-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-Cyclopropyl-N-(2-methyl-2H-pyrazol-3-ylmethyl)
amine in place of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-
acetic
acid in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd
for
C24H24N4~: 385.2023; found: 385.2023 (FAB).
EXAMPLE 31
Preparation of (+)-N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)
propanamide
N
Me
Me (\'
H
The above-titled compound was prepared according to the procedures
of Example 3 using 2-methyl-4-oxo-4-phenylbutanoic acid in place of 4-oxo-4-
phenylbutanoic acid in Step A and phenylhydrazine hydrochloride in place of
ortho-
tolylhydrazine hydrochloride in Step B. The (+)-enantiomer was obtained in
optically
pure form by chiral HPLC. Exact mass calcd for C2(H2~N30: 398.2227; found:
398.2228 (FAB).
EXAMPLE 32
Preparation of N-(2-fur 1y, methyl)-N-isoprop 1-y 2-(2-phenyl-1H-indol-3-
yl)acetamide
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O Me
(\,
Me
H
Step A: Preparation of N-Furan-2-ylmethyl-N-isopropylamine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 3=furaldehyde in place of pyridine-4-
carboxaldehyde.
Step B: Preparation of N-(2-furylmethyl)-N-isopropyl-2-(2-phenyl-1H-indol-3-
~rLl)acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-Furan-2-ylmethyl-N-isopropylamine in place of
N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 373 (M+1).
EXAMPLE 33
Preparation of N-isopropyl-N-(2-methyl-2H-pyrazol-3-ylmethyl)-2-(2-phenyl-1H-
indol-3 yl)-acetamide
Me
i
N,N Me
N/ Me
O
N
H
_ 8~ _
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Step A: Preparation of N-isopropyl-N-(2-methyl-2H-pyrazol-3-ylmeth~rl)amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 1-methyl-1H-pyrazole-5-carboxaldehyde in place of
pyridine-4-carboxaldehyde.
Step B: Preparation of N-isopropyl-N-(2-methyl-2H-pyrazol-3-ylmethyl)-2-(2-
phenyl-1 H-indol-3-yl)-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-isopropyl-N-(2-methyl-2H-pyrazol-3-ylmethyl)amine
in place of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic
acid
in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e
387
(M+1)
EXAMPLE 34
N-c~propyl-2-(2-phenyl-1H-indol-3-~)-N-(pyrazin-2-ylmeth~rl)acetamide
N
~~''N-~
N
O
N
H
Step A: Preparation of N-cyclopr~~pyrazin-2- l~yl)amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using pyrazine-2-carboxaldehyde in place of pyridine-4-
carboxaldehyde and cyclopropylamine in place of isopropylamine.
Step B: Preparation of N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyrazin-
2- lmethyl)acetamide
The above-titled compound was prepared according to the procedure
of Example 2~ Step D using N-cyclopropyl-N-(pyrazin-2-ylmethyl)amine in place
of
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N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of
(2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd for C24H22N40:
383.1869; found: 383.1866 (FAB).
EXAMPLE 35
Preparation of N-cyclopropyl-N-(3-furylmethyl)-2-(2-phenyl-1H-indol-3-yl)
acetamide
Step A: Preparation of N-Furan-3-ylmethyl-N-c~pro~ylamine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 3-furaldehyde in place of pyridine-4-carboxaldehyde
and
cyclopropylamine in place of isopropylamine.
Step B: Preparation of N-cyclopropyl-N-(3-furylmethyl)-2-(2-phenyl-1H-
indol-3-Xl)acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-Furan-3-ylmethyl-N-cyclopropylamine in place of
N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd for C24H22N202:
371.1744; found: 371.1754 (FAB).
EXAMPLE 36
Preparation of N-(2-cyanoethyl)-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-3-
ylmethyl)acetamide
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N
N~-CN
O
\ -
N
H
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-(2-cyanoethyl)-N-(pyridin-3-ylmethyl)amine in
place
of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in
place
of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 395 (M+1)
EXAMPLE 37
Preparation of N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyrazin-2-ylmethyl)
acetamide
N Me
~~''N-~
N Me
O
N
H
Step A: Preparation of N-isopropyl-N-(pyrazin-2-ylmethyl)amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using pyrazine-2-carboxaldehyde in place of pyridine-4-
carboxaldehyde.
Step B: Preparation of N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyrazin-2-
xlmethyl)acetamide
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The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-isopropyl-N-(pyrazin-2-ylmethyl)amine in place of
N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of
(2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd for C24H24N4~:
385.2035; found: 385.2023 (FAB).
EXAMPLE 3 8
N-isopropyl-N-[(1-methyl-1H-pyrazol-4-yl)methyl]-2-(2-phenyl-1H-indol-3-yl)
acetamide
Me~N ~ Me
~ ~~ N ~~
N Me
O
N
H
Step A: Preparation of N-isopropyl-N-(1-meth~rl-1H-pyrazol-4-ylmethyl)amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 1-methyl-1H-pyrazole-4-carboxaldehyde in place of
pyridine-4-carboxaldehyde.
Step B: Preparation of N-isopropyl-N-(1-methyl-1H-pyrazol-4-ylmethyl)-2-(2-
phenyl-1 H-indol-3-Xl)-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-isopropyl-N-(1-methyl-1H-pyrazol-4-ylmethyl)amine
in place of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic
acid
in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd for
C24H26N4~: 387.2174; found: 387.2179 (FAB).
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EXAMPLE 39
N-isopropyl-N-[(1-methyl-1H-pyrazol-3-yl)methyl]-2-(2-phenyl-1H-indol-3-yl)
acetamide
Me~N-N\ Me
~N'
Me
O
N
H
Step A: Preparation of N-isopropyl-N-(1-meth,1-~ 1H-Pyrazol-3-, l~yl)amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 1-methyl-1H-pyrazole-3-carboxaldehyde in place of
pyridine-4-carboxaldehyde.
Std: Preparation of N-isopropyl-N-(1-methyl-1H-pyrazol-3-ylmethyl)-2-(2-
phenyl-1H-indol-3-yl)-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-isopropyl-N-(1-methyl-1H-pyrazol-3-ylmethyl)amine
in place of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic
acid in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd
for
C24H26N40: 387.2156; found: 387.2179 (FAB).
EXAMPLE 40
Preparation of N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)
acetamide .
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N Me
N
Me
O
\ ~ -
N
H
Step A: Preparation of N-(pyridin-2-ylmeth~propan-2-amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using pyridine-2-carboxaldehyde in place of pyridine-4-
carboxaldehyde.
Step B: Preparation of N-isopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-
l~yl)acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D N-(pyridin-2-ylmethyl)propan-2-amine in place of N-
(pyridin-
4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place of (2-
ortho-
tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 384 (M+1)
EXAMPLE 41
Preparation of N-(2-cyanoethyl)-N-cyclopropyl-2-[2-(2-methylphenyl)-1H-indol-3-
yll acetamide
NC~
N
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The above-titled compound was prepared according to the
procedure of Example 2, Step D using 1-(cyclopropylamino)-propionitrile in
place of N-(pyridin-4-ylmethyl)propan-2-amine. ES mass spectrum m/e 344 (M+1)
EXAMPLE 42
Preparation of N-(2-cyanoethXl)-N-isoprop 1-~phenyl-1H-indol-3-yl)acetamide
NC~ Me
N
Me
O
\ ~ -
N
H
The above-titled compound was prepared according to the procedure
of Example 2, Step D, using using 1-(isopropylamino)-propionitrile in place of
N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 346 (M+1)
EXAMPLE 43
Preparation of N-cyclopropyl-N-[(1-methyl-1H-pyrazol-4-yl)methyl]-2-(2-phenyl-
1H-indol-3-yl) acetamide
Me~N
' N
N
O
N
H
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Step A: Preparation of N-cyclopropyl-N-(1-methyl-1H-pyrazol-4-ylmethyl)
amore
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 1-methyl-1H-pyrazole-4-carboxaldehyde in place of
pyridine-4-carboxaldehyde and cyclopropylamine in place of isopropylamine.
Step B: Preparation of N-cyclopropyl-N-(1-methyl-1H-pyrazol-4-ylmethyl)-2-
(2-phenyl-1H-indol-3-yl)-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-cyclopropyl-N-(1-methyl-1H-pyrazol-4-ylmethyl)
amine in place of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-
acetic
acid in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd
for
C24H24N4~: 385.2031; found: 385.2023 (FAB).
EXAMPLE 44
Preparation of N-cyclopropyl-N-[(1-methyl-1H-pyrazol-3-yl)methyl]-2-(2-phenyl-
1H-indol-3-yl) acetamide
Me~N.N~
Step A: Preparation of N-cyclopropyl-N-(1-methyl-1H-pyrazol-3-ylmethyl)
amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 1-methyl-1H-pyrazole-3-carboxaldehyde in place of
pyridine-4-carboxaldehyde and cyclopropylamine in place of isopropylamine.
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Std: Preparation of N-cyclopropyl-N-(1-methyl-1H-pyrazol-3-ylmethyl)-
2-(2-phenyl-1H-indol-3-yl)-acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-cyclopropyl-N-(1-methyl-1H-pyrazol-3-ylmethyl)
amine in place of N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-
acetic
acid in place of (2-ortho-tolyl-1H-indol-3-yl)-acetic acid. Exact mass calcd
for
C24H24N4~: 385.2028; found: 385.2023 (FAB).
EXAMPLE 45
Preparation of N-cyclobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)
acetamide
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-2-carboxaldehyde in place of
pyridine-
4-carboxaldehyde and cyclobutylamine in place of isopropylamine. Exact mass
calcd
for C26H25N3~: 396.2066; found: 396.2070 (FAB).
EXAMPLE 46
Preparation of N-cyclopropyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)
acetamide
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N
N
O
\ -
N
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-2-carboxaldehyde in place of
pyridine-
s 4-carboxaldehyde and cyclopropylamine in place of isopropylamine. Exact mass
calcd for C25H23N30: 382.1903; found: 382.1914 (FAB).
EXAMPLE 47
Preparation of N-cyclopropyl-N-(2-furylmethyl)-2-(2-phenyl-1H-indol-3-yl)
acetamide
H
Step A: Preparation of N-Furan-2-ylmeth~yclopropylamine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using 2-furaldehyde in place of pyridine-4-carboxaldehyde
and
cyclopropylamine in place of isopropylamine.
Step B: Preparation of N-cyclopropyl-N-(2-furylmethyl)-2-(2-phenyl-1H-
indol-3-girl acetamide
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The above-titled compound was prepared according to the procedure
of Example 2, Step D using N-Furan-2-ylmethyl-N-cyclopropylamine in place of N-
(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of (2-
ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 317 (M+1)
EXAMPLE 48
Preparation of N-isopropyl-2-[2-(2-methoxyphenyl)-1H-indol-3-yl]-N-(pyridin-4-
ylmeth,~) acetamide
N~ ~ ~ a
N
/ Me
Me
The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 2-methoxyphenyl boronic acid in place of ortho-
tolyl
boronic acid. ES mass spectrum m/e 414 (M+1)
EXAMPLE 49
Preparation of N-isobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)
acetamide
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Me
N ~Me
N
O
N
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-2-carboxaldehyde in place of
pyridine-
s 4-carboxaldehyde and isobutylamine in place of isopropylamine. Exact mass
calcd
for C26H27N30: 398.2218; found: 398.2227 (FAB).
EXAMPLE 50
Preparation of 2-[2-(2-chlorophenyl)-1H-indol-3-yl]-N-(2-cyanoethyl)-N-
c~propylacetamide
NCB
" CI
The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 2-chlorophenyl boronic acid in place of ortho-
tolyl
boronic acid and using 1-(cyclopropylamino)-propionitrile in place of N-
(pyridin-4-
ylmethyl)propan-2-amine. ES mass spectrum m/e 378 (M+1)
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EXAMPLE 51
Preparation of N-(tert-butyl)-N-(2-cyanoeth 1)-~ 2(2-phenyl-1H-indol-3-
yl)acetamide
NC Me
_~ Me
H
The above-titled compound was prepared according to the procedure
of Example 2, Step D using 1-(tent-butylamino)-propionitrile in place of N-
(pyridin-
4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place of (2-
ortho-
tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 360 (M+1)
EXAMPLE 52
Preparation of N-isobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-3-ylmethyl)
acetamide
Me
N ~ ~Me
JN
O
\ -
N
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-3-carboxaldehyde in place of
pyridine-
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4-carboxaldehyde and isobutylamine in place of isopropylamine. Exact mass
calcd
for C26H27N30: 398.2242; found: 398.2227 (FAB).
EXAMPLE 53
Preparation of Methyl N-cyclopropyl-N-[(2-phenyl-1H-indol-3-yl)acetyl]-beta-
alaninate
O
MeO
N'
O
N
H
A solution of N-(2-Cyano-ethyl)-N-cyclopropyl-2-(2-phenyl-1H-
indol-3-yl)-acetamide (0.100 g, 0.291 mmol) in MeOH : conc. HCl (5:1) was
heated
to reflux for 16 hours. The reaction was cooled to room temperature and poured
into CH2C12, washed with saturated aqueous NaHC03 and water, and then dried
(MgS04), filtered, and concentrated in vacuo. The material was then purified
by
reverse phase HPLC to yield the titled compound. ES mass spectrum m/e 377
(M+1).
EXAMPLE 54
Preparation of N-isopropyl-2-(6-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylrnethyl)acetamide and N-isopropyl-2-(4-methyl-2-phenyl-1H-indol-3-yl)-N-
(~yridin-4-ylmethyl) acetamide
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N~ a
n
/ ~ Me
N~N
Me
N
H
The above-titled compounds were prepared according to the procedure
of Example 3, Step B using meta-tolylhydrazine hydrochloride in place of ortho-
tolylhydrazine hydrochloride. The titled compounds were isolated as an
inseparable
mixture of regioisomers. Exact mass calcd for C2(H27N30: 398.2227; found:
398.2213 (FAB).
EXAMPLE 55
Preparation of N-isobutyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)
acetamide
Me Me
N
H
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The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using isobutylamine in place of isopropylamine.
Exact mass calcd for C2gH27N30: 398.2247; found: 398.2227 (FAB).
EXAMPLE 56
Preparation of N-cyclopentyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-ylmethyl)
acetamide
N N
O
N
H
The above-titled compound was prepared according to the procedures
of Example l, Steps A and B, using cyclopentylamine in place of
isopropylamine.
Exact mass calcd for C27H27N30: 410.2258; found: 410.2227 (FAB).
EXAMPLE 57
Preparation of N-[(1R)-1-methylpropyl]-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
1y methyl) acetamide
Me
N~~N~Me
~ 0
N 1
H
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Step A: Preparation of fR)-N-(pyridin-3- l~meth~)sec-butyl-2-amine
The above-titled compound was prepared according to the procedure
of Example 1, Step A using (R)-N-sec-butylamine in place of isopropylamine.
Step B: Preparation of N-[(1R)-1-methylpropyl]-2-(2-phenyl-1H-indol-3-yl)-
N-(pyridin-4- 1~~) acetamide
The above-titled compound was prepared according to the procedure
of Example 2, Step D using (R)-N-(pyridin-3-ylmethyl)sec-butyl-2-amine in
place of
N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of
(2-ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum mle 398 (M+1)
EXAMPLE 58
Preparation of N,N-bis(2-methoxyethYl)-2-(2-phenyl-1H-indol-3-yl)acetamide
Me~~
N~O,
Me
N
H
The above-titled compound was prepared according to the procedure
of Example 2, Step D using 2-methoxy-N-(2-methoxyethyl) ethanamine in place of
N-(pyridin-4-ylmethyl)propan-2-amine and 2-phenylindole-3-acetic acid in place
of
(2-ortho-tolyl-1H-indol-3-yl)-acetic acid. ES mass spectrum m/e 367 (M+1)
EXAMPLE 59
Preparation of N-isopropyl-2-(5-methyl-2-phenyl-1H-indol-3-yl)-N-(pyridin-4-
ylmethyl)acetamide
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Me
N~N
Me
O
Me
N
H
The above-titled compound was prepared according to the procedure
of Example 3, Step B using pare-tolylhydrazine hydrochloride in place of ortho-
tolylhydrazine hydrochloride. Exact mass calcd for C26H27N30: 398.2227; found:
398.2218 (FAB).
EXAMPLE 60
Preparation of N-(2-cyanoethyl)-N-cyclopropyl-2-[2-(2-methoxyphenyl)-1H-indol-
3-
y11 acetamide
Nc~
N'
" O
Me
The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 2-methoxyphenyl boronic acid in place of ortho-
tolyl
boronic acid and using 1-(cyclopropylamino)-propionitrile in place of N-
(pyridin-4-
ylmethyl)propan-2-amine. ES mass spectrum m/e 374 (M+1)
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EXAMPLE 61
Preparation of N-cyclopentyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-2-ylmethyl)
acetamide
N
N
O
N
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-2-carboxaldehyde in place of
pyridine-
4-carboxaldehyde and cyclopentylamine in place of isopropylamine. Exact mass
calcd for C27H27N30: 410.2238; found: 410.2227 (FAB).
EXAMPLE 62
Preparation of N-cyclopentyl-2-(2-phenyl-1H-indol-3-yl)-N-(pyridin-3-ylmethyl)
acetamide
N
/ v
N
O
\ -
N
H
The above-titled compound was prepared according to the procedures
of Example 1, Steps A and B, using pyridine-3-carboxaldehyde in place of
pyridine-
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4-carboxaldehyde and cyclopentylamine in place of isopropylamine. Exact mass
calcd for C27H27N30: 410.2254; found: 410.2227 (FAB).
EXAMPLE 63
Preparation of N-cyclopro~)rl-N-f(2-phenyl-1H-indol-3-,1)~ acet,1~1-beta-
alanine
O
HO~ ~
N' "
O
\ -
N
H
A solution of N-(2-cyano-ethyl)-N-cyclopropyl-2-(2-phenyl-1H-indol-
3-yl)-acetamide (0.050 g, 0.146 mmol) in 40% aqueous NaOH (1m1) was heated to
reflux for 5 hours and then cooled to room temperature. The reaction was
poured into
CH2C12 and washed. with 3 N HCI. The aqueous solution was then washed with
CH2C12 (3 x). The organic solutions were combined, dried (MgSO4), filtered and
concentrated in vacuo. Purification by reverse phase HPLC provided the titled
product. ES mass spectrum m/e 363 (M+1).
EXAMPLE 64
Preparation of N-Isopropyl-2-(2-pyridin-3-yl-1H-indol-3-yl)-N-pyridin-4-
ylmethyl-
acetamide
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a
N ~N
Me
O
N
The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 3-pyridine boronic acid in place of ortho-tolyl
boronic
acid. ES mass spectrum m/e 385 (M+1).
EXAMPLE 65
Preparation of N-Isopropyl-2-(2-pyridin-4-yl-1H-indol-3-yl)-N-pyridin-3-
ylmethyl-
acetamide
Me
i ~ N-~
Me
O
~N
~N
H
The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 4-pyridine boronic acid in place of ortho-tolyl
boronic
acid and using N-(pyridin-3-ylmethyl)propan-2-amine in place of N-(pyridin-4
ylmethyl)propan-2-amine. ES mass spectrum m/e 385 (M+1).
EXAMPLE 66
Preparation of N-Benzyl-N-isoprop 1-~pyridin-4-yl-1H-indol-3-yl)-acetamide
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~e
Me
H
The above-titled compound was prepared according to the procedures
of Example 2, Steps B-D using 4-pyridine boronic acid in place of ortho-tolyl
boronic
acid and using benzyl isopropylamine in place of N-(pyridin-4-ylmethyl)propan-
2-
amine. ES mass spectrum mle 384 (M+1).
EXAMPLE 67
In 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 wL): [3H]farnesyl diphosphate, Ras protein, 50 mM HEPES, pH 7.5,
a modulating anion (for example 5mM ATP), 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 minutes) by the addition of 1 M
HCl
in ethanol (1 mL). The quenched reactions are allowed to stand for 15 minutes
(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.
For inhibition studies, assays are run as described above, except
inhibitors are prepared as concentrated solutions in 100% dimethyl sulfoxide
and then
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diluted 20-fold into the enzyme assay mixture. Substrate concentrations for
inhibitor
IC50 determinations are as follows: FTase, 650 nM Ras-CVLS (SEQ.ID.NO.: 2),
100
nM farnesyl diphosphate.
The compounds of the instant invention are tested for inhibitory
activity against human FPTase by the assay described above.
EXAMPLE 68
Modified In vitro GGTase inhibition assay
The modified geranylgeranyl-protein transferase inhibition assay is
carned out at room temperature. A typical reaction contains (in a final volume
of
50 pL): [3H]geranylgeranyl diphosphate, biotinylated Ras peptide, 50 mM HEPES,
pH 7.5, a modulating anion (for example 10 mM glycerophosphate or 5mM ATP),
5 mM MgCl2, 10 ~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 minutes) 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. IC50 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 69
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 ~,Ci[35S]methionine (1000
Ci/mmol). After an additional 20 hours, the cells are lysed in 1 ml lysis
buffer (1%
NP40/20 mM HEPES, pH 7.515 mM MgCl2/1mM DTT/10 mg/ml aprotinen/2 mg/ml
leupeptin/2 mg/ml antipain/0.5 mM PMSF) and the Iysates cleared by
centrifugation
at 100,000 x g for 45 minutes. 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 ~,1 of a 25% suspension of protein A-Sepharose coated with rabbit anti rat
IgG
is added for 45 minutes. 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 NaCI) boiled in SDS-PAGE sample buffer and loaded on 13%
acrylamide gels. When the dye front reached the bottom, the gel is fixed,
soaked in
Enlightening, dried and autoradiographed. The intensities of the bands
corresponding
to farnesylated and nonfarnesylated ras proteins are compared to determine the
percent inhibition of farnesyl transfer to protein.
EXAMPLE 70
Cell-based in 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 71
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 (Merck)
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 arid 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. coli 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 plasrnid 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 Polymerase I. The "blunt ended" DNA containing the truncated SEAP
gene was isolated by electrophoresing the.digest in an agarose gel and cutting
out the
1910 base pair fragment. This 1910 base pair fragment was ligated into the
plasmid
pCMV-RE-AKI which had been cut with Bgl-II and filled in with E. coli Klenow
fragment DNA polymerase. Recombinant plasmids were screened for insert 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.NO.:S)
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.m.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-terminal PCR product containing HpaI and HindlII
restriction
sites. The sense strand C-terminal oligo (SEQ.)D.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-ligated 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 constitutive) expressin Sg EAP lap smid 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 Kieff, 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 religated. The resulting plasmid is named pCMV-AKI. Next, the
cytomegalovirus intron A nucleotide sequence was inserted downstream of the
CMV IEl promter in pCMV-AKI. 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 Hiigwood, 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 p 16T-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 SacI and Bgl-II and the larger vector
fragment isolated by agarose gel electrophoresis. The two gel isolated
fragments are
ligated at their respective SacI and Bgl-II sites to create plasmid pCMV-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 polymerise and the 1970 base pair
fragment
isolated from the vector fragment by agarose gel electrophoresis. The pCMV-AKI-
InA vector is prepared by digesting with Bgl-II and filling in the ends with
Klenow
DNA polymerise. 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 Sg_ EAP lap smid 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 religated. 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
polymerise
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 polymerise. The final construct is
generated
by blunt end ligating the SEAP fragment into the vector and transforming the
ligation
reaction into E. cola DHSacells. 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, IE1, and
upstream of a bovine growth hormone poly-A sequence. The plasmid would express
SEAP in a constitutive nammer when transfected into mammalian cells.
Clonin of a Myristylated 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:
5'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 CVLL 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.ID.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.1D.N0.: 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.
Cloning of 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.lD.NO.: 20)
Antisense strand:
5'-CTCTGTCGACGTATTTACATAATTACACACTTTGTC-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
Kpnl -Sal I cut mutagenesis vector pAlter-1 (Promega). Mutation of cysteine-12
to
a valine can be accomplished using the manufacturer's protocols and the
following
oligonucleotide:
5'-GTAGTTGGAGCTGTTGGCGTAGGC-3' (SEQ.ID.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.ID.NO.:23)
Antisense strand:
5'-CTCTGTCGACAGATTACATTATAATGCATTTTTTAATTTTCACAC-3'
(SEQ.B~.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 NEAA. Cells are grown at 37°C in a
5% C02
atmosphere until they reach 50-80% of confluency.
The transient transfection is performed by the CaPO~ method
(Sambrook 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 l Ocm plates 600 ~.1 of CaCl2-DNA solution is added
dropwise
while vortexing to 600 ~,1 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 hours at 37°C under a 5%
C02
atmosphere.
Following the DNA incubation period, the cells are washed with PBS
and trypsinized with lml of 0.05% trypsin. The 1 ml of trypsinized cells is
diluted
into 10 ml of phenol red free DMEM + 0.2% charcoal stripped calf serum + 1X
(Pen/Strep, Glutarnine and NEAA ). Transfected cells are plated in a 96 well
micro-
titer plate (100 ~.llwell) to which drug, diluted in media, has already been
added in a
volume of 100 ~,1. The final volume per well is 200 p1 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 C02.
At
the end of the incubation period, cells are examined micro-scopically for
evidence of
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cell distress. Next, 100 ,u1 of media containing the secreted alkaline
phosphatase is
removed from each well and transferred to a microtube array for heat treatment
at
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 w1 media is combined with 200 p,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-CaP~4 recipitate for lOcm. plate of cells
Ras expression plasmid (1 p,g/~,1) 10 ~1
DSE-SEAP Plasmid (1 p,g/p,l) 2 p,1
Sheared Calf Thymus DNA (1 pg/~,1) 8 w1
2M CaCl2 74 p,1
dH20 506 p,1
2X HBS Buffer
280mM NaCI
lOmM KCl
l.5mM Na2HP04 2H20
l2mM dextrose
50mM HEPES
Final pH = 7.05
Luminesence Buffer (26m1)
Assay Buffer 20m1
Emerald ReagentTM (Tropix) 2.5m1
100mM homoarginine 2.5m1
CSPD Reagent~ (Tropix) l.Oml
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Assay Buffer
Add 0.05M Na2C03 to 0.05M NaHC03 to obtain pH 9.5.
Make 1mM in MgCl2
EXAMPLE 72
The processing assays employed are modifications of that described by
DeClue et al [Cancer Research 51, 712-717, 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 ~,Ci/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 seconds at
room
temperature), and lysed by vortexing in 1 ml of lysis buffer (1% Nonidet P-40,
20
mM HEPES, pH 7.5, 150 mM NaCI, 1 mM EDTA, 0.5% deoxycholate, 0.1% SDS,
1 mM DTT, 10 ~,g/ml AEBSF, 10 ~,g/ml aprotinin, 2 ~,g/ml leupeptin and 2
~.g/ml
antipain). The lysate is then centrifuged at 15,000 x g for 10 minutes 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
,ug of
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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 p,1 elution buffer (10 mM Tris pH
7.4, 1%
SDS). The Ras is eluted from the beads by heating at 95°C for 5
minutes, after which
the beads are pelleted by brief centrifugation (15,000 x g for 30 seconds 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 pg Kirsten-ras specific
monoclonal antibody, c-K-ras Ab-1 (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 assax
PSN-1 cells are seeded in 24-well assay plates. For 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 for 24 hours at
37°C / 5% CO2.
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-
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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
densitometry. Dose-response curves and ECSp values are generated using
4-parameter curve fits in SigmaPlot software.
EXAMPLE 73
Rapt Processing Inhibition Assay
Protocol A:
Cells are labeled, incubated and lysed as described in Example 72.
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
p,g of
the Rapl antibody, Rapl/Krevl (121) (Santa Cruz Biotech), is added. The
protein/
antibody mixture is incubated on ice at 4°C for 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 p,1 elution buffer (10 mM Tris pH 7.4, 1% SDS). The Rapl is
eluted from the beads by heating at 95°C for 5 minutes, after which the
beads are
pelleted by brief centrifugation (15,000 x g for 30 seconds 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 Rapl antibody,
Rap1/Krev1 (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 Rapl visualized by fluorography.
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Protocol B:
PSN-1 cells are passaged every 3-4 days in lOcm plates, splitting
near-confluent plates 1:20 and 1:40. The day before the assay is set up, 5x
106 cells
are plated on l5cm plates to ensure the same stage of confluency in each
assay. The
media for these cells is RPM1 1640 (Gibco), with 15% fetal bovine serum and lx
Pen/Strep antibiotic mix. The day of the assay, cells are collected from the
l5cm
plates by trypsinization and diluted to 400,000 cells/ml in media. 0.5m1 of
these
diluted cells are added to each well of 24-well plates, for a final cell
number of
200,000 per well. The cells are then grown at 37°C overnight.
The compounds to be assayed are diluted in DMSO in 1/2-log
dilutions. The range of final concentrations to be assayed is generally 0.1-
100 ~,M.
Four concentrations per compound is typical. The compounds are diluted so that
each concentration is 1000x of the final concentration (i.e., for a 10 p.M
data point,
a 10 mM stock of the compound is needed).
2 ~uL of each 1000x compound stock is diluted into 1 ml media to
produce a 2X stock of compound. A vehicle control solution (2 ~,L DMSO to lml
media), is utilized. 0.5 ml of the 2X stocks of compound are added to the
cells.
After 24 hours, the media is aspirated from the assay plates. Each
well is rinsed with lml PBS, and the PBS is aspirated. 180 p,L SDS-PAGE sample
buffer (Novex) containing 5% 2-mercapto-ethanol is added to each well. The
plates
are heated to 100°C for 5 minutes using a heat block containing an
adapter for assay
plates. The plates are placed on ice. After 10 minutes, 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-HCl 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 ~,1 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
29 kd
(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
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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.
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 mI 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 p.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 (Rap1/Krev-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-
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ated protein by virtue of the greater apparent molecular weight of the
prenylated
protein. Dose-response curves and EC50 values are generated using 4-parameter
curve fits in SigmaPlot software.
EXAMPLE 75
In 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 vzvo
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.
Trotter, B. Wesley
Quigley, Amy G.
Dinsmore, Christopher J.
Lemma, William C., Jr.
Sisko, John T.
<120> Inhibitors of Prenyl-Protein Transferase
<130> 20731
<150> 60/237,241
<151> 2000-20-02
<160> 25
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 1
Cys Val Leu Leu
1
<210> 2
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 2
Cys Val Leu Ser
1
<210> 3
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 3
Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met
Z 5 10 15
<210> 4
-1-
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<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 4
gagagggaat tcgggccctt cctgcatgct gctgctgctgctgctgctgg gc 52
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 5
gagagagctc gaggttaacc cgggtgcgcg gcgtcggtggt ~ 41
<210> 6
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 6
gagagagtct agagttaacc cgtggtcccc gcgttgcttcct 42
<210> 7
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 7
gaagaggaag cttggtaccg ccactgggct gtaggtggtgget 43
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 8
ggcagagctc gtttagtgaa ccgtcag 27
<210> 9
<211> 27
<212> DNA
<213> Artificial Sequence
-2-
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<220>
<223> Completely synthetic sequence
<400> 9
gagagatctc aaggacggtg actgcag 27
<210> 10
<211> 86
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 10
tctcctcgag gccaccatgg ggagtagcaa gagcaagcct aaggacccca 60
gccagcgccg
gatgacagaa tacaagcttg tggtgg 86
<210> 11
<221> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 11
cacatctaga tcaggacagc acagacttgc agc 33
<210> 12
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 12
tctcctcgag gccaccatga cagaatacaa gcttgtggtg g 41
<210> 13
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 13
cactctagac tggtgtcaga gcagcacaca cttgcagc 38
<210> 14
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
-3-
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<400> 14
gagagaattc gccaccatga cggaatataa gctggtgg 38
<210> 15
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 15
gagagtcgac gcgtcaggag agcacacact tgc 33
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 16
ccgccggcct ggaggagtac ag 22
<210> 17
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 17
gagagaattc gccaccatga ctgagtacaa actggtgg 38
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 18
gagagtcgac ttgttacatc accacacatg gc 32
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 19
gttggagcag ttggtgttgg g 21
<210> 20
_ø_
CA 02424222 2003-03-31
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<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 20
gagaggtacc gccaccatga ctgaatataa acttgtgg 38
<210> 21
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 21
ctctgtcgac gtatttacat aattacacac tttgtc 36
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 22
gtagttggag ctgttggcgt aggc 24
<210> 23
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 23 -
gagaggtacc gccaccatga ctgaatataa acttgtgg 38
<210> 24
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Completely synthetic sequence
<400> 24
ctctgtcgac agattacatt ataatgcatt ttttaatttt cacao 45
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
-5-
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<220>
<223> Completely synthetic sequence
<400> 25
gtagttggag ctgttggcgt aggc 24
-6-
